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.

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

In the original publication [...].

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.

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.

Belatacept in renal transplant recipient with mild immunologic risk factor: A pilot prospective study (BELACOR).

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.

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.

Intravenous immunoglobulin therapy in kidney transplant recipients with de novo DSA: Results of an observational study.

BACKGROUND: Approximately 25% of kidney transplant recipients develop de novo anti-HLA donor-specific antibodies (dnDSA) leading to acute antibody-mediated rejection (ABMR) in 30% of patients. Preemptive therapeutic strategies are not available. METHODS: We conducted a prospective observational study including 11 kidney transplant recipients. Inclusion criteria were dnDSA occurring within the first year after transplant and normal allograft biopsy. All patients were treated with high-dose IVIG (2 g/kg 0, 1 and 2 months post-dnDSA). The primary efficacy outcome was incidence of clinical and subclinical acute ABMR within 12 months after dnDSA detection as compared to a historical control group (IVIG-). RESULTS: Acute ABMR occurred in 2 or 11 patients in the IVIG+ group and in 1 of 9 patients in the IVIG- group. IVIG treatment did not affect either class I or class II DSA, as observed at the end of the follow-up. IVIG treatment significantly decreased FcγRIIA mRNA expression in circulating leukocytes, but did not affect the expression of any other markers of B cell activation. CONCLUSIONS: In this first pilot study including kidney allograft recipients with early dnDSA, preemptive treatment with high-dose IVIG alone did not prevent acute ABMR and had minimal effects on DSA outcome and B cell phenotype.

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.

CD40 engagement strongly induces CD25 expression on porcine dendritic cells and polarizes the T cell immune response toward Th1.

Orientation of the immune response toward Th1, Th2, Th17 or Treg plays an important role in self-tolerance and defence against pathogens and tumors. However, this orientation has not been fully characterised in the pig and little is known about the influence of maturation stimulus on the capacity of dendritic cells selectively to direct different types of Th cell responses. Dendritic cell (DC) maturation can be induced by different agents such as inflammatory cytokines, TLR ligands and CD40L. However, the role of the latter in the maturation of pig DC has never been reported. In this study we analysed how different maturation agents influence the capacity of DC to skew the immune response. Monocyte-derived porcine DCs were matured with human CD40L-transfected L-cells, Lipopolysaccharide (LPS) alone or LPS in combination with Tumor necrosis factor-alpha (TNFalpha) and interferon-alpha (IFNalpha). We found that human CD40L induced DC maturation characterised by increased expression of co-stimulatory CD80/86 molecules, high production of IL-12p40 in DC and induction of IFNgamma and t-bet mRNA in T cells, suggesting a Th1 orientation. Moreover we report for the first time the appearance of CD25 after activation of porcine DC. Furthermore, DC activated with TNF+LPS+IFN showed the highest allo-stimulatory capacity of allogeneic lymphocytes and induced IL-17 mRNA in T lymphocytes, suggesting a Th17 orientation that has never been previously reported in the pig. We also showed that immature DCs did not produce any IL-10 or IL-12 and induced both GATA-3 and IL-13 transcription in allogeneic MLR suggesting a Th2 orientation. This study therefore underlines that the nature of the stimulus strongly influences the capacity of DC to steer the immune response in the pig.