Kensuke Takada

KLF2 Transcription-Factor Deficiency in T Cells Results in Unrestrained Cytokine Production and Upregulation of Bystander Chemokine Receptors

The transcription factor KLF2 regulates T cell trafficking by promoting expression of the lipid-binding receptor S1P(1) and the selectin CD62L. Recently, it was proposed that KLF2 also represses the expression of chemokine receptors. We confirmed the upregulation of the chemokine receptor CXCR3 on KLF2-deficient T cells. However, we showed that this was a cell-nonautonomous effect, as revealed by CXCR3 upregulation on wild-type bystander cells in mixed bone-marrow chimeras with KLF2-deficient cells. Furthermore, KLF2-deficient T cells overproduced IL-4, leading to the upregulation of CXCR3 through an IL-4-receptor- and eomesodermin-dependent pathway. Consistent with the increased IL-4 production, we found high concentrations of serum IgE in mice with T cell-specific KLF2 deficiency. Our findings support a model where KLF2 regulates T cell trafficking by direct regulation of S1P(1) and CD62L and restrains spontaneous cytokine production in naive T cells.

Self–class I MHC molecules support survival of naive CD8 T cells, but depress their functional sensitivity through regulation of CD8 expression levels

Previous studies have suggested that naive CD8 T cells require self-peptide–major histocompatability complex (MHC) complexes for maintenance. However, interpretation of such studies is complicated because of the involvement of lymphopenic animals, as lymphopenia drastically alters naive T cell homeostasis and function. In this study, we explored naive CD8 T cell survival and function in nonlymphopenic conditions by using bone marrow chimeric donors and hosts in which class I MHC expression is absent or limited to radiosensitive versus radioresistant cells. We found that long-term survival of naive CD8 T cells (but not CD4 T cells) was impaired in the absence of class I MHC. However, distinct from this effect, class I MHC deprivation also enhanced naive CD8 T cell responsiveness to low-affinity (but not high-affinity) peptide–MHC ligands. We found that this improved sensitivity was a consequence of up-regulated CD8 levels, which was mediated through a transcriptional mechanism. Hence, our data suggest that, in a nonlymphopenic setting, self-class I MHC molecules support CD8 T cell survival, but that these interactions also attenuate naive T cell sensitivity by dynamic tuning of CD8 levels.

β5t-containing thymoproteasome: specific expression in thymic cortical epithelial cells and role in positive selection of CD8+ T cells.

Proteasomes are multisubunit proteolytic complexes that degrade cytoplasmic and nuclear proteins in eukaryotes. Proteasome-dependent proteolysis contributes to various cellular processes, including misfolded protein degradation, signal transduction, and antigen presentation. The thymoproteasome is a form of proteasome that contains the vertebrate-specific catalytic subunit β5t specifically expressed by cortical epithelial cells in the thymus. The thymoproteasome is essential for the positive selection of CD8+ T cells that carry an immunocompetent repertoire of antigen recognition specificity. Here we summarize the structure and expression of the thymoproteasome and discuss how it regulates the positive selection of CD8+ T cells.

Kruppel-Like Factor 2 Is Required for Trafficking but Not Quiescence in Postactivated T Cells

The transcription factor Kruppel-like factor 2 (KLF2) was proposed to regulate genes involved in cell cycle entry and T cell trafficking; however, the physiological role of its expression in postactivated T cells is not well defined. Previous studies suggested that the cytokines IL-2 and IL-15 differentially regulate KLF2 re-expression in postactivation T cells and that these cytokines also influence effector versus memory T cell differentiation. Using conditional and inducible KLF2-knockout model systems, we tested the specific role of KLF2 expression in activated CD8+ T cells cultured with these cytokines. KLF2 was required for effective transcription of sphingosine-1-phosphate receptor-1 (S1P1) and CD62L in postactivation T cells. However, although different cytokines dramatically altered the expression of cell-cycle–related genes, endogenous KLF2 had a minimal impact. Correspondingly, KLF2-deficient T cells showed dysregulated trafficking but not altered proliferative characteristics following in vivo responses to Ag. Thus, our data help to define KLF2-dependent and -independent aspects of activatedCD8+ T cell differentiation and argue against a physiological role in cell cycle regulation.

Positive selection optimizes the number and function of MHCII-restricted CD4+ T cell clones in the naive polyclonal repertoire

T cell receptors (TCRs) on T lymphocytes in an individual bind foreign peptides bound to major histocompatibility complex (MHC) molecules expressed in that individual (designated MHCA). Results from radiation bone marrow chimeras and TCR transgenic mice indicate that this complex form of antigen recognition is the result of positive selection of clones with low affinity for self peptide:MHCA complexes during development. Here we used a sensitive peptide:MHC tetramer enrichment method to quantify the role of positive selection in the generation of the preimmune polyclonal T cell repertoire in normal individuals. We made the surprising observation that mouse and human naive T cells capable of binding to foreign peptide:MHCA were present at the same frequency in hosts that expressed MHCA or a different MHC isoform (MHCB). However, most of the clones in MHCB hosts also recognized self peptide:MHCA complexes. When these “alloreactive” T cells were removed from the MHCB repertoire via negative selection in an MHCA host, the number of foreign peptide:MHCA-binding T cells was reduced to one fifth and many of the remaining cells did not respond to the peptide. Therefore, although positive selection on MHCA was not required to produce foreign peptide:MHCA-binding clones, it had a large effect on selecting responsive clones.

Thymoproteasomes produce unique peptide motifs for positive selection of CD8+ T cells

Positive selection in the thymus provides low-affinity T-cell receptor (TCR) engagement to support the development of potentially useful self-major histocompatibility complex class I (MHC-I)-restricted T cells. Optimal positive selection of CD8+ T cells requires cortical thymic epithelial cells that express β5t-containing thymoproteasomes (tCPs). However, how tCPs govern positive selection is unclear. Here we show that the tCPs produce unique cleavage motifs in digested peptides and in MHC-I-associated peptides. Interestingly, MHC-I-associated peptides carrying these tCP-dependent motifs are enriched with low-affinity TCR ligands that efficiently induce the positive selection of functionally competent CD8+ T cells in antigen-specific TCR-transgenic models. These results suggest that tCPs contribute to the positive selection of CD8+ T cells by preferentially producing low-affinity TCR ligand peptides.

TCR affinity for thymoproteasome-dependent positively selecting peptides conditions antigen responsiveness in CD8+ T cells

In the thymus, low-affinity T cell antigen receptor (TCR) engagement facilitates positive selection of a useful T cell repertoire. Here we report that TCR responsiveness of mature CD8+ T cells is fine tuned by their affinity for positively selecting peptides in the thymus and that optimal TCR responsiveness requires positive selection on major histocompatibility complex class I–associated peptides produced by the thymoproteasome, which is specifically expressed in the thymic cortical epithelium. Thymoproteasome-independent positive selection of monoclonal CD8+ T cells results in aberrant TCR responsiveness, homeostatic maintenance and immune responses to infection. These results demonstrate a novel aspect of positive selection, in which TCR affinity for positively selecting peptides produced by thymic epithelium determines the subsequent antigen responsiveness of mature CD8+ T cells in the periphery.

Gimap3 and Gimap5 cooperate to maintain T-cell numbers in the mouse

Gimap3 (IAN4) and Gimap5 (IAN5) are highly homologous GTP‐binding proteins of the Gimap family. Gimap3 and Gimap5, whose transcripts are abundant in mature lymphocytes, can associate with antiapoptotic Bcl‐2 family proteins. While it is established that Gimap5 regulates T‐cell survival, the in vivo role of Gimap3 is unclear. Here we report the preparation and characteristics of mouse strains lacking Gimap3 and/or Gimap5. We found that the number of T cells was markedly reduced in mice deficient in both Gimap3 and Gimap5. The defects in T‐cell cellularity were more severe in mice lacking both Gimap3 and Gimap5 than in mice lacking only Gimap5. No defects in the cellularity of T cells were detected in mice lacking only Gimap3, whereas bone marrow cells from Gimap3‐deficient mice showed reduced T‐cell production in a competitive hematopoietic environment. Moreover, retroviral overexpression and short hairpin RNAs‐mediated silencing of Gimap3 in bone marrow cells elevated and reduced, respectively, the number of T cells produced in irradiated mice. These results suggest that Gimap3 is a regulator of T‐cell numbers in the mouse and that multiple Gimap family proteins cooperate to maintain T‐cell survival.

Positive-selection-inducing self-peptides displayed by cortical thymic epithelial cells.

A repertoire of antigen recognition specificities in mature T cell pool is formed by the selection during T cell development in the thymus. Positive selection is an essential process for the development of functionally competent T cells and is dependent on the interaction between T cell antigen receptors (TCRs) that newly generated thymocytes express and self-peptide-associated major histocompatibility complex (pMHC) molecules that cortical thymic epithelial cells (cTECs) express. Characterization of positive-selection-inducing peptides has revealed that the low-affinity TCR engagement by the positive-selection-inducing pMHC complexes initiates intracellular signals that induce the survival of immature thymocytes and their differentiation into mature T cells. Recent studies suggest unique mechanisms of antigen processing in cTECs for the production of positively selecting MHC-bound self-peptides.

Generation of Peptides That Promote Positive Selection in the Thymus

To establish an immunocompetent TCR repertoire that is useful yet harmless to the body, a de novo thymocyte repertoire generated through the rearrangement of genes that encode TCR is shaped in the thymus through positive and negative selection. The affinity between TCRs and self-peptides associated with MHC molecules determines the fate of developing thymocytes. Low-affinity TCR engagement with self-peptide–MHC complexes mediates positive selection, a process that primarily occurs in the thymic cortex. Massive efforts exerted by many laboratories have led to the characterization of peptides that can induce positive selection. Moreover, it is now evident that protein degradation machineries unique to cortical thymic epithelial cells play a crucial role in the production of MHC-associated self-peptides for inducing positive selection. This review summarizes current knowledge on positive selection–inducing self-peptides and Ag processing machineries in cortical thymic epithelial cells. Recent studies on the role of positive selection in the functional tuning of T cells are also discussed.