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

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

Foxp3+ Tregs suppress other T cells by stripping DCs of specific antigen/MHC complexes

Ag-specific or not Ag-specific, that is [maybe was] question for #Foxp3+ Tregs mechanism of action. New paper in @NatImmunol from #Shevach's lab @NIH says definitely antigen-specific. Lets dive in. BTW, ~20 years ago his lab developed assays that showed it was non-specific 🤫

— David Usharauli (@3DiMMUNE) January 16, 2019

Now his lab made U-turn and concluded that #suppression by #Tregs is actually #antigen-specific and difference between now and then comes down to in #vitro versus in #vivo assays 🤔

— David Usharauli (@3DiMMUNE) January 16, 2019

2. This #antigen-specific #suppression was still active when antigen-specific dendrtic cells were removed from Treg co-culture (1st culture) and exposed to T cells (2nd culture). pic.twitter.com/dj1pzDuNNv

— David Usharauli (@3DiMMUNE) January 16, 2019

So, from now on if someone asks you how Treg suppress other T cells don't hesitate to reply "antigen-specific manner", add Ethan Shevach said so and no one will argue with you. got it 🤨 https://t.co/I4pAYIIGy1

— David Usharauli (@3DiMMUNE) January 16, 2019

New auto[cross-reactive]-antigen, GDP-l-fucose synthase, identified in MS patients

Multiple sclerosis (MS) is a debilitating autoimmune disease affecting nervous system. Several auto-antigens have been traditionally suspected for MS pathology, mostly derived from neural tissues such myelin. However, more recently the list of relevant auto-antigens expanded to include such member as RAS guanyl releasing protein (RASGRP1–4) family. Similarly, a new paper in Science Translational Medicine presented evidence that yet another auto-antigen, GDP-l-fucose synthase, is a target antigen in a subset of MS patients.

The authors has used modified version of positional scanning epitope library to identify epitope derived from auto-antigen, GDP-L-fucose synthase, as a target epitope for CD4+ T cell clone, TCC21.1, derived from MS patients with DR15 haplotype. Peptides sequences matching GDP-l-fucose synthase was detected in brain tissue.

MS patients whose T cells displayed high response to peptides from GDP-L-fucose synthase in stimulation assay showed high responses to myelin proteins as well.

Curiously, almost all high responders carried DRB3*02:02 allele. 

Finally, the authors speculated that sequence similarity between human and microbiota-derived evolutionary conserved GDP-L-fucose synthase could be a factor that initiates MS pathology in these patients.

In summary, this study has a lot of nice data that support idea that GDP-L-fucose synthase is a  new auto-antigen that could be relevant in molecular diagnosis of MS pathology.

There are several questions about this study. GDP-L-fucose synthase expression is not restricted to brain tissue. So, it should be relevant to understand if other tissues were affected in those MS patients. Second, cross-reactivity per se is not sufficient to explain how and why autoimmune responses are being initiated. Microbiota expressing GDP-L-fucose synthase most likely reside in patients long before MS, maybe even since birth, and T cells are tolerant to them. So, what has to be changed, in a antigen-specific manner, to make T cells less tolerant to initiate specific autoimmune attack on nervous system and not a total autoimmunity targeting all available auto-antigens? 

posted by David Usharauli

Effort to identify tumor-specific antigens: The University Industrial Complex study results

New study in journal Cell is prime example why utilization of sophisticated high-throughput methods and computer technologies does not guarantee generation of clinically useful results. I imagine the only reason this study was even accepted in Cell was the fact that list of authors included many well-known scientists with links to both academia and silicon valley (Stanford University School of Medicine, Chan Zuckerberg Biohub, Parker Institute for Cancer Immunotherapy).   

Idea of this study was to develop techniques to quickly identify tumor-specific antigens (most likely mutated antigens) that could be used in immunotherapy (though there is no evidence that any cancer vaccines based on mutant protein sequences actually work in humans using available practices). 

For this task, the authors took advantage of yeast-display library expressing random peptide covalently linked to the HLA-A*02:01 molecule, an allele which is present in up to 50% of a number of populations. The authors estimated that "approximately 400 million unique peptides ranging from 8 to 11 amino acids are represented in the combined [yeast-display] libraries."

To validate this approach, they used three recombinant 'blinded' positive control TCRs derived from a melanoma patient (their antigen specificity had been identified independently by exome sequencing, tetramer staining and binding prediction algorithms). However, antigen-specificity of only 1 TCR (NKI 2) could be validated using their yeast-display library. As the authors said "targets of NKI 1 and NKI 3 could not be unambiguously identified through this blinded validation."

Of note, in these validation experiments with NKI 2 (specific for ALDPHSGHFV, a peptide neoantigen derived from CDK4 and other DMF5 TCR specific for EAAGIGILTV derived from the MART-1 melanoma antigen, successful validation [specific enrichment + TCR staining] occurred when HA tagged 10-mer epitope library were used. 

The authors anyway went ahead with this "less than perfect" approach to try to identify tumor antigen specificity of T cells derived from 2 patients with colorectal adenocarcinoma and homozygous for the HLA-A*02 allele. The authors focused on 20 TCR most enriched in tumor tissues (based on frequency of occurrence of the same TCR genes). 

Out of these 20, only 4 TCRs could enrich peptide from the library (only with c-Myc tagged 9-mer epitope library) and only 3 TCR could stain yeast samples.  

Next, the authors try to identify epitopes from potential landscape of sequences for each TCR. Several algorithms were deployed (at least 3 or more such as a modified variant of the previous statistical method using a position weight matrix and a method utilizing a two-layer convolutional neural network). They found 1 peptide sequence EYGVSYEW, which closely matches the peptide motif for TCR 1A, however, neither this exome peptide or the anchor-modified exome peptide (EMGVSYEM), nor the human peptide predictions stimulated the cell line modified to express the TCR 1A. TCR 4B was stimulated with several peptides and as the authors write "true in vivo specificity cannot be unambiguously identified without additional tumor information". Regarding TCR 2A and 3B, only 1 peptide stimulated cell line expressing these TCRs. This peptide was MMDFFNAQM, which is derived from U2AF2, a protein involved in an RNA splicing complex. However, in both patients, no mutations were found in U2AF2.

In summary, the authors wrote "although we cannot definitively determine an immune response targeting the peptide derived from U2AF2, the evidence from the yeast-display screen, prediction algorithm, and in vitro stimulation identify this peptide as the likely target". However, when reading this study it is clear that none of the components worked: yeast-display screen performed suboptimally, prediction algorithms provide little clue and in vitro stimulation made it even more confusing. So, what have we learned from all of these? I would say maybe don't do what they did.

posted by David Usharauli    

IBD converts tolerant antigens into immunogenic

Inflammatory bowel disease (IBD) is a pathological condition wherein body's immune cells wrongly attack its own or commensal microbiota-derived antigens that initiates a vicious cycles of permanent inflammation.

 

However, it is still not clear whether immune system attacks "new" antigens from microbiota or simply it loses adaptive tolerance to "old" ones. 

 

New study in Science Immunology tried to answer it to the extent it was possible to do in mouse model.

 

First, the authors generated IBD condition in mice by treating them with DSS + anti-IL10R. Keep in mind this is highly artificial model. Then, they transferred naïve T cells from previously established transgenic T cell lines specific for unknown commensal antigens that were known to drive Treg phenotype. Naïve T cells transferred into control WT mice generated Tregs while the naïve T cells transferred into IBD-conditioned mice preferably developed into effector T cells.

 

 

 

When analyzed for antigen specificity, the authors found that transgenic naïve T cells were reactive to antigens derived from Helicobacter species that have expanded during IBD-conditioning.

 

 

 

In vivo studies also confirmed that Helicobacter species could induce Treg generation from naïve transgenic T cells in "normal" condition.

 

 

 

Interestingly, transfer of T cells specific for other microbiota species that also underwent expansion during IBD-conditioning did not produce T cell expansion.

 

 

 

Finally, transfer of Treg-TCR transgenic naïve T cells into RAG-KO mice produced IBD when co-injected with Helicobacter species.

 

 

 

What these data indicate? In my view the authors made one correct and one wrong interpretation. First, they were correct to conclude that T cell response to IBD could be directed to "old" microbiota antigens rather than "new" never before seen microbiota-derived antigens. So basically in IBD we are losing tolerance rather than gaining immunity to microbiota antigens.

 

However, they made wrong conclusion that naïve T cells are converted into Tregs in vivo based on context (normal versus IBD). In their study loss of Treg generation is inhibited either during IBD-conditioning or in RAG KO hosts which could argue alternatively that such outcome has to do with failure of naïve T cells to interact with existing Tregs specific for the same or similar antigens in these scenarios (IBD or RAG-KO).

 

posted by David Usharauli

 

 

        

Identification of prostate-antigen specific natural Tregs (in mice)

Foxp3+ Tregs are central player in maintaining tolerance to self and other environmental antigens. However, till to this date we know little of their antigen specificity. It is because unlike conventional CD4+ T cells, Tregs do not secrete [upon antigen recognition] any cytokine that uniquely identifies them. The best marker is still Foxp3 molecule, an intracellular transcription factor.   

So it is always interesting to see new study that could identify Treg epitope, such as this new paper in Immunity that provided evidence that in mice peptide spanning residues 646–658 of prostate-specific TRPM8 channel-associated factor 3 protein (Tcaf3) is a natural epitope for thymic MJ23 TCR transgenic Treg development.

The authors showed that development of MJ23+ Tregs from adoptively transferred MJ23+ thymocytes (un-differentiated T cells) were only supported in hosts expressing intact Tcaf3 (and not in Tcaf3 KO mice).   

Next, using sensitive tetramer based antigen-specific T cell detection, the authors showed that WT mice also harbored Tcaf3[646–658]-tetramer specific T cells that were enriched in Tregs compared to other antigen-specific T cells (2W1S). Interestingly, Aire-KO mice which do not efficiently express peripheral antigens in the thymus harbored reduced numbers of Tcaf3[646–658]-tetramer specific Tregs.

Finally, the authors showed that prostate tissue from Aire KO mice harbored significantly more Tcaf3[646–658]-tetramer specific Tregs compared to prostate tissue from normal mice. I found these particular results problematic because should not normal mice prostate supposed to contain Tregs to prevent autoimmunity? Or are Tregs keeping autoreactive T cells in check in draining lymph nodes? 

In summary, this study showed that in mice prostate-specific Tcaf3[646–658] epitope is a natural ligand that selects Tregs in a Aire-dependent manner.

posted by David Usharauli

       

Combination of checkpoint inhibitor and IL-21-primed melanoma-specific T cells produced durable response, "cure", in melanoma patient

This week Journal of Experimental Medicine published a case study of a single melanoma patient who achieved durable response (>5 years), "cure", after combination therapy (IL-21 primed MART1-reactive "polyclonal" CD8+ T cells and Yervoy). 

Of note, earlier attempts to stop melanoma progression in this patient with IL-2/IL-7/IL-15 primed MART1-reactive "monoclonal" CD8+ T cells and/or Yervoy were not successful. As seen before, successful anti-melanoma immunotherapy produced autoimmune skin/hair disorder, vitiligo (loss of melanocytes). 

As the cellular level, IL-21 priming resulted in better survival of infused polyclonal T cells and epitope spreading targeting other melanoma antigens such as NY-EOS1, gp100, tyrosinase and MAGE-A3.

In summary, this study clearly shows the vast [not yet fully tapped] potential of cancer immunotherapy.

David Usharauli

 

Time dictates tolerance to skin microbiota

Yesterday journal Immunity published a very interesting study showing that early wave of thymic Foxp3-positive T cells specifically populate neonatal skin between days 7-13 and contribute to tolerance establishment to skin microbiota.   

To study relationship between skin microbiota and skin immune response, the authors modified human skin commensal microbe, Staphylococcus Epidermidis, by introducing into it a nominal peptide antigen 2W [Epi-2W]. This allowed tracking of 2W-tetramer⁺ CD4 T cells. Primary application of Epi-2W to adult mouse skin for the first time induced recruitment of 2W-tetramer⁺ CD4 T cells in skin draining lymph nodes without inducing any skin inflammation [proof-of-principle experiment].

Next, the authors tested how adult mice immune system would respond to secondary skin challenge with Epi-2W. They showed that adult mice exposed to skin Epi-2W do not develop tolerance to it upon secondary challenge. These mice displayed (a) skin inflammation

(b) no accumulation of 2W-tetramer⁺ Foxp3⁺ CD4 T cells, implying failure of tolerance.

However, if skin of neonatal day 7 mice were exposed to Epi-2W and later challenged again, these mice showed (a) minimal skin inflammation 

(b) and robust accumulation of 2W-tetramer⁺ Foxp3⁺ CD4 T cells, implying active tolerance to Epi-2W.

Followup experiments found that between neonatal day 6-13 there was a sudden increase of thymic Foxp3⁺ CD4 T cell numbers specifically in skin.

Finally, the authors showed that when this increase of neonatal skin Foxp3⁺ CD4 T cells were blocked, it resulted in tolerance failure towards Epi-2W challenge and skin inflammation.

In summary, these results indicate that timely [neonatal] exposure to skin commensals is necessary to establish antigen-specific Foxp3⁺ CD4 T cell-mediated immune tolerance to skin microbiota and to prevent pathological skin inflammation later on. It is possible that in near future human neonates will be artificially exposed to the defined sets of human commensals via skin application, nasal inhalations and oral application to specifically train their neonatal immune system for tolerance towards commensals and hence better distinguish between beneficial and pathogenic microorganims.

David Usharauli

Anergic B cells respond to polyvalent antigens via IgD receptor

Naive B cells express IgM and IgD receptors. Both receptors share the same unique immunoglobulin variable region but differ in immunoglobulin constant regions. IgM is secreted in response to antigenic signaling but not much is known regarding the role of IgD in immune response.

New study in Nature Immunology provided very interesting data about the function of IgD. It turned out that unlike IgM, IgD receptors only respond to polyvalent antigens.

The authors, led by Hassan Jumaa at the Institute of Immunology (Ulm, Germany), first showed that unlike IgM receptor, cell line expressing IgD receptors specific for hapten (NIP) or antigen (HEL) responded only to polyvalent antigenic forms.

Next, the authors find that these difference in response between IgM and IgD was related to the difference in hinge region (that connects variable and constant regions). IgD with no hinge region (IgDΔhinge) responded as if IgM and IgM with IgD hinge region responded as if IgD.

The authors observed that anergic B cells obtained from antigen (HEL)-specific B cell double transgenic mouse expressing soluble HEL (s-HEL) could still respond to polyvalent HEL antigen.

The authors showed that monovalent antigen could competitively inhibit IgD signaling in response to polyvalent antigens.

Next, the authors observed that functionally, absence of IgM receptor could compromise innate B-1 cells scavenging response (natural antibody response) to a soluble auto-antigen phosphatidylcholine (PtC).

Indeed, IgD receptor itself were unable to respond to a soluble phosphatidylcholine (PtC).

Finally, the authors also showed that the absence of IgM signaling compromised IgG scavenging functions against multiple auto-antigens (oxidized LDL, etc) as well.

In summary, this study revealed that anergic B cells (IgM^lowIgD^high) can respond to antigen when stimulated with polyvalent antigens. Probably the role of IgD receptor is to prevent improper activation of naive B cells in response to soluble antigens. I wonder what is the (a) phenotype of IgD KO mice or (b) whether auto-antigens targeted by natural antibodies are mono or polyvalent in nature?

David Usharauli