Tocilizumab in combination with a standard induction chemotherapy in acute myeloid leukaemia patients (TOCILAM study): a single-centre, single-arm, phase 1 trial.

Background: In acute myeloid leukaemia (AML), interleukin-6 (IL-6) promotes chemo-resistance and its levels correlate with poor prognosis. IL-6 blockade may represent a promising therapeutic strategy. We aimed to test, tocilizumab, an anti-IL-6 receptor (R) monoclonal antibody in combination with standard intensive AML induction chemotherapy. Methods: This investigator-initiated single-centre phase 1 trial was conducted at Nantes University Hospital in France. According to a continual reassessment method, three escalating doses were tested of intravenous (IV) tocilizumab (4, 6, and 8 mg/kg) administered at day (d) 8 of a standard AML induction chemotherapy (IV idarubicine 8 mg/m2 d1 to d5 + IV cytarabine 100 mg/m2 d1 to d7). All adults (aged ≥ 18 years) with an Eastern Cooperative Oncology Group performance status of 0-2 and with a newly diagnosed (excluding patients with a favourable risk according to ELN-2017 classification if <60 year-old) or a relapsed/refractory AML were eligible. The primary objective was to determine the maximum tolerated dose of tocilizumab to administrate with a standard intensive AML induction. Safety outcomes were continuously monitored for at each participant contact. This trial is registered with ClinicalTrials.gov, NCT04547062. Findings: Between Dec 29, 2020 and Dec 1, 2022, 12 patients were enrolled, of whom 75% had an ELN-2017 high-risk profile, and were treated with tocilizumab- two patients at 4 mg/kg, two at 6 mg/kg and eight at 8 mg/kg of tocilizumab. No dose-limiting toxicity related to tocilizumab was documented. There were nine serious adverse events, none of which were related to tocilizumab, and there was no treatment-related deaths. MTD was thus not reached. Two deaths occurred during induction. In the remaining ten evaluable patients, nine responded to treatment. Interpretation: The combination of tocilizumab with standard AML intensive induction appears to be safe and resulting responses are encouraging. A dose of 8 mg/kg of tocilizumab given at day 8 of induction could be used for further phase 2/3 studies. Funding: The Leucémie Espoir Atlantique Famille (LEAF)-"Tous avec Fabien" association.

The risk of COVID-19 in IBD patients is increased by urban living and is not influenced by disease activity or intravenous biologics.

Background: Patients with inflammatory bowel disease (IBD) may have a modified immune response to SARS-CoV-2. The objectives were to evaluate the prevalence of COVID-19 in patients treated with infliximab or vedolizumab, to analyze the factors associated with the infection, the impact of treatments and trough levels. Methods: Patients with IBD treated with intravenous biologics in 14 French centers were included between March and June 2020 and followed-up for 6 months. Blood samples were collected for serologies and trough levels. The analysis of factors associated with COVID-19 was conducted in a matched 1:1 case-control sub-study with positive patients. Results: In total, 1026 patients were included (74.9% infliximab). Over the follow-up period, 420 patients reported the occurrence of COVID-19 symptoms; 342 had been tested of whom 18 were positive. At the end of follow-up, 38 patients had a positive serology. Considering both nasal tests and serologies together, 46 patients (4.5%) had been infected. The risk of COVID-19 was related neither to the use of treatments (whatever the trough levels) nor to disease activity. Infections were more frequent when using public transport or living in flats in urban areas. Conclusions: The prevalence rate of COVID-19 in this IBD population treated with intravenous infliximab or vedolizumab was the same as the one in the French population before the start of the vaccination campaign. The risk was increased by urban living and was not influenced by disease activity or biologics. Sanitary barrier measures remain the best way to protect against SARS-CoV-2 in patients with IBD in biological therapy.

Kidney allograft rejection is associated with an imbalance of B cells, regulatory T cells and differentiated CD28-CD8+ T cells: analysis of a cohort of 1095 graft biopsies.

Introduction: The human immune system contains cells with either effector/memory or regulatory functions. Besides the well-established CD4+CD25hiCD127lo regulatory T cells (Tregs), we and others have shown that B cells can also have regulatory functions since their frequency and number are increased in kidney graft tolerance and B cell depletion as induction therapy may lead to acute rejection. On the other hand, we have shown that CD28-CD8+ T cells represent a subpopulation with potent effector/memory functions. In the current study, we tested the hypothesis that kidney allograft rejection may be linked to an imbalance of effector/memory and regulatory immune cells. Methods: Based on a large cohort of more than 1000 kidney graft biopsies with concomitant peripheral blood lymphocyte phenotyping, we investigated the association between kidney graft rejection and the percentage and absolute number of circulating B cells, Tregs, as well as the ratio of B cells to CD28-CD8+ T cells and the ratio of CD28-CD8+ T cells to Tregs. Kidney graft biopsies were interpreted according to the Banff classification and divided into 5 biopsies groups: 1) normal/subnormal, 2) interstitial fibrosis and tubular atrophy grade 2/3 (IFTA), 3) antibody-mediated rejection (ABMR), 4) T cell mediated-rejection (TCMR), and 5) borderline rejection. We compared group 1 with the other groups as well as with a combined group 3, 4, and 5 (rejection of all types) using multivariable linear mixed models. Results and discussion: We found that compared to normal/subnormal biopsies, rejection of all types was marginally associated with a decrease in the percentage of circulating B cells (p=0.06) and significantly associated with an increase in the ratio of CD28-CD8+ T cells to Tregs (p=0.01). Moreover, ABMR, TCMR (p=0.007), and rejection of all types (p=0.0003) were significantly associated with a decrease in the ratio of B cells to CD28-CD8+ T cells compared to normal/subnormal biopsies. Taken together, our results show that kidney allograft rejection is associated with an imbalance between immune cells with effector/memory functions and those with regulatory properties.

Antibody-mediated allograft rejection is associated with an increase in peripheral differentiated CD28-CD8+ T cells - Analyses of a cohort of 1032 kidney transplant recipients.

BACKGROUND: CD28-CD8+ T cells represent a differentiated CD8+ T cell subset that is found to be increased in various conditions associated with chronic antigenic stimulation such as aging, chronic viral infections, autoimmune diseases, cancers, and allotransplantation. METHODS: Using multivariate models, we analyzed a large cohort of 1032 kidney transplant patients in whom 1495 kidney graft biopsies were performed concomitant with a peripheral blood leukocyte phenotyping by flow cytometry. We investigated the association between the level of CD28-CD8+ T cells in the blood and the diagnosis of graft rejection according to the recent Banff classification of renal allograft pathology. FINDINGS: We found that antibody-mediated rejection (ABMR) was associated with a significant increase in the percentage as well as the absolute number of CD28-CD8+ T cells in the peripheral blood of kidney transplant patients at the time of biopsy. The confounder-adjusted mean difference of log percentage and log absolute value between the ABMR group and the normal/subnormal histology group were 0.29 (p=0.0004) and 0.38 (p=0.0004), respectively. Moreover, we showed that CD28-CD8+ T cells from the patients diagnosed with ABMR responded more rigorously to TCR and FcγRIIIA (CD16) engagement compared to their CD28+ counterparts as evidenced by an increase in the expression of IFNγ, TNFα, and CD107a. INTERPRETATION: Collectively, our data suggest that differentiated CD28-CD8+ T cells, with increased frequency, number, and function, may participate in the pathobiology of ABMR. Further studies are warranted to clarify the immunological role of this T cell subset in kidney graft rejection. FUNDING: Agence nationale de la recherche (France).

B Cell Aplasia Is the Most Powerful Predictive Marker for Poor Humoral Response after BNT162b2 mRNA SARS-CoV-2 Vaccination in Recipients of Allogeneic Hematopoietic Stem Cell Transplantation.

Little is known about the immune response to SARS-CoV-2 vaccination in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, several studies have reported that adequate protection could be provided to this population. The purpose of this study was to evaluate which factors can predict the efficacy of SARS-CoV-2 vaccination in these specifically immunosuppressed patients. Specific anti-Spike (S) antibody responses were assessed in a cohort of 117 allo-HSCT recipients after 2 injections of BNT162b2 mRNA SARS-CoV-2 vaccine (V1 and V2). Factors considered liable to influence the antibody response and analyzed in this series were the interval between allo-HSCT and V1, donor source, recipient and donor age, current immunosuppressive/chemotherapy (I/C) treatment, and levels of CD4+and CD8+ T cells, B cells, and natural killer cells at the time of V1. Overall, the S-antibody response rate, evaluated at a median of 35 days after V2, was 82.9% for the entire cohort, with 71 patients (61%) reaching the highest titer. In univariate analysis, a lower pre-V1 median total lymphocyte count, lower CD4+ T cell and B cell counts, ongoing I/C treatment, and a haploidentical donor were characteristic of nonhumoral responders. However, multiparameter analysis showed that B cell aplasia was the sole factor predicting the absence of a specific immune response (odds ratio, 0.01; 95% confidence interval, 0.00 to 0.10; P < 10-3). Indeed, the rate of humoral response was 9.1% in patients with B cell aplasia versus 95.9% in patients with a B cell count >0 (P < 10-9). These results advocate for the administration of anti-SARS-CoV-2 vaccination in allo-HSCT recipients as early as peripheral B cell levels can be detected, and also suggest the need for close monitoring of B-cell reconstitution after Allo-HSCT.

An easy and reliable whole blood freezing method for flow cytometry immuno-phenotyping and functional analyses.

BACKGROUND: Immune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Furthermore, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent to which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods. METHODS: In this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor® CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC. RESULTS: Though partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells, and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly, we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa). CONCLUSIONS: In conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.

Monocytic Human Leukocyte Antigen DR Expression in Young Infants Undergoing Cardiopulmonary Bypass.

BACKGROUND: Monocytic human leukocyte antigen DR (mHLA-DR) expression levels have been reported to be a marker of immunosuppression and a predictor of sepsis and mortality. There are, however, scant data regarding mHLA-DR monitoring in young infants after cardiopulmonary bypass. Our objectives were to investigate the kinetics of mHLA-DR expression and to determine whether mHLA-DR levels are associated with healthcare-associated infection (HAI) after cardiopulmonary bypass in young infants. METHODS: mHLA-DR levels were analyzed by flow cytometry using a standardized method in 49 infants (<3 months old) with congenital heart disease before and after cardiopulmonary bypass. Results are expressed as the number of anti-HLA-DR antibodies per cell (AB/c). RESULTS: Postoperative mHLA-DR expression was reduced in all infants. Eleven patients (22%) developed HAI, and 4 patients (8%) died during the 30-day follow-up. mHLA-DR expression was significantly lower on postoperative day 4 in the HAI group compared with those who without HAI (3768 AB/c [range, 1938-6144] vs 13,230 AB/c [range, 6152-19,130], P = .014). Although mHLA-DR expression was associated with postoperative severity, mHLA-DR ≤4500 AB/c in the first 72 hours among patients with higher postoperative severity (extracorporeal membrane oxygenation and/or corticoids and/or delayed closure of sternum) was associated with occurrence of HAI in the univariate analysis (odds ratio, 6.3; 95% confidence interval, 1.0-38.7; P = .037). CONCLUSIONS: Cardiopulmonary bypass induces a profound decrease in mHLA-DR expression in young infants. Among patients with higher postoperative severity, low level of mHLA-DR in the early postoperative period is associated with the development of HAI.

Concomitant decrease of double-positive lymphocyte population CD4CD8αα and Faecalibacterium prausnitzii in patients with colorectal cancer.

INTRODUCTION AND AIMS: Changes in the composition of the gut microbiota in patients with colorectal cancer (CRC) compatible with a contribution of the gut microbiota in carcinogenesis have been reported. In particular, a decrease Faecalibacterium prausnitzii has been identified. A CD4CD8αα, double-positive lymphocyte population (DP8α) has recently been demonstrated in the human colon and blood with regulatory functions and specificity for F. prausnitzii. Here, we aimed to detect dysbiosis in the fecal microbiome of patients with CRC by metagenomic analysis, and to look for changes in the levels of DP8α circulating T cells specific for F. prausnitzii in these patients. PATIENTS AND METHODS: Patients with CRC and control subjects were prospectively included. None had received antibiotics in the previous month or any anti-tumor treatment. A stool sample was collected for each participant, and analyzed by shotgun sequencing. The DP8α T cell population was identified and quantified on fresh whole blood by flow cytometry with anti-CD45, anti-CD3, anti-CD4 and anti-CD8α co-labeling. RESULTS: Twenty-one patients with CRC and 20 controls subjects were included. We found that mean relative abundance of five species was significantly decreased in CRC patients compared with controls, including F. prausnitzii, Barnesiella intestinihominis, Alistipes finegoldii, Bacteroides eggerthii and Eubacterium siraeum. We also found that the DP8α T cell population was significantly decreased in the blood of CRC patients compared with controls. CONCLUSION: In our work, we showed that a reduced abundance of F. prausnitzii in CRC patients was associated to a significant decrease in the circulating DP8α Treg population, suggesting a potential involvement of reduced activity of DP8α T cells in colonic carcinogenesis. These findings open new diagnostic and therapeutic strategies for CRC.

Dampening of CD8+ T Cell Response by B Cell Depletion Therapy in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: To compare the effects of rituximab (RTX) and conventional immunosuppressants (CIs) on CD4+ T cells, Treg cells, and CD8+ T cells in antineutrophil cytoplasmic antibody-associated vasculitis (AAV). METHODS: A thorough immunophenotype analysis of CD4+, Treg, and CD8+ cells from 51 patients with AAV was performed. The production of cytokines and chemokines by CD8+ T cells stimulated in vitro was assessed using a multiplex immunoassay. The impact of AAV B cells on CD8+ T cell response was assessed using autologous and heterologous cocultures. RESULTS: CD4+ and Treg cell subsets were comparable among RTX-treated and CI-treated patients. In contrast, within the CD8+ T cell compartment, RTX, but not CIS, reduced CD45RA+CCR7- (TEMRA) cell frequency (from a median of 39% before RTX treatment to 10% after RTX treatment [P < 0.01]) and efficiently dampened cytokine/chemokine production (e.g., the median macrophage inflammatory protein 1α level was 815 pg/ml in patients treated with RTX versus 985 pg/ml in patients treated with CIs versus 970 pg/ml in those with active untreated AAV [P < 0.01]). CD8+ T cell subsets cocultured with autologous B cells produced more proinflammatory cytokines in AAV patients than in controls (e.g., for tumor necrosis factor-producing effector memory CD8+ T cells: 14% in AAV patients versus 9.2% in controls [P < 0.05]). In vitro disruption of AAV B cell-CD8+ T cell cross-talk reduced CD8+ T cell cytokine production, mirroring the reduced CD8+ response observed ex vivo after RTX treatment. CONCLUSION: The disruption of a pathogenic B cell/CD8+ T cell axis may contribute to the efficacy of RTX in AAV. Further studies are needed to determine the value of CD8+ T cell immunomonitoring in B cell-targeted therapies.

Newborn Screening for Severe Combined Immunodeficiency: Analytic and Clinical Performance of the T Cell Receptor Excision Circle Assay in France (DEPISTREC Study).

Severe combined immunodeficiency (SCID) is characterized by a major T cell deficiency. Infants with SCID are asymptomatic at birth but die from infections in the first year of life if not treated. Survival rates are better for early treatment. SCID therefore meets criteria for newborn screening (NBS). T cell receptor excision circle (TREC) quantification is a reliable marker of T cell deficiency and can be performed using Guthrie cards. The DEPISTREC project was designed to study the feasibility, clinical utility, and cost-effectiveness of generalized SCID screening in France. About 200,000 babies from all over the country were screened at birth with a commercial kit. We determined assay performance and proposed a cutoff for classification of results. Our findings suggest that, given clearly established validation rules and decision-making procedures, the TREC assay is a suitably specific and sensitive method for high-throughput SCID screening. Clinical Trials: NCT02244450.

Bronchiectasis is highly prevalent in anti-MPO ANCA-associated vasculitis and is associated with a distinct disease presentation.

OBJECTIVES: To assess the prevalence of bronchiectasis in a Western cohort with ANCA-positive granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA) and its correlations with disease presentation and outcome. METHODS: Retrospective study of ANCA-associated vasculitis (AAV) patients followed at Nantes University Hospital (2005-2015). Clinical, biological, and follow-up data were collected through chart review. Two experienced radiologists blinded to the clinical data interpreted chest high-resolution CTs according to the Feischner Society criteria. RESULTS: Fifty-eight patients were included: 30 had MPA (51.7%) and 28 had GPA (48.3%). The median age at AAV diagnosis was 65.5 years. Anti-MPO-ANCA and anti-PR3-ANCA were present in 39 (67.2%) and 19 (32.8%) patients, respectively. Overall, bronchiectasis was found in 22 patients (37.9%), all of whom had anti-MPO ANCA. In multivariate analysis, bronchiectasis was independently associated with anti-MPO-ANCA, female gender and age at AAV diagnosis. Furthermore, anti-MPO ANCA patients with bronchiectasis had more frequent peripheral nerve involvement (54.5 vs. 17.6%, p = 0.019) and less frequent renal involvement than those without bronchiectasis (40.9% vs. 82.3%, p = 0.009). Disease course, survival and risk of severe pulmonary infection were similar in patients with and without bronchiectasis on chest CT. CONCLUSIONS: This study shows that bronchiectasis is a highly prevalent pre-existing respiratory condition in Caucasian patients with anti-MPO AAV. This subset of patients exhibits a distinct presentation. Further studies are needed to confirm these findings and clarify the clinical implications of this association. Whether the respiratory tract could be the site of initiation of anti-MPO auto-immunity remains to be investigated.

Local Modulation of Antigen-Presenting Cell Development after Resolution of Pneumonia Induces Long-Term Susceptibility to Secondary Infections.

Lung infections cause prolonged immune alterations and elevated susceptibility to secondary pneumonia. We found that, after resolution of primary viral or bacterial pneumonia, dendritic cells (DC), and macrophages exhibited poor antigen-presentation capacity and secretion of immunogenic cytokines. Development of these "paralyzed" DCs and macrophages depended on the immunosuppressive microenvironment established upon resolution of primary infection, which involved regulatory T (Treg) cells and the cytokine TGF-ß. Paralyzed DCs secreted TGF-ß and induced local Treg cell accumulation. They also expressed lower amounts of IRF4, a transcription factor associated with increased antigen-presentation capacity, and higher amounts of Blimp1, a transcription factor associated with tolerogenic functions, than DCs present during primary infection. Blimp1 expression in DC of humans suffering sepsis or trauma correlated with severity and complicated outcomes. Our findings describe mechanisms underlying sepsis- and trauma-induced immunosuppression, reveal prognostic markers of susceptibility to secondary infections and identify potential targets for therapeutic intervention.

Is pre-transplant sensitization against angiotensin II type 1 receptor still a risk factor of graft and patient outcome in kidney transplantation in the anti-HLA Luminex era? A retrospective study.

We aimed to assess the correlation of anti-angiotensin II type 1 receptor antibodies (anti-AT1R-Abs) before transplantation on a multicentric cohort of kidney transplant recipients (2008-2012), under tacrolimus and mycophenolate mofetil (MMF), screened by Luminex technology for anti-HLA immunization. Anti-AT1R antibody levels were measured by ELISA in pretransplantation sera of 940 kidney recipients from three French centers of the DIVAT cohort. Multivariable Cox models estimated the association between pretransplant anti-angiotensin II type 1 receptor antibodies and time to acute rejection episodes (ARE) or time to graft failure. Within our cohort, 387 patients (41.2%) had pretransplant AT1R-Abs higher than 10 U/ml and only 8% (72/970) greater than 17 U/ml. The cumulative probability of clinically relevant (cr)-ARE was 22.5% at 1 year post-transplantation [95% CI (19.9-25.4%)]. The cumulative probability of graft failure and patient death were 10.6% [95% CI (8.4-13.3%)] and 5.7% [95% CI (4.0-8.1%)] at 3 years post-transplantation, respectively. Multivariate Cox models indicated that pretransplant anti-AT1R antibody levels higher than 10 U/ml were not significantly independently associated with higher risks of acute rejection episodes [HR = 1.04, 95% CI (0.80-1.35)] nor with risk of graft failure [HR = 0.86, 95% CI (0.56-1.33)]. Our study did not confirm an association between pretransplant anti-AT1R antibody levels and kidney transplant outcomes.

Impact of stem cell graft on early viral infections and immune reconstitution after allogeneic transplantation in adults.

BACKGROUND: Viral infections are well-known complications after allogeneic stem cell transplant (allo-SCT). OBJECTIVES: We compared prospectively incidences of DNAemia and active infections (AI) for five opportunistic viruses (Human Herpesvirus 6 (HHV-6), Epstein-Barr virus (EBV), BK polyomavirus (BKPyV), Cytomegalovirus (CMV) and Adenovirus (ADV)) and kinetics of immune reconstitution (IR) in adults receiving either double umbilical cord blood (dUCB group) or unrelated peripheral blood stem cell (uPBSC group) allo-SCT after a reduced-intensity conditioning (RIC) regimen. STUDY DESIGN: Whole blood samples were collected at transplant, every 15days during the first 3 months and at 4, 5 and 6 months post-transplant. RESULTS: Sixty-five patients were enrolled (uPBSC n=34; dUCB n=31). Incidences of HHV-6 and BKPyV DNAemia were significantly higher for dUCB (97% vs 23.5% and 58% vs 32%, respectively) while EBV DNAemia was more frequently detected in uPBSC (71% vs 26%). The incidence of CMV DNAemia was similar between both groups. ADV AI developed only in dUCB. HHV-6 AI were also higher in dUCB (84% vs 21%). In multivariate analysis, dUCB graft was the only independent factor associated with HHV-6 DNAemia (OR: 19.0; 95%CI: 5.2-69.1; p<0.0001) while EBV DNAemia were significantly associated with uPBSC (OR: 29.9; 95%CI: 5.68-158; p <0.0001). dUCB graft was also the only factor associated with HHV-6 AI. Finally, higher counts and faster recoveries of B lymphocytes (p<0.0001) and monocytes (p=0.0007) were observed in the dUCB group. CONCLUSION: We demonstrate a strong correlation between sources of graft and patterns of viral DNAemia and AI and IR after RIC allo-SCT.

Dysregulated Responsiveness of Circulating Dendritic Cells to Toll-Like Receptors in ANCA-Associated Vasculitis.

OBJECTIVE: Dendritic cells (DCs) are critical effectors of innate and adaptive immunity playing crucial roles in autoimmune responses. We previously showed that blood DC numbers were reduced in autoimmune antineutrophil cytoplasmic autoantibody-associated vasculitis (AAV). Here, we assessed toll-like receptor (TLR) responsiveness of blood DCs from patients with granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA). METHODS: Blood samples from healthy controls (HCs), GPA, or MPA patients, without treatment, during acute phase (AP) or remission phase (RP) were analyzed. Cytokine production by DCs and T cells was assessed on whole blood by flow cytometry after TLRs or polyclonal stimulation, respectively. RESULTS: We first showed that GPA and MPA are associated with a decreased blood DC number during AP. Conventional DCs (cDCs) from patients with GPA and MPA in AP exhibited a profound decrease of IL-12/IL-23p40 production after TLR3, 4, or 7/8 stimulation compared to patients in remission and HC, with a return to normal values in RP. TNFα secretion was also affected, with a decrease in cDCs from GPA patients in AP after TLR3 stimulation but an increase after TLR7/8 stimulation. By contrast, the responsiveness of plasmacytoid DCs to TLR7 and 9 was only marginally affected. Finally, we observed that IFNγ-producing CD4+ T cell frequency was significantly lower in AP-GPA patients than in HC. CONCLUSION: We describe, for the first time, a dysregulated response to TLRs of circulating DCs in AAV patients mostly affecting cDCs that exhibit an unexpected reduced inflammatory cytokine secretion possibly contributing to an altered Th cell response.

Persistent deficiency of circulating mucosal-associated invariant T (MAIT) cells in ANCA-associated vasculitis.

OBJECTIVE: Mucosal associated invariant T cells (MAIT) and innate lymphoid cells (ILCs) have immunoregulatory functions at mucosal sites and have been involved in various inflammatory and autoimmune diseases. The aim of this study was to assess their frequencies in blood in ANCA-associated vasculitis (AAV). METHODS: The frequencies and function of MAIT cells, ILCs, γδT, iNKT, NK cells were analyzed by flow cytometry on PBMC of patients with granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) without any treatment, in acute (AP) and remission phase (RP) and compared with healthy controls (HC). RESULTS: The frequencies of MAIT cells were strongly decreased in GPA and MPA in AP compared to HC, both in never treated and in relapsing patients and independently of patient age. This was associated with an activated phenotype of patient MAIT cells, as shown by increased expression of CD69 and IFNγ. MAIT cells remained decreased during RP in AAV patients. The frequencies of iNKT and γδT cells were unaffected compared to HC, whereas those of NK cells were slightly reduced during AP in MPA. We also observed a significant decrease in frequencies of total ILCs with decreased ILC2 and ILC3 and increased ILC1 during AP in both GPA and MPA compared to HC. These frequencies normalized during RP. Interestingly, we observed a significant correlation between the frequency of total ILCs and BVAS. CONCLUSION: We show for the first time that AAV are associated with a major decrease and an activated phenotype of blood MAIT cell. These features persisted during remission suggesting a role for MAIT cells in the pathogenesis of AAV.

Long-lasting HHV-6 reactivation in long-term adult survivors after double umbilical cord blood allogeneic stem cell transplantation.

Higher incidence of human herpesvirus 6 (HHV-6) infection has been documented after umbilical cord blood allo-transplant in adults. Here we demonstrate that HHV-6 reactivation persists for a very long time in half of the patients after this type of graft. Long-term immune reconstitution does not explain this event, which remains to be explained.

Altered innate function of plasmacytoid dendritic cells restored by enzyme replacement therapy in Gaucher disease.

BACKGROUND: Gaucher disease (GD) is caused by an autosomal-recessive deficiency of ß-glucocerebrosidase leading to an accumulation of glucosylceramide in monocytes/macrophage lineage. We analyzed immune cells and especially the function of dendritic cells to evaluate the potential impact of glucosylceramide accumulation in these cells and its possible role in infections and malignancies usually described in this pathology. These analyses were performed for each patient without and under enzyme replacement therapy. METHODS: Seven GD patients were studied and compared with healthy volunteers. Immune cells (B cells, T cells, NK, dendritic cells), were analyzed by flow cytometry directly on whole blood. Cytokine production by blood dendritic cells was assessed after stimulation by toll-like receptor ligands. Cytokines in sera were measured using a multiplex assay. RESULTS: GD patients displayed decreased numbers of NK cells, γδ2 T cells and increased frequency of memory CD4(+)CD45RO(+) T cells, when compared to healthy controls. Numbers of dendritic cells (myeloid (mDC) and plasmacytoid (pDC) dendritic cells) were also decreased. We demonstrated that pDC from GD patients exhibited a decrease in IFNα production after TLR9 stimulation compared to controls. Importantly, enzyme replacement therapy restored pDC function. Finally, we observed an increase of IL-8 and IL-18 in GD patient sera, which were reduced under enzyme replacement therapy. CONCLUSIONS: Our data confirm that patients with GD exhibit altered numbers of innate and T lymphocytes and show for the first time that pDC from GD patients exhibit altered responsiveness to TLR9. These alterations could contribute to a decreased response to pathogens and could favor the development of malignancies.

Elevated soluble Flt1 inhibits endothelial repair in PR3-ANCA-associated vasculitis.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis exhibits endothelial damage, but the capacity for vessel repair in this disorder is not well understood. Here, we observed a marked increase in serum levels of soluble Flt1 (sFlt1), a potent inhibitor of vascular endothelial growth factor, in patients with active ANCA-associated vasculitis compared with patients during remission and other controls. Serum levels of sFlt1 correlated with C5a, an anaphylatoxin released after complement activation. Serum from patients with acute ANCA-associated vasculitis disrupted blood flow in the chicken chorioallantoic membrane assay, suggesting an antiangiogenic effect. Preincubation with excess human vascular endothelial growth factor prevented this effect. Anti-proteinase-3 (PR3) mAb and serum containing PR3-ANCA from patients with active vasculitis both induced a significant and sustained release of sFlt1 from monocytes, whereas anti-myeloperoxidase (MPO) mAb or polyclonal antibodies did not. However, the serum containing polyclonal PR3-ANCA did not induce release of sFlt1 from cultured human umbilical vein endothelial cells. In summary, these data suggest that anti-PR3 antibodies, and to a much lesser extent anti-MPO antibodies, increase sFlt1 during acute ANCA-associated vasculitis, leading to an antiangiogenic state that hinders endothelial repair.

Decreased numbers of blood dendritic cells and defective function of regulatory T cells in antineutrophil cytoplasmic antibody-associated vasculitis.

BACKGROUND: Dendritic cells (DC) and regulatory cells (Treg) play pivotal roles in controlling both normal and autoimmune adaptive immune responses. DC are the main antigen-presenting cells to T cells, and they also control Treg functions. In this study, we examined the frequency and phenotype of DC subsets, and the frequency and function of Treg from patients with ANCA-associated vasculitis (AAV). METHODOLOGY/PRINCIPAL FINDINGS: Blood samples from 19 untreated patients with AAV during flares and before any immunosuppressive treatment were analyzed, along with 15 AAV patients in remission and 18 age-matched healthy controls. DC and Treg numbers, and phenotypes were assessed by flow cytometry, and in vitro suppressive function of Treg was determined by co-culture assay. When compared to healthy volunteers, absolute numbers of conventional and plasmacytoid DC were decreased in AAV patients. During the acute phase this decrease was significantly more pronounced and was associated with an increased DC expression of CD62L. Absolute numbers of Treg (CD4(+)CD25(high)CD127(low/-) Tcells) were moderately decreased in patients. FOXP3 and CD39 were expressed at similar levels on Treg from patients as compared to controls. The suppressive function of Treg from AAV patients was dramatically decreased as compared to controls, and this defect was more pronounced during flares than remission. This Treg functional deficiency occurred in the absence of obvious Th17 deviation. CONCLUSION: In conclusion, these data show that AAV flares are associated with both a decrease number and altered phenotype of circulating DC and point to a role for Treg functional deficiency in the pathogenesis of AAV.