Stage-dependent reactivity of thymocytes to self-peptide--MHC complexes.
ABSTRACT: In mice that express a transgene for the 2C T cell antigen-receptor (TCR) and lack a recombinase-activating gene (2C(+)RAG(-/-) mice) most of the peripheral T cells are CD8(+), a few are CD4(+), and a significant fraction are CD4(-)CD8(-) [double negative (DN)]. The DN 2C cells, like DN T cells that are abundant in various other alphabeta TCR-transgenic mice, appear to be derived directly from DN thymocytes that prematurely express the TCR transgene. The DN 2C cells are virtually absent in mice deficient in major histocompatibility complex class II (MHC-II) but more abundant in mice deficient in MHC-I, suggesting that the DN 2C thymocytes are positively selected by self-peptide-MHC-II (pMHC-II) complexes and negatively selected by self-pMHC-I complexes. The pMHC-I complexes, however, positively select CD8(+) 2C T cells in the same mice. The different effects of thymic pMHC-I on DN and CD8(+) thymocytes are consistent with the finding that DN 2C thymocytes are more sensitive than more mature CD4(+)CD8(+) [double positive (DP)] thymocytes to a weak pMHC-I agonist for the 2C TCR. Together with previous evidence that DP thymocytes respond more sensitively than T cells in the periphery to weak pMHC agonists, the findings suggest progressive decreases in responsiveness to self-pMHC-I complexes as thymocytes develop from DN to DP thymocytes and then to mature naïve T cells in the periphery.
Project description:CD4(+)CD8(+) "double positive" (DP) thymocytes differentiate into diverse ?? T cell sub-types using mechanistically distinct programs. For example, conventional ?? T cells develop from DP cells after partial-agonist T cell receptor (TCR) interactions with self-peptide/MHC, whereas unconventional ?? T cells, such as TCR??(+)CD8??(+) intraepithelial lymphocytes (IELs), require full-agonist TCR interactions. Despite this, DP cells appear homogeneous, and it remains unclear how distinct TCR signalling instructs distinct developmental outcomes. Moreover, whether TCR signals at earlier stages of development, for example in CD4(-)CD8(-) double negative (DN) cells, impact on later fate decisions is presently unknown. Here, we assess four strains of mice that display altered TCR signal strength in DN cells, which correlates with altered generation of unconventional TCR??(+)CD8??(+) IELs. FVB/n mice (compared to C57BL/6 animals) and mice with altered preTCR? (pT?) expression, both displayed weaker TCR signalling in DN cells, an inefficient DN-to-DP transition, and reduced contribution of TCR??(+)CD8??(+) IELs to gut epithelium. Conversely, TCR??(+)CD8??(+) IEL development was favoured in mice with increased TCR signal strength in DN cells. Collectively, these data suggest TCR signal strength in DN cells directly impacts on subsequent DP cell differentiation, fundamentally altering the potential of thymocyte progenitors to adopt conventional versus unconventional T cell fates.
Project description:Developmental checkpoints in stem/progenitor cells are critical to the determination, commitment and differentiation into distinct lineages. Cancer cells often retain expression of lineage-specific checkpoint proteins, but their potential impact in cancer remains elusive. T lymphocytes mature in the thymus following a highly orchestrated developmental process that entails the successive rearrangements and expression of T-cell receptor (TCR) genes. Low affinity recognition of self-peptide/MHC complexes (self-pMHC) presented by thymic epithelial cells by the TCR of CD4+CD8+ (DP) cortical thymocytes transduces positive selection signals that ultimately shape the developing T cell repertoire. DP thymocytes not receiving these signals die by lack of stimulation whereas those that recognize self-pMHC with high affinity undergo TCR-mediated apoptosis and negative selection. In T-cell acute lymphoblastic leukaemia (T-ALL), leukaemic transformation of maturating thymocytes results from the acquisition of multiple genetic and epigenetic alterations in oncogenes and tumour suppressor genes, that disrupt the normal regulatory circuits and drive clonal expansion of differentiation-arrested lymphoblasts. We show here that TCR triggering by negatively-selecting self-pMHC prevented T-ALL development and leukaemia maintenance in mice. Induction of TCR signalling by high affinity self-pMHC or treatment with monoclonal antibodies to the CD3 signalling chain (anti-CD3) caused massive leukaemic cell death and a gene expression program resembling that of thymocyte negative selection. Importantly, anti-CD3 treatment hampered leukaemogenesis in mice transplanted with either mouse or patient-derived T-ALLs. These data provide a rationale for targeted therapy based on anti-CD3 treatment of T-ALL patients and demonstrate that endogenous developmental checkpoint proteins are amenable to therapeutic intervention in cancer cells. Gene expression data from four culture conditions was performed for the following cells: ALL-SIL-TCRÎ±/Î²-GFP co-cultured on OP9-DL1 for 48 h, ALL-SIL-TCRÎ±/Î²-GFP without co-culture, ALL-SIL cells transduced with TLX shRNA (sh-TLX), and ALL-SIL transduced with sh-control vectors. All conditions are performed in two replicates.
Project description:Genetic inactivation of Notch signaling in CD4(-)CD8(-) double-negative (DN) thymocytes was previously shown to impair T cell receptor (TCR) gene rearrangement and to cause a partial block in CD4(+)CD8(+) double-positive (DP) thymocyte development in mice. In contrast, in vitro cultures suggested that Notch was absolutely required for the generation of DP thymocytes independent of pre-TCR expression and activity. To resolve the respective role of Notch and the pre-TCR, we inhibited Notch-mediated transcriptional activation in vivo with a green fluorescent protein-tagged dominant-negative Mastermind-like 1 (DNMAML) that allowed us to track single cells incapable of Notch signaling. DNMAML expression in DN cells led to decreased production of DP thymocytes but only to a modest decrease in intracellular TCRbeta expression. DNMAML attenuated the pre-TCR-associated increase in cell size and CD27 expression. TCRbeta or TCRalphabeta transgenes failed to rescue DNMAML-related defects. Intrathymic injections of DNMAML(-) or DNMAML(+) DN thymocytes revealed a complete DN/DP transition block, with production of DNMAML(+) DP thymocytes only from cells undergoing late Notch inactivation. These findings indicate that the Notch requirement during the beta-selection checkpoint in vivo is absolute and independent of the pre-TCR, and it depends on transcriptional activation by Notch via the CSL/RBP-J-MAML complex.
Project description:The binding of oligomeric peptide-MHC (pMHC) complexes to cell surface TCR can be considered to approximate TCR-pMHC interactions at cell-cell interfaces. In this study, we analyzed the equilibrium binding of streptavidin-based pMHC oligomers (tetramers) and their dissociation kinetics from CD8(pos) T cells from 2C-TCR transgenic mice and from T cell hybridomas that expressed the 2C TCR or a high-affinity mutant (m33) of this TCR. Our results show that the tetramers did not come close to saturating cell-surface TCR (binding only 10-30% of cell-surface receptors), as is generally assumed in deriving affinity values (K(D)), in part because of dissociative losses from tetramer-stained cells. Guided by a kinetic model, the oligomer dissociation rate and equilibrium constants were seen to depend not only on monovalent association and dissociation rates (k(off) and k(on)), but also on a multivalent association rate (?) and TCR cell-surface density. Our results suggest that dissociation rates could account for the recently described surprisingly high frequency of tetramer-negative, functionally competent T cells in some T cell responses.
Project description:<h4>Background</h4>The binding of the T cell receptor (TCR) to major histocompatibility complex (MHC) molecules in the thymus determines fates of TCRalphabeta lymphocytes that subsequently home to secondary lymphoid tissue. TCR transgenic models have been used to study thymic selection and lineage commitment. Most TCR transgenic mice express the rearranged TCRalphabeta prematurely at the double negative stage and abnormal TCRalphabeta populations of T cells that are not easily detected in non-transgenic mice have been found in secondary lymphoid tissue of TCR transgenic mice.<h4>Methodology and principal findings</h4>To determine developmental pathways of TCR-transgenic thymocytes, we used Cre-LoxP-mediated fate mapping and show here that premature expression of a transgenic TCRalphabeta diverts some developing thymocytes to a developmental pathway which resembles that of gamma delta cells. We found that most peripheral T cells with the HY-TCR in male mice have bypassed the RORgammat-positive CD4(+)8(+) (double positive, DP) stage to accumulate either as CD4(-)8(-) (double negative, DN) or as CD8alpha(+) T cells in lymph nodes or gut epithelium. Likewise, DN TCRalphabeta cells in lymphoid tissue of female mice were not derived from DP thymocytes.<h4>Conclusion</h4>The results further support the hypothesis that the premature expression of the TCRalphabeta can divert DN thymocytes into gamma delta lineage cells.
Project description:Although natural killer T cells (NKT cells) are thought to be generated from CD4+CD8+ (DP) thymocytes, the developmental origin of CD4-CD8- (DN) NKT cells has remained unclear. In this study, we found the level of NK1.1 expression was highest in DN cells, followed by CD4 and CD8 (SP) and DP cells. The level of NK1.1 expression was highest in CD44+CD25- (DN1) cells, after that CD44+CD25+ (DN2), finally, CD44-CD25- (DN3) and CD44- CD25+ (DN4) cells. Unexpectedly, cytoplasmic CD3 was not only expressed in SP and DP thymocytes but also in most DN thymocytes at various stages. The mean fluorescence of cytoplasmic and surface CD3 in DN cells was significantly lower than in mature (SP) T and NKT cells in the thymus and spleen. Interestingly, there were more NKT cells in DN-cytoplasmic CD3 expression cells was higher than in DN-surface CD3 expression cells. There were more CD3-NKT cells in DN1 thymocytes than in TCR-β-NKT cells. NKT cells expressed higher levels of IL-7Rα which was correlated with CD44 expression in the thymus. Our data suggest that T cells and NKT cells follow similar patterns of expression with respect to cytoplasmic and surface CD3. Cytoplasmic CD3 could be used as a marker for early stage T cells. Both cytoplasmic CD3 and surface CD3 were expressed in mature T cells and immature T cells, including the immature cytoplasmic CD3+ surface CD3- and surface CD3+TCR-β- cells in DN1-NKT thymocytes. CD44 could be used as an additional marker of NKT cells which may originate from cytoplasmic CD3-positive DN thymocytes that express CD44 and IL-7Rα in mice.
Project description:CD4 and CD8 T cells are vital components of the immune system. We found that histone deacetylase 3 (HDAC3) is critical for the development of CD4 T cells, as HDAC3-deficient DP thymocytes generate only CD8SP thymocytes in mice. In the absence of HDAC3, MHC Class II-restricted OT-II thymocytes are redirected to the CD8 cytotoxic lineage, which occurs with accelerated kinetics. Analysis of histone acetylation and RNA-seq reveals that HDAC3-deficient DP thymocytes are biased towards the CD8 lineage prior to positive selection. Commitment to the CD4 or CD8 lineage is determined by whether persistent TCR signaling or cytokine signaling predominates, respectively. Despite elevated IL-21R/?c/STAT5 signaling in HDAC3-deficient DP thymocytes, blocking IL-21R does not restore CD4 lineage commitment. Instead, HDAC3 binds directly to CD8-lineage promoting genes. Thus, HDAC3 is required to restrain CD8-lineage genes in DP thymocytes for the generation of CD4 T cells.
Project description:T cell receptors (TCRs) recognize peptides presented by MHC molecules (pMHC) on an antigen-presenting cell (APC) to discriminate foreign from self-antigens and initiate adaptive immune responses. In addition, T cell activation generally requires binding of this same pMHC to a CD4 or CD8 co-receptor, resulting in assembly of a TCR-pMHC-CD4 or TCR-pMHC-CD8 complex and recruitment of Lck via its association with the co-receptor. Here we review structural and biophysical studies of CD4 and CD8 interactions with MHC molecules and TCR-pMHC complexes. Crystal structures have been determined of CD8?? and CD8?? in complex with MHC class I, of CD4 bound to MHC class II, and of a complete TCR-pMHC-CD4 ternary complex. Additionally, the binding of these co-receptors to pMHC and TCR-pMHC ligands has been investigated both in solution and in situ at the T cell-APC interface. Together, these studies have provided key insights into the role of CD4 and CD8 in T cell activation, and into how these co-receptors focus TCR on MHC to guide TCR docking on pMHC during thymic T cell selection.
Project description:The pre-T cell receptor (TCR) functions as a critical checkpoint during alphabeta T cell development. Signaling through the pre-TCR controls the differentiation of immature CD4(-)CD8(-)CD25(+)CD44(-) [double-negative (DN)3] thymocytes into CD4(+)CD8(+) double-positive (DP) cells through the CD4(-)CD8(-)CD25(-)CD44(-)(DN4) stage. In addition, pre-TCR activity triggers expansion and survival of thymocytes and inhibits TCRbeta gene rearrangement through a process referred to as allelic exclusion. Whereas many proteins involved in the pre-TCR transduction cascade have been identified, little is known about the nuclear factors associated with receptor function. Here, we use gene targeting to inactivate the Ets-1 transcription factor in mice and analyze pre-TCR function in developing Ets-1-deficient (Ets-1(-/-)) thymocytes. We find that inactivation of Ets-1 impairs the development of DN3 into DP thymocytes and induces an elevated rate of cell death in the DN4 subset. This defect appears specific to the alphabeta lineage because gammadelta T cells maturate efficiently. Finally, the percentage of thymocytes coexpressing two different TCRbeta chains is increased in the Ets-1(-/-) background and, in contrast with wild type, forced activation of pre-TCR signaling does not block endogenous TCRbeta gene rearrangement. These data identify Ets-1 as a critical transcription factor for pre-TCR functioning and for allelic exclusion at the TCRbeta locus.
Project description:Cytotoxic T lymphocytes (CTL) can respond to a few viral peptide-MHC-I (pMHC-I) complexes among a myriad of virus-unrelated endogenous self pMHC-I complexes displayed on virus-infected cells. To elucidate the molecular recognition events on live CTL, we have utilized a self-assembled biosensor composed of semiconductor nanocrystals, quantum dots, carrying a controlled number of virus-derived (cognate) and other (noncognate) pMHC-I complexes and examined their recognition by antigen-specific T cell receptor (TCR) on anti-virus CD8(+) T cells. The unique architecture of nanoscale quantum dot/pMHC-I conjugates revealed that unexpectedly strong multivalent CD8-MHC-I interactions underlie the cooperative contribution of noncognate pMHC-I to the recognition of cognate pMHC-I by TCR to augment T cell responses. The cooperative, CD8-dependent spread of signal from a few productively engaged TCR to many other TCR can explain the remarkable ability of CTL to respond to virus-infected cells that present few cognate pMHC-I complexes.