Mixed Leukocyte Reaction Assay Research Articles

Pretransplant, Th17 dominant alloreactivity in highly sensitized kidney transplant candidates.

Sensitization to donor human leukocyte antigen (HLA) molecules prior to transplantation is a significant risk factor for delayed access to transplantation and to long-term outcomes. Memory T cells and their cytokines play a pivotal role in shaping immune responses, thereby increasing the risk of allograft rejection among highly sensitized patients. This study aims to elucidate the precise contribution of different CD4+ memory T cell subsets to alloreactivity in highly sensitized (HS) kidney transplant recipients. Stimulation of peripheral blood mononuclear cells (PBMC) with various polyclonal stimulating agents to assess non-specific immune responses revealed that HS patients exhibit elevated immune reactivity even before kidney transplantation, compared to non-sensitized (NS) patients. HS patients' PBMC displayed higher frequencies of CD4+ T cells expressing IFNγ, IL4, IL6, IL17A, and TNFα and secreted relatively higher levels of IL17A and IL21 upon stimulation with PMA/ionomycin. Additionally, PBMC from HS patients stimulated with T cell stimulating agent phytohemagglutinin (PHA) exhibited elevated expression levels of IFNγ, IL4 and, IL21. On the other hand, stimulation with a combination of resiquimod (R848) and IL2 for the activation of memory B cells demonstrated higher expression of IL17A, TNFα and IL21, as determined by quantitative real-time PCR. A mixed leukocyte reaction (MLR) assay, employing third-party donor antigen presenting cells (APCs), was implemented to evaluate the direct alloreactive response. HS patients demonstrated notably higher frequencies of CD4+ T cells expressing IL4, IL6 and IL17A. Interestingly, APCs expressing recall HLA antigens triggered a stronger Th17 response compared to APCs lacking recall HLA antigens in sensitized patients. Furthermore, donor APCs induced higher activation of effector memory T cells in HS patients as compared to NS patients. These results provide an assessment of pretransplant alloreactive T cell subsets in highly sensitized patients and emphasize the significance of Th17 cells in alloimmune responses. These findings hold promise for the development of treatment strategies tailored to sensitized kidney transplant recipients, with potential clinical implications.

Abstract 1356: The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation

Abstract VISTA, a predominantly myeloid immune checkpoint, promotes an immunosuppressive tumor microenvironment and has been associated with resistance to approved checkpoint therapies. While blockade of VISTA with antibodies has shown anti-tumor efficacy in preclinical models, the biological mechanisms that underlie this effect remain poorly understood. To investigate the mechanistic basis of anti-tumor responses, we performed single-cell RNA sequencing of tumor infiltrating leukocytes from the cell-derived murine colon carcinoma xenograft model, CT26, which is known to be sensitive to VISTA blockade. We used HMBD-002, a uniquely blocking but non-depleting clinical-stage IgG4 anti-VISTA antibody, to identify the key immune cells and pathways impacted by VISTA blockade in the absence of Fc-mediated cell depletion. The translatability of these findings was validated in a human PBMC mixed leukocyte reaction assay. Subsequently, targeted depletion or inhibition of the identified immune populations and pathways was performed to confirm their contribution to the anti-tumor efficacy of HMBD-002.The blockade of VISTA by HMBD-002 significantly increased CD8+ T cells in the CT26 tumors, promoting an activated cytotoxic Tc1 program without inducing exhaustion. Concurrently, a substantial increase in proinflammatory M1 tumor-associated macrophages (TAMs) was noted within treated tumors, while other immune cell populations remained unaffected. Among the signaling pathways, the type-I interferon (IFN) response exhibited a significant upregulation, particularly in TAMs, indicating a type-I IFN-driven M1 reprogramming by HMBD-002. Validation experiments further demonstrated that the depletion of TAMs or CD8+ T cells, but not CD4+ T cells, abrogated the efficacy of HMBD-002. Moreover, blocking the type-I IFN signaling effectively impeded HMBD-002-induced TAM reprogramming and subsequent anti-tumor efficacy, thus confirming the pivotal roles of CD8+ T cells, TAMs, and type-I IFN signaling in driving the anti-tumor response to VISTA blockade. In conclusion, VISTA blockade with HMBD-002 promotes an anti-tumor response by type-I IFN mediated reprogramming of TAMs and activation of cytotoxic CD8+ T cells. Citation Format: Bhushan Dharmadhikari, Olga Zharkova, Dipti Thakkar, Roberto Tirado-Magallanes, Konrad Paszkiewicz, Piers J. Ingram, Jerome D. Boyd-Kirkup. The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1356.

Nicotine- and tar-removed cigarette smoke extract modulates the antigen presentation function of mouse bone marrow-derived dendritic cells.

Dendritic cells (DCs) take up antigens derived from pathogens such as bacteria and viruses, and from tumor cells and induce the activation of antigen-specific T cells through major histocompatibility complex (MHC)-mediated antigen presentation. Mainstream cigarette smoke extract (CSE) has various effects, and the effects of its major components, nicotine and tar, have been extensively analyzed. Recently, the physiological effects of nicotine- and tar-removed CSE (cCSE) have also been reported. However, the effects of cCSE on DC-mediated immune responses remain unknown. In this study, we found that cCSE enhanced lipopolysaccharide (LPS)-stimulated induction of the expression of MHC-I and MHC-II on the cell surface of mouse bone marrow-derived DCs (BMDCs). In contrast, cCSE suppressed the induction of CD86 induced by stimulation with curdlan and interferon-gamma (IFN-γ). In addition, cCSE suppressed the production of IL-12, IL-23, and IL-10 by LPS and curdlan stimulation. In the presence of cCSE, LPS-stimulated BMDCs showed enhanced activation of CD4 and CD8 T cells and increased IL-2 production from T cells by antigen presentation in a mixed-leukocyte reaction assay. In contrast, cCSE did not affect the activation of T cells by curdlan- or IFN-γ-stimulated BMDCs, and curdlan-stimulated BMDCs suppressed IL-17 production from T cells and enhanced IFN-γ production. These results suggest that cCSE has different effects on the activation signals induced by LPS, curdlan, and IFN-γ in BMDCs and modulates the antigen presentation function of BMDCs. This article is protected by copyright. All rights reserved.

ATP and its metabolite adenosine cooperatively upregulate the antigen-presenting molecules on dendritic cells leading to IFN-γ production by T cells

Dendritic cells (DCs) present foreign antigens to T cells via the major histocompatibility complex (MHC), thereby inducing acquired immune responses. ATP accumulates at sites of inflammation or in tumor tissues, which triggers local inflammatory responses. However, it remains to be clarified how ATP modulates the functions of DCs. In this study, we investigated the effects of extracellular ATP on mouse bone marrow-derived dendritic cells (BMDCs) as well as the potential for subsequent T cell activation. We found that high concentrations of ATP (1mM) upregulated the cell surface expression levels of MHC-I, MHC-II, and co-stimulatory molecules CD80 and CD86 but not those of co-inhibitory molecules PD-L1 and PD-L2 in BMDCs. Increased surface expression of MHC-I, MHC-II, CD80, and CD86 was inhibited by a pan-P2 receptor antagonist. In addition, the upregulation of MHC-I and MHC-II expression was inhibited by an adenosine P1 receptor antagonist and by inhibitors of CD39 and CD73, which metabolize ATP to adenosine. These results suggest that adenosine is required for the ATP-induced upregulation of MHC-I and MHC-II. In the mixed leukocyte reaction assay, ATP-stimulated BMDCs activated CD4 and CD8T cells and induced interferon-γ (IFN-γ) production by these T cells. Collectively, these results suggest that high concentrations of extracellular ATP upregulate the expression of antigen-presenting and co-stimulatory molecules but not that of co-inhibitory molecules in BMDCs. Cooperative stimulation of ATP and its metabolite adenosine was required for the upregulation of MHC-I and MHC-II. These ATP-stimulated BMDCs induced the activation of IFN-γ-producing T cells upon antigen presentation.

Immunosuppressive effect of Columbianadin on maturation, migration, allogenic T cell stimulation and phagocytosis capacity of TNF-α induced dendritic cells

Ethnopharmacological relevanceAngelicae pubescentis radix (APR) has a long history in the treatment of rheumatoid arthritis (RA) in China. It has the effects of dispelling wind to eliminate dampness, removing arthralgia and stopping pain in the Chinese Pharmacopeia, but its mechanisms was unclear. Columbianadin (CBN) was one of the main bioactive compounds of APR, and has many pharmacological effects. But the immunosuppressive effect of CBN on DCs and the potential mechanism needed to be explored. Aim of the studyThe study was aimed to clarify the immunosuppressive effect of CBN on maturation, migration, allogenic T cell stimulation and phagocytosis capacity of TNF-α induced DCs. Materials and methodsBone marrow-derived DCs were obtained and cultured from C57BL/6 mice in accordance with protocol. The phenotypic study (CD11c, CD40, CD80, CD86 and MHC Ⅱ) were measured by flow cytometry. FITC-dextran were uptaked by DCs and the change of endocytosis activity were mediated by acquired mannose receptor. Transwell chambers were used to detect the migration ability of DCs. Mixed leukocyte reaction (MLR) assay was used to detect the allostimulatory ability of CBN on TNF-α stimulated DCs. The secretion of cytokines and chemokines was measured by ELISA Kit. TLRs gene and MAPKs/NF-κB protein expression were checked by qRT-PCR and Western blot. ResultsCBN inhibited the maturation of TNF-α-induced DCs while maintaining phagocytosis capabilities. Additionally, CBN inhibited the migration of TNF-α stimulated DCs, which related to reduce the production of chemokines (MCP-1, MIP-1α). Notably, CBN could suppress the proliferation of CD4+T cells by inhibiting DCs maturation, and decrease the proinflammatory cytokines IL-6 production. Furthermore, CBN inhibited mRNA expression of TLR2, TLR7 and TLR9 in TNF-α-activated DCs. Meanwhile, the phosphorylation of p38, JNK1/2 and NF-κB protein were significantly inhibited in CBN treated DCs. ConclusionsThese findings provided novel insights into the pharmacological activity of CBN. They also indicated that inhibition DCs maturation owning to the immunosuppressive effect of CBN. CBN was expected as a potential immunosuppressant and TLRs/MAPKs/NF-κB pathway may be an important mechanism for CBN's immunosuppressive activity.

Abstract 1879: CIT-801: a bifunctional anti-PD1xIL15 agent demonstrated synergy and enhanced in vivo anti-tumor efficacy

Abstract PD1/PD-L1 antibody has revolutionized cancer therapy. However, only a subset of cancer patients are responsive to anti-PD1 antibody treatment. Lack of immune effector cells in the tumor microenvironment (cold tumor) is considered a major reason for such a resistance. Interleukin 15 (IL15) is a cytokine critical for the survival and proliferation of T-cells and NK cells. We hypothesized that a combination of IL15 with anti-PD1 antibody might turn a cold tumor into a hot tumor and overcome tumor resistance to PD1 therapy, and therefore benefit a larger cancer patient population. We have constructed a homodimeric bifunctional agent (CIT-801) that is composed of an anti-PD1 antibody (CIT-100) and IL15 plus the sushi domain of IL15Rα. In vitro, this bifunctional anti-PD1xIL15 agent retained the ability of its anti-PD1 component to enhance T-cell activation in both mixed leukocyte reaction (MLR) and PD1 luciferase reporter assays. It also retained the ability of its IL15 component to stimulate the proliferation of T-cells. Moreover, the bifunctional agent was able to enhance the activation, proliferation and cytotoxicity of NK cells in vitro. In a syngeneic MC38 tumor model with human-PD1 knock-in mice, the bifunctional anti-PD1xIL15 agent strongly inhibited tumor growth, superior to anti-PD1 antibody treatment alone. In summary, anti-PD1xIL15 bifunctional compound CIT-801 has shown a great therapeutic potential and is worth further development. Citation Format: Yiwen Li, Yuxiang Hu, Welson Wang, Wei Wu. CIT-801: a bifunctional anti-PD1xIL15 agent demonstrated synergy and enhanced in vivo anti-tumor efficacy [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 1879.

Δ42PD1-TLR4 Augments γδ-T Cell Activation of the Transitional Memory Subset of CD4+ T Cells.

SummaryTLR ligands can contribute to T cell immune responses by indirectly stimulating antigen presentation and cytokines and directly serving as co-stimulatory signals. We have previously reported that the human endogenous surface protein, Δ42PD1, is expressed primarily on (Vγ9)Vδ2 cells and can interact with TLR4. Since Vδ2 cells possess antigen presentation capacity, we sought to further characterize if the Δ42PD1-TLR4 interaction has a role in stimulating T cell responses. In this study, we found that stimulation of Vδ2 cells not only upregulated Δ42PD1 expression but also increased MHC class II molecules necessary for the antigen presentation. In a mixed leukocyte reaction assay, upregulation of Δ42PD1 on Vδ2 cells elevated subsequent T cell proliferation. Furthermore, the interaction between Δ42PD1-TLR4 augments Vδ2 cell stimulation of autologous CMV pp65-or TT-specific CD4+ T cell proliferation and IFN-γ responses, which was specifically and significantly reduced by blocking the Δ42PD1-TLR4 interaction. Furthermore, confocal microscopy analysis confirmed the interaction between Δ42PD1+HLA-DR+Vδ2 cells and TLR4+CD4 T cells. Interestingly, the subset of CD4+ T cells expressing TLR4 appears to be PD-1+ CD45RO+CD45RA+ transitional memory T cells and responded to Δ42PD1+HLA-DR+Vδ2 cells. Overall, this study demonstrated an important biological role of Δ42PD1 protein exhibited by Vδ2 antigen-presenting cells in augmenting T cell activation through TLR4, which may serve as an additional co-stimulatory signal.

Direct activation of aryl hydrocarbon receptor by benzo[a]pyrene elicits T-helper 2-driven proinflammatory responses in a mouse model of allergic dermatitis.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that binds to various environmental chemicals and contributes to numerous toxicological effects. However, the direct effects of AhR on the development of allergic diseases are not fully understood. The main aim of this study was to elucidate the action of AhR in the development of cutaneous allergies. Initially, the potential for a direct relationship between AhR and the immune cells was investigated in vitro, using murine bone marrow-derived dendritic cells, human epidermal keratinocytes, and the mixed leukocyte reaction assay. Benzo[a]pyrene (BaP) and 6-formylindolo[3,2-b]carbazole were used as selective ligands for the AhR. Pretreatment with BaP and/or 6-formylindolo[3,2-b]carbazole significantly induced cytokine release by activated keratinocytes and T-cell proliferation, whereas interleukin-12 production in bone marrow-derived dendritic cells was reduced by AhR activation. To confirm the in vitro results, in vivo experiments were also performed in T-helper (Th)2-type hapten toluene-2,4-diisocyanate- and Th1-type hapten dinitrochlorobenzene-induced mouse models of allergic dermatitis. Mice were orally administered BaP at 48, 24 and 4hours before the final allergen challenge. In the Th2 model, ear-swelling response and scratching behavior were promoted by BaP exposure, which supported the observed significant increases in local cytokine secretion. The infiltration of helper T cells, B cells and dendritic cells into the auricular lymph node was significantly enhanced by BaP administration, although Th1-type immune responses were not influenced by AhR activation. Our findings demonstrate that AhR activation directly activates keratinocytes and T cells, which leads to the exacerbation of Th2-type cutaneous allergy.

Abstract 2730: Preclinical characterization of the anti-PD-L1 monoclonal antibody LY3300054

Abstract Modulating the PD-L1/PD-1 axis in man through function blocking antibodies can release potent anti-tumor immunity, leading to durable objective responses across multiple tumor types. Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ antibody capable of binding human PD-L1 with high affinity and inhibiting binding to its two cognate receptors, PD-1 and CD80. LY3300054 is an antagonist monoclonal antibody recognizing human PD-L1 with high affinity (KD 0.08 nM), selected and derived from a scFv phage library. LY3300054 was engineered and expressed as an IgG1-Fc null monoclonal antibody to ablate immune effector function. Multiple in vitro assays, including mixed leukocyte reaction (MLR) and tetanus toxoid recall assay were utilized to demonstrate LY3300054 potent functional activity in enhancing the activation of primary human T cells in culture. The biological activity of LY3300054 on T cells was also shown to be enhanced by co-administration of anti-CTLA4 mAb (ipilimumab) in MLR. Further, we evaluated LY3300054 activity in vivo using xenograft mouse tumor models reconstituted with human immune cells. LY3300054 demonstrated anti-tumor activity in both NCI-H292 xenografts co-implanted with human PBMCs and HCC827 xenograft model upon infusion of human T cells. LY3300054 in vivo activity was also tested in HCC827- and Ov79-bearing immunodeficient NSG/NOG mice reconstituted with human hematopoietic stem cells. In this setting, LY3300054 displayed robust anti-tumor and immunomodulatory effects exemplified by T cell inflamed phenotype in the tumor and peripheral tissues. High-content molecular profiling identified distinct gene expression changes indicative of T cell activation in all models tested. A mutational strategy based on integrating the PD-1/PD-L1 structure data with the orthologous sequence data of PD-L1 has identified a residue on PD-L1, which is part of the PD-L1/PD-1 interface, that is critical for the species specificity of LY3300054. This study demonstrates that LY3300054 is novel anti-PD-L1 monoclonal antibody, capable of potently enhancing human T cell function both in vitro and in vivo, and provides previously not described insights into the effects of PD-L1 blockade on the intra- and extra-tumoral immune response. LY3300054 is currently under clinical evaluation in monotherapy and combination with other therapeutic modalities in multiple tumor types (NCT02791334; NCT03099109; NCT02791334; NCT02791334) Citation Format: Carmine Carpenito, Yiwen Li, George X. Wang, Maria S. Malabunga, Jaafar N. Haidar, Amelie Forest, Mary Y. Murphy, Gerald E. Hall, Cindy Wang, Leyi Shen, Andreas Sonyi, Darin Chin, Anthony L. Pennello, Ivan V. Inigo, David Surguladze, Yung-mae Yao, Douglas Burtrum, Ruslan D. Novosiadly, Kris Persaud, Dale L. Ludwig, Michael D. Kalos. Preclinical characterization of the anti-PD-L1 monoclonal antibody LY3300054 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2730.

Early passaging of mesenchymal stem cells does not instigate significant modifications in their immunological behavior

BackgroundBone marrow-derived allogeneic mesenchymal stem cells (MSCs) from young healthy donors are immunoprivileged and their clinical application for regenerative medicine is under evaluation. However, data from preclinical and initial clinical trials indicate that allogeneic MSCs after transplantation provoke a host immune response and are rejected. In the current study, we evaluated the effect of an increase in passage number in cell culture on immunoprivilege of the MSCs. Since only limited numbers of MSCs can be sourced at a time from a donor, it is imperative to expand them in culture to meet the necessary numbers required for cell therapy. Presently, the most commonly used passages for transplantation include passages (P)3–7. Therefore, in this study we included clinically relevant passages, i.e., P3, P5, and P7, for evaluation.MethodsThe immunoprivilege of MSCs was assessed with the mixed leukocyte reaction assay, where rat MSCs were cocultured with peripheral blood leukocytes for 72 h. Leukocyte-mediated cytotoxicity, apoptosis (Bax/Bcl-xl ratio), leukocyte proliferation, and alterations in cellular bioenergetics in MSCs were assessed after the coculture. Furthermore, the expression of various oxidized phospholipids (oxidized phosphatidylcholine (ox-PC)) was analyzed in MSCs using a lipidomic platform. To determine if the ox-PCs were acting in tandem with downstream intracellular protein alterations, we performed proteome analysis using a liquid chromatography/mass spectrometry (LC/MS) proteomic platform.ResultsOur data demonstrate that MSCs were immunoprivileged at all three passages since coculture with leukocytes did not affect the survival of MSCs at P3, P5, and P7. We also found that, with an increase in the passage number of MSCs, leukocytes did not cause any significant effect on cellular bioenergetics (basal respiration rate, spare respiratory capacity, maximal respiration, and coupling efficiency). Interestingly, in our omics data, we detected alterations in some of the ox-PCs and proteins in MSCs at different passages; however, these changes were not significant enough to affect their immunoprivilege.ConclusionsThe outcome of this study demonstrates that an increase in passage number (from P3 to P7) in the cell culture does not have any significant effect on the immunoprivilege of MSCs.

Abstract 36: Application of “Omics” to Assess the Inter Passage Variability in the Immunoprivilege of Mesenchymal Stem Cells

Introduction: Bone marrow derived allogeneic mesenchymal stem cells (MSCs) from young healthy donors are immunoprivileged. However recent data from preclinical and clinical studies indicate that allogeneic MSCs provoke immune response in the recipient heart and are rejected after transplantation. We recently observed that stem cell surface oxidized phospholipids (ox-PCs) modulate allogeneic immune cell response. In most of the allogeneic MSCs based clinical trials in cardiac patients, cells from a single donor were expanded in cultures and transplanted. However, the effect of increase in the passage number on stem cell membrane lipidome and its association with immunogenicity of MSCs has not been investigated yet, the present study was carried out to address this Methods: MSCs isolated from Wistar rats were allowed to grow for different passages namely Passage 3, 5 and 7 (P 3, 5 and 7). The immunoprivilege of MSCs was assessed by mixed leukocyte reaction assay. Briefly, MSCs were co-cultured with mixed leukocytes derived from SD rats for 72 h. Leukocyte mediated cytotoxicity (lactate dehydrogenase release) and apoptosis (Bax/Bcl-xl ratio) in MSCs was assessed after the co-culture. Furthermore, MSCs at different passages were analyzed by LC/MS/MS -“lipidomic platform” for cell surface ox-PCs levels and “proteomic platform” for intracellular proteins. Results and conclusions: We observed a decrease in lymphocyte mediated cytotoxicity and Bax/ Bcl-xl ratio in MSCs at P5 and7 compared to P3. The lipidome analysis showed that the levels of immunomodulatory ox-PCs increased in MSCs at P5 and P7. Inhibition of ox-PCs was associated with the loss of immunoprivilege of MSCs. To find if the ox-PCs were acting in tandem with downstream intracellular protein alterations, we performed proteome analysis. There were significant changes in proteins related to intracellular immune response, lipid metabolism and mitochondrial dynamics. In depth analysis of the ‘omics’ data is now going on to pinpoint associations between cell surface ox-PCs and the candidate intracellular proteins involved in immune modulation. To the best of our knowledge this is the first study integrating membrane lipidomic with intracellular proteins to assess immunogenicity of MSCs.

Impaired CD98 signaling protects against graft-versus-host disease by increasing regulatory T cells

Graft-versus-host disease (GvHD) is a major barrier to the broader use of allogenic hematopoietic stem cell transplantation for non-malignant clinical applications. A murine model of C57BL/6 to B6D2F1 acute GvHD was employed with T lymphocytes harboring a deletion of the CD98 heavy chain (CD98hc−/−) as donor cells. The CD98hc−/− resulted in lower responses to alloantigen stimulation in a mixed leukocyte reaction assay, and prevented the mortality associated with disease progression. The percentage of donor CD8 T lymphocytes was significantly decreased, while the percentage of Foxp3-positive regulatory T cells (Tregs) in recipients was increased by CD98hc−/−. Decreased expression of FAS, FASL, ICOS, ICOSL, PD-1 and PD-L1 by donor CD8 T cells, and mRNA expression of cytotoxic T cell-related cytokines in the recipients were shown in those with CD98hc−/−. Fewer infiltrated cells are found in the lungs, liver, tongue and skin of recipients with CD98hc−/− compared with the wild type recipients. Taken together, our data indicate that T cell-specific deletion of CD98hc can contribute to the prevention of GvHD development due to the attenuation of lymphocyte migration and by increasing the generation of Treg cells. These findings are expected to make it possible to develop novel approaches for the prevention of GvHD.

Abstract 272: Checkpoint inhibitor combinations in a human mixed leukocyte reaction

Abstract In an effort to identify effective target combinations, molecules specific for several immuno-oncology targets were combined in vitro using a mixed leukocyte reaction (MLR) assay. Briefly, 20 different healthy human peripheral blood mononuclear cell donor pairs were tested for proliferation and cytokine release in the presence of each molecule alone and in combination. The collection of agents targeted CTLA-4, PD-1, PD-L1, and CD73. Examples of significant (p<0.05) synergy as well as antagonism and additivity were observed. In addition the phenotype and genotype of the MLR donor PBMCs were characterized. The results from this study are significant for at least two reasons: 1) In contrast to murine syngeneic models they represent aspects of multiple different human donor “immune responses” and 2) In contrast to purified T-cell assays they represent an assay system in which both the lymphoid and myeloid/innate immune compartments play a crucial role, and so better recapitulate a physiological T-cell response. Furthermore, these results provide information at the human population level, offering a glimpse of “responder” and “non-responder” population profiles. This information is impossible to obtain from the currently available collection of syngeneic mouse models. Citation Format: Erin Sult, Carl Hay, Qihui Huang, Stacy Fuhrmann, Robert Hollingsworth, Kris F. Sachsenmeier. Checkpoint inhibitor combinations in a human mixed leukocyte reaction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 272. doi:10.1158/1538-7445.AM2015-272

Key Immune Cell Subsets Are Dysregulated in Patients with Myelofibrosis

Introduction: Myelofibrosis (MF) is a clonal disorder associated mainly with JAK2V617F and MPL mutations. Recently, a new mutation in the gene encoding calreticulin (CALR) was discovered in the majority of JAK2/MPL negative patients. MF is burdened by a high rate of potentially life-threatening infections. The issue of recurrent and opportunistic infections is increased after the introduction in clinical practice of JAK inhibitors with immunosuppressive activity. However, the role of crucial immune cell subsets is still poorly characterized. Here, we investigated the phenotype/function of selected immune cells in MF. Specifically, we focused on circulating regulatory (Tregs) and IL-17-producing T cells (Th17 cells), monocytes and dendritic cells (DCs). Monocyte-derived DCs were also characterized.Methods: We characterized circulating Th17 cells, Tregs, monocytes and DCs of 17 untreated MF patients and 8 healthy controls (HC) by flow cytometry. Th17 cells were identified as CD4+ CD161+ CD196+ cells while Tregs were enumerated as CD4+ CD25high CD127low T cells. We also tested the in vitro suppressive activity of circulating CD4+ CD25+ Tregs with a mixed leukocyte reaction assay. Two subpopulations of circulating DCs, myeloid CD11c+ and plasmacytoid CD123+cells, were enumerated as well. In addition, after immunomagnetic selection, we tested both phenotype of circulating monocytes and their capacity to differentiate into CD14-derived immature and mature DCs, using a specific cytokines cocktail. JAK2V617F and MPL mutations were detected with RT-PCR while the presence of CALR mutations were tested with Exon 9 Next Generation Sequencing assay.Results: JAK2V617F (11 cases), MPL (3 cases), and CARL (3 cases) mutations were detected. We found that circulating CD4+CD25highCD127low Tregs were reduced in MF patients as compared with healthy controls (p=0.043), although their suppressive ability was maintained. We also found a lower number of circulating Th17 cells (p=0.0026) in MF patients. This finding was particularly evident in JAK2V617F+(p=0.008) and CARL+(p=0.03) patients. Despite their number was in the normal range, circulating monocytes from MF patients showed reduced expression of the CD86 co-stimulatory molecule. Moreover, as compared with the normal counterparts, immature monocytes-derived DCs from patients maintained low CD14 expression without upregulating the CD80 co-stimulatory molecule expression (p=0.0063). Interestingly, at variance with plasmacytoid DCs, a reduced number of circulating myeloid DCs was observed in MF patients as compared with that of HC (p=0.01).Conclusions: Here we demonstrated that specific crucial subsets of immune cells show quantitative and/or qualitative abnormalities in MF patients. These findings may be useful to better understand the increased susceptibility of these patients to infections, since Th17 cells play a role in bacterial and fungal infections while myeloid DCs regulate Th1 activity. Of note, DCs inhibition might result in increased propensity to infections and compromised immune response to cancer.In addition, since monocytes are DC precursors, alterations in their differentiation pathway may contribute to develop defective immune responses. DisclosuresMartinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Porcine Neonatal Blood Dendritic Cells, but Not Monocytes, Are More Responsive to TLRs Stimulation than Their Adult Counterparts

The neonatal immune system is often considered as immature or impaired compared to the adult immune system. This higher susceptibility to infections is partly due to the skewing of the neonatal immune response towards a Th2 response. Activation and maturation of dendritic cells (DCs) play an important role in shaping the immune response, therefore, DCs are a target of choice for the development of efficient and protective vaccine formulations able to redirect the neonatal immune response to a protective Th1 response. As pigs are becoming more important for vaccine development studies due to their similarity to the human immune system, we decided to compare the activation and maturation of a subpopulation of porcine DCs in adult and neonatal pigs following stimulation with different TLR ligands, which are promising candidates for adjuvants in vaccine formulations. Porcine blood derived DCs (BDCs) were directly isolated from blood and consisted of a mix of conventional and plasmacytoid DCs. Following CpG ODN (TLR9 ligand) and imiquimod (TLR7 ligand) stimulation, neonatal BDCs showed higher levels of expression of costimulatory molecules and similar (CpG ODN) or higher (imiquimod) levels of IL-12 compared to adult BDCs. Another interesting feature was that only neonatal BDCs produced IFN-α after TLR7 or TLR9 ligand stimulation. Stimulation with CpG ODN and imiquimod also induced enhanced expression of several chemokines. Moreover, in a mixed leukocyte reaction assay, neonatal BDCs displayed a greater ability to induce lymphoproliferation. These findings suggest that when stimulated via TLR7 or TLR9 porcine DCs display similar if not better response than adult porcine DCs.

5-Aminolevulinic acid induces permanent acceptance of murine cardiac allografts via induction of regulator dendritic cells and expansion of alloantigen-specific Tregs. (P2176)

Abstract 5-aminolevulinic acid (5-ALA), an intermediate in heme synthesis is fundamentally important in aerobic energy metabolism. Hemeoxygenase-1 (HO-1) cleaves heme to form biliverdin, carbon monoxide (CO) and iron (Fe2+), and the pathway is accelerated by 5-ALA. In the present study, we examined the effects of 5-ALA on the maturation and function of dendritic cells (DCs), a potent class of APCs, and the downstream cell-cell mediating immunosuppressive activity. 5-ALA inhibited LPS-stimulated maturation of murine bone marrow DCs in vitro as reflected by reduced expression of MHC class-II (I-A/I-E), co-stimulatory molecules (CD80, CD40) and proinflammatory cytokines, IFN-g, IL-12 mRNA expression. In mixed cultures, DCs pretreated with 5-ALA impaired the proliferation of allogeneic T cells while promoting the expansion of CD4+CD25+ regulatory T cells (Tregs). Using an in vitro mixed leukocyte reaction (MLR) assay system, we found that the DCs pretreated with 5-ALA have more regulatory function to suppress the both CD4+ and CD8+ T cells proliferation. Furthermore, 5-ALA treatment impaired T cells proliferation in response to alloantigens and increased Tregs, resulting in permanent acceptance of murine cardiac allografts. Thus we conclude that 5-ALA triggers a tolerogenic state in DCs that allows increasing alloantigen-specific Tregs and the suppression of allograft rejection in vivo.

Role of Regulatory T Cells in Transferable Immunological Tolerance to Bone Marrow Donor in Murine Mixed Chimerism Model

Constructing a bone marrow chimera prior to graft transplantation can induce donor-specific immune tolerance. Mixed chimerism containing hematopoietic cells of both recipient- and donor-origin has advantages attributed from low dose of total body irradiation. In this study, we explored the mechanism of mixed chimerism supplemented with depletion of Natural Killer cells. Mixed chimerism with C57BL/6 bone marrow cells was induced in recipient BALB/c mice which were given 450 cGy of γ-ray irradiation (n = 16). As revealed by reduced proliferation and cytokine production in mixed leukocyte reaction and ELISpot assay (24.6 vs 265.5), the allo-immune response to bone marrow donor was reduced. Furthermore, the induction of transferable immunological tolerance was confirmed by adoptive transfer and subsequent acceptance of C57BL/6 skin graft (n = 4). CD4+FoxP3+ regulatory T cells were increased in the recipient compartment of the mixed chimera (19.2% → 33.8%). This suggests that regulatory T cells may be therapeutically used for the induction of graft-specific tolerance by mixed chimerism.

Deletion of CD98 Heavy Chain in T Cells Results in Cardiac Allograft Acceptance by Increasing Regulatory T Cells

Little is known about the CD98 heavy chain (CD98hc) in the T lymphocyte-mediated immune response to alloantigen. We used an in vitro mixed leukocyte reaction assay and a cardiac transplantation model to evaluate the mechanisms of CD98hc in regulating alloimmune responses. A T cell-specific deficiency of CD98hc resulted in lower responses to alloantigen stimulation in a mixed leukocyte reaction assay, and CD98hc-deficient mice accepted full major histocompatibility complex-mismatched cardiac allografts. Consistent with graft survival, the infiltration of the graft by immune cells in CD98hc-deficient mice was significantly lower than that in wild-type mice. A chemotaxis assay revealed the migration of CD98hc-deficient lymphocytes to decrease in the presence of CCL5 compared with wild-type cells. Moreover, the proportion of CD4/Foxp3-positive cells and Foxp3 messenger RNA increased significantly in CD98hc-deficient recipients, consistent with the down-regulation of mammalian target of rapamycin and PS6 kinase; and allograft permanent acceptance was shortened by the depletion of antibody-induced regulatory T cells. Finally, neutralizing antibody against CD98hc prolonged the cardiac allograft survival. Taken together, our data indicate that T cell-specific deficiency in CD98hc can contribute to cardiac allograft permanent acceptance correlating with the attenuation of lymphocyte migration and by increasing the generation of regulatory T cells. These findings are expected to make it possible to develop novel approaches for treating allograft rejection and promoting transplantation tolerance.

Characterization of Human Umbilical Cord Lining-Derived Epithelial Cells and Transplantation Potential

In this study we describe the derivation and immunological characterization of a primary epithelial cell type from the human umbilical cord membrane. These cord lining epithelial cells (CLECs) expressed and/or secreted isoforms of the nonclassical human leukocyte antigen class I (HLA-1b) glycoproteins, HLA-G and E. Conditioned media from CLECs inhibited mitogen-stimulated T-lymphocyte responses, and in a mixed leukocyte reaction (MLR) assay, cocultured CLECs inhibited allogeneic responses with a concomitant reduction in proinflammatory cytokines. Using a transwell coculture system, it was demonstrated that these immunoregulatory effects were mediated by soluble factors secreted by CLECs, in a dose-dependent manner. Functional studies using HLA-G blocking antibody showed that the effects of CLEC-secreted products could be inhibited, thus demonstrating a significant and important role for soluble HLA-G. In vivo, we show that transplanted CLECs could be maintained for extended periods in immunocompetent mice where xenorejection rapidly destroyed primary keratinocytes, a control human epithelial cell type. Additionally, CLECs delayed the rejection of keratinocytes and extended their survival when cotransplanted, indicating an ability to protect adjacent human cell types that would otherwise be rejected if transplanted alone. We also show that CLECs transduced with a modified human proinsulin gene were transplanted intraperitoneally into streptozotocin (STZ)-induced diabetic mice, resulting in significantly lower levels of serum glucose compared to control mice. This study has characterized the immunological properties of CLECs and tested a potential therapeutic application in the treatment of a type 1 diabetes mouse model.

Evidence that FoxP3+ regulatory T cells may play a role in promoting long-term acceptance of composite tissue allotransplants.

FoxP3/CD4/CD25 regulatory T cells (Treg) play an important role in maintaining peripheral tolerance and are potent suppressors of T-cell activation. In this study, we evaluated the role of Treg in peripheral tolerance to composite tissue allografts (CTA). Mixed allogeneic chimeric rats were prepared by preconditioning recipients with anti-αβ-T-cell receptor monoclonal antibody followed by total body irradiation. Animals received T-cell-depleted August Copenhagen Irish bone marrow cells followed by antilymphocyte serum and FK-506. A modified osteomyocutaneous hindlimb flap composed of bone and all limb tissue components was placed in animals with chimerism greater than or equal to 1% on day 28. Recipients with CTA surviving more than or equal to 6 months were evaluated for Treg. Skin samples from tolerant long-term allogeneic transplanted, syngeneic transplanted, rejected, and naïve animals were immunostained with fluorochrome-conjugated anti-FoxP3 and anti-CD4 monoclonal antibody and visualized under a laser confocal microscope. Significant CD4/FoxP3 Treg infiltrates were observed in tolerant donor-allograft skin samples. No graft infiltrating FoxP3 cells were observed in rejector, naïve, or skin from syngeneic CTA. In parallel experiments, mixed leukocyte reaction assays were performed to investigate the suppressor function of Treg cells. Splenocytes from tolerant, rejected, and naïve rats were sorted by flow cytometry for CD4/CD25 T cells. Treg demonstrated similar suppressive levels between the three groups. These data suggest that Treg may play an important role in maintenance of tolerance and promoting graft acceptance in long-term CTA acceptors and may explain the favorable outcomes observed in clinical CTA recipients.