Allogeneic Mixed Leukocyte Reaction Research Articles

GM-CSF, Flt3-L and IL-4 affect viability and function of conventional dendritic cell types 1 and 2.

Conventional type 1 dendritic cells (cDC1) and conventional type 2 dendritic cells (cDC2) have attracted increasing attention as alternatives to monocyte-derived dendritic cells (moDCs) in cancer immunotherapy. Use of cDCs for therapy has been hindered by their low numbers in peripheral blood. In the present study, we found that extensive spontaneous apoptosis and cDC death in culture within 24hrs represent an additional challenge. Different media conditions that maintain cDC viability and function were investigated. CD141+ cDC1 and CD1c+ cDC2 were isolated from healthy blood donor buffy coats. Low viabilities were found with CellGenix DC, RPMI-1640, and X-VIVO 15 standard culture media and with several supplements at 24hrs and 48hrs. Among multiple factors it was found that GM-CSF improved both cDC1 and cDC2 viability, whereas Flt3-L and IL-4 only increased viability of cDC1 and cDC2, respectively. Combinations of these three cytokines improved viability of both cDCs further, both at 24hrs and 48hrs time points. Although these cytokines have been extensively investigated for their role in myeloid cell differentiation, and are also used clinically, their effects on mature cDCs remain incompletely known, in particular effects on pro-inflammatory or tolerogenic cDC features. HLA-DR, CD80, CD83, CD86, PD-L1 and PD-L2 cDC membrane expressions were relatively little affected by GM-CSF, IL-4 and Flt3-L cytokine supplements compared to the strong induction following Toll-like receptor (TLR) stimulation for 24hrs. With minor exceptions the three cytokines appeared to be permissive to the TLR-induced marker expression. Allogeneic mixed leukocyte reaction showed that the cytokines promoted T-cell proliferation and revealed a potential to boost both Th1 and Th2 polarizing cytokines. GM-CSF and Flt3-L and their combination improved the capability of cDC1 for dextran uptake, while in cDC2, dextran capture was improved by GM-CSF. The data suggest that GM-CSF, IL-4 and Flt3-L and combinations might be beneficial for DC viability and function in vitro. Limited viability of cDCs could be a confounding variable experimentally and in immunotherapy.

Abstract 601: IO-108, A fully human therapeutic antibody blocking the myeloid checkpoint LILRB2/ILT4, promotes innate and adaptive anti-cancer immunity in preclinical studies

Abstract Tumor-associated myeloid cells inhibit anti-cancer immune responses systemically and in the tumor microenvironment (TME), thereby limiting the efficacy of T cell checkpoint inhibitors. However, the plasticity of myeloid cells may enable therapeutic intervention. The immune inhibitory receptor LILRB2 (also known as ILT4) is expressed primarily by myeloid cells (monocytes, macrophages, dendritic cells and neutrophils), and has emerged as a key myeloid checkpoint contributing to the tolerogenic activity of myeloid cells associated with cancer. LILRB2 has several ligands (classical and non-classical MHC-I, ANGPTL2/5, SEMA4A and CD1) and most of these are known to contribute to immune suppression in the TME. Using computational biology approaches, we found that high LILRB2 expression is associated with macrophage infiltration in many solid tumor types from TCGA. Therefore, LILRB2 is a compelling target to overcome immune suppressive activity of cancer-associated myeloid cells. IO-108 is a fully human IgG4 therapeutic antibody that binds to LILRB2 with high affinity and specificity. IO-108 binds to all myeloid cells in the solid TME and periphery. In vitro studies support that blockade of LILRB2 interaction with its various ligands is the anticipated mechanism of action of IO-108. The LILRB2 antagonist activity of IO-108 produces the desired pro-inflammatory (re)polarization of myeloid cells. IO-108 treatment results in increased pro-inflammatory responses and an enhanced antigen-presenting cell (APC) phenotype to multiple stimuli (e.g., T cell activators, STING and TLR agonists) in ex vivo assays. In addition, IO-108 precludes the anti-inflammatory myeloid cell phenotype resulting from “tumor conditioning” and promotes the differentiation of monocytes and immature dendritic cells (DC) into pro-inflammatory DC. IO-108 enhances the effect of PD-1 blocking antibodies in allogeneic mixed leukocyte reactions of CD4+ T cells and macrophages. Moreover, IO-108 polarized primary myeloid cells isolated from solid tumor patient blood and ovarian cancer-associated ascites towards a proinflammatory phenotype and attenuated their suppressive effect on autologous T cell proliferation and production of proinflammatory cytokines. In vivo, IO-108 inhibits tumor growth in mouse models, which is associated with immune cell activation. Importantly, IO-108 presents a favorable pharmacokinetic and safety profile in preclinical models. Collectively, the preclinical characterization of IO-108 enabled a comprehensive clinical biomarker plan and lends rationale to the clinical study (NCT05054348) of IO-108 as a novel immunotherapy for multiple solid tumor types, including those relapsed/refractory to standard of care therapies. Citation Format: Jing-Tyan Ma, Xiaoye Liu, Ryan Stafford, Krista McCutcheon, Heyu Chen, Tao Huang, Hongyu Tian, Kyu Hong, ChengCheng Zhang, Luke Chung, An Song, Paul Woodard, XCharlene Liao, MariaJose Costa. IO-108, A fully human therapeutic antibody blocking the myeloid checkpoint LILRB2/ILT4, promotes innate and adaptive anti-cancer immunity in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 601.

Oncolytic adenovirus ORCA-010 increases the type 1 T cell stimulatory capacity of melanoma-conditioned dendritic cells.

SummaryImmune checkpoint blockade has resulted in durable responses in patients with metastatic melanoma, but only in a fraction of treated patients. For immune checkpoint inhibitors (ICI) to be effective, sufficient infiltration with tumor‐reactive T cells is essential. Oncolytic viruses (OV) selectively replicate in and lyse tumor cells and so induce an immunogenic form of cell death, providing at once a source of tumor‐associated (neo)antigens and of danger signals that together induce effective T cell immunity and tumor infiltration. Melanoma‐associated suppression of dendritic cell (DC) differentiation effectively hampers OV‐ or immune checkpoint inhibitor (ICI)‐induced anti‐tumor immunity, due to a consequent inability to prime and attract anti‐tumor effector T cells. Here, we set out to study the effect of ORCA‐010, a clinical stage oncolytic adenovirus, on DC differentiation and functionality in the context of human melanoma. In melanoma and monocyte co‐cultures, employing a panel of five melanoma cell lines with varying origins and oncogenic mutation status, we observed clear suppression of DC development with apparent skewing of monocyte differentiation to a more M2‐macrophage‐like state. We established the ability of ORCA‐010 to productively infect and lyse the melanoma cells. Moreover, although ORCA‐010 was unable to restore DC differentiation, it induced activation and an increased co‐stimulatory capacity of monocyte‐derived antigen‐presenting cells. Their subsequent ability to prime effector T cells with a type I cytokine profile was significantly increased in an allogeneic mixed leukocyte reaction. Our findings suggest that ORCA‐010 is a valuable immunotherapeutic agent for melanoma.

Human CD83 Targeted Chimeric Antigen Receptor T Cell for the Prevention of Graft Versus Host Disease and Treatment of Myeloid Leukemia

Distinct from pharmacologic immunosuppression, we designed a programmed cytolytic effector T cell that prevents graft versus host disease (GVHD). CD83 is expressed on allo-activated conventional T cells (Tconv) and pro-inflammatory dendritic cells (DCs), which are implicated in GVHD pathogenesis. Therefore we developed a novel human CD83 targeted chimeric antigen receptor (CAR) T cell for GVHD prophylaxis. Here we demonstrate that human CD83 CAR T cells eradicate cell mediators of GVHD, significantly increase the ratio of regulatory T cells (Treg) to allo-activated Tconv, and provide lasting protection from xenogeneic GVHD. Further, we show human, acute myeloid leukemia (AML) expresses CD83 and can be targeted by CD83 CAR T cells. A 2nd generation CD83 CAR was generated with CD3ζ and 41BB costimulatory domain that was retrovirally transduced in human T cells to generate CD83 CAR T cells. The CD83 CAR construct exhibited a high degree of transduction efficiency of about 60%. The CD83 CAR T cells demonstrated robust IFN-γ and IL-2 production, killing, and proliferation when cultured with CD83+ target cells. To test whether human CD83 CAR T cells reduce alloreactivity in vitro, we investigated their suppressive function in allogeneic mixed leukocyte reactions (alloMLR). CD83 CAR T cells were added to 5-day alloMLRs consisting of autologous T cells and allogeneic monocyte-derived DCs at ratios ranging from 3:1 to 1:10. The CD83 CAR T cells potently reduced alloreactive T cell proliferation compared to mock transduced and CD19 CAR T cells. We identified that CD83 is differentially expressed on alloreactive Tconv, compared to Tregs. Moreover, the CD83 CAR T cell efficiently depletes CD83+ Tconv and proinflammatory DCs with 48 hours of engagement. To test the efficacy of human CD83 CAR T cells in vivo, we used an established xenogeneic GVHD model, where mice were inoculated with human PBMCs (25x106) and autologous CD83 CAR (1-10x106) or mock transduced T cells. The CD83 CAR T cells were well tolerated by the mice, and significantly improved survival compared to mock transduced T cells (Figure 1A). Mice treated with CD83 CAR T cells exhibited negligible GVHD target organ damage at day +21 (Figure 1B). Mice inoculated with CD83 CAR T cells demonstrated significantly fewer CD1c+, CD83+ DCs (1.7x106 v 6.2x105, P=0.002), CD4+, CD83+ T cells (4.8x103 v 5.8x102, P=0.005), and pathogenic Th1 cells (3.1x105 v 1.1x102, P=0.005) at day +21, compared to mice treated with mock transduced T cells. Moreover, the ratio of Treg to alloreactive Tconv (CD25+ non-Treg) was significantly increased among mice treated with CD83 CAR T cells (78 v 346, P=0.02), compared to mice injected with mock transduced T cells. Further, CD83 appears to be a promising candidate to target myeloid malignancies. We observed CD83 expression on malignant myeloid K562, Thp-1, U937, and MOLM-13 cells. Moreover, the CD83 CAR T cells effectively killed AML cell lines. Many AML antigens are expressed on progenitor stem cells. Thus, we evaluated for stem cell killing in human colony forming unit (CFU) assays, which demonstrated negligible on-target, off-tumor toxicity. Therefore, the human CD83 CAR T cell is an innovative cell-based approach to prevent GVHD, while providing direct anti-tumor activity against myeloid malignancies. Figure Disclosures Blazar: Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Davila:Atara: Research Funding; Celgene: Research Funding; Precision Biosciences: Consultancy; Bellicum: Consultancy; GlaxoSmithKline: Consultancy; Adaptive: Consultancy; Anixa: Consultancy; Novartis: Research Funding.

Regulation of p38MAPK-mediated dendritic cell functions by the deubiquitylase otubain 1.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that represent the essential link between innate and acquired immunity. Otubain (OTUB) 1 is shown to deubiquitinate TRAFs to suppress virus-induced inflammatory response. MAPK, a downstream molecule of TRAFs, is involved in regulating LPS-induced immune reactions and its activation is sensitive to the presence of OTUB1. Little is known about contributions of OTUB1 to changes in biological properties of DCs. The present study, therefore, explored whether DC functions are influenced by OTUB1. To this end, DCs were isolated and cultured with GM-CSF to attain bone marrow-derived DCs (BMDCs) and followed by treatment with lipopolysaccharide (LPS) in the presence or absence of OTUB1 siRNA. Expression of markers of cellular maturation and proliferation were analyzed by flow cytometry, and secretion of inflammatory cytokines and ability to stimulate CD4+ T-cells in allogenic mixed leukocyte reaction (allo-MLR) by ELISA, cell migration by a transwell migration assay and phagocytic capacity by FITC-dextran uptake measurement. As a result, treatment of the cells with OTUB1 siRNA prolonged activation of p38MAPK, increased CD54 expression and IL-6 release and reduced FITC-dextran uptake. Moreover, cytokine release produced from CD4+ T-cells in allo-MLR was different. The enhanced level of IFN-γ, but not other cytokine production was observed in the presence of siRNA OTUB1. All the effects were completely abolished when the cells were exposed with p38MAPK inhibitor SB203580. In conclusion, OTUB1 prevents the prolonged activation of p38MAPK, which in turn compromises DC functions.

Induction of PIR-A/B+ DCs in the in vitro inflammatory condition and their immunoregulatory function.

Dendritic cells (DCs), primary antigen-presenting cells, are now well known as an immunoregulator of many aspects of immune responses including inflammatory bowel diseases (IBDs) such as Crohn's disease and ulcerative colitis. We have reported that PIR-A/Bhigh cDCs (conventional DCs) appeared in dextran sodium sulfate (DSS)-induced colitis and serve as a negative immunoregulator in an animal model of IBD. The immunoregulatory role of PIR-A/B+ cDCs was confirmed in both an in vitro culture system and an in vivo transfer experiment. Here, we have investigated the differentiation process of PIR-A/B+ cDCs in an in vitro inflammatory environment and examined their functions. cDCs were isolated from the large intestinal lamina propria from C57BL/6 mice and cultured in an inflammatory environment (IL-1, IL-6, TNFα, and LPS). The appearance of PIR-A/B+ cDCs was determined after 24h, and the in vitro-induced PIR-A/B+ cDCs were functionally and genetically examined. PIR-A/B+ cDCs were detected after a 24-h culture only in the inflammatory environment, and the cells acted as a negative immunoregulator when examined in an allogenic mixed leukocyte reaction (MLR). The message level of IL-27 was highly upregulated in PIR-A/B+ cDCs, while that of high mobility group box 1 protein (HMGB1) was downregulated in these cells. This was well in accordance with the fact that PIR-A/B+ cDCs showed a suppressive function against activated T cells. We found that PIR-A/B+ cDCs produced IL-27, as verified by an ELISA assay, and that the inhibitory effect by PIR-A/B+ cDCs was, at least partially, due to IL-27. Furthermore, CD85d+ cells, a human counterpart of mouse PIR-A/B+ cDCs, were found in the lamina propria of the colon of the patients with ulcerative colitis, but not in the similar part of the non-inflammatory area of colon specimens from patients with colon cancer. PIR-A/B+ cDCs induced in an in vitro inflammatory environment model showed a suppressive function against activated T cells by producing an inhibitory cytokine.

1,25-Dihydroxyvitamin D3 modulates the phenotype and function of Monocyte derived dendritic cells in cattle

BackgroundThe active form of the vitamin D3, 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to have major effects not only on physiological processes but also on the regulation of the immune system of vertebrates. Dendritic cells are specialised antigen presenting cells which are in charge of the initiation of T-cell dependant immune responses and as such are key regulators of responses towards pathogens. In this study we set out to evaluate the effects of 1,25-(OH)2D3 on the phenotype of cattle monocyte-derived dendritic cells (MoDCs) and how the conditioning with this vitamin affects the function of these myeloid cells.ResultsMoDCs were generated from CD14+ monocytes with bovine IL-4 and GM-CSF with or without 1,25-(OH)2D3 supplementation for 10 days. Vitamin D conditioned MoDCs showed a reduced expression of co-stimulatory and antigen presenting molecules, as well as a reduced capability of endocytose ovalbumin. Furthermore, the capacity of MoDCs to induce proliferation in an allogeneic mixed leukocyte reaction was abolished when MoDCs were generated in presence of 1,25-(OH)2D3. LPS induced maturation of 1,25-(OH)2D3conditioned MoDCs resulted in lower secretion of IL-12 and higher IL-10 than that observed in MoDCs.ConclusionsThe typical immunotolerant phenotype observed in cattle DCs after exposure to 1,25-(OH)2D3 has a significant effect on the functionality of these immune cells, inhibiting the T-cell stimulatory capacity of MoDCs. This could have profound implications on how the bovine immune system deals with pathogens, particularly in diseases such as tuberculosis or paratuberculosis.

TAK1 inhibition ameliorates survival from graft-versus-host disease in an allogeneic murine marrow transplantation model.

Acute graft-versus-host disease(GVHD) isa major cause of morbidity and mortality in allogeneic hematopoietic cell transplantation (allo-HCT). Majority of the current immunosuppressive strategies targeting donor T cells to prevent or treat acute GVHD are only partially effective, and often require escalated immunosuppressive therapy. Recent studies have revealed that activation of antigen-presenting cells in the proinflammatory milieu is important for the priming and promotion of GVHD. This activation is mediated by innate immune signaling pathways, which therefore potentially represent new targets in addressing GVHD. Using gene expression analysis of peripheral monocytes from patients' post-allo-HCT, we detected an upregulation of TGF-β-activated kinase 1 (TAK1), a key regulator of the toll-like receptor signaling pathway. 5Z-7-oxozeaenol, a selective inhibitor of TAK1, reduced proinflammatory cytokine production by activated monocytes under lipopolysaccharide stimulation and T cell proliferation in allogeneic-mixed leukocyte reactions with monocyte-derived dendritic cells. In an experimental mouse model of GVHD, 5Z-7-oxozeaenol administration after allo-HCT ameliorated GVHD severity and mortality, with significant reduction in serum TNFα, IL-1β, and IL-12 levels. Our findings suggest that altering the activation status of innate immune cells by TAK1 inhibition may be a novel therapeutic approach for acute GVHD.

Immunomodulatory Effects of 1,25-Dihydroxyvitamin D3 on Dendritic Cells Promote Induction of T Cell Hyporesponsiveness to Myelin-Derived Antigens.

While emerging evidence indicates that dendritic cells (DC) play a central role in the pathogenesis of multiple sclerosis (MS), their modulation with immunoregulatory agents provides prospect as disease-modifying therapy. Our observations reveal that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment of monocyte-derived DC results in a semimature phenotype and anti-inflammatory cytokine profile as compared to conventional DC, in both healthy controls and MS patients. Importantly, 1,25(OH)2D3-treated DC induce T cell hyporesponsiveness, as demonstrated in an allogeneic mixed leukocyte reaction. Next, following a freeze-thaw cycle, 1,25(OH)2D3-treated immature DC could be recovered with a 78% yield and 75% viability. Cryopreservation did not affect the expression of membrane markers by 1,25(OH)2D3-treated DC nor their capacity to induce T cell hyporesponsiveness. In addition, the T cell hyporesponsiveness induced by 1,25(OH)2D3-treated DC is antigen-specific and robust since T cells retain their capacity to respond to an unrelated antigen and do not reactivate upon rechallenge with fully mature conventional DC, respectively. These observations underline the clinical potential of tolerogenic DC (tolDC) to correct the immunological imbalance in MS. Furthermore, the feasibility to cryopreserve highly potent tolDC will, ultimately, contribute to the large-scale production and the widely applicable use of tolDC.

Analysis of T Cell Proliferating and Polarizing Potential of Murine Dendritic Cells in Allogeneic-mixed Leukocyte Reaction

Analysis of T Cell Proliferating and Polarizing Potential of Murine Dendritic Cells in Allogeneic-mixed Leukocyte Reaction

Levamisole promotes murine bone marrow derived dendritic cell activation and drives Th1 immune response in vitro and in vivo

Our lab previously found that levamisole (LMS) as an adjuvant enhanced the efficacy of vaccine against infectious pathogens. However, the cellular and molecular mechanisms remain to be defined. In this study, we showed that BALB/c bone marrow-derived DC stimulated with LMS resulted in enhanced cell-surface expression of CD80, CD86, CD40 and MHC class II, as well as enhanced production of IL-12p70, TNF-α and IL-1β. Interestingly, the LMS activated DCs were able to stimulate CD4+ T cell proliferation and facilitated Th1 differentiation by increasing the secretion of IFN-γ in an allogeneic mixed leukocyte reaction. Furthermore, to confirm the in vitro data, we investigated the effect of LMS on antigen-specific antibody and cytokine production in BALB/c mice. Immunization with LMS plus OVA showed that anti-OVA IgG2a and IFN-γ were increased significantly compared with OVA alone in BALB/c mice. In conclusion, our results suggested that murine bone marrow-derived DC, played a crucial role in the effect of LMS on the induction of Th1 responses, which probably was due to its ability to promote DC maturation and secrete proinflammatory cytokines.

Targeting JAK2 By Gene Knockout or Pacritinib Treatment Reduces Gvhd and Xenograft Rejection By Promoting Induced Treg Differentiation

Janus kinase 2 (JAK2) signal transduction is a critical mediator of immune response. JAK2 activation promotes T-cell allosensitization, as well as Th1 and Th17 differentiation. JAK2-mediated STAT3 phosphorylation limits the generation of induced regulatory T cells (iTregs) by disrupting interactions between STAT5 and Foxp3. JAK2 is implicated in the onset of graft-versus-host disease (GVHD), which is a significant cause of transplant-related mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Using murine models of allo-HCT we show here that transfer of donor JAK2-/- T cells is associated with significantly less GVHD, compared with wild-type or JAK2 replete donors (Figure 1, P =.003 and 0.01, respectively). Th1 differentiation among JAK2-/- T-cells is dramatically decreased, compared with controls. Conversely, iTreg polarization and stability are significantly increased among the JAK2 deficient T cells. To investigate the effect of pharmacologic JAK2 inhibition on T-cell alloresponses, pacritinib (supplied by CTI BioPharma), was chosen as it does not induce myelosuppression or increase risk for opportunistic infections in myelofibrosis patients - unlike JAK1/JAK2 inhibitors. Pacritinib potently inhibits JAK2, but also has suppressive activity toward JAK3, CSF1R, and IRAK1. Pacritinib was administered at 100mg/kg twice a day by oral gavage for 4 weeks beginning on the day of MHC-disparate allo-HCT, significantly reducing GVHD in recipient mice compared with methylcellulose vehicle control. In allogeneic mixed leukocyte reactions using human cells, pacritinib (2.5μM) significantly reduces T-cell proliferation after 5 days of culture (P <.0001). In vitro studies verified pacritinib eliminates STAT3 activity by IL-6 in human CD4+ T-cells, while permitting IL-2 induced STAT5 phosphorylation despite its effects on JAK3. This offers a platform to reduce Th17 development, while promoting iTreg populations. As such, pacritinib dramatically suppresses Th17 differentiation reflected by RORgammaT expression among naïve CD4+ T cells stimulated by allogeneic dendritic cells (DC), compared with DMSO (P <.0001). iTregs were generated with allogeneic DCs in the presence of pacritinib or DMSO for 5 days, and then cultured with self T-cell responders and fresh DCs without additional drug exposure. The suppressive potency of either pacritinib- or DMSO-treated iTregs was similar, suggesting JAK2 is not required for iTreg function. We then evaluated whether JAK2 inhibition could prevent human skin graft rejection in an NSG mouse xenograft model. A 1x1 cm split-thickness human skin graft was transplanted onto the animal dorsally. The mice rested for 30 days after surgery to permit engraftment, then received 5x106 allogeneic peripheral blood mononuclear cells (PBMC) by intraperitoneal injection. Pacritinib was administered at the same dose and schedule for 2 weeks beginning at time of PBMC injection as tolerated by the NSG mice. The treatment significantly delayed allograft rejection by the human donor PBMCs, compared with vehicle control (Figure 2: Median graft survival 32.5 v 51 days, Day +60 survival 0% v 33.3%, pooled data from 2 experiments, n=5-6 mice per arm, P =.0011). Collectively, these data clearly identify JAK2 as a therapeutic target to control donor alloreactivity and promote iTreg responses after HCT or solid organ transplantation.*BCB and DB contributed equally to this work. [Display omitted] [Display omitted] DisclosuresSinger:CTI BioPharma, Corp: Employment, Equity Ownership.

T Cell Exhaustion/Senescence in Relapsed Multiple Myeloma after Autologous Stem Cell Transplantation

▪BACKGROUND: Multiple myeloma (MM) is the most common indication for high-dose chemotherapy and autologous stem cell transplantation (ASCT). Post-transplant lenalidomide maintenance therapy doubles progression-free survival, but almost all patients eventually relapse. The immune system participates in the control of MM, whereas compromised immunity contributes to its evolution. Post-transplant immunotherapy to induce or restore antitumor immunity offers a promising approach to target residual MM and improve patient outcomes. The rational development of immunotherapeutic interventions after ASCT, however, requires a comprehensive understanding of the immunologic milieu. We therefore evaluated lymphocyte composition and function after ASCT to guide optimal timing of immunotherapy and to identify potential markers of relapse.METHODS: Fifty-five MM patients undergoing ASCT were evaluated for at least one year. Peripheral blood from patients was obtained before ASCT and on d +12, +30, +90, +180, and +365 after ASCT, and at the time of relapse where applicable. Leukocyte concentrates were used as a source of healthy donor cells. Mononuclear cells were analyzed by flow cytometry for phenotypic assessment of lymphocyte subset composition. Functional assessment of dendritic cell and T cell activity in vitro was assayed in autologous and allogeneic mixed leukocyte reactions, cytotoxic T lymphocyte (CTL) lysis assays, and PD-1 blockade experiments.RESULTS: CD3+ CD4+ CD25bright CD127neg regulatory T cells (Tregs) decline as CD8+ T cells expand during early lymphocyte recovery after ASCT, markedly reducing the Treg:CD8+ effector T-cell ratio (Fig 1A) and providing a critical early window for the introduction of immune-based post-transplant consolidation therapies. CD8+ T cells can respond to autologous dendritic cells presenting tumor antigen in vitro as early as day +12 post-transplant, becoming antigen-specific CTL effectors and thereby demonstrating preservation of cellular reactivity (Fig 1B). CD4+ and CD8+ T cells express the negative regulatory molecules, CTLA-4, PD-1, LAG-3, and TIM-3, before and after ASCT (data not shown). A subpopulation of exhausted/senescent CD8+ T cells, however, down-regulates CD28 (Fig 2A) and up-regulates CD57 (Fig 2B) and PD-1 (Fig 2C), characterizing immune impairment and relapse after ASCT. CD4+ T cells show the same trends in the frequencies of CD28neg, CD28neg CD57+, and CD28neg PD-1+ cells, albeit at lower levels of expression (Fig 2D). Relapsing patients have higher numbers of CD8+ CD28neg PD-1+ T cells at +3 months after transplant (Fig 2E), but before detection of clinical disease, indicating their applicability in identifying patients at higher risk of relapse. PD-1 blockade revives the proliferation and cytokine secretion of the hyporesponsive, CD8+ CD28neg PD-1+ T cells in vitro (Fig 3).CONCLUSION: These results identify T cell exhaustion/senescence as a distinguishing feature of relapse and support early introduction of immunotherapy to stimulate antitumor immunity after ASCT. [Display omitted] [Display omitted] DisclosuresLesokhin:Genentech: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Aduro: Consultancy; Janssen: Consultancy, Research Funding; Efranat: Consultancy. Giralt:CELGENE: Consultancy, Honoraria, Research Funding; SANOFI: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Research Funding; JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; TAKEDA: Consultancy, Honoraria, Research Funding.

Characterisation of Human CD83 Expression on Immune Cells and Their Targeting with CD83 Antibodies to Prevent Graft Versus Host Disease in Allogeneic Haematopoietic Cell Transplantation

Introduction: CD83 is an important marker of activated dendritic cells (DC) but it is also expressed on other immune cells. Polyclonal anti-CD83 antibody depletes activated DC and prevents human peripheral blood mononuclear cell (PBMC) induced xenogeneic graft versus host disease (GVHD) in immunosuppressed SCID mice (J Exp Med 2009;206;387). We therefore generated a potential therapeutic human anti-CD83 mAb (3C12C), which had similar efficacy and T cell sparing effects in the same model (Leukemia 2015; in press). To investigate the specific immunosuppressive effect of 3C12C further, we undertook a comprehensive analysis of CD83 expression and its glycosylation pattern on various immune cell populations and tested the effect of 3C12C on T cell function using preclinical models, including a human CD83 (hCD83) knock in (KI) mouse.Methods: A panel of mouse and recombinant mAbs to hCD83 were used to analyse its expression by flow cytometry on resting and activated healthy donor PBMC. The expression of potential CD83 splice variants was examined by PCR. T cell expression was examined by flow cytometry and confocal microscopy after PHA, CD3/CD28 beads and allogeneic mixed leukocyte reaction (alloMLR) culture. Control human IgG1 (trastuzumab) and 3C12C mAbs were tested (0.125mg d-1) in a xenogeneic model of GVHD utilizing human PBMC transplanted into total body irradiation and anti-NK conditioned SCID mice. The genetically engineered hCD83 KI mouse was shown to be immune-competent and used to test the effect of 3C12C on LPS activated DC and T cells.Results: There were distinct CD83 splice variants (full length CD83, splicing variant CD83a, CD83b and CD83c) in different immune cells. CD83 glycosylation status also differed with high glycosylation required for surface expression on activated DC, whereas its expression on activated B cells and monocytes was resistant to de-glycosylation. Increases in CD83 expression on T cells occurred early with different kinetics, underlining the distinct signal pathway involved. The 3C12C mAb reduced T cell proliferation in the alloMLR but did not affect cytomegalovirus (CMV) or influenza (Flu) specific CD8+T cell numbers. Treatment with 3C12C prevented GVHD in human PBMC transplanted SCID mice, which otherwise developed histological GVHD between d8-13. Human DC were activated by d2 and expressed the CMRF-44 activation marker plus CD83, CD80 and CD86. Treatment with 3C12C mAb eliminated CD83+ CMRF44+ DC early post-transplant and reduced T cell activation. Further studies, established CMV and Flu specific T cells were retained and responded to antigen by IFNg production. Furthermore, Treg numbers were preserved. The 3C12C mAb depleted LPS activated DC in hCD83 KI mice in experiments performed prior to commencing transplant studies.Conclusion: These findings suggest that the potential therapeutic human anti-CD83 mAb induced significant immune suppression, by depletion of activated DC and consequential modulation of T cell activation. The reduction in allo/xeno activated T cells may result in part from a direct effect of anti-CD83 on early T cell responses. This apparently selective immunosuppressive effect preserves anti-viral T cell immunity and Treg pathways, suggesting that 3C12C merits further investigation as a novel agent for GVHD prophylaxis. DisclosuresHart:DendroCyte BioTech Pty Ltd: Equity Ownership. Clark:DendroCyte BioTech Pty Ltd: Equity Ownership.

Bidirectional polarization of T cell function via CD43 (IRC8P.437)

Abstract Co-receptors, being either co-stimulatory or co-inhibitory, play a pivotal role in T cell immunity, as they decide the fate of T cell function. CD43 is one of the abundant cell surface glycoproteins expressed on T cells. CD43 has been demonstrated to act as not only a potent co-receptor but also a negative regulator for T cell activation. To further investigate role of CD43 in T cell activation and subsequent function, peripheral blood T cells were activated via two distinct CD43 epitopes recognized by monoclonal antibodies (mAbs) 6E5 (T6E5-act) and 10G7 (T10G7-act) along with TCR signalling. Both the CD43 mAbs could potently induce T cell activation when immobilized but exerted differential downstream effects including differential activation of signalling pathways, T cell cytokine production and also distinct effector function. T10G7-act poorly responded in re-stimulation assay and further acquired suppressive function compared to T6E5-act or to T cells activated via CD28 (TCD28-act). Furthermore, T10G7-act did not directly inhibit responder T cells, but rather exhibited their effect via dendritic cells when added to allogenic mixed leukocyte reaction. Together our data suggests a unique role of CD43 in bidirectional polarization of T cell immunity, a positive co-stimulatory role and a negative role in down-modulation of immune response, depending on its targeted epitope.

PD-1/PD-L1 expression on CD(4+) T cells and myeloid DCs correlates with the immune pathogenesis of atrial fibrillation.

Although immuno-inflammatory response contributes to pathogenesis of AF, molecular and cellular mechanism in this process remains poorly understood. Recently, increasing evidence suggests that Programmed death-1 (PD-1)/PD-1 ligand (PD-L) pathway may be a potential pathway participating in AF pathogenesis. In this study, we detected the PD-1 and PD-L1, 2 expression on peripheral blood function cells by flow cytometry in 91 atrial fibrillation (AF) patients and 35 healthy volunteers. The expression of PD-1 on CD4+ T cells and PD-L1 on myeloid dendritic cells (mDCs) in AF patients is significantly down-regulated compared with healthy volunteers. In addition, the extent of PD-1/PD-L1 down-regulation is closely related with AF burden. More importantly, Allogeneic mixed leukocyte reactions (MLR) shows that the mDCs PD-L1 down-regulation is associated with increased T cell (CD4+ and CD8+) proliferation, increased type 1 effector cytokines (IL-2 and IFN-γ) secretion, and decreased type 2 effector cytokine (IL-10) secretion. Then, PD-L1 up-regulation by the stimulation of IFN-α can significantly convert this representation. Collectively, our report suggest that T(CD4+)/mDCs-associated PD-1/PD-L1 pathway plays a key role in AF immune regulation. PD-1/PD-L1 down-regulation in AF patients promotes T cells function and may contribute to AF pathogenesis.

High Functional CD70 Expression on α‐Type 1‐Polarized Dendritic Cells from Patients with Chronic Lymphocytic Leukaemia

Antigen-loaded dendritic cells (DCs) used as anticancer vaccine holds promise for therapy, but needs to be optimized. The most frequently described DC vaccine is being matured with a cocktail containing prostaglandin E2 (PGE2 DC). However, even though PGE2 DCs express both costimulatory and migratory receptors, their IL-12p70-prodcution is low, leading to an insufficient Th1 immune response. As an alternative, α-type-1 polarized DCs (αDC1s) have shown a superior production of IL-12p70 and subsequent activation of effector cells. From chronic lymphocytic leukaemia (CLL) patients, αDC1s can be generated to induce a functional Th1-immune response. Yet, another costimulatory receptor, CD70, appears to be essential for optimal DC function by promotion of T cell survival and function. So far, PGE2 is suggested as one of the most important factors for the induction of CD70 expression on DCs. Therefore, we wanted to investigate whether αDC1s have the ability to express functional CD70. We found that CD70 expression on αDC1s could be upregulated in the same manner as PGE2 DCs. In an allogeneic mixed leucocyte reaction, we found that antibody-blocking of CD70 on αDC1s from controls reduced effector cell proliferation although this could not be found when using CLL αDC1s. Nevertheless, CD70-blocking of αDC1s from both controls and patients with CLL had a negative influence on the production of both IL-12p70 and the Th1 cytokine IFN-γ, while the production of the Th2 cytokine IL-5 was enhanced. Together, this study further suggests that αDC1s should be considered as a suitable candidate for clinical antitumour vaccine strategies in patients with CLL.

Hepatitis B e Antigen Seroconversion Is Related with the Function of Dendritic Cells in Chronic Hepatitis B Virus Infection.

Aim. To investigate the relationship between hepatitis B e antigen seroconversion and the function of dendritic cells (DC) in patients with hepatitis B virus. Methods. The peripheral blood mononuclear cells (PBMC) from 21 chronic HBV patients in immune tolerance state, 23 patients in inactive HBsAg carrier state, and 10 healthy HBV-naive blood donors were incubated and induced into DC in presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), respectively. The expressions of surface markers on DC were detected by flow cytometry, and the stimulatory capacity of DC in allogenic mixed leukocyte reaction (MLR) was tested by CCK-8, and the level of cytokines released by DC was analyzed by enzyme-linked immunosorbent assay (ELISA). Results. DC from patients in immune tolerance showed a remarkably lower surface expression of CD80, CD86, and HLA-DR and exhibited an impaired stimulatory capacity in MLR and reduced secretion of IL-12, as compared to the patients in inactive HBsAg carrier state. There was no significant difference between the indicators from the patients in inactive HBsAg carrier state and healthy subjects. There was a significant difference of HBV DNA level between immune tolerance and inactive HBsAg carrier group (P < 0.01) and a negative correlation between HBV DNA level and the expressions of dendritic cells in both groups, respectively (P = 0.01). Conclusion. DC from patients in inactive HBsAg carrier state shows stronger function in comparison with patients in immune tolerance, the expressions of dendritic cells correlate with HBV DNA level, and the function stage of DC may play an important role in HBeAg seroconversion.

Ouabain affects the expression of activation markers, cytokine production, and endocytosis of human monocytes.

Ouabain is a steroid capable of binding to and inhibiting Na+,-K+-ATPase. Studies have demonstrated some actions of ouabain on immune cells, which indicated both pro- and anti-inflammatory properties of this molecule. Nevertheless, its effects on human monocytes are still poorly understood. Thus, the present work investigated effects of ouabain in the activation and function of human adherent monocytes. Our results show that there is an increase in intracellular calcium levels already 5 minutes following monocyte treatment with 10−7 M of ouabain. Furthermore, monocytes expressed increased amounts of surface activation markers such as CD69, HLA-DR, CD86, and CD80 and also presented an augmented endocytic activity of dextran-FITC particles after 24 hours of culture in the presence of ouabain. However, monocytes treated with ouabain did not have an increased stimulatory capacity in allogeneic mixed leukocyte reaction. Ouabain-treated monocytes produced higher levels of IL-1β and TNF-α as reported before. A novel observation was the fact that ouabain induced IL-10 and VEGF as well. Collectively, these results suggest that ouabain impacts monocyte activation and modulates monocyte functions, implying that this steroid could act as an immunomodulator of these cells.

Oral vitamin D3 supplementation reduces monocyte-derived dendritic cell maturation and cytokine production in Crohn’s disease patients

Low serum vitamin D levels may provoke or aggravate Crohn's disease (CrD). Vitamin D3 is a well-known immune modulator that affects immune functions in vitro and may prevent CrD flares. Dendritic cells (DC) are key mediators of vitamin D3 effects. In this study, we describe changes in monocyte-derived DC (mo-DC) maturation marker expression and cytokine production following 26 weeks of oral vitamin D3 supplementation in CrD patients. Ten CrD patients who had increased serum 25-hydroxy vitamin D levels after oral vitamin D3 and calcium treatment and ten seasonally matched placebo-treated patients were selected for this study. Mo-DC were generated before and after the 26 weeks and induced to mature upon lipopolysaccharide (LPS) stimulation. Maturation marker expression and cytokine production were analysed. Mo-DC function was analysed in a mixed leucocyte reaction (MLR). Compared with baseline values, LPS-matured mo-DC exhibited reduced expression of CD80 and reduced production of the cytokines IL-10, IL-1β, and IL-6 following 26 weeks of oral vitamin D3 supplementation. Mo-DC performance in an allogeneic MLR was unchanged after vitamin D3 supplementation. Treatment with the placebo did not affect maturation markers, cytokine production, or the MLR. Vitamin D3 treatment in CrD patients led to hypo-responsive LPS-stimulated mo-DC. This finding indicates that vitamin D3 levels have an impact on the monocytic precursors of mo-DC in vivo and may explain the positive effects of vitamin D3 supplementation on CrD patients. Alternatively, CrD patients with high serum vitamin D3 levels may represent a subgroup with low disease activity.