Mixed Lymphocyte Culture Reaction Research Articles

Amelioration of graft versus host disease in humanized mice by exopolysaccharide from a commensal bacterium

Abstract Graft versus host disease (GVHD) is the major and often lethal complication of allogenic bone marrow transplantation. Prophylactic regimens for GVHD are given as standard pre-transplantation therapy; however, up to 60% of these patients develop GVHD, and require additional immunosuppressive intervention. We identified a probiotic exopolysaccharide (EPS) that induces inhibitory macrophages and tolerogenic dendritic cells (DCs) that inhibit T cell proliferation. We tested if EPS-treated human DCs, derived in vitrofrom CD34 +cord blood cells, would inhibit allogeneic T cell proliferation in a mixed lymphocyte reaction (MLR) and found that they did, demonstrating that EPS can induce tolerogenic DCs in vitro.We translated these studies in vivousing a humanized mouse model of aGvHD and demonstrated that EPS-treated human DCs administered at the time of transfer of human peripheral blood mononuclear cells (hPBMCs) significantly increased survival of these mice compared to mice given PBS-treated DCs, or no DCs. Several subsets of DCs have been described, e.g., cDCs, moDCs and plasmacytoid DCs, each with different phenotypes and functions. Using labeled EPS, we showed that the tolerogenic CD34-derived DCs generated by EPS treatment are conventional DCs (cDCs). We found that EPS binds to two cDC populations, conventional DC1 (cDC1) and conventional DC2 (cDC2), but only cDC1 were inhibitory in our MLR cultures. Our data indicate that EPS-DCs, potentially cDC1, can be used as a cell-based therapy to mitigate aGvHD in humans. NIH 1R41AI155281-01

Gene Expression Profiling and Signaling Pathway Analysis of Tolerant T Cells Circulating in Adult Patients after Ptcy Haplotransplantation

The mechanisms of T cell-dependent tolerance, especially its signaling pathways after haploidentical hematopoietic stem cell transplantation (haplo-HSCT), are incompletely understood. Both central and peripheral regulation mechanisms are suspected. Subjects and Methods: We tested 8 patients with leukemia (5) or myelodysplastic syndromes (MDS, 3) (age 35-73-years old, median: 51-years old) following IRB approved consenting. All received Flu+TBI (+/- Cy) pre-conditioning and haplo-HSCT with post-transplant Cytoxan (PTCy) and FK506 as GvHD prophylaxis. Chimerism at tolerance studies was donor dominant (97-100%, mean: 99% for both CD33+ myeloid cells and CD3+T cells). Immunosuppression drug (ISD) was withdrawn ~6 months before the time of the in vitro tolerance study described below. We described previously Treg depletion from purified T cells with IL2-immunotoxin (Denileukin Diftitox, Seragen Inc™) to test the involvement of these cells in long-term tolerance. The contribution of Tr1 or anergy was examined by either blocking IL10R or adding low dose IL2, respectively. T-cell exhaustion was tested by blocking the PD1 pathway with anti-PD1 MoAb (OPDIVO, Bristol Myers Squibb™). Purified T cell responses to recipient dendritic cells (DC) were measured in mixed lymphocyte reaction (MLR) assay, and further tested by quantifying cytokine secretion in MLR culture supernatants using Bio-Plex immunoassays (Bio-Rad Lab™). To test for clonal deletion, alloreactive T-cell clones with frequency >0.1% identified in vitro from the graft pre-infusion were tracked by monitoring their T-cell receptor CDR3 sequence serially, in an ImmunoSEQ™ assay (Adaptive Biotech™). Gene expression profiling and signaling pathway analysis on alloreactive or tolerant T-cells were performed by collecting T cells from in vitro MLR reactions, purifying total RNAs from these cells, running RNA-sequencing and analyzing with software of CLC Genomic Workbench®v21, GSEA®v4.1.0 and IPA®. Results: We have previously shown that circulating T cells were hyporeactive to recipient DC in patients at 1yr (or 2yrs in 1 patient) post-haplo-HSCT [6 months, (or 2-5 months in 3 patients) post-ISD withdrawal], while these T cells responded vigorously to 3rd party APC, As positive control, host DC triggered robust 3rd party T-cell response. Neither depletion of Tregs, nor blockade of IL10R or PD1 could reverse T-cell hypo-reactivity. Low-dose IL2 triggered mild anti-host proliferation responses. Similar scenarios were detected in graft T cells against self-DC, (negative control in autologous set up), while vigorous responses were shown in the graft T cells against host-DC pre-haplo-HSCT. Cytokine profiling further confirmed the MLR responses. ImmunoSEQ™ revealed disappearance of alloreactive T-cell clones from peripheral blood with time. Low dose IL2 had minimal impact to reverse hyporeactivity. While most alloreactive T clones were undetectable, a few remained in the peripheral pool in tolerant patients tested (Nee, T et al, Transplantation and Cellular Therapy, 2022;28(3):S44-S45). Unlike graft T-cell responses against host DC, tolerant T-cells in circulation (including remaining alloreactive T cells) against hDC showed distinct patterns of gene expressions, with relative downregulation of allograft rejection (GSEA-Hallmark) (Fig 1A, B, C) and T-helper response, co-stimulation, cell proliferation pathway, that was paired with upregulation of co-inhibition, PD1-PDL1, Ferroptosis, PTEN and CREB pathways (Fig1D). The profiles suggested systemic alterations in the signaling pathways on tolerant T cells post-haplo-HSCT. Conclusion: Hypo-reactivity of donor-derived circulating T cells to recipient DC and graft T cells to graft DC (negative control) were evident. We found Treg, Tr1 activity or T-cell exhaustion to be insignificant as their blockade or removal did not lead to 'flare’ in circulating tolerant T-cell reactivity against patient DC. T-cell clonal deletion appears to be the dominant mechanism explaining the in vitro hyporeactive proliferation, cytokine secretion, and alloreactive TCR clonotype attrition, along with systemically altered signaling pathways in tolerant T-cells including the alloreactive T cells remained. Anergy may play a minor supportive role. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Off-the-Shelf, iPSC-Derived CAR-NK Cells Multiplexed-Engineered for the Avoidance of Allogeneic Host Immune Cell Rejection

Allogeneic off-the-shelf cell therapies offer distinct advantages over conventional autologous cell therapies in terms of scaled manufacturing, on-demand availability and optimization of cellular starting material. A unique consideration in the use of allogeneic cell therapies is the potential for immune cell-mediated recognition of the allogeneic cell product by the patient's immune system. CAR T-cell therapies are commonly combined with conditioning chemotherapies that suppress a patient's immune system, creating a suitable window of activity to elicit clinical response. However, protracted lympho-conditioning also affects immune reconstitution and can negatively impact the rate of infection. Alternative approaches to prevent allorejection may therefore help to enhance the efficacy of the therapy while preserving the immune system of the patient.Elimination of cell-surface human leukocyte antigen (HLA) molecule expression by genetic knockout (KO) has long been known to abrogate T-cell reactivity. However, loss of class I HLA elicits NK cell-mediated recognition and clearance, and therefore must be combined with other immune-modulating strategies to limit host NK cell reactivity. Allogeneic models combining class I HLA deletion with NK cell inhibitory molecules, such as HLA-E and CD47, have been shown to abrogate NK cell reactivity in mouse models. However, HLA-E is the canonical activator of NKG2C, a dominant activating receptor found on human NK cells. Likewise, the expression of signal regulatory protein alpha (SIRPα), the major interactor for CD47, is mostly restricted to macrophages and dendritic cells and not human NK cells, and the observed effects of this immune-modulating strategy in the mouse system may only offer partial or incomplete immune evasion in the human system.In this study, we provide details of a bona fide off-the-shelf strategy where iPSC-derived NK (iNK) cell therapy is multiplexed engineered with a novel combination of immune-evasion modalities; beta 2 microgobulin (B2M) KO to prevent CD8 T-cell mediated rejection; class II transactivator (CIITA) KO to prevent CD4 T-cell mediated rejection; and CD38 KO to enable combination with anti-CD38 mAbs, which can be administered to deplete host alloreactive lymphocytes, including both NK and T cells. In vitro mixed lymphocyte reaction (MLR) data demonstrated that upon co-culture with allogeneic PBMCs, B2M KO iNK cells stimulated less T-cell activation than their B2M sufficient counterparts as evidenced by reduced CD38, 41BB, and CD25 levels on T cells. Additionally, B2M KO iNK cells impaired T-cell expansion over the duration of co-culture, resulting in a 50% decrease in expansion at the peak of the control response. However, B2M KO iNK cells were depleted over time, suggesting activation of an NK cell “missing self” response by the peripheral blood NK (pbNK) cells.In contrast, when the assay was performed in the presence of anti-CD38 mAb, depletion of B2M KO iNK cells was blocked, and instead B2M KO iNK cell numbers increased by 3.5-fold, comparable to the iNK cell numbers found in the control arm (cultured without allogeneic PBMCs). Interestingly, pbNK cell numbers decreased, while T-cell activation and expansion remained lower than in B2M-sufficient MLR cultures. Furthermore, when B2M KO iNK cells were cocultured with tumor cells and anti-CD38 mAb in vitro, ADCC was comparable to the B2M sufficient cells, indicating uncompromised effector function. Finally, in vivo studies suggested that co-administration of anti-CD38 mAbs can significantly enhance the persistence of B2M KO iNK cells in the presence of allogeneic pbNK cells as seen in the spleen and bone marrow (Figure 1).Together these data demonstrate that the combination of triple-gene knockout of CD38, B2M and CIITA with a CD38-targeting mAb is an effective strategy to avoid host immune rejection, and highlights the potential advantages of multiplexed engineered iPSCs to facilitate large-scale manufacture of complex engineered, off-the-shelf cellular therapies. [Display omitted] DisclosuresWilliams: Fate Therapeutics: Current Employment. Malmberg: Merck: Research Funding; Vycellix: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Lee: Fate Therapeutics, Inc.: Current Employment. Bjordahl: Fate Therapeutics: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.

Novel Variant Ig Domain (vIgD) Proteins Generated Via Directed Evolution of Igsf Domains Have Therapeutic Efficacy in Animal Models of Graft Versus Host Disease

The immunoglobulin superfamily (IgSF) is a large, diverse family of proteins extensively targeted for treatment of cancers and autoimmune diseases. Most of the therapeutic strategies targeting this family have focused on high affinity antibodies binding a single receptor. Wild-type IgSF receptors typically exhibit low affinities for their counter-structures, limiting their utility in therapeutic modulation of immune responses. We have developed a novel variant Ig domain™ (vIgD™) directed evolution platform to affinity mature human IgSF extracellular domains. In this platform, libraries of mutagenized IgSF domains are selected for altered affinity to specific recombinant protein counterstructures. Fc fusion proteins incorporating the resulting engineered IgSF domains are then tested in vitro for their ability to either agonize or antagonize T cell responses.We successfully engineered the IgSF protein ICOSL, which binds to the costimulatory molecule ICOS on T cells and triggers T cell activation. We used our directed evolution platform to engineer a first-in-class single vIgD domain with higher affinity for ICOS and high affinity binding to the costimulatory molecule CD28, which ICOSL does not normally bind. Following the creation of dual ICOS/CD28 antagonism, the resulting domains were used to generate individual constructs encoding Fc-fusion proteins. These enhanced ICOS/CD28 dual antagonist Fc-fusion proteins were transfected into HEK-293 cells and protein produced by these cells was purified by protein A affinity chromatography. The resultant purified proteins were then tested in a FACS based binding assay on cells transfected with each costimulatory receptor showing up to a 2-3-fold increase in ICOS binding and high affinity binding to CD28. Various ICOS/CD28 dual antagonist Fc-fusion proteins were then tested in soluble format over a range of concentrations for functional activity in mixed lymphocyte reaction cultures (MLR) where allogeneic dendritic cells (DC) were used to stimulate purified human T cells. Most Variants showed significant ability to inhibit the MLR response as read out measuring proliferation and cytokine production. The potency of the Fc-fusion proteins varied somewhat depending on their relative affinities for CD28 and ICOS, with all being superior to wild type ICOSL protein and many showing superiority to the CD28 blocking reagent belatacept (CTLA4-Fc). The level of inhibition correlated well with binding of the Fc-fusion proteins to the T cells in culture.To assess the function of the ICOS/CD28 dual antagonist Fc-fusion proteins in vivo, a subset were tested in the human PBMC-NSG™ GvHD mouse model. This model monitors graft versus host disease generated by xenogeneic responses of T cells in human PBMCs transferred into NSG™. Administration of the dual ICOS/CD28 antagonist Fc-fusion proteins significantly protected mice from the effects of xenogeneic T cell activation in vivo with treated animals showing dramatically enhanced survival and greatly reduced disease scores. Interestingly, the level of protection correlated with the potency of the molecules in the in vitro MLR assay with less potent molecules in MLR assays failing to protect while more potent molecules protected well. Belatacept was also effective in protecting animals from GvHD, but not as well as the ICOS/CD28 dual antagonists produced by our vIgD platform. Collectively, these data demonstrate the vIgD directed evolution platform can generate IgSF proteins with altered affinities and improved binding properties. Variants engineered for desired immunological properties can effectively perturb T cell costimulation and affect T cell responses both in vitro and in vivo with the in vitro potency of the proteins correlating to in vivo potency. The dual ICOS/CD28 antagonists produced with the vIgD platform can be used therapeutically in disease settings where attenuation of T cell responses is beneficial, including GvHD and potentially other autoimmune diseases. Phase I enabling studies are in progress. [Display omitted] DisclosuresLevin:Alpine Immune Sciences: Employment, Equity Ownership. Evans:Alpine Immune Sciences: Employment, Equity Ownership. Rickel:Alpine Immune Sciences: Employment, Equity Ownership. Wolfson:Alpine Immune Sciences: Employment, Equity Ownership. Dillon:Alpine Immune Sciences: Employment, Equity Ownership. Kornacker:Alpine Immune Sciences: Employment, Equity Ownership, Patents & Royalties. Swanson:Alpine Immune Sciences: Employment, Equity Ownership, Patents & Royalties. Peng:Alpine Immune Sciences: Employment, Equity Ownership.

Repurposing a novel anti-cancer RXR agonist to attenuate murine acute GVHD and maintain graft-versus-leukemia responses

AbstractThe nuclear receptor (NR) subclass, retinoid X receptors (RXRs), exert immunomodulatory functions that control inflammation and metabolism via homodimers and heterodimers, with several other NRs, including retinoic acid receptors. IRX4204 is a novel, highly specific RXR agonist in clinical trials that potently and selectively activates RXR homodimers, but not heterodimers. In this study, in vivo IRX4204 compared favorably with FK506 in abrogating acute graft-versus-host disease (GVHD), which was associated with inhibiting allogeneic donor T-cell proliferation, reducing T-helper 1 differentiation, and promoting regulatory T-cell (Treg) generation. Recipient IRX4204 treatment reduced intestinal injury and decreased IFN-γ and TNF-α serum levels. Transcriptional analysis of donor T cells isolated from intestines of GVHD mice treated with IRX4204 revealed significant decreases in transcripts regulating proinflammatory pathways. In vitro, inducible Treg differentiation from naive CD4+ T cells was enhanced by IRX4204. In vivo, IRX4204 increased the conversion of donor Foxp3− T cells into peripheral Foxp3+ Tregs in GVHD mice. Using Foxp3 lineage-tracer mice in which both the origin and current FoxP3 expression of Tregs can be tracked, we demonstrated that IRX4204 supports Treg stability. Despite favoring Tregs and reducing Th1 differentiation, IRX4204-treated recipients maintained graft-versus-leukemia responses against both leukemia and lymphoma cells. Notably, IRX4204 reduced in vitro human T-cell proliferation and enhanced Treg generation in mixed lymphocyte reaction cultures. Collectively, these beneficial effects indicate that targeting RXRs with IRX4204 could be a novel approach to preventing acute GVHD in the clinic.

Siplizumab Induces NK Cell Fratricide Through Antibody-Dependent Cell-Mediated Cytotoxicity.

The glycoprotein CD2 is expressed on T and NK cells and contributes to cell-cell conjugation, agonistic signaling and actin cytoskeleton rearrangement. CD2 has previously been shown to have an important function in natural NK cell cytotoxicity but to be expendable in antibody-mediated cytotoxicity. Siplizumab is a monoclonal anti-CD2 IgG1 antibody that is currently undergoing clinical trials in the field of transplantation. This study investigated the effect of CD2 binding and Fc γ receptor binding by siplizumab (Fc-active) and Fc-silent anti-CD2 monoclonal antibodies in allogeneic mixed lymphocyte reaction and autologous lymphocyte culture. Further, induction of NK cell fratricide and inhibition of natural cytotoxicity as well as antibody-dependent cytotoxicity by these agents were assessed. Blockade of CD2 via monoclonal antibodies in the absence of Fc γ receptor binding inhibited NK cell activation in allogeneic mixed lymphocyte reaction. In contrast, siplizumab increased NK cell activation in both mixed lymphocyte reaction and autologous lymphocyte culture due to FcγRIIIA binding. However, experiments using purified NK cells did not show an inhibitory effect of CD2 blockade on natural cytotoxicity or antibody-dependent cytotoxicity. Lastly, it was shown that siplizumab induces NK cell fratricide. Concluding, siplizumab is a promising biopharmaceutical drug candidate for depletion of T and NK cells with minimal off-target effects.

IL-10 and TGF-β induce alloreactive CD4+CD25– T cells to acquire regulatory cell function

AbstractWe previously reported that interleukin-10 (IL-10) and transforming growth factor (TGF)–β treatment of primary mixed lymphocyte reaction (MLR) cultures resulted in secondary alloantigen-specific hyporesponsiveness and protection from graft-versus-host disease (GVHD) lethality. Here, we report that CD4+ T cells recovered from the IL-10– and TGF-β–treated primary MLR cultures have immunoregulatory function. Tolerized cells significantly inhibited proliferation of naive alloreactive CD4+ T cells in a primary MLR. Inhibition of the naive alloresponse was observed with as few as 1 tolerized cell to 10 naive responder cells. Tolerized cells were able to significantly reduce GVHD lethality when injected with naive alloreactive CD4+ T cells into major histocombatibility class (MHC) II disparate recipients. Rigorous CD25 depletion of the primary MLR had no effect on generation of a regulatory capacity, suggesting that the regulatory cells likely originated from CD4+CD25– T cells. Immune suppression was mediated independently of IL-10 and TGF-β production, as neutralizing antibodies for IL-10, IL-10R, and TGF-β were unable to revert suppression, and IL-10– deficient CD4+ T cells were able to mediate in vitro and in vivo suppression. The generation of immunoregulatory cells from a CD4+CD25– population during tolerization with IL-10 and TGF-β provides an additional mechanism to prevent GVHD lethality by T cells that may escape full tolerance induction.

Improved Function and Maturation of Dendritic Cells Stimulated by Recombinant pp65 Protein: An in-vitro Design.

Stimulated dendritic cells (DCs) have been shown to be effective in the induction of specific immune cells. Also, the CMV pp65 plays an important role in CMV life cycle and immune recognition. To assess the effect of CMV pp65 on the maturity and function of dendritic cells. Splenic DCs were treated with non-cytotoxic concentrations of the pp65 and analyzed for MHC II, CD86, and CD40 expression by flow cytometry. Then, ROR-γ, GATA3, T-bet, and FOXP3 gene expression levels were evaluated in T cells co-cultured with DCs using Real time-PCR. Finally, the effects of pp65 on allogenic T-cell responses in mixed lymphocyte culture (MLR), and the release of cytokines were investigated by ELISA and flow cytometry. The phagocytosis rate was significantly lower in the pp65-treated DCs than the non-stimulated DCs. There were significant differences in the raised level of CD40, CD86, and CCR7 in DCs as maturation markers. Furthermore, ROR-γ, and T-bet overexpression in T cells of the pp65-treated group compared with the non-stimulated group was observed. Significant differences were observed in the levels of IL-2, IL-6, IL-17, IL-22, TNF-α, and IFN-γ in pp65-stimulated groups compared with the non-stimulated DCs. The pp65-treated DCs can induce differentiation and functional activity of the cellular immune system, including Th17, and Th1, but not other major T-cell subsets such as Tregs, and Th2 population.

Suppression effect on IFN-γ of adipose tissue-derived mesenchymal stem cells isolated from β2-microglobulin-deficient mice.

Administration of bone marrow-derived mesenchymal stem cells (MSCs) is a possible treatment for graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation and other inflammatory conditions. To address the mechanism of immunosuppression by MSCs, in particular those derived from adipose tissue (AMSCs), AMSCs were isolated from three different mouse strains, and the suppressive capacity of the AMSCs thus obtained to suppress interferon (IFN)-γ generation in mixed lymphocyte reaction cultures serving as an in vitro model of GVHD were assessed. It was revealed that the AMSCs had a potent capacity to suppress IFN-γ production regardless of their strain of origin and that such suppression was not associated with production of interleukin-10. In addition, the results demonstrated that β2-microglobulin (β2m)-deficient AMSCs from β2m-/- mice were also potent suppressor cells, verifying the fact that the mechanism underlying the suppression by AMSCs is independent of major histocompatibility complex (MHC) class I expression or MHC compatibility. As AMSCs appear to have immunosuppressive properties, AMSCs may be a useful source of biological suppressor cells for the control of GVHD in humans.

The Suppressive Function of the Human Regulatory Macrophages In-Vitro have more Potency but not In-Vivo

Centre for Transplant & Renal Research. Introduction Human regulatory macrophages (Mregs) have been shown to suppress mitogen-driven T cell proliferation in vitro and are under evaluation as a cellular therapy in renal transplantation in THE ONE STUDY clinical trial. In this study, we investigated whether human Mregs were capable of suppressing the allogeneic response in vitro and protecting islet xeno-grafts in humanizing mice in vivo. Methods Human CD14+ cells were isolated from healthy donor peripheral blood monocytes (PBMCs) using CD14 magnetic beads. Mregs induction was performed by culturing CD14+ cells with M-CSF for 6 days and be stimulated with IFN-γ for a further 24 hr. The phenotype of Mregs was characterized by flow cytometry, and their suppressive activity was tested by mixed-lymphocyte reaction (MLR) using human PBMCs as responder cells, autologous Mregs as suppressor cells and irradiated allogeneic PBMCs as stimulator cells. In a separate transwell MLR setting, responder and allogeneic stimulator cells were cultured in the upper chamber and autologous Mregs were seeded in the lower chamber. Supernatants collected from MLR cultures were used for cytokine detection. Isolated the neonatal porcine islet cell clusters (NICCs) and cultured for 6 days. In vivo, every 5,000 IEQ NICCs were transplanted into NOD-SCID IL2R gamma -/- mice under the two sides of renal capsules. Adoptive transfer of human PBMCs and Mregs were suspended in PBS and injected into mice at doses of 10×106 and 2×106 by tail vein within 7 days after transplant. Graft rejection was defined as little or no intact graft with a heavy lymphocytic infiltrate on histological examination, survival was analyzed by H&E staining and endocrine secreting cells was analyzed by insulin staining. Results The resulting induced human Mregs demonstrated a typical Mregs phenotype, CD14lowCD80lowCD86+HLA-DR+CD83-/lowCD16-, and as a result, potent suppression of proliferating alloreactive PBMC even at Mregs: responder cells ratios >1:16. Separation of Mregs from responder and allogeneic stimulators in a transwell MLR system had the mild impact on their suppressive potency in the allogeneic response at all ratios of Mregs: responder cells tested. Upregulated levels of Mregs function related molecules TGF-b and IDO were detected in the supernatants collected from both non-transwell and transwell MLR cultures containing Mregs. In treated mice, the human PBMC were identified in the porcine-NICC-xenografts, as well as systemically in the spleens, blood, and kidneys. However, injected Mregs cannot be detected by flow cytometry, and there were no intact xenografts were found with mid-term. Conclusions In vitro induced human Mregs showed the capacity to suppress the allogeneic response in vitro and that was not cell-cell contact dependent. The porcine-NICC-xenografts cannot be protected by Human-Mregs in vivo as Mregs cannot proliferation. The suppressive function of the Mregs have more potency in vitro, but not in vivo.

Study on the immune functions of dendritic cells regulated by histone deacetylase inhibitor Belinostat

目的探讨组蛋白去乙酰化酶（HDAC）抑制剂Belinostat对小鼠骨髓来源树突状细胞（DC）免疫功能的影响及初步机制。方法体外诱导培养C57BL/6小鼠骨髓来源的DC，诱导培养第5天为未成熟DC（imDC）组，设0、50、100 nmol/L Belinostat作用组；imDC以脂多糖作用24 h为成熟DC（mDC），设0、50、100 nmol/L Belinostat作用组。从细胞形态、超微结构、免疫表型进行鉴定。流式细胞术检测各组DC免疫表型及趋化因子受体CCR7表达水平，趋化实验检测DC的体外迁移率。单向混合淋巴细胞培养法检测各组DC刺激下异基因淋巴细胞增殖率。ELISA法检测各组DC培养上清中TNF-α、IL-12及IL-10的表达水平。RQ-PCR检测Belinostat对DC中RelB mRNA表达水平的影响。结果成功诱导培养出imDC及mDC并鉴定。50和100 nmol/L Belinostat+imDC组CCR7表达水平均低于imDC组[（25.82±7.25）%对（50.44±5.61）%、（18.71±2.00）%对（50.44±5.61）%]；50 nmol/L Belinostat+mDC组CCR7表达水平高于mDC组[（71.14±1.96）%对（64.90±1.47）%]。Belinostat作用下imDC和mDC的迁移率均下降，但在imDC中组间比较差异无统计学意义。当刺激细胞∶反应细胞比例为1∶2时，100 nmol/L Belinostat+imDC刺激下淋巴细胞增殖率低于imDC组[（227.09±13.49）%对（309.49±53.69）%]。Belinostat作用下mDC所分泌的TNF-α、IL-12和IL-10较mDC组均明显下降，差异均有统计学意义（P值均<0.01）。Belinostat+imDC及Belinostat+mDC中RelB mRNA表达水平较imDC组及mDC组均有降低（P值均<0.05）。结论Belinostat可调节DC迁移、抑制T淋巴细胞增殖及细胞因子分泌，一定程度上抑制DC成熟，可能与其下调DC中NF-κB的转录因子RelB mRNA水平有关。

Human regulatory macrophages are potent in suppression of the xenoimmune response via indoleamine-2,3-dioxygenase-involved mechanism(s).

For xenotransplantation to truly succeed, we must develop immunomodulatory strategies to suppress the xenoimmune response but by minimizing immunosuppression over the long term. Regulatory macrophages (Mreg) have been shown to suppress polyclonal T-cell proliferation in vitro and prolong allograft survival in vivo. However, the question of whether they are capable of suppressing xenoimmune responses remains unknown. This study assessed the potential of human Mreg to be used as an effective immunomodulatory method in xenotransplantation. CD14+ monocytes selected from human peripheral blood mononuclear cells (PBMC) were cultured with macrophage colony-stimulating factor (M-CSF) for 7days with IFN-γ added at day 6 for Mreg induction. Mreg phenotyping was performed by flow cytometric analysis, and the in vitro suppressive function was assessed by mixed lymphocyte reaction (MLR) using irradiated pig PBMC as the xenogeneic stimulator cells, human PBMC as responder cells, and autologous Mreg as suppressor cells. To assess mRNA expression of Mreg functional molecules indoleamine-2,3-dioxygenase (IDO), IL-10, inducible nitric oxide synthase (iNOS) and TGF-β were measured by real-time PCR. Supernatants were collected from the MLR cultures for IDO activity assay by high-performance liquid chromatography (HPLC). The effects of the IDO inhibitor 1-D/L-methyl-tryptophan (1-MT), iNOS inhibitor NG -monomethyl-l-arginine (L-NMMA), and anti-IFN-γ or anti-TGF-β monoclonal antibody (mAb) treatment on Mreg suppressive capacity were tested from the supernatants of the MLR assays. We demonstrated that induced Mreg with a phenotype of CD14low CD16-/low CD80low CD83-/low CD86+/hi HLA-DR+/hi were capable of suppressing proliferating human PBMC, CD4+, and CD8+ T cells, even at a higher responder:Mreg ratio of 32:1 in a pig-human xenogeneic MLR. The strong suppressive potency of Mreg was further correlated with their upregulated IDO expression and activity. The IDO upregulation of Mreg was associated with an increased production of IFN-γ, an IDO stimulator, by xenoreactive responder cells in the xenogeneic MLR. While no effect on Mreg suppressive potency was detected by addition of the iNOS inhibitor L-NMMA or anti-TGF-β mAb into the MLR assays, inhibition of IDO activity by neutralizing IFN-γ or by IDO inhibitor 1-MT substantially impaired the capacity of Mreg to suppress the xenogeneic response, indicating the importance of upregulated IDO activity in Mreg-mediated suppression of the xenogeneic response in vitro. This study demonstrates that human Mreg are capable of suppressing the xenoimmune response in vitro via IDO-involved mechanism(s), suggesting their potential role as an effective immunomodulatory tool in xenotransplantation.

Immunomodulatory properties of titanium dioxide nanostructural materials.

OBJECTIVES:Although titanium dioxide (TiO2) nanostructural materials have been widely used in Biology and Medicine, very little is known about immunomodulation mechanism of these materials. Objectives of this study are to investigate in vitro immunomodulatory effects of TiO2. Immunosuppressant may lower immune responses and are helpful for the treatment of graft versus host diseases and autoimmune disorders.MATERIALS AND METHODS:In this study, we used H2Ti3O7 titanium dioxide nanotubes (TNT) nanotubes along with commercial TiO2 nanoparticles (TNP) and TiO2 fine particles (TFP). We investigated the in vitro immunomodulatory effects of TNP, TNT, and TFP using mixed lymphocyte reaction (MLR). Suppression was studied by 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Cytokine profile was measured by enzyme-linked immunosorbent assay (ELISA).RESULTS AND CONCLUSIONS:The results from this study illustrated that the TiO2 nanostructural materials strongly suppressed splenocytes proliferation in MLR. For TNP and TNT, at 50 μg/ml suppression of 20%–25% and 30%–35%, respectively, and for TFP at 100 μg/ml suppression was 25%–30% was observed. Suppression of splenocytes proliferation in the presence of TNP, TNT, and TFP demonstrated that these nanostructural materials probably block T-cell-mediated responses in vitro. Our ELISA results confirmed that significantly lower levels of Th1 type cytokines (interleukin-2, interferon-γ) in the 48 h MLR culture supernatants. Our data suggest that TiO2 nanostructural materials suppress splenocytes proliferation by suppressing Th1 cytokines.

In vitro inhibitory effects of thymol and carvacrol on dendritic cell activation and function

Context: Thyme has been used in traditional medicine for medicinal purposes since ancient times.Objective: The objective of this study was to investigate the effects of thymol and carvacrol as two major constituents of thyme on dendritic cells (DCs) maturation and T cell activation.Materials and methods: Splenic DCs were treated with non-cytotoxic concentrations of the components and then analyzed for MHC II, CD86, and CD40 expression by flow cytometry. The effects of compounds on mitogenic, as well as allogenic T cell responses in mixed lymphocyte culture (MLR) and the release of cytokines were investigated.Results: At 0.1 µg/ml, reduced mean fluorescent intensity (MFI) of CD86 for thymol (80.3 ± 0.2% of untreated control) and CD40 for carvacrol (79.5 ± 0.14%) was observed (p < 0.001). Decreased mitogenic T cell proliferation by thymol [proliferation index (PI) from 0.93 ± 0.11 at 1 µg/ml to 0.42 ± 0.16 at 100 µg/ml (p < 0.01)] and carvacrol [PI from 1.08 ± 0.3 at 1 µg/ml to 0.28 ± 0.1 at 100 µg/ml (p < 0.001)] was seen. Ten micrograms/ml thymol (PI, 0.85 ± 0.04) and carvacrol (PI, 0.89 ± 0.03) inhibited allogenic T cell response (p < 0.05). Decreased IFN-γ level in MLR supernatant from 1441 ± 27.7 pg/ml in untreated cells to 944 ± 32.1 at 10 µg/ml of thymol and of carvacrol (886 ± 31.7 pg/ml) (p < 0.01) was found. IL-4 levels were decreased in the presence of both compounds (p < 0.01).Conclusion: These data showed the suppressive effects of thymol and carvacrol on DCs maturation and function, as well as T cell responses.

Female third party lymphocytes are effective for immunotherapy of patients with unexplained primary recurrent spontaneous abortion: A retrospective analysis of outcomes

Objectives Allogeneic lymphocytes of paternal origin or supplied by a male third party have been used for the treatment of recurrent spontaneous abortion. Few studies, however, have examined the use of female third party lymphocytes. Our purpose was to determine whether female third party lymphocytes could be used for immunotherapy of women with recurrent spontaneous abortion.Methods In this retrospective non-randomised cohort-controlled study, the medical records of patients with three or more spontaneous abortions who received immunotherapy with lymphocytes from their partner, a male third party or a female third party, as well as those who received no immunotherapy, from 1996 to 2012 were reviewed. All patients were negative for mixed lymphocyte culture reaction (MLR)-blocking antibodies. Immunotherapy was performed in 302 patients in two courses, while 53 patients received no immunotherapy.Results The pregnancy rates in patients who received lymphocytes from their partners, a male third party or a female third party, and in those not immunised, were 85.6%, 87.3%, 89.7%, and 79.3%, respectively (p = 0.523);the live birth rates were 87.3%, 75.8%, 84.6%, and 40.5%, respectively (p < 0.001).Conclusions We conclude that female third party lymphocytes can be used for immunotherapy in patients with recurrent spontaneous abortion.Chinese 摘要目的 父源同种异体淋巴细胞或第三方男性供给淋巴细胞用于复发性自然流产的治疗。少数研究调查了女性第三方淋巴细胞的应用。我们的目的是验证女性第三方淋巴细胞是否可以用于复发性自然流产的免疫治疗。方法 在这项回顾性非随机队列对照研究中回顾了自1996年至2012年,接受配偶淋巴细胞,男性第三方淋巴细胞或接受女性第三方淋巴细胞免疫治疗,或未接受免疫治疗的三次或更多次自然流产的患者病历资料。所有患者的混合淋巴细胞培养反应(MLR)-封闭抗体均为阴性。302名患者接受了两个疗程的免疫治疗,而53名患者未接受免疫治疗。结果 接受配偶淋巴细胞,男性第三方淋巴细胞,女性第三方淋巴细胞和未接受免疫治疗的患者妊娠率分别为85.6%，87.3%，89.7%，和79.3% (p = 0.523)。活产率分别为87.3%, 75.8%, 84.6%和40.5% (p < 0.001) 。结论 我们推断,女性第三方淋巴细胞可用于复发性自然流产患者的免疫治疗。关键词免疫疗法；淋巴细胞；反复自然流产

A CB2-Selective Cannabinoid Suppresses T-Cell Activities and Increases Tregs and IL-10.

We have previously shown that agonists selective for the cannabinoid receptor 2 (CB2), including O-1966, inhibit the Mixed Lymphocyte Reaction (MLR), an in vitro correlate of organ graft rejection, predominantly through effects on T-cells. Current studies explored the mechanism of this immunosuppression by O-1966 using mouse spleen cells. Treatment with O-1966 dose-relatedly decreased levels of the active nuclear forms of the transcription factors NF-κB and NFAT in wild-type T-cells, but not T-cells from CB2 knockout (CB2R k/o) mice. Additionally, a gene expression profile of purified T-cells from MLR cultures generated using a PCR T-cell activation array showed that O-1966 decreased mRNA expression of CD40 ligand and CyclinD3, and increased mRNA expression of Src-like-adaptor 2 (SLA2), Suppressor of Cytokine Signaling 5 (SOCS5), and IL-10. The increase in IL-10 was confirmed by measuring IL-10 protein levels in MLR culture supernatants. Further, an increase in the percentage of regulatory T-cells (Tregs) was observed in MLR cultures. Pretreatment with anti-IL-10 resulted in a partial reversal of the inhibition of proliferation and blocked the increase of Tregs. Additionally, O-1966 treatment caused a dose-related decrease in the expression of CD4 in MLR cultures from wild-type, but not CB2R k/o, mice. These data support the potential of CB2-selective agonists as useful therapeutic agents to prolong graft survival in transplant patients, and strengthens their potential as a new class of immunosuppressive agents with broader applicability.

Defining the Alloreactive T Cell Repertoire Using High-Throughput Sequencing of Mixed Lymphocyte Reaction Culture

The cellular immune response is the most important mediator of allograft rejection and is a major barrier to transplant tolerance. Delineation of the depth and breadth of the alloreactive T cell repertoire and subsequent application of the technology to the clinic may improve patient outcomes. As a first step toward this, we have used MLR and high-throughput sequencing to characterize the alloreactive T cell repertoire in healthy adults at baseline and 3 months later. Our results demonstrate that thousands of T cell clones proliferate in MLR, and that the alloreactive repertoire is dominated by relatively high-abundance T cell clones. This clonal make up is consistently reproducible across replicates and across a span of three months. These results indicate that our technology is sensitive and that the alloreactive TCR repertoire is broad and stable over time. We anticipate that application of this approach to track donor-reactive clones may positively impact clinical management of transplant patients.

Hepatitis B Virus Is Associated With Low Rejection Rates Following Liver Transplant Independent of Hepatitis B Immunoglobulin Use.

Historical data suggests that Hepatitis B virus positive (HBV+) liver transplant (LT) recipients have less acute cellular rejection (ACR) than non-HBV+. It is unclear if this persists in the current era with reduced use of hepatitis B immunoglobulin (HBIG), as it might have immunosuppressive effects akin to pooled immunoglobulin. Thus, we examined the relationship between HBIG use on ACR in both in vitro and clinical studies. We performed a case-control study from the OPTN (2000-2011) comparing ACR rates of primary LT recipients with HBV, Hepatitis C, steatohepatitis (nonalcoholic or alcoholic), and immune-mediated liver disease. ACR was assessed with univariate analysis and multivariate logistic regression. Patient and graft survival was analyzed with the Kaplan-Meier method. The in vitro effect of HBIG on immune and regulatory cell responses was assessed with serial dilutions of HBIG using mixed lymphocyte reaction (MLR) cultures of laboratory volunteers. Of the 40,952 patients identified, 21% had at least one episode of ACR over a median follow up of 3.9 (range: 1.4 to 6.7) years. On univariate analysis, HBV+, Asian race and presence of hepatocellular carcinoma were associated with lower ACR (p<0.001). In multivariate analysis adjusted for age, gender, and immunosuppression, HBV status remained a strong predictor of freedom from ACR (OR 0.58, 95% CI:1.5-2.1). In patients with HBV, there was no effect of HBIG use on ACR status (p=0.09). Overall graft survival (60.8% vs 69.1%) and patient survival (67.7% vs 72.3%) were lower in patients with ACR, except for those with HBV where there was no significant difference in either outcome regardless of ACR status. In vitro, HBIG at all clinically applicable concentrations (e.g. akin to intravenous and intramuscular dosing) did not appear to inhibit immune responses or promote regulatory cell development and expansion. These results suggest that in the current era HBV+ LT recipients are at lower risk for ACR compared to other causes of liver disease independent of HBIG use. Furthermore, HBIG added to MLR cultures did not have direct inhibitory or regulatory effects at clinical concentrations. These findings support reducing immunosuppressive requirements in HBV+ LT recipients without an increase risk of ACR.

Anti-CD200R2, Anti-IL-9, Anti-IL-35, or Anti-TGF-β Abolishes Increased Graft Survival and Treg Induction Induced in Cromolyn-Treated CD200R1KO.CD200tg Mice

Rejection is associated with early degranulation (≥80%) of graft-infiltrating CD200R1 receptor-positive mast cells (MCs). Survival is increased, and MC degranulation is decreased, in CD200 mice but not in CD200R1KO mice. CD200 engagement of CD200R2 (not present on MCs) alters dendritic cell differentiation and enhances induction of Foxp3 regulatory T cells (Tregs). We investigated whether attenuation of MC degranulation by sodium cromoglycate allowed CD200 to increase survival in CD200R1KO mice. C57BL/6 control, CD200R1KO, CD200, or CD200R1KO.CD200 mice received BALB/c grafts with or without treatment with cromoglycate. Survival was monitored daily from day 10, with mixed lymphocyte culture responses measured on day 14 or 21 and graft immunohistology performed on day 14. Decreased MC degranulation and increased graft Foxp3 Treg infiltration/survival occurred in CD200 mice and in CD200-treated control mice or CD200R1KO.CD200 mice receiving cromoglycate. Neutralizing anti-CD200 or anti-CD200R1/R2 monoclonal antibody caused graft rejection, as did anti-interleukin (IL)-9, anti-IL-35, or anti-transforming growth factor-β antibodies, with the latter also decreasing graft-infiltrating Tregs. These data imply a coordinated effect of MCs and Tregs on increased graft survival induced by CD200, with a critical role for IL-9, IL-35, and transforming growth factor-β in the development/function of Tregs.

Increased expression of herpesvirus entry mediator in 1,25-dihydroxyvitamin D3-treated mouse bone marrow-derived dendritic cells promotes the generation of CD4+CD25+Foxp3+ regulatory T cells

Dendritic cells (DCs) can initiate immune responses or induce immune tolerance. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is a secosteroid hormone that can induce tolerogenic dendritic cells favoring the induction of regulatory T cells (Treg). The present study revealed a tolerogenic effect of 1,25(OH)2D3 on the phenotype and function of ovalbumin (OVA)‑activated mouse bone marrow‑derived DCs. Three inhibitory molecules associated with tolerogenic DCs, programmed death‑ligand-1 (PD‑L1), PD‑L2 and herpesvirus entry mediator (HVEM) and the expression of co‑stimulatory molecules (CD80, CD86 and CD40) on 1,25(OH)2D3‑treated DCs were examined. The levels of interleukin (IL)‑2, IL‑6 and IL‑10 secreted by 1,25(OH)2D3‑treated DCs were analyzed. The capability of 1,25(OH)2D3‑treated DCs to induce CD4+CD25+Foxp3+ Treg and the stimulation of allogeneic CD4+ T‑cell proliferation in mixed lymphocyte reaction (MLR) was studied. 1,25(OH)2D3‑treated DCs induced up to 21.0‑fold upregulation of the HVEM expression and 4.1‑fold enhancement of the HVEM expression upon activation with OVA [OVA‑D3/immature (im)DCs]. PD‑L1 was not affected by 1,25(OH)2D3‑treated DCs and downregulated PD‑L2 expression on 1,25(OH)2D3‑treated DCs and OVA‑D3/imDC. The expression of co‑stimulatory molecules (CD80, CD86 and CD40) was downregulated in 1,25(OH)2D3‑treated DCs and OVA‑D3/imDC. Furthermore, 1,25(OH)2D3‑treated DCs secreted much higher levels of IL‑10, however lower levels of IL‑2 and IL‑6 compared with the activated control DCs. Together with this pattern of cytokines, 1,25(OH)2D3‑treated DCs exhibited low allogeneic CD4+ T‑cell stimulatory activity and a higher number of CD4+CD25+Foxp3+ cells in the MLR cultures but not in the activated control DCs. These findings indicate that 1,25(OH)2D3 possesses the immunosuppressive properties by upregulating the expression of the inhibitory molecules, HVEM, which may be therapeutically useful in controlling chronic immune and/or inflammatory diseases.