Tracking deletion of autoreactive clones versus Treg generation for thymically expressed epitopes

So far 3 different outcomes have been identified for developing T cells in the thymus: to develop into naive T cells, get deleted or become Foxp3+ Treg. Both deletion and Treg path require the presence of specific epitopes. However, how a given T cell decides between these pathways is not well understood. 

Here is a new paper in PNAS that tries to tackle this question using the tetramer tracking approach. The authors are using PLP (brain-specific protein) as an endogenous antigen expressed in the thymus. Surprisingly both PLP^WT and PLP^KO mice showed near similar numbers of tetramer-positive T cells in peripheral tissue. However, as expected, only PLP^WT mice that express PLP epitopes in the thymus but not PLP^KO mice that do not express the same epitopes showed Treg development.

   


 Similar results were obtained when thymus tissue was analyzed.

  

To make tetramer tracking for reliable the authors used transgenic mice expressing a fixed TCR beta chain. These mice also showed a similar phenotype.  

As in PLP^WT and PLP^KO mice, fixed:TCR beta mice on PLP^WT but not on PLP^KO background harbored Tregs in the periphery. Notable, the rest of the tetramer-positive Foxp3-negative T cells displayed an anergic phenotype (CD73^HiFR4^Hi).

A similar phenotype was found in the thymus. Note, there was an unexpected and significant reduction of tetramer-positive T cells from the thymus to the periphery in fixed:TCR beta mice on PLP^KO background compared to fixed:TCR beta mice on PLP^WT background. 

So far these data indicated that there is almost no deletion of PLP specific T cells in the thymus on WT mice [compaed KO] but ~2-fold reduction in fixed:TCR beta mice on PLP^WT compared to KO. Almost half of the tetramer-positive T cells ended up in the Treg pool on the WT background. The remaining T cells showed an anergic phenotype. However the dramatic reduction of tetramer-positive T cells from the thymus to the periphery in KO mice raises some serious unanswered questions.

Finally, to find some correlation between TCR specificity and Treg/anergy/deletion phenotype, the authors selected 4 PLP-specific TCRs (denoted here as A, B, C, D). Their analysis showed that some (clone "A") but not other PLP-specific TCRs (clone "C") were able to generate Tregs in the thymus. Notable, TCR "C" displayed the highest affinity to PLP epitope. Also, there is a substantial reduction of clone "C" from the thymus to the periphery in the Foxp3-negative compartment. This possibly reflects the fact that most clones in "C" are anergic and slowly disappear from the periphery.  

In summary, this study re-confirms that tolerance to self-antigens is mostly controlled via Treg generation and that not all antigens/epitopes and their corresponding TCRs are able to participate in this process. There are few unexplained observations in this paper though as discussed above. 

posted by David Usharauli

A physiological T cells' "weaning reaction" to microbiota and solid foods in newborn pups requires FOXP3+ Tregs

1. "weaning reaction" is described as a #physiological CD4+ T cell response to to #microbiota+solid #foods in mouse pups that occurs between 2-4 weeks after birth measured by analysis of #TNF-alpha and #IFN-gamma levels. It does not occur in #germfree pups devoid of microbiota. pic.twitter.com/pSGWXOUBoC

— David Usharauli (@3DiMMUNE) March 21, 2019

3. More importantly, however, #microbiota exposure between 2-4 weeks after birth but not later could restore #physiological "#weaning reaction" in T cells in #germfree pups. #tolerance #immunology pic.twitter.com/9KUtk3MT7R

— David Usharauli (@3DiMMUNE) March 21, 2019

5. Interestingly, "weaning reaction" in #newborn pups were dependent on presence of gut residing RORgt+ FOXP3+ T regulatory cells (Tregs). #tolerance pic.twitter.com/s1nq2HPShK

— David Usharauli (@3DiMMUNE) March 21, 2019

2nd Correction (#6): giving #SCFAs to #antibiotic-treated newborn pups could prevent #inflammation later. It appears this effect of SCFAs requires #Tregs and #microbiota (it does not work in GF pups) that provides #antigens to Tregs to keeps them in #functioning state. pic.twitter.com/2ewMoDRobh

— David Usharauli (@3DiMMUNE) March 21, 2019

This suggests that the effect of SCFAs is mediated via Tregs and that artifically generated "weaning reaction" with SCFAs without proper Treg contribution (driven by microbiota and their antigens) is not optimal.

— David Usharauli (@3DiMMUNE) March 21, 2019

An antigen from gut commensal Bacteroides thetaiotaomicron (B. theta) is recognized by Foxp3+ Tregs

1. B.theta-specific T cells when transferred into #antibiotic-treated and B.theta #colonized T cell-deficient mice (#RAG1-KO) develop into #Foxp3+ #Tregs as well as effector T cells. It is not clear if donor tg T cells were on RAG1-KO background as well. Host mice stay healthy. pic.twitter.com/TD0pnnxgAR

— David Usharauli (@3DiMMUNE) February 9, 2019

3. The authors were able to identify a #specific #epitope in B.theta recognized by B.theta-specific T cells. The #sequence, EEFNLPTTNGGHAT, were mapped in BT4295 predicted to be a SusE/SusF #lipoprotein in outer #membrane (OM). pic.twitter.com/rH5cQHQcVN

— David Usharauli (@3DiMMUNE) February 9, 2019

In #summary, this study showed that certain gut #commensal #microbiota species, in this case, #Bacteroides thetaiotaomicron (B. theta) are recognized by #Tregs and even induce #effector T cell differentiation though the host stays healthy because Tregs regulate effector T cells.

— David Usharauli (@3DiMMUNE) February 9, 2019

Another point is whether #microbiota-specific Tregs are #denovo induced or #amplified from existing ones. I personally think it is a latter. Most #Tregs develop from #CD25-negative #precursors poised for Foxp3 expression & such precursors are all #thymus derived, in my opinion.

— David Usharauli (@3DiMMUNE) February 9, 2019

Can microbial innate signaling generate peripheral FOXP3+ regulatory T cells?

In the past several years few papers have been published suggesting that some commensal microbiota species acting via their conserved molecules [i.e. polysaccharide A (PSA) from Bacteroides fragilis (Bf)] and metabolic products [i.e. short-chain fatty acids (SCFAs)] could induce peripheral FOXP3+ Tregs. The mechanisms are still controversial due to fact that there is no consensus whether thymus-derived and peripherally generated FOXP3+ Tregs (pTregs) represent two distinct lineages or distinct differentiation phases of unique thymus-derived FOXP3+ Tregs.

In this regard, a new paper in Science Immunology is of interest. In this study the authors tried to produce evidence that microbial cell surface β-glucan/galactan (CSGG) polysaccharides of Bifidobacterium bifidum [B. bifidum], a probiotic strain PRI1, could induce generation of FOXP3+ pTregs specific for diverse set of antigens. Lets review it.

First, the authors showed that monocolonization of germ-free mice with B. bifidum dramatically increased frequency of colonic Tregs compared to control species. Of note, Bacteroides fragilis (Bf) showed no effect thus putting some doubt on original observation.

Second, to nominally distinguish between conversion vs. expansion, the authors transferred naive CD4+Foxp3− T cells sorted from Foxp3^GFP reporter mice into germ-free mice and analyzed for GFP expression 3 weeks later. Around 5% of donor T cells up-regulated FOXP3 in colon tissue.

Of note, monocolonization of germ-free mice with B. bifidum induced quite robust IFN-γ response too.

In addition, transfer of OT-II TCR transgenic T cells [on RAG1 KO background] recognizing OVA into OVA-fed B. bifidum treated mice or transfer of CBir TCR transgenic T cells that recognize bacterial flagellin into B. bifidum treated mice on SPF RAG1 KO background could augment their differentiation into FOXP3+ Tregs. Of note, first, OT-II were transferred into germ-free mice that harbor natural, thymus-derived Tregs that could direct naive OT-II conversion into FOXP3+ Tregs in a antigen[epitope]-specific manner (OVA cross-reactivity). Second, there is some inconsistency regarding CBir T cell experiment. In the figure legend it says they were transferred into SPF RAG1 KO hosts but in the supplemental data it says "naïve CBir CD4+CD62LhiCD44loFoxp3GFP- T cells (1x106) were transferred intravenously to the C57BL/6 mice maintained in SPF conditions". These difference could make huge difference in data interpretation. Third, CBir T cells specificity to flagellin has now been questioned in new study.

In vitro co-culture of DCs and T cells pretreated with B. bifidum or its cell surface β-glucan/galactan (CSGG) polysaccharides could induce FOXP3+ Treg phenotype in a TGF-β1 dependent manner. Again, Bacteroides fragilis (Bf) could not do it.

Also, TLR2 appears to be involved in the action of B. bifidum cell surface β-glucan/galactan (CSGG) polysaccharides.

In summary, this study tries to suggest that B. bifidum conserved microbial wall component, β-glucan/galactan (CSGG) polysaccharides could convert naive T cells into FOXP3+ Tregs. It is certainly obvious that β-glucan/galactan (CSGG) polysaccharides could increase frequency of FOXP3+ Tregs but whether it is genuine conversion vs. expansion of pre-committed proto-Tregs via direction of existing thymus-derived Tregs were not cleanly verified. And lets remember that B. bifidum monocolonization increased frequency of IFN-γ producing T cells too. How these data fit to tolerance model? I am also puzzled by the fact that the authors primarily or exclusively showing frequency and not the actual numbers of FOXP3+ Tregs without providing any guidance.

Finally, lets step back and think a little bit more about general premise of such papers. The authors concluded that because B. bifidum and its microbial wall component, β-glucan/galactan (CSGG) polysaccharides could increase frequency of FOXP3+ Tregs it indicates it is a tolerogenic effect. But what's biological function or context that would require increase in frequency of Tregs? Lets assume it is to control inflammation. So, without causing inflammation what is the point of increasing the numbers of Tregs? So, does it mean that B. bifidum is actually 'inflammatory' and Treg expansion is a compensatory mechanism to counteract to such inflammation? Second, expansion and maintenance of Tregs would require increase of those enigmatic cells that supply IL-2 to Tregs. So, by definition, expansion of Tregs cannot happen without concurrent expansion of IL-2 producing cells. So what kind of signaling could drive such concurrent expansion of both Tregs and IL-2 producing cells? 

posted by David Usharauli

Access to self antigens during germinal center reaction improves self/nonself discrimination against mimicry antigens

This week journal Science published short paper from Chis Goodnow's lab that raises very interesting question about biological significance for existence of anergic self-reactive B cells. Ordinarily, developing B cells when encountering self-antigens undergo deletion, receptor editing or physiological receptor signaling down-regulation that makes such 'anergic' B cells refractory to presence of normal level of self antigens. However, anergic B cells could be re-energized if challenged with high density self antigens or antigens sharing epitope similarity with self antigen.

Now, new study indicates that rather than developing into full blown auto-reactive immune response, anergic B cells when challenged with mimicry antigens mutates its receptors in a such a way, during process of hypermutation, as to achieve a high degree of discrimination between mimicry antigen and actual self antigen.   

The experimental set up itself is quite simple, only complex aspect was to analyze single cell B cell receptor mutation and their binding affinity recovered after antigen challenge. Two type of hosts were used here. Both groups harbor small numbers of self-reactive B cells (CD45.1+ SW_HEL B cells)  but only one group also harbored a specific antigen detected by these transgenic SW_HEL B cells and expressed "as as an integral membrane protein, mHEL^3X, encoded by a transgene with a ubiquitin promoter".

As expected SW_HEL B cells in double transgenic hosts were anergic with decreased surface immunoglobulin M (IgM) expression. However, these anergic B cells could be re-activated in germinal centers when challenged with Sheep red blood cells (SRBCs) covalently coupled with self antigen, HEL^3X, at high density.

Next set of experiments however showed very unusual results. When challenged with mimicry antigen DEL which slightly differs from self HEL antigen anergic B cell receptors in double transgenic hosts rapidly accumulated mutations that decreased binding affinity to self HEL antigen.

In fact, single cell BCR receptor analysis clearly showed that presence of self antigens dramatically enhanced anergic B cell receptor mutations that allowed up to 5,000-fold better discrimination capacity between self and mimicry antigen (pre vs. post comparison). This is based on assumption that starting affinity to self are the same for both normal and anergic SW_HEL B cells population. 

In summary, this study suggests that during germinal center reaction where B cell receptors undergo hypermutation, anergic B cell repertoire, in presence of self antigen, could be salvaged (redeemed) by accelerated accumulation of mutations that modifies their original specificity away from self antigens and allowing more fine discrimination between self and mimicry, cross-reactive nonself antigen. In this scenario, self antigens serve as negative-feedback templates that hypermutating receptors interacts repeatedly in real time to achieve minimal level of binding.

In my view such negative-feedback loop to B cells can only delivered by specialized cell type in germinal center that maintains, keeps memory of host's unadulterated "self antigen collection'' visible to B cells, a task somewhat similar to Foxp3+ Tregs. So, it is possible that new cell type need to be discovered that does it or it is also possible that the same Foxp3+ Tregs localized in germinal centers, referred as follicular Foxp3+ T regs, do it too. 

What are the global implication for such mechanism: It could explain why anergic B cells hang around and how their repertoire could be salvaged without compromising tolerance. The authors also puts forward another intriguing idea that commensal mcrobes and their antigens could serve as negative-feedback loop 'self' templates for anergic B cells that allows them to discriminate between self and mimicking nonself during immune response. 

posted by David Usharauli       

Tolerance to insulin is maintained by Foxp3+ Tregs

A new study in Journal of Immunology suggests that tolerance to insulin is maintained by Foxp3+ Tregs rather than by deletion of insulin-reactive T cell clones. 

 

Here, the authors reconstituted mice with T cells on scid background transduced either with high (4-8) or low (12-4.1) affinity TCR specific for native insulin peptide (insulin epitope B:9–23). In addition, each of TCR construct were fused with either native insulin (INS) or modified insulin carrying super-affinity peptide (R22E). All mice expressing either INS or R22E but not irrelevant HEL were protected from developing diabetes.

The authors showed that while R22E deleted developing insulin-specific T cell clones in the thymus, native INS did not.

In fact, the authors showed that if the T cells also lacked Foxp3 molecule (scid-scurfy), then protection against diabetes was lost in mice exposed to native INS.

This study could be interpreted to show that with the exception of  epitopes which are able to delete (purge) cognate T cell clones in the thymus, tolerance to self in the periphery is maintained by thymic-derived Foxp3+ Tregs.

posted by David Usharauli 





    

Maternal HBV infection induces HBVeAg/PD-L1 dependent tolerance in offspring

Hepatitis B virus (HBV) can be vertically transmitted from mother to babies. Unlike exposure at adulthood, immunological consequences of such exposure to HBV in newborns is "tolerance" to chronic HBV infection.

Ordinarily mice are not naturally infected with HBV. New paper in Immunity reported development of mouse model to study immunology of vertical transmission of HBV.

This model is a combination of two independent processes: 1st, use of HBV transgenic mice and 2nd, hydrodynamic injection of plasmid that contained 1.3-mer HBV genomic DNA. Initially, the authors showed mice born to HBV Tg females (referred here as TGD mouse), but not controls, developed "chronic HBV infection" when exposed to HBV plasmid. 

  

Analysis of HBV-specific CD8 T cells from HBV-DNA injected TGD mice liver showed up-regulation of checkpoint inhibitor PD-1 on T cells.

Treatment of TGD mice with anti-PDL1 antibody enabled them to control HBV-DNA infection.

Moreover, the authors found that clodronate-liposome depletion of macrophages from TGD mice also enabled them to control HBV-DNA infection.

Finally, the authors found that HBVeAg played important role in viral persistence and tolerance by observing that (a) mice born to HBV-mut Tg females (HBV lacking eAg) do not develop persistent HVB-DNA infection, and (b) TGD mice exposed to HBV-DNA lacking eAg also were able to eliminate virus.

In summary, this study suggests that HBVeAg controls newborn's tolerance to HBV via inhibitory PD-L1 signaling.

Few additional thoughts: first, the authors reported that after HBV-DNA injection, around 15% of liver macrophages, called Kupffer cells, expressed viral antigen. However, they also found that >75% of Kupffer cells expressed PD-L1 upon HBV-DNA injection. Second, since HBV virus from HBV Tg pregnant females cannot directly infect newborns, it is not clear how HBVeAg is able to modulate macrophages and tolerize newborn's CD8 T cells (the authors also acknowledged this inconsistency).

David Usharauli

Gut flora antigen packaged within outer membrane vesicles mediates natural tolerance to microbiota

Crohn’s disease (CD) and ulcerative colitis (UC) are gut inflammatory conditions of unknown etiology. Current hypothesis suggests that these conditions develop due to failure of body's immune system to tolerate antigen processed or generated within gut. 

New paper in Science has pointed to one of the mechanisms for such failure. This study showed that CD's risk gene, ATG16L1, is involved in response to microbiota and promotes gut tolerance via IL-10 producing Foxp3+ Tregs cells.

Initially the authors showed that DCs deficient for ATG16L1 failed to induce IL-10 producing Foxp3+ regulatory T cells in response to WT PSA-OMV (Polysaccharide A derived from gut commensal Bacteroides fragilis and packaged within outer membrane vesicles). Conversely, WT DCs failed to induce IL-10 producing regulatory T cells in response to OMVs harvested from isogenic B. fragilis mutant lacking PSA (ΔPSA-OMV).

Similarly, NOD2-deficient DCs pulsed with WT PSA-OMV failed to support IL-10 production from Foxp3+ Tregs during in vitro co-cultures (NOD2 has been shown to physically interact with ATG16L1).

In vivo experiments confirmed that unlike WT mice, mice deficient for DC-specific ATG16L1 or NOD2 were not protected by WT PSA-OMV from chemical-induced colitis (though it is not clear whether chemically-induced mouse colitis models, i.e. 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis or dextran sulfate sodium (DSS)-induced colitis, are representative of inflammations in Crohn's disease)

.

Finally, the authors showed that monocyte-derived dendritic cells (MoDC) from Crohn's patients harboring ATG16L1 T300A risk variant did not respond to WT PSA-OMV and failed to induce IL-10 production from Foxp3+ Tregs.

In summary, this study showed that failure to sense gut microbiota components by NOD2-ATG16L1 axis could predispose individuals to gut inflammation.

David Usharauli

Behavior of recent thymic emigrant T cells supports 60 year old hypothesis

This week Journal of Experimental Medicine published new study that showed that recent thymic emigrant T cells are highly susceptible to tolerance upon antigen encounter in the periphery.

I selected to review this article because its topic underlies one of the fundamental questions of immunology, namely how T cells are tolerized to peripheral antigens [not expressed in the thymus]?

Let's start from the beginning. T cells generated in the thymus express T cell receptor (TCR) with all kind of specificity. Most of T cells specific for self-antigens are deleted within thymus. Thymus also express some of the peripheral antigens, such as insulin and other proteins, under the guidance of two genes AIRE and Fezl. However, it is conceivable that some of the peripheral antigens are not expressed at all in the thymus or are expressed too little to completely delete auto-reactive T cells. So what those auto-reactive T cells would do once they leave the thymus and encounter self-antigens in the periphery?  

So, this new study suggests that those young T cells leaving thymus for the first time [they are called recent thymic emigrants, RTEs] are highly sensitive to tolerization and display anergic phenotype when stimulated by antigen. Basically there is a "window of opportunity" for peripheral tolerance for RTEs. These results support the hypothesis proposed by Joshua Lederberg 1950s to save Burnet's clonal selection theory (though it was disappointing to see that this study did not refer to it).

To distinguish between RTEs and conventional naive T cells the authors used RAG2p-GFP mouse model that marks RTEs with GFP for ~3 weeks [after leaving thymus]. When OVA-specific RTEs and naive T cells were transferred in OVA-expressing hosts and later re-stimulated, RTEs showed less proliferation and less effector differentiation.

Functional changes in RTEs upon encountering specific antigen in the periphery was confirmed by absence or delay in development of diabetes in OVA-expressing hosts (OVA is expressed in pancreas).

RTEs were also more susceptible to suppression by Tregs.

Finally, the authors showed that if RTEs were exposed to peripheral antigens in context of inflammation (alum or CFA), then they could undergo full effector differentiation equal to that of conventional naive T cells.

In summary, this short paper suggests that RTEs and naive T cells are functionally distinct and that 3 weeks of post-thymus period play physiological role in peripheral tolerance.

I also want to highlight some of the deficiencies of this paper: 

1st, the authors did not test whether RTEs indeed "become" conventional naive T cells in 3 weeks after transfer in antigen-free hosts. For example, I would have transferred RTEs first in antigen-free host and then 3 weeks later transfer again those T cells into antigen-expressed hosts. Prediction would be that 2nd transfer T cells should behave as conventional naive T cells.    

2nd, the authors used alum and CFA immunization to recreate inflammatory context. However, antigen presentation and inflammation in the context of alum or CFA are quite different from inflammation in natural context such as infection. I would have used bacterial or viral infection and then transfer RTEs in infected hosts to see how more natural inflammatory context would have affected their behavior (later I was informed that such experiment was indeed done with the same outcome).

David Usharauli

OX40/CD30 lay off Foxp3

 Since it's discovery in 2001, a transcription factor called Foxp3 has been recognized as a master regulator of autoimmune disease. It's total deficiency in CD4 T cells has such a profound effect that mouse that lacks it usually die of inflammatory multi-organ failure within 3 weeks of birth.

We don't know how exactly Foxp3+ CD4 T cells prevent autoimmunity. Currently, there is no mechanistic model that could satisfactorily explain their function. Even at the theory level, Foxp3+ T cell role is either totally ignored/dismissed (for example, in SNS model) or characterized as an immune class-specific effector/memory T cells (for example, in danger model). In short, the viable concept of negative regulation of immune system by antigen-specific Foxp3+ CD4 T cells is yet to come.

If you are interested in Foxp3+ T cell biology, I recommend reading a new study recently published in Journal of Experimental Medicine (1). This study, by Fabrina M. Gaspal and et al, made an interesting observation that mice triple deficient in OX40/CD30/Foxp3 are healthy. It appears that OX40/CD30 pathways control autoimmune potential of self-specific T cells. The simple explanation I favor is that in the absence of OX40/CD30 signaling, effector/memory T cell differentiation is impaired in general and as a consequence, self-specific T cells loose the capability to damage the tissue. Of course, the one caveat of the paper is that we have no idea whether those triple deficient mice are capable of mounting a proper immune response to non-self antigenic challenge or infection. If they cannot, then the absence of autoimmunity is the direct consequence of immunodeficiency (similar to gamma-c receptor deficient mouse model). However, if they could respond to non-self antigenic challenge or infection, then this model has made an unique contribution to our understanding of Foxp3 biology.

David Usharauli

(1) http://jem.rupress.org/content/208/8/1579.abstract