Correct stimulation of CD28H arms NK cells against tumor cells.

Tumor evasion has recently been associated with a novel member of the B7 family, HERV-H LTR-associating 2 (HHLA2), which is mostly overexpressed in PDL-1neg tumors. HHLA2 can either induce a costimulation signal when bound to CD28H or inhibit it by binding to KIR3DL3 on T- and NK cells. Given the broad distribution of CD28H expression on NK cells and its role, we compared two monoclonal antibodies targeting this novel NK-cell engager in this study. We show that targeting CD28H at a specific epitope not only strongly activates Ca2+ flux but also results in NK-cell activation. CD28H-activated NK cells further display increased cytotoxic activity against hematopoietic cell lines and bypass HHLA2 and HLA-E inhibitory signals. Additionally, scRNA-seq analysis of clear cell renal cancer cells revealed that HHLA2+ clear cell renal cancer cell tumors were infiltrated with CD28H+ NK cells, which could be targeted by finely chosen anti-CD28H Abs.

Confocal laser endomicroscopy as predictive biomarker of clinical and endoscopic efficacy of vedolizumab in ulcerative colitis: The DETECT study.

AIMS: In patients with ulcerative colitis (UC), no biomarker is available to help the physician to choose the most suitable biotherapy. The primary objective of this pilot study was to assess the feasibility of identification of α4ß7- and TNF-expressing cells, to predict the response to vedolizumab using confocal laser endoscopy (CLE). METHODS: Patients with moderate-to-severe UC, naïve of biotherapy, received vedolizumab. Clinical evaluation was performed at each infusion. Endoscopic evaluation was performed before inclusion and at week 22. Fresh colonic biopsies were stained using FITC-labelled vedolizumab and Alexa fluor-labelled adalimumab and ex vivo dual-band CLE images were acquired. Blood samples were collected to measure trough concentrations of vedolizumab and to determine absolute counts of T and B cells subpopulations, NK cells and monocytes. RESULTS: Nineteen patients were enrolled in the study and received at least one dose of vedolizumab. Clinical remission and endoscopic improvement were observed in 58% of whom 5 patients (45%) had an endoscopic subscore of 0. In terms of clinical response and remission, endoscopic improvement and histologic response, FITC-conjugated vedolizumab staining tended to be higher in responder patients compared to non-responders at week 22. A threshold value of 6 positive FITC-vedolizumab staining areas detected by CLE seemed informative to discriminate the responders and non-responders. The results were similar in terms of clinical remission and endoscopic improvement with a sensitivity of 78% and a specificity of 85% (p = 0.05). Trough concentrations and blood immune cells were not associated with responses to vedolizumab. CONCLUSION: This pilot study demonstrate that dual-band CLE is feasible to detect α4ß7- and TNF-expressing cells. Positive α4ß7 staining seems to be associated with clinical and endoscopic remission in UC patients treated by anti-α4ß7-integrin, subject to validation by larger-scale studies. Clinical-trial.gov: NCT02878083.

IL-22BP production is heterogeneously distributed in Crohn's disease.

Crohn's disease (CD), a form of inflammatory bowel disease (IBD), is characterized by impaired epithelial barrier functions and dysregulated mucosal immune responses. IL-22 binding protein (IL-22BP) is a soluble inhibitor regulating IL-22 bioactivity, a cytokine proposed to play protective roles during CD. We and others have shown that IL-22BP is produced in IBD inflamed tissues, hence suggesting a role in CD. In this work, we extended the characterization of IL-22BP production and distribution in CD tissues by applying enzyme-linked immunosorbent assays to supernatants obtained from the culture of endoscopic biopsies of patients, and reverse transcription-quantitative polymerase chain reaction on sorted immune cell subsets. We reveal that IL-22BP levels are higher in inflamed ileums than colons. We observe that in a cell-intrinsic fashion, populations of mononuclear phagocytes and eosinophils express IL-22BP at the highest levels in comparison to other sources of T cells. We suggest the enrichment of intestinal eosinophils could explain higher IL-22BP levels in the ileum. In inflamed colon, we reveal the presence of increased IL-22/IL22BP ratios compared to controls, and a strong correlation between IL-22BP and CCL24. We identify monocyte-derived dendritic cells (moDC) as a cellular subtype co-expressing both cytokines and validate our finding using in vitro culture systems. We also show that retinoic acid induces the secretion of both IL-22BP and CCL24 by moDC. Finally, we report on higher IL-22BP levels in active smokers. In conclusion, our work provides new information relevant to therapeutic strategies modulating IL-22 bioactivity in CD, especially in the context of disease location.

Transfusion-Related Renal Dysfunction After Cardiac Surgery: The Role of Myeloid-Related Protein_14 in Neutrophil-Mediated Tubular Damage.

Transfusion is a specific cause of acute kidney injury (AKI) after cardiac surgery. Whether there is an association between the composition of blood products and the onset of AKI is unknown. The present study suggests that the transfusion of packed red blood cells containing a high amount of myeloid-related protein 14 (MRP_14) could increase the incidence of AKI after cardiac surgery. In a mouse model, MRP_14 increased the influx of neutrophils in the kidney after ischemia-reperfusion and their ability to damage tubular cells. Higher concentrations of MRP_14 were found in packed red blood cells from female donors or prepared by whole blood filtration.

Non-permissive human conventional CD1c+ dendritic cells enable trans-infection of human primary renal tubular epithelial cells and protect BK polyomavirus from neutralization.

The BK polyomavirus (BKPyV) is a ubiquitous human virus that persists in the renourinary epithelium. Immunosuppression can lead to BKPyV reactivation in the first year post-transplantation in kidney transplant recipients (KTRs) and hematopoietic stem cell transplant recipients. In KTRs, persistent DNAemia has been correlated to the occurrence of polyomavirus-associated nephropathy (PVAN) that can lead to graft loss if not properly controlled. Based on recent observations that conventional dendritic cells (cDCs) specifically infiltrate PVAN lesions, we hypothesized that those cells could play a role in BKPyV infection. We first demonstrated that monocyte-derived dendritic cells (MDDCs), an in vitro model for mDCs, captured BKPyV particles through an unconventional GRAF-1 endocytic pathway. Neither BKPyV particles nor BKPyV-infected cells were shown to activate MDDCs. Endocytosed virions were efficiently transmitted to permissive cells and protected from the antibody-mediated neutralization. Finally, we demonstrated that freshly isolated CD1c+ mDCs from the blood and kidney parenchyma behaved similarly to MDDCs thus extending our results to cells of clinical relevance. This study sheds light on a potential unprecedented CD1c+ mDC involvement in the BKPyV infection as a promoter of viral spreading.

Preclinical Assessment of Autologous Tolerogenic Dendritic Cells From End-stage Renal Disease Patients.

BACKGROUND: Kidney transplantation is the therapeutic of choice for patients with kidney failure. While immunosuppressive drugs can control graft rejection, their use is associated with increased infections and cancer, and they do not effectively control chronic graft rejection. Cell therapy is an attractive strategy to minimize the use of pharmacological drugs. METHODS: We recently developed a protocol to generate human monocyte-derived autologous tolerogenic dendritic cells (ATDCs) from healthy volunteers. Herein, we transferred the ATDC manufacturing protocol to a Good Manufacturing Practice (GMP)-compliant facility. Furthermore, we compared the phenotype and in vitro functions of ATDCs generated from patients with end-stage renal disease to those generated from healthy volunteers. RESULTS: We describe the critical steps for GMP-compliant production of ATDCs and define the quality criteria required to allow release of the cell products. Furthermore, we showed that ATDCs generated from healthy volunteers and patients with kidney failure display the same tolerogenic profile based on their phenotype, resistance to maturation, and ability to modulate T-cell responses. CONCLUSIONS: Together, these results allowed us to define the production process and the quality criteria for the release of ATDCs before their administration in patients receiving a kidney transplant.

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.

PD-1 and TIGIT coexpression identifies a circulating CD8 T cell subset predictive of response to anti-PD-1 therapy.

BACKGROUND: Clinical benefit from programmed cell death 1 receptor (PD-1) inhibitors relies on reinvigoration of endogenous antitumor immunity. Nonetheless, robust immunological markers, based on circulating immune cell subsets associated with therapeutic efficacy are yet to be validated. METHODS: We isolated peripheral blood mononuclear cell from three independent cohorts of melanoma and Merkel cell carcinoma patients treated with PD-1 inhibitor, at baseline and longitudinally after therapy. Using multiparameter flow cytometry and cell sorting, we isolated four subsets of CD8+ T cells, based on PD-1 and TIGIT expression profiles. We performed phenotypic characterization, T cell receptor sequencing, targeted transcriptomic analysis and antitumor reactivity assays to thoroughly characterize each of these subsets. RESULTS: We documented that the frequency of circulating PD-1+TIGIT+ (DPOS) CD8+ T-cells after 1 month of anti-PD-1 therapy was associated with clinical response and overall survival. This DPOS T-cell population was enriched in highly activated T-cells, tumor-specific and emerging T-cell clonotypes and T lymphocytes overexpressing CXCR5, a key marker of the CD8 cytotoxic follicular T cell population. Additionally, transcriptomic profiling defined a specific gene signature for this population as well as the overexpression of specific pathways associated with the therapeutic response. CONCLUSIONS: Our results provide a convincing rationale for monitoring this PD-1+TIGIT+ circulating population as an early cellular-based marker of therapeutic response to anti-PD-1 therapy.

Transient antibody targeting of CD45RC inhibits the development of graft-versus-host disease.

Allogeneic bone marrow transplantation (BMT) is a widely spread treatment of many hematological diseases, but its most important side effect is graft-versus-host disease (GVHD). Despite the development of new therapies, acute GVHD (aGVHD) occurs in 30% to 50% of allogeneic BMT and is characterized by the generation of effector T (Teff) cells with production of inflammatory cytokines. We previously demonstrated that a short anti-CD45RC monoclonal antibody (mAb) treatment in a heart allograft rat model transiently decreased CD45RChigh Teff cells and increased regulatory T cell (Treg) number and function allowing long-term donor-specific tolerance. Here, we demonstrated in rat and mouse allogeneic GVHD, as well as in xenogeneic GVHD mediated by human T cells in NSG mice, that both ex vivo depletion of CD45RChigh T cells and in vivo treatment with short-course anti-CD45RC mAbs inhibited aGVHD. In the rat model, we demonstrated that long surviving animals treated with anti-CD45RC mAbs were fully engrafted with donor cells and developed a donor-specific tolerance. Finally, we validated the rejection of a human tumor in NSG mice infused with human cells and treated with anti-CD45RC mAbs. The anti-human CD45RC mAbs showed a favorable safety profile because it did not abolish human memory antiviral immune responses, nor trigger cytokine release in in vitro assays. Altogether, our results show the potential of a prophylactic treatment with anti-human CD45RC mAbs in combination with rapamycin as a new therapy to treat aGVHD without abolishing the antitumor effect.

Regulatory cell therapy in kidney transplantation (The ONE Study): a harmonised design and analysis of seven non-randomised, single-arm, phase 1/2A trials.

BACKGROUND: Use of cell-based medicinal products (CBMPs) represents a state-of-the-art approach for reducing general immunosuppression in organ transplantation. We tested multiple regulatory CBMPs in kidney transplant trials to establish the safety of regulatory CBMPs when combined with reduced immunosuppressive treatment. METHODS: The ONE Study consisted of seven investigator-led, single-arm trials done internationally at eight hospitals in France, Germany, Italy, the UK, and the USA (60 week follow-up). Included patients were living-donor kidney transplant recipients aged 18 years and older. The reference group trial (RGT) was a standard-of-care group given basiliximab, tapered steroids, mycophenolate mofetil, and tacrolimus. Six non-randomised phase 1/2A cell therapy group (CTG) trials were pooled and analysed, in which patients received one of six CBMPs containing regulatory T cells, dendritic cells, or macrophages; patient selection and immunosuppression mirrored the RGT, except basiliximab induction was substituted with CBMPs and mycophenolate mofetil tapering was allowed. None of the trials were randomised and none of the individuals involved were masked. The primary endpoint was biopsy-confirmed acute rejection (BCAR) within 60 weeks after transplantation; adverse event coding was centralised. The RTG and CTG trials are registered with ClinicalTrials.gov, NCT01656135, NCT02252055, NCT02085629, NCT02244801, NCT02371434, NCT02129881, and NCT02091232. FINDINGS: The seven trials took place between Dec 11, 2012, and Nov 14, 2018. Of 782 patients assessed for eligibility, 130 (17%) patients were enrolled and 104 were treated and included in the analysis. The 66 patients who were treated in the RGT were 73% male and had a median age of 47 years. The 38 patients who were treated across six CTG trials were 71% male and had a median age of 45 years. Standard-of-care immunosuppression in the recipients in the RGT resulted in a 12% BCAR rate (expected range 3·2-18·0). The overall BCAR rate for the six parallel CTG trials was 16%. 15 (40%) patients given CBMPs were successfully weaned from mycophenolate mofetil and maintained on tacrolimus monotherapy. Combined adverse event data and BCAR episodes from all six CTG trials revealed no safety concerns when compared with the RGT. Fewer episodes of infections were registered in CTG trials versus the RGT. INTERPRETATION: Regulatory cell therapy is achievable and safe in living-donor kidney transplant recipients, and is associated with fewer infectious complications, but similar rejection rates in the first year. Therefore, immune cell therapy is a potentially useful therapeutic approach in recipients of kidney transplant to minimise the burden of general immunosuppression. FUNDING: The 7th EU Framework Programme.

Immunophenotype of a Rat Model of Duchenne's Disease and Demonstration of Improved Muscle Strength After Anti-CD45RC Antibody Treatment.

Corticosteroids (CS) are standard therapy for the treatment of Duchenne's muscular dystrophy (DMD). Even though they decrease inflammation, they have limited efficacy and are associated with significant side effects. There is therefore the need for new protolerogenic treatments to replace CS. Dystrophin-deficient rats (Dmdmdx ) closely resemble the pathological phenotype of DMD patients. We performed the first Immunophenotyping of Dmdmdx rats and showed leukocyte infiltration in skeletal and cardiac muscles, which consisted mostly of macrophages and T cells including CD45RChigh T cells. Muscles of DMD patients also contain elevated CD45RChigh T cells. We treated Dmdmdx rats with an anti-CD45RC MAb used in previous studies to deplete CD45RChigh T cells and induce immune tolerance in models of organ transplantation. Treatment of young Dmdmdx rats with anti-CD45RC MAb corrected skeletal muscle strength and was associated with depletion of CD45RChigh T cells with no side effects. Treatment of young Dmdmdx rats with prednisolone resulted in increase in skeletal muscle strength but also severe growth retardation. In conclusion, anti-CD45RC MAb treatment has potential in the treatment of DMD and might eventually result in reduction or elimination of CS use.

IL-7 receptor influences anti-TNF responsiveness and T cell gut homing in inflammatory bowel disease.

It remains unknown what causes inflammatory bowel disease (IBD), including signaling networks perpetuating chronic gastrointestinal inflammation in Crohn's disease (CD) and ulcerative colitis (UC), in humans. According to an analysis of up to 500 patients with IBD and 100 controls, we report that key transcripts of the IL-7 receptor (IL-7R) pathway are accumulated in inflamed colon tissues of severe CD and UC patients not responding to either immunosuppressive/corticosteroid, anti-TNF, or anti-α4ß7 therapies. High expression of both IL7R and IL-7R signaling signature in the colon before treatment is strongly associated with nonresponsiveness to anti-TNF therapy. While in mice IL-7 is known to play a role in systemic inflammation, we found that in humans IL-7 also controlled α4ß7 integrin expression and imprinted gut-homing specificity on T cells. IL-7R blockade reduced human T cell homing to the gut and colonic inflammation in vivo in humanized mouse models, and altered effector T cells in colon explants from UC patients grown ex vivo. Our findings show that failure of current treatments for CD and UC is strongly associated with an overexpressed IL-7R signaling pathway and point to IL-7R as a relevant therapeutic target and potential biomarker to fill an unmet need in clinical IBD detection and treatment.

HLA-DR expression in neonates after cardiac surgery under cardiopulmonary bypass: a pilot study.

Monocyte HLA-DR expression has been reported as a marker of immunosuppression and a predictor of sepsis development. However, to date, there is no report on monocyte HLA-DR monitoring exclusively in neonates (< 28 days of life) who underwent cardiac surgery under cardiopulmonary bypass (CPB), which have a high risk of nosocomial infection. In this pilot study, we studied nine neonates with a diagnosis of congenital heart disease requiring surgery under CPB. There was a significant reduction in monocyte HLA-DR expression for the first two postoperative days, as compared to preoperatively (p = 0.004). Moreover, neonates who displayed an episode of NI had a dramatically lower HLA-DR expression at day 4, as compared to neonates without NI (4257 AB/c [2220-5895] vs 14,947 AB/c [9858-16,960]; p = 0.04). Our preliminary results could indicate that HLA-DR expression may be a useful biomarker of immunosuppression-induced secondary infection after CPB in neonates.

A standardized flow cytometry procedure for the monitoring of regulatory T cells in clinical trials.

BACKGROUND: Quantification of regulatory T cells (Tregs) is crucial in immunomonitoring in clinical trials as this cell population has been shown to be involved in a wide range of diseases, including cancers, autoimmune diseases, infections, and allergies. Human Tregs are defined as CD4+ CD25+ CD127low FoxP3+ cells, and the standardization of their staining by flow cytometry is a challenge, especially in multicenter clinical trials, notably because of the intracellular location of FoxP3. METHOD: A flow cytometry staining procedure was settled and standardized to measure human Tregs in peripheral whole blood using precoated dried antibodies in ready-to-use tubes. It was compared with reference methods and implemented and validated to be suitable with different cytometer platforms. RESULTS: The standardized protocol developed with dried antibodies and reduced volumes of whole blood allows an optimal identification of Tregs. Compared with classical staining procedure, it reduces the number of steps required, in a very fast and simple technique. The accuracy of the method was confirmed by a multicenter comparison with different cytometer brands. CONCLUSIONS: Our results highlight the reliability of this high-standard protocol that could become a reference method for the monitoring of Tregs in clinical trials. © 2018 International Clinical Cytometry Society.

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.

Receptor activating NF-κB ligand (RANKL) is a constitutive intracellular protein in resting human basophils and is strongly induced on their surface by interleukin 3.

Receptor activating NF-κB ligand (RANKL) is a member of the TNF superfamily that plays a pivotal role in bone homeostasis as being the major osteoclastogenesis factor. RANKL also has pleiotropic effects in the immune system in which it is expressed by activated T and B cells and some innate lymphoid cells. RANKL-RANK interactions mediate lymph node organogenesis and immunoregulatory functions in autoimmune disease and carcinogenesis as well as cross talk between the immune system and bone. In this study, we show that basophils were the strongest RANKL mRNA-expressing cells amongst major leukocyte subsets in human blood. RANKL was preformed as an intracellular protein in resting basophils and was rapidly and strongly expressed on their surface upon stimulation with IL-3, but not other stimuli. This expression was stable for at least 6 days. Activated basophils could also release soluble RANKL in small quantities upon interaction with DCs or monocytes. In the blood, basophils were the sole cells to express membrane RANKL in response to IL-3. This study indicates that basophils should be considered as new players in the pleiotropic and complex RANKL-RANK interaction system and suggests a role for RANKL in the interaction between basophils and immune cells in inflammatory allergic tissues and secondary lymphoid organs.

Involvement of the CX3CL1 (fractalkine)/CX3CR1 pathway in the pathogenesis of acute graft-versus-host disease.

This study investigated the role of cytokines and chemokines in aGVHD incidence and severity in 109 patients who underwent reduced-intensity conditioning allogeneic stem cell transplantation (HSCT). Among the 42 cytokines tested at d 0 HSCT, only CX3CL1 levels at d 0 HSCT were significantly associated with Grades II-IV aGVHD development (P = 0.04). Increased levels of CX3CL1 at d 20-30 and 50 post-HSCT were also significantly associated with aGVHD (P = 0.02 and P = 0.03, respectively). No such association was found before the conditioning regimen or at d 100-120 post-HSCT. As the receptor for CX3CL1 is CX3CR1, the number of CX3CR1(+) cells was determined by flow cytometry. The CX3CR1(+)CD8(+) T cell proportion was significantly higher in patients with aGVHD than those without aGVHD (P = 0.01). To investigate the distribution of the CX3CL1/CX3CR1 axis in the anatomic sites of aGVHD, CX3CL1 and CX3CR1 levels were studied by use of an in situ immunohistochemical analysis on GI biopsies of patients with intestinal aGVHD. CX3CL1 expression was increased significantly in the epithelial cells and mononuclear cells of the lamina propria. CX3CR1(+) mononuclear cells were identified in close contact with epithelial cells. These findings strongly suggest the implication of the CX3CL1/CX3CR1 axis in the pathogenesis of aGVHD.

Hydrocortisone prevents immunosuppression by interleukin-10+ natural killer cells after trauma-hemorrhage.

OBJECTIVE: Trauma induces a state of immunosuppression, which is responsible for the development of nosocomial infections. Hydrocortisone reduces the rate of pneumonia in patients with trauma. Because alterations of dendritic cells and natural killer cells play a central role in trauma-induced immunosuppression, we investigated whether hydrocortisone modulates the dendritic cell/natural killer cell cross talk in the context of posttraumatic pneumonia. DESIGN: Experimental study. SETTINGS: Research laboratory from an university hospital. SUBJECTS: Bagg Albino/cJ mice (weight, 20-24 g). INTERVENTIONS: First, in an a priori substudy of a multicenter, randomized, double-blind, placebo-controlled trial of hydrocortisone (200 mg/d for 7 d) in patients with severe trauma, we have measured the blood levels of five cytokines (tumor necrosis factor-α, interleukin-6, interleukin-10, interleukin-12, interleukin-17) at day 1 and day 8. In a second step, the effects of hydrocortisone on dendritic cell/natural killer cell cross talk were studied in a mouse model of posttraumatic pneumonia. Hydrocortisone (0.6 mg/mice i.p.) was administered immediately after hemorrhage. Twenty-four hours later, the mice were challenged with Staphylococcus aureus (7 × 10 colony-forming units). MEASUREMENTS AND MAIN RESULTS: Using sera collected during a multicenter study in patients with trauma, we found that hydrocortisone decreased the blood level of interleukin-10, a cytokine centrally involved in the regulation of dendritic cell/natural killer cell cluster. In a mouse model of trauma-hemorrhage-induced immunosuppression, splenic natural killer cells induced an interleukin-10-dependent elimination of splenic dendritic cell. Hydrocortisone treatment reduced this suppressive function of natural killer cells and increased survival of mice with posthemorrhage pneumonia. The reduction of the interleukin-10 level in natural killer cells by hydrocortisone was partially dependent on the up-regulation of glucocorticoid-induced tumor necrosis factor receptor-ligand (TNFsf18) on dendritic cell. CONCLUSIONS: These data demonstrate that trauma-induced immunosuppression is characterized by an interleukin-10-dependent elimination of dendritic cell by natural killer cells and that hydrocortisone improves outcome by limiting this immunosuppressive feedback loop.

Impaired blood dendritic cell numbers and functions after aneurysmal subarachnoid hemorrhage.

PREVIOUS PRESENTATION: Portions of this study were presented at the Annual Congress of Société Française d'Anesthésie et de Réanimation in Paris, September 2012. BACKGROUND: Toll-like receptor (TLR) agonists are promising therapy for the prevention of nosocomial infections in critical ill patients. We aimed to analyze the TLR-reactivity of circulating dendritic cells (DC) as assessed by cytokine production after an ex vivo challenge with TLR agonists in aneurysmal subarachnoid hemorrhage (SAH) patients. METHODS AND FINDINGS: A single-center prospective observational study took place in one intensive care unit of a teaching hospital. Blood samples were harvested on days 2, 5 and 10 in 21 severe SAH patients requiring mechanical ventilation and 17 healthy controls. DC production of cytokines (Tumour Necrosis Factor, TNF-α; Interleukin, IL-12; and Interferon, IFN-α) was assessed by intracellular immunostaining on TLR-3, 4, 7/8 and 9 stimulations. SAH patients had decreased numbers of blood myeloid (mDCs) and plasmacytoid DCs (pDCs) on days 2, 5 and 10. Compared with the healthy controls, the frequency of mDCs producing TNF-α after TLR-3 stimulation was decreased in the SAH patients. The frequency of myeloid DCs producing IL-12 after TLR-3 and 4 stimulations was also decreased in the SAH patients. In contrast, the mDCs response to TLR-7/8 was not impaired in the SAH patients. The frequency of pDCs producing TNF-α(+) and IFN-α(+) on TLR-7/8 stimulation were reduced at all of the tested times in the SAH patients, whereas reactivity to TLR-9 was preserved. On day 2, the pDCs from non-survivor patients (n=8) had a decreased ability to produce IFN-α on TLR-9 stimulation compared with the survivors. CONCLUSIONS: These data suggest functional abnormalities of circulating pDCs and mDCs that could be important for immunomodulation after SAH.