SHP1‐ing thymic selection

Thymocyte development and maintenance of peripheral T‐cell numbers and functions are critically dependent on T‐cell receptor (TCR) signal strength. SHP1 (Src homology region 2 domain‐containing phosphatase‐1), a tyrosine phosphatase, acts as a negative regulator of TCR signal strength. Moreover, germline SHP1 knockout mice have shown impaired thymic development. However, this has been recently questioned by an analysis of SHP1 conditional knockout mice, which reported normal thymic development of SHP1 deficient thymocytes. Using this SHP1 conditional knockout mice, in this issue of the European Journal of Immunology, Martinez et al. [Eur. J. Immunol. 2016. 46: 2103–2110] show that SHP1 indeed does have a role in the negative regulation of TCR signal strength in positively selected thymocytes, and in the final maturation of single positive thymocytes. They report that thymocyte development in such mice shows loss of mature, post‐selection cells. This is due to increased TCR signal transduction in thymocytes immediately post positive‐selection, and increased cell death in response to weak TCR ligands. Thus, SHP1‐deficiency shows strong similarities to deficiency in the T‐cell specific SHP1‐associated protein Themis.     

Two separable T cell receptor signals reconstitute positive selection of CD4 lineage T cells in vivo

Positive selection is an obligatory step during intrathymic T cell differentiation. It is associated with rescue of short-lived, self major histocompatibility complex (MHC)-restricted thymocytes from programmed cell death, CD4/CD8 T cell lineage commitment, and induction of lineage-specific differentiation programs. T cell receptor (TCR) signaling during positive selection can be closely mimicked by targeting TCR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. We show that selection of CD4 T cell lineage cells in mice deficient for MHC class I and MHC class II expression can be reconstituted in vivo by two separable T cell receptor signaling steps, whereas a single TCR signal leads only to induction of short-lived CD4⁺CD8^la intermediates. These intermediates remain susceptible to a second TCR signal for 12-48 h providing an estimate for the duration of positive selection in situ. While both TCR signals induce differentiation steps, only the second one confers long-term survival on immature thymocytes. In further support of the two-step model of positive selection we provide evidence that CD4 T cell lineage cells rescued by a single hybrid antibody pulse in MHC class II-deficient mice are pre-selected by MHC class 1.     

CD3-induced apoptosis of CD4+CD8+ thymocytes in the absence of clonotypic T cell antigen receptor

Clonal selection of T cells mediated through the T cell antigen receptor (TCR) mostly occurs at the CD4⁺CD8⁺ double positive thymocyte stage. Immature CD4⁺CD8⁺ thymocytes expressing self-reactive TCR are induced to die upon clonotypic engagement of TCR by self antigens. CD3 engagement by antibody of the surface TCR-CD3 complex is known to induce apoptosis of CD4⁺CD8⁺ thymocytes, a process that is generally thought to represent antigen-induced negative selection in the thymus. The present study shows that the CD3-induced apoptosis of CD4⁺CD8⁺ thymocytes can occur even in TCRα^? mutant mice which do not express the TCRαβ/CD3 antigen receptor. Anti-CD3 antibody induces death of CD4⁺CD8⁺ thymocytes in TCRα^? mice either in cell cultures or upon administration in vivo. Interestingly, most surface CD3 chains expressed on CD4⁺CD8⁺ thymocytes from TCRα^? mice are not associated with clonotypic TCR chains, including TCRβ. Thus, apoptosis of CD4⁺CD8⁺ thymocytes appear to be induced through the CD3 complex even in the absence of clonotypic antigen receptor chains. These results shed light on previously unknown functions of the clonotype-independent CD3 complex expressed on CD4⁺CD8⁺ thymocytes, and suggest its function as an apoptotic receptor inducing elimination of developing thymocytes.     

Common gamma chain cytokines promote regulatory T cell development and survival at the CD4+ CD8+ stage in the human thymus

Thymic commitment of human FOXP 3⁺ regulatory T cells begins at the double‐positive (DP ) CD 4⁺ CD 8⁺ stage. In the current study, we show that interleukin‐2 promotes the development of FOXP 3⁺ thymocytes and enhances their survival at the DP phase. IL ‐2 increases the frequency of FOXP 3⁺ cells and promotes the Treg phenotype after TCR ‐mediated positive selection at the most mature DP stage. However, it has no effect on FOXP 3⁺ cells at the earlier maturation steps before positive selection. DP FOXP 3⁺ thymocytes are highly susceptible to cell death but IL ‐2 promotes their survival. The anti‐apoptotic protein BCL ‐2 (B Cell Lymphoma 2) is also upregulated by IL ‐2 at the most mature DP stage. In addition to IL ‐2, we identify IL ‐15 to have a significant role in the upregulating FOXP 3 and survival of Tregs at the DP phase. IL ‐7 also increases the expression of BCL ‐2 in the DP FOXP 3⁺ thymocytes. Our results indicate that common gamma chain cytokines IL ‐2, IL ‐7 and IL ‐15 promote the development of regulatory T cells at the most mature DP stage after TCR ‐mediated positive selection through suppressing cell death.     

Phospholipase Cγ1 is required for pre‐TCR signal transduction and pre‐T cell development

Pre‐T cell receptor (TCR) signaling is required for pre‐T cell survival, proliferation, and differentiation from the CD4 and CD8 double negative (DN) to the double positive (DP) stage. However, the pre‐TCR signal transduction pathway is not fully understood and the signaling molecules involved have not been completely identified. Phospholipase Cγ (PLCγ) 1 is an important signaling molecule that generates two second messengers, diacylglycerol and inositol 1,4,5‐trisphosphate, that are important to mediate PKC activation and intracellular Ca²⁺ flux in many signaling pathways. Previously, we have shown that PLCγ1 is important for TCR‐mediated signaling, development and T‐cell activation, but the role of PLCγ1 in pre‐TCR signal transduction and pre‐T cell development is not known. In this study, we demonstrated that PLCγ1 expression level in pre‐T cells was comparable to that in mature T cells. Deletion of PLCγ1 prior to the pre‐TCR signaling stage partially blocked the DN3 to DN4 transition and reduced thymic cellularity. We also demonstrated that deletion of PLCγ1 impaired pre‐T cell proliferation without affecting cell survival. Further study showed that deficiency of PLCγ1 impaired pre‐TCR mediated Ca²⁺ flux and Erk activation. Thus our studies demonstrate that PLCγ1 is important for pre‐TCR mediated signal transduction and pre‐T cell development.     

Pertussis toxin can replace T cell receptor signals that induce positive selection of CD8 T cells

CD4⁺ helper T lymphocytes and CD8⁺ killer T lymphocytes are both generated in the thymus from common precursor cells expressing CD4 and CD8. The development of immature CD4⁺ CD8⁺ thymocytes into mature ‘single-positive’ T cells requires T cell antigen-receptor (TCR)-mediated positive selection signals. Although it is known that the recognition specificity of TCR expressed by CD4⁺ CD8⁺ thymocytes determines their fate to become either CD4⁺ or CD8⁺ T cells, the molecular signals that direct precursor thymocytes to become CD4⁺ and CD8⁺ T cells are unclear. By using ZAP-70^? mutant thymus organ cultures in which T cell development is arrested at the CD4⁺ CD8⁺ thymocyte stage, the present study shows that distinct biochemical treatments can selectively restore the generation of mature CD4⁺ and CD8⁺ T cells, bypassing TCR-induced positive selection signals. The combination of phorbol ester and ionomycin selectively restores the generation of CD4⁺CD8^? TCR^high cells consistent with previous results. On the other hand, we find that the generation of CD4^? CD8⁺ TCR^high cells is selectively induced by pertussis toxin. Interestingly, the signals generated by pertussis toxin, which increase Notch expression, can dominate the signals by phorbol ester and ionomycin, steering thymocyte development to CD8 lineage. These results indicate that distinct biochemical signals replace TCR signals that selectively induce positive selection of CD4⁺ and CD8⁺ T cells, and that biochemical treatment can manipulate the development and choice of CD4⁺ and CD8⁺ T cells.     

Developmentally regulated expression of P-glycoprotein (multidrug resistance) activity in mouse thymocytes

P-glycoprotein (P-gly) is the transmembrane efflux pump responsible for multidrug resistance in tumor cells. The activity of P-gly in mature peripheral lymphocytes is lineage specific, with CD8⁺ T cells and natural killer (NK) cells expressing high levels as compared to CD4⁺ T cells and B cells. We have now investigated P-gly activity in immature and mature subsets of mouse thymocytes. Our data indicate that P-gly activity is undetectable in immature CD4^?8^? and CD4⁺8⁺ thymocyte subsets. Among mature thymocytes, P-gly activity is absent in the CD4⁺ subset but present in the more mature (HSA^low) fraction of CD8⁺ cells. Furthermore, while thymic CD4^?8^? T cell receptor (TCR) γδ cells have little P-gly activity, a minor subset of CD4^?8^? or CD4⁺ TCR αβ⁺ thymocytes bearing the NK1.1 surface marker expresses high levels of P-gly activity. Collectively, our results indicate that P-gly activity arises late during thymus development and is expressed in a lineage-specific fashion.     

CD 69 expression during selection and maturation of CD4+8+ thymocytes

We have analyzed the inducibility of protein kinase C (PKC)-dependent expression of CD 69 molecules in T cell receptor (TCR) transgenic thymocytes developing in the presence or absence of selecting, class I major histocompatibility complex (MHC) molecules. Small CD4⁺8⁺ thymocytes developing in the absence of selecting MHC molecules could not be induced to express CD 69 by TCR cross-linking even after spontaneous in vitro up-regulation of their TCR level which resulted in enhanced Ca⁺⁺ flux. In contrast, a small proportion of CD4⁺8⁺TCR^low and most TCR^high (CD4⁺8⁺ and CD4⁺8⁺) thymocytes developing in the presence of selecting MHC ligands could be induced to express CD 69 upon TCR cross-linking. Unlike the anti-TCR antibody, phorbol 12-myristate 13-acetate - a direct activator of PKC - induced the expression of CD 69 on all thymocytes. These results suggest that positive selection of CD4⁺8⁺ thymocytes results in coupling of TCR-mediated signals to the CD 69 expression pathway. In vitro analysis of thymocytes before and after positive selection suggests that (1) positive selection does not immediately result in resistance to deletion and (2) that sustained TCR ligation is needed to promote maturation of positively selected CD4⁺8⁺ thymocytes resulting in gradual loss of the sensitivity to deletion and acquisition of the ability to proliferate in response to TCR-mediated signals.     

Conditional deletion of SLP-76 in mature T cells abrogates peripheral immune responses

The adaptor protein Src homology 2 domain‐containing leukocyte‐specific protein of 76 kDa (SLP‐76) is central to the organization of intracellular signaling downstream of the T‐cell receptor (TCR). Evaluation of its role in mature, primary T cells has been hampered by developmental defects that occur in the absence of WT SLP‐76 protein in thymocytes. Here, we show that following tamoxifen‐regulated conditional deletion of SLP‐76, mature, antigen‐inexperienced T cells maintain normal TCR surface expression but fail to transduce TCR‐generated signals. Conditionally deficient T cells fail to proliferate in response to antigenic stimulation or a lymphopenic environment. Mice with induced deletion of SLP‐76 are resistant to induction of the CD4⁺ T‐cell‐mediated autoimmune disease experimental autoimmune encephalomyelitis. Altogether, our findings demonstrate the critical role of SLP‐76‐mediated signaling in initiating T‐cell‐directed immune responses both in vitro and in vivo and highlight the ability to analyze signaling processes in mature T cells in the absence of developmental defects.     

Strength of TCR signal from self‐peptide modulates autoreactive thymocyte deletion and Foxp3+ Treg‐cell formation

Autoreactive CD4⁺CD8^? (CD4SP) thymocytes can be subjected to deletion when they encounter self‐peptide during their development, but they can also undergo selection to become CD4SPFoxp3⁺ Treg cells. We have analyzed the relationship between these distinct developmental fates using mice in which signals transmitted by the TCR have been attenuated by mutation of a critical tyrosine residue of the adapter protein SLP‐76. In mice containing polyclonal TCR repertoires, the mutation caused increased frequencies of CD4SPFoxp3⁺ thymocytes. CD4SP thymocytes expressing TCR Vβ‐chains that are subjected to deletion by endogenous retroviral superantigens were also present at increased frequencies, particularly among Foxp3⁺ thymocytes. In transgenic mice in which CD4SP thymocytes expressing an autoreactive TCR undergo both deletion and Treg‐cell formation in response to a defined self‐peptide, SLP‐76 mutation abrogated deletion of autoreactive CD4SP thymocytes. Notably, Foxp3⁺ Treg‐cell formation still occurred, albeit with a reduced efficiency, and the mutation was also associated with decreased Nur77 expression by the autoreactive CD4SP thymocytes. These studies provide evidence that the strength of the TCR signal can play a direct role in directing the extent of both thymocyte deletion and Treg‐cell differentiation, and suggest that distinct TCR signaling thresholds and/or pathways can promote CD4SP thymocyte deletion versus Treg‐cell formation.     

Separately expressed T cell receptor α and β chain transgenes exert opposite effects on T cell differentiation and neoplastic transformation

Two aspects of T cell differentiation in T cell receptor (TCR)-transgenic mice, the generation of an unusual population of CD4^?CD8^?TCR⁺ thymocytes and the absence of γδ cells, have been the focus of extensive investigation. To examine the basis for these phenomena, we investigated the effects of separate expression of a transgenic TCR α chain and a transgenic TCR β chain on thymocyte differentiation. Our data indicate that expression of a transgenic TCR α chain causes thymocytes to differentiate into a CD4^?CD8^?TCR⁺ lineage at an early developmental stage, depleting the number of thymocytes that differentiate into the αβ lineage. Surprisingly, expression of the TCR α chain transgene is also associated with the development of T cell lymphosarcoma. In contrast, expression of the transgenic TCR β chain causes immature T cells to accelerate differentiation into the αβ lineage and thus inhibits the generation of γδ cells. Our observations provide a model for understanding T cell differentiation in TCR-transgenic mice.     

Nature and nurture: T-cell receptor-dependent and T-cell receptor-independent differentiation cues in the selection of the memory T-cell pool

The initiation of a T‐cell response begins with the interaction of an individual T‐cell clone with its cognate antigen presented by MHC. Although the strength of the T‐cell receptor (TCR) –antigen–MHC (TCR‐pMHC) interaction plays an important and obvious role in the recruitment of T cells into the immune response, evidence in recent years has suggested that the strength of this initial interaction can influence various other aspects of the fate of an individual T‐cell clone and its daughter cells. In this review, we will describe differences in the way CD4⁺ and CD8⁺ T cells incorporate antigen‐driven differentiation and survival signals during the response to acute infection. Furthermore, we will discuss increasing evidence that the quality and/or quantity of the initial TCR‐pMHC interaction can drive the differentiation and long‐term survival of T helper type 1 memory populations.     

Diversity and clonotypic composition of influenza‐specific CD8+ TCR repertoires remain unaltered in the absence of Aire

TCR repertoire diversity is important for the protective efficacy of CD8⁺ T cells, limiting viral escape and cross‐reactivity between unrelated epitopes. The exact mechanism for selection of restricted versus diverse TCR repertoires is far from clear, although one thought is that the epitopes resembling self‐peptides might select a limited array of TCR due to the deletion of autoreactive TCR. The molecule Aire promotes the expression of tissue‐specific Ag on thymic medullary epithelial cells and the deletion of autoreactive cells, and in the absence of Aire autoreactive cells persist. However, the contribution of Aire‐dependent peptides to the selection of the Ag‐specific TCR repertoire remains unknown. In this study, we dissect restricted (D^bNP₃₆₆%⁺CD8⁺) and diverse (D^bPA₂₂₄%⁺CD8⁺, K^dNP₁₄₇%⁺CD8⁺) TCR repertoires responding to three influenza‐derived peptides in Aire‐deficient mice on both B6 and BALB/c backgrounds. Our study shows that the number, qualitative characteristics and TCR repertoires of all influenza‐specific, D^bNP₃₆₆%⁺CD8⁺, D^bPA₂₂₄%⁺CD8⁺ and K^dNP₁₄₇%⁺CD8⁺ T cells are not significantly altered in the absence of Aire. This provides the first demonstration that the selection of an Ag‐specific T‐cell repertoire is not significantly perturbed in the absence of Aire.     

Role of TCR specificity in CD4+CD25+ regulatory T-cell selection

Summary: CD4⁺CD25⁺ regulatory T cells play a crucial role in preventing autoimmune disease and can also modulate immune responses in settings such as transplantation and infection. We have developed a transgenic mouse system in which the role that T‐cell receptor (TCR) specificity for self‐peptides plays in the formation of CD4⁺CD25⁺ regulatory T cells can be examined. We have shown that interactions with a single self‐peptide can induce thymocytes bearing an autoreactive TCR to undergo selection to become CD4⁺CD25⁺ regulatory T cells and that thymocytes bearing TCRs with low affinity for the selecting peptide do not appear to undergo selection into this pathway. In addition, thymocytes with identical specificity for the selecting self‐peptide can undergo overt deletion versus abundant selection to become CD4⁺CD25⁺ regulatory T cells in response to variations in expression of the selecting peptide in different lineages of transgenic mice. Finally, we have shown that CD4⁺CD25⁺ T cells proliferate in response to their selecting self‐peptide in the periphery, but these cells do not proliferate in response to lymphopenia in the absence of the selecting self‐peptide. These studies are determining how the specificity of the TCR for self‐peptides directs the thymic selection and peripheral expansion of CD4⁺CD25⁺ regulatory T cells.     

Polarity gene discs large homolog 1 regulates the generation of memory T cells

Mammalian ortholog of Drosophila cell polarity protein, Dlg1, plays a critical role in neural synapse formation, epithelial cell homeostasis, and urogenital development. More recently, it has been proposed that Dlg1 may also be involved in the regulation of T‐cell proliferation, migration, and Ag‐receptor signaling. However, a requirement for Dlg1 in development and function of T lineage cells remains to be established. In this study, we investigated a role for Dlg1 during T‐cell development and function using a combination of conditional Dlg1 KO and two different Cre expression systems where Dlg1 deficiency is restricted to the T‐cell lineage only, or all hematopoietic cells. Here, using three different TCR models, we show that Dlg1 is not required during development and selection of thymocytes bearing functionally rearranged TCR transgenes. Moreover, Dlg1 is dispensable in the activation and proliferative expansion of Ag‐specific TCR‐transgenic CD4⁺ and CD8⁺ T cells in vitro and in vivo. Surprisingly, however, we show that Dlg1 is required for normal generation of memory T cells during endogenous response to cognate Ag. Thus, Dlg1 is not required for the thymocyte selection or the activation of primary T cells, however it is involved in the generation of memory T cells.     

Antigen-specific and nonspecific deletion of immature cortical thymocytes caused by antigen injection

Analysis of antigen-induced negative selection of thymocytes in T cell receptor (TCR)-transgenic mice is complicated by the presence of an antigen-responsive peripheral T cell compartment. Our experiments address the question of whether and how peripheral T cell activation can affect immature thymocytes. Following three daily injections of peptide antigen into mice expressing a peptide-specific transgenic TCR and deficient for TAP1, we and others have found profound deletion of the CD4⁺CD8⁺ (DP) thymocyte subset. However, our work shows that even though mature CD8⁺ T cells are inefficiently selected in TAP1-deficient mice, there was a striking degree of peripheral expansion and activation of CD8⁺ peripheral T cells. Furthermore, when cells from TCR-transgenic mice were adoptively transferred, we found that deletion of non-transgenic DP thymocytes occurred in Thy-1-congenic and even more efficiently in TAP1-deficient recipients after repeated peptide injection resulting in peripheral T cell activation. In the adoptive transfer experiments the degree of deletion of immature bystander thymocytes was decreased upon blocking of TNF. These data show that deletion of DP thymocytes can result from excessive peripheral T cell activation and identify TNF as an important effector molecule for this process. When steps are taken to avoid peripheral T cell activation, peptide antigen can induce TCR-mediated thymocyte deletion, presumably in the thymus cortex, since injection of TAP1-deficient TCR-transgenic mice resulted in deletion of immature DP thymocytes prior to detectable peripheral T cell expansion and activation. This effect was not blocked by inhibiting tumor necrosis factor activity. In addition, DP depletion was seen in the absence of peripheral T cell activation when antibody-mediated depletion of CD8⁺ T cells was performed. Our work clearly shows that two mechanisms for deletion of DP thymocytes exist: deletion induced by antigen presentation in the thymus and deletion as a consequence of repeated stimulation of mature peripheral T cells.