Mixed Lymphocyte Reaction Assay Research Articles

Anti-Donor Regulatory T-Cell Therapy in Adult-to-Adult Living Donor Liver Transplantation: A Case Report

We report on the case of a 50-year-old female patient with symptomatic polycystic liver disease who underwent living donor liver transplantation (LDLT) using right liver graft from her ABO-identical husband. To achieve operational tolerance, regulatory T-cell (T-reg)-based cell therapy was applied, following the protocol introduced by Todo et al. Briefly, donor lymphocytes were collected by leukapheresis 20 days before LDLT without any adverse events, and the cells were irradiated with a dose of 30 Gy and kept frozen. Lymphopheresis of the recipient was conducted in a similar manner 1 day before LDLT, and donor cells and recipient cells were cultured with anti-CD80/86 antibodies to induce the donor-specific T-reg. At 14 days of culture, the CD4+CD25+Foxp3+ cells had increased from 1.51% to 5.21%, and mixed lymphocyte reaction assay using an intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester-labeling technique revealed donor-specific hyporesponsiveness of CD4-positive lymphocytes. On postoperative day (POD) 13 (14 days of culture), these cells were infused to the recipient intravenously without any adverse events. Initial immunosuppression consisted of tacrolimus, steroid and mycophenolate mofetil (MMF), and cyclophosphamide (40 mg/kg) administered on POD 5. Both the steroid and MMF were continued until 4 weeks after LDLT, and the patient was discharged on POD 30 with normal liver function. On POD 52, the patient developed acute cellular rejection and received appropriate reinforcement of immunosuppressive therapy and is currently doing well with normal liver function 30 months after LDLT with reduced anti-donor allo-activity. In summary, T-reg therapy was safely performed in adult LDLT, and we are following the patient carefully to determine whether she can achieve operational tolerance in the future.

T cell exhaustion drives osteosarcoma pathogenesis.

BackgroundOsteosarcoma (OS) is a rare cancer with a bimodal age distribution that peaks in children and young adults. It has been shown that the expression of programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) on tumor-infiltrating immune cells negatively correlates with prognosis of OS patients. However, a comprehensive assessment of the tumor-infiltrating immune cells in OS and their function has not been performed.MethodsCD8+ T cells were isolated from biopsy tissue samples collected from OS patients and control subjects. Mass cytometry, Treg suppression assay, mixed lymphocyte reaction assay, and effector T cell functional assay were performed to analyze the function of tumor-infiltrating T cells. A xenograft metastasis model was established in BALB/c nude mice.ResultsMacrophages and CD3+ T cells comprised most of the tumor-infiltrating immune cells in OS, with a disproportionately higher number of helper CD4+ T cells than effector CD8+ T cells. Whereas the tumor-infiltrating regulatory T cells were functionally intact, the CD8+ T cells showed increased expression of the immune checkpoint receptor (ICR) PD-1 and T cell immunoglobulin and mucin-domain containing 3 (TIM3) and were functionally inactive. TIM3 blockade using a monoclonal antibody restored the T cell alloreactive function of the CD8+ T cells ex vivo. TIM3 blockade in a xenograft model of OS impaired tumor growth in vivo. TIM3 blockade decreased the number of tumor-infiltrating CD4+ T cells while increasing the numbers and functional activation of tumor-infiltrating CD8+ T cells in vivo.ConclusionsThese results highlight that TIM3 blockade might be a viable therapeutic option and should be tested in additional preclinical models.

Generation of Monkey Induced Pluripotent Stem Cell-Derived Cartilage Lacking Major Histocompatibility Complex Class I Molecules on the Cell Surface.

Due to the poor capacity for articular cartilage to regenerate, its damage tends to result in progressively degenerating conditions such as osteoarthritis. To repair the damage, the transplantation of allogeneic human induced pluripotent stem cell (iPSC)-derived cartilage is being considered. However, although allogeneic cartilage transplantation is effective, immunological reactions can occur. One hypothetical solution is to delete the expression of major histocompatibility complex (MHC) class I molecules to reduce the immunological reactions. For this purpose, we deleted the β2 microglobulin (B2M) gene in a cynomolgus monkey (crab-eating monkey [Macaca fascicularis]) iPS cells (cyiPSCs) to obtain B2M−/− cyiPSCs using the CRISPR/Cas9 system. Western blot analysis confirmed B2M−/− cyiPSCs lacked B2M protein, which is necessary for MHC class I molecules to be transported to and expressed on the cell surface by forming multimers with B2M. Flow cytometry analysis revealed no B2M−/− cyiPSCs expressed MHC class I molecules on their surface. The transplantation of B2M−/− cyiPSCs in immunodeficient mice resulted in teratoma that contained cartilage, indicating that the lack of MHC class I molecules on the cell surface affects neither the pluripotency nor the chondrogenic differentiation capacity of cyiPSCs. By modifying the chondrogenic differentiation protocol for human iPSCs, we succeeded at differentiating B2M+/+ and B2M−/− cyiPSCs toward chondrocytes followed by cartilage formation in vitro, as indicated by histological analysis showing that B2M+/+ and B2M−/− cyiPSC-derived cartilage were positively stained with safranin O and expressed type II collagen. Flow cytometry analysis confirmed that MHC class I molecules were not expressed on the cell surface of B2M−/− chondrocytes isolated from B2M−/− cyiPSC-derived cartilage. An in vitro mixed lymphocyte reaction assay showed that neither B2M+/+ nor B2M−/− cyiPSC-derived cartilage cells stimulated the proliferation of allogeneic peripheral blood mononuclear cells. On the contrary, osteochondral defects in monkey knee joints that received allogeneic transplantations of cyiPSC-derived cartilage showed an accumulation of leukocytes with more natural killer cells around B2M−/− cyiPSC-derived cartilage than B2M+/+ cartilage, suggesting complex mechanisms in the immune reaction of allogeneic cartilage transplanted in osteochondral defects in vivo.Impact statementThe transplantation of allogeneic induced pluripotent stem cell (iPSC)-derived cartilage is expected to treat articular cartilage damage, although the effects of major histocompatibility complex (MHC) in immunological reactions have not been well studied. We succeeded at creating B2M−/− cynomolgus monkey (cy)iPSCs and cyiPSC-derived cartilage that lack MHC class I molecules on the cell surface. B2M−/− cyiPSC-derived cartilage cells did not stimulate the proliferation of allogeneic peripheral blood mononuclear cells in vitro. On the contrary, the transplantation of B2M−/− cyiPSC-derived cartilage into osteochondral defects in monkey knee joints resulted in survival of transplants and accumulation of leukocytes, including natural killer cells, suggesting complex mechanisms for the immune reaction.

The Immunomodulatory Effect of Triptolide on Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are known to have immunosuppressive ability and have been used in clinical treatment of acute graft-versus-host disease, one of severe complications of the hematopoietic stem cell transplantation. However, MSCs are activated to suppress the immune system only after encountering an inflammatory stimulation. Thus, it will be ideal if MSCs are primed to be activated and ready to suppress the immune reaction before being administered. Triptolide (TPL) is a diterpene triepoxide purified from a Chinese herb-Tripterygium wilfordii Hook.f. It has been shown to possess anti-inflammatory and immunosuppressive properties in vitro. In this study, we aimed to use TPL to prime umbilical cord-derived MSCs (TPL-primed UC-MSCs) to enter a stronger immunosuppressive status. UC-MSCs were primed with TPL, which was washed out thoroughly, and the TPL-primed UC-MSCs were resuspended in fresh medium. Although TPL inhibited the proliferation of UC-MSCs, 0.01 μM TPL for 24 h was tolerable. The surface markers of TPL-primed UC-MSCs were identical to those of non-primed UC-MSCs. TPL-primed UC-MSCs exhibited stronger anti-proliferative effect for activated CD4+ and CD8+ T cells in the allogeneic mixed lymphocyte reaction assay than the non-primed UC-MSCs. TPL-primed UC-MSCs promoted the expression of IDO-1 in the presence of IFN-γ, but TPL alone was not sufficient. Furthermore, TPL-primed UC-MSCs showed increased expression of PD-L1. Conclusively, upregulation of IDO-1 in the presence of IFN-γ and induction of PD-L1 enhances the immunosuppressive potency of TPL-primed UC-MSCs on the proliferation of activated T cells. Thus, TPL- primed MSCs may provide a novel immunosuppressive cell therapy.

Preclinical Characterization of a Novel Anti-Cancer PD-L1 Inhibitor RPH-120.

RPH-120 is a novel fully human anti-PD-L1 IgG1 monoclonal antibody with specifically designed Asn300Ala mutation in Fc fragment. Surface plasmon resonance assay showed that affinity of the RPH-120 to the dimeric form of human PD-L1-Fc fusion protein was much higher than affinity to the monomeric His-tagged PD-L1. Further binding studies demonstrated that RPH-120 is able to bind to human and monkey but not mouse PD-L1. Tissue cross-reactivity study showed good comparability of human and Cynomolgus monkeys tissue staining. Bioactivity was assessed using mixed lymphocyte reaction assay. This study revealed that RPH-120 was able to activate T cells preventing PD1/PD-L1 interaction. Antitumor efficacy was analyzed in HCC-827 lung cancer xenografts in humanized CD34+ mice at three dosage levels: 20, 80, and 200 mg/kg. RPH-120 demonstrated significant tumor growth inhibition, and this inhibition was comparable to that of atezolizumab. In a single dose toxicity, toxicokinetic and dose range finding study performed in Cynomolgus monkeys, RPH-120 was administered via intravenous (IV) bolus or 60-min IV infusion, followed by 8-weeks recovery period. An acceptable toxicokinetic profile was demonstrated and administration at doses of up to 200 mg/kg was well tolerated by all animals. In conclusion, RPH-120 revealed promising in vitro and in vivo activity and safety. RPH-120 is a potent anti-PD-L1 drug candidate for cancer immunotherapy.

Abstract 2451: A novel single domain antibody targeting TIGIT for cancer use in combination therapies

Abstract T-cell immunoglobulin and ITIM domains (TIGIT) has emerged as another popular checkpoint receptor that negatively regulates T-cell functions. TIGIT is upregulated by immune cells, such as activated T cells, natural killer cells, and regulatory T cells. TIGIT binds to two ligands, CD155 (PVR) and CD112 (PVRL2, nectin-2), that are expressed by tumor cells and antigen-presenting cells in the tumor microenvironment. Beyond CTLA4 and PD1, TIGIT therapies recently demonstrated great combo potential in the clinic along with well-tolerated toxicity profile. Here we present HLX53, a novel single domain antibody (sdAb) targeting TIGIT with differentiated format, binding epitope, and functional activity. HLX53 binds to TIGIT and blocks CD155 and CD112 with sub-nanomolar affinity. In mixed lymphocyte reaction assay (MLR), HLX53 significantly increased IL2 secretion compared to Tiragolumab and Vibostolimab analogues. HLX53's IL2 induction seems to require an adequate bridging effect between T-cell and antigen-presenting cell via Fc-FcγR engagement, further promoting the maturation of antigen-specific T-cell. In vivo, HLX53 exhibits a better tumor inhibition than Tiragolumab analogue and shows synergy in combination with anti-PD-L1 antibody in MC38 model. In addition to its unique epitope/MOA, HLX53 being a sdAb has a shorter half-life and thus potentially a better tumor penetration and a differentiated PK/PD profile. Understanding pharmacokinetic and pharmacodynamic relationships enables a dosing regimen providing adequate target coverage and superior tumor eradication. Citation Format: Botong Hua, Ming Yang, Jie Xue, Chen Dong, Yi-Ting Mao, Ou Li, Eric Cheung, Hassan Issafras, Wenfeng Xu, Weidong Jiang. A novel single domain antibody targeting TIGIT for cancer use in combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2451.

Abstract 1444: TPX-0022, a potent MET/SRC/CSF1R inhibitor that modulates the tumor immune microenvironment in preclinical models

Abstract Within the tumor microenvironment (TME), CSF1R (colony stimulating factor 1 receptor) signaling plays a pivotal role in recruitment and enrichment of M2 tumor-associated macrophages (TAM) that dampen the anti-tumor immune response and promote tumor progression. Although CSF1R inhibitors have shown promise in the clinic, tumor cells and TAMs can bypass CSF1R inhibition and expand M2 TAMs through other growth factor receptors, such as MET (hepatocyte growth factor receptor) to confer resistance to targeted therapies and immune checkpoint inhibitors. Through autocrine signaling pathways, CSF1R and MET activation in tumors contribute to tumor invasiveness and metastasis. Therefore, inhibitors that target both CSF1R and MET, such as TPX-0022 (a compact macrocyclic inhibitor of MET/SRC/CSF1R), may more effectively suppress CSF1R mediated signaling in tumor cells and the stroma. In the current study, the role of TPX-0022 in the anti-tumor immune response is evaluated in the context of both normal immune cells and immunocompetent syngeneic murine models. TPX-0022 blocks both HGF and CSF1 mediated signaling and proliferation of MET and CSF1R dependent cell lines. In the MC-38 syngeneic model, TPX-0022 treatment repolarized TAMs towards M1 anti-tumorigenic macrophages, increased CD8 T cells, and inhibited tumor growth. In a mixed lymphocyte reaction assay, we demonstrated that pretreatment of macrophages with TPX-0022 enhances macrophage function in driving the activation and proliferation of CD8 T cells. Furthermore, TPX-0022 treatment of anti-C3/CD28 and or SIINFEKL peptide stimulated murine T cells had no deleterious effects on T cell activation and proliferation. Additionally, TPX-0022 enhanced T cell cytotoxic function towards cancer cells in a dose-dependent manner. Detailed analysis of the TME in CT-26 animals treated with TPX-0022 showed a significant reduction in TAMs, rebalancing both immunosuppressive Treg and M2 macrophages towards anti-tumorigenic CTL (CD8+ CD69+ GnzB+) and M1 macrophages. The combination of TPX-0022 and a PD-1 antibody resulted in significant downregulation of pro-tumorigenic cytokines IL-10, IP-10, IL-1β, and HGF, without impacting the anti-tumor cytokine IFN-γ. In the MC-38 model, TPX-0022 demonstrated single-agent activity and a trend to enhanced efficacy in combination with a PD-1 antibody. Taken together, the preclinical data demonstrates that the MET/SRC/CSF1R inhibitor TPX-0022 can reprogram tumor associated macrophages as well as support antigen presentation and activation of T cells within the TME. A TPX-0022 combination with a PD-1 therapy yielded more anti-tumor activity than single agent treatments. Together, the in vitro and in vivo preclinical data provide a mechanistic rationale for the potential utility of TPX-0022 in combination with immune checkpoint inhibitors. Citation Format: Vikas K. Goel, Wei Deng, Dayong zhai, Nathan V. Lee, Ana Parra, Brion W. Murray. TPX-0022, a potent MET/SRC/CSF1R inhibitor that modulates the tumor immune microenvironment in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1444.

Abstract 1274: CCX559 is a potent orally-administered small molecule PD-L1 inhibitor that induces anti-tumor immunity

Abstract Introduction: Cancer cells can escape tumor-specific T cell responses via engagement of inhibitory immune checkpoints. PD-L1/PD-1 interaction is one of the major checkpoints that limit effector T cell function against cancer cells, and monoclonal antibodies that block this interaction have been approved as therapies in multiple tumor indications. As a next generation therapy, small molecule inhibitors of PD-L1 potentially have advantageous properties compared to approved monoclonal antibodies, such as better penetration into solid tumors, reduced immunogenicity, lack of Fc-mediated side effects, convenience of oral administration, and lower cost of goods. We therefore embarked on an effort to identify and develop an orally available small molecule capable of targeting PD-L1/PD-1 interactions. Methods: Inhibition of the PD-L1/PD-1 interaction was measured using a binding assay, followed by a cell culture system assessing PD-1 inhibition of T cell receptor (TCR) activation. Human T cell responses were assessed in vitro using the mixed lymphocyte reaction (MLR) assay, and a human peripheral blood mononuclear cell (PBMC)-mediated tumor cell killing assay. For in vivo studies CCX559 was dosed orally in a syngeneic tumor model and in a human tumor cell/PBMC co-implantation model in immune deficient mice. Results: Using structural information and focused medicinal chemistry, we identified CCX559 as a potent inhibitor of PD-L1 interaction with PD-1. CCX559 prevented PD-L1/PD-1 inhibition of TCR signaling in a cell-based reporter assay, increased IFNγ secretion in allogeneic MLR assays, and increased tumor cell killing by human PBMCs. We demonstrated that CCX559 potentially employs multiple mechanisms to inhibit PD-L1, which are distinct from those published for human anti-PD-L1 antibodies. In murine tumor models, orally administered CCX559 reduced tumor growth similarly to a clinically-approved anti-human PD-L1 antibody. Summary: CCX559 is a highly potent, small molecule PD-L1 inhibitor that can be orally administered. CCX559 enhanced primary human T cell activity in vitro and demonstrated anti-tumor efficacy in two murine tumor models. Based on its unique mechanism of PD-L1 inhibition, strong anti-tumor activity, desirable drug properties, and good safety profile, we plan to advance CCX559 into clinical development in the first half of 2021. Citation Format: Chris Li, Marta Vilalta, Linda S. Ertl, Yu Wang, Yibin Zeng, Pingchen Fan, Christopher Lange, Darren McMurtrie, Ju Yang, Rebecca Lui, Ryan Scamp, Vicky Chhina, Alice Kumamoto, Ryan Ong, Ton Dang, Ashton Easterday, Niky Zhao, Shirley Liu, Rajinder Singh, Israel Charo, Kathleen Sullivan, Thomas J. Schall, Penglie Zhang. CCX559 is a potent orally-administered small molecule PD-L1 inhibitor that induces anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1274.

Abstract 1743: IL12 heterodimeric Fc-fusions engineered for reduced potency exhibit strong anti-tumor activity and improved therapeutic index compared to native IL12 agents

Abstract Interleukin-12 (IL12) is a proinflammatory cytokine produced by activated antigen-presenting cells that induces differentiation of Th1 cells and increased proliferation and cytotoxicity of T and NK cells. Stimulation of these cells by IL12 leads to production of high levels of IFNγ. These immune-stimulating aspects of IL12 may help to establish an inflammatory tumor microenvironment critical for anti-tumor responses. Preclinical studies in mice revealed that native IL12 can dramatically shrink syngeneic tumors, however clinical studies in humans resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Applying similar principles to IL12, we created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency to improve tolerability, slow receptor-mediated clearance, and extend half-life. IL12 is a heterodimeric protein consisting of two subunits, so we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and the IL12p40 subunit to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity of human IL12-Fc were assessed in a human PBMC engrafted mouse MCF7 tumor model. Surrogate mouse IL12-Fc were evaluated in additional murine tumor models. Tolerability and pharmacodynamic activity were assessed in non-human primates. IL12-Fc were produced with good yield and purity. An IL12-Fc potency series was created, and variants had up to a 10,000-fold reduction in STAT4 signaling potency and IFNγ production in an MLR assay compared to native IL12-Fc. Anti-tumor activity was achieved with potency-reduced IL12-Fc as a single-agent and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum indicated up to 200-fold increases in IFNγ levels. Combined, these data indicate that potency-reduced IL12-Fc retain strong anti-tumor activity, while potentially overcoming safety and tolerability issues related to narrow therapeutic index associated with recombinant native IL12 or IL12-Fc agents. Citation Format: Matthew J. Bernett, Ke Liu, Christine Bonzon, Rumana Rashid, Nicole Rodriguez, Nargess Hassanzadeh-Kiabi, Connie Ardila, Katrina Bykova, Michael Hackett, Norm Barlow, Irene Leung, Duc-Hanh Nguyen, Araz Eivazi, Seung Y. Chu, Rajat Varma, Umesh S. Muchhal, John R. Desjarlais. IL12 heterodimeric Fc-fusions engineered for reduced potency exhibit strong anti-tumor activity and improved therapeutic index compared to native IL12 agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1743.

Investigating the Efficacy of Anti-CD200 Immunotherapy in MRD Low and Risk B Cell Precursor ALL

Despite remission rates of >90% in childhood B cell precursor acute lymphoblastic leukaemia (BCP ALL) using frontline treatment, the incidence of relapse remains high. Immunotherapeutic approaches, including chimeric antigen receptor (CAR)-T cells targeting CD19 have resulted in improved outcomes for refractory cases. However, such therapies do not target CD19- ALL cells, a population known to have leukaemia initiating ability, and there are several reports of patients relapsing with CD19- leukaemia after CAR-T cell therapy. CD200 (OX-2) is an immunosuppressive molecule overexpressed in BCP ALL. In low risk minimal residual disease (MRD) ALL samples, only CD200+ cells have leukaemia initiating capacity in NOD.Cg- Prkdcscid Il2r gtm1Wjl/Sz (NSG) mice, suggesting that CD200 may be a suitable target for therapy in these cases. When CD200 binds to its receptor (CD200R) the immune response is suppressed. Consequently, malignant cells that overexpress CD200 may capitalise on this immune suppression and avoid destruction. Anti-CD200 antibodies (Ab) may be used to prevent such cancer cell evasion by binding to CD200 antigen and blocking its interaction with CD200R. The aim of this study was to investigate the expression of CD200 in MRD low and risk BCP ALL cases and assess the effects of using monoclonal (Mo) TTI-CD200 Ab in vitro and in vivo. Samples from MRD low and risk BCP ALL cases were stained with anti-CD200 Ab to quantify (i) the proportion of cells expressing CD200, (ii) the median fluorescence intensity (MFI) of CD200 and (iii) the number of CD200 antigen binding sites. Results were compared to those obtained using normal bone marrow (NBM) controls. The proportion of CD200+ cells was higher in ALL cases (median 40.0%, range 5.4-74.4%) compared to NBM cells (median 0.001%, range 0-0.003%, P =0.0004). There was no significant difference between the MRD low (median 33.8%, range 18.1-74.1%) and risk (median 44.93%, range 36.35-74.4%) ALL cases. However, CD200 MFI levels were significantly higher in the low risk cases (median 593, range 469-1313) compared to those observed in MRD risk cases (median 191, range 138-572, P =0.04). CD200 MFI levels in the low risk group were also significantly higher than in NBM cells (median 0.9, range 0-27, P =0.001). Additionally, the number of CD200 antigen sites on ALL cells was 5-fold higher in MRD low risk cases (median 1372, range 324-2179) than in risk cases (median 264, range 87-1500), while the number of CD200 sites on BM cells was only 17 (range 13-112, P =0.006 vs low risk cases). Monocyte-derived macrophages and CD4+ T cells from healthy donors were cultured with low risk BCP ALL cells ± TTI-CD200 in a mixed lymphocyte reaction assay. The production of interleukin (IL)-2 was measured as an indicator of immune system activation using an enzyme-linked immunosorbent assay. Median CD200 expression in these cases was 46.9% (range 0.7-94.2%) and MFI was 249 (34.8-1659). The median IL-2 increase observed on addition of TTI-CD200 was 16-fold (range 1.7-120) and it did not correlate with the proportion of CD200+ cells or MFI. In these cases, samples with very low proportions of CD200+ cells (0.9±0.4%) demonstrated a 2.5-fold increase in IL-2, whereas samples with higher proportions of CD200+ cells (91.1±4.4%) produced a 17.2-fold rise. To assess the effects of TTI-CD200 on ALL cells in vivo, NSG were inoculated with BCP ALL cells from MRD low and risk cases. Once leukaemia levels exceeded 0.5% in peripheral blood, animals received 4 doses of TTI-CD200 (20mg/kg i.v.) over 10 days. Consistent with previous findings, TTI-CD200 treatment of mice engrafted with MRD risk cases had no effect on disease burden. This may be due to the lower proportion of CD200+ cells in these cases (34.1±11.7% CD200+) and also that CD200- cells contribute to engraftment in risk cases. In contrast, in low risk ALL cases a 47.5±29% reduction in leukaemia burden was observed after treatment with TTI-CD200. The proportion of inoculated CD200+ cells was higher in these low risk cases (74.9±30.3% CD200+) and had significantly reduced by 99.4±0.6% (P =0.04) in ALL cells recovered from the murine BM on termination. The dose and schedule of TTI-CD200 used was much less intensive than standard chemotherapy. It may be possible to improve the in vivo efficacy using another round of therapy. These findings demonstrate the potential of using anti-CD200 MoAb therapy for low risk BCP ALL. DisclosuresPetrova:Trillium Therapeutics: Employment, Equity Ownership. Uger:Trillium Therapeutics: Employment, Equity Ownership, Patents & Royalties.

NKG2D Negatively Regulates CD4 T Cells in Allo-Response in Mice

NKG2D is a C-type lectin-like receptor which mainly expresses on mouse NK, activated CD8 and some γδ T cells. NKG2D is also found to express on human and mouse CD4 T cells, but the role of NKG2D in CD4 T cells is essentially unknown. We asked what subsets of CD4 T cells could express NKG2D; we activated and polarized CD4 T cells into Th0, Th1,Th2, Th17 and Foxp3+ Tregs in vitro and measured NKG2D expression on those subsets. We found that only a proportion of Th1 cells expressed NKG2D, whereas little or no NKG2D was expressed on other subsets of T cells. In a mixed lymphocyte reaction (MLR) assay, NKG2D was induced on a subset of CD4 T cells after stimulation with allogeneic antigen presenting cells (APCs) (Figure 1A). NKG2D+CD4+ T cells expressed higher levels of LAG-3, CD49b, CD80, FasL and PD-L1 (Figure 1B) and secreted more IL-10 and IFN-γ than NKG2D- CD4+ T cells (Figure 1C).Previous reports by others showed that NKG2D on NK and activated CD8 T cells contributing to graft versus host disease (GVHD) pathogenesis have been well-established, but the expression and function of NKG2D on CD4 T cells in GVHD development are unknown.To address these questions, we utilized a CD4-driven, MHC-mismatched aGVHD model of allogeneic bone marrow transplantation (allo-BMT), C57BL/6 to BALB/c. We monitored the development of NKG2D+CD4+ T cells in allogeneic recipients. We observed that NKG2D was continuously induced on CD4 T cells during the whole aGVHD process - gradually increased, peaked on day 14 and then slowly decreased after allo-BMT. By comparing the severity of aGVHD in recipients transferred with WT versus NKG2D-/- CD4 T cells. We observed that recipients transferred with NKG2D-/- CD4 T cells showed more severe GVHD demonstrated by a higher lethality (Figure 1D) and more severe diarrhea compared to WT counterpart. Mechanistically, NKG2D-/- CD4 T cells had a higher capacity of migrating to gut and liver than WT CD4 T cells reflected by the higher expression of α4β7 and CXCR3. Furthermore, NKG2D+CD4+ T cells secreted less proinflammatory cytokine IL-17 and more anti-inflammatory cytokine IL-10. Strikingly, NKG2D expression coincided with T-bet, but excluded RORγt or Foxp3 expression of CD4 T cells. Given NKG2D+CD4+ T cells expressed regulatory markers and secreted IL-10 and IFN-γ; we hypothesized that NKG2D+CD4+ T cells may serve as suppressive cells (e.g. Tr1 cells). To test this hypothesis, we sorted NKG2D+CD4+ and NKG2D-CD4+ T cells from recipients of allogeneic BMT, and performed in vitro suppressive assay. We observed that NKG2D+CD4+ T cells had a higher suppressive activity than NKG2D-CD4+ T cells. (Figure 1E)Taken together, the current work demonstrates that NKG2D may serve as a negative regulator on CD4 T cells, and NKG2D+CD4+ subset can function as a regulatory cell subset in the development of GVHD after allo-BMT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Withaferin A inhibits lymphocyte proliferation, dendritic cell maturation in vitro and prolongs islet allograft survival

The immunosuppressive regimen for clinical allogeneic islet transplantation uses beta cell–toxic compounds such as tacrolimus that cause islet graft loss. Previously we reported that the plant-derived steroidal lactone Withaferin A (WA) can protect islet grafts by inhibiting nuclear factor-kappa B (NF-κB). Since the NF-κB signaling pathway is essential for T-cell activation, we hypothesized that long-term WA administration may also provide an immunosuppressive effect. Treatment of BALB/c donor islets and C57BL/6N recipients with WA alone resulted in 80% islet graft long-term survival vs. 40% in low-dose FK506-treated mice. In vitro, WA significantly blocked mouse and human T-cell proliferation by CD3/CD28 bead stimulation and in mixed lymphocyte reaction assay. Treatment of immature dendritic cells with WA prevented their maturation in response to inflammatory stimuli, as seen by decreased expression of CD83 and human leukocyte antigen–DR isotype. Exosomes released by islets treated with WA contained significantly fewer proinflammatory molecules interleukin-6, interleukin-8, monocyte chemoattractant protein-1, interferon-gamma-induced protein-10, inducible nitric oxide synthase, and cyclooxygenase-2. In conclusion, WA treatment not only reduced inflammation but also prolonged allograft survival, possibly through suppression of dendritic cell maturation and T-cell proliferation. WA has the potential to inhibit both the innate and adaptive immune response to prolong allograft survival.

HMBD-002 is a novel, neutralizing, anti-VISTA antibody exhibiting strong preclinical efficacy and safety, being developed as a monotherapy and in combination with pembrolizumab.

e14569 Background: Inhibition of immune checkpoints has been an effective strategy against several cancers, but efficacy gaps remain. VISTA is an immunoregulatory protein expressed on both hematopoietic and tumor cells and has been shown to induce an immunosuppressive environment by repressing T-cell activation and cytokine production. VISTA expression has been shown to be upregulated following treatment with immune checkpoint inhibitors. Hummingbird Bioscience is developing HMBD-002, an IgG4 antibody against VISTA with high binding specificity across species (human, cyno and rodent) and low pM affinity to the region of VISTA required for engagement with interaction partners, including VSIG3. As HMBD-002 is an IgG4 isotype, it uniquely blocks the inhibitory function of VISTA and releases immune suppression without depleting VISTA+ cells. Here we establish the preclinical efficacy and safety of HMBD-002 along with the rationale for development of HMBD-002 as a monotherapy and in combination with PD1 inhibitors in triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC). Methods: The effect of HMBD-002 in an in vitro model of human immune activation was evaluated using an allogenic Mixed Lymphocyte Reaction (MLR) assay. Tumor growth inhibition studies were conducted in syngeneic murine cell-derived xenograft (CDX) models. The PK and tolerability of HMBD-002 was evaluated in rats and cynomolgus monkeys with a 21 or 28-day observation period. The expression profile of VISTA was examined in different cancers by IHC staining of FFPE TMAs of various solid cancers. Results: HMBD-002 induced a significant dose dependent increase in the levels of inflammatory cytokines, IFN-γ and TNF- α and suppressed the expansion of CD14+ monocytes. Treatment with HMBD-002 significantly inhibited tumor growth and improved survival in CDX models. HMBD-002 is well tolerated in rats and cynomolgus monkeys with acceptable serum half-life, no HMBD-002 related morbidity/mortality or clinical signs, and no HMBD-002 related changes were observed in body weight, food consumption, clinical chemistry, or hematology parameters. HMBD-002 did not induce cytokine release in human PBMCs or whole blood. TNBC and NSCLC patients were found to harbor the highest VISTA expression across cancer types tested, providing a rationale for exploring these indications in upcoming clinical studies. Conclusions: These studies provide the rationale for development of HMBD-002 as a monotherapy and in combination with pembrolizumab to treat patients with solid tumors expressing VISTA in the tumor microenvironment. A Phase 1 study will be initiated in 2021.

Long Non-coding RNAs and mRNAs Expression Profiles of Monocyte-Derived Dendritic Cells From PBMCs in AR.

ObjectiveThe objective of this study is to explore the long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) expression profiles of monocyte-derived dendritic cells (DCs) obtained from peripheral blood mononuclear cells (PBMCs). DCs are known to play a major role in the regulating function of allergic rhinitis (AR).MethodsPBMCs were separately isolated from the human peripheral blood of patients with AR and normal person (NP). The mixed lymphocyte reaction (MLR) assay was used to evaluate the function of DCs. Flow cytometry was used to determine the immune regulatory function of immature DCs (imDCs) and mature DCs (mDCs). lncRNAs and mRNAs in the NP group (DCs isolated from NP) and the test group (DCs isolated from patients with AR) were identified via chip technology and bioinformatic analyses. Moreover, bioinformatic analyses were employed to identify the related biological functions of monocyte-derived DCs and construct the functional networks of lncRNAs and mRNAs that are differentially expressed (DE) in imDCs and mDCs.ResultsMLR was significantly higher in the mDCs group than that in the imDCs group. CD14 was highly expressed in imDCs, whereas HLA-DR, CD80, and CD86 were highly expressed in mDCs (p < 0.001). We identified 962 DE lncRNAs and 308 DE mRNAs in the imDCs of NP and patients with AR. Additionally, there were 601 DE lncRNAs and 168 DE mRNAs in the mDCs in the NP and test groups. Quantitative RT-qPCR was used to study the significant fold changes of lncRNAs and mRNAs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found 16 significant regulated pathways in imDCs and 10 significant regulated pathways in mDCs, including the phagosome, cell adhesion signaling pathway, and inflammatory mediator regulation of TRP channels pathway.ConclusionOur research studied the lncRNA and mRNA expression profiles of monocyte-derived DCs and demonstrated the functional networks that are involved in monocyte-derived DCs-mediated regulation in AR. These results provided possible molecular mechanisms of monocyte-derived DCs in the immunoregulating function and laid the foundation for the molecular therapeutic targets of AR.

A novel CD4+ CTL subtype characterized by chemotaxis and inflammation is involved in the pathogenesis of Graves\u2019 orbitopathy

Graves’ orbitopathy (GO), the most severe manifestation of Graves’ hyperthyroidism (GH), is an autoimmune-mediated inflammatory disorder, and treatments often exhibit a low efficacy. CD4+ T cells have been reported to play vital roles in GO progression. To explore the pathogenic CD4+ T cell types that drive GO progression, we applied single-cell RNA sequencing (scRNA-Seq), T cell receptor sequencing (TCR-Seq), flow cytometry, immunofluorescence and mixed lymphocyte reaction (MLR) assays to evaluate CD4+ T cells from GO and GH patients. scRNA-Seq revealed the novel GO-specific cell type CD4+ cytotoxic T lymphocytes (CTLs), which are characterized by chemotactic and inflammatory features. The clonal expansion of this CD4+ CTL population, as demonstrated by TCR-Seq, along with their strong cytotoxic response to autoantigens, localization in orbital sites, and potential relationship with disease relapse provide strong evidence for the pathogenic roles of GZMB and IFN-γ-secreting CD4+ CTLs in GO. Therefore, cytotoxic pathways may become potential therapeutic targets for GO.

Human invariant natural killer T cells promote tolerance by preferential apoptosis induction of conventional dendritic cells.

Graft-versus-host disease (GvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. We recently showed in murine studies and in vitro human models that adoptively transferred invariant natural killer T (iNKT) cells protect from GvHD and promote graft-versus-leukemia effects. The cellular mechanisms underlying GvHD prevention by iNKT cells in humans, however, remain unknown. In order to study relevant cellular interactions, dendritic cells (DC) were either generated from monocytes or isolated directly from blood of healthy donors or GvHD patients and co-cultured in a mixed lymphocyte reaction (MLR) with T cells obtained from healthy donors or transplantation bags. Addition of culture-expanded iNKT cells to the MLR-induced DC apoptosis in a cell contact-dependent manner, thereby preventing T-cell activation and proliferation. Annexin V/propidium iodide staining and image stream assays showed that CD4+CD8–, CD4–CD8+ and double negative iNKT cells are similarly able to induce DC apoptosis. Further MLR assays revealed that conventional DC (cDC) but not plasmacytoid DC (pDC) could induce alloreactive T-cell activation and proliferation. Interestingly, cDC were also more susceptible to apoptosis induced by iNKT cells, which correlates with their higher CD1d expression, leading to a bias in favor of pDC. Remarkably, these results could also be observed in GvHD patients. We propose a new mechanism how ex vivo expanded human iNKT cells prevent alloreactivity of T cells. iNKT cells modulate T-cell responses by selective apoptosis of DC subsets, resulting in suppression of T-cell activation and proliferation while enabling beneficial immune responses through pDC.

Immunomodulatory Activity of Human Bone Marrow and Adipose-Derived Mesenchymal Stem Cells Prolongs Allogenic Skin Graft Survival in Nonhuman Primates.

Objective In the present study, we examined the tolerance-inducing effects of human adipose-derived mesenchymal stem cells (hAD-MSCs) and bone marrow-derived MSCs (hBM-MSCs) on a nonhuman primate model of skin transplantation. Materials and Methods In this experimental study, allogenic and xenogeneic of immunomodulatory properties of human AD-MSCs and BM-MSCs were evaluated by mixed lymphocyte reaction (MLR) assays. Human MSCs were obtained from BM or AD tissues (from individuals of either sex with an age range of 35 to 65 years) and intravenously injected (2×106 MSCs/kg) after allogeneic skin grafting in a nonhuman primate model. The skin sections were evaluated by H&E staining for histopathological evaluations, particularly inflammation and rejection reaction of grafts after 96 hours of cell injection. At the mRNA and protein levels, cellular mediators of inflammation, such as CD4+IL-17+ (T helper 17; Th17) and CD4+INF-γ+ (T helper 1, Th1) cells, along with CD4+FoxP3+ cells (Treg), as the mediators of immunomodulation, were measured by RT-PCR and flow cytometry analyses. Results A significant Treg cells expansion was observed in MSCs-treated animals which reached the zenith at 24 hours and remained at a high concentration for 96 hours; however, Th1 and Th17 cells were significantly decreased. Our results showed that human MSCs significantly decrease Th1 and Th17 cell proliferation by decreasing interleukin-17 (IL-17) and interferon-γ (INF-γ) production and significantly increase Treg cell proliferation by increasing FoxP3 production. They also extend the allogenic skin graft survival in nonhuman primates. Histological evaluations showed no obvious presence of inflammatory cells or skin redness or even bulging after MSCs injection up to 96 hours, compared to the group without MSCs. There were no significant differences between hBM-MSCs and hAD-MSCs in terms of histopathological scores and inflammatory responses (P<0.05). ConclusionIt seems that MSCs could be regarded as a valuable immunomodulatory tool to reduce the use of immunosuppressive agents.

Hypoxia, Serum Starvation, and TNF-α Can Modify the Immunomodulation Potency of Human Adipose-Derived Stem Cells.

Adipose-derived stem cells (ADSCs) are mesenchymal stem cells (MSCs) that are found in adipose tissues, which are easily obtained from liposuction procedures using an enzyme mixture. The adhering cells are then selectively cultivated. ADSCs have great potential in regenerative medicine because they are plentiful, easily accessible, and less invasive. They also have an impressive proliferation ability and can be differentiated into mesenchymal lineages and trans-differentiating into many other cell types. In particular, they have extraordinary abilities in immunomodulation. This study aimed to investigate the effects of culture conditions (hypoxia, starvation, and TNF-α treatment) on the immunomodulation of human ADSCs. Human ADSCs were expanded in vitro in the standard condition before they were cultured in different stress conditions. ADSCs from passages fifth were confirmed as MSCs by some standard assays suggested by the International Society for Cell and Gene Therapy. These MSCs were used to culture in four different stress conditions: hypoxia, serum starvation, and TNF-α treatment in 48h. After treatments, MSCs were used to evaluate their immunomodulation capacity using MSCs mixed lymphocyte reaction assay, and the concentrations of IDO, PGE2, IL-6, and IL-10 were secreted in the culture medium. In different stress conditions, ADSCs exhibited different responses related to their immunomodulation. In serum starvation, ADSCs exerted a strong secretion of IDO and PGE2, whereas they showed strong IL-6 secretion in the TNF-α-supplemented medium. When exposed to lymphocytes, ADSCs caused an increase in the ratio of regulatory T cells (Tregs), and co-culture lymphocytes with ADSCs induced in hypoxic malnutrition conditions increased the IL-10 level the most. In addition, when exposed to dendritic cells (DCs), ADSCs inhibited the mature marker expressions of the DCs. The current research showed that ADSCs change their immunomodulation properties to survive in in vitro culture environments. Treatment of ADSCs in the starvation medium for 48h can increase the immunomodulation of ADSCs.

HLA-A*02:01-directed chimeric antigen receptor/forkhead box P3-engineered CD4+ T cells adopt a regulatory phenotype and suppress established graft-versus-host disease

Background aimsTo investigate the feasibility of using CD4 + T cells genetically modified to express an allo-HLA directed CAR and FOXP3 to suppress T cell proliferation and cytokine secretion in GvHD. Methods: Human CD4+ T cells from A*02:01 negative donors were transduced to express A*02 CAR and FOXP3 and co-cultured in mixed lymphocyte reaction assays to demonstrate T cell suppression. A*02- CAR/FOXP CD4+ T cells were then injected into mice engrafted with allogeneic T cells in a GvHD mouse model. ResultsCD4+ T cells genetically modified to express allo-HLA-directed CAR and FOXP3 proliferate rapidly, downregulate CD127 and interferon-γ, express high CD25 and Helios and convert to a stable antigen-dependent suppressive phenotype. In mixed lymphocyte reaction assays, these cells potently suppressed T-cell proliferation and secreted IL-10. In a graft-versus-host disease model, A*02-CAR/FOXP3 CD4+ T cells outperformed polyclonal Tregs by reducing liver and lung inflammation, inhibiting pro-inflammatory cytokine production and limiting grafted CD3+ T-cell expansion. ConclusionsCD4 + T cells expressing allo-antigen directed HLA-specific CAR and FOXP3 act as potent, specific and stable suppressors of inflammation that out-perform their Treg counterparts both in vitro and in vivo.

A streamlined proliferation assay using mixed lymphocytes for evaluation of human mesenchymal stem cell immunomodulation activity

BackgroundMesenchymal stromal cells (MSCs) have been proposed for treatment of acute respiratory distress syndrome (ARDS), graft versus host disease (GVHD), wound healing and trauma. A consensus is building that immunomodulation by MSCs is important for therapeutic potential. MSCs suppress peripheral blood mononuclear cell (PBMC) proliferation in vitro, potentially reflecting an ability to suppress PBMC inflammatory responses in vivo. Current mixed lymphocyte reaction (MLR) assays commonly used to evaluate MSC potency generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated PBMCs in the presence of mitotically inactive MSCs. Proliferation of PBMCs is monitored by several methods, including incorporation of radiolabeled nucleotides, BRDU labeling and ELISA assay or flow cytometry of carboxyfluorescein labeled PBMCs. Here we present a streamlined assay using MSCs in a direct co-culture system with unmodified MSCs using a luminescent ATP assay to evaluate both PBMC and MSC proliferation/survival. MethodsPBMCs were isolated from fresh anti-coagulated whole blood by centrifugation over Ficoll-Paque in LeucoSep tubes. Isolated PBMCs from 8 to 10 donors were pooled and cryopreserved at 1 × 107/ml in 50% RPMI medium,10% DMSO, 40% human AB serum. MSCs derived from bone marrow, adipose tissue or umbilical cord (BM-MSC, Ad-MSC, UC-MSC, respectively) were serially diluted starting at 50–60,000 cells/well and cultured in 96 well plates for 4–48 h in their respective medium. On Day 0, MSCs were washed, resuspended in PBMC media (RPMI with 10% FBS, 2 mM Glutamine, 10 mM HEPES, pH 7.4) and incubated with or without 150,000 freshly thawed pooled PBMCs/well, in the presence or absence of phytohemagglutinin A (PHA, 0–5 μg/ml). Proliferation of both MSCs (adherent) and PBMCs (non-adherent) was assessed by quantitation of ATP levels using the bioluminescent reagent Cell Titer-Glo (Promega). Culture supernatant contained PBMC, while washed adherent cells were primarily MSCs. Both cell types were incubated for 30 min with an equal volume of Cell Titer-Glo reagent and then assayed in white plates on a luminescence plate reader. ResultsPBMC proliferation in response to PHA stimulation resulted in a robust increase in ATP by 72 h, with >6 fold increase over unstimulated PBMCs, which showed no increase. MSC proliferation was decreased <20% at the highest PHA concentrations. Co-culture with MSCs suppressed PBMC proliferation dependent upon MSC passage number, source, and prior growth conditions. Total time to complete the ATP assay was under an hour including incubations. With minimal manipulations in the assay, intra- and inter- assay variations averaged 11.1 and 15.7% respectively. ConclusionsDirect co-culture of live unmodified MSCs with freshly thawed pooled PBMCs gives a robust determination of immunosuppression by MSCs with unparalleled ease. Graded responses can be determined, allowing comparison of potency between MSC preparations as in comparisons between freshly thawed and cultured MSCs as well as interferon-γ licensed MSCs. With the 96 well plate assay, far fewer PBMCs are generally required than in a typical flow cytometry determination. This streamlined assay can be performed within 72 h, without irradiating cells and without specialized equipment.