If T cell clones are so diverse, what prevents anti-tumor immune response?

Identifying cancer and pathogen-specific epitopes or TCRs may sound intuitive, but it is a futile approach. Diversity of T cell or B cell clones guarantees that the adaptive immune system will always have relevant clones to detect cancer or pathogen.  

Epitopes have no meaning attached to them with one exception.  It is when the said epitope is self. Each body will have different sets of self-epitopes. Every self-epitopes relevant for host's survival are encoded in the thymus, and thymic Tregs are trained to prevent any T cell activity against those epitopes in the periphery. This is called tolerance, and it is antigen[epitope]-specific. 

Then what prevents effective responses to cancers or pathogens? It is commonly but mistakenly believed that Tregs prevent effective T or B cell responses to cancers or pathogens. But Tregs only prevent anti-self response, and it is epitope-specific action.  So, by definition, if Tregs do their job as required, we cannot blame them. But it has nothing to do with cancer or pathogens, which obviously have other epitopes different from self, we call nonself. So, if cancer cells or pathogens express nonself epitopes that are always detected by adaptive immune system, why not everyone can fight it off effectively?

This is because T cells themselves prevent it. Yes, T cells, not Tregs, prevent effective response to cancers or pathogens in certain conditions. What are those conditions? These are condition when polarized T helper cells prevent other T cells functions. Polarization is a pathological state. A Polarized T cell's effect on other T cells is epitope non-specific, meaning, a polarized T helper cell specific to cancer or pathogen nonself epitope A will prevent T cells specific to epitope B, C, D, E, F, etc., to function properly. It is exactly Treg's job to shut down those polarized T helper cells to allow other T cells to manifest their functions and get rid of either cancer or pathogen. And Treg do it, as we already said, epitope-specific manner.

For this reason, it is not so important to identify any cancer or pathogen-specific nonself epitope, but rather to identify an epitope that could activate polarized T helper cells.

To make things even more complicated, one may ask if Tregs are self-specific and act epitope-specific manner, how can Tregs shut down polarized T helper cells that are nonself-specific? It is possible because Tregs are cross-reactive and can inhibit only those polarized T helper cells which share TCR specificity with Tregs.

In other words, control of self-tolerance and control of effective anti-nonself response are one and the same.  

These are 3 papers that together provide a full discussion related to the SPIRAL model we have developed to explain how Tregs work within an adaptive immune system:

 

 

Concurrent cross-reactivity of microbiota-derived epitopes to both self and pathogens may underlie the "Hygiene hypothesis"  

 

https://onlinelibrary.wiley.com/doi/10.1111/sji.12708 

 

Could cross-reactivity rescue Foxp3+ regulatory T cell precursors from thymic deletion? 

 

https://onlinelibrary.wiley.com/doi/10.1111/sji.12940

 

 

 

Microbiota-Specific Foxp3+ Regulatory T Cells Could Control Pathological T Helper Responses

 

https://www.dl.begellhouse.com/journals/2ff21abf44b19838,40a32b197f7156bc,7988864072d2c7c6.html

 

 

   

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

The auto-reactive CD4+ T cells provide IL-2 to proto-Tregs in the thymus

The T cells expressing the transcription factor Foxp3 called regulatory T cells, abbreviated as Tregs, are the most important cell type in the immune system. Without them, the whole immune system goes haywire. As a result, the body simply dies in a very short time.

The Tregs develop in the thymus and require two things: TCR signaling and IL-2. The thymus expresses a very diverse set of epitopes including that from peripheral tissues such as the pancreas or prostate. The high-affinity interaction between TCR and epitope/MHC II makes proto-Treg sensitive to local IL-2, a necessary step to complete a Treg formation loop.

But what cell provides that crucial IL-2 to proto-Tregs? There hasn't been any consensus with this regard but a new paper in the Journal of Experimental Medicine from Sasha Rudensky's lab indicates that it is mature CD4+ T cells and CD25+Foxp3- CD4+ single-positive (SP) T cells that are the main source of thymic IL-2 required for Treg development.

For this study, they used an IL-2 reporter mouse wherein cells expressing or having a history of the expression of IL-2 are genetically labeled and analyzed. They found that IL-2 expression was restricted to TCRbeta expressing CD4+ population.

Out of CD4+ T cells, the most IL-2 was made by mature CD4 SP and CD25+Foxp3- CD4+ T cell population. Of note, CD25+Foxp3- T cell population contains proto-Tregs.

Interestingly, the authors also detected mature Tregs with the history of IL-2 expression. It implies that bifurcation between Tregs versus IL-2 producer is a stochastic process.

As expected, TCR signaling together with IL-2 was essential for Treg formation. A "bystander" effect on Foxp3 upregulation on antigen-independent proto-Tregs (Vbeta 8- T cells) could be explained by the fact that these T cells were likely TCR activated in vivo before harvesting for ex vivo experimentation.

Based on these data, the authors suggested the following model: among mature SP CD4 T cells, a small pool produces IL-2 that in the context of high-affinity TCR/epitope interaction and CD25 upregulation promotes Foxp3+ Treg formation either autocrine or paracrine manner. Since the thymus is expressing self epitopes we can conclude that those IL-2 producing T cells are auto-reactive T cells.

The following questions remain unanswered:

1. What determines Treg, IL-2-producer or deletion pathways? All three options are open for high-affinity TCR+ CD4 SP cells.

2. Do TCR specificity overlaps between Tregs and IL-2 producers?

3. What cells provide IL-2 to Tregs in the periphery?

4. Is IL-2 delivery TCR/epitope-specific or non-specific event?


We have recently published a new model, called SPIRAL, that provides answers to these questions. The SPIRAL is based on the principle of epitope cross-reactivity.


Shared TCR epitope cross-reactivity could permit dyads of Foxp3+ regulatory and IL-2-producing T cell precursors to escape thymic purge 


posted by David Usharauli


 

A neonatal temporal window for thymic epitope-specific Foxp3+ Treg formation

The thymus-derived Foxp3+ Tregs are indisputably the most important immune cell type. Surprisingly, little has been done to found out their antigen specificity. One reason for this lack of interest to study it has to do with the fact most scientists thought Tregs inhibited unwanted T cell responses antigen non-specific manner. So, they reasoned why to bother with TCR specificity. More recently however they started to pay close attention to antigen-specificity of Tregs since it became clear that antigen-specific Tregs showed superior, maybe even exclusive, therapeutic effect in animal models.  

So any study that advances our understanding of the formation of antigen-specific Tregs is immensely valuable. Below I will review one such research published in Nature Immunology from Eric Huseby's lab at the University of Massachusetts Medical School, Worcester, MA, USA.

In this study, they cloned several hundred TCRs from Foxp3+ GFP+ Tregs and screened their specificity in an in vitro IL-2 bioassay using standard hybridoma technology and library of ~1,750 unique self-peptides (it is astounding that so few labs have used this readily available approach). About 17 peptides showed a positive response. They chose to focus on 2 peptides derived from peptidyl arginine deiminase type IV (Padi4_92–105) and Adducin 2 (Add2_606–621). TCR specificity for Padi4_92–105 or Add2_606–621 was confirmed with respective KO mice.

Curiously, they noticed that thymic development of  Padi4_92–105 specific Tregs was time restricted and their formation rapidly went down after 3 weeks post birth.

More importantly, specific antigen expression was primarily responsible for both initial Treg formation and later its reduction.

However, even if there was initially an age-related decline in the frequency of Padi4_92–105 specific Tregs both in the thymus and periphery, their absolute numbers were maintained at a constant level in the periphery afterward. This is important to highlight.

Notably, this age-related antigen-dependent Treg reduction could be reversed in chimera where only thymic stromal but not bone-marrow derived cells expressed specific epitope. It could mean dose effect or specialized antigen-presentation pathway contributes to age-related decline in Padi4_92–105 specific Tregs formation.

Furthermore, out of several Padi4_92–105 specific TCRs with different antigen response potency, only moderate potency responders were enriched in Tregs in the periphery (the highest potency T cells were lost in the thymus and the lowest potency T cells ended up in Tconv spleen pool). In my view, this is conveniently too clean to my liking.

Also, the authors found Treg formation best correlated with the TCR:self-MHC half-life (t_1/2).

In summary, this study identified several self epitopes that drive mouse Treg formation in the thymus and this process is restricted to a few weeks post birth. It is not clear why or how the cessation of Treg formation is happening in the thymus here. As absolute numbers of such epitope-specific Tregs that seeded the periphery stayed constant it could indicate that a saturation feedback loop may exist between periphery and thymus that adjust Treg numbers. Additionally, the authors suggest that Treg formation could be predicted based solely on TCR:self-MHC dwell half-life (t_1/2). However, dwell time cannot explain their own observation about the age-related decline of Treg formation. What has changed in 8-week versus 3-week thymus to upend dwell time so dramatically? Besides, this paper did not address a mechanism of bifurcation that determines deletion versus Treg formation at the single thymocyte level that has been shown to occur independently of TCR affinity. 

Of note, these results could explain why some CD4+ TCR transgenic mice don't show thymic Foxp3+ Treg formation but still harbor them in the periphery, for example, marilyn CD4+ TCR transgenic mouse. As such mice are ordinarily examined when they are adults (>8 weeks) it will miss the thymic phase.  

posted by David Usharauli

Two non-overlapping precursors generate Foxp3+ regulatory T cells in the thymus

New study in @NatImmunol compared #Treg differentiation potential of #CD25+ versus Foxp3(low) precursors in the ##thymus. https://t.co/rIFWqybNnm pic.twitter.com/9ibTGBeHF9

— David Usharauli (@3DiMMUNE) January 22, 2019

2. One interesting #difference between CD25+ vs. Foxp3(low) #precursors was dependency on #IL4 #signaling to generate Tregs only from Foxp3(low) precursors (both responded to IL-2). pic.twitter.com/hNoLxVBfIP

— David Usharauli (@3DiMMUNE) January 22, 2019

in summary, two types of thymic precursors can generate Tregs. Since their #TCR did not overlap with each other but both of them overlapped with mature #Foxp3+ #Tregs it suggests certain #functional bifurcation considering differences in protection against tissue #inflammation.

— David Usharauli (@3DiMMUNE) January 22, 2019

First-born advantage of early produced CD8 T cells

During immune response to infection or vaccine antigen-specific T cells differentiate into various categories of effector/memory population. This is a stochastic process that follows some not yet fully understood "rules". New study in Cell suggests that one of those rules is a "date of birth" of individual CD8 T cells that participate in immune response. 

For this study the authors used genetically modified mice where T cells could be permanently color-labeled after injection of tamoxifen (CD4 promoter-driven tamoxifen-inducible CRE mice, CD4^cre-ERT2). When compared to each other, CD8 T cells produced early on during neonatal stage (day 1-7) harbored high proportion of cells with innate-like phenotype (CD44⁺/CD122⁺) than those CD8 T cells produced after day 28.

Similar phenotype was maintained even in mice where both day 1 and day 28 CD8 T cells were produced in the same mouse (following neonatal RFP⁺ thymus transplantation into YFP⁺ adult mice and analyzed at indicated time period post transplantation).

Notably, early-born CD8 T cells were characterized by heightened sensitivity to innate cytokines such IL-18 and IL-12 and rapid initial response to cognate antigen (between day 1-7 post-infection).

The authors opined that various categories of effector/memory T cells observed in other studies should be re-interpreted in light of "layered" CD8 T cells wherein CD8 T cells of different "date of birth" are producing different effector/memory T cell population.

It is certainly interesting observation. But there are several missing opportunities in this study. First, due to their innate-like phenotype, the authors should have looked at the role of microbiota in imprinting these features of early-born CD8 T cells. Second, it is not clear and the authors did not examined if biologically, lack of early-born vs. adult-born CD8 T cells, would have modified the host's response to infection. Third, the authors did not analyze whether early-born versus adult-born CD8 T cells differed in their TCR profile (even using transgenic CD8 T cells on WT background is not proper control). Fourth, different effector/memory categories have been produced by injection of a single CD8 T cells so individual CD8 T cells can indeed produce diverse phenotype of effector/memory T cells. 

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

       

Without thymic assistance peripheral tolerance to self is easily breached

Yesterday journal Immunity published an important paper about mechanisms of immune tolerance. This study showed that peripheral immune tolerance, as we knew it, doesn't really exist. Here is my analysis of main findings from this study.

The authors have used 4 types of mouse strains for this study, each of them expressing nominal neo-self protein, Cre recombinase, under the guidance of ubiquitous or tissue-specific promoters (UBC-Cre (everywhere), RIP-Cre (insulin producing cells), CC10-Cre (lung tissue), Vil-Cre (intestine tissue). To track endogenous Cre:I-A^bspecific CD4 T cells the authors designed CD4 T cell Cre [pp61-71]:I-A^b tetramers. Staining for Cre:I-A^b tetramer positive CD4 T cells revealed that all strains, except UBC-Cre mice, harbored equal number of Cre:I-A^b specific CD4 T cells.

To test the functionality of Cre:I-A^b tetramer positive CD4 T cells mice were immunized with Cre peptide/CFA. Both WT and RIP-Cre mice showed similar expansion of Cre:I-A^b tetramer positive CD4 T cells. UBC-Cre mice showed minimal expansion as expected [due to low frequency]. Interestingly, expansion of Cre:I-A^b tetramer positive CD4 T cells from CC10-Cre and Vil-Cre were minimal too, even though these mice harbored similar frequency of Cre:I-A^b tetramer positive CD4 T cells as WT or RIP-Cre mice.

These results suggested that in CC10-Cre and Vil-Cre mice Cre:I-A^b tetramer positive CD4 T cells were actively "contained". Indeed, CC10-Cre and Vil-Cre mice harbored large proportion of Cre:I-A^b tetramer positive Foxp3⁺ CD4 T cells

and depletion of Foxp3⁺ CD4 T cells in these mice [but not in WT, RIP-Cre or UBC-Cre mice] allowed further expansion of Foxp3-negative Cre:I-A^b tetramer positive CD4 T cells upon Cre peptide/CFA immunization.

Additional experiments revealed that development of Cre:I-A^b tetramer positive Foxp3⁺ CD4 T cells were not affected in Foxp3^ΔCNS1 BM chimera mice, implying that Cre:I-A^b tetramer positive Foxp3⁺ CD4 T cells were mostly thymic Foxp3⁺ CD4 T cells.

However, peripheral antigen expression promoted tonic signaling and active cycling of Cre:I-A^b tetramer positive Foxp3⁺ CD4 T cells in CC10-Cre and Vil-Cre mice.

Finally, the authors showed that multiple immunization [Lm-Cre infection followed by Cre/CFA immunization] was able to overcome Foxp3⁺ CD4 T cell-mediated non-deletional tolerance in RIP-Cre, CC10-Cre and Vil-Cre mice [but not in UBC-Cre mice] that resulted in antigen-specific tissue retention of Cre:I-A^b tetramer positive CD4 T cells and could even induce a transient increase in blood glucose level in RIP-Cre mice [though none of these mice showed any overt sustained autoimmunity].

In summary, these results indicate that (a) peripheral tolerance is primarily maintained by non-deletional action of thymus derived Foxp3⁺ CD4 T cells, and (b) without ectopic expression of peripheral antigen in the thymus [via Aire or Fezl], such non-deletional tolerance could be easily breached by recurrent infection resulting in sustained autoimmunity.

David Usharauli