Evidence for distinct CD4 silencer functions at different stages of thymocyte differentiation

An intronic silencer within the CD4 gene is the critical cis regulatory element for T cell subset-specific expression of CD4. We have combined transfection studies with gene targeting in mice to identify several key sequences within the silencer core that are required for gene silencing during thymocyte development. In mice, mutations in individual sites resulted in variegated, but heritable, derepression of CD4 in mature CD8(+) T lymphocytes, whereas compound mutations resulted in full derepression. These results indicate that there is partial redundancy in recruiting a chromatin remodeling machinery that results in epigenetic silencing. Mutations in single sites also resulted in partial derepression of CD4 in immature double-negative thymocytes, but there was no apparent variegation. These findings suggest two distinct modes of CD4 silencer function at different developmental stages: active repression in CD4(-)CD8(-) thymocytes, in which silencing must be reversible, and epigenetic gene silencing upon differentiation to the CD8(+) cytotoxic T cell lineage.     

Distinct BMI-1 and EZH2 expression patterns in thymocytes and mature T cells suggest a role for Polycomb genes in human T cell differentiation

BMI-1 and EZH2 Polycomb-group (PcG) proteins belong to two distinct protein complexes involved in the regulation of hematopoiesis. Using unique PcG-specific antisera and triple immunofluorescence, we found that mature resting peripheral T cells expressed BMI-1, whereas dividing blasts were EZH2(+). By contrast, subcapsular immature double-negative (DN) (CD4(-)/CD8(-)) T cells in the thymus coexpressed BMI-1 and EZH2 or were BMI-1 single positive. Their descendants, double-positive (DP; CD4(+)/CD8(+)) cortical thymocytes, expressed EZH2 without BMI-1. Most EZH2(+) DN and DP thymocytes were dividing, while DN BMI-1(+)/EZH2(-) thymocytes were resting and proliferation was occasionally noted in DN BMI-1(+)/EZH2(+) cells. Maturation of DP cortical thymocytes to single-positive (CD4(+)/CD8(-) or CD8(+)/CD4(-)) medullar thymocytes correlated with decreased detectability of EZH2 and continued relative absence of BMI-1. Our data show that BMI-1 and EZH2 expression in mature peripheral T cells is mutually exclusive and linked to proliferation status, and that this pattern is not yet established in thymocytes of the cortex and medulla. T cell stage-specific PcG expression profiles suggest that PcG genes contribute to regulation of T cell differentiation. They probably reflect stabilization of cell type-specific gene expression and irreversibility of lineage choice. The difference in PcG expression between medullar thymocytes and mature interfollicular T cells indicates that additional maturation processes occur after thymocyte transportation from the thymus.     

The transient expression of C-C chemokine receptor 8 in thymus identifies a thymocyte subset committed to become CD4+ single-positive T cells

Developing T cells journey through the different thymic microenvironments while receiving signals that eventually will allow some of them to become mature naive T cells exported to the periphery. This maturation can be visualized by the phenotype of the developing cells. CCR8 is a ss-chemokine receptor preferentially expressed in the thymus. We have developed 8F4, an anti-mouse CCR8 mAb that is able to neutralize the ligand-induced activation of CCR8, and used it to characterize the CCR8 protein expression in the different thymocyte subsets. Taking into account the intrathymic lineage relationships, our data showed that CCR8 expression in thymus followed two transient waves along T cell maturation. The first one took place in CD4(-) CD8(-) double-negative thymocytes, which showed a low CCR8 expression, and the second wave occurred after TCR activation by the Ag-dependent positive selection in CD4(+) CD8(+) double-positive cells. From that maturation stage, CCR8 expression gradually increased as the CD4(+) cell differentiation proceeded, reaching a maximum at the CD4(+) CD8(-) single-positive stage. These CD4(+) cells expressing CCR8 were also CD69(high) CD62L(low) thymocytes, suggesting that they still needed to undergo some differentiation step before becoming functionally competent naive T cells ready to be exported from the thymus. Interestingly, no significant amounts of CCR8 protein were detectable in CD4(-) CD8(+) thymocytes. Our data showing a clear regulation of the CCR8 protein in thymus suggest a relevant role for CCR8 in this lymphoid organ, and identify CCR8 as a possible marker of thymocyte subsets recently committed to the CD4(+) lineage.     

Bone morphogenetic protein 2/4 signaling regulates early thymocyte differentiation

Bone morphogenetic protein (BMP)2 and BMP4 are involved in the development of many tissues. In this study, we show that BMP2/4 signaling is involved in thymocyte development. Our data suggest that termination of BMP2/4 signaling is necessary for differentiation of CD44(+)CD25(-)CD4(-)CD8(-) double negative (DN) cells along the T cell lineage. BMP2 and BMP4 are produced by the thymic stroma and the requisite BMP receptor molecules (BMPR-1A, BMPR-1B, BMPR-II), and signal transduction molecules (Smad-1, -5, -8, and -4) are expressed by DN thymocytes. BMP4 inhibits thymocyte proliferation, enhances thymocyte survival, and arrests thymocyte differentiation at the CD44(+)CD25(-) DN stage, before T cell lineage commitment. Neutralization of endogenous BMP2 and BMP4 by treatment with the antagonist Noggin promotes and accelerates thymocyte differentiation, increasing the expression of CD2 and the proportion of CD44(-)CD25(-) DN cells and CD4(+)CD8(+) double-positive cells. Our study suggests that the BMP2/4 pathway may function in thymic homeostasis by regulating T cell lineage commitment and differentiation.     

CD83 expression influences CD4+ T cell development in the thymus

T lymphocyte selection and lineage commitment in the thymus requires multiple signals. Herein, CD4+ T cell generation required engagement of CD83, a surface molecule expressed by thymic epithelial and dendritic cells. CD83-deficient (CD83-/-) mice had a specific block in CD4+ single-positive thymocyte development without increased CD4+CD8+ double- or CD8+ single-positive thymocytes. This resulted in a selective 75%-90% reduction in peripheral CD4+ T cells, predominantly within the naive subset. Wild-type thymocytes and bone marrow stem cells failed to differentiate into mature CD4+ T cells when transferred into CD83-/- mice, while CD83-/- thymocytes and stem cells developed normally in wild-type mice. Thereby, CD83 expression represents an additional regulatory component for CD4+ T cell development in the thymus.     

Distinct mechanisms contribute to generate and change the CD4:CD8 cell ratio during thymus development: a role for the Notch ligand, Jagged1

In adult life, the high CD4:CD8 cell ratio observed in peripheral lymphoid organs originates in the thymus. Our results show that the low peripheral CD4:CD8 cell ratio seen during fetal life also has an intrathymic origin. This distinct production of CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes is regulated by the developmental age of the thymic stroma. The differential expression of Notch receptors and their ligands, especially Jagged1, throughout thymus development plays a key role in the generation of the different CD4:CD8 cell ratios. We also show that the intrathymic CD4:CD8 cell ratio sharply changes from fetal to adult values around birth. Differences in the proliferation and emigration rates of the mature thymocyte subsets contribute to this change.     

Phosphatidylinositol 3-kinase regulates the CD4/CD8 T cell differentiation ratio

The signaling pathways that control T cell differentiation have only begun to be elucidated. Using T cell lines, it has been shown that class IA phosphatidylinositol 3-kinase (PI3K), a heterodimer composed of a p85 regulatory and a p110 catalytic subunit, is activated after TCR stimulation. Nonetheless, the contribution of p85/p110 PI3K isoforms in T cell development has not been described. Mice deficient in the other family of class I PI3K, p110gamma, which is regulated by G protein-coupled receptors, exhibit reduced thymus size. Here we examine T cell development in p110gamma-deficient mice and in mice expressing an activating mutation of the p85 regulatory subunit, p65(PI3K), in T cells. We show that p110gamma-deficient mice have a partial defect in pre-TCR-dependent differentiation, which is restored after expression of the p65(PI3K) activating mutation. Genetic alteration of both PI3K isoforms also affects positive selection; p110gamma deletion decreased and p65(PI3K) expression augmented the CD4(+)/CD8(+) differentiation ratio. Finally, data are presented showing that both PI3K isoforms influenced mature thymocyte migration to the periphery. These observations underscore the contribution of PI3K in T cell development, as well as its implication in determining the CD4(+)/CD8(+) T cell differentiation ratio in vivo.     

Inefficient cell spreading and cytoskeletal polarization by CD4+CD8+ thymocytes: regulation by the thymic environment

CD4(+)CD8(+) double-positive (DP) thymocytes express a lower level of surface TCR than do mature T cells or single-positive (SP) thymocytes. Regulation of the TCR on DP thymocytes appears to result from intrathymic signaling, as in vitro culture of these cells results in spontaneous TCR up-regulation. In this study, we examined cell spreading and cytoskeletal polarization responses that have been shown to occur in response to TCR engagement in mature T cells. Using DP thymocytes stimulated on lipid bilayers or nontransgenic thymocytes added to anti-CD3-coated surfaces, we found that cell spreading and polarization of the microtubule organizing center and the actin cytoskeleton were inefficient in freshly isolated DP thymocytes, but were dramatically enhanced after overnight culture. SP (CD4(+)) thymocytes showed efficient responses to TCR engagement, suggesting that releasing DP thymocytes from the thymic environment mimics some aspects of positive selection. The poor translation of a TCR signal to cytoskeletal responses could limit the ability of DP thymocytes to form stable contacts with APCs and may thereby regulate thymocyte selection during T cell development.     

Cutting Edge: A possible role for CD4+ thymic macrophages as professional scavengers of apoptotic thymocytes

A vast majority of thymocytes are eliminated during T cell development by apoptosis. However, apoptotic thymocytes are not usually found in the thymus, indicating that apoptotic thymocytes must be eliminated rapidly by scavengers. Although macrophages and dendritic cells are believed to play such role, little is known about scavengers in the thymus. We found that CD4(+)/CD11b(+)/CD11c(-) cells were present in the thymus and that they expressed costimulatory molecules for T cell selection and possessed Ag-presenting activity. Moreover, these CD4(+)/CD11b(+) cells phagocytosed apoptotic thymocytes much more efficiently than thymic CD4(-)/CD11b(+) cells as well as activated peritoneal macrophages. CD4(+)/CD11b(+) cells became larger along with thymus development, while no such change was observed in CD4(-)/CD11b(+) cells. Finally, engulfed nuclei were frequently found in CD4(+)/CD11b(+) cells. These results strongly suggest that thymic CD4(+)/CD11b(+) cells are major scavengers of apoptotic thymocytes.     

The role of LFA-1/ICAM-1 interactions during murine T lymphocyte development.

We have examined the expression and function of the cell adhesion molecules LFA-1 (CD11a/CD18), ICAM-1 (CD54), and ICAM-2 in murine fetal thymic ontogeny and in the adult thymus. On fetal days 14 and 15, 40 to 50% of thymocytes coexpress high levels of LFA-1 and ICAM-1, as determined by flow cytometry. By day 16, more than 90% of fetal thymocytes are LFA-1+ ICAM-1hi, and all IL-2R+ cells are located in this population. Although LFA-1 expression remains unchanged thereafter, ICAM-1 expression appears to be differentially regulated in different thymocyte subpopulations, with CD4+8+ cells being ICAM-1lo and CD4-8- thymocytes remaining ICAM-1hi. ICAM-2 surface expression is dull on both fetal and adult thymocytes. Surprisingly, the expression of ICAM-1 is differentially up-regulated on T cells having a mature phenotype in thymus and in peripheral lymphoid organs, with CD8+ T cells bearing the highest amount of surface ICAM-1. Addition of anti-ICAM-1 or anti-LFA-1 antibodies to fetal thymic organ cultures results in the impaired generation of CD4+8+ cells. These results indicate that LFA-1/ICAM-1 interactions facilitate murine thymic development and suggest that cell adhesion molecules mediate important events in T cell differentiation.     

Targeting CD4 coreceptor expression to postselection thymocytes reveals that CD4/CD8 lineage choice is neither error-prone nor stochastic

The mechanism by which CD4/CD8 lineage choice is coordinated with TCR specificity during positive selection remains an unresolved problem in immunology. The stochastic/selection model proposes that CD4/CD8 lineage choice in TCR-signaled CD4(+)CD8(+) thymocytes occurs randomly and therefore is highly error-prone. This perspective is strongly supported by "coreceptor rescue" experiments in which transgenic CD4 coreceptors were ectopically expressed on thymocytes throughout their development and caused significant numbers of cells bearing MHC-II-specific TCR to differentiate into mature, CD8 lineage T cells. However, it is not known if forced coreceptor expression actually rescued positively selected thymocytes making an incorrect lineage choice or if it influenced developing thymocytes into making an incorrect lineage choice. We have now reassessed coreceptor rescue and the concept that lineage choice is highly error-prone with a novel CD4 transgene (referred to as E8(I)-CD4) that targets expression of transgenic CD4 coreceptors specifically to thymocytes that have already undergone positive selection and adopted a CD8 lineage fate. Unlike previous CD4 transgenes, the E8(I)-CD4 transgene has no effect on early thymocyte development and cannot itself influence CD4/CD8 lineage choice. We report that the E8(I)-CD4 transgene did in fact induce expression of functional CD4 coreceptor proteins on newly arising CD8 lineage thymocytes precisely at the point in thymic development that transgenic CD4 coreceptors would putatively rescue MHC-II-specific thymocytes that incorrectly adopted the CD8 lineage. However, the E8(I)-CD4 transgene did not reveal any MHC-II-selected thymocytes that adopted the CD8 lineage fate. These results demonstrate that CD4/CD8 lineage choice is neither error-prone nor stochastic.