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

Tumor elimination requires simultaneous expression of both class I and II neo-epitopes

The most tumors express mutant epitopes that could be detected by T cells. According to current paradigm, CD4+ T cells provides help to CD8+ T cells that in turn attack tumors. As tumor cells ordinarily express class I recognized by CD8+ T cells but not class II molecules recognized by CD4+ T cells, primary focus on CD8+ T cell epitopes made a lot of sense. But what about CD4+ T cell 'help' to CD8 T cells? 

Indeed, a new 'classically-done' immunology study from Robert Schreiber's lab clearly showed that irrespective class II expression, tumor cells must express both CD8+ and CD4+ T cell neo-epitopes to achieve efficient local tumor control following immunotherapy.

As a starting point, they used nonimmunogenic oncogene-driven KP9025 sarcoma cells (KP), which lack mutational neoantigens. Next they re-expressed in KP cells 2 mutant epitopes, one for class I, mLAMA4, and another for class II, mITGB1 (identified using a hidden Markov model (HMM)-based MHC binding predictor the authors claim is better than other available algorithms). 

 

A mutant but not wild-type version of ITGB1 was detected by CD4+ TILs.

  

Next, the authors showed that only KP tumors expressing both neo-epitopes but not single expressors, could be eliminated by T cells following immunotherapy.

  

As expected, presence of CD4+ T cell epitope enhanced CD8+ T cell response.

Interestingly, both class I and II  neo-epitopes must be expressed by the same tumor to mediate protection when used as immunized agents (mixing of single expressor tumors was not enough).

And notably, expression of both class I and II neo-epitopes were necessary to mediate efficient local tumor control (single expressor tumors were resistant against CD8+ or CD4+  T cells)

In summary, this is a simple, easy to follow experments that indicate the authors' thought process.  It shows that CD4+ T cells 'help' to CD8+ T cells are required both at priming and as well as at effector stage. It is not clear if it is simply a quantitative or rather qualitative issue. It is not known either whether CD4+ T cells do something directly against tumor beyond simply helping CD8+ T cells here. 

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