Natural Antibodies and Alloreactive T Cells Long after Kidney Transplantation

Background. The relationship between circulating effector memory T and B cells long after transplantation and their susceptibility to immunosuppression are unknown. To investigate the impact of antirejection therapy on T cell-B cell coordinated immune responses, we assessed IFN-γ-producing memory cells and natural antibodies (nAbs) that potentially bind to autoantigens on the graft. Methods. Plasma levels of IgG nAbs to malondialdehyde (MDA) were measured in 145 kidney transplant recipients at 5–7 years after transplantation. In 54 of these patients, the number of donor-reactive IFN-γ-producing cells was determined. 35/145 patients experienced rejection, 18 of which occurred within 1 year after transplantation. Results. The number of donor-reactive IFN-γ-producing cells and the levels of nAbs were comparable between rejectors and nonrejectors. The nAbs levels were positively correlated with the number of donor-reactive IFN-γ-producing cells (rs = 0.39, p = 0.004 ). The positive correlation was only observed in rejectors (rs = 0.53, p = 0.003 ; nonrejectors: rs = 0.24, p = 0.23 ). Moreover, we observed that intravenous immune globulin treatment affected the level of nAbs and this effect was found in patients who experienced a late ca-ABMR compared to nonrejectors ( p = 0.008 ). Conclusion. The positive correlation found between alloreactive T cells and nAbs in rejectors suggests an intricate role for both components of the immune response in the rejection process. Treatment with intravenous immune globulin impacted nAbs.

The Effects of Interferons on Allogeneic T Cell Response in GVHD: The Multifaced Biology and Epigenetic Regulations

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematological malignancies. This beneficial effect is derived mainly from graft-versus-leukemia (GVL) effects mediated by alloreactive T cells. However, these alloreactive T cells can also induce graft-versus-host disease (GVHD), a life-threatening complication after allo-HSCT. Significant progress has been made in the dissociation of GVL effects from GVHD by modulating alloreactive T cell immunity. However, many factors may influence alloreactive T cell responses in the host undergoing allo-HSCT, including the interaction of alloreactive T cells with both donor and recipient hematopoietic cells and host non-hematopoietic tissues, cytokines, chemokines and inflammatory mediators. Interferons (IFNs), including type I IFNs and IFN-γ, primarily produced by monocytes, dendritic cells and T cells, play essential roles in regulating alloreactive T cell differentiation and function. Many studies have shown pleiotropic effects of IFNs on allogeneic T cell responses during GVH reaction. Epigenetic mechanisms, such as DNA methylation and histone modifications, are important to regulate IFNs’ production and function during GVHD. In this review, we discuss recent findings from preclinical models and clinical studies that characterize T cell responses regulated by IFNs and epigenetic mechanisms, and further discuss pharmacological approaches that modulate epigenetic effects in the setting of allo-HSCT.

Regulation of Alloantibody Responses

The control of alloimmunity is essential to the success of organ transplantation. Upon alloantigen encounter, naïve alloreactive T cells not only differentiate into effector cells that can reject the graft, but also into T follicular helper (Tfh) cells that promote the differentiation of alloreactive B cells that produce donor-specific antibodies (DSA). B cells can exacerbate the rejection process through antibody effector functions and/or B cell antigen-presenting functions. These responses can be limited by immune suppressive mechanisms mediated by T regulatory (Treg) cells, T follicular regulatory (Tfr) cells, B regulatory (Breg) cells and a newly described tolerance-induced B (TIB) cell population that has the ability to suppress de novo B cells in an antigen-specific manner. Transplantation tolerance following costimulation blockade has revealed mechanisms of tolerance that control alloreactive T cells through intrinsic and extrinsic mechanisms, but also inhibit alloreactive B cells. Thus, the control of both arms of adaptive immunity might result in more robust tolerance, one that may withstand more severe inflammatory challenges. Here, we review new findings on the control of B cells and alloantibody production in the context of transplant rejection and tolerance.

Regulatory Dendritic Cells Induced by Bendamustine Are Associated With Enhanced Flt3 Expression and Alloreactive T-Cell Death

The growth factor Flt3 ligand (Flt3L) is central to dendritic cell (DC) homeostasis and development, controlling survival and expansion by binding to Flt3 receptor tyrosine kinase on the surface of DCs. In the context of hematopoietic cell transplantation, Flt3L has been found to suppress graft-versus-host disease (GvHD), specifically via host DCs. We previously reported that the pre-transplant conditioning regimen consisting of bendamustine (BEN) and total body irradiation (TBI) results in significantly reduced GvHD compared to cyclophosphamide (CY)+TBI. Pre-transplant BEN+TBI conditioning was also associated with greater Flt3 expression among host DCs and an accumulation of pre-cDC1s. Here, we demonstrate that exposure to BEN increases Flt3 expression on both murine bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs (moDCs). BEN favors development of murine plasmacytoid DCs, pre-cDC1s, and cDC2s. While humans do not have an identifiable equivalent to murine pre-cDC1s, exposure to BEN resulted in decreased plasmacytoid DCs and increased cDC2s. BEN exposure and heightened Flt3 signaling are associated with a distinct regulatory phenotype, with increased PD-L1 expression and decreased ICOS-L expression. BMDCs exposed to BEN exhibit diminished pro-inflammatory cytokine response to LPS and induce robust proliferation of alloreactive T-cells. These proliferative alloreactive T-cells expressed greater levels of PD-1 and underwent increased programmed cell death as the concentration of BEN exposure increased. Alloreactive CD4+ T-cell death may be attributable to pre-cDC1s and provides a potential mechanism by which BEN+TBI conditioning limits GvHD and yields T-cells tolerant to host antigen.

Berberine Prolongs Mouse Heart Allograft Survival by Activating T Cell Apoptosis via the Mitochondrial Pathway

Berberine, which is a traditional Chinese medicine can inhibit tumorigenesis by inducing tumor cell apoptosis. However, the immunoregulatory of effects berberine on T cells remains poorly understood. Here, we first examined whether berberine can prolong allograft survival by regulating the recruitment and function of T cells. Using a major histocompatibility complex complete mismatch mouse heterotopic cardiac transplantation model, we found that the administration of moderate doses (5 mg/kg) of berberine significantly prolonged heart allograft survival to 19 days and elicited no obvious berberine-related toxicity. Compared to that with normal saline treatment, berberine treatment decreased alloreactive T cells in recipient splenocytes and lymph node cells. It also inhibited the activation, proliferation, and function of alloreactive T cells. Most importantly, berberine treatment protected myocardial cells by decreasing CD4+ and CD8+ T cell infiltration and by inhibiting T cell function in allografts. In vivo and in vitro assays revealed that berberine treatment eliminated alloreactive T lymphocytes via the mitochondrial apoptosis pathway, which was validated by transcriptome sequencing. Taken together, we demonstrated that berberine prolongs allograft survival by inducing apoptosis of alloreactive T cells. Thus, our study provides more evidence supporting the potential use of berberine in translational medicine.

Dimethyl Fumarate Ameliorates Graft-Versus-Host Disease By Negatively Regulating Aerobic Glycolysis in Alloreactive T-Cells

Background Dimethyl fumarate (DMF), a fumaric acid derivative, is currently used worldwide as a therapeutic agent for autoimmune diseases, such as multiple sclerosis and psoriasis. As an activator of Nrf-2, DMF protects cells from oxidative stress by inducing anti-oxidant enzymes. In addition, a recent report in Science has shown that DMF catalytically inactivates GAPDH, thereby reduces glycolytic activity, and results in immune modulation in activated CD4+ T-cells. We have previously shown that DMF and its metabolite monomethyl fumarate (MMF) significantly inhibit 3H-thymidine uptake in activated T-cells. DMF also decreased the expression of proliferation marker Ki-67 and intracellular IFN-γ of activated T-cells in a dose dependent manner. These findings prompted us to investigate whether DMF can be used for the treatment of graft-versus host disease (GVHD) after hematopoietic stem cell transplantation. In the current study, we investigated whether, and if so, how DMF inhibits human T-cell immune response and suppress acute GVHD in vivo using a xenogeneic GVHD mouse model. Methods To induce acute GVHD, human peripheral blood mononuclear cells (hPBMCs) were intravenously injected into sublethally irradiated (250 cGy) NOG mice. We allocated the mice into two groups; DMF treatment and non-treatment (control mice). Mice in the DMF group were administered DMF orally (100 mg/kg) for consecutive 7 days (day -3 to +3), and compared with the control mice treated with the same volume of vehicle. Results First, we observed that DMF treatment prolonged the survival of mice (Figure 1). Supporting the result, histopathological analysis showed that the number of hPBMCs infiltrated in the lungs and liver was decreased in the DMF group. Next, to identify the alteration of donor human cell populations after DMF treatment, hPBMCs were retrieved from the lungs on day 9 after transplantation and were analyzed by flow cytometry. Consistent with the histological findings, the absolute number of hPBMCs (hCD45+), and also T-cells (hCD45+hCD3+), in the lungs was significantly lower in the DMF group compared with the control (p < 0.01) (Figure 2). Notably, the number of CD4+ T-cells, but not CD8+ T-cells, was decreased by the DMF treatment. The proportion of regulatory T-cells (Tregs) (hCD45+CD4+CD25+Foxp3+) was elevated in the DMF group, and this finding is consistent with existing reports that DMF may increase the proportion of Tregs. Furthermore, the expression level of PD-1 on hCD4+ T-cells was significantly lower in the DMF group. These results suggest that DMF treatment mainly regulates cell proliferation and functional differentiation of donor human CD4+ T-cells, leading to reduced severity of GVHD. Given that GAPDH and aerobic glycolysis have been shown as potential targets of DMF, we then measured glycolytic activity in human T-cells obtained from mice during GVHD. Extracellular acidification rate, an indicator of glycolytic activity, was monitored under basal conditions followed by sequential treatment with glucose, oligomycin, and 2-deoxy-D-glucose (a competitive inhibitor of glucose). Glycolytic activity after the addition of glucose was significantly lower in the T-cells of DMF group than in those of the control group (Figure 3). DMF treatment also led to a significant reduction in glycolytic capacity and glycolytic reserve. Furthermore, the oxygen consumption rate, an indicator of oxidative phosphorylation, was decreased in the DMF group, indicating that DMF disrupts mitochondrial energy production in T-cells, either directly or indirectly. Similar results were obtained from CD4+ T-cells. These results suggest that DMF treatment can negatively regulate aerobic glycolysis in alloreactive T-cells, leading to the mitigation of GVHD. Conclusion Oral administration of DMF ameliorates GVHD and prolongs the survival of mice by reducing donor CD4+ T-cell proliferation, while the number of Tregs is maintained. Our data suggests that DMF treatment drives donor T-cells into a metabolically inactive state by inhibiting aerobic glycolysis. This investigation provides pre-clinical data to use oral DMF as a prophylactic agent for acute GVHD. Disclosures Kanda: Daiichi Sankyo: Honoraria; Shire: Honoraria; Alexion Pharmaceuticals: Honoraria; Takeda Pharmaceuticals: Honoraria; Novartis: Honoraria; Kyowa Kirin: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Celgene: Honoraria; Otsuka: Honoraria, Research Funding; Chugai Pharma: Honoraria, Research Funding; Janssen: Honoraria; Astellas Pharma: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Merck Sharp & Dohme: Honoraria; Mochida Pharmaceutical: Honoraria; Mundipharma: Honoraria; Sanofi: Honoraria, Research Funding; Meiji Seika Kaisha: Honoraria; Bristol-Myers Squibb: Honoraria; Shionogi: Research Funding; Ono Pharmaceutical: Honoraria; Nippon Shinyaku: Honoraria, Research Funding.

A Bank of CD30.CAR-Modified, Epstein-Barr Virus-Specific T Cells That Lacks Host Reactivity and Resists Graft Rejection for Patients with CD30-Positive Lymphoma

While autologous T cell therapies can effectively treat B-cell leukemia and lymphoma, the personalized manufacturing process is difficult to scale, expensive and may fail. Even when autologous products are successfully manufactured, they are not immediately available to acutely ill patients. "Off-the-shelf" T cell products derived from healthy donors that can rapidly be administered, would improve accessibility and reduce the cost of T cell therapy. However, major obstacles to successful allogeneic T cell products include their potential for graft-versus-host disease (GVHD) and graft rejection, mediated by host and recipient alloreactive T cells respectively. To address GVHD, we are using Epstein-Barr Virus-specific T cells (EBVSTs) as our platform since they are virus specific rather than allospecific and have not produced GVHD in more than 300 allogeneic recipients. To prevent graft rejection we have introduced into these EBVSTs, a chimeric antigen receptor for CD30 (CD30.CAR). CD30 is upregulated during the activation of alloreactive T cells, which leads to them becoming targets. The CD30.CAR provides the additional advantage of targeting CD30-positive lymphoma and has proved safe and effective in prior clinical trials (NCT02917083) using autologous CAR-T cells. Hence, we expect off-the-shelf CD30.CAR EBVSTs to eliminate the alloreactive T cells they elicit in allogeneic hosts, and therefore persist for sufficient time to eliminate CD30-positive lymphoma, without causing GVHD. Here we show that CD30.CAR-EBVSTs resist fratricide by masking their own CD30 molecules expressed in cis, but are nonetheless protected from rejection when co-cultured with alloreactive T cells expressing CD30 in trans. Notably, CD30.CAR EBVSTs preserve the function of both their TCR and the CD30.CAR, with retention of EBV specificity and the ability to eliminate CD30-positive tumor cells. We have manufactured a bank of clinical grade CD30.CAR EBVSTs from donors with HLA types designed to provide a partial HLA match for our diverse recipients. Clinical grade CD30.CAR EBVST cultures readily expanded to sufficient numbers for a planned clinical trial and expressed the CD30.CAR on 77% to 99% of cells. All of the lines passed functional release criteria of having greater than 100 IFNɣ spot-forming units (SFU) per 105 cells in response to both latent and lytic EBV antigens, and greater than 20% specific cytolysis against a CD30-positive Hodgkin lymphoma cell line, HDLM2, at an effector to target ratio of 20:1. Although CD30.CAR killing is not HLA restricted, we will select the CD30.CAR EBVST product for each recipient, based on the best HLA class I and class II match. This will allow endogenous EBV to boost the in vivo activity of CD30.CAR EBVSTs, and will provide additional reactivity for patients with CD30-positive and EBV-positive tumors. The IND for the clinical trial (NCT04288726) has been approved and we will recruit patients with CD30-positive lymphomas including Hodgkin lymphoma, diffuse large B cell lymphoma and NK/T cell lymphoma. In summary, we present an approach to making an off-the-shelf T cell therapy that can rapidly translate to the clinic, requires no gene editing, and can serve as a platform for other CAR/TCRs to target a multiplicity of malignancies. Disclosures Quach: Tessa Therapeutics: Research Funding. Brenner:Memmgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Maker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Tumstone: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Heslop:Tessa Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; Gilead Biosciences: Consultancy; PACT Pharma: Consultancy; Kiadis: Consultancy; AlloVir: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ramos:Novartis: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding; Kuur Therapeutics: Research Funding. Rouce:Tessa Therapeutics: Other, Research Funding; Novartis: Honoraria. Rooney:Marker Therapeutics: Current equity holder in publicly-traded company, Other: co-founder; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Allovir: Current equity holder in publicly-traded company, Other: co-founder.