Selection of T-cell receptors with a recurrent CDR3β peptide-contact motif within the repertoire of alloreactive CD8(+) T cells

Peptide/MHC complexes recognized by alloreactive T lymphocytes (TLs) have been identified, but their contribution to in vivo allo‐rejection is not known. We previously characterized the peptide pBM1, highly represented among endogenous H‐2K^b (K^b)‐associated peptides and critically required to induce full activation of H‐2^k monoclonal CD8⁺ TLs expressing the cognate TCR‐BM3.3. Here, we asked whether a pBM1/K^b‐specific TL subset could be detected within a polyclonal TL population rejecting allogeneic cells in vivo. We show that the proportion of pBM1/K^b‐binding CD8⁺ TLs increased from <0.04% in naïve mice to 3% of activated CD44⁺ CD8⁺ TLs in H‐2^k mice rejecting K^b‐expressing cells. Among these, TCR‐Vβ2 usage was greatly enriched, and 75% of them shared a TCR‐Vβ2 CDR3β motif with the prototype TCR‐BM3.3. Fewer than 5% of K^b‐reactive CD44⁺ CD8⁺ TLs not binding pBM1/K^b displayed this CDR3β motif. We found that the recurrent CDR3β motif of pBM1/K^b‐binding TLs was assembled from distinct V/D/J recombination events, suggesting that it is recruited upon immunization for its optimal TCR‐peptide/MHC fit. Thus, a CDR3β motif generated by a process akin to “convergent recombination” accounts for a sizable fraction of the alloreactive anti‐K^b TCR repertoire.     

TCR down-regulation boosts T-cell-mediated cytotoxicity and protection against poxvirus infections

Cytotoxic T (Tc) cells play a key role in the defense against virus infections. Tc cells recognize infected cells via the T-cell receptor (TCR) and subsequently kill the target cells by one or more cytotoxic mechanisms. Induction of the cytotoxic mechanisms is finely tuned by the activation signals from the TCR. To determine whether TCR down-regulation affects the cytotoxicity of Tc cells, we studied TCR down-regulation-deficient CD3γLLAA mice. We found that Tc cells from CD3γLLAA mice have reduced cytotoxicity due to a specific deficiency in exocytosis of lytic granules. To determine whether this defect was reflected in an increased susceptibility to virus infections, we studied the course of ectromelia virus (ECTV) infection. We found that the susceptibility to ECTV infection was significantly increased in CD3γLLAA mice with a mortality rate almost as high as in granzyme B knock-out mice. Finally, we found that TCR signaling in CD3γLLAA Tc cells caused highly increased tyrosine phosphorylation and activation of the c-Cbl ubiquitin ligase, and that the impaired exocytosis of lytic granules could be rescued by the knockdown of c-Cbl. Thus, our work demonstrates that TCR down-regulation critically increases Tc cell cytotoxicity and protection against poxvirus infection.     

Typing of the immunological system in human embryos by coelocentesis

Coelocentesis offers a new opportunity for gaining access to the human embryos from 28 d postfertilization. However, while some studies about its biochemical composition have been reported, our knowledge about immunological pattern of this compartment is still limited. For this reason, we studied the human coelomic fluids sampled from 6.6 to 10 wk of gestation. The majority of cellular population consisted in mesenchymal/epithelial cells. In fluids sampled before 10 wk we found only a preT Cell Receptor expression and an absence or a very low frequency of B lymphocytes, T lymphocytes and NK (natural killer) antigens. These preliminary data suggest that the immunological system in human embryos could be in the ideal conditions to start a process of tolerance induction.     

Analysis of the Conservation of T Cell Receptor Alpha and Beta Chain Variable Regions Gene in pp65 Peptide-Specific HLA-A＊0201-Restricted CD8^＋ T Cells

Many viral epitope specific T cell receptors （TCRs） in MHC-matched individuals have been demonstrated to involve conserved amino acid motifs in β chain complementarity-determining region 3 （CDR3）. However, it is not sure whether the conserved motifs can also be found in TCR β chain. In previous studies, we developed a modified method to enlarge the percentage of cytomegalovirus （CMV） pp65 peptide-specific CD8^＋ T cells in PBMC by continuous peptide stimulation in vitro, which provides sufficient number of specific T cells for detection. In this study, we further analyzed the restrictive usage of TCR Vα and Vβ gene families and investigated the CDR3 gene sequence of pp65 peptide-specific CD8β T cells. Analysis of CDR3 spectratypes suggested a restricted usage of TCR α chain AV8, AV12, AV21, AV31 families and TCR βchain BV3, BV14, BV21, BV23, BVll families in donor CD8^＋ T cells stimulated by pp65 peptide. The sequences of these T cells involved similar sequence （TX） G （X） A in CDR3 region of TCR α chain and L （XT） G （X） A in TCR β chain.     

Division‐linked differentiation can account for CD8+ T‐cell phenotype in vivo

The CD8⁺ T‐cell response to infection involves a large initial expansion in the numbers of responding cells, accompanied by differentiation of these cells. Expression of the adhesion molecule CD62L is high on naïve cells and rapidly downregulated on the surface of the majority (～90%) of cells during the ‘effector’ phase of acute infection. Adoptive transfer studies have been used to study differentiation in this system; however, relatively little work has investigated the phenotype of cells in the endogenous repertoire. We demonstrate that the extent of CD62L down‐regulation is positively correlated with clone size in vivo, consistent with division‐linked differentiation of responding cells. Other features of the endogenous CD62L^hi and CD62L^lo repertoire are that the CD62L^lo repertoire is less diverse than the CD62L^hi repertoire and represents a subset of clonotypes found in the CD62L^hi repertoire. To test whether these observations are compatible with a mechanism of division‐linked differentiation, we developed a mathematical model, where there is a probability of CD62L down‐regulation associated with cell division. Comparison of model results with experimental data suggests that division‐linked differentiation provides a simple mechanism to explain the relationship between clone size and phenotype of CD8⁺ T cells during acute infection.     

Cytogenetic and molecular characterization of a hepatosplenic T-cell lymphoma: report of a novel chromosomal aberration

Hepatosplenic T-cell lymphomas (HSTCL) are rare cancers and comprise 5% of peripheral T-cell lymphomas. These well-characterized extranodal lymphomas have a disguised onset, secondary to intrasinusoidal infiltration of the spleen, liver, and bone marrow, with a rapidly progressive course that is poorly responsive to chemotherapy and often ensues in the setting of immune system suppression. We describe the clinical, immunophenotypic, cytogenetic, fluorescence in situ hybridization, and molecular analyses for T cell receptor gene rearrangement in a 21-year-old man diagnosed with HSTCL. Immunophenotypic analysis revealed negativity for CD5 as well as double negativity for CD4/CD8 mature T-cell immunophenotype, which suggested the diagnosis of hepatosplenic T-cell lymphoma. Molecular analysis confirmed a TCR gene rearrangement, thereby verifying the common T-cell origin of the present HSTCL case. Furthermore, cytogenetic analysis revealed a novel chromosomal rearrangement, t(7;15)(p22;q21). Metaphase fluorescence in situ hybridization analysis confirmed the translocation of a chromosomal segment from 15q21 to 7p22.     

Cadmium and T cell differentiation: limited impact in vivo but significant toxicity in fetal thymus organ culture

DNA lesions, including oxydated bases, nucleotide damage and double strand breaks, are continuously produced in living cells and represent a threat for genetic stability. Highly conserved repair processes have evolved to maintain DNA integrity. Cadmium (Cd) is an environmental carcinogenic pollutant known to inactivate several proteins involved in DNA repair systems while at the same time creating an oxidative stress that can result in additional DNA lesions. Cd also has potent immunotoxic effects. DNA repair by non-homologous end joining (NHEJ) is absolutely required for T lymphocyte differentiation. In this study, we examined the impact of Cd on non-homologous end joining pathway by analyzing T cell development in the thymus of mice that received Cd-supplemented drinking water. In vivo, the absence of major alteration indicates that Cd does not affect NHEJ, despite its accumulation in the thymus. Cd contamination affects only a discrete population of developing thymocytes. However, these cells are functional as the cellular response observed in mice following gamma-radiation exposure is identical in treated and control mice. Furthermore, Cd diet did not perturb the redox status in thymocytes and more importantly did not generate significant DNA lesions in organs that accumulate the highest concentration of Cd. Our results show that in vivo, Cd does not affect NHEJ or base and nucleotide repair, and that Cd toxicity to T cells is rather linked to cell cycle perturbations.     

Thapsigargin对SLE患者T细胞TCR／CD3复合物介导的[Ca^2＋]i反应的影响

目的证实SLE患者T细胞功能异常是否与TCR/CD3复合物介导的[Ca2+]i反应异常有关,以及[Ca2+]i反应异常的原因.方法用CD3单抗与羊抗鼠二抗IgG相关联刺激T细胞,并用Thapsigargin干预后,分别用粘附细胞仪连续观察10 min T细胞[Ca2+]i的变化,并评价[Ca2+]i反应与CD3分子表达的相关性.结果正常人和SLE患者T细胞[Ca2+]i反应的基准值相似(P=0.105);SLE患者T细胞的[Ca2+]i反应高峰值、平台值明显高于正常对照(P＜0.001,P＜0.001);加入Thapsigargin后二者[Ca2+]i反应无显著差异,二者的T细胞CD3阳性率无差异(P=0.665).结论SLE患者T细胞TCR/CD3介导的[Ca2+]i反应存在异常,并且这种异常不是因为胞内Ca2+库排空后跨膜Ca2+内流不同所致.