High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2.

Heterologous polyclonal antibodies might represent an alternative to the use of convalescent plasma or monoclonal antibodies (mAbs) in coronavirus disease (COVID-19) by targeting multiple antigen epitopes. However, heterologous antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrates, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the α1,3-galactose, potentially leading to serum sickness or allergy. Here, we immunized cytidine monophosphate-N-acetylneuraminic acid hydroxylase and α1,3-galactosyl-transferase (GGTA1) double KO pigs with the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor binding domain to produce glyco-humanized polyclonal neutralizing antibodies lacking Neu5Gc and α1,3-galactose epitopes. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10 000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized spike/angiotensin converting enzyme-2 interaction at a concentration <1 µg/mL, and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. We also found that pig GH-pAb Fc domains fail to interact with human Fc receptors, thereby avoiding macrophage-dependent exacerbated inflammatory responses and a possible antibody-dependent enhancement. These data and the accumulating safety advantages of using GH-pAbs in humans warrant clinical assessment of XAV-19 against COVID-19.

The inhibitory receptor CD94/NKG2A on CD8+ tumor-infiltrating lymphocytes in colorectal cancer: a promising new druggable immune checkpoint in the context of HLAE/ß2m overexpression.

We previously demonstrated that HLA-E/ß2m overexpression by tumor cells in colorectal cancers is associated with an unfavorable prognosis. However, the expression of its specific receptor CD94/NKG2 by intraepithelial tumor-infiltrating lymphocytes, their exact phenotype and function, as well as the relation with the molecular status of colorectal cancer and prognosis remain unknown. Based on a retrospective cohort of 234 colorectal cancer patients, we assessed the expression of HLA-E, ß2m, CD94, CD8, and NKp46 by immunohistochemistry on tissue microarray. The expression profile of HLA-E/ß2m on tumor cells and the density of tumor-infiltrating lymphocytes were correlated to the clinicopathological and molecular features (Microsatellite status, BRAF and RAS mutations). Then, from the primary tumors of 27 prospective colorectal cancers, we characterized by multiparameter flow cytometry the nature (T and/or NK cells) and the co-expression of the inhibitory NKG2A or activating NKG2C chain of ex vivo isolated CD94+ tumor-infiltrating lymphocytes. Their biological function was determined using an in vitro redirected cytolytic activity assay. Our results showed that HLA-E/ß2m was preferentially overexpressed in microsatellite instable tumors compared with microsatellite stable ones (45% vs. 19%, respectively, p = 0.0001), irrespective of the RAS or BRAF mutational status. However, HLA-E/ß2m+ colorectal cancers were significantly enriched in CD94+ intraepithelial tumor-infiltrating lymphocytes in microsatellite instable as well as in microsatellite stable tumors. Those CD94+ tumor-infiltrating lymphocytes mostly corresponded to CD8+ αß T cells, and  to a lesser extent to NK cells, and mainly co-expressed a functional inhibitory NKG2A chain. Finally, a high number of CD94+ intraepithelial tumor-infiltrating lymphocytes in close contact with tumor cells was independently associated with a worse overall survival. In conclusion, these findings strongly suggest that HLA-E/ß2m-CD94/NKG2A represents a new druggable inhibitory immune checkpoint, preferentially expressed in microsatellite instable tumors, but also in a subgroup of microsatellite stable tumors, leading to a new opportunity in colorectal cancer immunotherapies.

HCMV triggers frequent and persistent UL40-specific unconventional HLA-E-restricted CD8 T-cell responses with potential autologous and allogeneic peptide recognition.

Immune response against human cytomegalovirus (HCMV) includes a set of persistent cytotoxic NK and CD8 T cells devoted to eliminate infected cells and to prevent reactivation. CD8 T cells against HCMV antigens (pp65, IE1) presented by HLA class-I molecules are well characterized and they associate with efficient virus control. HLA-E-restricted CD8 T cells targeting HCMV UL40 signal peptides (HLA-EUL40) have recently emerged as a non-conventional T-cell response also observed in some hosts. The occurrence, specificity and features of HLA-EUL40 CD8 T-cell responses remain mostly unknown. Here, we detected and quantified these responses in blood samples from healthy blood donors (n = 25) and kidney transplant recipients (n = 121) and we investigated the biological determinants involved in their occurrence. Longitudinal and phenotype ex vivo analyses were performed in comparison to HLA-A*02/pp65-specific CD8 T cells. Using a set of 11 HLA-E/UL40 peptide tetramers we demonstrated the presence of HLA-EUL40 CD8 αßT cells in up to 32% of seropositive HCMV+ hosts that may represent up to 38% of total circulating CD8 T-cells at a time point suggesting a strong expansion post-infection. Host's HLA-A*02 allele, HLA-E *01:01/*01:03 genotype and sequence of the UL40 peptide from the infecting strain are major factors affecting the incidence of HLA-EUL40 CD8 T cells. These cells are effector memory CD8 (CD45RAhighROlow, CCR7-, CD27-, CD28-) characterized by a low level of PD-1 expression. HLA-EUL40 responses appear early post-infection and display a broad, unbiased, Vß repertoire. Although induced in HCMV strain-dependent, UL4015-23-specific manner, HLA-EUL40 CD8 T cells are reactive toward a broader set of nonapeptides varying in 1-3 residues including most HLA-I signal peptides. Thus, HCMV induces strong and life-long lasting HLA-EUL40 CD8 T cells with potential allogeneic or/and autologous reactivity that take place selectively in at least a third of infections according to virus strain and host HLA concordance.

Viral and tumor antigen-specific CD8 T-cell responses in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous cancer, which is immunogenic, regardless of the presence of MCPyV (80% of cases). The identification of MCC-specific epitopes recognized by CD8 T cells is crucial to expand the arsenal of immunotherapeutic treatments. Until now, most efforts focused on the identification of virus-specific epitopes, whereas immune responses directed against shared cellular tumor-specific antigens have not been evidenced. In this study, we measured T-cell responses against viral (n = 3) and tumor antigens (n = 47) from TILs derived from 21 MCC tumors. Virus-specific CD8 T-cell responses dominated MCC-specific immune responses, and we identified two new HLA-peptide complexes derived from the LT antigen, located in a region encompassing 3 previously identified epitopes. Finally, we show that MAGE-A3 antigen, frequently expressed by MCC tumors, was recognized by CD8 TILs from a virus-negative MCC tumor and thus could be a target for immunotherapy in this setting.

IL-9 promotes the survival and function of human melanoma-infiltrating CD4(+) CD8(+) double-positive T cells.

We previously demonstrated an accumulation of tumor-reactive CD4(+) CD8(+) double positive (DP) T cells within melanoma-infiltrating lymphocytes, supporting their role in the regulation of anti-tumor immune responses. Similarly to their CD8(+) counterparts, intra-tumor DP T cells are MHC class-I restricted but differed by a limited lytic activity against autologous melanoma cells. Based on these observations and to further characterize DP T cells, both populations were compared at the transcriptional level. Our results revealed the overexpression of the IL-9 receptor (IL-9R) by DP T cells and prompted us to investigate the impact of IL-9 on their biology. We show that IL-9 favors DP T-cell survival by protecting them from apoptosis and by promoting their proliferation. In addition, IL-9 enhances their ability to produce cytokines and increased their levels of granzyme B/perforin as well as degranulation capacity, leading to a strengthened cytotoxic activity against melanoma cells. Taken together, the IL-9R(high) DP T-cell population could be a new preferential target for IL-9, which could take part in their retention within the melanoma infiltrate while also favoring their anti-tumor activity. More generally, our results extend the pleiotropic effects of IL-9 to IL-9R-expressing intra-tumor T cells, which could further potentiate anti-tumor immune responses.

Soluble HLA-I/peptide monomers mediate antigen-specific CD8 T cell activation through passive peptide exchange with cell-bound HLA-I molecules.

Accumulating evidence that serum levels of soluble class I HLA molecules (sHLA-I) can, under various pathological conditions, correlate with disease stage and/or patient survival, has stimulated interest in defining whether sHLA-I can exert immunological functions. However, despite a mounting number of publications suggesting the ability of sHLA-I to affect immune effectors in vitro, the precise underlying mechanism still remains controversial. In this article, we address potential functions of both classical and nonclassical sHLA-I, using soluble recombinant HLA-I/peptide monomers, and clearly demonstrate their ability to trigger Ag-specific activation of CD8 T cells in vitro. Furthermore, we provide strong evidence that this behavior results from the passive transfer of peptides from monomers to T cell-bound HLA-I molecules, allowing for fratricide representation and activation. Hence, we proposed a unifying model of T cell activation by HLA-I/peptide monomers, reappraising the potential involvement of sHLA-I molecules in the immune response.

A microbiota-modulated checkpoint directs immunosuppressive intestinal T cells into cancers.

Antibiotics (ABX) compromise the efficacy of programmed cell death protein 1 (PD-1) blockade in cancer patients, but the mechanisms underlying their immunosuppressive effects remain unknown. By inducing the down-regulation of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the ileum, post-ABX gut recolonization by Enterocloster species drove the emigration of enterotropic α4ß7+CD4+ regulatory T 17 cells into the tumor. These deleterious ABX effects were mimicked by oral gavage of Enterocloster species, by genetic deficiency, or by antibody-mediated neutralization of MAdCAM-1 and its receptor, α4ß7 integrin. By contrast, fecal microbiota transplantation or interleukin-17A neutralization prevented ABX-induced immunosuppression. In independent lung, kidney, and bladder cancer patient cohorts, low serum levels of soluble MAdCAM-1 had a negative prognostic impact. Thus, the MAdCAM-1-α4ß7 axis constitutes an actionable gut immune checkpoint in cancer immunosurveillance.

Tumor-reactive CD4+ CD8αß+ CD103+ αßT cells: a prevalent tumor-reactive T-cell subset in metastatic colorectal cancers.

High level of T-cell infiltration in colorectal carcinomas (CRCs) is a good prognostic indicator, but the tumor reactivity of this infiltrate (tumor infiltrating lymphocytes [TIL]) is poorly documented. This study examined the presence, phenotype and functional features of tumor-reactive lymphocytes in human CRC. Freshly dissociated TIL and T cell lines were isolated from CRC samples and from some paired normal colonic mucosa. Four tumor cell lines were obtained. Autologous tumor reactivity of CRC TIL and tumor-reactive cell features were analyzed. We demonstrate the presence among CRC TIL of variable fractions (up to 18%) of double positive CD4(+) CD8αß(+) (DP) αß T cells. Interestingly, a high proportion (16-20%) of this TIL subset displayed tumor reactivity, whilst this was the case for no or few single positive TIL. Low levels of DP TIL were found in most CRC samples and in normal colonic mucosa, but these cells were higher in metastatic CRC. Furthermore, we showed that DP TIL were polyclonal, restricted by HLA class-I, proliferated poorly and secreted higher amounts of IL-4 and IL-13 than single positive T cells, on cognate or CD3 stimulation. DP CRC TIL also expressed CD103, confirming their mucosal origin. Increased frequencies of tumor-reactive DP TIL in metastatic CRC suggest that these cells play a role in the metastatic process of this cancer. Based on their high secretion of IL-4 and IL-13 and on previously described roles of these cytokines in cancers, we postulate that DP TIL could favor CRC growth or metastasis and/or downmodulate immune responses to these tumors.

A novel and efficient approach to high-throughput production of HLA-E/peptide monomer for T-cell epitope screening.

Over the past two decades, there has been a great interest in the study of HLA-E-restricted αß T cells during bacterial and viral infections, including recently SARS-CoV-2 infection. Phenotyping of these specific HLA-E-restricted T cells requires new tools such as tetramers for rapid cell staining or sorting, as well as for the identification of new peptides capable to bind to the HLA-E pocket. To this aim, we have developed an optimal photosensitive peptide to generate stable HLA-E/pUV complexes allowing high-throughput production of new HLA-E/peptide complexes by peptide exchange. We characterized the UV exchange by ELISA and improved the peptide exchange readout using size exclusion chromatography. This novel approach for complex quantification is indeed very important to perform tetramerization of MHC/peptide complexes with the high quality required for detection of specific T cells. Our approach allows the rapid screening of peptides capable of binding to the non-classical human HLA-E allele, paving the way for the development of new therapeutic approaches based on the detection of HLA-E-restricted T cells.

High-Density of FcγRIIIA+ (CD16+) Tumor-Associated Neutrophils in Metastases Improves the Therapeutic Response of Cetuximab in Metastatic Colorectal Cancer Patients, Independently of the HLA-E/CD94-NKG2A Axis.

Antibody-dependent cellular cytotoxicity (ADCC) in the anti-tumor effect of cetuximab in metastatic colorectal cancer (mCRC) is only based on the impact of FcγRIIIA (CD16) polymorphisms as predictive of therapeutic response. However, nature, density and therapeutic impact of FcγRIIIA+ (CD16) effector cells in tumor remain poorly documented. Moreover, the inhibition of cetuximab-mediated ADCC induced by NK cells by the engagement of the new inhibitory CD94-NKG2A immune checkpoint has only been demonstrated in vitro. This multicentric study aimed to determine, on paired primary and metastatic tissue samples from a cohort of mCRC patients treated with cetuximab: 1) the nature and density of FcγRIIIA+ (CD16) immune cells, 2) the expression profile of HLA-E/ß2m by tumor cells as well as the density of CD94+ immune cells and 3) their impact on both objective response to cetuximab and survival. We demonstrated that FcγRIIIA+ (CD16) intraepithelial immune cells mainly correspond to tumor-associated neutrophils (TAN), and their high density in metastases was significantly associated with a better response to cetuximab, independently of the expression of the CD94/NKG2A inhibitory immune checkpoint. However, HLA-E/ß2m, preferentially overexpressed in metastases compared with primary tumors and associated with CD94+ tumor infiltrating lymphocytes (TILs), was associated with a poor overall survival. Altogether, these results strongly support the use of bispecific antibodies directed against both EGFR and FcγRIIIA (CD16) in mCRC patients, to boost cetuximab-mediated ADCC in RAS wild-type mCRC patients. The preferential overexpression of HLA-E/ß2m in metastases, associated with CD94+ TILs and responsible for a poor prognosis, provides convincing arguments to inhibit this new immune checkpoint with monalizumab, a humanized anti-NKG2A antibody, in combination with anti- FcγRIIIA/EGFR bispecific antibodies as a promising therapeutic perspective in RAS wild-type mCRC patients.

The Caspase-1/IL-18 Axis of the Inflammasome in Tumor Cells: A Modulator of the Th1/Tc1 Response of Tumor-Infiltrating T Lymphocytes in Colorectal Cancer.

In colorectal cancer (CRC), a high density of T lymphocytes represents a strong prognostic marker in subtypes of CRC. Optimized immunotherapy strategies to boost this T-cell response are still needed. A good candidate is the inflammasome pathway, an emerging player in cancer immunology that bridges innate and adaptive immunity. Its effector protein caspase-1 matures IL-18 that can promote a T-helper/cytotoxic (Th1/Tc1) response. It is still unknown whether tumor cells from CRC possess a functional caspase-1/IL-18 axis that could modulate the Th1/Tc1 response. We used two independent cohorts of CRC patients to assess IL-18 and caspase-1 expression by tumor cells in relation to the density of TILs and the microsatellite status of CRC. Functional and multiparametric approaches at the protein and mRNA levels were performed on an ex vivo CRC explant culture model. We show that, in the majority of CRCs, tumor cells display an activated and functional caspase-1/IL-18 axis that contributes to drive a Th1/Tc1 response elicited by TILs expressing IL-18Rα. Furthermore, unsupervised clustering identified three clusters of CRCs according to the caspase-1/IL-18/TIL density/interferon gamma (IFNγ) axis and microsatellite status. Together, our results strongly suggest that targeting the caspase-1/IL-18 axis can improve the anti-tumor immune response in subgroups of CRC.

Transcriptomic features of tumour-infiltrating CD4lowCD8high double positive αß T cells in melanoma.

Peripheral CD4+CD8+ double positive (DP) T cells are a phenotypically and functionally heterogeneous population depending on their origin and pathologic context. We previously identified among tumour infiltrating lymphocytes in melanoma, a tumour-reactive MHC class-I restricted CD4lowCD8high DP αß T-cell subpopulation with CD4-like function. In this study, we used an in-depth comparative transriptomic analysis of intra-melanoma DP T cells and CD4 and CD8 single positive (SP) T cells, to better comprehend the origin of this DP phenotype, and define the transcriptomic signature of activated DP T cells. We observed that intra-melanoma DP T cells were transcriptome-wise closer to their CD8 SP T-cell counterparts in terms of number of genes differentially expressed (97 in common with CD8 SP T cells and 15 with CD4 SP T cells) but presented hallmarks of a transition to a CD4-like functional profile (CD40LG) with a decreased cytotoxic signature (KLRC1) in favour of an increased cytokine-receptor interaction signature (IL4, IL24, IL17A…). This unleashed CD4-like program could be the results of the observed unbalanced expression of the THPOK/Runx3 transcription factors in DP T cells. Overall, this study allow us to speculate that intra-melanoma DP T cells arise from CD8 SP T cells being reprogrammed to a helper function.

High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2.

Perfusion of convalescent plasma (CP) has demonstrated a potential to improve the pneumonia induced by SARS-CoV-2, but procurement and standardization of CP are barriers to its wide usage. Many monoclonal antibodies (mAbs) have been developed but appear insufficient to neutralize SARS-CoV-2 unless two or three of them are being combined. Therefore, heterologous polyclonal antibodies of animal origin, that have been used for decades to fight against infectious agents might represent a highly efficient alternative to the use of CP or mAbs in COVID-19 by targeting multiple antigen epitopes. However, conventional heterologous polyclonal antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrate epitopes, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the Gal α1,3-galactose (αGal), ultimately forming immune complexes and potentially leading to serum sickness or allergy. To circumvent these drawbacks, we engineered animals lacking the genes coding for the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) and α1,3-galactosyl-transferase (GGTA1) enzymes to produce glyco-humanized polyclonal antibodies (GH-pAb) lacking Neu5Gc and α-Gal epitopes. We found that pig IgG Fc domains fail to interact with human Fc receptors and thereby should confer the safety advantage to avoiding macrophage dependent exacerbated inflammatory responses, a drawback possibly associated with antibody responses against SARS-CoV-2 or to avoiding a possible antibody-dependent enhancement (ADE). Therefore, we immunized CMAH/GGTA1 double knockout (DKO) pigs with the SARS-CoV-2 spike receptor-binding domain (RBD) to elicit neutralizing antibodies. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10,000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized Spike/angiotensin converting enzyme-2 (ACE-2) interaction at a concentration < 1µg/mL and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. These data and the accumulating safety advantages of using glyco-humanized swine antibodies in humans warranted clinical assessment of XAV-19 to fight against COVID-19.

Increased frequency of nonconventional double positive CD4CD8 alphabeta T cells in human breast pleural effusions.

Breast cancer remains a leading cause of cancer-related death within the female population. Immunotherapy is expected to provide additional therapeutic benefits but has met so far limited success. This may be due in part to the poor understanding of immune responses to breast cancer. Although CD4(+) and CD8(+) T lymphocytes infiltrate these tumors, the phenotype and functions of these cells remain ill defined. This study was designed to investigate further about these questions, taking advantage of multiparameter flow cytometry on lymphocytes derived from peripheral blood, solid tumors, metastatic lymph nodes and pleural effusions samples of patients with breast cancer. Results showed that, in addition to conventional CD4(+) and CD8(+) alphabeta T cells, individual tumors and most pleural effusions contained significant fractions of unconventional double positive (DP) CD4(+)CD8(+) alphabeta T cells. These DP T cells displayed the phenotype and cytotoxic potential of effector/memory activated CD8(+) T cells but differed essentially from these cells by a high production of IL-5 and IL-13. The increased frequency of DP T cells in advanced breast cancer and their high lytic potential and original cytokine profile suggest that this T-cell subset may play a specific role in the regulation of immune responses to human breast cancer.

[Physiopathological mechanisms of immune-related adverse events induced by anti-CTLA-4, anti-PD-1 and anti-PD-L1 antibodies in cancer treatment]. / Mécanismes physiopathologiques des effets secondaires des immunothérapies par anticorps anti-CTLA-4, anti-PD-1 et anti-PD-L1 dans le traitement du cancer.

Recently, the emergence of anti-CTLA-4 and anti-PD-1/PD-L1 antibodies called immune check-point inhibitors (ICPI) has modified the landscape of anti-cancer treatments. These therapeutics are associated with immune related adverse events that affect many organs, most commonly skin, digestive tract, endocrine glands and lungs. This review summarizes the main physiopathological hypotheses on the mechanisms of these toxicities. In most cases, the T lymphocytes hyperactivation induced by ICPI generates a specific response directed against tumor antigens, leading to anti-tumor activity in tumor tissues but also side effects in normal tisues called "on-target". The CD8+ cytotoxic T lymphocytes-mediated cell lysis induces the release of neoantigens, tumor antigens and auto-antigens from normal tissues, respectively. This phenomenon called "epitope spreading" leads to diversification of the T cell repertoire and thus to reduced immune tolerance, which is exacerbated by inhibition of regulator T lymphocytes. Furthermore, the predominant activation of Th1 and Th17T lymphocytes mediated by ICPI induced an increased production of pro-inflammatory cytokines such as interferon-γ (IFNγ) and interleukine-17 (IL-17). These two mechanisms are responsible for the so called "off-target" toxicities. The roles of cross-reactivity with the intestinal microbiota, hypersensitivity and the specific effect of PD-L2 remain to be determined. Better knowledge of these mechanisms will improve patient care and help predict patients at risk of developing severe toxicities to ICPIs.

Corrigendum: Broad Impairment of Natural Killer Cells From Operationally Tolerant Kidney Transplanted Patients.

[This corrects the article on p. 1721 in vol. 8, PMID: 29312288.].

Synthetic anticancer vaccine candidates: rational design of antigenic peptide mimetics that activate tumor-specific T-cells.

A rational design approach was followed to develop peptidomimetic analogues of a cytotoxic T-cell epitope capable of stimulating T-cell responses as strong as or stronger (heteroclytic) than those of parental antigenic peptides. The work described herein focused on structural alterations of the central amino acids of the melanoma tumor-associated antigenic peptide Melan-A/MART-1(26-35) using nonpeptidic units. A screening was first realized in silico to select altered peptides potentially capable of fitting at the interface between the major histocompatibilty complex (MHC) class-I HLA-A2 molecule and T-cell receptors (TCRs). Two compounds appeared to be high-affinity ligands to the HLA-A2 molecule and stimulated several Melan-A/MART-1 specific T-cell clones. Most remarkably, one of them even managed to amplify the response of one clone. Together, these results indicate that central TCR-contact residues of antigenic peptides can be replaced by nonpeptidic motifs without loss of binding affinity to MHC class-I molecules and T-cell triggering capacity.

Emergence of High-Avidity Melan-A-Specific Clonotypes as a Reflection of Anti-PD-1 Clinical Efficacy.

Therapeutic strategies using anti-PD-1-blocking antibodies reported unparalleled effectiveness for melanoma immunotherapy, but deciphering immune responses modulated by anti-PD-1 treatment remains a crucial issue. Here, we analyzed the composition and functions of the large Melan-A-specific T-cell repertoire in the peripheral blood of 9 melanoma patients before and after 2 months of treatment with anti-PD-1. We observed amplification of Melan-A-specific Vß subfamilies undetectable before therapy (thereafter called emerging Vß subfamilies) in responding patients, with a predominant expansion in patients with a complete response. These emerging Vß subfamilies displayed a higher functional avidity for their cognate antigen than Vß subfamilies not amplified upon anti-PD-1 therapy and could be identified by a sustained coexpression of PD-1 and TIGIT receptors. Thus, in addition to the emergence of neoantigen-specific T cells previously documented upon anti-PD-1 therapy, our work describes the emergence of high-avidity Melan-A-specific clonotypes as a surrogate marker of treatment efficacy. Cancer Res; 77(24); 7083-93. ©2017 AACR.

Broad Impairment of Natural Killer Cells from Operationally Tolerant Kidney Transplanted Patients.

The role of natural killer (NK) cells in organ transplantation is controversial. This study aims to decipher their role in kidney transplant tolerance in humans. Previous studies highlighted several modulated genes involved in NK cell biology in blood from spontaneously operationally tolerant patients (TOLs; drug-free kidney-transplanted recipients with stable graft function). We performed a phenotypic, functional, and genetic characterization of NK cells from these patients compared to kidney-transplanted patients with stable graft function under immunosuppression and healthy volunteers (HVs). Both operationally TOLs and stable patients harbored defective expression of the NKp46 activator receptor and lytic molecules perforin and granzyme compared to HVs. Surprisingly, NK cells from operationally TOLs also displayed decreased expression of the CD16 activating marker (in the CD56Dim NK cell subset). This decrease was associated with impairment of their functional capacities upon stimulation, as shown by lower interferon gamma (IFNγ) production and CD107a membranous expression in a reverse antibody-dependent cellular cytotoxicity (ADCC) assay, spontaneous lysis assays, and lower target cell lysis in the 51Cr release assay compared to HVs. Conversely, despite impaired K562 cell lysis in the 51Cr release assay, patients with stable graft function harbored a normal reverse ADCC and even increased amounts of IFNγ+ NK cells in the spontaneous lysis assay. Altogether, the strong impairment of the phenotype and functional cytotoxic capacities of NK cells in operationally TOLs may accord with the establishment of a pro-tolerogenic environment, despite remaining highly activated after transplantation in patients with stable graft function.