Reduced thymic output and peripheral naïve CD4 T-cell alterations in primary progressive multiple sclerosis (PPMS)
David G. HaegertJessica D. HackenbrochDanielle A. DuszczyszynLeslie Fitz-GeraldEvelyn ZastepaHelen MasonYves LapierreJack P. AntelAmit Bar‐Or
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Double negative
Homeostasis
Cd4 t cell
Abstract TCR gene rearrangement and expression are central to the development of clonal T lymphocytes. The pre-TCR complex provides the first signal instructing differentiation and proliferation events during the transition from CD4−CD8−TCR− double negative (DN) stage to CD4+CD8+ double positive (DP) stage. How the pre-TCR signal leads to downstream gene expression is not known. HeLa E-box binding protein (HEB), a basic helix-loop-helix transcription factor, is abundantly detected in thymocytes and is thought to regulate E-box sites present in many T cell-specific gene enhancers, including TCR-α, TCR-β, and CD4. Targeted disruption of HEB results in a 5- to 10-fold reduction in thymic cellularity that can be accounted for by a developmental block at the DN to DP stage transition. Specifically, a dramatic increase in the CD4low/−CD8+CD5lowHSA+TCRlow/− immature single positive population and a concomitant decrease in the subsequent DP population are observed. Adoptive transfer test shows that this defect is cell-autonomous and restricted to the αβ T cell lineage. Introduction of an αβ TCR transgene into the HEBko/ko background is not sufficient to rescue the developmental delay. In vivo CD3 cross-linking analysis of thymocytes indicates that TCR signaling pathway in the HEBko/ko mice appears intact. These findings suggest an essential function of HEB in early T cell development, downstream or parallel to the pre-TCR signaling pathway.
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The pre–T-cell receptor (TCR) is crucial for the early T-cell development, but the ligand for pre-TCR remains unidentified. We recently proposed a model that pre-TCR complexes oligomerize spontaneously through interactions of the pre-TCRα chain. To investigate the mechanism underlying this ligand-independent signaling in vivo, we established knock-in mice that express a pre-TCRα mutant lacking charged amino acids (D 22 R 24 R 102 R 117 to A 22 A 24 A 102 A 117 ; 4A). CD4 + CD8 + thymocyte number was significantly reduced in invariant pre-TCRα (pTα 4A/4A ) mice, whereas CD4 − CD8 − thymocytes were unaffected. The percentages of double-negative 3 (DN3) cells and γδ T cells were increased in the pTα 4A/4A thymus, indicating that β-selection is impaired in pTα 4A/4A mice. Pre-TCR–mediated tyrosine phosphorylation and clonal expansion into double-positive thymocytes were also defective in the knock-in mice. Pre-TCR was expressed at higher levels on pTα 4A/4A cell surfaces than on those of the wild type, suggesting that the charged residues in pTα are critical for autonomous engagement and subsequent internalization of pre-TCR. Pre-TCR–mediated allelic exclusion of the TCRβ gene was also inhibited in pTα 4A/4A mice, and thereby, dual TCRβs were expressed on pTα 4A/4A T cells. Furthermore, the TCRβ chain variable region (Vβ) repertoire of mature T cells was significantly altered in pTα 4A/4A mice. These results suggest that charged residues of pTα are critical for β-selection, allelic exclusion, and TCRβ repertoire formation.
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During αβ thymocyte development, the clonotypic αβ–T cell receptor (TCR) is preceded by sequentially expressed immature versions of the TCR–CD3 complex: the pre-TCR, containing a clonotypic TCR-β chain and invariant pre-Tα, is expressed on pre-T cells before rearrangement of the TCR-α locus. Moreover, clonotype-independent CD3 complexes (CIC) appear on pro-T cells before VDJ rearrangements of TCR-β genes. The pre-TCR is known to mediate TCR-β selection, the prerequisite for maturation of CD4−8− double negative (DN) thymocytes to the CD4+8+ double positive stage. A developmental function of CIC has so far not been delineated. In mice single deficient and double deficient for CD3ζ/η and/or p56lck, we observe a pronounced reduction in the proportions of CD25+ DN thymocytes that express intracellular TCR-β chains. TCR-β transcripts are reduced in parallel with TCR-β polypeptide chains whereas no reduction in TCR-β locus rearrangements could be detected. Wild-type levels of TCR-β transcripts and of cells expressing TCR-β polypeptide chains are induced by treatment with anti-CD3ε mAb. The data suggest that the initial expression of rearranged TCR-β VDJ genes in pro-T cell to pre-T cell progression is dependent on CD3 complex signaling, and thus define a putative developmental function for CIC.
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During T cell development in the thymus, pre-T cell receptor (TCR) complexes signal CD4(-) CD8(-) (double negative [DN]) thymocytes to differentiate into CD4(+) CD8(+) (double positive [DP]) thymocytes, and they generate such signals without apparent ligand engagements. Although ligand-independent signaling is unusual and might be unique to the pre-TCR, it is possible that other TCR complexes such as alphabeta TCR or alphagamma TCR might also be able to signal the DN to DP transition in the absence of ligand engagement if they were expressed on DN thymocytes. Although alphagamma TCR complexes efficiently signal DN thymocyte differentiation, it is not yet certain if alphabeta TCR complexes are also capable of signaling DN thymocyte differentiation, nor is it certain if such signaling is dependent upon ligand engagement. This study has addressed these questions by expressing defined alphabeta TCR transgenes in recombination activating gene 2(-/-) pre-Talpha(-/-) double deficient mice. In such double deficient mice, the only antigen receptors that can be expressed are those encoded by the alphabeta TCR transgenes. In this way, this study definitively demonstrates that alphabeta TCR can in fact signal the DN to DP transition. In addition, this study demonstrates that transgenic alphabeta TCRs signal the DN to DP transition even in the absence of their specific MHC-peptide ligands.
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Abstract It is well documented that in wildtype mice TCRγδ+ cells differentiate along a double negative (DN) pathway whereas TCRαβ+ cells differentiate along the double positive (DP) pathway, suggesting that the TCR itself induces lineage differentiation. Under experimental conditions and in genetically modified mice, however, evidence was presented suggesting that rather than TCR itself, TCR signal strength and notch determine lineage choice. In the human thymus, a “chimeric” DP TCRγδ+ cell population is present and constitutes a sizeable fraction of the γδ population. We asked the question whether these cells belong to the αβ or γδ lineage or whether these cells are bipotent. We found that TCRγδ DP cells are bipotent cells since strong TCR signals induces differentiation to TCRγδ cells, whereas Notch activation induces TCRαβ lineage differentiation. We furthermore could show that Notch signaling diverts TCRγδ DN cells to the DP pathway and induces strong proliferation. In line with these findings, TCRγδ+ acute lymphoblastic leukemias (ALL) with activating Notch1 mutations follow the DP differentiation pathway, whereas the DN ALL cells are devoid of these activating Notch1 mutations. We were able to confirm that also in vivo TCRγδ DP have rearranged TCRβ chains, actively rearrange the TCRα locus and delete the TCRδ locus (αβ lineage). Using TCRα rearrangements as a lineage marker, we could show that a subpopulation of mature TCRγδ cells is derived from DP cells.
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Immature CD4+CD8+ double-positive (DP) thymocytes are positively selected for further development if they express TCR reacting with thymic ligands of low affinity. However, the majority of DP thymocytes express low TCR levels. This low level of TCR may be insufficient to recognize thymic ligands. To understand the basis for the low expression of TCR on DP thymocytes, we determined the density of TCR expression at various stages of their development using TCR transgenic (TCR-Tg) mice. We found that TCR expression was high in the thymocytes that had recently transited into the DP stage but then gradually decreased on DP cells if they were not selected by TCR interaction with MHC molecules. However, such TCR suppression was not observed in positively selected DP cells and in the non-selected DP cells obtained from CD45 deficient mice or from mice receiving anti-CD4 mAb. These findings suggest that the once highly expressed TCR at the DP stage is suppressed by CD45 and/or CD4 on non-selected thymocytes. Furthermore, TCR suppression is prevented by TCR-mediated signals. The maintenance of high TCR levels on positively selected DP thymocytes may facilitate their selection.
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A rearranged T cell receptor (TCR) Vα and Jα gene from a cytochrome c- specific T cell hybridoma was introduced into the genomic Jα region. The introduced TCR α chain gene is expressed in a majority of CD3 positive and CD4 CD8 double-negative immature thymocytes. However, only a few percent of the double-positive and single-positive thymocytes express this TCR α chain. This decrease is caused by a rearrangement of TCR α chain locus, which deletes the introduced TCR gene. Analysis of the mice carrying the introduced TCR α chain and the transgenic TCR β chain from the original cytochrome c- specific T cell hybridoma revealed that positive selection efficiently rescues double-positive thymocytes from the loss of the introduced TCR α chain gene. In the mice with negatively selecting conditions, T cells expressing the introduced TCR αβ chains were deleted at the double-positive stage. However, a large number of thymocytes escape negative selection by using an endogenous TCR α chain created by secondary rearrangement maintaining normal thymocyte development. These results suggest that secondary rearrangements of the TCR α chain gene play an important role in the formation of the T cell repertoire.
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Significance Expression of a productively rearranged T-cell receptor (TCR)-β chain induces a program of αβ T-lineage differentiation, whereas thymocytes that productively rearranged TCR-γ and TCR-δ typically give rise to γδ-lineage T cells. However, given that all three TCR gene loci simultaneously undergo gene rearrangements, the possibility exists that a developing thymocyte may express a γδ-TCR together with a TCR-β or pre-TCR complex, and it is not clear to what this outcome would give rise in terms of T-lineage differentiation. Our findings point to a striking conclusion, in that rather than transmitting signals that exclusively promote αβ-lineage commitment/differentiation, the pre-TCR can function in concert with the γδ-TCR to promote γδ commitment/differentiation, a result that supports a signal strength model of αβ/γδ-lineage choice.
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