A cluster of broadly neutralizing IgG against BK polyomavirus in a repertoire dominated by IgM.

The BK polyomavirus (BKPyV) is an opportunistic pathogen, which is only pathogenic in immunosuppressed individuals, such as kidney transplant recipients, in whom BKPyV can cause significant morbidity. To identify broadly neutralizing antibodies against this virus, we used fluorescence-labeled BKPyV virus-like particles to sort BKPyV-specific B cells from the PBMC of KTx recipients, then single-cell RNAseq to obtain paired heavy- and light-chain antibody sequences from 2,106 sorted B cells. The BKPyV-specific repertoire was highly diverse in terms of both V-gene usage and clonotype diversity and included most of the IgM B cells, including many with extensive somatic hypermutation. In two patients where sufficient data were available, IgM B cells in the BKPyV-specific dataset had significant differences in V-gene usage compared with IgG B cells from the same patient. CDR3 sequence-based clustering allowed us to identify and characterize three broadly neutralizing "41F17-like" clonotypes that were predominantly IgG, suggesting that some specific BKPyV capsid epitopes are preferentially targeted by IgG.

Contribution of the SYK Tyrosine kinase expression to human iNKT self-reactivity.

Invariant Natural Killer T (iNKT) cells are particular T lymphocytes at the frontier between innate and adaptative immunities. They participate in the elimination of pathogens or tumor cells, but also in the development of allergic reactions and autoimmune diseases. From their first descriptions, the phenomenon of self-reactivity has been described. Indeed, they are able to recognize exogenous and endogenous lipids. However, the mechanisms underlying the self-reactivity are still largely unknown, particularly in humans. Using a CD1d tetramer-based sensitive immunomagnetic approach, we generated self-reactive iNKT cell lines from blood circulating iNKT cells of healthy donors. Analysis of their functional characteristics in vitro showed that these cells recognized endogenous lipids presented by CD1d molecules through their TCR that do not correspond to α-glycosylceramides. TCR sequencing and transcriptomic analysis of T cell clones revealed that a particular TCR signature and an expression of the SYK protein kinase were two mechanisms supporting human iNKT self-reactivity. The SYK expression, strong in the most self-reactive iNKT clones and variable in ex vivo isolated iNKT cells, seems to decrease the activation threshold of iNKT cells and increase their overall antigenic sensitivity. This study indicates that a modulation of the TCR intracellular signal contributes to iNKT self-reactivity.

NKG2D Controls Natural Reactivity of Vγ9Vδ2 T Lymphocytes against Mesenchymal Glioblastoma Cells.

PURPOSE: Cellular immunotherapies are currently being explored to eliminate highly invasive and chemoradioresistant glioblastoma (GBM) cells involved in rapid relapse. We recently showed that concomitant stereotactic injections of nonalloreactive allogeneic Vγ9Vδ2 T lymphocytes eradicate zoledronate-primed human GBM cells. In the present study, we investigated the spontaneous reactivity of allogeneic human Vγ9Vδ2 T lymphocytes toward primary human GBM cells, in vitro and in vivo, in the absence of any prior sensitization. EXPERIMENTAL DESIGN: Through functional and transcriptomic analyses, we extensively characterized the immunoreactivity of human Vγ9Vδ2 T lymphocytes against various primary GBM cultures directly derived from patient tumors. RESULTS: We evidenced that GBM cells displaying a mesenchymal signature are spontaneously eliminated by allogeneic human Vγ9Vδ2 T lymphocytes, a reactivity process being mediated by γδ T-cell receptor (TCR) and tightly regulated by cellular stress-associated NKG2D pathway. This led to the identification of highly reactive Vγ9Vδ2 T lymphocyte populations, independently of a specific TCR repertoire signature. Moreover, we finally provide evidence of immunotherapeutic efficacy in vivo, in the absence of any prior tumor cell sensitization. CONCLUSIONS: By identifying pathways implicated in the selective natural recognition of mesenchymal GBM cell subtypes, accounting for 30% of primary diagnosed and 60% of recurrent GBM, our results pave the way for novel targeted cellular immunotherapies.

Impact on early outcomes and immune reconstitution of high-dose post-transplant cyclophosphamide vs anti-thymocyte globulin after reduced intensity conditioning peripheral blood stem cell allogeneic transplantation.

We have compared prospectively the outcome and immune reconstitution of patients receiving either post-transplant cyclophosphamide (PTCY) (n = 30) or anti-thymocyte globulin ATG (n = 15) as Graft-versus-host disease (GVHD) prophylaxis after reduced-intensity conditioning (RIC) allogeneic peripheral blood stem cell (PBSC) transplantation (allo-SCT). The outcome and immune reconstitution of patients receiving either of these two regimens were compared prospectively. This study allowed also to investigate the impact of PTCY between haplo-identical vs matched donors and of clofarabine as part of the RIC regimen. The γ/δ T-cells, α/ß T-cells (CD8+ and CD4+), NK T-cells, NK cells, B-cells, Tregs and monocytes were analyzed by flow cytometry from a total of 583 samples. In the PTCY group significant delayed platelets recovery, higher CD3+ donor chimerism, higher HHV-6 and lower EBV reactivations were observed. Early survival advantage for CD4+ T-cells, Tregs and α/ß T-cells was documented in the PTCY group while it was the case for α/ß T-cells, NK cells and monocytes in the ATG group. Higher counts of NK and monocytes were observed at days +30 and/or day+60 in the ATG group. Both results were retained even in the case of mismatched donors. However, higher percentages of CD4+ T-cells, α/ß T-cells and Tregs were observed with haplo-identical donors in the PTCY group. Finally, clofarabine was responsible for early survival advantage of NK T-cells in the PTCY group while it abrogated the early survival advantage of γ/δ T-cells in the ATG group. In conclusion, there are marked differences in the immunological effects of ATG vs PTCY as GVHD prophylaxis for RIC PBSC allo-SCT.

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

Antigen-specific single B cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies.

BACKGROUND: There is an ever-increasing need of monoclonal antibodies (mAbs) for biomedical applications and fully human binders are particularly desirable due to their reduced immunogenicity in patients. We have applied a strategy for the isolation of antigen-specific B cells using tetramerized proteins and single-cell sorting followed by reconstruction of human mAbs by RT-PCR and expression cloning. RESULTS: This strategy, using human peripheral blood B cells, enabled the production of low affinity human mAbs against major histocompatibility complex molecules loaded with peptides (pMHC). We then implemented this technology using human immunoglobulin transgenic rats, which after immunization with an antigen of interest express high affinity-matured antibodies with human idiotypes. Using rapid immunization, followed by tetramer-based B-cell sorting and expression cloning, we generated several fully humanized mAbs with strong affinities, which could discriminate between highly homologous proteins (eg. different pMHC complexes). CONCLUSIONS: Therefore, we describe a versatile and more effective approach as compared to hybridoma generation or phage or yeast display technologies for the generation of highly specific and discriminative fully human mAbs that could be useful both for basic research and immunotherapeutic purposes.

Single-cell analysis of thymocyte differentiation: identification of transcription factor interactions and a major stochastic component in αß-lineage commitment.

T cell commitment and αß/γδ lineage specification in the thymus involves interactions between many different genes. Characterization of these interactions thus requires a multiparameter analysis of individual thymocytes. We developed two efficient single-cell methods: (i) the quantitative evaluation of the co-expression levels of nine different genes, with a plating efficiency of 99-100% and a detection limit of 2 mRNA molecules/cell; and (ii) single-cell differentiation cultures, in the presence of OP9 cells transfected with the thymus Notch1 ligand DeltaL4. We show that during T cell commitment, Gata3 has a fundamental, dose-dependent role in maintaining Notch1 expression, with thymocytes becoming T-cell-committed when they co-express Notch1, Gata3 and Bc11b. Of the transcription factor expression patterns studied here, only that of Bcl11b was suggestive of a role in Pu1 down-regulation. Individual thymocytes became αß/γδ lineage-committed at very different stages (from the TN2a stage onwards). However, 20% of TN3 cells are not αß/γδ-lineage committed and TN4 cells comprise two main subpopulations with different degrees of maturity. The existence of a correlation between differentiation potential and expression of the pre-TCR showed that 83% of αß-committed cells do not express the pre-TCR and revealed a major stochastic component in αß-lineage specification.

Clonal analysis reveals uniformity in the molecular profile and lineage potential of CCR9(+) and CCR9(-) thymus-settling progenitors.

The entry of T cell progenitors to the thymus marks the beginning of a multistage developmental process that culminates in the generation of self-MHC-restricted CD4(+) and CD8(+) T cells. Although multiple factors including the chemokine receptors CCR7 and CCR9 are now defined as important mediators of progenitor recruitment and colonization in both the fetal and adult thymi, the heterogeneity of thymus-colonizing cells that contribute to development of the T cell pool is complex and poorly understood. In this study, in conjunction with lineage potential assays, we perform phenotypic and genetic analyses on thymus-settling progenitors (TSP) isolated from the embryonic mouse thymus anlagen and surrounding perithymic mesenchyme, including simultaneous gene expression analysis of 14 hemopoietic regulators using single-cell multiplex RT-PCR. We show that, despite the known importance of CCL25-CCR9 mediated thymic recruitment of T cell progenitors, embryonic PIR(+)c-Kit(+) TSP can be subdivided into CCR9(+) and CCR9(-) subsets that differ in their requirements for a functional thymic microenvironment for thymus homing. Despite these differences, lineage potential studies of purified CCR9(+) and CCR9(-) TSP reveal a common bias toward T cell-committed progenitors, and clonal gene expression analysis reveals a genetic consensus that is evident between and within single CCR9(+) and CCR9(-) TSP. Collectively, our data suggest that although the earliest T cell progenitors may display heterogeneity with regard to their requirements for thymus colonization, they represent a developmentally homogeneous progenitor pool that ensures the efficient generation of the first cohorts of T cells during thymus development.