Treg-targeted IL-2/anti-IL-2 complex controls graft-versus-host disease and supports anti-tumor effect in allogeneic hematopoietic stem cell transplantation.

Modulating an immune response in opposite directions represents the holy grail in allogeneic hematopoietic stem cell transplantation (allo-HSCT) to avoid insufficient reactivity of donor T cells and hematologic malignancy relapse while controlling the potential development of graft-versus-host disease (GVHD), in which donor T cells attack the recipient's tissues. IL-2/anti-IL-2 complexes (IL-2Cx) represent a therapeutic option to selectively accentuate or dampen the immune response. In dedicated experimental models of allo-HSCT, including also human cells injected in immunodeficient NSG mice, we evaluated side-by-side the therapeutic effect of two IL-2Cx designed either to boost regulatory T cells (Treg) or alternatively to activate effector T cells (Teff), on GVHD occurrence and tumor relapse. We also evaluated the effect of the complexes on the phenotype and function of immune cells in vivo. Unexpectedly, both pro-Treg and pro-Teff IL-2Cx prevented GVHD development. They both induced Treg expansion and reduced CD8+ T-cell numbers, compared to untreated mice. However, only mice treated with the pro-Treg IL-2Cx, showed a dramatic reduction of exhausted CD8+ T cells, consistent with a potent anti-tumor effect. When evaluated on human cells, pro-Treg IL-2Cx also preferentially induced Treg expansion in vitro and in vivo, while allowing the development of a potent anti-tumor effect in NSG mice. Our results demonstrate the clinical relevance of using a pro-Treg, but not a pro-Teff IL2Cx to modulate alloreactivity after HSCT, while promoting a graft-versus-leukemia effect.

Effect of lethal total body irradiation on bone marrow chimerism, acute graft-versus-host disease, and tumor engraftment in mouse models: impact of different radiation techniques using low- and high-energy X-rays.

PURPOSE: Effects of X­ray energy levels used for myeloablative lethal total body irradiation (TBI) delivery prior to bone marrow transplantation (BMT) in preclinical mouse models were examined. MATERIALS AND METHODS: In mouse models, single-fraction myeloablative TBI at a lethal dose was delivered using two different X­ray devices, either low (160 kV cabinet irradiator) or high energy (6 MV linear accelerator), before semi-allogeneic hematopoietic stem-cell transplantation (HSCT) to ensure bone marrow (BM) chimerism, graft-versus-host disease (GVHD), and tumor engraftment. Recipient mice were clinically followed for 80 days after bone marrow transplantation (BMT). Flow cytometry was performed to assess donor chimerism and tumor engraftment in recipient mice. RESULTS: Both X­ray irradiation techniques delivered a 10 Gy single fraction of TBI, presented a lethal effect, and could allow near-complete early donor chimerism on day 13. However, low-energy irradiation increased T cells' alloreactivity compared to high-energy irradiation, leading to clinical consequences for GVHD and tumor engraftment outcomes. The alloreactive effect differences might be attributed to the distinction in inflammatory status of irradiated recipients at donor cell infusion (D0). Delaying donor cell administration (D1 after lethal TBI) attenuated T cells' alloreactivity and clinical outcomes in GVHD mouse models. CONCLUSION: Different X­ray irradiation modalities condition T cell alloreactivity in experimental semi-allogeneic BMT. Low-energy X­ray irradiator induces a post-TBI inflammatory burst and exacerbates alloreactive reactions. This technical and biological information should be considered in interpreting GVHD/ graft-versus-leukemia effect results in mice experimental models of BMT.

Radiation therapy for triple-negative breast cancer: emerging role of microRNAs as biomarkers and radiosensitivity modifiers. A systematic review.

PURPOSE: Radiation therapy (RT) for triple-negative breast cancer (TNBC) treatment is currently delivered in the adjuvant setting and is under investigation as a booster of neoadjuvant treatments. However, TNBC radioresistance remains an obstacle, so new biomarkers are needed to select patients for any integration of RT in the TNBC therapy sequence. MicroRNAs (miRs) are important regulators of gene expression, involved in cancer response to ionizing radiation (IR) and assessable by tumor tissue or liquid biopsy. This systematic review aimed to evaluate the relationships between miRs and response to radiation in TNBC, as well as their potential predictive and prognostic values. METHODS: A thorough review of studies related to miRs and RT in TNBC was performed on PubMed, EMBASE, and Web of Science. We searched for original English articles that involved dysregulation of miRs in response to IR on TNBC-related preclinical and clinical studies. After a rigorous selection, 44 studies were chosen for further analysis. RESULTS: Thirty-five miRs were identified to be TNBC related, out of which 21 were downregulated, 13 upregulated, and 2 had a double-side expression in this cancer. Expression modulation of many of these miRs is radiosensitizing, among which miR-7, -27a, -34a, -122, and let-7 are most studied, still only in experimental models. The miRs reported as most influencing/reflecting TNBC response to IR are miR-7, -27a, -155, -205, -211, and -221, whereas miR-21, -33a, -139-5p, and -210 are associated with TNBC patient outcome after RT. CONCLUSION: miRs are emerging biomarkers and radiosensitizers in TNBC, worth further investigation. Dynamic assessment of circulating miRs could improve monitoring and TNBC RT efficacy, which are of particular interest in the neoadjuvant and the high-risk patients' settings.

Costimulatory blockade molecules and B-cell-mediated immune response: current knowledge and perspectives.

There is an urgent need for therapeutic agents that target humoral alloimmunity in solid organ transplantation. This includes sensitized patients with preformed donor-specific human leukocyte antigen antibodies and patients who develop de novo donor-specific antibodies, both of which are associated with acute and chronic antibody-mediated rejection and allograft loss. In the last decade, both experimental and clinical studies highlighted the major impact of costimulation molecules in the control of immune responses both in the field of transplantation and autoimmune disease. Although these molecules have been initially developed to control the early steps of T-cell activation, recent evidence also supports their influence at several steps of the humoral response. In this review, we aim to provide an overview of the current knowledge of the effects of costimulatory blockade agents on humoral responses in both autoimmune and allogeneic contexts. We first present the effects of costimulatory molecules on the different steps of alloantibody production. We then summarize mechanisms and clinical results observed using cytotoxic T lymphocyte antigen-4 (CTLA4)-Ig molecules both in transplantation and autoimmunity. Finally, we present the potential interest and implications of other costimulatory family members as therapeutic targets, with emphasis on combinatorial approaches, for the optimal control of the alloantigen-specific humoral response.

Control of Humoral Response in Renal Transplantation by Belatacept Depends on a Direct Effect on B Cells and Impaired T Follicular Helper-B Cell Crosstalk.

Generation of de novo donor-specific antibodies (dnDSAs) after renal transplant is recognized as the leading cause of late transplant failure. Hence, the optimal immunosuppressive strategies to limit dnDSA development need to be defined. Recent clinical trials using the novel costimulatory blockade agent CTLA4-Ig (Belatacept) have shown that kidney transplant recipients (KTRs) treated with Belatacept have better graft survival and function and a lower proportion of dnDSAs than control-treated KTRs. Mechanisms involved in the control of humoral responses by Belatacept remain to be investigated. Here, we analyzed the effect of Belatacept on different steps of the B cell-mediated response in humans. In vitro, Belatacept reduced plasmablast differentiation, Ig production, and the expression of the major transcription factor involved in plasma cell function, Blimp-1, in a T cell-independent manner. Moreover, Belatacept induced activation of the STAT3 transcription factor in stimulated B cells and reduced the expression of CD86. Additionally, Belatacept blocked CD28-mediated activation of T follicular helper cells (Tfhs) in an autologous Tfh-memory B cells model. We then validated these observations in KTRs treated with Belatacept, who had a reduced proportion of blood effector B cells and activated Tfh (PD1+ICOS+) compared with control-treated KTRs. Our in vitro and in vivo results suggest that Belatacept modulates B cell function directly and at the level of B cell-Tfh interaction. These mechanisms likely account for the optimal control of humoral responses observed in KTRs treated with Belatacept.

Control of GVHD by regulatory T cells depends on TNF produced by T cells and TNFR2 expressed by regulatory T cells.

Therapeutic CD4(+)Foxp3(+) natural regulatory T cells (Tregs) can control experimental graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HCT) by suppressing conventional T cells (Tconvs). Treg-based therapies are currently tested in clinical trials with promising preliminary results in allo-HCT. Here, we hypothesized that as Tregs are capable of modulating Tconv response, it is likely that the inflammatory environment and particularly donor T cells are also capable of influencing Treg function. Indeed, previous findings in autoimmune diabetes revealed a feedback mechanism that renders Tconvs able to stimulate Tregs by a mechanism that was partially dependent on tumor necrosis factor (TNF). We tested this phenomenon during alloimmune response in our previously described model of GVHD protection using antigen specific Tregs. Using different experimental approaches, we observed that control of GVHD by Tregs was fully abolished by blocking TNF receptor type 2 (TNFR2) or by using TNF-deficient donor T cells or TNFR2-deficient Tregs. Thus, our results show that Tconvs exert a powerful modulatory activity on therapeutic Tregs and clearly demonstrate that the sole defect of TNF production by donor T cells was sufficient to completely abolish the Treg suppressive effect in GVHD. Importantly, our findings expand the understanding of one of the central components of Treg action, the inflammatory context, and support that targeting TNF/TNFR2 interaction represents an opportunity to efficiently modulate alloreactivity in allo-HCT to either exacerbate it for a powerful antileukemic effect or reduce it to control GVHD.

In Vivo Expansion of Activated Foxp3+ Regulatory T Cells and Establishment of a Type 2 Immune Response upon IL-33 Treatment Protect against Experimental Arthritis.

IL-33 is strongly involved in several inflammatory and autoimmune disorders with both pro- and anti-inflammatory properties. However, its contribution to chronic autoimmune inflammation, such as rheumatoid arthritis, is ill defined and probably requires tight regulation. In this study, we aimed at deciphering the complex role of IL-33 in a model of rheumatoid arthritis, namely, collagen-induced arthritis (CIA). We report that repeated injections of IL-33 during induction (early) and during development (late) of CIA strongly suppressed clinical and histological signs of arthritis. In contrast, a late IL-33 injection had no effect. The cellular mechanism involved in protection was related to an enhanced type 2 immune response, including the expansion of eosinophils, Th2 cells, and type 2 innate lymphoid cells, associated with an increase in type 2 cytokine levels in the serum of IL-33-treated mice. Moreover, our work strongly highlights the interplay between IL-33 and regulatory T cells (Tregs), demonstrated by the dramatic in vivo increase in Treg frequencies after IL-33 treatment of CIA. More importantly, Tregs from IL-33-treated mice displayed enhanced capacities to suppress IFN-γ production by effector T cells, suggesting that IL-33 not only favors Treg proliferation but also enhances their immunosuppressive properties. In concordance with these observations, we found that IL-33 induced the emergence of a CD39(high) Treg population in a ST2L-dependent manner. Our findings reveal a powerful anti-inflammatory mechanism by which IL-33 administration inhibits arthritis development.

Belatacept inhibit human B cell germinal center development in immunodeficient mice.

The humoral response mediated by alloantibodies directed against donor HLA molecules (DSAs) is one of the main causes of graft loss in kidney transplantation. Understanding the pathophysiology leading to humoral kidney rejection as the development of therapeutic tools is therefore a main objective in the field of solid organ transplantation and necessitate adapted experimental models. Among the immunosuppressive agents used in renal transplantation, belatacept, a fusion protein targeting T costimulatory molecules has shown its ability to prevent more efficiently the secretion of DSA by different mechanisms including a direct action on plasma cells but also on B lymphocytes and follicular helper T lymphocytes (Tfh) cooperation. This cellular cooperation occurs within germinal centers (GC), the seat of B lymphocytes differentiation. Here, we aimed to develop a dedicated mouse model in which human GC would be functional to study the effect of belatacept on GC formation and the ability of B lymphocytes to secrete immunoglobulin. We next demonstrate that belatacept inhibits the formation of these GCs, by inhibiting the frequency of Tfh and B lymphocytes. This alters the B maturation and therefore the generation of plasma cells and consequently, immunoglobulin secretion.

Pathologic Response to Neoadjuvant Sequential Chemoradiation Therapy in Locally Advanced Breast Cancer: Preliminary, Translational Results from the French Neo-APBI-01 Trial.

BACKGROUND: Radiation therapy (RT), a novel approach to boost the anticancer immune response, has been progressively evaluated in the neoadjuvant setting in breast cancer (BC). PURPOSE: We aimed to evaluate immunity-related indicators of response to neoadjuvant chemoradiation therapy (NACRT) in BC for better treatment personalization. PATIENTS AND METHODS: We analyzed data of the first 42 patients included in the randomized phase 2 Neo-APBI-01 trial comparing standard neoadjuvant chemotherapy (NACT) and NACRT regimen in locally advanced triple-negative (TN) and luminal B (LB) subtype BC. Clinicopathological parameters, blood counts and the derived parameters, total tumor-infiltrating lymphocytes (TILs) and their subpopulation, as well as TP53 mutation status, were assessed as predictors of response. RESULTS: Twenty-one patients were equally assigned to each group. The pathologic complete response (pCR) was 33% and 38% in the NACT and NACRT groups, respectively, with a dose-response effect. Only one LB tumor reached pCR after NACRT. Numerous parameters associated with response were identified, which differed according to the assigned treatment. In the NACRT group, baseline hemoglobin of ≥13 g/dL and body mass index of <26 were strongly associated with pCR. Higher baseline neutrophils-to-lymphocytes ratio, total TILs, and T-effector cell counts were favorable for pCR. CONCLUSION: This preliminary analysis identified LB and low-TIL tumors as poor responders to the NACRT protocol, which delivered RT after several cycles of chemotherapy. These findings will allow for amending the selection of patients for the trial and help better design future trials of NACRT in BC.

TNFR2 blockade of regulatory T cells unleashes an antitumor immune response after hematopoietic stem-cell transplantation.

BACKGROUND: Targeting immune checkpoints that inhibit antitumor immune responses has emerged as a powerful new approach to treat cancer. We recently showed that blocking the tumor necrosis factor receptor-type 2 (TNFR2) pathway induces the complete loss of the protective function of regulatory T cells (Tregs) in a model of graft-versus-host disease (GVHD) prevention that relies on Treg-based cell therapy. Here, we tested the possibility of amplifying the antitumor response by targeting TNFR2 in a model of tumor relapse following hematopoietic stem-cell transplantation, a clinical situation for which the need for efficient therapeutic options is still unmet. METHOD: We developed appropriate experimental conditions that mimic patients that relapsed from their initial hematological malignancy after hematopoietic stem-cell transplantation. This consisted of defining in allogeneic bone marrow transplantation models developed in mice, the maximum number of required tumor cells and T cells to infuse into recipient mice to develop a model of tumor relapse without inducing GVHD. We next evaluated whether anti-TNFR2 treatment could trigger alloreactivity and consequently antitumor immune response. In parallel, we also studied the differential expression of TNFR2 on T cells including Treg from patients in post-transplant leukemia relapse and in patients developing GVHD. RESULTS: Using experimental conditions in which neither donor T cells nor TNFR2-blocking antibody per se have any effect on tumor relapse, we observed that the coadministration of a suboptimal number of T cells and an anti-TNFR2 treatment can trigger alloreactivity and subsequently induce a significant antitumor effect. This was associated with a reduced percentage of activated CD4+ and CD8+ Tregs. Importantly, human Tregs over-expressed TNFR2 relative to conventional T cells in healthy donors and in patients experiencing leukemia relapse or cortico-resistant GVHD after hematopoietic stem cell transplantation. CONCLUSIONS: These results highlight TNFR2 as a new target molecule for the development of immunotherapies to treat blood malignancy relapse, used either directly in grafted patients or to enhance donor lymphocyte infusion strategies. More widely, they open the door for new perspectives to amplify antitumor responses against solid cancers by directly targeting Tregs through their TNFR2 expression.

Nucleolin Therapeutic Targeting Decreases Pancreatic Cancer Immunosuppression.

Background: The pancreatic ductal adenocarcinoma (PDAC) microenvironment is highly fibrotic and hypoxic, with poor immune cell infiltration. Recently, we showed that nucleolin (NCL) inhibition normalizes tumour vessels and impairs PDAC growth. Methods: Immunocompetent mouse models of PDAC were treated by the pseudopeptide N6L, which selectively inhibits NCL. Tumour-infiltrating immune cells and changes in the tumour microenvironment were analysed. Results: N6L reduced the proportion of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) and increased tumour-infiltrated T lymphocytes (TILs) with an activated phenotype. Low-dose anti-VEGFR2 treatment normalized PDAC vessels but did not modulate the immune suppressive microenvironment. RNAseq analysis of N6L-treated PDAC tumours revealed a reduction of cancer-associated fibroblast (CAF) expansion in vivo and in vitro. Notably, N6L treatment decreased IL-6 levels both in tumour tissues and in serum. Treating mPDAC by an antibody blocking IL-6 reduced the proportion of Tregs and MDSCs and increased the amount of TILs, thus mimicking the effects of N6L. Conclusions: These results demonstrate that NCL inhibition blocks the amplification of lymphoid and myeloid immunosuppressive cells and promotes T cell activation in PDAC through a new mechanism of action dependent on the direct inhibition of the tumoral stroma.

Correction: Ponzo et al. Nucleolin Therapeutic Targeting Decreases Pancreatic Cancer Immunosuppression. Cancers 2022, 14, 4265.

In the original publication [...].

CD8+T cell responsiveness to anti-PD-1 is epigenetically regulated by Suv39h1 in melanomas.

Tumor-infiltrating CD8 + T cells progressively lose functionality and fail to reject tumors. The underlying mechanism and re-programing induced by checkpoint blockers are incompletely understood. We show here that genetic ablation or pharmacological inhibition of histone lysine methyltransferase Suv39h1 delays tumor growth and potentiates tumor rejection by anti-PD-1. In the absence of Suv39h1, anti-PD-1 induces alternative activation pathways allowing survival and differentiation of IFNγ and Granzyme B producing effector cells that express negative checkpoint molecules, but do not reach final exhaustion. Their transcriptional program correlates with that of melanoma patients responding to immune-checkpoint blockade and identifies the emergence of cytolytic-effector tumor-infiltrating lymphocytes as a biomarker of clinical response. Anti-PD-1 favors chromatin opening in loci linked to T-cell activation, memory and pluripotency, but in the absence of Suv39h1, cells acquire accessibility in cytolytic effector loci. Overall, Suv39h1 inhibition enhances anti-tumor immune responses, alone or combined with anti-PD-1, suggesting that Suv39h1 is an "epigenetic checkpoint" for tumor immunity.

Macrophage autophagy protects against hepatocellular carcinogenesis in mice.

Autophagy is a lysosomal degradation pathway of cellular components that regulates macrophage properties. Macrophages are critically involved in tumor growth, metastasis, angiogenesis and immune suppression. Here, we investigated whether macrophage autophagy may protect against hepatocellular carcinoma (HCC). Experiments were performed in mice with deletion of the autophagy gene Atg5 in the myeloid lineage (ATG5Mye-/- mice) and their wild-type (WT) littermates. As compared to WT, ATG5Mye-/- mice were more susceptible to diethylnitrosamine (DEN)-induced hepatocarcinogenesis, as shown by enhanced tumor number and volume. Moreover, DEN-treated ATG5Mye-/- mice exhibited compromised immune cell recruitment and activation in the liver, suggesting that macrophage autophagy invalidation altered the antitumoral immune response. RNA sequencing showed that autophagy-deficient macrophages sorted from DEN mice are characterized by an enhanced expression of immunosuppressive markers. In vitro studies demonstrated that hepatoma cells impair the autophagy flux of macrophages and stimulate their expression of programmed cell death-ligand 1 (PD-L1), a major regulator of the immune checkpoint. Moreover, pharmacological activation of autophagy reduces hepatoma cell-induced PD-L1 expression in cultured macrophages while inhibition of autophagy further increases PD-L1 expression suggesting that autophagy invalidation in macrophages induces an immunosuppressive phenotype. These results uncover macrophage autophagy as a novel protective pathway regulating liver carcinogenesis.

Intra-articular electrotransfer of mouse soluble tumour necrosis factor receptor in a murine model of rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes inflammation and destruction of the joints. In the collagen-induced arthritis mouse model of RA, we developed a nonviral gene therapy method designed to block in situ the main cytokine tumour necrosis factor (TNF)-alpha METHODS: Electrotransfer was used to deliver a plasmid encoding extracellular domain of mouse soluble TNF-alpha receptor type I fused to the Fc fragment of mouse immunoglobulin (Ig)G1 (pTNFR-Is) corresponding to a dimeric TNF-alpha soluble receptor fusion protein (mTNFR-Is/Ig). RESULTS: Delivery of the plasmid into the knees at symptom onset improved the histological inflammation and destruction not only at the knees, but also at the ankles, indicating a local and a regional therapeutic effect. The plasmid was detected in synovial membrane and meniscus specimens from the injected joints. In the synovial membrane, 15 days post-injection, interleukin (IL)-17 and TNF-alpha mRNAs expression were increased, whereas IL-10 mRNA was unchanged. However, the empty plasmid exerted a pro-inflammatory effect 30 days post-injection. CONCLUSIONS: These data indicate that local nonviral gene therapy against TNF-alpha is effective, although further work is needed to decrease plasmid induced inflammation.

Phenotypic and Transcriptomic Lymphocytes Changes in Allograft Recipients After Intravenous Immunoglobulin Therapy in Kidney Transplant Recipients.

High dose intravenous immunoglobulin (IVIG) are widely used after kidney transplantation and its biological effect on T and B cell phenotype in the context of maintenance immunosuppression was not documented yet. We designed a monocentric prospective cohort study of kidney allograft recipients with anti-HLA donor specific antibodies (DSA) without acute rejection on screening biopsies treated with prophylactic high-dose IVIG (2 g/kg) monthly for 2 months. Any previous treatment with Rituximab was an exclusion criterion. We performed an extensive analysis of phenotypic and transcriptomic T and B lymphocytes changes and serum cytokines after treatment (day 60). Twelve kidney transplant recipients who completed at least two courses of high-dose IVIG (2 g/kg) were included in a median time of 45 (12-132) months after transplant. Anti-HLA DSA characteristics were similar before and after treatment. At D60, PBMC population distribution was similar to the day before the first infusion. CD8+ CD45RA+ T cells and naïve B-cells (Bm2+) decreased (P = 0.03 and P = 0.012, respectively) whereas Bm1 (mature B-cells) increased (P = 0.004). RORγt serum mRNA transcription factor and CD3 serum mRNA increased 60 days after IVIG (P = 0.02 for both). Among the 25 cytokines tested, only IL-18 serum concentration significantly decreased at D60 (P = 0.03). In conclusion, high dose IVIG induced limited B cell and T cell phenotype modifications that could lead to anti-HLA DSA decrease. However, no clinical effect has been isolated and the real benefit of prophylactic use of IVIG after kidney transplantation merits to be questioned.

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.

Human Apoptotic Cells, Generated by Extracorporeal Photopheresis, Modulate Allogeneic Immune Response.

The induction of specific and sustainable tolerance is a challenging issue in organ transplantation. The discovery of the immunosuppressive properties of apoptotic cells in animal models has paved the way for their use in human transplantation. In this work, we aimed to define a stable, reproducible, and clinically compatible production procedure of human apoptotic cells (Apo-cells). Using a clinically approved extracorporeal photopheresis technique, we have produced and characterized phenotypically and functionally human apoptotic cells. These Apo-cells have immunosuppressive properties proved in vitro and in vivo in NOD/SCID/γC mice by their capacity to modulate an allogeneic response following both a direct and an indirect antigen presentation. These results brought the rationale for the use of Apo-cells in tolerance induction protocol for organ transplantation.

Simple, Reproducible, and Efficient Clinical Grading System for Murine Models of Acute Graft-versus-Host Disease.

Acute graft-versus-host disease (aGVHD) represents a challenging complication after allogeneic hematopoietic stem cell transplantation. Despite the intensive preclinical research in the field of prevention and treatment of aGVHD, and the presence of a well-established clinical grading system to evaluate human aGVHD, such a valid tool is still lacking for the evaluation of murine aGVHD. Indeed, several scoring systems have been reported, but none of them has been properly evaluated and they all share some limitations: they incompletely reflect the disease, rely on severity stages that are distinguished by subjective assessment of clinical criteria and are not easy to discriminate, which could render evaluation more time consuming, and their reproducibility among different experimenters is uncertain. Consequently, clinical murine aGVHD description is often based merely on animal weight loss and mortality. Here, we propose a simple scoring system of aGVHD relying on the binary (yes or no) evaluation of five important visual parameters that reflect the complexity of the disease without the need to sacrifice the mice. We show that this scoring system is consistent with the gold standard histological staging of aGVHD across several donor/recipient mice combinations. This system is also a strong predictor of survival of recipient mice when used early after transplant and is highly reproducible between experimenters.