around the world that have been vaccinated with BCG put Hofstra and the US healthcare system to shame.
If you suffer from an autoimmune disease or wish to prevent asthma or allergies in newborns shoot BCG.
If you have relapsing remitting MS shoot BCG. If you have cancer and are about ready to die swallow metformin and aspirin, a combination with a scientific basis that will never be widely applied because it is inexpensive. Money is like spray paint for some. Read and think and you will do better without ever going to medical school. Sadly Hiram Maxim continues to be the greatest doctor ever. There is not a single condition that he could not cure, cheaply, inexpensively and with a guarantee.
Hofstra North Shore-LIJ med school gets $10M endowment
Originally published: January 27, 2014 6:37 PM
Updated: January 27, 2014 9:42 PM
By
RIDGELY OCHS
ridgely.ochs@newsday.com
Hofstra University welcomed its first class of
medical students Monday as it opened a new medical school in conjunction
with North Shore-Long Island Jewish Health System. Videojournalist:
Katie Currid (July 25, 2011)
Hofstra North Shore-LIJ School of
Medicine has received a $10-million endowment for a scholarship fund --
the largest single gift to the medical school and among the largest
single gifts ever to the university.
The Louis Feil Charitable Lead Annuity
Trust, a charity with a history of giving to medical facilities,
research and education, made the endowment. It's named in memory of
Gertrude and Louis Feil, parents of Jeffrey Feil, chief executive of The
Feil Organization, a Manhattan real estate company.
Feil is a Rockville Centre resident and a trustee of the charity.
BLOG: The Daily Apple |
PHOTOS: Dropping LBs
DATA: Explore hospital rankings | Compare hospital charges |
Uninsured people in NY |
Compare hospital infection data
WEIGH IN: Ask your fitness questions
Dr. Lawrence Smith, the medical school dean, said the
scholarship fund would mean that more students would be able to graduate
with less debt from the medical school, where annual costs are close to
$50,000.
"Our commitment is that when a student
graduates from medical school, the amount of financial debt should not
be so great that it will pervert their ability to follow their
enthusiasm and not choose the field they want to go in," Smith said.
Last year, the Feil trust gave $5
million to Peconic Bay Medical Center in Riverhead toward a new
ambulatory care center in Manorville. In 2011, it donated $3 million to
South Nassau Communities Hospital in Oceanside. Louis and Gertrude Feil
were also longtime supporters of Weill Cornell Medical College in
Manhattan.
"The Feil trust has long supported
community-based health care facilities and programs that improve access
to high-quality medical care," Hofstra president Stuart Rabinowitz said
in a statement. "We are honored by this extraordinary support for the
students."
The medical school, with an enrollment
of 180, opened in 2011 to its first class, which will graduate next
year. Last summer, the school broke ground on a $35.9 million,
65,000-square-foot addition that will more than double its size.
Ristori
G, Romano S, Cannoni S, Visconti A, Tinelli E, Mendozzi L, Cecconi P,
Lanzillo R, Quarantelli M, Buttinelli C, Gasperini C, Frontoni M,
Coarelli G, Caputo D, Bresciamorra V, Vanacore N, Pozzilli C, Salvetti
M.
Neurology. 2014 Jan 7;82(1):41-8. doi: 10.1212/01.wnl.0000438216.93319.ab. Epub 2013 Dec 4.
- PMID:
- 24306002
- [PubMed - in process]
-
-
-
Front Immunol. 2013; 4: 478.
TNF Receptor 2 and Disease: Autoimmunity and Regenerative Medicine
Abstract
The
regulatory cytokine tumor necrosis factor (TNF) exerts its effects
through two receptors: TNFR1 and TNFR2. Defects in TNFR2 signaling are
evident in a variety of autoimmune diseases. One new treatment strategy
for autoimmune disease is selective destruction of autoreactive T cells
by administration of TNF, TNF inducers, or TNFR2 agonism. A related
strategy is to rely on TNFR2 agonism to induce T-regulatory cells (Tregs)
that suppress cytotoxic T cells. Targeting TNFR2 as a treatment
strategy is likely superior to TNFR1 because of its more limited
cellular distribution on T cells, subsets of neurons, and a few other
cell types, whereas TNFR1 is expressed throughout the body. This review
focuses on TNFR2 expression, structure, and signaling; TNFR2 signaling
in autoimmune disease; treatment strategies targeting TNFR2 in
autoimmunity; and the potential for TNFR2 to facilitate end organ
regeneration.
Keywords: TNF, TNF receptor 2, autoimmune disease, type 1 diabetes, regeneration
Introduction
Tumor
necrosis factor (TNF) is a pleiotropic cytokine involved in regulating
diverse bodily functions including cell growth modulation, inflammation,
tumorigenesis, viral replication, septic shock, and autoimmunity (
1).
These functions hinge upon TNF’s binding to two distinct membrane
receptors on target cells: TNFR1 (also known as p55 and TNFRSF1A) and
TNFR2 (also known as p75 and TNFRSF1B). TNFR1 is ubiquitously expressed
on the lymphoid system and nearly all cells of the body, which likely
accounts for TNF’s wide-ranging functions. TNFR2 is expressed in a more
limited manner on certain populations of lymphocytes, including
T-regulatory cells (T
regs) (
2,
3), endothelial cells, microglia, neuron subtypes (
4,
5), oligodendrocytes (
6,
7), cardiac myocytes (
8), thymocytes (
9,
10), islets of Langerhans (personal communication, Faustman Lab), and human mesenchymal stem cells (
11).
Its more restricted cellular expression makes TNFR2 more attractive
than TNFR1 as a molecular target for drug development. Activation of
TNFR1 alone by exogenous TNF is systemically toxic (
12,
13).
As
a general rule, TNF depends on TNFR1 for apoptosis and TNFR2 for any
function related to cell survival, although there is some degree of
overlapping function depending upon the activation state of the cell and
a variety of other factors (
14). Likewise, TNFR1 and TNFR2 have distinct intracellular signaling pathways, although there is some overlap and crosstalk (
15).
TNF binding to TNFR1 triggers apoptosis through two pathways, by
activation of the adaptor proteins TNFR1-associated death domain (TRADD)
and Fas-associated death domain (FADD). In contrast, TNFR2 signaling
relies on TRAF2 and activation and nuclear entry of the pro-survival
transcription factor nuclear factor-kB (NFkB) (
16–
18). TNFR2 expression on T
regs is induced upon T-cell receptor activation (
19).
While
the etiologies of autoimmune disorders vary, there is some degree of
overlap in their genetic, post-translational, and environmental origins.
One overlapping feature is that various defects in TNF signaling
pathways, acting through the TNF receptors and NFkB in autoreactive T
cells, occur in both human and mouse models of various autoimmune
disorders, including Crohn’s disease, Sjogren’s syndrome, multiple
sclerosis, ankylosing spondylitis, and type I diabetes (
20–
39).
The defects range from defects in the proteasome in both the non-obese
diabetic (NOD) mouse model and humans with Sjogren’s syndrome, to
specific polymorphisms in the TNFR1 or TNFR2 receptors themselves, to
punitive interruptions in genes that control the ubiquitination of the
NFkB pathway.
TNFR Expression, Structure, and Signaling
As
noted above, TNFR1 and TNFR2 possess different patterns of expression.
TNFR1 is found on nearly all bodily cells, whereas TNFR2 is largely
found on certain immune cells (CD4+ and CD9+ lymphocytes), certain CNS
cells, and endothelial cells, among others. Neither receptor is located
on erythrocytes. Typically, cells that express TNFR2 also express TNFR1,
with the ratio of expression varying according to cell type and
functional role. Because TNFR1 typically signals cell death, while TNFR2
typically signals cell survival, the ratio of their co-expression will
shift the balance between cellular survival and apoptosis.
TNFR1
and TNFR2 have extracellular, transmembrane, and cytoplasmic
components. The extracellular component of both receptors is rich in
cysteine, which is characteristic of the TNF superfamily. TNFR1 contains
434 amino acids. Its intracellular region of 221 amino acids contains a
death domain that binds TRADD or FADD. In T cells, activation of TRADD
or FADD activates the caspases, resulting in apoptosis (Figure ). A second apoptotic pathway relies on TRADD’s activation of RIP (receptor interacting protein) (Figure A).
In contrast to TNFR1, TNFR2 does not have a cytoplasmic death domain.
The receptor consists of 439 amino acids. Its extracellular domain is
formed by the first 235 amino acids, its transmembrane domain is formed
by 30 amino acids, while its cytoplasmic domain is formed by 174 amino
acids. TNFR2’s cytoplasmic domain has a TRAF2 binding site. TRAF2, in
turn, binds TRAF1, TRAF3, cIAP1, and cIAP2 (
17,
18). These signaling proteins activate several other signaling proteins, yielding cell survival (Figure A).
Cell survival is ensured when the transcription factor NFkB is
liberated from its inhibitor protein IkBα in the cytoplasm and
translocates to the nucleus where it activates pro-survival target genes
(
40).
Both TNFR1 and TNFR2 can bind monomeric TNF or trimeric soluble TNF
although soluble TNF induces no or weak signaling for TNFR2. This may be
related to altered association or dissociation kinetics or more optimal
kinetics with pre-formed transmembrane TNF (
41). TNFR2 also preferentially binds transmembrane TNF (
42).
Transmembrane TNF is a trimer on the cell surface and transmits signals
to the cell where it is contained, i.e., reverse signaling. It is
thought that TNFR2 preferentially binds transmembrane bound TNF (
43).
Solution of the crystal structure of the TNF-TNFR2 complexes
demonstrated that these interactions also result in the formation of
aggregates on the cell surface and this likely promotes signaling (
44).
TNF Signals through
TNFR1 andTNFR2
receptors (A) but abnormalities in this signaling pathway in
autoimmunity (B) can favor a pathway of selective apoptosis due to a
variety of protein signaling defects.
Transgenic mice have been produced to try to understand better the function of TNFR2 (
45). TNFR2
−/− mice homozygous for TNFR2
−/−
are viable and fertile. They also show normal T-cell development and
activity and are resistant to TNF-induced death. The T-cell
proliferation responses are diminished and they also show abnormal
central nervous system regeneration (JAX Mice Database – 00260).
TNF in Development and Autoimmunity
Tumor
necrosis factor, and its signaling through the two receptors, plays
several crucial roles during normal development. It shapes the efficacy
of the immune system and protects against infectious disease, cancer,
and autoimmune disease (
46).
Upon release, TNF proceeds throughout the lifecycle to exert regulatory
roles over immune cells by triggering transcription of genes
responsible for inflammation, proliferation, differentiation, and
apoptosis. To counter a pathological infection, TNF facilitates
proliferation of immune cell clones. To continue to fight against the
infection, TNF stimulates differentiation and recruitment of naïve
immune cells. Subsequently, TNF orchestrates destruction of superfluous
immune cell clones to reduce inflammation and tissue damage once the
infection is resolved.
In the process
of developing autoimmunity, abnormal progenitors to T cells and other
immune cell types proliferate and begin to mature in the thymus. T-cell
education occurs through two parallel pathways for CD4 and CD8 T cells
through either HLA class II or HLA class I cell surface structures. For
almost all autoimmune disease there is strong genetic linkage to the HLA
class II region. This genetic region is rich in immune response genes
and contains not only the class II genes themselves but also the HLA
class I assembly genes such as the tap transporters (Tap1/Tap2) and
proteasome genes (that control self peptide presentation) such as LMP2
(PSMB9), LMP7 (PSMB8), and LMP10 (PSMB10) (
47).
During T-cell education, the vast majority of immature immune cells die
by apoptosis, which serves to remove defective progenitors. The process
is not foolproof, however. Failures in T-cell education in humans
perhaps driven by defective antigen presentation allow autoreactive but
still immature T cells defined as CD45RA+ (2H4) and lesser numbers of
CD45RO+ (4B4) to enter the circulation (
36,
48,
49).
In humans and autoimmune animal models diverse mutations and
polymorphisms drive altered proteasome function with varying phenotypes
of autoimmunity (
50–
55).
Once in the circulation, the cells differentiate into mature
autoreactive T cells when they encounter specific self-antigens (
56).
The failure of T-cell education of autoreactive CD8 T cells, due to HLA
class I interruption, yields self-reactive T cells directed at specific
self-antigens. This failure underlies various immune diseases,
including type I diabetes, Crohn’s disease, multiple sclerosis, and
Sjogren’s syndrome (
50).
TNFR2 Signaling and Benefits to Health
TNFR2
signaling pathways appear to offer protective roles in several
disorders, including autoimmune disease, heart disease, demyelinating
and neurodegenerative disorders, and infectious disease. According to
in vitro and
in vivo studies, TNF or TNFR2 agonism is associated with pancreatic regeneration (
57–
59), cardioprotection (
60,
61), remyelination (
5,
6), survival of some neuron subtypes (
5,
62,
63), and stem cell proliferation (
11,
64–
66).
Knockout of the
tnfr2 gene in a mouse model produces a higher rate of heart failure and reduced survival after myocardial infarction (
60).
TNFR1 signaling is deleterious and TNFR2 signaling is protective in
regeneration and repair processes following infarcted myocardium in
female mice (
61).
An
agonist for TNFR2 selectively destroys autoreactive T cells but not
healthy T cells in blood samples from type I diabetes patients, as well
as multiple sclerosis, Graves, Sjogren’s autoreactive T cells (
57).
Animal models of type I diabetes exhibit massive regeneration of the
pancreas after elimination of autoreactive T cells with low-dose TNF (
58,
59). TNFR2 is crucial for TNF-induced regeneration of oligodendrocyte precursors that make up myelin (
6),
a finding that may be important in the treatment of multiple sclerosis
and other demyelinating disorders, regardless of whether they have an
autoimmune etiology. In viral encephalitis-infected knockout mice, the
TNFR2 pathway is relied upon to repair the brain’s hippocampus, and
TNFR1 is relied upon to repair the brain’s striatum (
63).
Oligodendrocyte regeneration appears to occur as a result of TNFR2
activation on astrocytes, which promotes oligodendrocyte proliferation
through the induction of chemokine CXCL12 in an animal model of
demyelination (
67).
Lastly, TNFR1 promotes neurodegeneration while TNFR2 promotes
neuroprotection in an animal model of retinal ischemia in knockout mice (
68).
TNF Receptor and Autoimmune Disease
A
variety of defects in TNFR2 and downstream NFKB signaling are found in
various autoimmune diseases. The defects include polymorphisms in the
TNFR2 gene, upregulated expression of TNFR2, and TNFR2 receptor
shedding. A recently published study implicates a new decoy splice
variant of the TNFR1 receptor in multiple sclerosis. This causes a
relative deficiency in TNF with inadequate TNFR2 signaling for
autoreactive T-cell selection and induction of beneficial T
regs (
39). Polymorphisms in TNFR2 have been identified in some patients with familial rheumatoid arthritis (
69–
71), Crohn’s disease (
72), ankylosing spondylitis (
38), ulcerative colitis (
73), and immune-related conditions such as graft versus host disease associated with scleroderma risk (
74).
Common to several autoimmune diseases, with the notable exception of
type I diabetes, is a polymorphism in which the amino acid methionine is
substituted for arginine at position 196 in exon 6 of chromosome 1p36 (
16).
This polymorphism may alter the binding kinetics between TNF and TNFR2,
the result of which may reduce signaling through NFkB.
Upregulated expression of TNFR2 is also found in several immune diseases (
16,
75).
Higher systemic levels of soluble TNFR1 (sTNFR1) and soluble TNFR2
(sTNFR2) are produced by administration of TNF to patients, likely by
shedding of receptors into the extracellular space (
76,
77).
The greater the TNF stimulation, the greater is the increase in sTNFR1
and sTNFR2. Higher levels of sTNFR2 but not sTNFR1 are found in serum
and bodily fluids of patients with familial rheumatoid arthritis (
78)
and systemic lupus erythematosus, both of which are marked by
polymorphisms in TNFR2. TNFR2, but not TNFR1, is upregulated in the
lamina propria of mice with Crohn’s disease, and it causes
in vivo experimental colitis (
79).
Decreasing the concentration of TNFR2, via receptor shedding or other
means, is a possible compensatory mechanism to lower inflammation. The
extracellular component of TNFR2 is proteolytically cleaved to produce
sTNFR2. This component binds to TNF in the extracellular space, yielding
lower concentrations of TNF available for binding to functional T cells
(
80,
81).
The development of the first anti-TNF medications, including soluble
TNFR2 fusion proteins like Enbrel, were therapeutic for some patients
with rheumatoid arthritis but consistently worsened or induced new
autoimmune diseases like type 1 diabetes, lupus, or multiple sclerosis.
The human data are consistent with past mouse data where overexpression
of TNFR2 triggered multi-organ inflammation especially in the presence
of TNF.
To achieve cell survival, the
final steps in the TNFR2 pathway rely on NFkB mobilization and
translocation to the nucleus. This can only occur with an intact
proteasome, which is responsible for cleaving the bond between NFkB and
its inhibitor protein IKBA. A defect that inhibits proteasomal-driven
cleavage of NFkB is seen in the type I diabetes-prone and Sjogren’s
syndrome-prone NOD mouse (
33). A protein subunit of the proteasome, LMP2, is lowered in all patients with Sjogren’s syndrome (
36,
52,
82). The LMP2 subunit of the proteasome is necessary for intracellular activation of NFkB in highly activated T cells (
33).
TNF as Treatment for Autoimmune Disease
Given
the commonality of TNFR signaling abnormalities in autoimmune diseases,
the administration of TNF has emerged as a common treatment strategy.
Low-dose TNF exposure, acting through its receptors, selectively
destroys autoreactive, but not healthy, CD8+ T cells in blood samples
from patients with type I diabetes (
57). Low-dose TNF also kills autoreactive T cells in an animal model of Sjogren’s syndrome (
83). A similar result with TNF exposure is achieved in blood samples from patients with scleroderma (
84).
A sustained effect need not require continuous dosing, unlike treatment
with anti-cytokines or immunosuppressive drugs: TNF can be effective
when administered intermittently (
33).
However, the administration of TNF is not feasible in humans because it
is systemically toxic when given to cancer patients who already have
high TNF levels due to an intrinsic defense system (
12,
13,
85).
The toxicity of TNF likely stems from the ubiquitous cellular
expression of TNFR1. Because TNFR2 is more restricted in its cellular
expression, TNFR2 agonism may offer a safer therapeutic approach than
administration of TNF. The possibility of intermittent exposure would
also enhance the safety profile. As noted earlier, upregulated
expression of TNFR2 in the target tissue is observed in several
autoimmune disorders on the target; this target tissue expression may be
responsible for the growth-promoting and regenerative properties of TNF
agonism. In a baboon study, TNFR2 agonism was generally safe but
exhibited adverse effects in the form of thymocyte proliferation, a
febrile reaction, and a small, transient inflammation caused by
mononuclear cell infiltration (
86).
Not all TNFR2 antibodies are the same, however, as some can bind to the
receptor without eliciting an immune response. It may well be the case
that tissue-specific or cell-specific therapies afford a better safety
profile. Many factors have profound effects on the nature of TNFR
signaling with antibody agonists. Their safety and efficacy are affected
by changes in the ligand, receptor, adapter proteins, or other members
of the signaling pathway. Findings may also vary depending on culture
conditions, origin of cells, and activation state.
The
rationale for TNFR2 agonism as therapy for autoimmune disease was first
shown in type I diabetes. TNFR2 agonism or induction of TNF is an
effective means of selectively killing autoreactive CD8+ T cells in
animal models, in human cells
in vitro (
33,
58,
83,
87,
88) and in blood samples taken from patients with type I diabetes (
57).
In the latter study, there was a dose-response relationship between
TNFR2 agonism and CD8+ T-cell toxicity. The CD8+ T cells were
autoreactive to insulin, a major autoantigen in type I diabetes.
How
is TNF effective at killing autoreactive T cells? A variety of TNFR2
signaling defects prevent liberation of NFkB from IkB, precluding
transcription of pro-survival genes. This in turn biases autoreactive T
cells to shift to the TRADD/FADD cell death signaling pathway which
leads to apoptosis (Figure B). In other words, NFkB dysregulation makes autoreactive T cells selectively vulnerable to TNF-induced apoptosis (
20).
T cells, unlike B cells and other immune cells, do not constitutively
express the active form of NFkB. Only this active form can translocate
to the nucleus in order to transcribe pro-survival genes.
Therapeutic Strategies for Autoimmune Disease
Small-molecule agonists
Medicinal
chemists have found it challenging to create receptor-specific agonists
for the TNF superfamily. Developing an antagonist is generally
accomplished more readily than developing an agonist. That said,
peptides, antibodies, and small molecules have been developed as TNFR2
agonists (
89,
90). Of these types, antibody agonists have been more effective at engaging a specific signaling pathway (
57).
In a labor-intensive process, TNFR2 agonists have been developed by
point mutations in the TNF protein by site-directed mutagenesis (
90). Our laboratory has recently generated a TNFR2 agonist that activates TNF signaling pathways and suppresses CD8 T cells (
91). The advantage of this agonist is that it also induced proliferation of T
reg
cells that exert an immunosuppressive function. TNFR2 agonists, while
less toxic than TNFR1 agonists, still may have toxicities, especially to
cells within the CNS (
16). For that reason it may be desirable to develop agonists that do not succeed at crossing the blood-brain barrier.
TNF inducers
The
foremost inducer of TNF is the mycobacterium bovis bacillus
Calmette–Guerin (BCG), which has been on the market for decades as a
vaccine for tuberculosis and as a treatment for bladder cancer. Its
chemical equivalent that does not meet FDA’s standards for purity is
complete Freund’s adjuvant (CFA). In an early double blinded
placebo-controlled Phase I clinical trial, BCG administration produced a
transient increase in TNF in the circulation (
92).
BCG or CFA have been successfully used in animal models of type I
diabetes to either prevent onset of diabetes or kill autoreactive T
cells, leading to the restoration of pancreatic islet cell function and
normoglycemia (
58,
59,
93–
95).
Furthermore, in a proof-of-concept randomized, controlled clinical
trial, BCG killed the insulin-autoreactive T cells in the circulation of
patients with type I diabetes (
92).
With the removal of insulin-autoreactive T cells, pancreatic islets
managed to regenerate to the extent that there was a transient rise in
C-peptide, a marker for insulin production. The transient rise in
C-peptide was striking, considering that patients in the trial averaged
15
years
of disease. This clinical trial data repudiated the presumption that
loss of pancreatic function is irreversible. Although BCG and CFA
release TNF and therefore are not specific for TNFR2, they have low
toxicity and thereby may be safe for treating autoimmune disease by
virtue of inducing low levels of TNF.
NFkB pathway modulation
Nuclear
factor-kB is thwarted from entering the nucleus to transcribe
pro-survival genes in autoimmune diseases featuring defects in TNF
signaling (
33,
34).
Instead of being cleaved, NFkB remains bound in the cytoplasm to its
inhibitory chaperone protein IkBa. A genetic defect in type I
diabetes-prone and Sjogren’s syndrome-prone NOD mouse blocks the
proteasome from cleaving NFKB from IkBa (
34). Patients with Sjogren’s syndrome also exhibit this defect (
52).
Consequently, inhibiting NFkB’s translocation to the nucleus offers
another therapeutic approach to autoimmune disease if it could be done
in the select cells that are disease causing.
TNFR1 antagonism
Tumor
necrosis factor binds to TNFR1 and TNR2. Another way to make TNF
selective for TNFR2 signaling, an effect that could promote tissue
regeneration and remove autoimmunity, is to create a TNFR1 antagonist.
This strategy would bias TNF to act solely through the TNFR2 receptor.
This strategy also appears promising for hepatitis or autoimmunity in
murine models (
96).
A humanized TNFR1-specific antagonistic antibody for selective
inhibition of TNF action has been tested with promising results (
96–
98).
Expansion of T-regulatory cells via TNFR2
T-regulatory
cells are a type of immunosuppressive cell that displays diverse
clinical applications in transplantation, allergy, infectious disease,
GVHD, autoimmunity, and cancer (
99). T
regs
co-express CD4+ and the interleukin-2 receptor alpha chain CD25 hi and
feature inducible levels of intracellular transcription factor forkhead
box P3 (FOXP3). Naturally occurring T
regs appear to express TNFR2 at a higher density than TNFR1 (
3,
100,
101). There is evidence from animal models that TNF signaling through TNFR2 promotes T
reg activity: TNFR2 activates and induces proliferation of T
regs (
100) and TNFR2 expression indicates maximally suppressive T
regs (
102).
T-regulatory
cells have been proposed to prevent or treat autoimmune disease, but
the rate-limiting problem has been obtaining sufficient quantities,
whether by generating them
ex vivo or stimulating their production
in vivo.
In vivo
stimulation turns out to be too toxic with standard expansion agents
IL-2, anti-CD3, and anti-CD28. These expansion agents can be used to
generate large quantities of T
regs
ex vivo, but the problem is that they produce heterogeneous
progeny consisting of mixed CD4+ populations. Heterogeneous progeny
carry risk: they are capable of releasing pro-inflammatory cytokines and
consist of cell populations with antagonistic properties. Some new
approaches are being attempted, including expansion of T
regs
in vivo with TL1A-Ig, a naturally occurring TNF receptor superfamily agonist (
103). Additionally, our laboratory has developed a method of
ex vivo
expansion using a newly synthesized TNFR2 monoclonal antibody agonist
that produces homogeneous progeny expressing a uniform phenotype of 14
cell surface markers (
91). The TNFR2-agonist expanded T
regs are capable of suppressing CD8+ T cells. In healthy humans, the TNF inducer BCG causes transient expansion of T
regs (
91). In a clinical trial, BCG triggers T
reg production in patients with type I diabetes (
92), which appears to contribute to the suppression of disease and temporary restoration of islet cell function.
Use of TNFR2 for tissue regeneration
When
type 1 diabetes was first reversed in end-stage diabetic mice with
boosting of TNF, the research showed an unexpected outcome (
59).
The pancreas of the treated diabetic mice had regenerated their islets
and the original islet transplants that were performed to restore blood
sugars were not needed (
59).
The histologic shape of the reappearing insulin secreting islets was
also remarkable. The newly regenerated islets were larger in size than
unaffected, untreated NOD mouse cohorts, and contrasted greatly from
islets of NOD mice that had received immunosuppressive drug strategies,
such as anti-lymphocyte serum or anti-CD4 or anti-CD3 antibodies, to
avert diabetes (
104,
105). Past autoimmune treatments of diabetic NOD mice worked almost only in pre-diabetic mice or early new-onset diabetic mice (
106).
Also the rescued islets of NOD mice, commonly treated with anti-CD3
immunosuppressive antibodies, were small in size, and demonstrated no or
limited regeneration. The immunosuppressive drug was best administered
to pre-diabetic mice or to mice with recent onset hyperglycemia. In
total, this data strongly suggested that administration of TNF directly
or boosting TNF indirectly with BCG or the heat-killed equivalent, CFA,
had a dual mechanism of action – a direct killing of the autoreactive T
cells and also a TNF effect directly on the target organ to promote
healing and regeneration. Also the TNF effect on the target tissue
indicated that even late stage diabetes could be reversed in large part
due to the regenerative effect in contrast to a pure rescue effect,
survival of existing islets without expansion, of standard
immunosuppressive strategies.
The
effect of TNF on the pancreas was not the only tissue showing possible
regeneration with TNF stimulation. In the field of neuroregeneration,
the Ting laboratory showed TNF similarly promoted proliferation of
oligodendrocytes progenitors and remyelination (
6).
Gradually the broader literature reported the regenerative effect of
TNF and TNFR2 agonism on heart regeneration, bone marrow stem cells, and
even neuron regeneration in the setting of Parkinson’s disease model in
mice (
11,
60,
66,
107).
Conclusion
An
overlapping feature across autoimmune disorders is various defects in
TNF signaling through its two receptors. TNFR2 is a more attractive
molecular target than TNFR1 because of its limited cellular expression. A
variety of strategies utilizing TNFR2 agonism can be pursued for
treatment of autoimmune disease and also used for regenerative medicine
therapies. TNFR2 agonism has been associated with selective death of
autoreactive T cells in type 1 diabetes and with induction of Tregs.
It holds promise for treating other autoimmune disorders featuring
dysregulation of NFkB, which is a key component of the TNFR2 signaling
pathway.
Conflict of Interest Statement
The
authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a
potential conflict of interest.
References
1.
Aggarwal BB, Gupta SC, Kim JH.
Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey. Blood (2012) 119:651–65.10.1182/blood-2011-04-325225 [PMC free article] [PubMed] [Cross Ref]
2.
Ware CF, Crowe PD, Vanarsdale TL, Andrews JL, Grayson MH, Jerzy R, et al.
Tumor necrosis factor (TNF) receptor expression
in T lymphocytes. Differential regulation of the type I TNF receptor
during activation of resting and effector T cells. J Immunol (1991) 147:4229–38. [PubMed]
3.
Annunziato F, Cosmi L, Liotta F, Lazzeri E, Manetti R, Vanini V, et al.
Phenotype, localization, and mechanism of suppression of CD4(+)CD25(+) human thymocytes. J Exp Med (2002) 196:379–87.10.1084/jem.20020110 [PMC free article] [PubMed] [Cross Ref]
4.
Yang L, Lindholm K, Konishi Y, Li R, Shen Y.
Target depletion of distinct tumor necrosis
factor receptor subtypes reveals hippocampal neuron death and survival
through different signal transduction pathways. J Neurosci (2002) 22:3025–32. [PubMed]
5.
McCoy MK, Tansey MG.
TNF signaling inhibition in the CNS: implications for normal brain function and neurodegenerative disease. J Neuroinflammation (2008) 5:45.10.1186/1742-2094-5-45 [PMC free article] [PubMed] [Cross Ref]
6.
Arnett HA, Mason J, Marino M, Suzuki K, Matsushima GK, Ting JP.
TNF alpha promotes proliferation of oligodendrocyte progenitors and remyelination. Nat Neurosci (2001) 4:1116–22.10.1038/nn738 [PubMed] [Cross Ref]
7.
Dopp JM, Sarafian TA, Spinella FM, Kahn MA, Shau H, de Vellis J.
Expression of the p75 TNF receptor is linked to TNF-induced NFkappaB translocation and oxyradical neutralization in glial cells. Neurochem Res (2002) 27:1535–42.10.1023/A:1021608724117 [PubMed] [Cross Ref]
8.
Irwin MW, Mak S, Mann DL, Qu R, Penninger JM, Yan A, et al.
Tissue expression and immunolocalization of tumor necrosis factor-alpha in postinfarction dysfunctional myocardium. Circulation (1999) 99:1492–8.10.1161/01.CIR.99.11.1492 [PubMed] [Cross Ref]
9.
Tartaglia LA, Weber RF, Figari IS, Reynolds C, Palladino MA, Jr, Goeddel DV.
The two different receptors for tumor necrosis factor mediate distinct cellular responses. Proc Natl Acad Sci USA (1991) 88:9292–6.10.1073/pnas.88.20.9292 [PMC free article] [PubMed] [Cross Ref]
10.
Grell M, Becke FM, Wajant H, Mannel DN, Scheurich P.
TNF receptor type 2 mediates thymocyte proliferation independently of TNF receptor type 1. Eur J Immunol (1998) 28:257–63.10.1002/(SICI)1521-4141(199801)28:01<257::AID-IMMU257>3.0.CO;2-G [PubMed] [Cross Ref]
11.
Böcker W, Docheva D, Prall WC, Egea V, Pappou E, Rossmann O, et al.
IKK-2 is required for TNF-alpha-induced invasion and proliferation of human mesenchymal stem cells. J Mol Med (2008) 86:1183–92.10.1007/s00109-008-0378-3 [PubMed] [Cross Ref]
12.
Sidhu RS, Bollon AP.
Tumor necrosis factor activities and cancer therapy – a perspective. Pharmacol Ther (1993) 57:79–128.10.1016/0163-7258(93)90037-E [PubMed] [Cross Ref]
13.
Hieber U, Heim ME.
Tumor necrosis factor for the treatment of malignancies. Oncology (1994) 51:142–53.10.1159/000227329 [PubMed] [Cross Ref]
14.
Pimentel-Muinos FX, Seed B.
Regulated commitment of TNF receptor signaling: a molecular switch for death or activation. Immunity (1999) 11:783–93.10.1016/S1074-7613(00)80152-1 [PubMed] [Cross Ref]
15.
Faustman D, Davis M.
TNF receptor 2 pathway: drug target for autoimmune diseases. Nat Rev Drug Discov (2010) 9:482–93.10.1038/nrd3030 [PubMed] [Cross Ref]
16.
Carpentier I, Coornaert B, Beyaert R.
Function and regulation of tumor necrosis factor type 2. Curr Med Chem (2004) 11:2205–12.10.2174/0929867043364694 [PubMed] [Cross Ref]
17.
Rothe M, Sarma V, Dixit VM, Goeddel DV.
TRAF2-mediated activation of NF-kappaB by TNF receptor 2 and CD40. Science (1995) 269:1424–7.10.1126/science.7544915 [PubMed] [Cross Ref]
18.
Rothe M, Wong SC, Henzel WJ, Goeddel DV.
A novel family of putative signal transducers
associated with the cytoplasmic domain of the 75 kDa tumor necrosis
factor receptor. Cell (1994) 78:681–92.10.1016/0092-8674(94)90532-0 [PubMed] [Cross Ref]
19.
Govindaraj C, Scalzo-Inguanti K, Scholzen A, Li S, Plebanski M.
TNFR2 expression on CD25(hi)FOXP3(+) T cells
induced upon TCR stimulation of CD4 T cells identifies maximal
cytokine-producing effectors. Front Immunol (2013) 4:233.10.3389/fimmu.2013.00233 [PMC free article] [PubMed] [Cross Ref]
20.
Kodama S, Davis M, Faustman DL.
The therapeutic potential of tumor necrosis factor for autoimmune disease: a mechanistically based hypothesis. Cell Mol Life Sci (2005) 62:1850–62.10.1007/s00018-005-5022-6 [PubMed] [Cross Ref]
21.
Abbott DW, Wilkins A, Asara JM, Cantley LC.
The Crohn’s disease protein, NOD2, requires RIP2 in order to induce ubiquitinylation of a novel site on NEMO. Curr Biol (2004) 14:2217–27.10.1016/j.cub.2004.12.032 [PubMed] [Cross Ref]
22.
Eckmann L, Karin M.
NOD2 and Crohn’s disease: loss or gain of function?
Immunity (2005) 22:661–7.10.1016/j.immuni.2005.06.004 [PubMed] [Cross Ref]
23.
Maeda S, Hsu LC, Liu H, Bankston LA, Iimura M, Kagnoff MF, et al.
Nod2 mutation in Crohn’s disease potentiates NF-kappaB activity and IL-1beta processing. Science (2005) 307:734–8.10.1126/science.1103685 [PubMed] [Cross Ref]
24.
Watanabe T, Kitani A, Murray PJ, Strober W.
NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper type 1 responses. Nat Immunol (2004) 5:800–8.10.1038/ni1092 [PubMed] [Cross Ref]
25.
Levine A, Shamir R, Wine E, Weiss B, Karban A, Shaoul RR, et al.
TNF promoter polymorphisms and modulation of growth retardation and disease severity in pediatric Crohn’s disease. Am J Gastroenterol (2005) 100:1598–604.10.1111/j.1572-0241.2005.41737.x [PubMed] [Cross Ref]
26.
van Heel DA, Udalova IA, De Silva AP, McGovern DP, Kinouchi Y, Hull J, et al.
Inflammatory bowel disease is associated with a
TNF polymorphism that affects an interaction between the OCT1 and
NF(-kappa)B transcription factors. Hum Mol Genet (2002) 11:1281–9.10.1093/hmg/11.11.1281 [PubMed] [Cross Ref]
27.
Karban AS, Okazaki T, Panhuysen CI, Gallegos T, Potter JJ, Bailey-Wilson JE, et al.
Functional annotation of a novel NFKB1 promoter polymorphism that increases risk for ulcerative colitis. Hum Mol Genet (2004) 13:35–45.10.1093/hmg/ddh008 [PubMed] [Cross Ref]
28.
Kammer GM, Tsokos GC.
Abnormal T lymphocyte signal transduction in systemic lupus erythematosus. Curr Dir Autoimmun (2002) 5:131–50.10.1159/000060555 [PubMed] [Cross Ref]
29.
Hegazy DM, O’Reilly DA, Yang BM, Hodgkinson AD, Millward BA, Demaine AG.
NFkappaB polymorphisms and susceptibility to type 1 diabetes. Genes Immun (2001) 2:304–8.10.1038/sj.gene.6363776 [PubMed] [Cross Ref]
30.
Deng GY, Muir A, MacLaren NK, She JX.
Association of LMP2 and LMP7 genes within the major histocompatibility complex with insulin-dependent diabetes mellitus: population and family studies. Am J Hum Genet (1995) 56:528–34. [PMC free article] [PubMed]
31.
Ding H, Cheng H, Fu Z, Yan L, Yang G.
Relationship of large multifunctional proteasome
7 gene polymorphism with susceptibility to type 1 diabetes mellitus and
DR3 gene. Chin Med J (Engl) (2001) 114:1263–6. [PubMed]
32.
Fu Y, Yan G, Shi L, Faustman D.
Antigen processing and autoimmunity. Evaluation of mRNA abundance and function of HLA-linked genes. Ann N Y Acad Sci (1998) 842:138–55.10.1111/j.1749-6632.1998.tb09642.x [PubMed] [Cross Ref]
33.
Hayashi T, Faustman D.
Essential role of HLA-encoded proteasome subunits in NF-kB activation and prevention of TNF-a induced apoptosis. J Biol Chem (2000) 275:5238–47.10.1074/jbc.275.7.5238 [PubMed] [Cross Ref]
34.
Hayashi T, Faustman D.
NOD mice are defective in proteasome production and activation of NF-kappaB. Mol Cell Biol (1999) 19:8646–59. [PMC free article] [PubMed]
35.
Hayashi T, Faustman DL.
Selected contribution: association of gender-related LMP2 inactivation with autoimmune pathogenesis. J Appl Physiol (2001) 91:2804–15. [PubMed]
36.
Yan G, Fu Y, Faustman DL.
Reduced expression of Tap1 and Lmp2 antigen processing genes in the non-obese diabetic (NOD) mouse due to a mutation in their shared bidirectional promoter. J Immunol (1997) 159:3068–80. [PubMed]
37.
Miterski B, Böhringer S, Klein W, Sindern E, Haupts M, Schimrigk S, et al.
Inhibitors in the NFkappaB cascade comprise
prime candidate genes predisposing to multiple sclerosis, especially in
selected combinations. Genes Immun (2002) 3:211–9.10.1038/sj.gene.6363846 [PubMed] [Cross Ref]
38.
Chatzikyriakidou A, Georgiou I, Voulgari PV, Drosos AA.
The role of tumor necrosis factor (TNF) and TNF receptor polymorphisms in susceptibility to ankylosing spondylitis. Clin Exp Rheumatol (2009) 27:645–8. [PubMed]
39.
Gregory AP, Dendrou CA, Attfield KE, Haghikia A, Xifara DK, Butter F, et al.
TNF receptor 1 genetic risk mirrors outcome of anti-TNF therapy in multiple sclerosis. Nature (2012) 488:508–11.10.1038/nature11307 [PubMed] [Cross Ref]
40.
Gehr G, Gentz R, Brockhaus M, Loetscher H, Lesslauer W.
Both tumor necrosis factor receptor types mediate proliferative signals in human mononuclear cell activation. J Immunol (1992) 149:911–7. [PubMed]
41.
Grell M, Wajant H, Zimmermann G, Scheurich P.
The type 1 receptor (CD120a) is the high-affinity receptor for soluble tumor necrosis factor. Proc Natl Acad Sci USA (1998) 95:570–5.10.1073/pnas.95.2.570 [PMC free article] [PubMed] [Cross Ref]
42.
Horiuchi T, Mitoma H, Harashima S, Tsukamoto H, Shimoda T.
Transmembrane TNF-alpha: structure, function and interaction with anti-TNF agents. Rheumatology (Oxford) (2010) 49:1215–28.10.1093/rheumatology/keq031 [PMC free article] [PubMed] [Cross Ref]
43.
Grell M, Douni E, Wajant H, Löhden M, Clauss M, Maxeiner B, et al.
The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80kDa tumor necrosis factor receptor. Cell (1995) 83:793–802.10.1016/0092-8674(95)90192-2 [PubMed] [Cross Ref]
44.
Mukai Y, Nakamura T, Yoshikawa M, Yoshioka Y, Tsunoda S, Nakagawa S, et al.
Solution of the structure of the TNF-TNFR2 complex. Sci Signal (2010) 3:ra83.10.1126/scisignal.2000954 [PubMed] [Cross Ref]
45.
Douni E, Kollias G.
A critical role of the p75 tumor necrosis factor
receptor (p75TNF-R) in organ inflammation independent of TNF,
lymphotoxin alpha, or the p55TNF-R. J Exp Med (1998) 188:1343–52.10.1084/jem.188.7.1343 [PMC free article] [PubMed] [Cross Ref]
46.
Aggarwal BB.
Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol (2003) 3:745–56.10.1038/nri1184 [PubMed] [Cross Ref]
47.
Kloetzel PM.
Antigen processing by the proteasome. Nat Rev Mol Cell Biol (2001) 2:179–87.10.1038/35056572 [PubMed] [Cross Ref]
48.
Faustman D, Li XP, Lin HY, Fu YE, Eisenbarth G, Avruch J, et al.
Linkage of faulty major histocompatibility complex class I to autoimmune diabetes. Science (1991) 254:1756–61.10.1126/science.1763324 [PubMed] [Cross Ref]
49.
Faustman D, Eisenbarth G, Daley J, Breitmeyer J.
Abnormal T lymphocyte subsets in type I diabetes mellitus: analysis with anti-2H4 and anti-4B4 antibodies. Diabetes (1989) 38:1462–8.10.2337/diabetes.38.11.1462 [PubMed] [Cross Ref]
50.
Fu Y, Nathan DM, Li F, Li X, Faustman DL.
Defective major histocompatibility complex class I expression on lymphoid cells in autoimmunity. J Clin Invest (1993) 91:2301–7.10.1172/JCI116459 [PMC free article] [PubMed] [Cross Ref]
51.
Hayashi T, Kodama S, Faustman DL.
Reply to ‘LMP2 expression and proteasome activity in NOD mice’. Nat Med (2000) 6:1065–6.10.1038/80353 [PubMed] [Cross Ref]
52.
Krause S, Kuckelkorn U, Dörner T, Burmester GR, Feist E, Kloetzel PM.
Immunoproteasome subunit LMP2 expression is deregulated in Sjogren’s syndrome but not in other autoimmune disorders. Ann Rheum Dis (2006) 65:1021–7.10.1136/ard.2005.045930 [PMC free article] [PubMed] [Cross Ref]
53.
Liu Y, Ramot Y, Torrelo A, Paller AS, Si N, Babay S, et al.
Mutations in proteasome subunit beta type 8
cause chronic atypical neutrophilic dermatosis with lipodystrophy and
elevated temperature with evidence of genetic and phenotypic
heterogeneity. Arthritis Rheum (2012) 64:895–907.10.1002/art.33368 [PMC free article] [PubMed] [Cross Ref]
54.
Mishto M, Bellavista E, Ligorio C, Textoris-Taube K, Santoro A, Giordano M, et al.
Immunoproteasome LMP2 60HH variant alters MBP
epitope generation and reduces the risk to develop multiple sclerosis in
Italian female population. PLoS One (2010) 5:e9287.10.1371/journal.pone.0009287 [PMC free article] [PubMed] [Cross Ref]
55.
Haroon N, Maksymowych WP, Rahman P, Tsui FW, O’Shea FD, Inman RD.
Radiographic severity of ankylosing spondylitis
is associated with polymorphism of the large multifunctional peptidase 2
gene in the Spondyloarthritis Research Consortium of Canada cohort. Arthritis Rheum (2012) 64:1119–26.10.1002/art.33430 [PubMed] [Cross Ref]
56.
Faustman DL, Davis M.
The primacy of CD8 T lymphocytes in type 1 diabetes and implications for therapies. J Mol Med (2009) 87:1173–8.10.1007/s00109-009-0516-6 [PubMed] [Cross Ref]
57.
Ban L, Zhang J, Wang L, Kuhtreiber W, Burger D, Faustman DL.
Selective death of autoreactive T cells in human diabetes by TNF or TNF receptor 2 agonism. Proc Natl Acad Sci USA (2008) 105:13644–9.10.1073/pnas.0803429105 [PMC free article] [PubMed] [Cross Ref]
58.
Ryu S, Kodama S, Ryu K, Schoenfeld DA, Faustman DL.
Reversal of established autoimmune diabetes by restoration of endogenous beta cell function. J Clin Invest (2001) 108:63–72.10.1172/JCI12335 [PMC free article] [PubMed] [Cross Ref]
59.
Kodama S, Kuhtreiber W, Fujimura S, Dale EA, Faustman DL.
Islet regeneration during the reversal of autoimmune diabetes in NOD mice. Science (2003) 302:1223–7.10.1126/science.1088949 [PubMed] [Cross Ref]
60.
Monden Y, Kubota T, Inoue T, Tsutsumi T, Kawano S, Ide T, et al.
Tumor necrosis factor-alpha is toxic via receptor 1 and protective via receptor 2 in a murine model of myocardial infarction. Am J Physiol Heart Circ Physiol (2007) 293:H743–53.10.1152/ajpheart.00166.2007 [PubMed] [Cross Ref]
61.
Wang M, Crisostomo PR, Markel TA, Wang Y, Meldrum DR.
Mechanisms of sex differences in TNFR2-mediated cardioprotection. Circulation (2008) 118:S38–45.10.1161/CIRCULATIONAHA.107.756890 [PMC free article] [PubMed] [Cross Ref]
62.
Marchetti L, Klein M, Schlett K, Pfizenmaier K, Eisel UL.
Tumor necrosis factor (TNF)-mediated
neuroprotection against glutamate-induced excitotoxicity is enhanced by
N-methyl-d-aspartate receptor activation. Essential role of a TNF
receptor 2-mediated phosphatidylinositol 3-kinase-dependent NF-kappa B
pathway. J Biol Chem (2004) 279:32869–81.10.1074/jbc.M311766200 [PubMed] [Cross Ref]
63.
Rodriguez M, Zoecklein L, Papke L, Gamez J, Denic A, Macura S, et al.
Tumor necrosis factor alpha is reparative via
TNFR1 in the hippocampus and via TNFR2 in the striatum after
virus-induced encephalitis. Brain Pathol (2009) 19:12–26.10.1111/j.1750-3639.2008.00151.x [PMC free article] [PubMed] [Cross Ref]
64.
Al-Lamki RS, Lu W, Wang J, Yang J, Sargeant TJ, Wells R, et al.
TNF, acting through inducibly expressed TNFR2,
drives activation and cell cycle entry of c-kit cardiac stem cells in
ischemic heart disease. Stem Cells (2013) 31(9):1881–92.10.1002/stem.1433 [PMC free article] [PubMed] [Cross Ref]
65.
Tan J, Weil BR, Abarbanell AM, Wang Y, Herrmann JL, Dake ML, et al.
Ablation of TNF-alpha receptors influences mesenchymal stem cell-mediated cardiac protection against ischemia. Shock (2010) 34:236–42.10.1097/SHK.0b013e3181d75ae3 [PubMed] [Cross Ref]
66.
Rezzoug F, Huang Y, Tanner MK, Wysoczynski M, Schanie CL, Chilton PM, et al.
TNF-alpha is critical to facilitate hemopoietic stem cell engraftment and function. J Immunol (2008) 180:49–57. [PubMed]
67.
Patel JR, Williams JL, Muccigrosso MM, Liu L, Sun T, Rubin JB, et al.
Astrocyte TNFR2 is required for CXCL12-mediated
regulation of oligodendrocyte progenitor proliferation and
differentiation within the adult CNS. Acta Neuropathol (2012) 124:847–60.10.1007/s00401-012-1034-0 [PMC free article] [PubMed] [Cross Ref]
68.
Fontaine V, Mohand-Said S, Hanoteau N, Fuchs C, Pfizenmaier K, Eisel U.
Neurodegenerative and neuroprotective effects of
tumor Necrosis factor (TNF) in retinal ischemia: opposite roles of TNF
receptor 1 and TNF receptor 2. J Neurosci (2002) 22:RC216. [PubMed]
69.
Barton A, John S, Ollier WE, Silman A, Worthington J.
Association between rheumatoid arthritis and
polymorphism of tumor necrosis factor receptor II, but not tumor
necrosis factor receptor I, in Caucasians. Arthritis Rheum (2001) 44:61–5.10.1002/1529-0131(200101)44:1<61::AID-ANR9>3.0.CO;2-Q [PubMed] [Cross Ref]
70.
Dieudé P, Petit E, Cailleau-Moindrault S, Osorio J, Pierlot C, Martinez M, et al.
Association between tumor necrosis factor
receptor II and familial, but not sporadic, rheumatoid arthritis:
evidence for genetic heterogeneity. Arthritis Rheum (2002) 46:2039–44.10.1002/art.10101 [PubMed] [Cross Ref]
71.
Orozco G, Abelson AK, González-Gay MA, Balsa A, Pascual-Salcedo D, García A, et al.
Study of functional variants of the BANK1 gene in rheumatoid arthritis. Arthritis Rheum (2009) 60:372–9.10.1002/art.24244 [PubMed] [Cross Ref]
72.
Sashio H, Tamura K, Ito R, Yamamoto Y, Bamba H, Kosaka T, et al.
Polymorphisms of the TNF gene and the TNF
receptor superfamily member 1B gene are associated with susceptibility
to ulcerative colitis and Crohn’s disease, respectively. Immunogenetics (2002) 53:1020–7.10.1007/s00251-001-0423-7 [PubMed] [Cross Ref]
73.
Pierik M, Vermeire S, Steen KV, Joossens S, Claessens G, Vlietinck R, et al.
Tumour necrosis factor-alpha receptor 1 and 2
polymorphisms in inflammatory bowel disease and their association with
response to infliximab. Aliment Pharmacol Ther (2004) 20:303–10.10.1111/j.1365-2036.2004.01946.x [PubMed] [Cross Ref]
74.
Ishikawa Y, Kashiwase K, Akaza T, Morishima Y, Inoko H, Sasazuki T, et al.
Polymorphisms in TNFA and TNFR2 affect outcome of unrelated bone marrow transplantation. Bone Marrow Transplant (2002) 29:569–75.10.1038/sj.bmt.1703409 [PubMed] [Cross Ref]
75.
Atreya R, Zimmer M, Bartsch B, Waldner MJ, Atreya I, Neumann H, et al.
Antibodies against tumor necrosis factor (TNF)
induce T-cell apoptosis in patients with inflammatory bowel diseases via
TNF receptor 2 and intestinal CD14(+) macrophages. Gastroenterology (2011) 141:2026–38.10.1053/j.gastro.2011.08.032 [PubMed] [Cross Ref]
76.
Diez-Ruiz A, Tilz GP, Zangerle R, Baier-Bitterlich G, Wachter H, Fuchs D.
Soluble receptors for tumour necrosis factor in clinical laboratory diagnosis. Eur J Haematol (1995) 54:1–8.10.1111/j.1600-0609.1995.tb01618.x [PubMed] [Cross Ref]
77.
Lantz M, Malik S, Slevin ML, Olsson I.
Infusion of tumor necrosis factor (TNF) causes an increase in circulating TNF-binding protein in humans. Cytokine (1990) 2:402–6.10.1016/1043-4666(90)90048-X [PubMed] [Cross Ref]
78.
Cope AP, Aderka D, Doherty M, Engelmann H, Gibbons D, Jones AC, et al.
Increased levels of soluble tumor necrosis factor receptors in the sera and synovial fluid of patients with rheumatic diseases. Arthritis Rheum (1992) 35:1160–9.10.1002/art.1780351008 [PubMed] [Cross Ref]
79.
Holtmann MH, Douni E, Schütz M, Zeller G, Mudter J, Lehr HA, et al.
Tumor necrosis factor-receptor 2 is up-regulated on lamina propria T cells in Crohn’s disease and promotes experimental colitis in vivo. Eur J Immunol (2002) 32:3142–51.10.1002/1521-4141(200211)32:11<3142::AID-IMMU3142>3.0.CO;2-4 [PubMed] [Cross Ref]
80.
Xanthoulea S, Pasparakis M, Kousteni S, Brakebusch C, Wallach D, Bauer J, et al.
Tumor necrosis factor (TNF) receptor shedding
controls thresholds of innate immune activation that balance opposing
TNF functions in infectious and inflammatory diseases. J Exp Med (2004) 200:367–76.10.1084/jem.20040435 [PMC free article] [PubMed] [Cross Ref]
81.
Engelmann H, Holtmann H, Brakebusch C, Avni YS, Sarov I, Nophar Y, et al.
Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity. J Biol Chem (1990) 265:14497–504. [PubMed]
82.
Li F, Guo J, Fu Y, Yan G, Faustman D.
Abnormal class I assembly and peptide presentation in the diabetic NOD mouse. Proc Natl Acad Sci U S A (1994) 91:11128–32.10.1073/pnas.91.23.11128 [PMC free article] [PubMed] [Cross Ref]
83.
Tran SD, Kodama S, Lodde BM, Szalayova I, Key S, Khalili S, et al.
Reversal of Sjogren’s-like syndrome in non-obese diabetic mice. Ann Rheum Dis (2007) 66:812–4.10.1136/ard.2006.064030 [PMC free article] [PubMed] [Cross Ref]
84.
Kessel A, Rosner I, Rozenbaum M, Zisman D, Sagiv A, Shmuel Z, et al.
Increased CD8+ T cell apoptosis in scleroderma is associated with low levels of NF-kappa B. J Clin Immunol (2004) 24:30–6.10.1023/B:JOCI.0000018060.36183.bb [PubMed] [Cross Ref]
85.
Balkwill F.
Tumour necrosis factor and cancer. Nat Rev Cancer (2009) 9:361–71.10.1038/nrc2628 [PubMed] [Cross Ref]
86.
Welborn MB, III, Van Zee K, Edwards PD, Pruitt JH, Kaibara A, Vauthey JN, et al.
A human tumor necrosis factor p75 receptor agonist stimulates in vitro T cell proliferation but does not produce inflammation or shock in the baboon. J Exp Med (1996) 184:165–71.10.1084/jem.184.1.165 [PMC free article] [PubMed] [Cross Ref]
87.
Qin HY, Chaturvedi P, Singh B.
In vivo apoptosis of diabetogenic T cells in NOD mice by IFN-gamma/TNF-alpha. Int Immunol (2004) 16:1723–32.10.1093/intimm/dxh173 [PubMed] [Cross Ref]
88.
Christen U, Wolfe T, Möhrle U, Hughes AC, Rodrigo E, Green EA, et al.
A dual role for TNF-alpha in type 1 diabetes:
islet-specific expression abrogates the ongoing autoimmune process when
induced late but not early during pathogenesis. J Immunol (2001) 166:7023–32. [PubMed]
89.
Sheehan KC, Pinckard JK, Arthur CD, Dehner LP, Goeddel DV, Schreiber RD.
Monoclonal antibodies specific for murine p55 and p75 tumor necrosis factor receptors: identification of a novel in vivo role for p75. J Exp Med (1995) 181:607–17.10.1084/jem.181.2.607 [PMC free article] [PubMed] [Cross Ref]
90.
Loetscher H, Stueber D, Banner D, Mackay F, Lesslauer W.
Human tumor necrosis factor alpha (TNF alpha) mutants with exclusive specificity for the 55-kDa or 75-kDa TNF receptors. J Biol Chem (1993) 268:26350–7. [PubMed]
91.
Okubo Y, Mera T, Wang L, Faustman DL.
Homogeneous expansion of human T-regulatory cells via tumor necrosis factor receptor 2. Sci Rep (2013) 3:3153.10.1038/srep03153 [PMC free article] [PubMed] [Cross Ref]
92.
Faustman DL, Wang L, Okubo Y, Burger D, Ban L, Man G, et al.
Proof-of-concept, randomized, controlled clinical trial of Bacillus-Calmette-Guerin for treatment of long-term type 1 diabetes. PLoS One (2012) 7:e41756.10.1371/journal.pone.0041756 [PMC free article] [PubMed] [Cross Ref]
93.
Harada M, Kishimoto Y, Makino S.
Prevention of overt diabetes and insulitis in NOD mice by a single BCG vaccination. Diabetes Res Clin Pract (1990) 8:85–9.10.1016/0168-8227(90)90017-N [PubMed] [Cross Ref]
94.
Sadelain MW, Qin HY, Sumoski W, Parfrey N, Singh B, Rabinovitch A.
Prevention of diabetes in the BB rat by early immunotherapy using Freund’s adjuvant. J Autoimmun (1990) 3:671–80.10.1016/S0896-8411(05)80034-4 [PubMed] [Cross Ref]
95.
McInerney MF, Pek SB, Thomas DW.
Prevention of insulitis and diabetes onset by treatment with complete Freund’s adjuvant in NOD mice. Diabetes (1991) 40:715–25.10.2337/diab.40.6.715 [PubMed] [Cross Ref]
96.
Kontermann RE, Münkel S, Neumeyer J, Müller D, Branschädel M, Scheurich P, et al.
A humanized tumor necrosis factor receptor 1
(TNFR1)-specific antagonistic antibody for selective inhibition of tumor
necrosis factor (TNF) action. J Immunother (2008) 31:225–34.10.1097/CJI.0b013e31816a88f9 [PubMed] [Cross Ref]
97.
Shibata H, Yoshioka Y, Ohkawa A, Abe Y, Nomura T, Mukai Y, et al.
The therapeutic effect of TNFR1-selective antagonistic mutant TNF-alpha in murine hepatitis models. Cytokine (2008) 44:229–33.10.1016/j.cyto.2008.07.003 [PubMed] [Cross Ref]
98.
Kitagaki M, Isoda K, Kamada H, Kobayashi T, Tsunoda S, Tsutsumi Y, et al.
Novel TNF-alpha receptor 1 antagonist treatment attenuates arterial inflammation and intimal hyperplasia in mice. J Atheroscler Thromb (2012) 19:36–46.10.5551/jat.9746 [PubMed] [Cross Ref]
99.
McMurchy AN, Bushell A, Levings MK, Wood KJ.
Moving to tolerance: clinical application of T regulatory cells. Semin Immunol (2011) 23:304–13.10.1016/j.smim.2011.04.001 [PMC free article] [PubMed] [Cross Ref]
100.
Chen X, Baumel M, Mannel DN, Howard OMZ, Oppenheim JJ.
Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4(+)CD25(+) T regulatory cells. J Immunol (2007) 179:154–61. [PubMed]
101.
Chen X, Subleski JJ, Hamano R, Howard OM, Wiltrout RH, Oppenheim JJ.
Co-expression of TNFR2 and CD25 identifies more of the functional CD4(+)FOXP3(+) regulatory T cells in human peripheral blood. Eur J Immunol (2010) 40:1099–106.10.1002/eji.200940022 [PMC free article] [PubMed] [Cross Ref]
102.
Chen X, Subleski JJ, Kopf H, Howard OM, Männel DN, Oppenheim JJ.
Cutting edge: expression of TNFR2 defines a
maximally suppressive subset of mouse CD4+CD25+FoxP3+ T regulatory
cells: applicability to tumor-infiltrating T regulatory cells. J Immunol (2008) 180:6467–71. [PMC free article] [PubMed]
103.
Khan SQ, Tsai MS, Schreiber TH, Wolf D, Deyev VV, Podack ER.
Cloning, expression, and functional characterization of TL1A-Ig. J Immunol (2013) 190:1540–50.10.4049/jimmunol.1201908 [PubMed] [Cross Ref]
104.
Maki T, Ichikawa T, Blanco R, Porter J.
Long-term abrogation of autoimmune diabetes in non-obese diabetic mice by immunotherapy with anti-lymphocyte serum. Proc Natl Acad Sci USA (1992) 89:3434–8.10.1073/pnas.89.8.3434 [PMC free article] [PubMed] [Cross Ref]
105.
Chatenoud L, Thervet E, Primo J, Bach JF.
Anti-CD3 antibody induces long-term remission of overt autoimmunity in non-obese diabetic mice. Proc Natl Acad Sci USA (1994) 91:123–7.10.1073/pnas.91.1.123 [PMC free article] [PubMed] [Cross Ref]
106.
Kuhtreiber WM, Hayashi T, Dale EA, Faustman DL.
Central role of defective apoptosis in autoimmunity. J Mol Endocrinol (2003) 31:373–99.10.1677/jme.0.0310373 [PubMed] [Cross Ref]
107.
Yong J, Lacan G, Dang H, Hsieh T, Middleton B, Wasserfall C, et al.
BCG vaccine-induced neuroprotection in a mouse model of Parkinson’s disease. PLoS One (2011) 6:e16610.10.1371/journal.pone.0016610 [PMC free article] [PubMed] [Cross Ref]
PLoS One. 2012; 7(8): e41756.
Proof-of-Concept, Randomized, Controlled Clinical Trial of Bacillus-Calmette-Guerin for Treatment of Long-Term Type 1 Diabetes
Denise L. Faustman,
1,* Limei Wang,
1 Yoshiaki Okubo,
1 Douglas Burger,
1 Liqin Ban,
1 Guotong Man,
1 Hui Zheng,
2 David Schoenfeld,
2 Richard Pompei,
3 Joseph Avruch,
3 and
David M. Nathan3
T. Mark Doherty, Editor
This article has been
cited by other articles in PMC.
Abstract
Background
No
targeted immunotherapies reverse type 1 diabetes in humans. However, in
a rodent model of type 1 diabetes, Bacillus Calmette-Guerin (BCG)
reverses disease by restoring insulin secretion. Specifically, it
stimulates innate immunity by inducing the host to produce tumor
necrosis factor (TNF), which, in turn, kills disease-causing autoimmune
cells and restores pancreatic beta-cell function through regeneration.
Methodology/Principal Findings
Translating
these findings to humans, we administered BCG, a generic vaccine, in a
proof-of-principle, double-blind, placebo-controlled trial of adults
with long-term type 1 diabetes (mean: 15.3 years) at one clinical center
in North America. Six subjects were randomly assigned to BCG or placebo
and compared to self, healthy paired controls (n
=
6) or reference subjects with (n
=
57) or without (n
=
16)
type 1 diabetes, depending upon the outcome measure. We monitored
weekly blood samples for 20 weeks for insulin-autoreactive T cells,
regulatory T cells (Tregs), glutamic acid decarboxylase (GAD) and other
autoantibodies, and C-peptide, a marker of insulin secretion.
BCG-treated patients and one placebo-treated patient who, after
enrollment, unexpectedly developed acute Epstein-Barr virus infection, a
known TNF inducer, exclusively showed increases in dead
insulin-autoreactive T cells and induction of Tregs. C-peptide levels
(pmol/L) significantly rose transiently in two BCG-treated subjects
(means: 3.49 pmol/L [95% CI 2.95–3.8], 2.57 [95% CI 1.65–3.49]) and the
EBV-infected subject (3.16 [95% CI 2.54–3.69]) vs.1.65 [95% CI 1.55–3.2]
in reference diabetic subjects. BCG-treated subjects each had more than
50% of their C-peptide values above the 95
th percentile of the reference subjects. The EBV-infected subject had 18% of C-peptide values above this level.
Conclusions/Significance
We
conclude that BCG treatment or EBV infection transiently modified the
autoimmunity that underlies type 1 diabetes by stimulating the host
innate immune response. This suggests that BCG or other stimulators of
host innate immunity may have value in the treatment of long-term
diabetes.
Introduction
A
long-standing goal of immunology is to develop targeted immune
therapies that eliminate the predominant cause of type 1 diabetes: the
autoimmune T lymphocytes (T cells) that destroy the insulin-secreting
cells of the pancreas. Current immune treatments for type 1 diabetes,
such as immunosuppressants and anti-cytokines, are non-specific, killing
or harming both the pathological T cells (i.e., insulin-autoreactive
cytotoxic T cells) and healthy cells.
Two decades of
autoimmune disease research in animal models, including the non-obese
diabetic (NOD) mouse model of type 1 diabetes, have uncovered
overlapping genetic and functional mechanisms of disease and led to the
identification of the cytokine tumor necrosis factor (TNF) as a
potential novel immunotherapy
[1]–
[7].
In the case of type 1 diabetes, the rationale for administering TNF is
that insulin-autoreactive T cells bear several intracellular signaling
defects that make them selectively vulnerable to death upon exposure to
TNF
[4]–
[7]. TNF destroys insulin-autoreactive T cells, but not healthy T cells, in
in vitro
studies of human diabetic blood samples and in the NOD mouse model. TNF
exposure may also augment production of beneficial regulatory T cells
(Tregs), a subset of T cells believed to suppress insulin-autoreactive T
cells. Interventions that have destroyed insulin-autoreactive T cells
and boosted beneficial types of T cells have led to regeneration of
insulin-producing islet cells in the pancreas of rodents with autoimmune
diabetes, resulting in restoration of normoglycemia, even in advanced
disease
[7],
[8].
TNF
treatment at high doses in humans is limited by its systemic toxicity.
An alternative approach is to test a safe, U.S. Food and Drug
Administration (FDA)-approved vaccine containing
Mycobacterium bovis bacillus- Calmette-Guerin (BCG), which has been known for over 20 years to induce TNF
[9]. This avirulent strain of
Mycobacterium is different from that which causes tuberculosis in humans (
Mycobacterium tuberculosis).
The
release of TNF after exposure to pathogens, such as BCG, is an example
of a first-line host defense commonly called the innate immune response
[9].
Similar results to those with TNF administration have been achieved
with BCG or its non-FDA approved variant, complete Freund’s adjuvant
(CFA), in rodent models of autoimmune diabetes
[7],
[8],
[10]–
[12].
Nearly
two decades ago, a single, low dose of BCG in humans with late-stage
pre-diabetes was initially found to successfully induce a clinical
remission in some patients
[13],
but when efficacy was re-evaluated in expanded trials, it could not be
observed a year after vaccination. At the time, the mechanisms behind
BCG’s failure were not understood and specific biomarkers or knowledge
of TNF action and autoimmunity were unavailable. In recent years,
however, the mechanism of action underlying the therapeutic potential of
BCG and TNF in autoimmune disease has been further elucidated
[1],
supporting the hypothesis based on animal data that BCG vaccination may
be beneficial in type 1 diabetes, especially if the mechanism of action
of BCG trigger TNF can be closely followed with sophisticated and early
biomarkers of safety.
We conducted a
proof-of-principle, double-blind, placebo-controlled trial, in which we
administered two low-dose BCG vaccinations to patients with long-term
type 1 diabetes. Here, we report on the safety of two low-dose BCG
vaccinations and their effects on four serially studied biomarkers in
long-term type 1 diabetes.
Frequent
blood sampling for up to 5 months was conducted to measure biomarkers of
immune and pancreatic function, including: (1) levels and viability of
cytotoxic autoreactive T cells against insulin, a known autoantigen in
diabetes; (2) induction of protective Tregs; (3) antibodies against the
autoantigen glutamic acid decarboxylase (GAD); and (4) levels of fasting
C-peptide, a marker of endogenous insulin production.
Methods
The
protocol for this trial and supporting CONSORT checklist are available
as supporting information: see Checklist S1 and Protocol S1.
Clinical Trial Participants
All
clinical trial participants were required to be adults, ages 18 to 50
years, with long-term diabetes treated continuously with insulin from
the time of diagnosis; have no demonstrable insulin secretion (fasting
and glucagon-stimulated C-peptide less than 0.2 pmol/L) as assessed by a
standard C-peptide assay by an outside vendor; be pancreatic GAD
autoantibody positive; have a normal complete blood count (CBC); and
have a negative purified protein derivative (PPD) test. Diabetic
patients were excluded if they were pregnant or not using acceptable
birth control; had a chronic infectious disease, including human
immunodeficiency virus (HIV); had a history of tuberculosis (TB) or
current TB infection; were currently receiving treatment with
glucocorticoids, chronic immunosuppressive medications or high dose
aspirin (>160 mg/day); or were currently living with an
immunosuppressed individual. Also excluded were type 1 diabetics with
keloid formation or hemoglobin A1C (HbA1C) values greater than 8%.
Non-diabetic Matched Controls
Healthy,
non-diabetic control subjects were included if they were 18 to 45 years
of age, with no history of autoimmune disease or diabetes, no history
of HIV, and no history of autoimmunity in first-degree family members.
These participants were paired weekly/bi-weekly to the diabetic patients
who were randomized to BCG or placebo.
Reference Groups and Subjects
The
study also included several reference groups: a reference group of type
1 diabetic individuals serially monitored for at least 20 weeks (n
=
57) and a one-time serial studied reference group of type 1 diabetics (n
=
17)
studied for one outcome measure (insulin-autoreactive T cells) and
matched in disease duration and age to the diabetic clinical trial
subjects. The clinical trial subjects were compared to one or more of
these groups, depending on the outcome measure as shown in .
The criteria for inclusion and exclusion of diabetic reference subjects
were the same as those for the clinical trial subjects as related to
age of onset, duration of diabetes and HbA1C values. The reference
subjects studied for insulin-autoreactive T cells were also matched for
human leukocyte antigen (HLA)-A2 status. The serial study of these
reference subjects was performed to expand the database of autoreactive T
cell variation and serially studied C-peptide values in single
subjects, i.e., these separate and sequential blood draws defined the
biological variation in assays in single cohorts and distinguished this
biological variation from variation possibly attributable to BCG
treatment in the randomized clinical trial subjects also studied in a
serial fashion.
CONSORT
flow chart (A) and flow diagraph (B) with depicts of treatment concept,
outcomes and subject comparison groups for the study.
Ethics
This
study was approved by the Human Studies Committee at Massachusetts
General Hospital, Boston, MA and by the FDA. All patients provided
written informed consent.
Trial Design
This
was a proof-of-principle, double-blinded, placebo-controlled clinical
trial that also included a paired healthy control population and
reference subjects. All interventions were administered and clinical
trial participants seen at one clinical center in North America
(Massachusetts General Hospital, Boston, MA, USA) between 2009 and 2011.
The FDA approved this protocol in 2007 and when funding was secured,
the enrollment was launched in 2009.
Intervention Population and Paired Healthy Controls
For
the double-blind, placebo-controlled portion of the study, diabetic
subjects were randomly assigned to BCG or placebo (saline) vaccinations
according to the randomization scheme prepared by the Massachusetts
General Hospital (MGH) research pharmacy. The BCG injection was prepared
by the research pharmacy from lyophilized BCG (TheraCys®,
Sanofi-Pasteur, Toronto, Ontario, Canada), and all syringes (BCG and
saline) were prefilled by the pharmacy. Randomized patients received two
0.1 ml intradermal injections into the deltoid area containing either
low-dose BCG (1.6–3.2×106 colony-forming units/injection) or
saline placebo, administered four weeks apart (Week 0 and Week 4).
Weekly blood sampling was performed until Week 8, followed by bi-weekly
blood sampling until Week 12 and then a final visit at Week 20. This
frequent blood monitoring was performed to validate outcomes and observe
any early effects of therapy. All subjects were seen in the morning and
were required to be fasting and normoglycemic prior to having their
blood drawn.
All injections were administered in the
MGH diabetes clinic. Staff who administered BCG or placebo injections
were not the same as those who examined the participants to grade any
reactions at the injection site. All blood was processed within two
hours of being drawn. All blood samples were blinded and simultaneously
sent to the laboratory for monitoring of T cell response and for storage
of serum for pancreas response tests (ultrasensitive C-peptide assay
and autoantibodies), which were performed by outside vendors at the
completion of the trial as described in “Assay methods”.
A
group of paired healthy control participants, receiving neither BCG nor
placebo, had blood samples obtained at the same time as diabetic
subjects. Their samples were analyzed immediately for T cells in a
masked fashion on the same day as the samples from diabetic subjects.
Masking and Unblinding
The
MGH research pharmacy performed all masking of BCG and saline
vaccinations. All blood samples that were collected were randomly coded
prior to blinded submission to the MGH lab or outside vendor lab for
processing. Unblinding did not occur until all samples were processed
and all data were downloaded into the central computers.
Primary Outcome Measures
We
monitored the safety of BCG in advanced type 1 diabetes and its action
on immune and pancreas outcomes, including levels of
insulin-autoreactive T cells, Treg cells, autoantibodies (including
GAD), and C-peptide, an indicator of endogenous insulin secretion.
T Cell Assay Methods
The two cell-based assays (Treg cells and autoreactive T cells) were performed through Week 12.
Cell isolation
CD4
and CD8 T cells were isolated from fresh human blood within 2 hours of
venipuncture using Invitrogen™ Dynal® CD4 positive isolation kit and
Dynal® CD8 positive isolation kit (Life Technologies Corporation,
Carlsbad, CA, USA). This method is unique in yielding cells both free of
magnetic particles and free of an attached positive selection antibody
to either the CD4 protein or the CD8 protein. The blood was drawn into
BD Vacutainer® tubes (BD, Franklin Lakes, NJ, USA) containing acid
citrate and dextrose or ethylenediaminetetraacetic acid (EDTA). The CD8
or CD4 cells extracted for these studies were selected from fresh blood
and were required, for standardization purposes, to be greater than 98%
pure, 95% viable, and 85% yield for the validated T cell assays
[4],
[14] described below.
Use of controls
For
all T cell assays in this study, a diabetic blood sample was always
drawn at the same time as blood from a paired healthy control to allow
assay standardization.
Detection of autoreactive CD8 T cells to a fragment of insulin
Insulin-autoreactive T cells were assayed by flow cytometry after fresh blood cell separations
[14]
to obtain high-yield and highly pure and viable CD8 T cells for
tetramer staining. Tetramers are T cell detection reagents composed of
the binding region of specific HLA class I proteins with loaded peptides
in the exterior binding grooves. The tetramers, which are made
fluorescent, bind to specific T cells with specific reactivity to the
presented peptide fragment, thereby allowing for cell identification. To
detect autoreactive T cells to insulin, we used tetramers to HLA-A2
*0210 insulin beta 10–18 with a fragment of HLVEALYLV (Beckman Coulter #
T02001)
[15].
To further confirm the specificity of insulin-autoreactive T cell
detection, cell samples were examined simultaneously with T cell
reagents to detect oncogene-specific human epidermal growth factor
receptor-2 (HER-2) or Epstein-Barr virus (EBV)-specific T cells of acute
infection. For simultaneously studied healthy controls, the following
tetramer reagents were used: HLA*0201 Her-2/neu with a sequence to
KIFGSLAFL (Beckman Coulter #
T02001), a breast cancer peptide; HLA*0201 null without a non-specific peptide fragment (Beckman Coulter #
T01044); or an EBV tetramer reagent HLA-A*0201 EBV with sequence of GLCTLVAML (Beckman Coulter #
T01010).
Tetramer
reagent staining was conducted on the highly pure CD8 T cells after 12
hours of culture at 26°C followed by 6 hours at 37°C and/or 1 hour rest
at 26°C followed by 12 hours at 37°C. Cells were then stained with
phycoerythrin-labeled class I tetramers (Beckman Coulter, Fullerton, CA)
and SYTOX green dye (MBL International, Woburn, MA) and/or CD8
antibodies (BD Biosciences, San Jose, CA). All CD8 T cells were stained
at 4°C in the dark for 30 minutes and then washed twice in Hanks
balanced salt solution with 2% heat inactivated bovine serum. On
average, 100,000 highly pure CD8 T cells were analyzed to ensure
optimized data points on the Becton Dickinson FACSCalibur using the Cell
Quest acquisition program and allow the detection of rare autoreactive T
cells. All cells were analyzed while fresh to prevent fixation
artifacts and enable quantification of dead versus viable cells. Prior
to tetramer staining, cells were neither frozen nor expanded.
Calculations of insulin-positive T cells were reported as the percentage
of insulin-autoreactive T cells to the total numbers of isolated pure
CD8 T lymphocytes.
Note that all diabetic treated
patients in the randomized portion of the study were HLA-A2+ except for
diabetic #iv. Although diabetic #iv was HLA-A2 negative, the formal
binding site for the HLA-A2 insulin-autoreactive T cell reagent was
HLA-A6802. HLA-A6802 is a subtype of the HLA-A2 family and has an
identical binding cleft to HLA-A2 and other common subtypes within the
HLA-A2 family. Therefore, if diabetic subject #iv were to have
detectable insulin-autoreactive T cells, those cells would stain
positive for the insulin-autoreactive T cell reagent. Three healthy
controls in this study (Control #ii, Control #iii and Control #v) were
also HLA-A2+.
Reference diabetics were
monitored over a three-year period for the presence or absence of
insulin-autoreactive T cells and compared to their paired healthy
reference controls.
Detection of Treg CD4+ cells
Treg cells were assayed by flow cytometry after fresh blood cell separations as described above and by Burger et al
[14]. Two different methods of cell detection were employed. Treg cells were detected as either CD4, CD25
bright with Foxp3 staining, or with CD4, CD25
bright and CD127
lowantibody
staining. Intracellular staining of Foxp3 was performed with Human Treg
Flow™ Kit (Biolegend, San Diego, CA, USA), according to the
manufacturer’s instructions. Isolated CD4 positive cells were incubated
briefly with CD4-PE-Cy5 (clone RPA-T4) and CD25-PE (clone BC96)
antibodies for 20 minutes at room temperature. After washing, cells were
fixed with Foxp3 Fix/Perm solution (Biolegend) for 20 minutes at room
temperature. Cells were washed again and permeabilized with Foxp3 Perm
Buffer (Biolegend) for 15 minutes at room temperature. Cells were then
stained with Foxp3 Alexa Fluor® 488 antibody (clone 259D, Biolegend) for
30 minutes. Isotype controls were done for each sample prior to flow
cytometric analysis. For detection of Treg cells, staining was performed
with a CD4 antibody (clone RPA-T4, BD Biosciences, San Jose, CA, USA), a
CD25 antibody (clone 4E3, Miltenyi Biotech, Auburn, CA, USA) and an
anti-human CD127 antibody (clone hIL-7R-M21, BD Biosciences).
Flow cytometry for T cell assays
For
the flow cytometry studies, the flow gates were set “open” for
inclusion of CD8 or CD4 T cells of all sizes, but exclusion of the
following: cell debris, red blood cells, fragmented cells, and apoptotic
bodies. The “open gate” was chosen for the purified CD8 or CD4 T cells
because T cells undergoing cell death, especially by apoptosis, can
display changes in light scattering properties. The goal was to ensure
accuracy by analyzing high numbers of cells per sample and to capture
dying cells of all shapes. Cell viability was quantified by either of
two stains that fluorescently labeled dead cells, i.e., Sytox (MBL
international Co., Woburn, MA, USA) or propidium iodine (PI). Purified
CD8 cells form distinct scatter pictures on forward versus side scatter
highlighted the shrunken size of dead versus viable cells.
With
open gating and inclusion of all purified CD8 T cells in each sample,
some reference diabetics consistently displayed insulin-autoreactive T
cells. In contrast, some reference diabetics consistently had
undetectable insulin-autoreactive T cells compared to healthy reference
controls, which were simultaneously studied at each monitoring time. The
data were collected over the multi-year time span. The signal for
insulin-autoreactive T cells was in the range of 0.06–0.09%. The healthy
control background signal is in the range of 0.04–0.05%
[15].
The reverse was also true: diabetics who initially lacked
insulin-autoreactive T cells, on repeat sampling, continued to lack
those cells.
Serum Assay Methods for Pancreas Monitoring
GAD
autoantibody and fasting C-peptide levels were assayed by radio-binding
and ELISA assays in diabetic subjects to assess whether the subjects
had a pancreas response to the BCG injection. For these serum assays,
fresh human blood was collected by venipuncture into red top tubes and
allowed to clot. The serum was then separated by centrifugation within 2
hours of venipuncture. Serum was stored at −80°C until analysis. The
C-peptide assay was undertaken through week 20.
Detection of C-peptide secretion
Measurement
of connecting peptide (C-peptide) co-secreted with insulin permits
direct estimation of any remaining insulin from the pancreas in contrast
with endogenous sources. The first, performed by the Mayo Clinic
(Rochester, MN, USA) utilizing the Roche Cobas C-peptide assay (Roche
Diagnostics, Indianapolis, IN, USA) for clinically detectable C-peptide,
was used for eligibility and had a lower limit of detection of 330–470
pmol/L. This insensitive but standard assay was applied to fasting and
glucagon-stimulated blood samples. After screening negative for
enrollment purposes, subjects’ serum was stored and freezer-banked. For
subsequent samples (baseline through Week 20), the saved serum was sent
to Sweden for analysis of serial C-peptide levels by an ultrasensitive
C-peptide assay with a lower level of detection of 1.5 pmol/L and an
assay range up to 285 pmol/L (Mercodia AB, Uppsala, Sweden). For
C-peptide values of 1.5–37 pmol/L, the within-assay coefficient of
variation was 3.8%; for values of 38–60 pmol/L, it was 2.6%; and for
values of 143–285 pmol/L, it was 2.5%. The Mercodia Ultrasensitive
C-peptide ELISA kit, which is an FDA-listed reagent and has a filed
document registration, has been evaluated for accuracy and is classified
in the United States as a class one device for ultrasensitive detection
of C-peptide levels. This assay is calibrated against the International
Reference Reagent for C-peptide, IRR C-peptide 84/510. All statistics
on C-peptide levels were performed using the lower level of detection of
the assay, i.e., 1.5 pmol/L.
Detection of GAD autoantibodies
GAD
autoantibodies provide evidence of diabetic autoimmunity since GAD
proteins are intracellular proteins specific to insulin secreting cells
and are released from T cell mediated beta cell destruction. The release
of intracellular GAD results in the immune response of autoantibodies.
Enrolled patients were required to be GAD autoantibody positive. Prior
to enrollment, a single serum sample for GAD autoantibody was sent
either to the Joslin Clinic in Boston, MA, USA (Subject #vi, Subject #i,
Subject #ii, Subject #iv) or to Quest Diagnostics (Cambridge, MA, USA)
(Subject #iii, Subject #iv). After the first BCG or placebo injection,
serum samples collected from baseline to Week 20 were sent to Germany
for diabetic autoantibody panels
[16]
at the laboratories of Drs. Ezio Bonifacio and Peter Achenbach of the
Diabetes Research Institute in Munich, Germany. The autoantibodies
studied were GAD, IA-2A (islet-specific protein tyrosine phosphatase),
and ZnT8Carg-A (pancreatic beta cell-specific zinc transporter)
[17].
The GAD assay sensitivity is 86%, specificity is 100%, and inter-assay
variation is 18%. For the IA-2 autoantibody assay, the sensitivity is
72%, the specificity is 100%, and the inter-assay variation is 16%. For
the ZnT8Carg-A assay, the sensitivity is 72%, the specificity is 99%,
and the inter-assay variation is 17%.
Sample Size
Sample
size for the randomized population was determined in conjunction with
the FDA and with the intense use of serial biomarker studies as outlined
by the Institute of Medicine guidelines for clinical trials
[18].
A sample size of 6 randomized patients was determined as appropriate
for the intense serial blood monitoring required in this
proof-of-concept trial for the placebo or BCG interventional limbs and
an expanded population of diabetics and non-diabetic controls for assay
validation that is referred to as reference subjects.
Statistical Analysis
Randomized
participants were compared to self, healthy paired controls, or
reference subjects with or without type 1 diabetes, depending on the
outcome measure, according to the schema depicted in . None of the analyses compared the BCG-treated to placebo-treated clinical trial subjects.
For
each randomized patient, a linear regression model with auto-correlated
errors was used for statistical comparisons between baseline and
post-treatment periods in autoantibody levels over the course of the
study. This was the appropriate test for this comparison because any
change in autoantibodies should be sustained over the monitoring period
of this trial, i.e., the t 1/2 of B cells that produce
antibodies exceeds 60 days. P-values compared the values of each person
to their post-baseline values by two-sided test based on a regression
model with auto-correlated errors. For C-peptide assays, a cut-off value
of 1.5 pmol/L was used since this value is the lower limit of detection
of the ultra-sensitive assay used in this study. C-peptide assays were
performed by the outside vendors in duplicate; figures are therefore
presented as the means +/− the SE. For the comparison of EBV-infected or
BCG-injected patients to the long-term diabetic reference samples, the
Kolmogorov-Smirnov two-sample test was used to compare the distribution
of each patient with the reference samples. We applied this method in a
conservative fashion by overestimating the variability of the clinical
trial sample, as a more exact comparison is difficult to obtain due to
the low sampling frequency and small numbers of measurements per patient
in the reference group. P-values of <0.05 were considered
statistically significant. SAS® version 9.2 was used for the statistical
analysis.
For serum samples sent out
to commercial sources for assay performance, both published inter-assay
and intra-assay variability was considered for the statistical analysis
of the clinical trial samples. We also verified that the inter-assay
variability was consistent in the plate for the clinical samples by
comparing the pre-treatment values with all post-treatment values of the
same patient to self in the same plate. This self-comparison analysis
was performed for serum assays such as C-peptide or autoantibodies. The
area under the curve (AUC) was calculated for all treatment and control
groups, although the control group varied according to the assay.
Results
Participant Enrollment and Characteristics
A total of 85 participants were studied: 63 type 1 diabetics and 22 non-diabetic controls (, .).
In the double-blinded, placebo-controlled portion of the study, a total
of six diabetic subjects were randomly assigned to BCG or placebo
vaccinations. The randomized clinical trial subjects had disease for a
mean duration of 15.3 years (range 7–23 years) and mean age of 35 years
(range 26–47) (), and were paired to healthy controls (n
=
6)
at each weekly blood drawing time for greater than 20 weeks of study.
In addition to these participants, 57 additional reference subjects with
long-term diabetes and 16 reference healthy subjects served as
reference subjects for both serial T cell assays and serum sample
comparisons. Diabetic reference patients had disease for a mean duration
20 years (range 8–53 years) and mean age of 39 years (range 21–65) ().
The intense serial monitoring of blood samples of all clinical trial
subjects resulted in a total of 1,012 blood samples from diabetic or
comparison subjects to quantify both T cell and pancreas changes. This
serial study of biomarkers and comparison groups for the subjects are
depicted in . This intensive study of novel T cell and pancreas biomarkers required different comparison groups ()
due to the lack of serial normative data on the four parameters chosen
to study BCG efficacy in advanced type 1 diabetes. The objective of the
trial was to test safety of multi-dosing BCG in long-term diabetics.
Four monitored endpoints of efficacy were studied as markers of disease
activity: death of insulin autoreactive T cells, induction of Treg
cells, changes in autoantibodies and the restoration of endogenous
insulin secretion through C-peptide levels.
Clinical characteristics of groups of clinical trial subjects and reference subjects.
Epstein-Barr Virus (EBV) Infection
At
screening for clinical trial enrollment, and unbeknownst to us, one
diabetic clinical trial subject had an acute undiagnosed case of EBV
infection. This patient presented with cold/flu symptoms at weeks 3–4
after the placebo injection ().
The presence of the new EBV infection in blood samples was detected
during our blinded laboratory protocols that required analysis of
EBV-reactive T cells (EBV-tetramer positive CD8 T cells) as a control
during the CD8 insulin-autoreactive T cell assays. Further confirmation
of this diagnosis of acute EBV infection was obtained at the end of the
trial with serology sent for commercial antibody testing (Quest
Diagnostics, Cambridge, MA, USA) ().
Clinical laboratory studies reveal acute EBV infection in placebo-treated diabetic.
This
placebo-treated EBV subject completed the five-month trial and was
subjected to the same types of statistical analyses and outcome studies
as other clinical trial subjects. The treatment team and subject
remained masked to treatment assignment.
The course of
the EBV infection was reconstructed from serially studied fresh T cell
samples and by standard clinical laboratory tests on stored serum
samples ().
To understand the precise time course of the EBV infection, this
diabetic’s serum was screened for EBV VCA antibody (IgM), an antibody
that is typically positive days after infection onset to 3–6 weeks
post-infection. The serum was also tested for EBV Early Antigen D Ab, an
antibody that is typically positive only in the infection window
running from 1 month after infection to 2 months post-infection ().
This
placebo-treated diabetic subject was early antigen D antibody-positive
at the first baseline sample at week 0, had CD8 lymphocytosis over 12
weeks of study ()
and demonstrated mildly elevated liver enzyme levels early in the trial
course, all consistent with an acute EBV infection. As the EBV
serologic studies show, Subject #vi had an acute infection that lasted
longer than one month but did not exceed two months in duration. The EBV
tetramer positive cells became vividly positive at week 6 in the T cell
assay and were still vividly positive at week 8, although declining
slightly ().
As a comparison, we include the EBV positive data from a long-term
diabetic that was not part of this clinical trial, but who had a very
distant past EBV infection, to show the low numbers of EBV memory cells
seen using the EBV tetramer methods when infection is not acute (
Fig. S1).
All
other clinical trial subjects in this study were negative for both
acute and past EBV infections throughout the duration of T cell
monitoring during the trial (
Fig. S1). EBV infections, like BCG, trigger innate immunity by inducing secretion of host TNF
[9].
The patient’s EBV status and receipt of placebo saline injections
fortuitously enabled us to compare the serial T cell and pancreas
effects of EBV- and BCG-triggered innate immune responses in the same
study
[9],
[19].
All other clinical trial subjects in this study were negative for both
acute and past EBV infections through T cell monitoring during the trial
(
Fig. S1).
The Majority of Insulin-autoreactive T Cells Released into the Blood after BCG Treatment or EBV Infection are Dead
At
baseline, all six clinical trial subjects lacked elevated levels of
insulin-autoreactive T cells compared to their paired non-diabetic
controls, with ≤0.4% as the upper limit of normal based on the reference
subjects and background staining ().
The presence of insulin-autoreactive T cells was not a requirement for
enrollment into this study, and past studies identified pathologic
autoreactive T cells reactive with this peptide in about 40% of
long-term diabetics
[4].
Within 1 to 4 weeks after BCG treatment, increased numbers of
insulin-autoreactive T cells appeared in the circulation of each
BCG-treated subject vs. their paired healthy control (
i).
Similar, if not greater elevations in circulating insulin-autoreactive T
cells were also seen in the EBV-infected placebo subject coincident
with the T cell and serologic immune response to an ongoing EBV
infection (
iii). Like the non-EBV infected placebo-treated subjects (ii), all paired healthy controls showed no change (
i–iii, blue lines).
Insulin-autoreactive T-cells released into the circulation are dead after BCG treatment or EBV infection.
Among
diabetic reference subjects, approximately 60% had no
insulin-autoreactive T cells. Their values ranged from 0.2–0.4% at all
determinations, levels essentially indistinguishable from their paired
non-diabetic controls (
iv,v).
The remaining 40% consistently had insulin-autoreactive T cell levels
ranging from 0.4–1% at all measurements, a range higher than their
paired non-diabetic controls (
iv,v).
None of the diabetic reference subjects followed longitudinally and
having baseline insulin-autoreactive T cells of <0.4% (n
=
8)
had subsequent values that rose above 0.4%. Thus, the presence or
absence of circulating insulin-autoreactive T cells was shown to be a
stable phenotype in serially studied and untreated type 1 diabetic
subjects with these monitoring methods.
The
insulin-autoreactive T cells appearing in the circulation after BCG or
EBV infection were more likely dead than alive compared to paired
healthy controls (,
Fig. S2, ),
probably indicating not only the rapid release of pre-formed
insulin-autoreactive T cells after BCG treatment or EBV infection but
also their redundant death by TNF induction. Also unlike the low
affinity insulin-autoreactive T cells observed with routine monitoring
of diabetics, the TNF-targeted death of pathogenic cells allowed the
identification of both low affinity as well as newly appearing, high
affinity subsets of autoreactive T cells not previously identified in
the circulation ().
For the three BCG-treated subjects, the AUC representing the cumulative
concentrations of insulin-autoreactive T cells over the course of study
were 2.22, 0.71 and 1.03 compared to their paired healthy control. The
two non-EBV infected placebo-treated subjects’ AUCs were 0.57 and 0.07,
while the EBV-infected subject had a strikingly elevated AUC of 5.69,
reflecting the large numbers of dead insulin-autoreactive T cells being
released into the circulation after the EBV infection. The transient
increases in the number of insulin-autoreactive cells seen in the
BCG-treated or EBV-infected clinical trial subjects (
i, iii) formed a pattern distinctly different than the stable levels observed in the two other placebo-treated subjects (
ii) and in reference diabetic subjects (
iv,v).
Cytometric study of dead and living insulin-autoreactive T cells
revealed that the pathogenic T cells captured in the blood had both the
common low affinity insulin-autoreactive cells as well as the
treatment-specific release of high affinity autoreactive T cells for the
insulin peptide fragments ().
Routine monitoring of diabetics for insulin autoreactive T cells by
diverse studies only reveals low affinity insulin-autoreactive T cells
in diabetes subjects without treatment
[4]. The TNF-induced death
in vivo of insulin-autoreactive T cells with BCG vaccinations or acute EBV infection was confined to the autoreactive T cells.
Two-color
flow pictures of the serial weekly blood monitoring of dead and live
insulin autoreactive T cells in a control subject (left) and BCG-treated
diabetic subject (right).
Regulatory T Cells are Induced by BCG and EBV
The
EBV-infected subject and two BCG-treated subjects appeared to exhibit
increases in the numbers of Treg cells compared to their paired healthy
controls studied simultaneously (ii, iii, vi); the other two placebo-control subjects had stable levels (iv, v).
A similar trend for elevations in Tregs in response to BCG or EBV was
observed by measuring the AUC, a measure of the total accumulation of
Treg ratios. The three BCG-treated subjects had cumulative Treg ratios
of patients compared to controls of 0.12, 0.42 and 0.30 compared to
placebo treated subject accumulations of 0.11 and 0.03. The EBV infected
subject had cumulative Tregs of 0.32.
TREG cells and GAD-autoantibodies change in response to BCG and EBV.
GAD Autoantibody Levels Show Sustained Change after BCG Treatment
At
baseline, GAD autoantibodies, ranging from 60 to 650 units, were
present in all diabetic clinical trial subjects except one BCG-treated
subject ().
There was a statistically significant and sustained change in GAD
autoantibody levels in two of the three BCG-treated subjects after
injections, with one diabetic showing a decrease and the other an
increase relative to self-baseline (p
=
0.0001 and p
=
0.0017, respectively (
ii,iii). In contrast, none of the other diabetic subjects showed any variations from their baseline values of GAD (
iv,v,vi).
The other islet-specific autoantibodies studied, tyrosine phosphatase
IA-2A and beta cell-specific zinc transporter (ZnT8A), were present in
some of the diabetic subjects at baseline ();
only ZnT8A had statistically significant decreases in one BCG treatment
subject. A similar trend for higher or lower acute elevations in GAD in
response to BCG was observed by measuring the AUC, a measure of the
total positive or negative accumulations of GAD autoantibody levels over
the course of the trial. The total raw levels of GAD autoantibodies
over the trial course were 0.00, −379 and +433 for the BCG-treated
subjects and −102 and −116 for the placebo treated subject. The
EBV-subject accumulated GAD autoantibodies of 245. Altered GAD
autoantibody levels have been documented to decrease after re-exposure
of the immune system to childhood BCG vaccinations and acutely increase
or decrease after islet transplantation although the clinical
significance is unknown
[20]–
[22].
IA-2A and ZnT8 autoantibodies in clinical trial subjects by study week.
Fasting Insulin Secretion Temporarily Increased as Measured by C-peptide after BCG and EBV Infection
At
baseline as a recruitment requirement, none of the six diabetic
clinical trial subjects had detectable levels of fasting or stimulated
C-peptide using a relatively low sensitivity C-peptide assay for
screening in the standard clinic setting. Serum from all clinical trial
patients was saved for subsequent insulin secretion studies with an
ultrasensitive C-peptide assay. When the baseline samples were
re-assayed with the ultrasensitive assay, all six clinical trial
subjects had detectable C-peptide above the lower range of sensitivity
of the ultrasensitive assay (>1.5 pmol/L) ().
Fasting C-peptide levels show transient increase in BCG-treated and EBV-infected clinical trial subjects.
Two
of the three BCG-treated subjects and the EBV-infected subject had
transient increases in fasting C-peptide levels by Week 20 compared to
either their baseline or to the values in 41 reference diabetic
subjects. Specifically, C-peptide levels transiently and significantly
rose with BCG administration in Subject #i (mean concentration 3.49
pmol/L [95% CI 2.95–3.8]), Subject #ii (2.57 pmol/L [95% CI 1.65–3.49]),
as well as in the EBV-infected placebo Subject #vi (3.16 pmol/L [95% CI
2.54–3.69]) relative to 41 reference diabetic subjects (mean
=
1.65 pmol/L [95% CI 1.55–3.2]), using the Kolmogorov-Smirnov two-sample test (). Subjects #i and #ii each had more than 50% of their C-peptide values above the 95
th
percentile of the reference levels. Subject #vi had 18% of C-peptide
values above this level. Neither non-EBV infected placebo-treated
diabetic subject (
iv and v) had C-peptide fluctuations of statistical significance
.
The biologic stability of low levels of fasting C-peptide levels with
serial monitoring in the ultra-sensitive assay is apparent in 41
reference diabetics ()
and confirmed in 17 additional diabetic subjects evaluated weekly for
12 weeks that were collected after the trial completion to further
confirm the stability of the ultrasensitive C-peptide assay in serially
studied long term diabetics with these low levels ().
AUC measurements of C-peptide, a measure of cumulative changes of
C-peptide levels over the 5-month trial, were higher in the two
BCG-treated and one EBV-infected subject than in the non-EBV infected
placebo clinical trial subjects.
C-peptide levels remain stable and near the lower limit of an ultrasensitive assay in a longterm diabetic group (N=17) sampled weekly for 12 weeks in a fasting state.
Other Clinical and Safety Monitoring
There
were no significant changes in any of the clinical trial patients in
any safety monitoring parameters, including routine chemistry and liver
function tests, hematologic studies, or HbA1c levels. Other than the
expected vaccination scars associated with BCG, no adverse effects
occurred. None of the participants dropped out of the clinical trial.
Discussion
The
goals of the current trial were to determine whether activation of the
innate immune system could be accomplished safely with repeated BCG
vaccinations and whether this treatment would ameliorate, for any time
period, the advanced autoimmune state of long-term type 1 diabetes. We
found that repeated BCG vaccination at low doses was safe and well
tolerated. We also found that BCG vaccination and an unexpected EBV
infection in a placebo-treated diabetic subject, both known triggers of
innate immunity, caused rapid increases in circulating
insulin-autoreactive T cells that were mostly dead. The rapid release of
dead insulin-autoreactive T cells supports the hypothesis, first
demonstrated in the NOD-mouse model of autoimmune diabetes, that BCG
ameliorates the advanced autoimmune process underlying type 1 diabetes
by stimulating TNF, which selectively kills only disease-causing cells
and, further, permits pancreas regeneration
[7],
[8] as evidenced by the transient increase in C-peptide secretion we observed using an ultrasensitive C-peptide assay.
The
response we observed in the placebo subject who experienced an acute
EBV infection provides evidence that infectious agents other than
Mycobacterium can activate innate immunity in long-term diabetic subjects and modify the host’s aberrant autoimmune response
[9].
The subjects EBV status and receipt of placebo saline injections
fortuitously enabled us to compare the serial T cell and pancreas
effects of EBV- and BCG-triggered innate immune responses in the same
study
[9],
[19]. EBV infections, like BCG, are known to trigger innate immunity by inducing a strong host TNF response
[9],
[19],
and the changes in autoimmune cells and beta cell responses we observed
in BCG-treated subjects were similar or sometimes even larger in the
EBV-infected subject, suggesting that a larger dose of BCG might be more
effective. The transient increases in C-peptide, found after both an
acute EBV infection and with BCG vaccinated subjects, suggests a
positive impact of these immune perturbations on beta cell function.
This
study may offer mechanistic insights into ongoing clinical trials of
broad-spectrum immunosuppressive drugs, such as anti-CD3 antibodies, in
new-onset type 1 diabetes. The administration of humanized anti-CD3
antibodies is associated with side effects, including re-activation of
EBV in recent-onset type 1 diabetes. as reported to the FDA. Lowering
the dose of anti-CD3 antibodies reduced EBV reactivation in clinical
studies, but also eliminated efficacy. In another trial of anti-CD3 in
new-onset diabetes, the release of greater numbers of
insulin-autoreactive-specific T cells correlated with the simultaneous
appearance in the circulation of EBV-specific T cells. Taken together,
findings from anti-CD3 trials and the trial reported in this paper
demonstrate that EBV infection or BCG vaccination marshals innate
immunity characterized by known elevations in TNF and that this leads to
potentially therapeutic benefits, especially death of
insulin-autoreactive T cells.
Drug development is
facilitated by understanding drug mechanism and by development of
biomarkers for monitoring early responses to therapy. One previous
uncontrolled study of a single dose BCG vaccination reported possibly
successful stabilization of blood sugars in 65% of pre-diabetic patients
[13].
Subsequent controlled clinical studies of a single low-dose BCG
vaccination in new-onset diabetic children did not show a benefit when
the patients were re-studied, typically a year later
[23]–
[25].
The current trial is unique in now understanding the mechanism of BCG
and the development of closely linked bio-markers to track mechanism. We
additionally utilized multi-dosing of BCG combined with frequent
monitoring for disease-specific biomarkers for up to 20 weeks to observe
any TNF-driven immune effects. Intensive monitoring uncovered
alterations in disease-specific T cells and changes in C-peptide
secretion that suggest brief functional improvement in the pancreas. Our
findings are consistent with trials showing BCG vaccination decreased
disease activity and prevented progression of brain lesions in advanced
multiple sclerosis, an autoimmune disease similarly sharing autoreactive
T cells vulnerable to TNF-triggered cell death
[26],
[27]. Recent findings also suggest repeat BCG administration, but not single BCG vaccinations in childhood prevents diabetes onset
[28] and childhood BCG vaccinations prevent autoantibody formation
[20].
In
the current study, BCG was expressly chosen as a treatment for its
induction of TNF, which has been shown to play a therapeutic role in at
least in four rodent models of five autoimmune diseases
[3],
[7],
[8],
[10],
[12],
[29],
[30] and
in vitro
[4].
In contrast to the clinical utility of anti-TNF therapies in rheumatoid
arthritis but worsening of symptoms when anti-TNF is used in most other
autoimmune diseases
[31]–
[37],
these experiments have repeatedly shown that TNF or TNF-inducers
protect against onset and progression of many forms of autoimmunity.
They also have reversed autoimmune disease, ameliorated advanced
autoimmune disease, if administered in newly transplanted islet tissues,
and/or permitted regeneration of the end organs. In some of these
diverse rodent and human models of autoimmunity, the underlying
mechanism of TNF’s therapeutic effect has been traced to various genetic
and functional errors in the proteasome or proteasome-activated
transcriptional factor NFκB (nuclear factor-κB) signaling pathway
[1],
[17],
[38]–
[55].
For
a therapeutic and sustained amelioration of the autoimmune state,
including a permanent elimination of insulin-autoreactive T cells in
diabetes, potentially leading to a sustained return of C-peptide
secretion, more frequent or higher dosing of BCG will likely be
required. Past human studies have established that even modest levels of
remaining C-peptide activity are beneficial in the reduced incidence of
retinopathy and nephropathy as well as the avoidance of hypoglycemia
[56].
Our findings provide proof-of-principle evidence that
insulin-autoreactive T cells can be specifically targeted and
eliminated, albeit briefly,
in vivo, even in long-standing
disease with a transient restoration of C-peptide. This paves the way
for either higher doses or more frequent BCG administered in future
trials for patients with advanced disease to maintain or restore
C-peptide levels.
Supporting Information
Figure S1
Levels of EBV-specific memory T-cells in placebo subject with latent EBV infection who was not part of this trial
(A) Negative levels of EBV-specific memory T-cells in clinical trial
subjects, both BCG-treated and placebo-treated clinical trial subjects.
(TIFF)
Figure S2
Flow cytometric methods used for the analysis of purified CD8 T-cells for quantifying the numbers of dead versus live cells.
Fresh CD8 T-cells cultured overnight can be demonstrated by forward
versus side scatter histograms on a flow cytometer to be either viable
or dead based on the placement on a side-scatter versus forward scatter
flow gate. The CD8 T cells can additionally be confirmed as dead or
alive based not only by the size of dying cells (scatter) but also by
staining with propidium iodide (PI), a reagent that stains dead cells.
With differential flow gating and/or staining with PI, the dead cells
are concentrated in the left upper quadrant and the viable cells are
concentrated in the right lower quadrant.
(TIFF)
Checklist S1
CONSORT Checklist.
(DOC)
Protocol S1
Trial Protocol.
(DOC)
Acknowledgments
We thank L. Murphy and M. Davis, PhD and D. Briscoe, MPH, for providing formatting and editorial assistance.
Funding Statement
The
Iacocca Foundation and philanthropic dollars supported this study. The
authors also reserve gratitude to the James B Pendleton Charitable
Trust. Finally, the authors extend their appreciation to the Friends
United for Juvenile Diabetes Research and Partnership for Cures. DMN was
supported in part by the Charlton Fund for Innovative Diabetes
Research. NIH support included #P30DK057521 to DLF. No drug company or
for-profit resources supported this trial. The funders had no role in
study design, data collection and analysis, decision to publish, or
preparation of the manuscript. This study was funded by philanthropic
grants only.
References
1.
Kodama S, Davis M, Faustman DL (2005) The therapeutic potential of tumor necrosis factor for autoimmune disease: a mechanistically based hypothesis. Cell Mol Life Sci 62: 1850–1862. [PubMed]
2.
Satoh J, Seino H, Abo T (1989) Recombinant human tumor necrosis factor a suppresses autoimmune diabetes in nonobese diabetic mice. J Clin Invest 84: 1345–1348. [PMC free article] [PubMed]
3.
Grewal IS, Grewal KD, Wong FS, Picarella DE, Janeway CA, et al. (1996) Local
expression of transgene encoded TNF alpha in islets prevents autoimmune
diabetes in non-obese diabetic (NOD) mice by preventing the development
of autoreactive islet specific T cells. J Exp Med 184: 1963–1974. [PMC free article] [PubMed]
4.
Ban L, Zhang J, Wang L, Kuhtreiber W, Burger D, et al. (2008) Selective death of autoreactive T cells in human diabetes by TNF or TNF receptor 2 agonism. Proc Natl Acad Sci U S A 105: 13644–13649. [PMC free article] [PubMed]
5.
Qin HY, Chaturvedi P, Singh B (2004) In vivo apoptosis of diabetogenic T cells in NOD mice by IFN-γ/TNF-α. Int Immunol 16: 1723–1732. [PubMed]
6.
Christen U, Von Herrath MG (2002) Apoptosis
of autoreactive CD8 lymphocytes as a potential mechanism for the
abrogation of type 1 diabetes by islet-specific TNF-alpha expression at a
time when the autoimmune process is already ongoing. Ann N Y Acad Sci 958: 166–169. [PubMed]
7.
Ryu S, Kodama S, Ryu K, Schoenfeld DA, Faustman DL (2001) Reversal of established autoimmune diabetes by restoration of endogenous beta cell function. J Clin Invest 108: 63–72. [PMC free article] [PubMed]
8.
Kodama S, Kuhtreiber W, Fujimura S, Dale EA, Faustman DL (2003) Islet regeneration during the reversal of autoimmune diabetes in NOD mice. Science 302: 1223–1227. [PubMed]
9.
Rahman MM, McFadden G (2006) Modulation of tumor necrosis factor by microbial pathogens. PLoS Pathog 2: e4. [PMC free article] [PubMed]
10.
Harada M, Kishimoto Y, Makino S (1990) Prevention of overt diabetes and insulitis in NOD mice by a single BCG vaccination. Diabetes Res Clin Prac 8: 85–89. [PubMed]
11.
Sadelain MWJ, Hui-Yu Q, Lauzon J, Singh B (1990) Prevention of type I diabetes in NOD mice by adjuvant immunotherapy. Diabetes 39: 583–589. [PubMed]
12.
McInerney MF, Pek SB, Thomas DW (1991) Prevention of insulitis and diabetes onset by treatment with complete Freund’s adjuvant in NOD mice. Diabetes 40: 715–725. [PubMed]
13.
Shehadeh N, Calcinaro F, Bradley BJ, Bruchlim I, Vardi P, et al. (1994) Effect of adjuvant therapy on development of diabetes in mouse and man [see comments]. Lancet 343: 706–707. [PubMed]
14.
Burger DE, Wang L, Ban L, Okubo Y, Kuhtreiber WM, et al. (2011) Novel automated blood separations validate whole cell biomarkers. PLoS One 6: e22430. [PMC free article] [PubMed]
15.
Pinkse GG, Tysma OH, Bergen CA, Kester MG, Ossendorp F, et al. (2005) Autoreactive CD8 T cells associated with beta cell destruction in type 1 diabetes. Proc Natl Acad Sci U S A 102: 18425–18430. [PMC free article] [PubMed]
16.
Achenbach P, Lampasona V, Landherr U, Koczwara K, Krause S, et al. (2009) Autoantibodies to zinc transporter 8 and SLC30A8 genotype stratify type 1 diabetes risk. Diabetologia 52: 1881–1888. [PubMed]
17.
Castiblanco J, Anaya J-M (2008) The I kappa BL gene polymorphism influences risk of acquiring systemic lupus erythematosus and Sjogren’s syndrome. Human Immunology 69: 45–51. [PubMed]
18. Trials CoSfS-N-PCR, Policy BoHS (2001) Small Clinical Trials: Issues and Challenges: The National Academies Press.
19.
Devergne O, Hatzivassiliou E, Izumi KM, Kaye KM, Kleijnen MF, et al. (1996) Association
of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain
important for B-lymphocyte transformation: role in NF-kappaB activation. Mol Cell Biol 16: 7098–7108. [PMC free article] [PubMed]
20.
Sanjeevi CB, Das AK, Shtauvere-Brameus A (2002) BCG vaccination and GAD65 and IA-2 autoantibodies in autoimmune diabetes in southern India. Ann N Y Acad Sci 958: 293–296. [PubMed]
21.
Braghi S, Bonifacio E, Secchi A, Di Carlo V, Pozza G, et al. (2000) Modulation
of humoral islet autoimmunity by pancreas allotransplantation
influences allograft outcome in patients with type 1 diabetes. Diabetes 49: 218–224. [PubMed]
22.
Bosi E, Braghi S, Maffi P, Scirpoli M, Bertuzzi F, et al. (2001) Autoantibody response to islet transplantation in type 1 diabetes. Diabetes 50: 2464–2471. [PubMed]
23.
Pozzilli P (1997) BCG vaccine in insulin-dependent diabetes mellitus. IMDIAB Group. Lancet 349: 1520–1521. [PubMed]
24.
Allen HF, Klingensmith GJ, Jensen P, Simoes E, Hayward A, et al. (1999) Effect of Bacillus Calmette-Guerin vaccination on new-onset type 1 diabetes. A randomized clinical study. Diabetes Care 22: 1703–1707. [PubMed]
25.
Elliott JF, Marlin KL, Couch RM (1998) Effect of bacille Calmette-Guerin vaccination on C-peptide secretion in children newly diagnosed with IDDM. Diabetes Care 21: 1691–1693. [PubMed]
26.
Ristori G, Buzzi MG, Sabatini U, Giugni E, Bastianello S, et al. (1999) Use of Bacille Calmette-Guerin (BCG) in multiple sclerosis. Neurology 53: 1588–1589. [PubMed]
27.
Paolillo A, Buzzi MG, Giugni E, Sabatini U, Bastianello S, et al. (2003) The
effect of Bacille Calmette-Guerin on the evolution of new enhancing
lesions to hypointense T1 lesions in relapsing remitting MS. Journal of Neurology 250: 247–248. [PubMed]
28. Khan M, Aydin M (2012) Effect of BCG vaccine in the prevention of type 1 diabetes mellitus. Contemp J Med 2: 1–8.
29.
Sadelain MW, Qin HY, Sumoski W, Parfrey N, Singh B, et al. (1990) Prevention of diabetes in the BB rat by early immunotherapy using Freund’s adjuvant. Journal of Autoimmunity 3: 671–680. [PubMed]
30. Okubo Y, Kanazawa Y, Oikawa Y, Miyazaki JI, Shimada A (2006) Islet hypertrophy observed in “reversed”. diabetic NOD mouse after pancreatic beta cell line administration Diabetes A 281: 1193.
31.
Enayati PJ, Papadakis KA (2005) Association of Anti-tumor Necrosis Factor Therapy With the Development of Multiple Sclerosis. J Clin Gastroenterol 39: 303–306. [PubMed]
32.
Sicotte NL, Voskuhl RR (2001) Onset of multiple sclerosis associated with anti-TNF therapy. Neurology 57: 1885–1888. [PubMed]
33.
Thomas CW Jr, Weinshenker BG, Sandborn WJ (2004) Demyelination during anti-tumor necrosis factor alpha therapy with infliximab for Crohn’s disease. Inflamm Bowel Dis 10: 28–31. [PubMed]
34.
van Oosten BW, Barkhof F, Truyen L, Boringa JB, Bertelsmann FW, et al. (1996) Increased
MRI activity and immune activation in two multiple sclerosis patients
treated with the monoclonal anti-tumor necrosis factor antibody cA2. Neurology 47: 1531–1534. [PubMed]
35.
Boulton JG, Bourne JT (2007) Unstable diabetes in a patient receiving anti-TNF-alpha for rheumatoid arthritis. Rheumatology 46: 178–179. [PubMed]
36.
Ramos-Casals M, Brito-Zeron P, Soto MJ, Cuadrado MJ, Khamashta MA (2008) Autoimmune diseases induced by TNF-targeted therapies. Best Practice & Research in Clinical Rheumatology 22: 847–861. [PubMed]
37.
Ko JM, Gottlieb AB, Kerbleski JF (2009) Induction and exacerbation of psoriasis with TNF-blockade therapy: A review and analysis of 127 cases. Journal of Dermatological Treatment 20: 100–108. [PubMed]
38.
Hayashi T, Faustman D (1999) NOD mice are defective in proteasome production and activation of NF- kappaB. Mol Cell Biol 19: 8646–8659. [PMC free article] [PubMed]
39.
Hayashi T, Faustman DL (2001) Selected contribution: Association of gender-related LMP2 inactivation with autoimmune pathogenesis. J Appl Physiol 91: 2804–2815. [PubMed]
40.
Hayashi T, Kodama S, Faustman DL (2000) Reply to ‘LMP2 expression and proteasome activity in NOD mice’. Nat Med 6: 1065–1066. [PubMed]
41.
Guo D, Li M, Zhang Y, Yang P, Eckenrode S, et al. (2004) A functional variant of SUMO4, a new I kappa B alpha modifier, is associated with type 1 diabetes. Nat Genet 36: 837–841. [PubMed]
42.
Wong HK, Kammer GM, Dennis G, Tsokos GC (1999) Abnormal
NF-kappa B activity in T lymphocytes from patients with systemic lupus
erythematosus is associated with decreased p65-RelA protein expression. J Immunol 163: 1682–1689. [PubMed]
43.
Kessel A, Rosner I, Rozenbaum M, Zisman D, Sagiv A, et al. (2004) Increased CD8+ T cell apoptosis in scleroderma is associated with low levels of NF-kappa B. J Clin Immunol. 24: 30–36. [PubMed]
44.
Valero R, Baron ML, Guerin S, Beliard S, Lelouard H, et al. (2002) A
defective NF-kappa B/RelB pathway in autoimmune-prone New Zealand black
mice is associated with inefficient expansion of thymocyte and
dendritic cells. J Immunol 169: 185–192. [PubMed]
45.
Zaiss DMW, Bekker CPJ, Grone A, Lie BA, Sijts AJAM (2011) Proteasome Immunosubunits Protect against the Development of CD8 T Cell-Mediated Autoimmune Diseases. Journal of Immunology 187: 2302–2309. [PMC free article] [PubMed]
46.
Bohren KM, Nadkarni V, Song JH, Gabbay KH, Owerbach D (2004) A
M55V polymorphism in a novel SUMO gene (SUMO-4) differentially
activates heat shock transcription factors and is associated with
susceptibility to type I diabetes mellitus. J Biol Chem 279: 27233–27238. [PubMed]
47.
Vereecke L, Beyaert R, van Loo G (2009) The ubiquitin-editing enzyme A20 (TNFAIP3) is a central regulator of immunopathology. Trends in Immunology 30: 383–391. [PubMed]
48.
Trynka G, Zhernakova A, Romanos J, Franke L, Hunt KA, et al. (2009) Coeliac disease-associated risk variants in TNFAIP3 and REL implicate altered NF-kappa B signalling. Gut 58: 1078–1083. [PubMed]
49.
Zhang G-L, Zou Y-F, Feng X-L, Shi H-J, Du X-F, et al. (2011) Association of the NFKBIA gene polymorphisms with susceptibility to autoimmune and inflammatory diseases: a meta-analysis. Inflammation Research 60: 11–18. [PubMed]
50.
Till A, Rosenstiel P, Krippner-Heidenreich A, Mascheretti-Croucher S, Croucher PJ, et al. (2005) The
Met-196 -> Arg variation of human tumor necrosis factor receptor 2
(TNFR2) affects TNF-alpha-induced apoptosis by impaired NF-kappaB
signaling and target gene expression. J Biol Chem 280: 5994–6004. [PubMed]
51.
Allcock RJN, de la Concha EG, Fernandez-Arquero M, Vigil P, Conejero L, et al. (1999) Susceptibility to multiple sclerosis mediated by HLA-DRB1 is influenced by a second gene telomeric of the TNF cluster. Human Immunology 60: 1266–1273. [PubMed]
52.
Allcock RJN, Christiansen FT, Price P (1999) The central MHC gene IKBL carries a structural polymorphism that is associated with HLA-A3,B7,DR15. Immunogenetics 49: 660–665. [PubMed]
53.
Miterski B, Bohringer S, Klein W, Sindern E, Haupts M, et al. (2002) Inhibitors
in the NFkappaB cascade comprise prime candidate genes predisposing to
multiple sclerosis, especially in selected combinations. Genes Immun 3: 211–219. [PubMed]
54.
Wandinger KP, Sturzebecher CS, Bielekova B, Detore G, Rosenwald A, et al. (2001) Complex
immunomodulatory effects of interferon-beta in multiple sclerosis
include the upregulation of T helper 1-associated marker genes. Annals of Neurology 50: 349–357. [PubMed]
55.
Fu Y, Yan G, Shi L, Faustman D (1998) Antigen processing and autoimmunity. Evaluation of mRNA abundance and function of HLA-linked genes. Annals of the New York Academy of Sciences 842: 138–155. [PubMed]
56.
Steffes MW, Sibley S, Jackson M, Thomas W (2003) Beta-cell function and the development of diabetes-related complications in the diabetes control and complications trial. Diabetes Care 26: 832–836. [PubMed]
Articles from PLoS ONE are provided here courtesy of Public Library of Science
Related citations in PubMed
Cited by other articles in PMC
America's health care reform through history
Dropping LBs: LIers share weight-loss success stories
Obamacare cartoons
The Daily Apple health blog
Health insurance rates for LI, NYC communities
Search LI's hospital rankings
No comments:
Post a Comment