Background Although anti-HLA antibodies (Abs) cause most antibody-mediated rejections of renal allografts, non-anti-HLA Abs have also been postulated to contribute. A better understanding of such Abs in rejection is needed. MethodsWe conducted a nationwide study to identify kidney transplant recipients without anti-HLA donor-specific Abs who experienced acute graft dysfunction within 3 months after transplantation and showed evidence of microvascular injury, called acute microvascular rejection (AMVR). We developed a crossmatch assay to assess serum reactivity to human microvascular endothelial cells, and used a combination of transcriptomic and proteomic approaches to identify non-HLA Abs. ResultsWe identified a highly selected cohort of 38 patients with early acute AMVR. Biopsy specimens revealed intense microvascular inflammation and the presence of vasculitis (in 60.5%), interstitial hemorrhages (31.6%), or thrombotic microangiopathy (15.8%). Serum samples collected at the time of transplant showed that previously proposed anti-endothelial cell Abs-angiotensin type 1 receptor (AT1R), endothelin-1 type A and natural polyreactive Abs-did not increase significantly among patients with AMVR compared with a control group of stable kidney transplant recipients. However, 26% of the tested AMVR samples were positive for AT1R Abs when a threshold of 10 IU/ml was used. The crossmatch assay identified a common IgG response that was specifically directed against constitutively expressed antigens of microvascular glomerular cells in patients with AMVR. Transcriptomic and proteomic analyses identified new targets of non-HLA Abs, with little redundancy among individuals. ConclusionsOur findings indicate that preformed IgG Abs targeting non-HLA antigens expressed on glomerular endothelial cells are associated with early AMVR, and that in vitro cell-based assays are needed to improve risk assessments before transplant.

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation (allo-HCT), limiting the success of this therapy. Many proinflammatory cytokines secreted following the conditioning regimen have been linked to aGVHD initiation. Interleukin-22 (IL-22) is a cytokine related to IL-10 for its structure and is secreted by T helper type 17 (TH17) cells and innate immune cells. Given the paradoxical role of IL-22 in inflammation with both protective or proinflammatory functions, we investigated whether IL-22 could have a role in aGVHD pathophysiology in a mouse allo-HCT model. In this study, we show that IL-22 deficiency in donor T cells can decrease the severity of aGVHD, while limiting systemic and local inflammation in aGVHD target organs. In addition, we found that Foxp3+ regulatory T cells (Treg cells) were increased in recipient mice that received IL-22-deficient T cells, suggesting that Treg were involved in the reduced severity of GVHD. Finally, we found that the graft-versus-leukemia (GVL) effect mediated by donor T cells was preserved in the absence of IL-22. Overall, these data suggest that targeting of IL-22 may represent a valid approach towards decreasing aGVHD severity after allo-HCT while preserving the GVL effect.

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Key Points• LXR activation inhibits BPDCN cell survival through the increase of cholesterol efflux, the inhibition of NF-kB, and IL-3 signaling.• Treatment with LXR agonists can be proposed as a new therapeutic approach for BPDCN.Blastic plasmacytoid dendritic cell (PDC) neoplasm (BPDCN) is an aggressive hematological malignancy with a poor prognosis that derives from PDCs. No consensus for optimal treatment modalities is available today and the full characterization of this leukemia is still emerging. We identified here a BPDCN-specific transcriptomic profile when compared with those of acute myeloid leukemia and T-acute lymphoblastic leukemia, as well as the transcriptomic signature of primary PDCs. This BPDCN gene signature identified a dysregulation of genes involved in cholesterol homeostasis, some of them being liver X receptor (LXR) target genes. LXR agonist treatment of primary BPDCN cells and BPDCN cell lines restored LXR target gene expression and increased cholesterol efflux via the upregulation of adenosine triphosphate-binding cassette (ABC) transporters, ABCA1 and ABCG1. LXR agonist treatment was responsible for limiting BPDCN cell proliferation and inducing intrinsic apoptotic cell death. LXR activation in BPDCN cells was shown to interfere with 3 signaling pathways associated with leukemic cell survival, namely: NF-kB activation, as well as Akt and STAT5 phosphorylation in response to the BPDCN growth/survival factor interleukin-3. These effects were increased by the stimulation of cholesterol efflux through a lipid acceptor, the apolipoprotein A1. In vivo experiments using a mouse model of BPDCN cell xenograft revealed a decrease of leukemic cell infiltration and BPDCN-induced cytopenia associated with increased survival after LXR agonist treatment. This demonstrates that cholesterol homeostasis is modified in BPDCN and can be normalized by treatment with LXR agonists which can be proposed as a new therapeutic approach. (Blood. 2016;128(23):2694-2707

The use of chimeric antigen receptor (CAR)-engineered regulatory T cells (Tregs) has emerged as a promising strategy to promote immune tolerance. However, in conventional T cells (Tconvs), CAR expression is often associated with tonic signaling, which can induce CAR-T cell dysfunction. The extent and effects of CAR tonic signaling vary greatly according to the expression intensity and intrinsic properties of the CAR. Here, we show that the 4-1BB CSD-associated tonic signal yields a more dramatic effect in CAR-Tregs than in CAR-Tconvs with respect to activation and proliferation. Compared to CD28 CAR-Tregs, 4-1BB CAR-Tregs exhibit decreased lineage stability and reduced in vivo suppressive capacities. Transient exposure of 4-1BB CAR-Tregs to a Treg stabilizing cocktail, including an mTOR inhibitor and vitamin C, during ex vivo expansion sharply improves their in vivo function and expansion after adoptive transfer. This study demonstrates that the negative effects of 4-1BB tonic signaling in Tregs can be mitigated by transient mTOR inhibition.

The exact composition of leukocyte infiltration during kidney allograft rejection is difficult to comprehend and visualize on the same biopsy slide. Using an innovative technology of multiplex immunofluorescence (mIF), we were able to detect simultaneously NK cells, macrophages, and T cells and to determine their intra‐ or extravascular localization using an endothelial marker. Twenty antibody‐mediated rejection (ABMR), 20 T cell–mediated rejection (TCMR), and five normal biopsies were labeled, with automatic leukocyte quantification and localization. This method was compared to a classic NKp46 immunohistochemistry (IHC) with manual quantification and to mRNA quantification. mIF automatic quantification was strongly correlated to IHC (r = .91, P < .001) and to mRNA expression levels (r > .46, P < .021). T cells and macrophages were the 2 predominant populations involved in rejection (48.0 ± 4.4% and 49.3 ± 4.4%, respectively, in ABMR; 51.8 ± 6.0% and 45.3 ± 5.8% in TCMR). NK cells constituted a rare population in both ABMR (2.7 ± 0.7%) and TCMR (2.9 ± 0.6%). The intravascular compartment was mainly composed of T cells, including during ABMR, in peritubular and glomerular capillaries. However, NK cell and macrophage densities were significantly higher during ABMR in glomerular and peritubular capillaries. To conclude, this study demonstrates the feasibility and utility of mIF imaging to study and better understand the kidney allograft rejection process.

ABSTRACTlabile group of amino acids to a diphtheria toxin (DT) payload that has been truncated at its receptor binding region. 20 Since IL-3, the natural ligand for IL-3R, binds with very high specificity and avidity, 21 SL-401 is able to transport DT efficiently and preferentially to cells that overexpress IL-3R, leading to internalization followed by receptor-mediated endocytosis and localization of SL-401 to early endosomes. After cleavage of the SL-401 DT constituent in the acidic medium of endosomes, DT translocates into the cytosol and binds to ADP-ribosylated elongation factor 2, leading to blockade of protein synthesis and cell death. 22 Given the ubiquitous and high expression of IL-3R by BPDCN and the lack of therapies available to treat BPDCN, SL-401 is a potential therapeutic for BPDCN. The present study evaluated the cytotoxicity of SL-401 against patient-derived BPDCN cell lines (CAL-1 and GEN2.2) and primary BPDCN cells isolated directly from 12 patients. The investigations were performed in vitro, as well as in vivo in a murine model of BPDCN. The aim of the study was to provide further support for the use of SL-401 in patients suffering from BPDCN. Methods Patients' cells and cell linesPeripheral blood or bone marrow cells were obtained for diagnostic purposes from 12 BPDCN patients (Table 1) from our national network that collects data and cells from cases diagnosed in France since (authorization number #DC-2008. BPDCN was diagnosed from the results of histopathology and immunostaining of cutaneous lesions, blood or bone marrow. 2,8 Two established cell lines derived from BPDCN patients were used (GEN 2.2, patent #0215927, Dr. Plumas, EFS Rhone-Alpes, Grenoble, France and CAL-1, Dr. Maeda, Nagasaki University, Japan) as well as TF/H-Ras (Prof. Frankel) and CD123 neg (MFI<800) Daudi cell lines (ACC78, DSMZ Braunschweig, Germany) as positive and negative controls, respectively. Other lymphoid and myeloid leukemic cells used to compare sensitivity to SL-401 are described in the Online Supplementary Appendix. Drug and cultureThe SL-401 drug (Stemline Therapeutics, New York, NY, USA) was stored at -80°C and tested at eight concentrations ranging from 365 pM to 0.08 fM (21 ng/mL to 0.4 ng/mL) in order to cover (including myeloperoxidase, CD13, CD33, CD117, CD15, CD65, CD14, CD64); T : T lymphoid markers (including membrane CD3, intracytoplasmic CD3, CD7, CD5, CD2, CD8) Cytotoxicity evaluation by flow cytometryFlow cytometry was performed using a CANTO II cytometer (BD Biosciences, San Jose, CA, USA) and DIVA 6.2 software (BD Biosciences). The cytotoxic effects of SL-401 and the various drugs were evaluated using annexin-V and 7-amino actinomycin D (AV/7AAD) and a panel of different monoclonal antibodies to gate the blastic population described in the Online Supplementary Appendix. In the mouse model, anti-mouse and anti-human CD45 plus anti-human CD123, CD4, CD56, CD304 were used to identify BPDCN human cells (Online Supplementary Appendix). A defined number of calibrated 3-mm latex beads (Flowco...

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation, limiting the success of this therapy. We previously reported that interleukin-22 (IL-22) participates to aGVHD development, but the underlying mechanisms of its contribution remain poorly understood. In this study, we analyzed the mechanism of the pathological function of IL-22 in intestinal aGVHD. Ex-vivo colon culture experiments indicated that IL-22 was able to induce Th1-like inflammation via signal transducer and activator of transcription factor-1 (STAT1) and CXCL10 induction in the presence of type I interferon (IFN). To evaluate a potential synergy between IL-22 and type I IFN in aGVHD, we transplanted recipient mice, either wild-type (WT) or type I IFN receptor deficient (IFNAR(-/-)), with bone marrow cells and WT or IL-22 deficient (IL-22(-/-)) T cells. We observed a decreased GVHD severity in IFNAR(-/-) recipient of IL-22(-/-) T cells, which was associated with a lower level of STAT1 activation and reduced CXCL10 expression in the large intestine. Finally, immunohistochemistry staining of STAT1 performed on gastrointestinal biopsies of 20 transplanted patients showed exacerbated STAT1 activation in gastrointestinal tissues of patients with aGVHD as compared with those without aGVHD. Thus, interfering with both IL-22 and type I IFN signaling may provide a novel approach to limit aGVHD.