Catastrophic antiphospholipid syndrome (CAPS) leads to organ dysfunction due to thrombotic microangiopathy (TMA). Complement may play a role in CAPS, and its blockade could prevent antiphospholipid syndrome (APS) complications after kidney transplantation (KT). Here, we report a case of APS recurrence after KT in a 38-year-old woman with early acute cortical kidney allograft necrosis despite preventive eculizumab treatment, probably because of insufficient complement blockade. The patient had recurrent but controlled CAPS for years with renal dysfunction, leading to preemptive KT. Anticoagulation and eculizumab were administered to prevent thrombosis and TMA after KT. She developed acute kidney injury (AKI) with incomplete biological TMA. Imaging revealed cortical necrosis in the renal allograft. In the absence of donor-specific anti-HLA antibodies, we concluded a relapse. Additional doses of eculizumab and plasma exchange allowed the normalization of biological tests and improvement of kidney allograft function. A retrospective complement analysis showed an incomplete blockade at the time of AKI. One year after KT, the renal allograft function was impaired. This suggests that inadequate complement blockade leads to a relapse of APS in the renal allograft with cortical necrosis and dysfunction. Our case highlights the importance of monitoring complement activity and adjusting the dose of eculizumab or ravulizumab.

Experiment models of xenogeneic graft-versus-host disease (xeno-GVHD), in which human immune cells are injected into immunodeficient mice, are increasingly used to study human immune cell behavior in vivo and to test therapeutic approaches. Today, the main, and more commonly accepted clinical parameters used to characterize xeno-GVHD are weight loss and mortality. These criteria do not provide an accurate and subtle assessment of the disease intensity, nor do they reflect the great variability of xeno-GVHD, which depends on the donor. Relying on previous work in which we described an original clinical grading system for assessing GVHD in mice, we propose an adaptation of this system for xeno-GVHD models. This simple, solid, and reproducible scoring system of xeno-GVHD is constituted of the binary (yes or no) evaluation of 4 easy-to-evaluate parameters that reflect the complexity of the disease without the need to sacrifice the mice. This scoring system is consistent with the gold standard histological grading of human GVHD and with numerous biomarkers characteristic of the disease. We propose this new clinical grading system to evaluate and compare the results obtained with a common tool, regardless of the experimenters and laboratories where the experiments would have been carried out and whatever the therapeutic strategy evaluated.

IntroductionAfter kidney transplantation (KT), there is no reliable assessment of the immunosuppressive state. We analysed pre-KT T-, B- and NK-cell populations in relation to the occurrence of opportunistic infections (OI) or acute rejection (AR) after KT.MethodsWe included 422 adult KT recipients from 01/2016 to 09/2020. Immune cells were analysed using flow cytometry in 283 recipients before KT in three groups: AR, OI or no event within 24 months after KT.ResultsThere were 49 recipients in the OI group, 44 in the AR group and 190 in the control group. Before KT, higher absolute counts and percentages of NK cells (p=0.001 and p=0.007 respectively), elevated absolute counts of plasmablasts and CD21-CD38- B cells (age-associated B cells) (p=0.045 and p=0.028 respectively), and a lower proportion of CD3+ T cells (p=0.022) were independently associated with the occurrence of OI within two years following kidney transplantation (KT). In recipients with OI occurring before three months, only absolute count of NK cells before KT remained independently associated with the occurrence of OI (p=0.002). None of the studied immune cell population was associated with AR.ConclusionOur results suggest that higher levels of pretransplant NK cells and age-associated B cells are correlated with the occurrence of OI within two years after KT. This result may improve stratification of individualized infectious risk prior to KT.

Introduction:The safety and benefit of the anti-CD38 monoclonal antibody daratumumab, which induces lysis of antibody-producing plasma cells in sensitized patients prior to kidney transplantation, remain to be determined. Methods:A 2-phase (1 and 2), monocentric open-label study was conducted to evaluate the month 6 (M6) safety and efficacy of daratumumab in kidney transplant candidates with calculated panel reactive antibody (cPRA) > 95%. In the first (safety) phase, we used 4-weekly escalating doses of daratumumab. Phase 2 tested desensitization with 8 weekly infusions of 16 mg/kg daratumumab. cPRA 10,000 was calculated considering only human leukocyte antigen (HLA) antibodies with mean fluorescence intensity (MFI) of > 10,000. Results:Nine patients were enrolled in phase 1 and 14 in phase 2. Safety analysis showed 4 serious non-treatment-emergent adverse events (non-TEAEs), 36 mild TEAEs, mostly infusion-related reactions, grade 1 and 2 (causing 2 temporary drug discontinuations), but no serious TEAEs. Significant reductions in anti-HLA antibodies were observed at month 3 (M3), with cPRA 10,000 (P = 0.003), number of anti-HLA (P < 0.001), maximum MFI (MFI max) (P = 0.053), and the sum of MFI (MFI sum) (P < 0.001), with complete return to baseline levels at month 12 (M12). At M6, 46.15% (19.22%-74.87%) and 76.92% (46.19%-94.96%) of patients showed sustained response (1% decrease in cPRA) for cPRA 2000 and 10,000, respectively. At month 1 (M1), immune cells (T-reg, CD8 + TEMRA, CD19 + CD138 + B cells, and NK cells) significantly decreased. At M3, other antibodies decreased significantly, but returned to baseline levels at M12, except for gamma globulins, without any infectious complications. Conclusion:The first use of daratumumab in desensitization demonstrated infusion-related adverse (AEs) events and rapid, albeit transient, reductions in anti-HLA antibodies, with less than 40% of durable responders, limiting its potential clinical use.

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.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, highly resistant to standard chemotherapy and immunotherapy. Regulatory T cells (Tregs) expressing tumor necrosis factor α receptor 2 (TNFR2) contribute to immunosuppression in PDAC. Treg infiltration correlates with poor survival and tumor progression in patients with PDAC. We hypothesized that TNFR2 inhibition using a blocking monoclonal antibody (mAb) could shift the Treg-effector T cell balance in PDAC, thus enhancing antitumoral responses.MethodTo support this hypothesis, we first described TNFR2 expression in a cohort of 24 patients with PDAC from publicly available single-cell analysis data. In orthotopic and immunocompetent mouse models of PDAC, we also described the immune environment of PDAC after immune cell sorting and single-cell analysis. The modifications of the immune environment before and after anti-TNFR2 mAb treatment were evaluated as well as the effect on tumor progression.ResultsPatients with PDAC exhibited elevated TNFR2 expression in Treg, myeloid cells and endothelial cells and lower level in tumor cells. By flow cytometry and single-cell RNA-seq analysis, we identified two Treg populations in orthotopic mouse models: Resting and activated Tregs. The anti-TNFR2 mAb selectively targeted activated tumor-infiltrating Tregs, reducing T cell exhaustion markers in CD8+T cells. However, anti-TNFR2 treatment alone had limited efficacy in activating CD8+T cells and only slightly reduced the tumor growth. The combination of the anti-TNFR2 mAb with agonistic anti-CD40 mAb promoted stronger T cell activation, tumor growth inhibition, and improved survival and immunological memory in PDAC-bearing mice.ConclusionOur data suggest that combining a CD40 agonist with a TNFR2 antagonist represents a promising therapeutic strategy for patients with PDAC.

High human leukocyte antigen (HLA) sensitization limits access to compatible transplantation. New CD38-targeting agents have been shown to reduce anti-HLA antibodies, although with important interpatient variability. Thus, pretreatment identification of responder and nonresponder (NR) patients is needed for treatment decision-making. We analyzed 26 highly sensitized (HS) patients from 2 desensitization trials using anti-CD38 monoclonal antibodies. Hierarchical clustering identified 3 serologic responder groups: high responders, low responders, and NR. Spectral flow cytometry and functional HLA-specific memory B cell (mBC) assessment were first conducted on peripheral blood mononuclear cells and bone marrow samples from 16 patients treated with isatuximab (NCT04294459). Isatuximab effectively depleted bone marrow plasma cells, peripheral CD38-expressing plasmablasts, plasma cells, transitional B cells, and class-switch mBCs, ultimately reducing frequencies of HLA-specific immunoglobulin G (IgG)-producing mBCs. Multidimensional spectral flow cytometry with partial least squares discriminant analysis revealed that pretreatment abundance of specific circulating mBC phenotypes, especially CD38neg class-switch mBCs, accurately distinguished between high serologic responders and low responders or NR (AUC 0.958, 0.860-1.000, P = .009), who also displayed significantly lower frequencies of HLA-specific IgG-producing mBCs (P < .0001). This phenotypical mBC signature predicting response to therapy was validated in an external HS patient cohort (n = 10) receiving daratumumab (NCT04204980). This study identifies critical circulating mBC subset phenotypes that distinguish HS patients with successful serologic responses to CD38-targeting desensitization therapies, potentially guiding treatment decision-making.

Effects of X‑ray energy levels used for myeloablative lethal total body irradiation (TBI) delivery prior to bone marrow transplantation (BMT) in preclinical mouse models were examined. In mouse models, single-fraction myeloablative TBI at a lethal dose was delivered using two different X‑ray 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. Both X‑ray 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. Different X‑ray irradiation modalities condition T cell alloreactivity in experimental semi-allogeneic BMT. Low-energy X‑ray 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.

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.

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.

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.

In the original publication [...].

BACKGROUND:The use of distant autografts to restore maxillary bone defects is clinically challenging and has unpredictable outcomes. This variation may be explained by the embryonic origin of long bone donor sites, which are derived from mesoderm, whereas maxillary bones derive from neural crest. Gingival stem cells share the same embryonic origin as maxillary bones. Their stemness potential and ease of access have been repeatedly shown. One limitation in human cell therapy is the use of foetal calf serum during cell isolation and culture. To overcome this problem, a new serum-free medium enriched with an alternative to foetal calf serum, i.e., platelet lysate, needs to be adapted to clinical grade protocols.METHODS:Different serum-free media enriched with platelet lysate at various concentrations and supplemented with different growth factors were developed and compared to media containing foetal calf serum. Phenotypic markers, spontaneous DNA damage, and stem cell properties of gingival stem cells isolated in platelet lysate or in foetal calf serum were also compared, as were the immunomodulatory properties of the cells by co-culturing them with activated peripheral blood monocellular cells. T-cell proliferation and phenotype were also assessed by flow cytometry using cell proliferation dye and specific surface markers. Data were analysed with t-test for two-group comparisons, one-way ANOVA for multigroup comparisons and two-way ANOVA for repeated measures and multigroup comparisons.RESULTS:Serum-free medium enriched with 10% platelet lysate and growth hormone yielded the highest expansion rate. Gingival stem cell isolation and thawing under these conditions were successful, and no significant DNA lesions were detected. Phenotypic markers of mesenchymal stem cells and differentiation capacities were conserved. Gingival stem cells isolated in this new serum-free medium showed higher osteogenic differentiation potential compared to cells isolated in foetal calf serum. The proportion of regulatory T cells obtained by co-culturing gingival stem cells with activated peripheral blood monocellular cells was similar between the two types of media.CONCLUSIONS:This new serum-free medium is well suited for gingival stem cell isolation and proliferation, enhances osteogenic capacity and maintains immunomodulatory properties. It may allow the use of gingival stem cells in human cell therapy for bone regeneration in accordance with good manufacturing practice guidelines.

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.

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.

The benefit of belatacept on antibody-mediated rejection (ABMR) incidence after kidney transplant with preformed donor-specific antibodies (DSAs) has never been assessed. Between 2014 and 2016, we conducted a multicenter prospective clinical trial with 49 patients to determine kidney allograft outcome in recipients with preformed DSAs (maximal mean fluorescence intensity 500 to 3000) treated with belatacept (BELACOR trial). Immunosuppressive strategy included antithymocyte globulin, belatacept, mycophenolate mofetil, and steroids. An ancillary control group was designed retrospectively, including patients fulfilling the same inclusion criteria treated with calcineurin inhibitors. In BELACOR group, no patient exhibited acute ABMR, patient and allograft survival at 1 year was 100% and 95.4%, respectively, and the estimated glomerular filtration rate was 53.2 mL/min/1.73 m2 . However, the 12-month incidence of acute T cell-mediated rejection was 25.4% (14.5% to 42.4%). Comparison with the control group showed significantly higher T cell-mediated rejection incidence only in the BELACOR group (P = .003). Considering the DSAs, the outcome was similar in the 2 groups except a significantly higher number of patients displayed a complete disappearance of class II DSAs in the BELACOR group (P = .001). Belatacept was not associated with an acute ABMR increased risk and may be considered as immunosuppressive strategy in transplant recipients with preformed DSAs (maximal mean fluorescence intensity 500 to 3000). Prospective randomized trials are needed to confirm these results.

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.