T1D target epitope from zinc transporter 8 (ZnT8) cross-reacts with commensal bacteria

Type 1 diabetes (T1D) is considered autoimmune disease. Of course, in humans, we don't have a direct evidence that islet-specific auto-reactive T cells and auto-antibodies found in peripheral blood from T1D patients are indeed responsible for tissue damage. Such evidence would require T/B cell and Ab depletion experiment that is not feasible. Autoimmune nature of T1D is basically extrapolated from mouse studies or in vitro antigen binding assays.   

Several islet antigens are known to represent targets in T1D, such as preproinsulin (PPI), glutamic acid decarboxylase (GAD), insulinoma-associated protein-2 (IA-2), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) and more recently described zinc transporter 8 (its peptide ZnT8_186–194).

New study in Science Immunology found that ZnT8_186–194-specific CD8 T cells are largely similarly present in both T1D patients and healthy controls and that ZnT8_186–194-specific CD8 T cells could recognize (cross-react) peptide derived from gut commensal microbe Bacteroides stercoris.

Staining with ZnT8_186–194-specific HLA class I multimers (MMr) and other functional antigen-specific assays found that T1D and healthy controls harbored largely similar number and functional ZnT8_186–194-specific CD8 T cells.

Interestingly, ZnT8_186–194-specific CD8 T cells could be double stained with HLA class I multimer + peptide derived from B. stercoris, a commensal bacterial species found in gut flora. It is not the first time such cross-reactivity has been observed between islet-specific CD8 T cells and commensal bacteria. Previously, at least two bacterial species have been identified to cross-react with IGRP-specific CD8 T cells.

In summary, this study indicates that deletion of auto-reactive CD8 T cells in the thymus is not sufficient to prevent autoimmunity and that regulatory mechanisms operating in the periphery is necessary to prevent initiation of auto-reactive attack by circulating ZnT8_186–194-specific CD8 T cells (probably Tregs the authors had in mind). Cross-reactive peptide derived from B. stercoris could play role in priming of ZnT8_186–194-specific CD8 T cells in absence of regulatory circuit. But how and why such antigen-specific tolerance breakdown happens in one and not in another is not clear at this stage.

posted by David Usharauli

Loss of microbiota depletes cross-reactive Foxp3+ Tregs leading to selective immunopathologies

Check out our follow-up manuscript in PeerJ Preprints that provides a brief guide to SPIRAL, a novel interpretive framework that demonstrates the central role of microbiota-Treg axis in the initiation of immune disorders.

Kamala T, Usharauli D. (2017)

 

Loss of microbiota depletes cross-reactive Foxp3+ Tregs leading to selective immunopathologies.

PeerJ Preprints 5:e3237v1

https://doi.org/10.7287/peerj.preprints.3237v1

 

The 'Hygiene hypothesis', a cornerstone model to account for the role of exogenous pathogens and later of endogenous microbiota in immune disorders, is currently presumed to operate at the innate immunity and metabolite levels to properly 'educate' the immune system. Doing so however fails to satisfactorily account for the antigen-specific nature of such disorders. SPIRAL is a novel interpretive framework that resolves this dilemma. It represents the periodic table of cross-reactive Foxp3+ regulatory T cell (Treg) epitopes selected from commensal microbiota over evolutionary time to mediate self-nonself discrimination and effector class regulation. Here, we utilize the SPIRAL's predictive power to provide a mechanistic antigen-specific basis for the initiation of allergies and autoimmune diseases as well as for the failure to mount effective anti-tumor and vaccine responses through selective loss of microbiota and corresponding cross-reactive Foxp3+ Tregs.



 

IL-27 protects against autoimmunity through its effect on Tregs

IL-27 is a heterodimeric cytokine composed of the p28 and Ebi3 subunits produced by APCs. It binds to IL-27 receptors (IL 27Rα:gp130) expressed on several cell types, including T lymphocytes. IL-27Ra-/- mice are shown to be highly susceptible to experimental autoimmune encephalomyelitis (EAE), a mouse of human MS. Earlier studies suggested that effect of IL-27 is mediated through its suppression of pro-inflammatory Th17 cells and generation of anti-inflammatory IL-10 producing Tr1 cells.

A new study in PNAS, however, showed that mice with Treg-specific IL-27Rα-deficiency displayed similar susceptibility to EAE as total IL-27Rα-deficient mice pointing to the role of Tregs in mediating IL-27 effect on EAE.

Compared to WT mice, mice with Treg-selective IL-27Rα-deficiency were not able to recover from EAE, a similar trend seen in total IL-27Rα-deficient mice. IL-10 levels were not different between WT and KO littermates.

Furthermore, in contrast to WT mice, injection of "therapeutic" dosage of IL-27 had no effect on EAE dynamics in Treg-selective IL-27Rα-deficient mice. Nor did antibody-blockade of IL-10 diminish effect of IL-27 on WT mice.

These results suggest that in addition to other cell types or even in contrast to other studies, the role of IL-27 in controlling severity of EAE could be solely mediated through its effect on Foxp3+ Tregs.

posted by David Usharauli 

 

MHC class II epitope presentation modulates microbiota and protects against autoimmunity

It is not clear how exactly microbiota modulates host's immune system. Evidence are largely based on empirical observations and nonspecific factors secreted by microbiota. Presently very little is known if microbiota-immune system interface is also controlled at the level of antigen-specific adaptive immune system.    

 

New study from Diane Mathis lab published in PNAS suggests it may be the case.

 

Her lab studies human type I diabetes (T1D) mouse model known as NOD. NOD mice lack MHC II allele, Eα. In this study they used NOD mice expressing Eα, referred as Eα16/NOD. In mating experiments, they noticed when Eα was expressed by female but not by male parent, baby mice with NOD genotype showed significant protection from developing T1D, suggesting protection was transmitted vertically from Eα16/NOD mother to NOD pups. Interestingly, this protection was lost when pregnant mothers (dams) were treated with antibiotics pointing towards role of microbiota.

Experiments with germ-free sterile mice confirmed this observation.

In summary, this study showed that MHC class II [epitope] presentation modulates composition of microbiota in such a way to harbor species protective against T1D. Again, the authors were unable to specifically pinpoint any specific mechanism of protection, though they reported increase in Foxp3+ Treg numbers in Eα16/NOD mice compared to NOD (but found no difference in microbiota bound to IgA between mouse strains). It is likely that epitope presentation at the level of adaptive CD4 T cells contributed to development of protective environment.

posted by David


    

 

Does TGF-β control T cell autoimmunity independent of Tregs?

When T cells attack body's own antigens its called autoimmunity. Each and every one with the adaptive immune system carry this potential. Mostly two mechanisms prevent autoimmunity: thymic deletion of overtly auto-reactive T cells (recessive tolerance) and Foxp3+ Tregs (dominant tolerance).

 

Within immune system, TGF-β plays important inhibitory role at T cell level. However, since TGF-β is involved in Treg biology, it is not clear if it has Treg-independent role in preventing autoimmunity.

 

New paper published in PNAS tried to answer this question.

 

The authors used OT-II RIP-mOva mice model (on RAG KO background) in which all CD4 T cells express OVA-specific T cell receptor and pancreas express OVA protein. These mice harbor OT-II Foxp3+ Tregs and they don't develop autoimmune diabetes.

To separate effect of Tregs versus TGF-β, the authors either compared TGF-βRII-KO mice vs. Foxp3KO (both on RAG1-KO OT-II RIP-mOva background) or adoptively transferred into RAG1-KO RIP-mOVA mice either Foxp3KO OT-II or OT-II T cells expressing TGF-βRII under the control of estrogen receptor. They noticed that OT-II T cell population lacking TGF-βRII but not Foxp3 could cause or accelerate autoimmune diabetes.

These two set of experiments are central for this paper. However, contrary to the authors' conclusions, these experiments do not fully answer Tregs versus TGF-β question. The main problem is that total TGF-βRII deficiency in all CD4 T cells affects both effector and Tregs (functionally at least if not number wise) while Foxp3 deficiency only affects Tregs. That is to say that if Tregs were TGF-βRII-sufficient and effector T cells TGF-βRII-deficient outcome could be different (WT Tregs might be able to stop effector OT-II cells). Another way to separate the role of Tregs versus TGF-β would be to specifically inactivate TGF-βRII in effector T cells leaving Tregs intact.

posted by David Usharauli


 

antigen-specific Foxp3+ Tregs maintain tolerance in HLA-linked autoimmunity

This week Nature published new paper that has features of textbook studies. In it the authors showed that in human HLA transgenic mice model of Goodpasture disease [HLA+antigen]-specific Foxp3+ Tregs protected against autoimmune disease development.

Goodpasture disease is an "HLA-linked autoimmune renal disorder characterized by an immunodominant CD4+ T-cells [reactive to] self-epitope derived from the α3 chain of type IV collagen (α3135–145)". In humans presence of HLA-DR15 allele increases disease risk, while presence of HLA-DR1 allele is shown to be dominantly protective in trans with HLA-DR15.

Interestingly enough the authors reported similar pattern of HLA dependency in mouse model of human Goodpasture disease. Here, DR15+ mice were susceptible to disease development. DR1+ mice were resistant to disease development and DR15+DR1+ double-positive mice were healthy except when Tregs were depleted (all mice were on Fcgr2b−/− background, +/- Treg depletion, + immunization with peptide α 3135–145). 

It is not clear how Treg specific for DR1+peptide protects against autoreactive T cells specific for DR15+peptide. It is possible that there is some similarities between these HLA+peptides (cross-reactivity).

David Usharauli

Minimal threshold for Treg repertoire diversity that prevents autoimmunity

If you have read enough of my blog you most likely noticed the preference for research articles that provide some new insight into Tregs' biology.

Few weeks back I analyzed new paper from Rudensky's lab that showed that 50% of mouse with Tregs expressing single TCR specificity were protected from lethal autoimmunity, though not from non-lethal or less lethal forms of  tissue autoimmunity. 

So what is the minimal Tregs TCRβ repertoire diversity required to suppress autoreactive T cells that escape thymic selection?

New paper from Thomas Malek's lab tried to found it out by serially transferring WT Tregs into Treg-deficient IL-2RβKO mice (1st and 2nd level recipients) and analyzing TCR repertoire pre and post transfer. They observed that after each transfer into IL-2RβKO Tregs TCR repertoire diversity got narrower and when it fell < 7000 unique Treg clonal specificity mice showed autoimmunity.

This threshold also correlated with pathological increase in CD62L^lowCD44^hi population that represents activated T cells typical to autoimmunity.

In summary, this study indicates that there is a minimal threshold for Tregs repertoire diversity that is essential to prevent non-lethal forms of autoimmunity.

David Usharauli
        

New synthetic molecule prevents autoimmunity without compromising anti-pathogen immunity

In December of 2016 Science Translational Medicine published new study where the authors introduced new, low-molecular weight compound that specifically inhibited autoimmune/allergic responses while sparing anti-pathogen immune response.

This new molecule, AX-024, specifically targets adaptor protein Nck that is involved in low affinity TCR signaling. 

It does not affect signaling delivered via IL-2, BCR or PMA/Ion stimulation.

AX-024 inhibited TLR7/8 agonist, imiquimod (IMQ), induced psoriasis skin symptoms in mice.

Also, AX-024 significantly reduced clinical symptoms associated with mouse model of human multiple sclerosis (MS). Such effect of AX-024 was superior to Fingolimod, a sphingosine 1-phosphate receptor modulator approved for the treatment of relapsing-remitting MS.

Notably, AX-024 in therapeutic dosage did not impair anti-pathogen immunity or memory generation.

In summary, by blocking Nck adaptor protein involved in low-affinity TCR signaling, AX-024 inhibited autoreactivity while preserving necessary capacity for anti-pathogen immunity. Such property makes it desirable candidate for clinical trials in humans.

David Usharauli 

Regulatory T cells with a single TCR specificity prevent lethal autoimmunity

Foxp3+ regulatory T cells, Tregs, prevent autoimmunity and their total or partial defects can cause lethal or variable autoimmunity. For most part, endogenous ligands recognized by Tregs are not known.

Based on data derived from artificial transgenic models it is suggested that development and maintenance of naturally derived Tregs could be antigen-specific, though it is not known how diverse Treg TCR repertoire should be to keep auto-reactive T cells in check.

To answer this question, Rudensky's research group conducted series of experiment on transgenic mice (G113TgFoxp3^YFP-CreTcra^FL/FL mice) which harbor normal repertoire of conventional T cells but where "all" naturally developed Treg cells would express single TCR specificity (derived from Vβ6+Vα2+ G113 TCR).

Surprisingly, unlike control mice (which did not survive past 4 wk of age), 50% of G113TgFoxp3^YFP-CreTcra^FL/FL mice survived to 4 mo of age (the end point of the experiment) though all of them displayed "visible evidence of significant autoimmunity".

So what does this result tell us? These are quite unexpected results. It would be highly unusual to imagine that a single antigen and single Treg specificity could be sufficient to prevent lethal autoimmunity in 50% of mice. We don't even know the source of the endogenous antigen G113 TCR supposed to recognize.

I personally would rather focus on technical aspect of this result: how robust is TCR modification in G113TgFoxp3^YFP-CreTcra^FL/FL mice? Maybe there is still "leakage" of endogenous TCRalpha chains? I wonder about this because of "50% data". Maybe in those 50% surviving mice Tregs were able to assemble TCR with endogenous TCRalphas?

David Usharauli  

Eye-specific autoimmunity in AIRE/Lyn double deficient animal

AIRE protein in the thymus directs expression of tissue-specific antigens that on one hand reduces number of auto-antigen specific T cell clones and on the other hand leads to generation of auto-antigen specific Foxp3+ regulatory T cells. However relationship between between central tolerance and its peripheral counterpart is not fully understood.

New study in Journal of Clinical Investigation examined relationship between hypomorphic AIRE function on Lyn^-/- background. The authors found that such combination selectively accentuated eye-specific autoimmunity.  

First, the authors observed that mice double deficient for AIRE^GW/+/Lyn^-/- functions (but not single deficient) developed spontaneous eye inflammation.

These AIRE^GW/+/Lyn^-/- mice showed expansion of eye-protein specific CD4 T cells and autoantibodies.

These auto-antigen specific T cells could infiltrate eye tissue causing inflammation.

The authors found that selective absence of Lyn in CD11c+ dendritic cells (Aire^GW/+CD11c-Cre Lyn^fl/fl) were sufficient to recapitulate this eye immunopathology.

Finally, the authors found that auto-antigen uptake by Lyn^-/- DCs in eye draining lymph nodes contributed to eye inflammation.

In summary, this study indicates that some of the clinically relevant autoimmune phenotypes could be linked to failure to establish both central and peripheral tolerance.

David Usharauli

PD-1 signaling assists regulatory T cells when Foxp3 is down

Foxp3⁺ regulatory T cells (Tregs) maintain peripheral tolerance to self. Several molecules expressed by Tregs, such as CTLA-4, play crucial role in maintaining this state of tolerance to self. PD-1 is another such molecule, though its role in Tregs function is less clear.

New paper in PNAS shed some light in PD-1 role in Tregs biology. It found that PD-1 keeps Tregs in shape when Foxp3 level is unstable.

This study arose from unexpected observation in new gene-modified mice where GFP was inserted in Foxp3 locus (mice carrying the IRES-GFPcre reporter KI at the 3′ untranslated region of the FoxP3 gene, FoxP3-GFPcreKI). When these mice were crossed with PD-1KO mice, it was found that male offspring of such cross showed early death, reminiscence of Foxp3KO mice. This was surprising since PD-1KO mice ordinarily do not show such phenotype.

Further experimentation found that GFP insertion affected Foxp3 stability thus resulted in reduced Foxp3 levels in FoxP3-GFPcreKI mice.

Indeed, FoxP3-GFPcreKI/PD-1KO male mice could be rescued with transfer of WT Tregs irrespective of their PD-1 expression indicating that PD-1 function were dispensable Tregs with normal level of Foxp3.

Finally, the authors showed that absence of PD-1 could further destabilize "Foxp3-low" Tregs function (conversion into ex-Foxp3 Tregs) resulting in lethal autoimmunity.

In summary, this study revealed that PD-1 could contribute to Tregs function in situations that affects Foxp3 stability.

David Usharauli

Hybrid peptides represent novel target for auto-reactive T cells in autoimmune diabetes

This week Science published intriguing study showing that in type I diabetic individuals hybrid peptides derived from two unrelated proteins represent novel auto-antigens.  

Initially, the authors showed that chemically cross-linked or synthetic peptide that is made one half of proinsulin C-peptide and another half from chromogranin A (ChgA) peptide (both β cell proteins), could activate diabetogenic mouse T cell clones.

Next, the authors showed that T cells specific for such hybrid peptides can be detected in un-manipulated diabetes susceptible NOD female mice (it would have been more informative if the authors have included data from non-susceptible WT mice as a control).

Finally, the authors showed in T cells from T1D individuals could also recognize hybrid peptide made of human proinsulin C-peptide and neuropeptide Y.

In summary, this study suggests that in T1D susceptible individuals, diabetogenic auto-antigens could be generated by fusion of peptide derived from two unrelated proteins (a side reaction of the proteolytic hydrolysis of peptide in secretory granules). Whether generation of such immunogenic hybrid peptides happens only T1D susceptible individuals remains to be determined.  

David Usharauli

Treg cells need a little Helios to function

Regulatory T cells (T_REG cells) are required to maintain tolerance to self and maybe even to foreign antigens, such as commensal bacteria-derived antigens. But how exactly T_REG cells keep other immune cells in check is not clear. 

Simply speaking, paradox with T_REG cells is that if T_REG cells are too potent or constantly "active" then they should prevent any immune response to any antigens. If T_REG cells are not constantly "active", then what signal(s) activate them? and if active T_REG cells are "inhibited" by infection and they regain their inhibitory functions once infection is cleared, how is such system coordinated? Basically, none of the available models can satisfactorily explain full spectrum of T_REG cells biology. Hence the reason why translation of basic knowledge about T_REG cells for clinical application is so slow.

So, every time we see new paper about T_REG cells we hope that we can acquire "some" missing information. In this regards, lets examine new paper in Science from Harvey Cantor's lab. There, the authors found that presence of transcription factor Helios is necessary to stabilize T_REG cells phenotype.

This is a simple, observation-type paper. Initially, the authors showed that starting from 5 months of age, mice deficient for Helios develop auto-antibodies to self antigens.

Next, using adoptive transfer experiments in combination with bone marrow chimera, the authors showed that Helios-deficient T_REG cells lacked suppressive function.

Finally, the authors showed that Helios-deficient T_REG cells displayed unstable phenotype and could be induced to express effector cytokines.

In summary, this study showed that Helios played an important role in stability of T_REG cells phenotype, especially during the ageing process. Of note, other studies with Helios-deficient mice did not observe the same phenotype. The authors claim that it has to do with the age difference of the experimental mice used in those earlier experiments. But genetic difference between mice colony itself or their gut flora could not be dismissed without proper experimentation.

David Usharauli    

Stimulating CTLA4-Ig mimetic abatacept relieves autoimmune inflammation

It is quite counter-intuitive to believe that human common immune deficiencies are characterized by autoimmune inflammations in various organs. Such observations suggest that what we call immune deficiencies are actually immune disregulations. Immune disregulation would imply that such patient is not able to produce for example sufficient amount of IgA to mucosal antigens but will have excessive response dominated with TNF-alpha that would appear as autoimmune inflammation.

New paper in journal Science provided an example for such immune deficiency, LRBA, and its rescue by abatacept, a drug that mimics natural CTLA4 action.       

The authors described several patients with deficiency in LRBA (lipopolysaccharide responsive beige-like anchor protein) expression and showing signs of autoimmune inflammation.

The authors found that these LRBA deficient patients had reduced CTLA4 protein level (but not other molecules such as CD40L, CD107a), a phenotype that could be reproduced in healthy cells with LRBA siRNA. 

Interestingly, chloroquine, a drug that reduces lysosomal degradation could improve CTLA4 expression in cells from LRBA patients implying that excessive degradation of CTLA4 protein in LRBA deficient patients.

Finally, the authors showed that CTLA4 and LRBA colocalize in endosomal vesicles, providing a mechanistic explanation for low levels of CTLA4 protein in LRBA patients.

In summary, these results provide a rationale for use of stimulating CTLA4-Ig mimetic in treatment of LRBA deficiency.

David Usharauli  

         

Determinants of T cell exhaustion during chronic viral infection and autoimmunity

Typically when we think about ordinary immune response, we imagine several finite steps involving T and B cells, such as antigen recognition and initiation of antigen-specific clonal expansion, control of antigenic spread and elimination of infected cells and finally return to quiescent state and memory establishment.    

This scenario represents what ordinary is called immune response to transient (acute) antigenic stimulation. However, how does host immune system respond to persistent (chronic) antigenic stimulation, for example, chronic viral presence (HIV, HVB, HCV, Malaria, TB) or autoimmune disease (self-antigens)? Are the mechanisms that control immune response similarly activated during acute or chronic antigenic presence?

As you can see many infectious diseases with no effective vaccines fall exactly in the category of chronic infections (HIV, HBV, HCV, Malaria, TB). This is not a random outcome. There should be some biological or immunological underpinning to account for our failure to develop such vaccines. 

This new paper in journal Nature provided some additional results that may help us to better understand mechanisms controlling chronic immune responses. To tell the truth, it is quite difficult-to-digest article with lot of large data set analyses. The paper was under review for more than 1 year and its main finding regarding molecule KAT2B isn't even mention in their abstract. Strange. So we just need to assume that their analyses are done correctly and are statistically valid.

In this paper the authors tried to correlate CD8 T cell exhaustion phenotype with the clinical outcome (flare-free survival) of patients suffering from various autoimmune diseases. Murine chronic LCMV infection-associated CD8 T cell exhaustion phenotype was used as a reference. 

First, the authors noticed that unlike coordinated up-regulation of several inhibitory receptors during murine chronic LCMV infection, CD8 T cell phenotype from patients with autoimmune diseases displayed distinct disease-selective up-regulation of exhaustion-associated inhibitory receptors.

Interestingly, for each patient with autoimmune diseases, CD8 T cell exhaustion phenotype correlated with a favorable prognosis.

Next, in vitro experiments showed that fine balance between incoming co-stimulatory (e.g. CD2) and co-inhibitory (e.g. PD-L1) signals may determine exhaustion phenotype of CD8 T cells during persistent antigenic stimulation (one caveat: anti-CD3, anti-CD28 or anti-CD2 antibody stimulation are not physiological mode of activation, at all).

Finally, the authors showed that level of expression of KAT2B (top-ranked CD4 T cell co-stimulation candidate) could predict (1) favorable response during chronic viral infection and/or positive host response to vaccination, and (2) poor prognosis during autoimmune diseases.

In summary, these results indicate that treatment of chronic viral infection and chronic autoimmune diseases may require activation of opposite receptors.

David Usharauli