CpG-ODN-induced sustained expression of BTLA mediating selective inhibition of human B cells

BTLA (B- and T-lymphocyte attenuator) is a prominent co-receptor that is structurally and functionally related to CTLA-4 and PD-1. In T cells, BTLA inhibits TCR-mediated activation. In B cells, roles and functions of BTLA are still poorly understood and have never been studied in the context of B cells activated by CpG via TLR9. In this study, we evaluated the expression of BTLA depending on activation and differentiation of human B cell subsets in peripheral blood and lymph nodes. Stimulation with CpG upregulated BTLA, but not its ligand: herpes virus entry mediator (HVEM), on B cells in vitro and sustained its expression in vivo in melanoma patients after vaccination. Upon ligation with HVEM, BTLA inhibited CpG-mediated B cell functions (proliferation, cytokine production, and upregulation of co-stimulatory molecules), which was reversed by blocking BTLA/HVEM interactions. Interestingly, chemokine secretion (IL-8 and MIP1β) was not affected by BTLA/HVEM ligation, suggesting that BTLA-mediated inhibition is selective for some but not all B cell functions. We conclude that BTLA is an important immune checkpoint for B cells, as similarly known for T cell

CD1d-antibody fusion proteins target iNKT cells to the tumor and trigger long-term therapeutic responses

Despite the well-established antitumor activity of CD1d-restricted invariant natural killer T lymphocytes (iNKT), their use for cancer therapy has remained challenging. This appears to be due to their strong but short-lived activation followed by long-term anergy after a single administration of the CD1d agonist ligand alpha-galactosylceramide (αGC). As a promising alternative, we obtained sustained mouse iNKT cell responses associated with prolonged antitumor effects through repeated administrations of tumor-targeted recombinant sCD1d-antitumor scFv fusion proteins loaded with αGC. Here, we demonstrate that CD1d fusion proteins bound to tumor cells via the antibody fragment specific for a tumor-associated antigen, efficiently activate human iNKT cell lines leading to potent tumor cell lysis. The importance of CD1d tumor targeting was confirmed in tumor-bearing mice in which only the specific tumor-targeted CD1d fusion protein resulted in tumor inhibition of well-established aggressive tumor grafts. The therapeutic efficacy correlated with the repeated activation of iNKT and natural killer cells marked by their release of TH1 cytokines, despite the up-regulation of the co-inhibitory receptor PD-1. Our results demonstrate the superiority of providing the superagonist αGC loaded on recombinant CD1d proteins and support the use of αGC/sCD1d-antitumor fusion proteins to secure a sustained human and mouse iNKT cell activation, while targeting their cytotoxic activity and cytokine release to the tumor sit

Identification of Urine Biomarkers to Improve Eligibility for Prostate Biopsy and Detect High-Grade Prostate Cancer

PCa screening is based on the measurements of the serum prostate specific antigen (PSA) to select men with higher risks for tumors and, thus, eligible for prostate biopsy. However, PSA testing has a low specificity, leading to unnecessary biopsies in 50–75% of cases. Therefore, more specific screening opportunities are needed to reduce the number of biopsies performed on healthy men and patients with indolent tumors. Urine samples from 45 patients with elevated PSA were collected prior to prostate biopsy, a mass spectrometry (MS) screening was performed to identify novel biomarkers and the best candidates were validated by ELISA. The urine quantification of PEDF, HPX, CD99, CANX, FCER2, HRNR, and KRT13 showed superior performance compared to PSA. Additionally, the combination of two biomarkers and patient age resulted in an AUC of 0.8196 (PSA = 0.6020) and 0.7801 (PSA = 0.5690) in detecting healthy men and high-grade PCa, respectively. In this study, we identified and validated novel urine biomarkers for the screening of PCa, showing that an upfront urine test, based on quantitative biomarkers and patient age, is a feasible method to reduce the number of unnecessary prostate biopsies and detect both healthy men and clinically significant PCa

ILC2-modulated T cell-to-MDSC balance is associated with bladder cancer recurrence.

Non-muscle-invasive bladder cancer (NMIBC) is a highly recurrent tumor despite intravesical immunotherapy instillation with the bacillus Calmette-Guérin (BCG) vaccine. In a prospective longitudinal study, we took advantage of BCG instillations, which increase local immune infiltration, to characterize immune cell populations in the urine of patients with NMIBC as a surrogate for the bladder tumor microenvironment. We observed an infiltration of neutrophils, T cells, monocytic myeloid-derived suppressor cells (M-MDSCs), and group 2 innate lymphoid cells (ILC2). Notably, patients with a T cell-to-MDSC ratio of less than 1 showed dramatically lower recurrence-free survival than did patients with a ratio of greater than 1. Analysis of early and later time points indicated that this patient dichotomy existed prior to BCG treatment. ILC2 frequency was associated with detectable IL-13 in the urine and correlated with the level of recruited M-MDSCs, which highly expressed IL-13 receptor α1. In vitro, ILC2 were increased and potently expressed IL-13 in the presence of BCG or tumor cells. IL-13 induced the preferential recruitment and suppressive function of monocytes. Thus, the T cell-to-MDSC balance, associated with a skewing toward type 2 immunity, may predict bladder tumor recurrence and influence the mortality of patients with muscle-invasive cancer. Moreover, these results underline the ILC2/IL-13 axis as a targetable pathway to curtail the M-MDSC compartment and improve bladder cancer treatment

TCRep 3D: An Automated In Silico Approach to Study the Structural Properties of TCR Repertoires

TCRep 3D is an automated systematic approach for TCR-peptide-MHC class I structure prediction, based on homology and ab initio modeling. It has been considerably generalized from former studies to be applicable to large repertoires of TCR. First, the location of the complementary determining regions of the target sequences are automatically identified by a sequence alignment strategy against a database of TCR Vα and Vβ chains. A structure-based alignment ensures automated identification of CDR3 loops. The CDR are then modeled in the environment of the complex, in an ab initio approach based on a simulated annealing protocol. During this step, dihedral restraints are applied to drive the CDR1 and CDR2 loops towards their canonical conformations, described by Al-Lazikani et. al. We developed a new automated algorithm that determines additional restraints to iteratively converge towards TCR conformations making frequent hydrogen bonds with the pMHC. We demonstrated that our approach outperforms popular scoring methods (Anolea, Dope and Modeller) in predicting relevant CDR conformations. Finally, this modeling approach has been successfully applied to experimentally determined sequences of TCR that recognize the NY-ESO-1 cancer testis antigen. This analysis revealed a mechanism of selection of TCR through the presence of a single conserved amino acid in all CDR3β sequences. The important structural modifications predicted in silico and the associated dramatic loss of experimental binding affinity upon mutation of this amino acid show the good correspondence between the predicted structures and their biological activities. To our knowledge, this is the first systematic approach that was developed for large TCR repertoire structural modeling

Quantitative multiparameter assays to measure the effect of adjuvants on human antigen-specific CD8 T-cell responses.

Large numbers and functionally competent T cells are required to protect from diseases for which antibody-based vaccines have consistently failed (1), which is the case for many chronic viral infections and solid tumors. Therefore, therapeutic vaccines aim at the induction of strong antigen-specific T-cell responses. Novel adjuvants have considerably improved the capacity of synthetic vaccines to activate T cells, but more research is necessary to identify optimal compositions of potent vaccine formulations. Consequently, there is a great need to develop accurate methods for the efficient identification of antigen-specific T cells and the assessment of their functional characteristics directly ex vivo. In this regard, hundreds of clinical vaccination trials have been implemented during the last 15 years, and monitoring techniques become more and more standardized

Expression and Release of HLA-E by Melanoma Cells and Melanocytes: Potential Impact on the Response of Cytotoxic Effector Cells

International audienceHLA-E are nonclassical MHC molecules with poorly characterized tissue distribution and functions. Because of their capacity to bind the inhibitory receptor, CD94/NKG2A, expressed by NK cells and CTL, HLA-E molecules might play an important role in immunomodulation. In particular, expression of HLA-E might favor tumor cell escape from CTL and NK immunosurveillance. To address the potential role of HLA-E in melanoma immunobiology, we assessed the expression of these molecules ex vivo in human melanoma biopsies and in melanoma and melanocyte cell lines. Melanoma cell lines expressed no or low surface, but significant intracellular levels of HLA-E. We also report for the first time that some of them produced a soluble form of this molecule. IFN-␥ significantly increased the surface expression of HLA-E and the shedding of soluble HLA-E by these cells, in a metalloproteinase-dependent fashion. In contrast, melanocyte cell lines constitutively expressed HLA-E molecules that were detectable both at the cell surface and in the soluble form, at levels that were poorly affected by IFN-␥ treatment. On tumor sections, a majority of tumor cells of primary, but a low proportion of metastatic melanomas (30-70 and 10-20%, respectively), expressed HLA-E. Finally, HLA-E expression at the cell surface of melanoma cells decreased their susceptibility to CTL lysis. These data demonstrate that HLA-E expression and shedding are normal features of melanocytes, which are conserved in melanoma cells of primary tumors, but become dependent on IFN-␥ induction after metastasis. The biological significance of these findings warrants further investigation

Identification of Five New HLA-B*3501-Restricted Epitopes Derived from Common Melanoma-Associated Antigens, Spontaneously Recognized by Tumor-Infiltrating Lymphocytes 1

International audienceWe previously described HLA-B35-restricted melanoma tumor-infiltrating lymphocyte responses to frequently expressed mela-noma-associated Ags: tyrosinase, Melan-A/MART-1, gp100, MAGE-A3/MAGE-A6, and NY-ESO-1. Using clones derived from these TIL, we identified in this study the corresponding epitopes. We show that five of these epitopes are new and that melanoma cells naturally present all the six epitopes. Interestingly, five of these epitopes correspond to or encompass melanoma-associated Ag epitopes presented in other HLA contexts, such as A2, A1, B51, and Cw3. In particular, the HLA-B35-restricted Melan-A epitope is mimicked by the peptide 26-35, already known as the most immunodominant melanoma epitope in the HLA-A*0201 context. Because this peptide lacked adequate anchor amino acid residues for efficient binding to HLA-B35, modified peptides were designed. Two of these analogues were found to induce higher PBL-and tumor-infiltrating lymphocyte-specific responses than the parental peptide, suggesting that they could be more immunogenic in HLA-B*3501 melanoma patients. These data have important implications for the formulation of polypeptide-based vaccines as well as for the monitoring of melanoma-specific CTL response in HLA-B*3501 melanoma patients