Regulation of the pro-apoptotic protein Bim by T cell receptor triggering in human T cells

Abstract: Bim is a significant pro-apoptotic member of Bcl-2 family of proteins; it is proved to be an important regulator of apoptosis: over the years it has been shown to play a pivotal role for the development and maintenance of homeostasis in many cellular systems and tissues. The Bcl-2 family of proteins is a group of key regulators of the mitochondrial, or intrinsic, pathway of apoptosis. It contains anti-apoptotic proteins, which promote cell survival, and pro-apoptotic ones, which induce cell death through the release of apoptotic factors from mitochondria. Proteins of the subfamily of BH-3 only proteins, which includes Bim, are important regulators of this process. Bim, as well as Bid were shown to bind the majority of anti-apoptotic and proapoptotic Bcl-2 family members and are believed to induce oligomerization of Bax and Bak proteins, which results in the outer membrane permeabilization and release of pro-apoptotic factors initiating cell death. In spite of its importance for different aspects of T-cell biology, regulation of Bim expression and activity by T-cell receptor (TCR) triggering is poorly understood. The focus of this work was to analyze how different modes of TCR-triggering affect Bim expression in human T-cells at different stages of their differentiation with particular emphasis on effector CD8+ cytotoxic T-lymphocytes (CTLs). We showed that expression of Bim is upregulated in activated antigen-specific human CTLs upon polyclonal or specific TCR triggering. Both activation of protein kinase C and induction of calcium influx appear to be necessary and sufficient for Bim upregulation after TCR-ligation. We also showed that Bim expression is differentially regulated by MHC:peptide ligands of different agonistic potency. Ligands which failed to induce Bim expression, failed to induce apoptosis, while induction of Bim accompanied by inefficient upregulation of Bcl-XL was associated with death receptor-independent activation induced cell death (AICD) triggered by partially agonistic ligands in specific CTLs. Cyclosporine A blocked Bim upregulation and rescued CTLs from death receptor independent AICD while rapamycin did not exhibit these activities, in accordance with known effects of the drugs on T-cell deletion in transplant recipients. These results defined a new pathway of Bim regulation, strongly implicated Bim as a mediator of AICD and suggested that Bim upregulation can be targeted to influence the outcome of specific immune responses. We also investigated in more detail the regulation of altered peptide ligands (APL) activity by immunologic help. We analyzed the capacity of exogenous IL-2 and IL-15, which are physiologically produced by cells of the adaptive and innate immune system, respectively, to modulate proliferation, responsiveness to repeated stimulation and apoptotic programs triggered in specific CTL by either fully or partially agonistic peptide ligands. We show 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. Some APL partially suppress the responsiveness of specific CTL to secondary stimulation, while this inhibitory effect is diminished if APLstimulated cells are cultured in the presence of either of the lymphokines. We also demonstrate that IL-2 and IL-15 suppress up-regulation of Bim and induction of a death receptor-independent apoptotic program triggered by partially agonistic APL. Our results suggest that under conditions of insufficient immunologic help, partially agonistic APL may actively suppress specific CTL responses and become especially advantageous for immune escape of tumors or viruses. We analyzed the expression of Bim in total peripheral blood lymphocytes (PBLs) and different subpopulations of ex vivo isolated human T-lymphocytes from healthy donors or patients with infectious mononucleosis. We showed that the majority of freshly isolated PBLs express relatively low levels of Bim, which are not modulated by TCRcrosslinking. However, TCR triggering induced significant Bim upregulation in some PBL samples. Longitudinal analysis demonstrated that responsiveness to TCR triggering, as revealed by Bim upregulation, varies over time in PBLs isolated from the same individual. Memory or naive T-cells enriched on the basis of CD45RO or CD45RA expression did not differ in their capacity to upregulate Bim in response to receptor crosslinking and were comparable, in this respect, to freshly isolated total lymphocytes. In contrast, lymphocytes isolated from patients with acute stage IM containing characteristic massive expansions of antigenspecific effector CD8+ T-cells expressed increased levels of Bim, which could not be accounted for by increased proportion of CD8+ cells. Moreover, lymphocytes from at least half of the analyzed IM patients exhibited strong upregulation of all major Bim isoforms in response to TCR triggering. These results support the notion that the activity of Bim in human CD8+ T-cells is regulated at the level of protein expression and the regulation of Bim promoter changes along with the process of Tlymphocyte differentiation enabling them to respond to TCR triggering by Bim upregulation. Our data support an important role of Bim in the regulation of T-cell homeostasis and T-cell mediated immunity and suggest that interference with TCR induced Bim upregulation may affect the outcome of natural immune responses, vaccination, autoimmunity and development of transplant tolerance.