Post-selection Thymocytes Research Articles

The Timing and Abundance of IL-2Rβ (CD122) Expression Control Thymic iNKT Cell Generation and NKT1 Subset Differentiation.

Invariant NKT (iNKT) cells are thymus-generated innate-like T cells, comprised of three distinct subsets with divergent effector functions. The molecular mechanism that drives the lineage trifurcation of immature iNKT cells into the NKT1, NKT2, and NKT17 subsets remains a controversial issue that remains to be resolved. Because cytokine receptor signaling is necessary for iNKT cell generation, cytokines are proposed to contribute to iNKT subset differentiation also. However, the precise roles and requirements of cytokines in these processes are not fully understood. Here, we show that IL-2Rβ, a nonredundant component of the IL-15 receptor complex, plays a critical role in both the development and differentiation of thymic iNKT cells. While the induction of IL-2Rβ expression on postselection thymocytes is necessary to drive the generation of iNKT cells, surprisingly, premature IL-2Rβ expression on immature iNKT cells was detrimental to their development. Moreover, while IL-2Rβ is necessary for NKT1 generation, paradoxically, we found that the increased abundance of IL-2Rβ suppressed NKT1 generation without affecting NKT2 and NKT17 cell differentiation. Thus, the timing and abundance of IL-2Rβ expression control iNKT lineage fate and development, thereby establishing cytokine receptor expression as a critical regulator of thymic iNKT cell differentiation.

Priming of lineage-specifying genes by Bcl11b is required for lineage choice in post-selection thymocytes

T-lineage committed precursor thymocytes are screened by a fate-determination process mediated via T cell receptor (TCR) signals for differentiation into distinct lineages. However, it remains unclear whether any antecedent event is required to couple TCR signals with the transcriptional program governing lineage decisions. Here we show that Bcl11b, known as a T-lineage commitment factor, is essential for proper expression of ThPOK and Runx3, central regulators for the CD4-helper/CD8-cytotoxic lineage choice. Loss of Bcl11b results in random expression of these factors and, thereby, lineage scrambling that is disconnected from TCR restriction by MHC. Initial Thpok repression by Bcl11b prior to the pre-selection stage is independent of a known silencer for Thpok, and requires the last zinc-finger motif in Bcl11b protein, which by contrast is dispensable for T-lineage commitment. Collectively, our findings shed new light on the function of Bcl11b in priming lineage-specifying genes to integrate TCR signals into subsequent transcriptional regulatory mechanisms.

Essential Roles of SATB1 in Specifying T Lymphocyte Subsets

T cell receptor (TCR) signaling by MHC class I and II induces thymocytes to acquire cytotoxic and helper fates via the induction of Runx3 and ThPOK transcription factors, respectively. The mechanisms by which TCR signaling is translated into transcriptional programs for each cell fate remain elusive. Here, we show that, in post-selection thymocytes, a genome organizer, SATB1, activates genes for lineage-specifying factors, including ThPOK, Runx3, CD4, CD8, and Treg factor Foxp3, via regulating enhancers in these genes in a locus-specific manner. Indeed, SATB1-deficient thymocytes are partially re-directed into inappropriate T lineages after both MHC class I-and II-mediated selection, and they fail to generate NKT and Treg subsets. Despite its essential role inactivating enhancers for the gene encoding ThPOKin TCR-signaled thymocytes, SATB1 becomes dispensable for maintaining ThPOK in CD4+ Tcells. Collectively, our findings demonstrate that SATB1 shapes the primary Tcell pool by directing lineage-specific transcriptional programs in the thymus.

Abstract A073: Epigenetic regulation of the Cd4 locus

Abstract CD4 helper and CD8 cytotoxic T cells originate from progenitor cells with binary potential. Transcriptional regulatory elements are key in establishing heritable epigenetic mechanisms to ensure gene-expression programs that maintain lineage identity. We show that, during thymocyte development, the co-receptor CD4 is regulated by a stage-specific regulatory element that ensures commitment to the CD4 lineage. Sustained CD4 expression in post-selection thymocytes requires this intronic element situated 3' to a silencer element previously shown to repress cd4 in CD8 cytotoxic T cells. Despite normal CD4 expression before selection, thymocytes lacking this intronic element failed to maintain CD4 expression following positive selection and were redirected to the CD8 lineage after MHC class II–restricted selection. We find that this regulatory element is key for propagating TET enzyme–mediated locus demethylation in CD4 T cells, a process that is likely initiated by another cis-element controlling CD4 expression at the double positive stage, before lineage commitment. This locus demethylation event is critical in ensuring heritable lineage identity and disruption of this process results in unstable CD4 expression and altered gene expression profiles. Cis and trans element interactions and molecular mechanisms governing heritable expression in response to distinct signals that dictate lineage ‘choice’ during T cell development will be presented. Citation Format: Priya Issuree, Kenny Day, Dan R. Littman. Epigenetic regulation of the Cd4 locus [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A073.

Targeted loss of SHP1 in murine thymocytes dampens TCR signaling late in selection.

SHP1 is a tyrosine phosphatase critical to proximal regulation of TCR signaling. Here, analysis of CD4-Cre SHP1(fl/fl) conditional knockout thymocytes using CD53, TCRβ, CD69, CD4, and CD8α expression demonstrates the importance of SHP1 in the survival of post selection (CD53(+) ), single-positive thymocytes. Using Ca(2+) flux to assess the intensity of TCR signaling demonstrated that SHP1 dampens the signal strength of these same mature, postselection thymocytes. Consistent with its dampening effect, TCR signal strength was also probed functionally using peptides that can mediate selection of the OT-I TCR, to reveal increased negative selection mediated by lower-affinity ligand in the absence of SHP1. Our data show that SHP1 is required for the survival of mature thymocytes and the generation of the functional T-cell repertoire, as its absence leads to a reduction in the numbers of CD4(+) and CD8(+) naïve T cells in the peripheral lymphoid compartments.

A silencer-proximal intronic region is required for sustained CD4 expression in postselection thymocytes.

It has been proposed that differential kinetics of CD4/CD8 coreceptors regulate fate choice of selected thymocytes. Sustained signals by interaction between MHC class II and TCR/CD4 is required for commitment to the CD4 helper lineage. Although prematurely terminated MHC-TCR/CD4 interaction in transgenic mouse models results in lineage redirection, it is unclear whether CD4 expression is actively maintained by endogenous cis-elements to facilitate prolonged signaling under physiological conditions. In this article, we show that sustained CD4 expression in postselection thymocytes requires an intronic sequence containing an uncharacterized DNase I hypersensitivity (DHS) site located 3' to the silencer. Despite normal CD4 expression before selection, thymocytes lacking a 1.5-kb sequence in intron 1 including the 0.4-kb silencer and the DHS, but not the 0.4-kb silencer alone, failed to maintain CD4 expression upon positive selection and are redirected to the CD8 lineage after MHC class II-restricted selection. Furthermore, CpG dinucleotides adjacent to the DHS are hypermethylated in CD8(+) T cells. These results indicate that the 1.5-kb cis-element is required in postselection thymocytes for helper lineage commitment, presumably mediating the maintenance of CD4 expression, and suggest that inactivation of the cis-element by DNA methylation may contribute to epigenetic Cd4 silencing.

Control of Thymic T Cell Maturation, Deletion and Egress by the RNA-Binding Protein HuR

HuR emerged as a posttranscriptional regulator of mRNAs involved in cellular control, stress, and immunity but its role in governing such responses remains elusive. In this study, we assessed HuR's role in the staged progression of thymic T cell differentiation by means of its genetic ablation. Mice with an early deletion of HuR in thymocytes possess enlarged thymi but display a substantial loss of peripheral T cells. We show that this discordant phenotype related to specific defects in thymic cellular processes, which demonstrated HuR's involvement in: 1) intrinsic checkpoint signals suppressing the cell cycle of immature thymocyte progenitors, 2) TCR and antigenic signals promoting the activation and positive selection of mature thymocytes, 3) antigenic and death-receptor signals promoting thymocyte deletion, and 4) chemokine signals driving the egress of postselection thymocytes to the periphery. The cellular consequences of HuR's dysfunction were underlined by the aberrant expression of selective cell cycle regulators, TCR, and death-receptor signaling components. Our studies reveal the signal-dependent context of HuR's cellular activities in thymocytes and its importance in the generation of a physiological T cell pool.

Antagonistic interplay between ThPOK and Runx in lineage choice of thymocytes

Differentiation of CD4 +CD8 + double-positive (DP) thymocytes into either CD4 +-helper or CD8 +-cytotoxic lineages involves several phases. It has been suggested that, following initial specification to one of the lineages by a set of lineage-specific genes during positive selection, stable cell identity is established during the commitment process by eliminating differentiation potential toward the other lineage. While the Runx3 transcription factor fixes the Cd4 gene into a silenced state during cytotoxic-lineage cell differentiation, the ThPOK transcription factor is both necessary and sufficient to generate a CD4 +CD8 − phenotype in post-selection thymocytes, regardless of the MHC specificity of the TCRs. Recent studies have revealed that a reciprocal antagonistic interplay between Runx3 and ThPOK is a central component in the transcription factor network governing the helper versus cytotoxic-lineage decision.

Dying for That Touch of MINK?

During development in the thymus, T cells with T cell receptors (TCRs) that bind major histocompatibility complex (MHC)-self peptide complexes with low affinity (which recognize foreign antigens) survive, and T cells with TCRs that bind MHC-self peptide complexes with high affinity (which recognize self-antigens) are eliminated. The survival pathways activated by low-affinity binding depend on activation of extracellular signal-regulated kinase (ERK), whereas the early steps through which high-affinity binding leads to apoptosis during "negative selection" are less well understood (see Palmer). McCarty et al. performed serial analysis of genes expressed in developing mouse thymocytes and found that expression of the misshapen-Nck-interacting kinase (NIK)-related kinase (MINK, a MAPKKKK) increased early in thymocyte development and then decreased after selection took place. Mice in which thymocyte MINK was knocked down through RNA interference showed increased numbers of postselection thymocytes; moreover, the deletion of autoreactive thymocytes was impaired. Western analysis combined with immunoprecipitation indicated that TCR activation in vitro promoted the association of MINK with Nck and CD3ϵ; moreover, MINK transfection or T cell activation in MINK-containing thymocytes promoted JNK (c-Jun N-terminal kinase) activation (which has been associated with TCR-dependent apoptosis). Similarly, administration of a negative selection peptide ligand in vivo promoted association of MINK, Nck, and CD3ϵ, whereas MINK was required for activation of JNK and an increase in the concentration of the proapoptotic BimEL downstream of the TCR. Thus, MINK appears to play a critical role in connecting TCR activation to apoptotic pathways in negative selection. N. McCarty, S. Paust, K. Ikizawa, I. Dan, X. Li, H. Cantor, Signaling by the MINK is essential in the negative selection of autoreactive thymocytes. Nat. Immunol. 6 , 65-72 (2005). [PubMed] E. Palmer, Signaling negative selection: Is MINK the missing link? Nat. Immunol. 6 , 9-10 (2005). [PubMed]

Thymic selection and peripheral activation of CD8 T cells by the same class I MHC/peptide complex.

Thymic selection is controlled by the interaction between TCR and MHC/peptide. Strength and quality of the signal determine whether thymocytes are selected or deleted. The factors that contribute to this signal remain poorly defined. Here we show that fetal thymic organ cultures (FTOCs) derived from OT-I transgenic mice (the OT-I TCR is restricted by K(b)-SIINFEKL) on a K(b)D(b-/-) background support positive selection, but only when provided with soluble H-2K(b)-SIINFEKL complexes. Selection of CD8 T cells is independent of the valency of the ligand or its capability to coengage CD8 molecules. Both CD8alphaalpha and CD8alphabeta T cells are selected by H-2K(b)-SIINFEKL, but only CD8alphabeta cells are capable of releasing IFN-gamma in response to the same ligand. The alpha(4)beta(7) integrin is up-regulated on postselection thymocytes from FTOCs. After adoptive transfer, FTOC-derived OT-I CD8 T cells divide in response to the agonist peptide SIINFEKL. These results establish that CD8 T cells responsive to their nominal peptide-Ag can be generated in FTOC supplemented with soluble MHC class I molecules equipped with the same peptide.

The majority of postselection CD4+ single-positive thymocytes requires the thymus to produce long-lived, functional T cells.

We have previously isolated, and characterized in vitro, two subsets of CD4hi T cell receptor (TCR)hi single positive (SP) thymocytes: CD8- and CD8lo. In this report, we have analyzed phenotypic, functional, and developmental characteristics of these "late" CD4hi SP thymocyte subsets. The TCRhi phenotype and the elimination of T cells expressing TCR V beta segments reactive with endogenous mouse mammary tumor virus (MMTV) products suggested that both subsets had undergone positive and negative selection. CD8-4hi thymocytes were functional, as judged by their ability to: (a) induce lethal graft versus host disease (GVHD); (b) survive and expand in peripheral lymphoid organs; and (c) proliferate, rather than undergo apoptosis, in response to in vitro TCR cross-linking. By contrast, CD8lo4hi cells could not induce GVHD, were unable to expand (and perhaps even survive) in peripheral organs and underwent apoptosis upon TCR cross-linking. However, when reintroduced into the thymus, these cells matured into functional, long-lived CD8-4hi lymphocytes. These results document an obligatory requirement for the thymic microenvironment in the final maturation of the majority of CD4hi SP postselection thymocytes, and demonstrate the existence of a previously unrecognized control point in T cell development.