Studies of suppression using monoclonal regulatory T cells and the importance of co-receptor Lck coupling ratios for negative selection

Normal physiology is not given without immunological tolerance. Depending on the origin of induction, tolerance can be divided into central and peripheral tolerance. Central tolerance comprises depletion of autoreactive T cells in the thymus (i.e. negative selection), and with this prevents autoimmunity. Peripheral tolerance critically depends on regulatory T cells (Tregs) maintaining lymphocyte homeostasis. Hallmark features of CD4+ Tregs are the expression of high surface IL-2-Rα (CD25) and the transcription factor Forkhead box protein P3 (Foxp3). Expression of non-functional Foxp3 results in lethal multi-organ lymphocytic infiltrations and cytokine secretions. Numerous data agree on an indispensable role for Tregs enabling physiological immunity. Yet an understanding of how Tregs function at a cellular and molecular level has not been fully elucidated.
In the first part of the thesis we made use of a RagKO TCR transgenic mouse expressing a Foxp3 transgene. This mouse provides a source of monoclonal CD4+, Foxp3+ T cells with a defined specificity. We show that monoclonal B3K506 Tregs are fully functional in vitro and in vivo and clearly require cognate antigen to be suppressive. We further show that the strength of Treg stimulation determines the strength of Treg mediated suppression. Finally we analysed various suppressive mechanisms used by monoclonal Tregs and found that Treg-Tconv proximity is an important parameter, which correlates with effective suppression.
In the second part of the thesis we aimed to understand the molecular mechanism underlying the affinity threshold for negative selection. We quantified the amount of Lck coupled to CD8 or CD4 coreceptors. We found that CD4 co-receptors have higher Lck coupling ratios than do CD8 co-receptors. In addition we determined the absolute numbers of surface molecules (i.e. CD8α, CD4 and CD3ε) on double positive (DP) thymocytes in B6 and MHC- class I restricted, TCR Tg mouse strains. A model, explaining how the TCR measures antigen affinity to initiate a negative selection signal, was generated. Here we show that the affinity threshold for different co-receptors depends on the probability that a peptide-MHC- TCR complex will collide with a co-receptor carrying Lck during the time pMCH binds to the TCR (Stepanek, O. et al. Cell, 2014)