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

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 





    

Thymic epitope expression determines pattern of CD4 T cell peripheral tolerance

A few months ago I posted my analysis of one of the important papers from journal of Immunity discussing mechanisms of CD4 T cell peripheral tolerance. In that paper, the authors led by J.J. Moon, showed that number and functionality of CRE recombinase-specific endogenous CD4 T cells were determined by antigen expression in the thymus. 

Today, journal Nature Immunology published very similar study from Marc Jenkins' lab (JJ Moon's former supervisor) where the authors showed that number and functionality of eGFP epitope-specific endogenous CD4 T cells were also determined by level of specific epitope expression in the thymus. These studies have important implications predicting the efficacy of vaccines and mechanisms of autoimmune diseases, so lets review it.

As some of you might know Marc Jenkins' lab pioneered technique for analysis of endogenous antigen[epitope]-specific T cells in WT mice. Here, the authors have analysed expansion of endogenous eGFP epitope:MHC II tetramer⁺ CD4 T cells in dozen of transgenic mice expressing eGFP protein under the guidance of different ubiquitous or tissue-specific promoters. 

This analysis revealed 3 patterns of T cell tolerance: ignorance, thymic Treg development and thymic deletion. Mechanistically, level of antigen expression in the thymus determined which out of these 3 tolerance patterns were operational.   

Tolerance by ignorance: Ins1^eGFP mice express eGFP exclusively in pancreas. Analysis showed that both Ins1^eGFP  and WT mice harbor similar number of eGFP-specific naive CD4 T cells, implying ignorance.

Tolerance by Treg induction: Ins2^eGFP mice express eGFP in pancreas and in the thymus in a AIRE-dependent manner.

Presence of eGFP in the thymus in Ins2^eGFP mice slightly reduced tet⁺ CD4 T cell numbers and correspondingly increased frequency of tet⁺ Foxp3⁺ CD4 T cells. 

Immunization with CFA-eGFP peptide showed that tolerance in Ins2^eGFP mice depended on AIRE-driven eGFP epitope expression in the thymus.

Tolerance by thymic deletion: UBC^eGFP mice displayed a profound thymic deletion of eGFP-specific tet⁺ CD4 T cells. 

The authors clearly showed that eGFP expression in the thymus inversely correlated with the number of tet⁺ CD4 T cells. 

Similar pattern of T cell "tolerance" were found for truly nonself- and self-epitope-specific T cells.

In summary, this study confirmed that level of antigen expression in the thymus (and not in periphery) determined overall T cell tolerance status. By analyzing the number of antigen-specific human T cells [in comparison to reference "self"-specific T cell numbers] the scientist could predict efficacy of vaccines, tumor vaccine for example, or predict the potential for development of autoimmune diseases.

David Usharauli