Ikaros null mice display defects in T cell selection and CD4 versus CD8 lineage decisions

Previous evidence suggested that the hemopoietic-specific nuclear factor Ikaros regulates TCR signaling thresholds in mature T cells. In this study, we test the hypothesis that Ikaros also sets TCR signaling thresholds to regulate selection events and CD4 vs CD8 lineage determination in developing thymocytes. Ikaros null mice were crossed to three lines of TCR-transgenic mice, and positive selection, negative selection, and CD4 vs CD8 lineage decisions were analyzed. Mice expressing a polyclonal repertoire or a MHC class II-restricted TCR transgene exhibited enhanced positive selection toward the CD4 lineage. Moreover, in the absence of Ikaros, CD4 development can occur with decreased thresholds of TCR signaling. In addition, CD4 single-positive thymocytes were detected in MHC class I-restricted TCR-transgenic Ikaros null mice. To assess the role of Ikaros in negative selection, we analyzed deletion of T cells induced by conventional Ag or by endogenous superantigen. Surprisingly, negative selection was impaired in Ikaros null thymocytes despite evidence of high levels of TCR signal and no intrinsic defect in apoptosis ex vivo. To our knowledge, these data identify Ikaros as the first nuclear factor that plays a critical role in regulating negative selection as well as CD4 vs CD8 lineage decisions during positive selection.     

Polarized development of memory cell-like IFN-gamma-producing cells in the absence of TCR zeta-chain

TCR/CD3 complex-mediated signals play critical roles in regulating CD4(+) Th cell differentiation. In this report, we have examined the in vivo role of a key TCR/CD3 complex molecule zeta-chain in regulating the differentiation of Th cells. We have studied T cells from zeta-chain-deficient mice (zetaKO mice), zeta-chain-bearing mice (zeta(+) mice), and from zetaKO mice expressing a FcRgamma chain transgene (FcRgammaTG, zetaKO mice). Our results demonstrated that, compared with those of control mice, CD4(+) T cells and not CD8(+) T cells from zetaKO mice were polarized into IFN-gamma-producing cells. Some of these IFN-gamma-producing cells could also secrete IL-10. Interestingly, zetaKO mouse T cells produced IFN-gamma even after they were cultured in a Th2 condition. Our studies to determine the molecular mechanisms underlying the polarized IFN-gamma production revealed that the expression level of STAT4 and T-bet were up-regulated in freshly isolated T cells from zetaKO mice. Further studies showed that noncultured zetaKO mice CD4(+) T cells and thymocytes bore a unique memory cell-like CD44(high), CD62L(low/neg) phenotype. Altogether, these results suggest that, in the absence of the zeta-chain, CD4(+) T cells develop as polarized IFN-gamma-producing cells that bear a memory cell-like phenotype. The zeta-chain-bearing T cells may produce a large amount of IFN-gamma only after they are cultured in a condition favoring Th1 cell differentiation. This study may provide important implications for the down-regulation of zeta-chain in T cells of patients bearing a variety of tumors, chronic inflammatory and infectious diseases.     

CCR7 expression in developing thymocytes is linked to the CD4 versus CD8 lineage decision

During thymic development, T cell progenitors undergo positive selection based on the ability of their T cell Ag receptors (TCR) to bind MHC ligands on thymic epithelial cells. Positive selection determines T cell fate, in that thymocytes whose TCR bind MHC class I (MHC-I) develop as CD8-lineage T cells, whereas those that bind MHC class II (MHC-II) develop as CD4 T cells. Positive selection also induces migration from the cortex to the medulla driven by the chemokine receptor CCR7. In this study, we show that CCR7 is up-regulated in a larger proportion of CD4(+)CD8(+) thymocytes undergoing positive selection on MHC-I compared with MHC-II. Mice bearing a mutation of Th-POK, a key CD4/CD8-lineage regulator, display increased expression of CCR7 among MHC-II-specific CD4(+)CD8(+) thymocytes. In addition, overexpression of CCR7 results in increased development of CD8 T cells bearing MHC-II-specific TCR. These findings suggest that the timing of CCR7 expression relative to coreceptor down-regulation is regulated by lineage commitment signals.     

Coreceptor signal strength regulates positive selection but does not determine CD4/CD8 lineage choice in a physiologic in vivo model

TCR signals drive thymocyte development, but it remains controversial what impact, if any, the intensity of those signals have on T cell differentiation in the thymus. In this study, we assess the impact of CD8 coreceptor signal strength on positive selection and CD4/CD8 lineage choice using novel gene knockin mice in which the endogenous CD8alpha gene has been re-engineered to encode the stronger signaling cytoplasmic tail of CD4, with the re-engineered CD8alpha gene referred to as CD8.4. We found that stronger signaling CD8.4 coreceptors specifically improved the efficiency of CD8-dependent positive selection and quantitatively increased the number of MHC class I (MHC-I)-specific thymocytes signaled to differentiate into CD8+ T cells, even for thymocytes expressing a single, transgenic TCR. Importantly, however, stronger signaling CD8.4 coreceptors did not alter the CD8 lineage choice of any MHC-I-specific thymocytes, even MHC-I-specific thymocytes expressing the high-affinity F5 transgenic TCR. This study documents in a physiologic in vivo model that coreceptor signal strength alters TCR-signaling thresholds for positive selection and so is a major determinant of the CD4:CD8 ratio, but it does not influence CD4/CD8 lineage choice.     

Fas modulates both positive and negative selection of thymocytes.

We studied the functional role of Fas (CD95) in thymic T cell development using the TCR transgenic mice homozygous for the lpr mutation, DO10 lpr/lpr mice. In DO10 lpr/lpr mice, the differentiation of CD4(+)CD8(+) double-positive (DP) thymocytes to CD4(+) single-positive (SP) thymocytes was markedly impaired, as indicated by decreased generation of CD4(+) SP thymocytes and reduced ratio of CD4(+) SP thymocytes to DP thymocytes in lpr/lpr mice compared with those of +/+ mice. Activation of DP thymocytes in the process of positive selection was also significantly inhibited in DO10 lpr/lpr mice, as shown by the lower levels of CD69 expression on DP thymocytes in lpr/lpr mice compared to +/+ mice. Furthermore, the deletion of DP thymocytes induced by in vivo administration of OVA peptide (up to 150 micrograms) and anti-TCR clonotype mAb did not occur in DO10 lpr/lpr mice, whereas these treatments significantly decreased DP thymocytes in DO10 +/+ mice. On the other hand, no significant difference in DO10 transgenic TCR expression on DP thymocytes was found between DO10 lpr/lpr and +/+ mice. Together, these results indicate that Fas is importantly involved in both positive and negative selection of thymocytes.     

Selective reduction of post-selection CD8 thymocyte proliferation in IL-15Rα deficient mice

Peripheral CD8(+) T cells are defective in both IL-15 and IL-15Rα knock-out (KO) mice; however, whether IL-15/IL-15Rα deficiency has a similar effect on CD8 single-positive (SP) thymocytes remains unclear. In this study, we investigated whether the absence of IL-15 transpresentation in IL-15Rα KO mice results in a defect in thymic CD8 single positive (SP) TCR(hi) thymocytes. Comparison of CD8SP TCR(hi) thymocytes from IL-15Rα KO mice with their wild type (WT) counterparts by flow cytometry showed a significant reduction in the percentage of CD69(-) CD8SP TCR(hi) thymocytes, which represent thymic premigrants. In addition, analysis of in vivo 5-bromo-2-deoxyuridine (BrdU) incorporation demonstrated that premigrant expansion of CD8SP TCR(hi) thymocytes was reduced in IL-15Rα KO mice. The presence of IL-15 transpresentation-dependent expansion in CD8SP TCR(hi) thymocytes was assessed by culturing total thymocytes in IL-15Rα-Fc fusion protein-pre-bound plates that were pre-incubated with IL-15 to mimic IL-15 transpresentation in vitro. The results demonstrated that CD8SP thymocytes selectively outgrew other thymic subsets. The contribution of the newly divided CD8SP thymocytes to the peripheral CD8(+) T cell pool was examined using double labeling with intrathymically injected FITC and intravenously injected BrdU. A marked decrease in FITC(+) BrdU(+) CD8(+) T cells was observed in the IL-15Rα KO lymph nodes. Through these experiments, we identified an IL-15 transpresentation-dependent proliferation process selective for the mature CD8SP premigrant subpopulation. Importantly, this process may contribute to the maintenance of the normal peripheral CD8(+) T cell pool.     

The regulated expression of a diverse set of genes during thymocyte positive selection in vivo

A signal initiated by the newly formed Ag receptor is integrated with microenvironmental cues during T cell development to ensure positive selection of CD4+CD8+ progenitors into functionally mature CD4+ or CD8+ T lymphocytes. During this transition, a survival program is initiated, TCR gene recombination ceases, cells migrate into a new thymic microenvironment, the responsiveness of the Ag receptor is tuned, and the cells commit to a specific T lineage. To determine potential regulators of these processes, we used mRNA microarray analysis to compare gene expression changes in CD4+CD8+ thymocytes from TCR transgenic mice that have received a TCR selection signal with those that had not received a signal. We found 129 genes with expression that changed significantly during positive selection, the majority of which were not previously appreciated. A large number of these changes were confirmed by real-time PCR or flow cytometry. We have combined our findings with gene changes reported in the literature to provide a comprehensive report of the genes regulated during positive selection, and we attempted to assign these genes to positive selection process categories.     

MEK activity regulates negative selection of immature CD4+CD8+ thymocytes

CD4+CD8+ thymocytes are either positively selected and subsequently mature to CD4 single positive (SP) or CD8 SP T cells, or they die by apoptosis due to neglect or negative selection. This clonal selection is essential for establishing a functional self-restricted T cell repertoire. Intracellular signals through the three known mitogen-activated protein (MAP) kinase pathways have been shown to selectively guide positive or negative selection. Whereas the c-Jun N-terminal kinase and p38 MAP kinase regulate negative selection of thymocytes, the extracellular signal-regulated kinase (ERK) pathway is required for positive selection and T cell lineage commitment. In this paper, we show that the MAP/ERK kinase (MEK)-ERK pathway is also involved in negative selection. Thymocytes from newborn TCR transgenic mice were cultured with TCR/CD3epsilon-specific Abs or TCR-specific agonist peptides to induce negative selection. In the presence of the MEK-specific pharmacological inhibitors PD98059 or UO126, cell recovery was enhanced and deletion of DP thymocytes was drastically reduced. Furthermore, development of CD4 SP T cells was blocked, but differentiation of mature CD8 SP T cells proceeded in the presence of agonist peptides when MEK activity was blocked. Thus, our data indicate that the outcome between positively and negatively selecting signals is critically dependent on MEK activity.     

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.     

Involvement of p21ras distinguishes positive and negative selection in thymocytes.

Small molecular weight GTP binding proteins of the ras family have been implicated in signal transduction from the T cell antigen receptor (TCR). To test the importance of p21ras in the control of thymocyte development, we generated mice expressing a dominant-negative p21ras protein (H-rasN17) in T lineage cells under the control of the lck proximal promoter. Proliferation of thymocytes from lck-H-rasN17 mice in response to TCR stimulation was nearly completely blocked, confirming the importance of p21ras in mediating TCR-derived signals in mature CD4+8- or CD8+4- thymocytes. In contrast, some TCR-derived signals proceeded unimpaired in the CD4+8+ thymocytes of mice expressing dominant-negative p21ras. Analysis of thymocyte development in mice made doubly transgenic for the H-Y-specific TCR and lck-H-rasN17 demonstrated that antigen-specific negative selection occurs normally in the presence of p21H-rasN17. Superantigen-induced negative selection in vivo also proceeded unhindered in H-rasN17 thymocytes. In contrast, positive selection of thymocytes in the H-Y mice was severely compromised by the presence of p21H-rasN17. These observations demonstrate that positive and negative selection, two conceptually antithetical consequences of TCR stimulation, are biochemically distinguishable.     

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.     

Analyzing expression of perforin, Runx3, and Thpok genes during positive selection reveals activation of CD8-differentiation programs by MHC II-signaled thymocytes

Intrathymic positive selection matches CD4-CD8 lineage differentiation to MHC specificity. However, it is unclear whether MHC signals induce lineage choice or simply select thymocytes of the appropriate lineage. To investigate this issue, we assessed thymocytes undergoing positive selection for expression of the CD8 lineage markers perforin and Runx3. Using both population-based and single-cell RT-PCR analyses, we found large subsets of MHC class II (MHC-II)-signaled thymocytes expressing these genes within the CD4+ 8+ and CD4+ 8(int), but not the CD4+ 8- populations of signaling competent mice. This indicates that MHC-II signals normally fail to impose CD4 differentiation and further implies that the number of mature CD8 single-positive (SP) thymocytes greatly underestimates CD8 lineage choice. We next examined whether MHC-II-restricted CD4+ 8- thymocytes remain competent to initiate CD8 lineage gene expression. In mice in which expression of the tyrosine kinase Zap70 and thereby TCR signaling were impaired selectively in SP thymocytes, MHC-II-signaled CD4+ 8- thymocytes expressed perforin and Runx3 and failed to up-regulate the CD4 marker Thpok. This indicated that impairing TCR signals at the CD4 SP stage switched gene expression patterns from CD4- to CD8-lineage specific. We conclude from these findings that MHC-II-signaled thymocytes remain competent to initiate CD8-specific gene expression even after CD8 down-regulation and that CD4 lineage differentiation is not fixed before the CD4 SP stage.     

Interchangeability of Themis1 and Themis2 in thymocyte development reveals two related proteins with conserved molecular function

Themis1, a recently identified T cell protein, has a critical function in the generation of mature CD4(+)CD8(-) and CD4(-)CD8(+) (CD4 and CD8 single-positive [SP]) thymocytes and T cells. Although Themis1 has been shown to bind to the adaptor proteins LAT and Grb2, previous studies have yielded conflicting results regarding whether thymocytes from Themis1(-/-) mice exhibit TCR-mediated signaling defects. In this study, we demonstrate that, in the absence of Themis1, TCR-mediated signaling is selectively impaired in CD4 SP and CD8 SP thymocytes but is not affected in CD4(+)CD8(+) double-positive thymocytes despite high expression of Themis1 in double-positive thymocytes. Like Themis1, Themis2, a related member of the Themis family, which is expressed in B cells and macrophages, contains two conserved cysteine-based domains, a proline-rich region, and a nuclear localization signal. To determine whether Themis1 and Themis2 can perform similar functions in vivo, we analyzed T cell development and TCR-mediated signaling in Themis1(-/-) mice reconstituted with either Themis1 or Themis2 transgenes. Notably, Themis1 and Themis2 exhibited the same potential to restore T cell development and TCR-mediated signaling in Themis1(-/-) mice. Both proteins were tyrosine phosphorylated and were recruited within Grb2 signaling complexes to LAT following TCR engagement. These results suggest that conserved molecular features of the Themis1 and Themis2 proteins are important for their biological activity and predict that Themis1 and Themis2 may perform similar functions in T and B cells, respectively.     

RasGRP1 transmits prodifferentiation TCR signaling that is crucial for CD4 T cell development

TCR signaling plays a governing role in both the survival and differentiation of bipotent double-positive thymocytes into the CD4(+) and CD8(+) single-positive T cell lineages. A central mediator of this developmental program is the small GTPase Ras, emitting cytoplasmic signals through downstream MAPK pathways and eventually affecting gene expression. TCR signal transduction orchestrates the activation of Ras by integrating at least two Ras-guanyl nucleotide exchange factors, RasGRP1 and Sos. In this study, we have characterized the relationship between RasGRP1 function and its potential roles in promoting ERK activity, cell survival, maturation, and lineage commitment. Investigations on RasGRP1(-/-) mice expressing a transgenic (Tg) MHC class II-restricted TCR revealed that the development of CD4 T cells expressing this Tg TCR is completely dependent on RasGRP1. Unexpectedly, a small number of functional CD8 single-positive thymocytes expressing the Tg MHC class II-restricted TCR exists in mutant mice. In addition, RasGRP1(-/-) double-positive thymocytes exhibit marked deficits in TCR-stimulated up-regulation of the positive selection marker CD69 and the antiapoptotic protein Bcl-2, whereas CD5 induction is unaffected. To evaluate the role of RasGRP1 in providing cellular survival signaling, we enforced Bcl-2 expression in RasGRP1(-/-) thymocytes. These studies demonstrate that RasGRP1 function cannot be fully complemented by Tg Bcl-2 expression. Therefore, we propose that RasGRP1 transmits differentiation signaling critically required for CD4 T cell development.     

Modeling TCR signaling complex formation in positive selection

T cell receptor signaling in the thymus can result in positive selection, and hence progressive maturation to the CD4(+)8(-) or CD4(-)8(+) stage, or induction of apoptosis by negative selection. Although it is poorly understood how TCR ligation at the CD4(+)8(+) stage can lead to such different cell fates, it is thought that the strength of signal may play a role in determining the outcome of TCR signaling. In this study, we have characterized the formation of an active signaling complex in thymocytes undergoing positive selection as a result of interaction with thymic epithelial cells. Although this signaling complex involves redistribution of cell surface and intracellular molecules, reminiscent of that observed in T cell activation, accumulation of GM1-containing lipid rafts was not observed. However, enforced expression of the costimulatory molecule CD80 on thymic epithelium induced GM1 polarization in thymocytes, and was accompanied by reduced positive selection and increased apoptosis. We suggest that the presence or absence of CD80 costimulation influences the outcome of TCR signaling in CD4(+)8(+) thymocytes through differential lipid raft recruitment, thus determining overall signal strength and influencing developmental cell fate.