Regulation of non-responsiveness and death in cytotoxic T cells by the agonistic potency of MHC : Peptide ligands

CD8+ T lymphocytes are important for immunological control of infections and tumors. The key interaction required to initiate the process of T cell activation is the engagement of the T cell receptor (TCR) with a major histocompatibility complex (MHC) class I/peptide complex on an antigen presenting cell (APC). Depending on the affinity of the interaction between the TCR and MHC class I molecule, different arrays of signaling pathways can be activated in the T cell. Molecular alterations in the peptide bound to the MHC class I can lead to a lower affinity of the MHC:TCR-interaction resulting in incomplete or qualitatively different T-cell responses. Altered peptide ligands (APLs) exhibiting such activity are referred to as partial agonists and often occur naturally through genetic instability, which affects T cell epitopes derived from rapidly mutating viruses or tumor-associated cellular antigens. We studied the molecular basis of partial agonism using MHC class I/peptide tetramer complexes. By using tetramers assembled with a fully agonist peptide or its synthetic variant we could study the relationship between tetramer staining, cytokine production and different pathways of activation induced cell death (AICD). We found that positive tetramer staining correlated with at least two different activation programs in CD8+ T lymphocytes: full scale activation associated with Fas-dependent AICD and an incomplete activation followed by Fas-independent AICD. Further, we used raft-disrupting agents to assess the role of lipid rafts in determining the agonistic potency of different peptide ligands. We showed that overall binding of specific tetramers to CTLs was reduced upon raft disruption, although the half-life of tetramer:TCR complexes formed under these conditions was not affected. These findings suggest that different TCR complexes on the surface of CTLs may have different requirements for cholesterol and CTLs may be heterogeneous in their raft structure. In addition we analyzed programs of negative regulation of CD8+ T lymphocytes and the capacity of APLs to activate and modulate such programs. Upon specific triggering CD8+ T-cells become refractory to a secondary stimulus; a condition referred to Activation induced Non-Responsiveness (AINR). We have shown that TCR-triggering results in a novel degradation pathway of Lck, a kinase which plays a critical role in the initiation of T cell activation. Down-regulation of Lck through degradation correlated with AINR in CTLs. By blocking Lck degradation we could prevent the development of AINR. We further investigated how activation of CTLs with APLs affected Lck expression. The capacity of different peptide variants to induce Lck degradation correlated with their agonistic potency. Inefficient recognition of APLs by specific T lymphocytes is believed to contribute to the failure of the immune system to control certain tumor types and progressive viral diseases. To better understand the regulation of APL activity by immunologic help, we analyzed the capacity of exogenous IL-2 and IL-15 to influence different aspects of activation triggered in CTLs by either fully or partially agonistic peptide ligands. We showed that signals induced by the lymphokines synergize with weak TCR signaling induced by partially agonistic APL, converting many of these peptides from inhibitory to stimulatory ligands. We also demonstrated that IL-2 and IL-15 suppress induction of a death receptor-independent apoptotic program triggered by partially agonistic APL. In conclusion, we have analyzed the molecular basis of partial agonism in CTL recognition of peptide epitopes and characterized molecular changes associated with death and AINR in specific CTLs. We have shown that structural changes in the sequence of CTL peptide epitopes may decrease the affinity of MHC/TCR interactions and generate APLs, which not only trigger incomplete activation programs but also induce and modulate negative regulation programs in CTLs. This APL induced signaling of suppressive nature appears to be more prominent in the absence of immunological help, suggesting that under conditions of immune deregulation APLs may actively suppress CTL responses against infectious agents or tumors. LIST OF PUBLICATIONS This thesis is based on the following papers, which will be referred to by their roman numerals: I. Wei C, Uhlin M, Masucci MG, Levitsky V Tetramer binding and secretion of interferon-gamma in response to antigenic stimulation are compatible with a range of affinities of MHC:TCR interaction and distinct programs of cytotoxic T-lymphocyte activation. Hum Immunol. 2002 Oct;63(10):821. II. Uhlin M, Masucci MG, Levitsky V. Pharmacological disintegration of lipid rafts decreases specific tetramer binding and disrupts the CD3 complex and CD8 heterodimer in human cytotoxic T-lymphocytes. Scand J Immunol. 2003 Feb;57(2):99-106 III. Uhlin M, Masucci MG, Levitsky V Regulation of Lck degradation and refractory state in CD8+ cytotoxic T-lymphocytes. Proc Natl Acad Sci U S A. 2005 Jun 28;102(26):9264-9. IV. Uhlin M, Sandalova E, Masucci MG, Levitsky V The activity of partially agonistic peptides recognized by cytotoxic T-Lymphocytes is modulated by help signals triggered by exogenous lymphokines. Eur J Immunol. 2005 Oct;35(10):2929-39.