Mixed Leukocyte Reaction Research Articles

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.

Cetuximab ameliorates suppressive phenotypes of myeloid antigen presenting cells in head and neck cancer patients.

BackgroundMyeloid-derived suppressor cells (MDSC) and M2 monocytes/macrophages are two types of suppressive myeloid antigen presenting cells that have been shown to promote tumor progression and correlate with poor prognosis in cancer patients. Tumor antigen specific monoclonal antibodies (mAb) have emerged as important agents for cancer therapy. In addition to the direct inhibition of tumor growth, the Fc portions of the therapeutic mAbs, such as the IgG1 portion of the anti-epidermal growth factor receptor (EGFR) mAb cetuximab, might interact with the Fc-gamma receptors (FcγR) on myeloid cells and modulate their suppressive activity.MethodsPatients with locally advanced head and neck squamous cell carcinoma (HNSCC) on the UPCI 08–013 NCT01218048 trial were treated with single-agent cetuximab before surgery. Blood were collected pre- and post-cetuximab treatment to analyze frequency of monocytic MDSC (CD11b+CD14+HLA-DRlo/-), granulocytic MDSC (LIN−CD11b+CD15+) and CD11b+CD14+HLA-DRhi monocytes by flow cytometry. Besides, CD11b+CD14+HLA-DRhi monocytes were sorted for qPCR analysis of IL-10 and IL-12B transcripts. MDSC were generated in vitro with or without coated hIgG1 and tested for suppressive activity in mixed leukocyte reaction (MLR). Naïve monocytes from HNSCC patients co-cultured with tumor cell lines in the presence of cetuximab or hIgG1 were analyzed for M1/2 surface markers and cytokines.ResultsWe observed significantly increased monocytic MDSC in non-responders and decreased granulocytic MDSC in responders after cetuximab treatment. In addition, circulating CD11b+CD14+HLA-DRhi monocytes of cetuximab responders displayed attenuated M2 polarization, with decreased CD163+ expression and IL-10 transcripts after cetuximab treatment. This beneficial effect appeared to be FcγR dependent, since CD16 ligation reproduced the reversal of suppressive activity of MDSC in vitro. CD14+ naïve monocytes from the co-cultures of tumor cells, cetuximab and HNSCC patient PBMC or purified monocytes were skewed to an M1-like phenotype, with increased expression of HLA-DR, CD86 and production of IL-12 p70. Likewise, reduced M2 features (expression of CD163 and production of IL-10) were found after crosslinking CD16 on the surface of monocytes to cetuximab-coated tumor cells.ConclusionOur studies demonstrate a novel function of cetuximab in ameliorating suppressive phenotypes of FcγR bearing myeloid cells in cancer patients, which is associated with better clinical outcome of cetuximab-treated patients.Clinical trial registry#NCT01218048. Registered 7 October 2010.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-015-0097-6) contains supplementary material, which is available to authorized users.

Placenta-derived mesenchymal stem cells possess better immunoregulatory properties compared to their cord-derived counterparts-a paired sample study.

Mesenchymal stem cells (MSCs) show immunoregulatory properties. Here, we compared MSCs obtained from placenta (P-MSCs) and umbilical cord (C-MSCs) from the same donor, for their immunomodulatory efficacy. P-MSCs and C-MSCs showed similar morphology and phenotypic profile, but different clonogenic ability. Importantly, they showed a significant difference in their immunosuppressive properties as assessed in mixed leukocyte reaction (MLR). The P-MSCs affected the antigen presenting ability of mononuclear cells (MNCs) and dendritic cells (DCs) significantly as compared to C-MSCs resulting in a reduced T-cell proliferation. P-MSC conditioned medium (CM) showed a significant reduction in T cell proliferation as compared to C-MSC CM, thus suggesting that a cell to cell contact is not essential. We found increased levels of IL-10 and TGFβ1 and reduction in levels of IFNγ in P-MSC MLRs as compared to C-MSC MLRs. Furthermore, the CD3+ CD4+ CD25+ T regulatory cells were enriched in case of P-MSCs in both, MSC-MNC and MSC-DC co-cultures. This observation was further supported by increased mRNA expression of FoxP3 in P-MSCs. Presently, cord-derived MSCs are being employed in transplantation therapies parallel to the bone marrow-derived MSCs. Our findings suggest that P-MSCs can be a better alternative to C-MSCs, to provide aid in immunological ailments.

Activated T cells enhance interferon-α production by plasmacytoid dendritic cells stimulated with RNA-containing immune complexes

ObjectivesPatients with systemic lupus erythematosus (SLE) have an ongoing interferon-α (IFN-α) production by plasmacytoid dendritic cells (pDCs). We investigated whether T cells can promote IFN-α production by pDCs.MethodsHuman pDCs were...

IL-10 restricts dendritic cell (DC) growth at the monocyte-to-monocyte-derived DC interface by disrupting anti-apoptotic and cytoprotective autophagic molecular machinery.

An evolving premise is that cytoprotective autophagy responses are essential to monocyte-macrophage differentiation. Whether autophagy functions similarly during the monocyte-to-dendritic cell (DC) transition is unclear. IL-10, which induces apoptosis in maturing human DCs, has been shown to inhibit starvation-induced autophagy in murine macrophage cell lines. Based on the strict requirement that Bcl-2-mediated anti-apoptotic processes are implemented during the monocyte-to-DC transition, we hypothesized that cytoprotective autophagy responses also operate at the monocyte-DC interface and that IL-10 inhibits both anti-apoptotic and cytoprotective autophagy responses at this critical juncture. In support of our premise, we show that levels of anti-apoptotic Bcl-2 and autophagy-associated LC3 and Beclin-1 proteins are coincidentally upregulated during the monocyte-to-DC transition. Autophagy was substantiated by increased autophagosome visualization after bafilomycin treatment. Moreover, the autophagy inhibitor 3-MA restricted DC differentiation by prompting apoptosis. IL-10 implemented apoptosis that was coincidentally associated with reduced levels of Bcl-2 and widespread disruption of the autophagic flux. During peak apoptosis, IL-10 produced the death of newly committed DCs. However, cells surviving the IL-10 apoptotic schedule were highly phagocytic macrophage-like cells displaying reduced capacity to stimulate allogeneic naïve T cells in a mixed leukocyte reaction, increased levels of LC3, and mature autophagosomes. Thus, IL-10's negative control of DC-driven adaptive immunity at the monocyte-DC interface includes disruption of coordinately regulated molecular networks involved in pro-survival autophagy and anti-apoptotic responses.

Rapamycin-conditioned dendritic cells induced immune tolerance through the regulation of Treg/Th17 cells in mice

To investigate the effect of tolerogenic dendritic cells (Tol-DC) generated by Rapamycin (Rapa) on the differentiation of Treg/Th17 cells and explore the possible mechanism of tolerance induction. DC progenitors from mouse bone marrow were propagated with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin (IL)-4 stimulation for 6 days in the presence or absence of Rapa (20 ng/ml). During DC culture, morphology of cell was observed under electron microscope. Cell surface expression of CD11c, CD40 and CD80 was analyzed by flow cytometry. The antigen-presenting function of DC was determined by one-way mixed leukocyte reactions. In vivo, the recipient BALB/c mice receiving transplantation of skin allograft from C57BL/6 mice were divided into control, Rapa, immature DC (imDC) and Tol-DC group. The survival time of the skin allograft was observed and Treg/Th17 cells were analyzed by flow cytometry in each group. The immunephenotypic analysis showed that in comparison with those in the control group and the LPS group the expression of the co-stimulatory molecules CD40 and CD80 were significantly lower in the Rapa-group and Rapa+LPS group. The ability to stimulate proliferation of T cells of the same genotype in the Rapa-group was significantly inhibited (P<0.01). In the in vivo experiment, the mice's survival time remarkably prolonged, the percentage of Treg cells was enhanced and Th17 cells was reduced in the mice's spleen in Tol-DC group. Tol-DC generated by Rapamycin can significantly induce immune tolerance through up-regulate Tregs and down-regulate Th17 cells. The present study highlights the therapeutic potential of preventing allograft rejection using in vitro-generated Tol-DCs, which can be loaded with donor antigen, and potentially used to promote organ transplant tolerance.

Phenotypic and functional analysis of CD1a+ dendritic cells from cats chronically infected with feline immunodeficiency virus.

Numerous studies suggest dendritic cell (DC) dysfunction is central to the dysregulated immune response during HIV infection; however, in vivo studies are lacking. In the present study we used feline immunodeficiency virus (FIV) infection of cats as a model for HIV-1 infection to assess the maturation and function of dendritic cells, in vivo and in vitro. We compared CD1a+ DC migration, surface phenotype, endocytosis, mixed leukocyte reaction (MLR) and regulatory T cell (Treg) phenotype induction by CD1a+ cells isolated from lymph nodes of FIV-infected and control cats. Results showed that resident CD1a+ DC in lymph nodes of chronically FIV-infected cats are phenotypically mature, can stimulate normal primary T cell proliferation, override Treg suppression and do not skew toward Treg induction. In contrast, FIV infection had deleterious effects on antigen presentation and migratory capacity of CD1a+ cells in tissues.

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&amp;lt;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

Alloantigen-specific T-cell hyporesponsiveness induced by dnIKK2 gene-transfected recipient immature dendritic cells

Immature dendritic cells (iDCs) have been shown to be able to induce peripheral T-cell tolerance through distinct pathways. Here, we investigated the tolerogenic property of recipient iDCs whose maturation was arrested by a dominant negative mutant of inhibitor of nuclear factor kappa-B kinase 2 (dnIKK2) gene. We found that dnIKK2-iDCs presented a typical semi-mature morphology and expressed lower levels of CD80 and CD86, slightly higher MHC-II than untransfected iDCs. The expression of these molecules had no significant change even dnIKK2-iDCs were pulsed by donor antigen. In primary mixed leukocyte reaction (MLR), dnIKK2-iDCs exhibited impaired ability to stimulate allogeneic T-cells, but induced CD4+CD25− T-cell formation. In co-culture MLR, these CD4+CD25− T-cells suppressed T-cell alloreaction in an antigen-specific manner. Besides, CD4+CD25− T-cells inhibited IL-2 and IFN-γ release, whereas promoted IL-10 and TGF-β secretion. These data suggested recipient dnIKK2-iDCs could maintain peripheral tolerance through down-regulating costimulatory molecule expressions and inducing CD4+CD25− T-cell formation.

Abstract 3184: Tumor-entrained dendritic cells promote ICOS/ICOSL-dependent Th17-like responses in pancreatic adenocarcinoma

Abstract BACKGROUND: Ranking 4th among cancer-related deaths worldwide, pancreatic adenocarcinoma (PDAC) boasts a dismal prognosis. The robust immune infiltrate that comprises its tumor microenvironment (TME) can influence disease progression. Tumor rejection by T lymphocytes relies on proper guidance by dendritic cells (DCs). This antigen-education is manifested by the DCs’ expression of co-stimulatory factors, such as ICOS-ligand (ICOSL). In this study, we plan to elucidate the mechanisms used by PDAC-entrained DC to modify T cell effector function and differentiation. METHODS: To establish our in vivo model, either PBS or 2.5e+5 “FC1242” murine PDAC cells derived from the tumor of a KPC mouse (LSL-KrasG12D; Trp53R172H; pdxCre/+) were injected into the distal pancreata of C57BL/6J (WT) mice. After 20 days, mice were euthanized and the spleen and tumor were assayed. DC phenotype was determined by flow cytometry (FC). DC function was assessed by mixed-leukocyte reactions (MLR) and peptide-specific proliferation assays, +/- neutralizing mAbs against ICOSL or ICOS. Supernatant cytokines were assessed at 84h using cytometric bead assays (CBA). Intracellular cytokines were analyzed by FC after 5h re-stimulation with PMA/IMN. To assess in vivo proliferation, 2.5e+6 of both OT1 and OT2 T cells labeled with CFSE or CPD, respectively, were co-injected into WT mice and challenged with 5e+5 OVA-loaded DC after 24h. Proliferation was assessed 4 days post-DC inoculation. RESULTS: Splenic FC1242-DC had lower expression of MHC2 and CD83, while tumor-infiltrating DC (TME-DC) displayed higher levels of each, relative to controls. TME-DC also exhibited higher expression of ICOSL, CD80, and CD86. Despite no difference in ICOSL expression between splenic groups, MHC2 and CD83 were substantially lower on ICOSL+ DC of FC1242 spleen, while highest on that of the TME, relative to controls. We found a higher fraction of CD11b+ myeloid DC (mDC) and a lower fraction of cross-presenting CD8α+ DC (xDC) in both tumor-bearing groups relative to controls. FC1242-mDC expressed higher CD80 and PDL1 than sham-mDC, while FC1242-xDC displayed lower CD80 and no difference in PDL1. Also, tumor-entrained DC had a diminished propensity for CD8+ T cell expansion in vitro and in vivo, resulting in lower Granzyme B, IFNγ, and TNFα. Interestingly, DC tumor-entrainment improved CD4+ T cell expansion and augmented IL-17A, IL-6, G-CSF secretion, while diminishing IFNγ, TNFα, IL-13, and IL-2. Blockade of the ICOS-pathway hindered IL17 production, and amplified production of IFNγ and TNFα by T cells. CONCLUSION: This study suggests that DC possess a vital role as liaisons between the TME and cell-mediated immunity. Furthermore, Th17 cells in the TME can accelerate PDAC progression. This makes the ICOS-pathway especially attractive, as it can modify anti-tumor immunity while circumventing the barriers of drug-delivery to the TME. Citation Format: Rocky M. Barilla, Raul Caso, Antonina Avanzi, Anjlee Panjwani, Xiaopei L. Zeng, Steve Matthews, Daniel M. Tippens, Lena Tomkoetter, Elliot M. Levie, Alejandro Torres-Hernandez, Donnele Daley, George Miller. Tumor-entrained dendritic cells promote ICOS/ICOSL-dependent Th17-like responses in pancreatic adenocarcinoma. [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 3184. doi:10.1158/1538-7445.AM2015-3184

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface.

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (>70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

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.

MP55-17 EFFECT OF EPIGENETIC MODIFICATION AND IMMUNOMODULATION ON MURINE PROSTATE CANCER AND DENDRITIC CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research III1 Apr 2015MP55-17 EFFECT OF EPIGENETIC MODIFICATION AND IMMUNOMODULATION ON MURINE PROSTATE CANCER AND DENDRITIC CELLS Jay Sulek, Shaoqing Zhou, Albert Petrossian, Samuel Robinson, Ekaterine Goliadze, and Georgi Guruli Jay SulekJay Sulek More articles by this author , Shaoqing ZhouShaoqing Zhou More articles by this author , Albert PetrossianAlbert Petrossian More articles by this author , Samuel RobinsonSamuel Robinson More articles by this author , Ekaterine GoliadzeEkaterine Goliadze More articles by this author , and Georgi GuruliGeorgi Guruli More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2060AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Decreased expression of highly immunogenic cancer-testis antigens (CTA) might help tumor to achieve low immunogenicity, escape immune surveillance and grow unimpeded. Our aim was to evaluate CTA expression in tumor and normal tissues and to investigate possible means of improving the immune response in a murine prostate cancer (CaP) model by using the combination of epigenetic modifier 5-azacitidine (5-AzaC) and immunomodulator lenalidomide. No study to date has examined these agents in combination to treat prostate cancer or their combined impact on antigen-presenting cells (dendritic cells). METHODS Gene microarrays, quantitative PCR (qPCR) and Western blots were performed and the expressions of different CTA were compared between murine tumor and normal prostate. Effect of 5-AzaC and lenalidomide was assessed on murine CaP (RM-1 cells) and immunocompetent dendritic cells (DC). Flow cytometry, ELISA and mixed leukocyte reaction were performed to evaluate the combined effect of 5-AzaC and lenalidomide on dendritic cells. RESULTS Gene arrays and qPCR demonstrated decreased expression for most CTA-encoding genes in CaP tissue compared to normal prostate. CTA expression increased in a dose dependent fashion in RM-1 cells exposed to 5-AzaC. Quantitative PCR results were confirmed by Western blot. Flow cytometry demonstrated increased expression of co-stimulatory molecules (CD40, CD80, CD86, CD205) on DC after their treatment with 5-AzaC and lenalidomide (15%). Production of pro-inflammatory cytokines IL-12 and IL-15 by DC were increased as well. Mixed leukocyte reaction demonstrated increased ability of dendritic cells to stimulate T cells after their exposure to 5-AzaC. CONCLUSIONS Decreased expression of CTA by prostate cancer cells can be a means of escaping immune monitoring. Combination of epigenetic modification and immunomodulation by 5-AzaC and lenalidomide increased tumor immunogenicity and enhanced DC function at the same time. This combination merits further evaluation for a possible role in the treatment of advanced prostate cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e679 Peer Review Report Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jay Sulek More articles by this author Shaoqing Zhou More articles by this author Albert Petrossian More articles by this author Samuel Robinson More articles by this author Ekaterine Goliadze More articles by this author Georgi Guruli More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

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.

Exposure to Δ9-Tetrahydrocannabinol Impairs the Differentiation of Human Monocyte-derived Dendritic Cells and their Capacity for T cell Activation.

The capacity for human monocytes to differentiate into antigen-presenting dendritic cells (DC) can be influenced by a number of immune modulating signals. Monocytes express intracellular cannabinoid type 1 (CB1) and 2 (CB2) receptors and we demonstrate that exposure to Δ9-tetrahydrocannabinol (THC) inhibits the forskolin-induced generation of cyclic adenosine monophosphate in a CB2-specific manner. In order to examine the potential impact of cannabinoids on the generation of monocyte-derived DC, monocytes were cultured in vitro with differentiation medium alone [containing granulocyte/macrophage-colony stimulating factor (GM-CSF) and Interleukin-4 (IL-4)] or in combination with THC. The presence of THC (0.25-1.0μg/ml) altered key features of DC differentiation, producing a concentration-dependent decrease in surface expression of CD11c, HLA-DR and costimulatory molecules (CD40 and CD86), less effective antigen uptake, and signs of functional skewing with decreased production of IL-12 but normal levels of IL-10. When examined in a mixed leukocyte reaction, DC that had been generated in the presence of THC were poor T cell activators as evidenced by their inability to generate effector/memory T cells or to stimulate robust IFN-γ responses. Some of these effects were partially restored by exposure to exogenous IL-7 and bacterial superantigen (S. aureus Cowans strain). These studies demonstrate that human monocytes express functional cannabinoid receptors and suggest that exposure to THC can alter their differentiation into functional antigen presenting cells; an effect that may be counter-balanced by the presence of other immunoregulatory factors. The impact of cannabinoids on adaptive immune responses in individuals with frequent drug exposure remains to be determined.

DC-Based Immunotherapy Combined with Low-Dose Methotrexate Effective in the Treatment of Advanced CIA in Mice.

We have previously demonstrated that semimature dendritic cell- (smDC-) based immunotherapy is effective for the treatment of collagen-induced arthritis (CIA) prior to disease onset. In the present study, we examined the efficacy of combination therapy with smDCs and methotrexate (MTX) in advanced CIA with a score of 2-3. Combination therapy with low-dose MTX and type II collagen- (CII-) pulsed smDCs (CII-smDCs) was more effective in inhibiting disease progression than high or low-dose MTX alone or a combination of high dose MTX and CII-smDCs. The effect of CII-smDCs alone was also comparable to the combination therapy. CD4+Foxp3+ Treg populations and IL-10 secretion markedly increased, and CII-specific autoreactive T cells decreased in mice treated with CII-smDCs alone or in combination with MTX. Combination therapy reduced the secretion of interferon-γ (IFN-γ) and IL-17 with little influence on the IL-4 secretion in the mixed leukocyte reaction. These results imply that the combination therapy with low-dose MTX and smDCs is effective in controlling advanced CIA by enhancing Treg population and suppresses antigen-specific Th1/Th17 immunity, rather than initiating Th1 to Th2 immune deviation. Our findings provide a better understanding of the DC therapy in combination with MTX for the treatment of patients with rheumatoid arthritis (RA).

Immunosuppressive drugs affect high-mannose/hybrid N-glycans on human allostimulated leukocytes.

N-glycosylation plays an important role in the majority of physiological and pathological processes occurring in the immune system. Alteration of the type and abundance of glycans is an element of lymphocyte differentiation; it is also common in the development of immune-mediated inflammatory diseases. The N-glycosylation process is very sensitive to different environmental agents, among them the pharmacological environment of immunosuppressive drugs. Some results show that high-mannose oligosaccharides have the ability to suppress different stages of the immune response. We evaluated the effects of cyclosporin A (CsA) and rapamycin (Rapa) on high-mannose/hybrid-type glycosylation in human leukocytes activated in a two-way mixed leukocyte reaction (MLR). CsA significantly reduced the number of leukocytes covered by high-mannose/hybrid N-glycans, and the synergistic action of CsA and Rapa led to an increase of these structures on the remaining leukocytes. This is the first study indicating that β1 and β3 integrins bearing high-mannose/hybrid structures are affected by Rapa and CsA. Rapa taken separately and together with CsA changed the expression of β1 and β3 integrins and, by regulating the protein amount, increased the oligomannose/hybrid-type N-glycosylation on the leukocyte surface. We suggest that the changes in the glycosylation profile of leukocytes may promote the development of tolerance in transplantation.

Magnesium corrosion particles do not interfere with the immune function of primary human and murine macrophages.

Magnesium is currently under investigation as a prospective biodegradable implant material. Biodegradation of magnesium causes a release of magnesium, hydroxide ions and hydrogen gas but it can also lead to the formation of particulate debris. Implant-derived particles may have immunotoxic effects. To investigate the influence of magnesium-derived particles on the immune functions of primary macrophages, up to 500 μg/ml magnesium or magnesium corrosion particles were added to the cell culture medium. No major effects were observed on cell viability and on the release of the proinflammatory cytokine tumor necrosis factor (TNF)α. In addition, the ability of macrophages to stimulate proliferation of allogenic lymphocytes in a mixed leukocyte reaction remained unaffected. When macrophages were incubated with magnesium particles and then infected with the apathogenic Mycobacterium smegmatis, infection-induced TNFα secretion from murine macrophages was inhibited but not from human macrophages. However, the bactericidal activity of either cell type was not influenced. In conclusion, magnesium-related particles did not restrict the immune function of macrophages, suggesting that magnesium implants and corrosion particles derived thereof are highly biocompatible and have a low inflammatory potential.Electronic supplementary materialThe online version of this article (doi:10.1007/s40204-014-0032-9) contains supplementary material, which is available to authorized users.