Requirement for CARMA1 in antigen receptor-induced NF-kappa B activation and lymphocyte proliferation.

Ligation of antigen receptors (TCR, BCR) on T and B lymphocytes leads to the activation of new transcriptional programs and cell cycle progression. Antigen receptor-mediated activation of NF-kappa B, required for proliferation of B and T cells, is disrupted in T cells lacking PKC theta and in B and T cells lacking Bcl10, a caspase recruitment domain (CARD)-containing adaptor protein. CARMA1 (also called CARD11 and Bimp3), the only lymphocyte-specific member in a family of membrane-associated guanylate kinase (MAGUK) scaffolding proteins that interact with Bcl10 by way of CARD-CARD interactions, is required for TCR-induced NF-kappa B activation in Jurkat T lymphoma cells. Here we show that T cells from mice lacking CARMA1 expression were defective in recruitment of Bcl10 to clustered TCR complexes and lipid rafts, in activation of NF-kappa B, and in induction of IL-2 production. Development of CD5(+) peritoneal B cells was disrupted in these mice, as was B cell proliferation in response to both BCR and CD40 ligation. Serum immunoglobulin levels were also markedly reduced in the mutant mice. Together, these results show that CARMA1 has a central role in antigen receptor signaling that results in activation and proliferation of both B and T lymphocytes.

The Th2 lymphoproliferation developing in LatY136F mutant mice triggers polyclonal B cell activation and systemic autoimmunity.

Lat(Y136F) knock-in mice harbor a point mutation in Tyr(136) of the linker for activation of T cells and show accumulation of Th2 effector cells and IgG1 and IgE hypergammaglobulinemia. B cell activation is not a direct effect of the mutation on B cells since in the absence of T cells, mutant B cells do not show an activated phenotype. After adoptive transfer of linker for activation of T cell mutant T cells into wild-type, T cell-deficient recipients, recipient B cells become activated. We show in vivo and in vitro that the Lat(Y136F) mutation promotes T cell-dependent B cell activation leading to germinal center, memory, and plasma cell formation even in an MHC class II-independent manner. All the plasma and memory B cell populations found in physiological T cell-dependent B cell responses are found. Characterization of the abundant plasmablasts found in secondary lymphoid organs of Lat(Y136F) mice revealed the presence of a previously uncharacterized CD93-expressing subpopulation, whose presence was confirmed in wild-type mice after immunization. In Lat(Y136F) mice, B cell activation was polyclonal and not Ag-driven because the increase in serum IgG1 and IgE concentrations involved Abs and autoantibodies with different specificities equally. Although the noncomplement-fixing IgG1 and IgE are the only isotypes significantly increased in Lat(Y136F) serum, we observed early-onset systemic autoimmunity with nephritis showing IgE autoantibody deposits and severe proteinuria. These results show that Th2 cells developing in Lat(Y136F) mice can trigger polyclonal B cell activation and thereby lead to systemic autoimmune disease.

Cytolytic T lymphocyte recognition of the murine cytomegalovirus nonstructural immediate-early protein pp89 expressed by recombinant vaccinia virus.

The murine immediate-early (IE) protein pp89 is a nonstructural virus-encoded phosphoprotein residing in the nucleus of infected cells, where it acts as transcriptional activator. Frequency analysis has shown that in BALB/c mice the majority of virus-specific CTL recognize IE antigens. The present study was performed to assess whether pp89 causes membrane antigen expression detected by IE-specific CTL. Site-directed mutagenesis has been used to delete the introns from gene ieI, encoding pp89, for subsequent integration of the continuous coding sequence into the vaccinia virus genome. After infection with the vaccinia recombinant, the authentic pp89 was expressed in cells that became susceptible to lysis by an IE-specific CTL clone. Priming of mice with the vaccinia recombinant sensitized polyclonal CTL that recognized MCMV-infected cells and transfected cells expressing pp89. Thus, a herpesviral IE polypeptide with essential function in viral transcriptional regulation can also serve as a dominant antigen for the specific CTL response of the host.

Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood.

To study telomere length dynamics in hematopoietic cells with age, we analyzed the average length of telomere repeat sequences in diverse populations of nucleated blood cells. More than 500 individuals ranging in age from 0 to 90 yr, including 36 pairs of monozygous and dizygotic twins, were analyzed using quantitative fluorescence in situ hybridization and flow cytometry. Granulocytes and naive T cells showed a parallel biphasic decline in telomere length with age that most likely reflected accumulated cell divisions in the common precursors of both cell types: hematopoietic stem cells. Telomere loss was very rapid in the first year, and continued for more than eight decades at a 30-fold lower rate. Memory T cells also showed an initial rapid decline in telomere length with age. However, in contrast to naive T cells, this decline continued for several years, and in older individuals lymphocytes typically had shorter telomeres than did granulocytes. Our findings point to a dramatic decline in stem cell turnover in early childhood and support the notion that cell divisions in hematopoietic stem cells and T cells result in loss of telomeric DNA.

Effects of epitope modification on T cell receptor-ligand binding and antigen recognition by seven H-2Kd-restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative.

We tested for antigen recognition and T cell receptor (TCR)-ligand binding 12 peptide derivative variants on seven H-2Kd-restricted cytotoxic T lymphocytes (CTL) clones specific for a bifunctional photoreactive derivative of the Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI). The derivative contained iodo-4-azidosalicylic acid in place of PbCS S-252 and 4-azidobenzoic acid on PbCS K-259. Selective photoactivation of the N-terminal photoreactive group allowed crosslinking to Kd molecules and photoactivation of the orthogonal group to TCR. TCR photoaffinity labeling with covalent Kd-peptide derivative complexes allowed direct assessment of TCR-ligand binding on living CTL. In most cases (over 80%) cytotoxicity (chromium release) and TCR-ligand binding differed by less than fivefold. The exceptions included (a) partial TCR agonists (8 cases), for which antigen recognition was five-tenfold less efficient than TCR-ligand binding, (b) TCR antagonists (2 cases), which were not recognized and capable of inhibiting recognition of the wild-type conjugate, (c) heteroclitic agonists (2 cases), for which antigen recognition was more efficient than TCR-ligand binding, and (d) one partial TCR agonist, which activated only Fas (C1)95), but not perforin/granzyme-mediated cytotoxicity. There was no correlation between these divergences and the avidity of TCR-ligand binding, indicating that other factors than binding avidity determine the nature of the CTL response. An unexpected and novel finding was that CD8-dependent clones clearly incline more to TCR antagonism than CD8-independent ones. As there was no correlation between CD8 dependence and the avidity of TCR-ligand binding, the possibility is suggested that CD8 plays a critical role in aberrant CTL function.

Impact of 3 different short-term chemotherapy regimens on lymphocyte-depletion and reconstitution in melanoma patients.

Recent immunotherapy trials have shown that lymphodepletion induced by short-term chemotherapy favors subsequent expansion of adoptively transferred T cells, by homeostatic mechanisms. To take advantage of this effect, novel regimens are being developed with the aim to enhance tumor immunity and reduce treatment toxicity. We have designed a clinical phase I trial combining chemotherapy, reinfusion of PBMC containing Melan-A(MART-1)-specific T cells, and vaccination with Melan-A peptide in Incomplete Freund's Adjuvant. Treatment with Busulfan plus Fludarabine depleted lymphocytes only weakly. Cyclophosphamide (CTX) plus Fludarabine depleted lymphocytes more profoundly, with a maximal effect using high doses of CTX. It is interesting to note that, the degree of homeostatic T-cell proliferation correlated tightly with the extent of lymphodepletion. As compared with CD4 T cells, CD8 T cells showed higher susceptibility to chemotherapy, followed by more rapid homeostatic proliferation and recovery, resulting in strong inversions of CD4/CD8 ratios. Despite efficient homeostatic proliferation of total CD4 and CD8 T cells, the frequency of CD8 T cells specific for Melan-A and cancer-testis antigens remained relatively low. In contrast, EBV-specific T cells expanded and reached high numbers. We conclude that short-term chemotherapy promoted homeostatic lymphocyte proliferation depending on the intensity of lymphocyte depletion, however without preferential expansion of tumor antigen-specific T cells.

NLRC5 deficiency impairs MHC class I-dependent lymphocyte killing by cytotoxic T cells

Purpose/Objective: NLRs are intracellular proteins involved in sensing pathogen- and danger-associated molecular patterns, thereby initiating inflammatory responses or cell death. The function of the family member NLRC5 remains a matter of debate, particularly with respect to NF-jB activation, type I IFN, and MHC class I expression. Materials and methods: To study the function of this NLR in vivo, we generated Nlrc5-deficient mice. Results: We found that NLRC5 deletion led to a mild reduction in MHC class I expression on DCs and an intermediate decrease on B cells, while MHC class I levels were dramatically lowered on T, NKT, and NK cells. Nlrc5-/- lymphocytes showed decreased H-2 gene transcript abundance and, accordingly, NLRC5 was sufficient to drive MHC class I expression in a human lymphoid cell line. Moreover, endogenous NLRC5 localized to the nucleus and occupied the proximal promoter region of H-2 genes. Notably, cytotoxic T cell-mediated elimination of Nlrc5-/- lymphocytes was markedly reduced. In addition, we observed low NLRC5 expression in several murine and human lymphoid-derived tumor cell lines. Conclusions: We found that NLRC5 acts as a key transcriptional regulator of MHC class I genes, in particular in lymphocytes. Loss of NLRC5 expression represents an advantage for evading CD8+ T cellmediated elimination by downmodulation of MHCI levels * a mechanism transformed cells may take advantage of. Therefore, our data support an essential role for NLRs in directing not only innate, but also adaptive immune responses (Staehli F et al. J Immunol 2012).

CD8+ cytotoxic T lymphocyte activation by soluble major histocompatibility complex-peptide dimers.

CD8+ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the molecular basis of this sensitive recognition process, we studied dimeric pMHC complexes containing linkers of different lengths. Although dimers containing short (10-30-A) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (> or = 80 A) did not. Based on this and on fluorescence resonance energy transfer experiments, we describe a dimeric binding mode in which two T cell receptors engage in an anti-parallel fashion two pMHC complexes facing each other with their constant domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, we demonstrated that pMHC dimers containing one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition.

Hes1 is a critical but context-dependent mediator of canonical Notch signaling in lymphocyte development and transformation.

Although canonical Notch signaling regulates multiple hematopoietic lineage decisions including T cell and marginal zone B cell fate specification, the downstream molecular mediators of Notch function are largely unknown. We showed here that conditional inactivation of Hes1, a well-characterized Notch target gene, in adult murine bone marrow (BM) cells severely impaired T cell development without affecting other Notch-dependent hematopoietic lineages such as marginal zone B cells. Competitive mixed BM chimeras, intrathymic transfer experiments, and in vitro culture of BM progenitors on Delta-like-expressing stromal cells further demonstrated that Hes1 is required for T cell lineage commitment, but dispensable for Notch-dependent thymocyte maturation through and beyond the beta selection checkpoint. Furthermore, our data strongly suggest that Hes1 is essential for the development and maintenance of Notch-induced T cell acute lymphoblastic leukemia. Collectively, our studies identify Hes1 as a critical but context-dependent mediator of canonical Notch signaling in the hematopoietic system.

An infrared spectral signature of human lymphocyte subpopulations from peripheral blood.

Metastatic melanomas are frequently refractory to most adjuvant therapies such as chemotherapies and radiotherapies. Recently, immunotherapies have shown good results in the treatment of some metastatic melanomas. Immune cell infiltration in the tumor has been associated with successful immunotherapy. More generally, tumor infiltrating lymphocytes (TILs) in the primary tumor and in metastases of melanoma patients have been demonstrated to correlate positively with favorable clinical outcomes. Altogether, these findings suggest the importance of being able to identify, quantify and characterize immune infiltration at the tumor site for a better diagnostic and treatment choice. In this paper, we used Fourier Transform Infrared (FTIR) imaging to identify and quantify different subpopulations of T cells: the cytotoxic T cells (CD8+), the helper T cells (CD4+) and the regulatory T cells (T reg). As a proof of concept, we investigated pure populations isolated from human peripheral blood from 6 healthy donors. These subpopulations were isolated from blood samples by magnetic labeling and purities were assessed by Fluorescence Activated Cell Sorting (FACS). The results presented here show that Fourier Transform Infrared (FTIR) imaging followed by supervised Partial Least Square Discriminant Analysis (PLS-DA) allows an accurate identification of CD4+ T cells and CD8+ T cells (>86%). We then developed a PLS regression allowing the quantification of T reg in a different mix of immune cells (e.g. Peripheral Blood Mononuclear Cells (PBMCs)). Altogether, these results demonstrate the sensitivity of infrared imaging to detect the low biological variability observed in T cell subpopulations.

Overexpression of GPC6 and TMEM132D in Early Stage Ovarian Cancer Correlates with CD8+ T-Lymphocyte Infiltration and Increased Patient Survival.

Infiltration of cytotoxic T-lymphocytes in ovarian cancer is a favorable prognostic factor. Employing a differential expression approach, we have recently identified a number of genes associated with CD8+ T-cell infiltration in early stage ovarian tumors. In the present study, we validated by qPCR the expression of two genes encoding the transmembrane proteins GPC6 and TMEM132D in a cohort of early stage ovarian cancer patients. The expression of both genes correlated positively with the mRNA levels of CD8A, a marker of T-lymphocyte infiltration [Pearson coefficient: 0.427 (p = 0.0067) and 0.861 (p < 0.0001), resp.]. GPC6 and TMEM132D expression was also documented in a variety of ovarian cancer cell lines. Importantly, Kaplan-Meier survival analysis revealed that high mRNA levels of GPC6 and/or TMEM132D correlated significantly with increased overall survival of early stage ovarian cancer patients (p = 0.032). Thus, GPC6 and TMEM132D may serve as predictors of CD8+ T-lymphocyte infiltration and as favorable prognostic markers in early stage ovarian cancer with important consequences for diagnosis, prognosis, and tumor immunobattling.

Analysis of BRAF and NRAS Mutation Status in Advanced Melanoma Patients Treated with Anti-CTLA-4 Antibodies: Association with Overall Survival?

Ipilimumab and tremelimumab are human monoclonal antibodies (Abs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4). Ipilimumab was the first agent to show a statistically significant benefit in overall survival in advanced melanoma patients. Currently, there is no proven association between the BRAFV600 mutation and the disease control rate in response to ipilimumab. This analysis was carried out to assess if BRAFV600 and NRAS mutation status affects the clinical outcome of anti-CTLA-4-treated melanoma patients. This is a retrospective multi-center analysis of 101 patients, with confirmed BRAF and NRAS mutation status, treated with anti-CTLA-4 antibodies from December 2006 until August 2012. The median overall survival, defined from the treatment start date with the anti-CTLA-4. Abs-treatment to death or till last follow up, of BRAFV600 or NRAS mutant patients (n = 62) was 10.12 months (95% CI 6.78-13.2) compared to 8.26 months (95% CI 6.02-19.9) in BRAFV600/NRASwt subpopulation (n = 39) (p = 0.67). The median OS of NRAS mutated patients (n = 24) was 12.1 months and although was prolonged compared to the median OS of BRAF mutated patients (n = 38, mOS = 8.03 months) or BRAFV600/NRASwt patients (n = 39, mOS = 8.26 months) the difference didn't reach statistical significance (p = 0.56). 69 patients were able to complete 4 cycles of anti-CTLA-4 treatment. Of the 24 patients treated with selected BRAF- or MEK-inhibitors, 16 patients received anti-CTLA 4 Abs following either a BRAF or MEK inhibitor with only 8 of them being able to finish 4 cycles of treatment. Based on our results, there is no difference in the median OS in patients treated with anti-CTLA-4 Abs implying that the BRAF/NRAS mutation status alone is not sufficient to predict the outcome of patients treated with anti-CTLA-4 Abs.

Leishmaniavirus : a possible target against metastatic leishmaniasis

Leishmaniasis is widely spread disease found in bath tropical and temperate regions but limited to the habitat of its sand fly vector. lt affects over 12 million people with 2 million new cases each year. As cutaneous leishmaniasis patients show varying levels of immunity to the disease after recovery, the development of a vaccine has much promise as a prevention strategy. Unfortunately however, existing anti-leishmanial vaccines are plagued by safety issues and have only ever shown limited efficacy .So, despite much effort, no effective vaccine is currently available. Recent studies suggest a correlation between the presence of Leishmania RNA virus (LRV) and the development of mucocutaneous leishmaniasis (MCL), which is characterised by the presence of secondary lesions in nasal and buccal mucosa, causing destructive and disfiguring facial lesions. Moreover, recent research has associated the viral presence to treatment fa ilure in patients. ln the first part of this work, we propose that these viral particles may serve as promising vaccine candidates due to their powerful TLR-3 antigenicity, launching an early cell-mediated attack on stimulated cells and thus eliminating their virulent complications. The second part of this work discusses a preliminary study on the lymphocyte immune response against Leishmania guyanensis infection. The lymphocyte response (and in particular, the raie of CDS+ T cells) is controversial and varies greatly between Leishmania species. Here, we illustrate the importance of a small CDS+ T cell subpopulation, expressing the CDSaa+ receptor. These intraepithelial lymphocytes are mainly present in the skin, vagina and intestinal tissue and are best known for their raie in the early immune response against pathogens. Similarly to traditional CDS+ cells, they secrete the tissue-destructive enzymes, perforin and granzyme, which can result in a hyper-inflammatory cutaneous lesion, raising a possibility for their raie in Leishmania infection. lndeed, our initial results in a murine mode( of Leishmania guyanensis infection suggest a pathogenic raie for CDSaa+ T cells. Further research into species-specific immune responses against the various Leishmania parasites is critical to realising the clinical potential of immunotherapy in the treatment and prevention of this disfiguring disease . -- La Leishmaniose est une maladie infectieuse causée par le parasite Leishmania. Elle est localisée dans les régions où son vecteur se reproduit, c'est-à-dire dans des régions tropicales ou tempérées. Cette pathologie affecte 12 millions des personnes dans le monde et 2 millions de nouveaux cas sont recensés chaque année. D'autres facteurs, tels la déforestation, les conditions d'hygiène ou encore l'accès limité aux médicaments, aggravent la pathologie et renforcent sa propagation. Les patients affectés par la leishmaniose et qui arrivent à en guérir, présentent une protection contre une réinfection. Pour cette raison, le développement d'un vaccin reste la meilleure solution pour combattre ce fléau. Mais, à ce jour, et malgré beaucoup d'efforts, aucun vaccin efficace n'a encore été développé. Un autre facteur responsable de l'aggravation de la pathologie et de la résistance de ces parasites aux drogues est un virus qui peut infecter certaines souches de Leishmania. Ce virus, appelé Leishmania RNA virus, peut induire une réponse inflammatoire exagérée, ce qui a comme résultat l'aggravation de la pathologie, la survie et la dissémination de ce parasite au sein de l'hôte infecté. Vu l'absence d'un vaccin contre ce parasite, Leishmania, nous proposons de développer un vaccin non pas contre le parasite lui- même mais contre l'agent qui provoque l'exacerbation de la pathologie, c'est-à-dire le virus. Dans cette étude, nous décrivons le développement d'un vaccin contre LRV, qui empêche le parasite d'induire des inflammations exagérées dans les souris. En d'autres mots, nous essayons de prévenir toutes les complications générées par cet hyperpathogène qu'est le LRV, en utilisant sa capside comme cible pour le développement d'un vaccin. Dans la deuxième partie de ce manuscrit, nous avons aussi étudié plus en détail la réponse immunitaire, et en particulier la réponse des lymphocytes T COB suite à l'infection du parasite Leishmania guyanensis porteur du LRV.

T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens.

T lymphocytes reactive with the product of the Mlsa-allele of the minor lymphocyte stimulating (Mls) locus use a predominant T-cell receptor beta-chain variable gene segment (V beta 6). Such V beta 6-bearing T cells are selectively eliminated in the thymus of Mlsa-bearing mice, consistent with a model in which tolerance to self antigens is achieved by clonal deletion.