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Abstract Invariant natural killer T (NKT) cell subsets are defined based on their cytokine-production profiles and transcription factors. Their distribution is different in C57BL/6 (B6) and BALB/c mice, with a bias for NKT1 and NKT2/NKT17 subsets, respectively. Here, we show that the non-classical class I-like major histocompatibility complex CD1 molecules CD1d2, expressed in BALB/c and not in B6 mice, could not account for this difference. We find however that NKT cell subset distribution is intrinsic to bone marrow derived NKT cells, regardless of syngeneic CD1d-ligand recognition, and that multiple intrinsic factors are likely involved. Finally, we find that CD1d expression levels in combination with T cell antigen receptor signal strength could also influence NKT cell distribution and function. Overall, this study indicates that CD1d-mediated TCR signals and other intrinsic signals integrate to influence strain-specific NKT cell differentiation programs and subset distributions.

Myelodysplastic syndromes are characterised by clonal haematopoiesis, with the affected cells often harbouring mutations in the TET2 gene, an important regulator of DNA methylation state. Here authors show that the same mutations are also found in NK cells, perturbing their DNA methylation pattern and cytolytic function.

Summary: Oxylipins are major immunomodulating mediators, yet studies of inflammation focus mainly on cytokines. Here, using a standardized whole-blood stimulation system, we characterized the oxylipin-driven inflammatory responses to various stimuli and their relationships with cytokine responses. We performed a pilot study in 25 healthy individuals using 6 different stimuli: 2 bacterial stimuli (LPS and live BCG), 2 viral stimuli (vaccine-grade poly I:C and live H1N1 attenuated influenza), an enterotoxin superantigen and a Null control. All stimuli induced a strong production of oxylipins but most importantly, bacterial, viral, and T cell immune responses show distinct oxylipin signatures. Integration of the oxylipin and cytokine responses for each condition revealed new immune networks improving our understanding of inflammation regulation. Finally, the oxylipin responses and oxylipin-cytokine networks were compared in patients with active tuberculosis or with latent infection. This revealed different responses to BCG but not LPS stimulation highlighting new regulatory pathways for further investigations.

Innate lymphoid cells (ILCs) – which include cytotoxic Natural Killer (NK) cells and helper-type ILC – are important regulators of tissue immune homeostasis, with possible roles in tumor surveillance. We analyzed ILC and their functionality in human lymph nodes (LN). In LN, NK cells and ILC3 were the prominent subpopulations. Among the ILC3s, we identified a CD56+/ILC3 subset with a phenotype close to ILC3 but also expressing cytotoxicity genes shared with NK. In tumor-draining LNs (TD-LNs) and tumor samples from breast cancer (BC) patients, NK cells were prominent, and proportions of ILC3 subsets were low. In tumors and TD-LN, NK cells display reduced levels of NCR (Natural cytotoxicity receptors), despite high transcript levels and included a small subset CD127− CD56− NK cells with reduced function. Activated by cytokines CD56+/ILC3 cells from donor and patients LN acquired cytotoxic capacity and produced IFNg. In TD-LN, all cytokine activated ILC populations produced TNFα in response to BC cell line. Analyses of cytotoxic and helper ILC indicate a switch toward NK cells in TD-LN. The local tumor microenvironment inhibited NK cell functions through downregulation of NCR, but cytokine stimulation restored their functionality.

Lympho-epithelial interactions between intestinal T resident memory cells (Trm) and the epithelium have been associated with inflammatory bowel disease (IBD) activity. We developed ex vivo autologous organoid-mucosal T cell cocultures to functionally assess lymphoepithelial interactions in Crohn’s Disease (CD) patients compared to controls. We demonstrate the direct epithelial cell death induced by autologous mucosal T cells in CD patients but not in controls. These findings were positively correlated with T cell infiltration of the organoids. This potential was inhibited by limiting lympho-epithelial interactions through CD103 and NKG2D blocking antibodies. These data directly demonstrate for the first time the direct deleterious effect of mucosal T cells on the epithelium of CD patients. Such ex-vivo models are promising techniques to unravel the pathophysiology of these diseases and the potential mode of action of current and future therapies.

Background Prostate cancer (PC) responds to androgen deprivation therapy (ADT) usually in a transient fashion, progressing from hormone-sensitive PC (HSPC) to castration-resistant PC (CRPC). We investigated a mouse model of PC as well as specimens from PC patients to unravel an unsuspected contribution of thymus-derived T lymphocytes and the intestinal microbiota in the efficacy of ADT.Methods Preclinical experiments were performed in PC-bearing mice, immunocompetent or immunodeficient. In parallel, we prospectively included 65 HSPC and CRPC patients (Oncobiotic trial) to analyze their feces and blood specimens.Results In PC-bearing mice, ADT increased thymic cellularity and output. PC implanted in T lymphocyte-depleted or athymic mice responded less efficiently to ADT than in immunocompetent mice. Moreover, depletion of the intestinal microbiota by oral antibiotics reduced the efficacy of ADT. PC reduced the relative abundance of Akkermansia muciniphila in the gut, and this effect was reversed by ADT. Moreover, cohousing of PC-bearing mice with tumor-free mice or oral gavage with Akkermansia improved the efficacy of ADT. This appears to be applicable to PC patients because long-term ADT resulted in an increase of thymic output, as demonstrated by an increase in circulating recent thymic emigrant cells (sjTRECs). Moreover, as compared with HSPC controls, CRPC patients demonstrated a shift in their intestinal microbiota that significantly correlated with sjTRECs. While feces from healthy volunteers restored ADT efficacy, feces from PC patients failed to do so.Conclusions These findings suggest the potential clinical utility of reversing intestinal dysbiosis and repairing acquired immune defects in PC patients.

Summary: Background: Advanced age is accompanied by a decline of immune functions, which may play a role in increased vulnerability to emerging pathogens and low efficacy of primary vaccinations in elderly people. The capacity to mount immune responses against new antigens is particularly affected in this population. However, its precise determinants are not fully understood. We aimed here at establishing the influence of persistent viral infections on the naive T-cell compartment and primary immune responsiveness in older adults. Methods: We assessed immunological parameters, related to CD8+ and CD4+ T-cell responsiveness, according to the serological status for common latent herpesviruses in two independent cohorts: 1) healthy individuals aged 19y to 95y (n = 150) and 2) individuals above 70y old enrolled in a primo-vaccination clinical trial (n = 137). Findings: We demonstrate a prevalent effect of age and CMV infection on CD8+ and CD4+ naive T cells, respectively. CMV seropositivity was associated with blunted CD4+ T-cell and antibody responses to primary vaccination. Interpretation: These data provide insights on the changes in adaptive immunity over time and the associated decline in vaccine efficacy with ageing. This knowledge is important for the management of emerging infectious diseases in elderly populations.

Abstract Objectives Allogeneic haematopoietic stem cell transplantation (allo‐HSCT) is the only currently available curative treatment for sickle cell disease (SCD). Here, we comprehensively evaluated the reconstitution of T‐ and B‐cell compartments in 29 SCD patients treated with allo‐HSCT and how it correlated with the development of acute graft‐versus‐host disease (aGvHD). Methods T‐cell neogenesis was assessed by quantification of signal‐joint and β‐chain TCR excision circles. B‐cell neogenesis was evaluated by quantification of signal‐joint and coding‐joint K‐chain recombination excision circles. T‐ and B‐cell peripheral subset numbers were assessed by flow cytometry. Results Before allo‐HSCT (baseline), T‐cell neogenesis was normal in SCD patients compared with age‐, gender‐ and ethnicity‐matched healthy controls. Following allo‐HSCT, T‐cell neogenesis declined but was fully restored to healthy control levels at one year post‐transplantation. Peripheral T‐cell subset counts were fully restored only at 24 months post‐transplantation. Occurrence of acute graft‐versus‐host disease (aGvHD) transiently affected T‐ and B‐cell neogenesis and overall reconstitution of T‐ and B‐cell peripheral subsets. B‐cell neogenesis was significantly higher in SCD patients at baseline than in healthy controls, remaining high throughout the follow‐up after allo‐HSCT. Notably, after transplantation SCD patients showed increased frequencies of IL‐10‐producing B‐regulatory cells and IgM+ memory B‐cell subsets compared with baseline levels and with healthy controls. Conclusion Our findings revealed that the T‐ and B‐cell compartments were normally reconstituted in SCD patients after allo‐HSCT. In addition, the increase of IL‐10‐producing B‐regulatory cells may contribute to improve immune regulation and homeostasis after transplantation.

Although the risk of developing lymphoma has decreased in the highly active antiretroviral therapy era, this cancer remains the major cause of mortality in HIV-infected patients. Autologous hematopoietic stem cell transplantation (ASCT) outcome does not differ for HIV-infected versus HIV-uninfected patients. We propose to develop a new treatment for HIV-associated high-risk lymphoma based on autologous transplantation of two genetically modified products: CD4+ T lymphocytes and CD34+ hematopoietic stem cells (HSPCs). The cells will be transduced ex vivo with the Cal-1 lentiviral vector encoding for both a short hairpin RNA (shRNA) against CCR5 (sh5) and the HIV-1 fusion inhibitor C46. The transduced cells will be resistant to HIV infection by two complementary mechanisms: impaired binding of the virus to the cellular CCR5 co-receptor and decreased fusion of the virus as C46 interacts with gp41 and inhibits HIV infection. This phase I/II pilot study, also entitled GENHIV, will involve two French participating centers: Saint Louis Hospital and Necker Hospital in Paris. We plan to enroll five HIV-1-infected patients presenting with high-risk lymphoma and require a treatment with ASCT. The primary objective of this study is to evaluate the safety, feasibility, and success of engraftment of Cal-1 gene-transduced CD4+ T lymphocytes and CD34+ HSPCs.

Abstract Background Immunotherapies still fail to benefit colorectal cancer (CRC) patients. Relevant functional assays aimed at studying these failures and the efficacy of cancer immunotherapy in human are scarce. 3D tumor cultures, called tumor organoids or spheroids, represent interesting models to study cancer treatments and could help to challenge these issues. Methods We analyzed heterotypic cocultures of human colon tumor-derived spheroids with immune cells to assess the infiltration, activation and function of T and NK cells toward human colorectal tumors in vitro. Results We showed that allogeneic T and NK cells rapidly infiltrated cell line-derived spheroids, inducing immune-mediated tumor cell apoptosis and spheroid destruction. NKG2D, a key activator of cytotoxic responses, was engaged on infiltrating cells. We thus assessed the therapeutic potential of an antibody targeting the specific ligands of NKG2D, MICA and MICB, in this system. Anti-MICA/B enhanced immune-dependent destruction of tumor spheroid by driving an increased NK cells infiltration and activation. Interestingly, tumor cells reacted to immune infiltration by upregulating HLA-E, ligand of the inhibitory receptor NKG2A expressed by CD8 and NK cells. NKG2A was increased after anti-MICA/B treatment and, accordingly, combination of anti-MICA/B and anti-NKG2A was synergistic. These observations were ultimately confirmed in a clinical relevant model of coculture between CRC patients-derived spheroids and autologous tumor-infiltrating lymphocytes. Conclusions Altogether, we show that tumor spheroids represent a relevant tool to study tumor-lymphocyte interactions on human tissues and revealed the antitumor potential of immunomodulatory antibodies targeting MICA/B and NKG2A.

Introduction: Burn injury is associated with a high risk of death. Whether a pattern of immune and inflammatory responses after burn is associated with outcome is unknown. The aim of this study was to explore the association between systemic immune and inflammatory responses and outcome in severely-ill burn patients.Materials and Methods: Innate immunity, adaptive immunity, activation and stress and inflammation biomarkers were collected at admission and days 2, 7, 14, and 28 in severely-ill adult burn patients. Primary endpoint was mortality at day 90, secondary endpoint was secondary infections. Healthy donors (HD) served as controls. Multiple Factorial Analysis (MFA) was used to identify patterns of immune response.Results: 50 patients were included. Age was 49.2 (44.2–54.2) years, total burn body surface area was 38.0% (32.7–43.3). Burn injury showed an upregulation of adaptive immunity and activation biomarkers and a down regulation of innate immunity and stress/inflammation biomarkers. High interleukin-10 (IL-10) at admission was associated with risk of death. However, no cluster of immune/inflammatory biomarkers at early timepoints was associated with mortality. HLA-DR molecules on monocytes at admission were associated with bacterial infections and septic shock. Later altered immune/inflammatory responses in patients who died may had been driven by the development of septic shock.Conclusion: Burn injury induced an early and profound upregulation of adaptive immunity and activation biomarkers and a down regulation of innate immunity and stress/inflammation biomarkers. Immune and inflammatory responses were associated with bacterial infection and septic shock. Absence of immune recovery patterns was associated with poor prognosis.

The LabEx Milieu Interieur (MI) project is a clinical study centered on the detailed characterization of the baseline and induced immune responses in blood samples from 1,000 healthy donors. Analyses of these samples has lay ground for seminal studies on the genetic and environmental determinants of immunologic variance in a healthy cohort population. In the current study we developed in vitro methods enabling standardized quantification of MI-cohort-derived primary fibroblasts responses. Our results show that in vitro human donor cohort fibroblast responses to stimulation by different MAMPs analogs allows to characterize individual donor immune-phenotype variability. The results provide proof-of-concept foundation to a new experimental framework for such studies. A bio-bank of primary fibroblast lines was generated from 323 out of 1,000 healthy individuals selected from the MI-study cohort. To study inter-donor variability of innate immune response in primary human dermal fibroblasts we chose to measure the TLR3 and TLR4 response pathways, both receptors being expressed and previously studied in fibroblasts. We established high-throughput automation compatible methods for standardized primary fibroblast cell activation, using purified MAMPS analogs, poly I:C and LPS that stimulate TLR3 and TLR4 pathways respectively. These results were in turn compared with a stimulation method using infection by HSV-1 virus. Our “Add-only” protocol minimizes high-throughput automation system variability facilitating whole process automation from cell plating through stimulation to recovery of cell supernatants, and fluorescent labeling. Images were acquired automatically by high-throughput acquisition on an automated high-content imaging microscope. Under these methodological conditions standardized image acquisition provided for quantification of cellular responses allowing biological variability to be measured with low system noise and high biological signal fidelity. Optimal for automated analysis of immuno-phenotype of primary human cell responses our method and experimental framework as reported here is highly compatible to high-throughput screening protocols like those necessary for chemo-genomic screening. In context of primary fibroblasts derived from donors enrolled to the MI-clinical-study our results open the way to assert the utility of studying immune-phenotype characteristics relevant to a human clinical cohort.

Chronic graft-versus-host disease (cGvHD) is a severe complication of allogeneic hematopoietic stem cell transplantation that affects various organs leading to a reduced quality of life. The condition often requires enduring immunosuppressive therapy, which can also lead to the development of severe side effects. Several approaches including small molecule inhibitors, antibodies, cytokines, and cellular therapies are now being developed for the treatment of cGvHD, and some of these therapies have been or are currently tested in clinical trials. In this review, we discuss these emerging therapies with particular emphasis on tyrosine kinase inhibitors (TKIs). TKIs are a class of compounds that inhibits tyrosine kinases, thereby preventing the dissemination of growth signals and activation of key cellular proteins that are involved in cell growth and division. Because they have been shown to inhibit key kinases in both B cells and T cells that are involved in the pathophysiology of cGvHD, TKIs present new promising therapeutic approaches. Ibrutinib, a Bruton tyrosine kinase (Btk) inhibitor, has recently been approved by the Food and Drug Administration (FDA) in the United States for the treatment of adult patients with cGvHD after failure of first-line of systemic therapy. Also, Janus Associated Kinases (JAK1 and JAK2) inhibitors, such as itacitinib (JAK1) and ruxolitinib (JAK1 and 2), are promising in the treatment of cGvHD. Herein, we present the current status and future directions of the use of these new drugs with particular spotlight on their targeting of specific intracellular signal transduction cascades important for cGvHD, in order to shed some light on their possible mode of actions.

Background Targeted therapies and immunotherapies are first-line treatments for patients with advanced melanoma. Serine–threonine protein kinase B-RAF (BRAF) and mitogen-activated protein kinase (MEK) inhibition leads to a 70% response rate in patients with advanced melanoma with a BRAFV600E/K mutation. However, acquired resistance occurs in the majority of patients, leading to relapse. Immunotherapies that activate immune cytotoxic effectors induce long-lasting responses in 30% of patients. In that context, combination of targeted therapies with immunotherapy (IT) is a promising approach. We considered boosting natural killer (NK) cell tumor immunosurveillance, as melanoma cells express stress-induced molecules and activate NK cell lysis.Methods Here we have generated vemurafenib (a BRAF inihibitor)-resistant (R) cells from BRAFV600E SK28 and M14-sensitive (S) melanoma cell lines and investigated how resistance interferes with immunogenicity to NK cells. We determined the levels of several soluble molecules including NK ligands in 61 melanoma patients at baseline and 6 months M post-treatment with targeted therapies or immunotherapies.Results Vemurafenib resistance involved activation of p-AKT in SK28R and of p-MEK/p-ERK in M14R cells and was accompanied by modulation of NK ligands. Compared with S cells, SK28R displayed an increased expression of natural killer group 2 D (NKG2D) receptor ligands (major histocompatibility complex class (MHC) I chain-related protein A (MICA) and UL16-binding protein 2 (ULBP2)) whereas M14R exhibited decreased ULBP2 . SK28R and M14R cells induced higher NK degranulation and interferon gamma secretion and were more efficiently lysed by donor and patient NK cells. SK28R showed increased tumor necrosis factor-related apoptosis-inducing ligand receptor II (TRAIL-RII) expression and TRAIL-induced apoptosis, and TRAIL-induced apoptosis of M14R was decreased. Combined BRAF/MEK inhibitors abrogated the growth of SK28S, M14S, and M14R cells, while growth of SK28R was maintained. BRAF/MEK inhibition attenuated NK activity but R cell lines activated polyfunctional NK cells and were lysed with high efficiency. We investigated the relationship of soluble NK ligands and response to treatment in a series of melanoma patients. Soluble NKG2D ligands known to regulate the receptor function have been associated to cancer progression. Serum analysis of patients treated with target therapies or IT indicates that soluble forms of NK ligands (MICA, B7H6, programmed cell death ligand 1, and carcinoembryonic antigen cell adhesion molecule 1) may correlate with clinical response.Conclusion These results support strategies combining targeted therapies and NK-based immunotherapies.

Hematopoietic stem cell transplantation (HSCT) is an effective treatment option for several malignant and non-malignant hematological diseases. The clinical outcome of this procedure relies to a large extent on optimal recovery of adaptive immunity. In this regard, the thymus plays a central role as the primary site for de novo generation of functional, diverse, and immunocompetent T-lymphocytes. The thymus is exquisitely sensitive to several insults during HSCT, including conditioning drugs, corticosteroids, infections, and graft-vs.-host disease. Impaired thymic recovery has been clearly associated with increased risk of opportunistic infections and poor clinical outcomes in HSCT recipients. Therefore, better understanding of thymic function can provide valuable information for improving HSCT outcomes. Recent data have shown that, besides gender and age, a specific single-nucleotide polymorphism affects thymopoiesis and may also influence thymic output post-HSCT, suggesting that the time of precision medicine of thymic function has arrived. Here, we review the current knowledge about thymic role in HSCT and the recent work of genetic control of human thymopoiesis. We also discuss different transplant-related factors that have been associated with impaired thymic recovery and the use of T-cell receptor excision circles (TREC) to assess thymic output, including its clinical significance. Finally, we present therapeutic strategies that could boost thymic recovery post-HSCT.

Abstract: To evaluate the immunological mechanisms associated with clinical outcomes after autologous hematopoietic stem cell transplantation (AHSCT), focusing on regulatory T- (Treg) and B- (Breg) cell immune reconstitution, 31 systemic sclerosis (SSc) patients underwent simultaneous clinical and immunological evaluations over 36-month posttransplantation follow-up. Patients were retrospectively grouped into responders (n = 25) and nonresponders (n = 6), according to clinical response after AHSCT. Thymic function and B-cell neogenesis were respectively assessed by quantification of DNA excision circles generated during T- and B-cell receptor rearrangements. At the 1-year post-AHSCT evaluation of the total set of transplanted SSc patients, thymic rebound led to renewal of the immune system, with higher T-cell receptor (TCR) diversity, positive correlation between recent thymic emigrant and Treg counts, and higher expression of CTLA-4 and GITR on Tregs, when compared with pretransplant levels. In parallel, increased bone marrow output of newly generated naive B-cells, starting at 6 months after AHSCT, renovated the B-cell populations in peripheral blood. At 6 and 12 months after AHSCT, Bregs increased and produced higher interleukin-10 levels than before transplant. When the nonresponder patients were evaluated separately, Treg and Breg counts did not increase after AHSCT, and high TCR repertoire overlap between pre- and posttransplant periods indicated maintenance of underlying disease mechanisms. These data suggest that clinical improvement of SSc patients is related to increased counts of newly generated Tregs and Bregs after AHSCT as a result of coordinated thymic and bone marrow rebound.

Abstract The determinants of clinical responses after autologous hematopoietic stem cell transplantation (aHSCT) in systemic sclerosis (SSc) are still unraveled. We analyzed long-term immune reconstitution (IR) and T cell receptor (TCR) repertoire diversity in 10 SSc patients, with at least 6 years simultaneous clinical and immunological follow-up after aHSCT. Patients were retrospectively classified as long-term responders (A, n = 5) or non-responders (B, n = 5), using modified Rodnan’s skin score (mRSS) and forced vital capacity (FVC%). All patients had similar severe SSc before aHSCT. Number of reinjected CD34+ cells was higher in group B versus A (P = 0.02). Long-term mRSS fall >25% was more pronounced in group A (P = 0.004), the only to improve long-term FVC% >10% (P = 0.026). There was an overall trend toward increased of T cell reconstitution in group B versus A. B cells had a positive linear regression slope in group A (LRS = 11.1) and negative in group B (LRS = −11.6). TCR repertoire was disturbed before aHSCT and the percentage of polyclonal families significantly increased at long-term (P = 0.046), with no difference between groups. Despite improved skin score after aHSCT in all SSc patients, pretransplant B cell clonal expansion and faster post-transplant T cell IR in long-term non-responder/relapsing patients call for new therapeutic protocols guided by IR analysis to improve their outcome.

The antitumor functions of NK cells are regulated by the integration of positive and negative signals triggered by numerous membrane receptors present on the NK cells themselves. Among the main activating receptors, NKG2D binds several stress-induced molecules on tumor targets. Engagement of NKG2D by its ligands (NKG2D-Ls) induces NK cell activation leading to production of cytokines and target cell lysis. These effects have therapeutic potential as NKG2D-Ls are widely expressed by solid tumors, whereas their expression in healthy cells is limited. Here, we describe the genetic and environmental factors regulating the NKG2D/NKG2D-L pathway in tumors. NKG2D-L expression is linked to cellular stress and cell proliferation, and has been associated with oncogenic mutations. Tumors have been found to alter their to NKG2D-L expression as they progress, which interferes with the antitumor function of the pathway. Nevertheless, this pathway could be advantageously exploited for cancer therapy. Various cancer treatments, including chemotherapy and targeted therapies, indirectly interfere with the cellular and soluble forms of NKG2D-Ls. In addition, NKG2D introduced into chimeric antigen receptors in T- and NK cells is a promising tumor immunotherapy approach.

Background: Characterization of the intracellular biochemical processes that regulate the generation and maintenance of effector and memory CD8+ T-cells from naïve precursors is essential for our understanding of adaptive immune responses and the development of immunotherapies. However, the metabolic determinants of antigen-driven activation and differentiation remain poorly defined, especially in humans.Methods: We used a variety of different approaches, including gene expression profiling and measurements of nutrient flux, to characterize the basal and activation-induced energetic requirements of naïve and phenotypically-defined subsets of human memory CD8+ T-cells.Findings: Profound metabolic differences were apparent as a function of differentiation status, both at rest and in response to stimulation via the T cell receptor (TCR). Of particular note, resting naïve CD8+ T cells were largely quiescent, but rapidly upregulated diverse energetic pathways after ligation of surface-expressed TCRs. Moreover, autophagy and the mechanistic target of rapamycin (mTOR)-dependent glycolytic pathway were identified as critical mediators of antigen-driven priming in the naïve CD8+ T cell pool, the efficiency of which was dampened by the presence of neutral lipids and fatty acids.Interpretation: These observations provide a metabolic roadmap of the CD8+ T-cell compartment in humans and reveal potentially selective targets for novel immunotherapies.

Systems approaches for the study of immune signaling pathways have been traditionally based on purified cells or cultured lines. However, in vivo responses involve the coordinated action of multiple cell types, which interact to establish an inflammatory microenvironment. We employed standardized whole-blood stimulation systems to test the hypothesis that responses to Toll-like receptor ligands or whole microbes can be defined by the transcriptional signatures of key cytokines. We found 44 genes, identified using Support Vector Machine learning, that captured the diversity of complex innate immune responses with improved segregation between distinct stimuli. Furthermore, we used donor variability to identify shared inter-cellular pathways and trace cytokine loops involved in gene expression. This provides strategies for dimension reduction of large datasets and deconvolution of innate immune responses applicable for characterizing immunomodulatory molecules. Moreover, we provide an interactive R-Shiny application with healthy donor reference values for induced inflammatory genes.

Natural killer group 2D (NKG2D) is a well-characterized activating receptor expressed on many immune cells, including invariant natural killer T (iNKT) cells. These cells were shown to be responsible of liver injury in the model of concanavalin A (Con A)-induced hepatitis, considered to be an experimental model of human autoimmune hepatitis. In this study, we investigated whether NKG2D plays a role in the hepatitis induced by iNKT cell-mediated immune response to Con A. By using killer cell lectin-like receptor subfamily K, member 1 deficient (Klrk1−/−) mice, we found that the absence of NKG2D reduced the hepatic injury upon Con A administration. This was not due to an intrinsic functional defect of NKG2D-deficient iNKT cells as mice missing NKG2D have normal distribution and function of iNKT cells. Furthermore, increased resistance to Con A-induced hepatitis was confirmed using neutralizing anti-NKG2D antibodies. The reduced pathogenic effect of Con A in the absence of NKG2D correlates with a reduction in pathogenic cytokine production and FAS-Ligand (FAS-L) expression by iNKT cells. We also found that Con A administration led to an increase in the retinoic acid early inducible (RAE-1) surface expression on wild-type hepatocytes. Finally, we found that Con A has no direct action on FAS-L expression or cytokine production by iNKT cells and thus propose that NKG2D-L expression on stressed hepatocytes promote cytotoxic activity of iNKT cells via its interaction with NKG2D contributing to hepatic injury. In conclusion, our results highlight NKG2D as an essential receptor required for the activation of iNKT cells in Con A-induced hepatitis and indicate that it represents a potential drug target for prevention of autoimmune hepatitis.

NKp46 is a major determinant of natural killer (NK) cell function and it is implicated in tumor immune surveillance in acute myeloid leukemia (AML). The purpose of this study was to investigate the prognostic significance of NKp46 expression in an independent cohort of patients with AML, and to investigate the impact of NKp46 on clinical outcome after allogeneic stem cell transplantation (allo-SCT). NKp46 expression was assessed at diagnosis on NK cells by flow cytometry (N = 180 patients). Clinical outcome was evaluated with regard to NKp46 expression. Patients with NKp46high phenotype at diagnosis had better progression-free survival (PFS) and overall survival (OS) than patients with NKp46low phenotype (74.3% vs. 46.6%, p = 0.014; 82.6% vs. 57.1%, p = 0.010, respectively). In multivariate analysis, high NKp46 was an independent factor for improved OS (HR = 0.409, p = 0.010) and PFS (HR = 0.335, p = 0.011). Subgroup analysis revealed that allo-SCT had a favorable impact on PFS in patients with NKp46high phenotype (p = 0.025). By contrast, allo-SCT did not impact PFS in patients with low NKp46 expression (p = 0.303). In conclusion, we validate the prognostic value of NKp46 expression at diagnosis in AML. However, the prognostic value of NKp46 expression is limited to patients treated with allo-SCT, thus suggesting that NKp46 status may be predictive for allo-SCT responsiveness.

Despite persistence of leukemic stem cells, patients with chronic myeloid leukemia who achieve and maintain deep molecular responses may successfully stop the tyrosine kinase inhibitor imatinib. However, questions remain unanswered regarding the biological basis of molecular relapse after imatinib cessation. In IMMUNOSTIM, we monitored 51 patients from the French Stop IMatinib trial for peripheral blood T cells and natural killer cells. Molecular relapse-free survival at 24 months was 45.1% (95% CI: 31.44%–58.75%). At the time of imatinib discontinuation, non-relapsing patients had significantly higher numbers of natural killer cells of the cytotoxic CD56dim subset than had relapsing patients, while CD56bright natural killer cells, T cells and their subsets did not differ significantly. Furthermore, the CD56dim natural killer-cell count was an independent prognostic factor of molecular-relapse free survival in a multivariate analysis. However, expression of natural killer-cell activating receptors, BCR-ABL1+ leukemia cell line K562-specific degranulation and cytokine-induced interferon-gamma secretion were decreased in non-relapsing and relapsing patients as compared with healthy individuals. After imatinib cessation, the natural killer-cell count increased significantly and stayed higher in non-relapsing patients than in relapsing patients, while receptor expression and functional properties remained unchanged. Altogether, our results suggest that natural killer cells may play a role in controlling leukemia-initiating cells at the origin of relapse after imatinib cessation, provided that these cells are numerous enough to compensate for their functional defects. Further research will decipher mechanisms underlying functional differences between natural killer cells from patients and healthy individuals and evaluate the potential interest of immunostimulatory approaches in tyrosine kinase inhibitor discontinuation strategies. (ClinicalTrial.gov Identifier NCT00478985)

Natural killer (NK) cells are potent antitumor effectors, involved in hematological malignancies and solid tumor immunosurveillance. They infiltrate various solid tumors, and their numbers are correlated with good outcome. The function of NK cells extends their lytic capacities toward tumor cells expressing stress-induced ligands, through secretion of immunoregulatory cytokines, and interactions with other immune cells. Altered NK cell function due to tumor immune escape is frequent in advanced tumors; however, strategies to release the function of NK infiltrating tumors are emerging. Recent therapies targeting specific oncogenic mutations improved the treatment of cancer patients, but patients often relapse. The actual development consists in combined therapeutic strategies including agents targeting the proliferation of tumor cells and others restorating functional antitumor immune effectors for efficient and durable efficacy of anticancer treatment. In that context, we discuss the recent results of the literature to propose hypotheses concerning the potential use of NK cells, potent antitumor cytotoxic effectors, to design novel antitumor strategies.

Accumulating evidence highlights natural killer (NK) cell parameters as potential prognostic factors in cancer patients, which provides a strong rationale for developing therapeutic strategies aiming at restoring NK cell. However, reaching this point warrants better characterization of tumor-induced NK cell alterations. Our group recently reported heterogeneous NK maturation in acute myeloid leukemia (AML) patients. However, the clinical significance of such observations remained to be assessed on a larger cohort of patients. NK maturation based on expression of CD56, CD57, and KIR was assessed by flow cytometry in newly diagnosed AML patients (N = 87 patients from GOELAMS-LAM-IR-2006 multicenter trial). Clinical outcome was evaluated with regard to NK maturation profiles. Unsupervised integrated analysis of NK maturation markers confirmed the existence of three distinct groups of patients [hypomaturation (24.1%), intermediate maturation (66.7%), and hypermaturation (9.2%)]. In univariate analysis, significant differences in overall survival (OS) (P = 0.0006) and relapse-free survival (RFS) (P

Chronic infection perturbs immune homeostasis. While prior studies have reported dysregulation of effector and memory cells, little is known about the effects on naïve T cell populations. We performed a cross-sectional study of chronic hepatitis C (cHCV) patients using tetramer-associated magnetic enrichment to study antigen-specific inexperienced CD8+ T cells (i.e., tumor or unrelated virus-specific populations in tumor-free and sero-negative individuals). cHCV showed normal precursor frequencies, but increased proportions of memory-phenotype inexperienced cells, as compared to healthy donors or cured HCV patients. These observations could be explained by low surface expression of CD5, a negative regulator of TCR signaling. Accordingly, we demonstrated TCR hyperactivation and generation of potent CD8+ T cell responses from the altered T cell repertoire of cHCV patients. In sum, we provide the first evidence that naïve CD8+ T cells are dysregulated during cHCV infection, and establish a new mechanism of immune perturbation secondary to chronic infection.

Immune reconstitution after allogeneic stem cell transplantation is a dynamic and complex process depending on the recipient and donor characteristics, on the modalities of transplantation, and on the occurrence of graft-versus-host disease. Multivariate methods widely used for gene expression profiling can simultaneously analyze the patterns of a great number of biological variables on a heterogeneous set of patients. Here we use these methods on flow cytometry assessment of up to 25 lymphocyte populations to analyze the global pattern of long-term immune reconstitution after transplantation. Immune patterns were most distinct from healthy controls at six months, and had not yet fully recovered as long as two years after transplant. The two principal determinants of variability were linked to the balance of B and CD8+ T cells and of natural killer and B cells, respectively. Recipient’s cytomegalovirus serostatus, cytomegalovirus replication, and chronic graft-versus-host disease were the main factors shaping the immune pattern one year after transplant. We identified a complex signature of under- and over-representation of immune populations dictated by recipient’s cytomegalovirus seropositivity. Finally, we identified dimensions of variance in immune patterns as significant predictors of long-term non-relapse mortality, independently of chronic graft-versus-host disease.

Natural killer cells are the first lymphocyte subset to reconstitute, and play a major role in early immunity after allogeneic hematopoietic stem cell transplantation. Cells expressing the activating receptor NKG2C seem crucial in the resolution of cytomegalovirus episodes, even in the absence of T cells. We prospectively investigated natural killer-cell reconstitution in a cohort of 439 adult recipients who underwent non-T-cell-depleted allogeneic hematopoietic stem cell transplantation between 2005 and 2012. Freshly collected blood samples were analyzed 3, 6, 12 and 24 months after transplantation. Data were studied with respect to conditioning regimen, source of stem cells, underlying disease, occurrence of graft-versus-host disease, and profiles of cytomegalovirus reactivation. In multivariate analysis we found that the absolute numbers of CD56bright natural killer cells at month 3 were significantly higher after myeloablative conditioning than after reduced intensity conditioning. Acute graft-versus-host disease impaired reconstitution of total and CD56dim natural killer cells at month 3. In contrast, high natural killer cell count at month 3 was associated with a lower incidence of chronic graft-versus-host disease, independently of a previous episode of acute graft-versus-host disease and stem cell source. NKG2C+CD56dim and total natural killer cell counts at month 3 were lower in patients with reactivation of cytomegalovirus between month 0 and month 3, but expanded greatly afterwards. These cells were also less numerous in patients who experienced later cytomegalovirus reactivation between month 3 and month 6. Our results advocate a direct role of NKG2C-expressing natural killer cells in the early control of cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation.

Immune recovery after profound lymphopenia is a major challenge in many clinical situations, such as allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recovery depends, in a first step, on hematopoietic lymphoid progenitors production in the bone marrow (BM). In this study, we characterized CD34+Lin-CD10+ lymphoid progenitors in the peripheral blood of allo-HSCT patients. Our data demonstrate a strong recovery of this population 3 months after transplantation. This rebound was abolished in patients who developed acute graft-versus-host disease (aGVHD). A similar recovery profile was found for both CD24+ and CD24- progenitor subpopulations. CD34+lin-CD10+CD24- lymphoid progenitors sorted from allo-HSCT patients preserved their T cell potentiel according to in vitro T-cell differentiation assay and the expression profile of 22 genes involved in T-cell differentiation and homing. CD34+lin-CD10+CD24- cells from patients without aGVHD had reduced CXCR4 gene expression, consistent with an enhanced egress from the BM. CCR7 gene expression was reduced in patients after allo-HSCT, as were its ligands CCL21 and CCL19. This reduction was particularly marked in patients with aGVHD, suggesting a possible impact on thymic homing. Thus, the data presented here identify this population as an important early step in T cell reconstitution in humans and so, an important target when seeking to enhance immune reconstitution.

Patients with Type 2 diabetes (T2D) are highly susceptible to infection and have an increased incidence of some tumors, possibly due to immune system dysfunction. In the innate cellular immune system, Natural Killer (NK) lymphocytes are important effectors responsible for controlling infections and combating tumor development. We analyzed NK cell subsets in 51 patients with long-standing T2D. Compared with healthy blood donors, diabetic patients showed a profound decrease in both NKG2D-positive NK cells (44% vs. 55.5%, P

Background Due to increased rates of secondary solid organ cancer in patients with severe aplastic anemia who received an irradiation-based conditioning regimen, we decided some years ago to use the combination of cyclophosphamide and antithymocyte globulin. We report the long-term follow up of patients who underwent hematopoietic stem cell transplantation from an HLA-matched sibling donor after this conditioning regimen.Design and Methods We analyzed 61 consecutive patients transplanted from June 1991 to February 2010, following conditioning with cyclophosphamide (200 mg/kg) and antithymocyte globulin (2.5 mg/kg/day × 5 days).Results Median age was 21 years (range 4–43); 41 of the 61 patients were adults. Median duration of the disease before hematopoietic stem cell transplantation was 93 days. All but 2 patients received bone marrow as the source of stem cells and all but 2 engrafted. Cumulative incidence of acute grade II–IV graft-versus-host disease was 23% (95%CI 13–34) and 18 developed chronic graft-versus-host disease (cumulative incidence 32% at 72 months, 95% CI 20–46). In multivariate analysis, a higher number of infused CD3 cells was associated with an increased risk of developing chronic graft-versus-host disease (P=0.017). With a median follow up of 73 months (range 8–233), the estimated 6-year overall survival was 87% (95% CI 78–97). At 72 months, the cumulative incidence of avascular necrosis was 21% and 12 patients presented with endocrine dysfunction (cumulative incidence of 19%). Only one patient developed a secondary malignancy (Hodgkin’s lymphoma) during follow up.Conclusions Cyclophosphamide and antithymocyte globulin is an effective conditioning regimen for patients with severe aplastic anemia and is associated with low treatment-related mortality. Long-term complications include avascular necrosis and endocrine dysfunction.

Immune reconstitution was analyzed in 140 consecutive patients who were 2-year disease-free and who underwent myeloablative allogeneic transplantation. A CD4 and CD8 defect was observed involving naive, terminally differentiated, memory and competent cells and above limits values for activated subsets. Natural killer cells normalize at six months while we observed expansion of CD19+/CD5+ B cells after three months and a persisting defect of memory B cells. Chronic graft-versus-host disease did not influence significantly those parameters for CD8 subsets while the naïve and competent CD4 subsets were strongly affected. But the most profound impact of chronic graft-versus-host disease was on B-cell subsets, especially on the memory B population. The cumulative incidence of late severe infections was low (14% at four years). Using Cox’s models, only low B-cell counts at 12 (P=0.02) and 24 (P=0.001) months were associated with the hazard of developing late infection, in particular if patients did not develop chronic graft-versus-host disease.

Completion of early stages of retrovirus infection depends on the cell cycle. While gammaretroviruses require mitosis for proviral integration, lentiviruses are able to replicate in post-mitotic non-dividing cells. Resting cells such as naive resting T lymphocytes from peripheral blood cannot be productively infected by retroviruses, including lentiviruses, but the molecular basis of this restriction remains poorly understood. We demonstrate that in G0 resting cells (primary fibroblasts or peripheral T cells), incoming foamy retroviruses accumulate in close proximity to the centrosome, where they lie as structured and assembled capsids for several weeks. Under these settings, virus uncoating is impaired, but upon cell stimulation, Gag proteolysis and capsid disassembly occur, which allows viral infection to proceed. The data imply that foamy virus uncoating is the rate-limiting step for productive infection of primary G0 cells. Incoming foamy retroviruses can stably persist at the centrosome, awaiting cell stimulation to initiate capsid cleavage, nuclear import, and viral gene expression.