Mixed Lymphocyte Culture Assay Research Articles

Interferon-gamma and tumor necrosis factor-alpha synergistically enhance the immunosuppressive capacity of human umbilical-cord-derived mesenchymal stem cells by increasing PD-L1 expression.

The immunosuppressive capacity of mesenchymal stem cells (MSCs) is dependent on the "license" of several proinflammatory factors to express immunosuppressive factors such as programmed cell death 1 ligand 1 (PD-L1), which determines the clinical therapeutic efficacy of MSCs for inflammatory or immune diseases. In MSCs, interferon-gamma (IFN-γ) is a key inducer of PD-L1 expression, which is synergistically enhanced by tumor necrosis factor-alpha (TNF-α); however, the underlying mechanism is unclear. To reveal the mechanism of pretreated MSCs express high PD-L1 and explore the application of pretreated MSCs in ulcerative colitis. We assessed PD-L1 expression in human umbilical-cord-derived MSCs (hUC-MSCs) induced by IFN-γ and TNF-α, alone or in combination. Additionally, we performed signal pathway inhibitor experiments as well as RNA interference experiments to elucidate the molecular mechanism by which IFN-γ alone or in combination with TNF-α induces PD-L1 expression. Moreover, we used luciferase reporter gene experiments to verify the binding sites of the transcription factors of each signal transduction pathway to the targeted gene promoters. Finally, we evaluated the immunosuppressive capacity of hUC-MSCs treated with IFN-γ and TNF-α in both an in vitro mixed lymphocyte culture assay, and in vivo in mice with dextran sulfate sodium-induced acute colitis. Our results suggest that IFN-γ induction alone upregulates PD-L1 expression in hUC-MSCs while TNF-α alone does not, and that the co-induction of IFN-γ and TNF-α promotes higher expression of PD-L1. IFN-γ induces hUC-MSCs to express PD-L1, in which IFN-γ activates the JAK/STAT1 signaling pathway, up-regulates the expression of the interferon regulatory factor 1 (IRF1) transcription factor, promotes the binding of IRF1 and the PD-L1 gene promoter, and finally promotes PD-L1 mRNA. Although TNF-α alone did not induce PD-L1 expression in hUC-MSCs, the addition of TNF-α significantly enhanced IFN-γ-induced JAK/STAT1/IRF1 activation. TNF-α up-regulated IFN-γ receptor expression through activation of the nuclear factor kappa-B signaling pathway, which significantly enhanced IFN-γ signaling. Finally, co-induced hUC-MSCs have a stronger inhibitory effect on lymphocyte proliferation, and significantly ameliorate weight loss, mucosal damage, inflammatory cell infiltration, and up-regulation of inflammatory factors in colitis mice. Overall, our results suggest that IFN-γ and TNF-α enhance both the immunosuppressive ability of hUC-MSCs and their efficacy in ulcerative colitis by synergistically inducing high expression of PD-L1.

In Vitro Model of Suppression of the Alloantigen Response by Tolerogenic Dendritic Cells Transfected with Personalized DNA Constructs Encoding HLA Epitopes.

A search for efficient graft rejection modulation techniques for the promotion of durable engraftment remains to be a matter of close study all over the world. Despite the variety of immunosuppressive drugs, the schemes currently used show a lack of selectivity and have a number of side effects. Here we investigated an approach for the induction of antigen-specific tolerance in a human "stimulator-responder" model in vitro, using dendritic cells (DCs) transfected with designed DNA constructs encoding the stimulator's major histocompatibility complex (MHC) epitopes. The object of the study is peripheral blood mononuclear cells (PBMCs) from 10 healthy donors. To induce antigen-specific tolerance, personalized DNA constructs were created for five responder-stimulator pairs, based on the sequences of donors' and recipients' MHCs. DNA sequencing was performed to select epitopes for incorporation into genetic constructs. A mixed lymphocyte culture assay was used (i) to assess the proliferative response in both directions for all possible stimulator-responder pairs (90 reactions) and (ii) to assess the tolerogenic properties of the generated transfected DCs (5 reactions). A significant increase in the amounts of FoxP3+ CD4+CD25+ cells and in IL-10 production was shown in culture of donor mononuclear cells after co-cultivation with the responder's dendritic cells transfected with donor-specific plasmids. The tolerogenic cultures generated using tolerogenic DCs transfected with MHC epitopes had a significantly greater ability to inhibit the proliferation of autologous MNCs in response to an allogeneic MHC stimulus. The produced DCs transfected with DNA constructs against HLA stimulating epitopes exhibited tolerogenic properties and may be used to develop antigen-specific tolerance. Thus, we proposed a perspective approach to the induction of antigen-specific tolerance, which should subsequently be studied for use in clinical practice.

Upregulation of microRNA-155 Enhanced Migration and Function of Dendritic Cells in Three-dimensional Breast Cancer Microenvironment

ABSTRACT Background: Dendritic cells (DCs) play an essential role in the induction and regulation of immune responses, including the activation of effector T lymphocytes for the eradication of cancers. However, the tumor microenvironment (TME) often leads to DCs dysfunction due to their immature state. MicroRNA-155 (miR-155) has emerged as a typical multifunctional gene regulator associated with immune system development and immune cell activation and differentiation. Methods: In this study, a three-dimensional TME model that closely mimics the microenvironment of breast cancer was prepared. MiR-155 overexpression and control vectors were constructed using lentivirus. The relative expression of miR-155 was determined by qRT-PCR. Cell viability, antigen uptake and cell surface marker expression were analyzed by live-dead staining and flow cytometry. The migration ability of bone marrow-derived DCs (BMDCs) was qualified by transwell assay. A mixed lymphocyte culture assay was used to assess T cell-specific proliferation. Cytokine levels were determined by ELISA. Results: We found that the expression of miR-155 in DCs was inhibited by the TME. Furthermore, upregulation of miR-155 enhanced the migration ability, uptake of antigen and elevated the expression of the mature DCs markers CD80 and MHCII. More importantly, overexpression of miR-155 in DCs significantly induced T cell proliferation and IFN-γ and IL-2 secretion. Conclusion: MiR-155 is a potential molecular regulator that may improve the efficacy of DCs-based tumor immunotherapy.

MiR-223-3p Inhibits Antigen Endocytosis and Presentation and Promotes the Tolerogenic Potential of Dendritic Cells through Targeting Mannose Receptor Signaling and Rhob.

Background The role of miR-223-3p in dendritic cells (DCs) is unknown. This study is aimed at investigating the effect of miR-223-3p on the antigen uptake and presentation capacities of DCs and the underlying molecular mechanism. Methods FITC-OVA antigen uptake and cell surface markers in bone marrow-derived DCs (BMDCs) were analyzed by flow cytometry. BMDCs were transfected with the miR-223-3p mimic or inhibitor. Cytokine levels were determined by ELISA. CD4+ T cell differentiation was determined by mixed lymphocyte culture assay. Results OVA treatment significantly downregulated miR-223-3p in BMDCs. The miR-223-3p mimic significantly inhibited OVA-induced antigen uptake and surface expression of MHC-II on BMDCs (P < 0.01). The miR-223-3p mimic increased TGF-β1 production in OVA-treated DCs (P < 0.01). Mixed lymphocyte reaction showed that the miR-223-3p mimic significantly promoted Treg cell differentiation. In addition, the miR-223-3p mimic significantly upregulated CD103 in DCs, indicating the promotion of tolerogenic DCs. The miR-223-3p mimic downregulated Rhob protein in OVA-induced DCs. Rhob knockdown significantly suppressed the ability of FITC-OVA endocytosis (P < 0.01) and surface MHC-II molecule expression (P < 0.01) in BMDCs, promoting promoted Treg cell differentiation. Mannose receptor (MR) knockdown significantly upregulated miR-223-3p, downregulated Rhob protein in OVA-treated DCs, inhibited the FITC-OVA endocytosis and surface MHC-II expression in BMDCs, and promoted Treg cell differentiation (all P < 0.01). Conclusion These data suggest that miR-223-3p has an inhibitory effect on the antigen uptake and presentation capacities of BMDCs and promotes Treg cell differentiation, which is, at least partially, through targeting MR signaling and Rhob.

MiR-146a Promotes Tolerogenic Properties of Dendritic Cells and through Targeting Notch1 Signaling

ABSTRACT Background MiR-146a has been shown to negatively regulate innate immune, inflammatory response and antiviral pathway, however, its role in the tolerogenic responses remains largely unknown. This study aimed to investigate the role of miR-146a in the OVA-induced allergic inflammation of dendritic cells (DCs). Methods Bone marrow-derived DCs (BMDCs) were treated with OVA (100 µg/ml) for 24 h. MiR-146a expressions were assessed by quantitative RT-PCR. BMDCs were transfected with miR-146a mimics or inhibitor. Cell surface markers were analyzed by flow cytometry. Cytokine levels were determined by ELISA assay. Mixed lymphocyte culture assay was adopted to assess CD4 + T-cell differentiation. The 3ʹ UTR luciferase reporter assay was utilized to determine the miRNA target sequence. Results OVA treatment significantly up-regulated miR-146a in BMDCs in a dose- and time-dependent manner. In the OVA-treated DCs, overexpression of miR-146a (mimics transfection) down-regulated the surface markers (CD80, CD86) and increased production of anti-inflammatory cytokines TGF-β1 and IL-10 but decreased pro-inflammatory cytokine IL-12. MiR-146a overexpression promoted immature DC to induce regulatory T cells (Treg) differentiation. By contrast, transfection of miR-146a inhibitor into DC exhibited the opposite trends. Notch1 was a direct target of miR-146a, and Notch1 knock-down induced similar effects as miR-146a mimics transfection in BMDCs. Moreover, the effect of miR-146a inhibitor on OVA-induced DC was attenuated by Notch1 knock-down. Conclusion miRNA-146a promoted tolerogenic properties of DCs, at least partially, through targeting Notch1 signaling.

OR35 A novel mixed lymphocyte culture method to evaluate donor HLA specific immunity in different HLA type mismatched donors and recipients

Aim A novel flow cytometry based mixed lymphocyte culture assay (Flow Cellular Crossmatch: Flow-CXM) was developed for detecting antigen or donor specific T cell immunity. The preliminary study demonstrated that the donor specific T cell response in recipient was correlated with HLA mismatch (MM) numbers between donors & recipients. The aim of this study is to further evaluate the significance of Flow-CXM in different HLA type MM donors and recipients. Methods CXM (Fig. 1): 0.1 × 106 irradiated donor PBMCs were incubated with 0.1 × 106 recipient’s PBMCs and Th1 (IFNγ & IL-2) or Th2 (IL-4, 5, 10) cytokine capture beads for 16 h. The cells were lysed and the captured cytokines were detected by corresponding PE-labeled 2nd antibodies. The beads with different fluorescence were gated and PE stained beads (Flowspot %) were counted to represent the relative number of donor specific responding T cells. HLA Typing High-resolution HLA typing of all donor and recipient samples were performed by NGS or SSO. Results A total 109 paired bone marrow donor and recipient samples were tested by Flow-CXM. Compared to the HLA identical group, IL-2 but not IFNγ response significantly increased in HLA-class I (CI) (P Conclusions The newly developed Flow-CXM is able to detect earlier T cell activation of donor HLA specific immunity in recipient within 24 h. The multiple T cell activation markers (Th1/Th2 cytokines) can be measured in a single reaction. The methodology of Flow-CXM is simple, sensitive, and HLA specific. It hold a potential to be used in clinical transplantation for predicting and monitoring GVHD/GVL in BMT and cellular rejection in solid organ transplantation. Download : Download high-res image (404KB) Download : Download full-size image

Immunomodulatory effects of mesenchymal stem cells derived from the bone marrow in acute leukemia patients

To study the immunomodulatory effects and mechanisms of mesenchymal stem cells (MSC) derived from the bone marrow in acute leukemia patients in vitro. Bone marrow mononuclear cells from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) were obtained and cultured in low serum medium. The immunophenotypes were assessed by FACS and immunol histochemistry. The levels of cytokines were evaluated by enzyme linked immunosorbant assay (ELISA). T-cell suppression ability was evaluated by Transwell chamber assay. Moreover, the immunoregulatory ability of AML- and ALL-derived MSC was detected by mixed lymphocyte culture assay. ALL-derived MSC showed a typical fibroblast-like morphology. They were positive for CD29, CD44 and CD105, the positive rate were 98.81%, 99.25% and 90.52%, respectively, while negative for CD31, CD45 and CD34. Moreover, ALL- and AML-derived MSC didn't express HLA-DR and co-stirnulatory molecules (CD40, CD80 and CD86). ALL and AML derived MSC could secret several cytokines, such as TGF-β1 (567.58 ± 52.64 and 357.15 ± 33.52), HGF (647.27 ± 102.54 and 219.67 ± 62.37), IL-6 (59.67 ± 15.69 and 54.35 ± 12.31) and IL-11 (102.58 ± 23.54 and 78.21 ± 9.67), the level of secretion of TGF-β1 and HGF were higher in ALL bone marrow derived MSC than that of in AML bone marrow derived MSC. ALL and AML derived MSC significantly suppressed T lymphocyte proliferation in a dose-dependent manner, the counts per minute (CPM) were (3.58 ± 0.54) × 10(4), (2.87 ± 0.33) × 10(4), (1.78 ± 0.51) × 10(4) and (1.15 ± 0.15) × 10(4) for AML derived MSC, and CPM were (1.96 ± 0.31) × 10(4), (1.57 ± 0.28) × 10(4), (0.91 ± 0.41) × 10(4) and (0.22 ± 0.11) × 10(4) for ALL derived MSC when MSC were 0.5 × 10(4), 1 × 10(4), 2 × 10(4) and 5 × 10(4). In addition, the CPM was (4.01 ± 0.72) × 10(4) in control group. The immunosuppressive ability was different between MSCs derived from AML and ALL. The immunosuppressive effect of ALL derived MSC could be reversed by anti-TGF-β1 and anti-HGF antibody. ALL-derived MSC show immunoregulatory effect in vitro and this effect is achieved through cytokines. But MSCs derived from AML display abnormal changes in T-cell suppression ability.

A study of the immune properties of human umbilical cord lining epithelial cells

Background aimsOur previous study has demonstrated the stem cell-like properties of human umbilical cord lining epithelial cells (CLECs) and their capability for epidermal reconstitution in organotypic skin culture; however, the immunogenicity of these cells has not been clearly defined. We assessed several aspects of the immune properties of CLECs in vitro. MethodsWe examined CLECs for their immunoregulatory function in a mixed lymphocyte culture experiment. We characterized the expression patterns of the major histocompatibility complex (MHC), co-stimulatory molecules and the pro-/anti-inflammatory cytokines and growth factors in CLECs by means of reverse transcription-polymerase chain reaction, Western blotting, flow cytometry and FlowCytomix multiple analyte detection assays. ResultsCLECs were found not to induce but to suppress the proliferation response of the peripheral blood mononuclear cells in a mixed lymphocyte culture assay. They did not express the MHC class II antigen HLA-DR but the non-classic MHC class I antigens HLA-G and HLA-E and lacked the expression of the co-stimulatory molecules CD40, CD80 and CD86. In addition, they produced less interleukin-1β and transforming growth factor-β1 but more interleukin-4 and hepatocyte growth factor than did adult keratinocytes, a pattern in favor of wound healing with less inflammation response. ConclusionsOur data suggest that CLECs have an immunosuppressive function in addition to their low immunogenicity. This could be at least partially explained by their expression of HLA-G and HLA-E molecules associated with immune tolerance and absence of HLA-DR and co-stimulatory molecules. The demonstration that CLECs produce a favorable pattern of cytokines and growth factors for wound healing provides further support for their potential clinical application in allogeneic cell therapy.

Human UCB CD133+ Cell Infusion In NOD-SCID Mice with Acute Hind Limb Ischemia Improves Reperfusion of An Ischemic Bed

AbstractAbstract 4697Clinical trials have shown intracoronary or intramuscular injections of hematopoietic stem cells (HSC) ameliorate vascular ischemia. HSCs are isolated from bone marrow (BM), umbilical cord blood (UCB), and adult peripheral blood (AB). Critical limb ischemia patients, injected with autologous HSC into ischemic tissue had improved perfusion and reduced amputations. However, autologous HSC effectiveness declines with age. We hypothesized that UCB CD133+ HSCs' are more efficacious than autologous HSC at revascularization for an ischemic bed.Immune reactive surface antigens on UCB CD133+ cells were determined using multi-parameter flow cytometry. UCB CD133+ allogeneic immune responses from 72 h co-cultures were analyzed using a mixed lymphocyte culture (MLR) proliferation assays, and induced cytokine production found using TH1(inflammatory)/TH2(anti-inflammatory) cytometric bead assays (CBA) (BD Biosciences (San Jose, CA). In vivo studies were done using NOD/SCID mice treated with low-dose irradiation (200cGy) then femoral ligation. Study mice were divided into 3 treatment groups and underwent intracoronary infusions of; 1×106 UCB CD133+, 0.5×106 UCB mononuclear cells (MNCs), or EGM media alone. Day 3 post-ligation and cell infusion, quadriceps from 3 mice in each group were harvested, total RNA isolated using Trizol™ and RNA for each treatment group pooled. Pooled RNA was converted to cDNA then assayed using mouse ABI Affymetrix™ Gene chip. Computational pathway analyzes were run for significant transcriptome changes. Gene array data was confirmed on select genes using quantitative reverse transcriptase real time PCR (qPCR). Treated mice were followed for ischemic bed reperfusion by Laser Doppler imaging for 28 days post-ligation. UCB CD133+ cells express MHC class I and II, but lack CD80 and CD86. Allogeneic MLR assays using UCB and AB MNCs showed UCB CD133+ cells were stimulatory to MNC proliferation. The MNCs alloresponse to UCB CD133+ cells produced high concentration of interleukin 4 (IL-4) and IL-10 indicative of a TH2 response. These results show allogeneic UCB CD133+ cells induce a TH2 immune response due to an absence of co-stimulation of immune cells. To verify whether the results are recapitulated in vivo, we treated acute limb ischemia in NOD/SCID mice with human UCB CD133+ cells and monitored revascularization by blood flow and Affymetrix mouse gene array. Computational analysis of Affymetrix mouse gene arrays from comparing all 3 treatment protocols indicated that ischemic quadriceps treated with UCB CD133+ cells exhibited a significant enrichment of anti-angiogeneic, chemotaxic, cytokine, and immune response genes. UCB CD133+ treated mice had increase expression of anti-inflammatory, and anti-angiogeneic genes versus controls. Conversely media and UCB MNC treated mice had equal to or higher pro-inflammatory gene expression. The anti-inflammatory gene response was confirmed for 2 of the 3 genes, arginase 1, CXCL7, and tumor growth factor beta binding protein 2 (TGFβBP2) comparing CD133+ treated mice to UCB MNC treated mice. TGFβBP2 was not observed to change in vivo. UCB CD133+ cells treated mice also had improved blood perfusion of the ischemic bed at 28 days post-ligation versus control protocols. The in vivo mouse model supports the in vitro observation, but demonstrates improved reperfusion over controls.Currently there are 2 non-exclusive hypotheses for the function of CD133+ cells in revascularization. First the CD133+ cells integrate and differentiate into new vascular endothelial cells and the second is that the CD133+ cells augment revascularization via a paracrine effect which does not require integration into the host tissue. In vitro studies suggest that UCB CD133+ cells promote an allogeneic TH2 type response because of a lack of co-stimulatory immune surface antigens, resulting in high concentrations of IL-4 and IL-10. This skewed TH2 response inhibits inflammation, the subsequent tissue damage, and may augment revascularization. Although these studies cannot exclude a role for UCB CD133+ cellular integration, results support the paracrine hypothesis as infusion of selected CD133+ cells appears to promote an anti-inflammatory, anti-angiogenic profile at day 3 post-ligation and at 1 month improved reperfusion compared with non-selected MNC. Disclosures:No relevant conflicts of interest to declare.

Clinical Expansion of Cord Blood-derived T Cells for Use as Donor Lymphocyte Infusion After Cord Blood Transplantation

Allogeneic stem cell transplantation (SCT) from cord blood (CB) as a stem cell source is a promising alternative when no human leukocyte antigen-matched donor is found. Donor lymphocyte infusion (DLI) is a possible treatment modality for threatening graft failure or relapse of an underlying malignancy after transplantation. Ethical and logistical reasons limit the possibility of DLI in the setting of CB SCT. To remedy this restriction, we performed expansion of donor T cells in vitro from CB grafts in a clinical setting for use as future DLI and characterized the expanded cells in comparison to T cells from CB acquired ex vivo and adult peripheral blood. T cells were expanded from grafts used for transplantation, upon CD3/CD28 crosslinking and culture in interleukin-2. Phenotype and function of T cells were assessed by flow cytometry and mixed lymphocyte culture assays. T-cell receptor repertoire distribution was evaluated with polymerase chain reaction-based spectratyping. We were able to amplify T cells to sufficient amounts for DLI in 13 out of 13 initiated expansions. Expanded T cells presented with an activated phenotype and could be induced to produce cytokines by a nonspecific stimulus. When exposed to allogeneic targets, expanded CB T cells proliferated at comparable levels to their ex vivo and adult blood counterparts. In summary, clinical expansion of CB T cells for DLI is feasible and may be a future modality for treatment of graft failure or relapse after SCT.

Matrix Metalloproteinase Proteolysis of the Myelin Basic Protein Isoforms Is a Source of Immunogenic Peptides in Autoimmune Multiple Sclerosis

BackgroundMatrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination leading to autoimmune multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The classic MBP isoforms are predominantly expressed in the oligodendrocytes of the CNS. The splice variants of the single MBP gene (Golli-MBP BG21 and J37) are widely expressed in the neurons and also in the immune cells. The relative contribution of the individual MMPs to the MBP cleavage is not known.Methodology/Principal FindingsTo elucidate which MMP plays the primary role in cleaving MBP, we determined the efficiency of MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP and MT6-MMP in the cleavage of the MBP, BG21 and J37 isoforms in the in vitro cleavage reactions followed by mass-spectroscopy analysis of the cleavage fragments. As a result, we identified the MMP cleavage sites and the sequence of the resulting fragments. We determined that MBP, BG21 and J37 are highly sensitive to redundant MMP proteolysis. MT6-MMP (initially called leukolysin), however, was superior over all of the other MMPs in cleaving the MBP isoforms. Using the mixed lymphocyte culture assay, we demonstrated that MT6-MMP proteolysis of the MBP isoforms readily generated, with a near quantitative yield, the immunogenic N-terminal 1–15 MBP peptide. This peptide selectively stimulated the proliferation of the PGPR7.5 T cell clone isolated from mice with EAE and specific for the 1–15 MBP fragment presented in the MHC H-2U context.Conclusions/SignificanceIn sum, our biochemical observations led us to hypothesize that MT6-MMP, which is activated by furin and associated with the lipid rafts, plays an important role in MS pathology and that MT6-MMP is a novel and promising drug target in MS especially when compared with other individual MMPs.

IVIG effects on immature dendritic cells of Common Variable Immunodeficiency Patients

Previous studies have suggested that lyophilized IGIV may inhibit dendritic cell (DC) maturation. We investigated the effects of a newer liquid formulation of IGIV on the peripheral blood monocyte (PBMC)-induced differentiation and function of DC in healthy subjects and CVID patients. Immature DCs were derived from PBMC by negative selection using magnetic beads from five adult CVID patients and six adult healthy subjects. PBMC were incubated with IL-4 and GM-CSF to induce DC maturation. IGIV (0.15 mM Gamunex), DMSO, or BSA was added to cultures. Flow cytometry was performed to analyze the differentiation of PBMC into immature DC using the following monoclonal antibodies: CD14, CD1a, CD40, CD83, CD80, CD86, HLA-DR. DC function was evaluated by lucifer yellow and dextran sulfate for antigen uptake, and allogeneic T-cell responses in mixed lymphocyte culture (MLC) assay for antigen processing. There were no significant differences in the expression of immature DC markers between controls vs. CVID patients. IGIV upregulated CD86 expression (p < 0.001) and CD83 (p < 0.001) in both groups. IGIV suppressed lucifer yellow uptake of imDC but enhanced allogeneic T-cell proliferation in MLC. We were unable to identify any defects in the DC differentiation pathway of PBMC in our CVID patients. Our results suggest that liquid 10% IGIV promotes monocyte-derived DC maturation in vitro to a more mature functional phenotype, in contrast to lyophilized IGIV, as shown by the upregulation of CD83 and CD86, and the enhanced antigen processing ability in MLC.

Human herpesvirus 8 (HHV-8) inhibits monocyte differentiation into dendritic cells and impairs their immunostimulatory activity

Several viruses interfere with the host immune response by infecting dendritic cells and by altering their functional activity. Here, we report that exposure to Human herpesvirus 8 (HHV-8) of human dendritic cell (DC) monocyte precursors resulted in impaired immature DC (iDC) formation as indicated by a reduced CD1a expression. In accordance, the immunostimulatory ability of such iDC was significantly reduced, as indicated by mixed lymphocyte culture (MLR) assays. The immunostimulatory functions of DCs were similarly inhibited by the UV inactivated viral stocks, suggesting that the virus binding is sufficient to determine the observed effect. Furthermore, HHV8 mediated inhibition of the DC allostimulatory function was present in lipopolysaccharide (LPS) matured DCs. A strong reduction of the expression of the costimulatory molecule CD80 on the surface of the virus-exposed cells was observed as well. Impairment of dendritic cell development and function might represent an important strategy used by HHV-8 to escape from the host defense mechanisms.

Survival of bone marrow‐derived mesenchymal stem cells in a xenotransplantation model

Mesenchymal stem cells (MSCs) are immunoprivileged and the allogeneic MSCs implantation has been used to facilitate tissue repairs such as bone and cartilage defect. The present study aimed to investigate the feasibility of xenogeneic MSCs implantation. Green fluorescent protein (GFP) transgenic rat bone marrow-derived MSCs were loaded into HA/TCP Skelite blocks and implanted intramuscularly into the quadriceps of the MF1 and SCID mice. After 11 weeks, the implants were harvested and processed for further examinations. The peripheral blood mononuclear cells of each animal were also collected to measure the in vitro immune responses using mixed lymphocyte culture and cytotoxic assay. In the MF1 mice, some surviving MSCs were found in the explants after 11 weeks of implantation, but there was no sign of new bone formation as neither osteocalcin mRNA nor osteoid tissues were detected in the explants; the lymphocyte proliferation and cytotoxicity against donor MSCs were significantly increased in the animals with the xenogeneic MSCs implantation compared with the control littermates without transplantation. In the control SCID mice, osteoid tissues derived from the implanted MSCs were found in the explants; no difference of lymphocyte proliferation and cytotoxicity against the donor MSCs was detected between the SCID mice with or without MSCs implantation. The data suggested that rat MSCs survived the 11 weeks of xenotransplantation in the MF1 mice, but the increased host immune sensitization led to the impaired in vivo osteogenesis potential of MSCs.

High-Dose Methotrexate (MTX) as Part of a Novel Immunosuppressive Regimen in Canine MHC-Haploidentical Hematopoietic Cell Transplantation (HCT).

In a preclinical canine model using major histocompatibility (MHC)-haploidentical littermate peripheral blood stem cell (PBSC) grafts, initial donor chimerism could be achieved after a nonmyeloablative conditioning regimen consisting of monoclonal antibody S5 (anti-CD44) from days −7 to −2, 200 cGy TBI on day 0, and MMF and cyclosporine for postgrafting immunosuppression. However, sustainable donor chimerisms could not be achieved in many of these dogs, with 25 of 44 (57%) rejecting their grafts at a median of 7 weeks after transplant. In order to improve the engraftment rate, a novel strategy to overcome the immunological barrier created by this MHC-haploidentical setting was developed. By adding single, progressively higher doses of MTX on day+3 after transplantation to the above regimen, we hoped to control host T and NK cells, both of which contribute to graft rejection in MHC-haploidentical HCT, and to target alloreactive donor T cells which are responsible for graft-versus-host disease (GVHD). Once dose toxicity is established, this system will also allow the study of transplants using gene-modified, MTX-resistant CD34 selected PBSC, which can provide additional intrinsic graft protection on exposure to MTX. To date, 11 dogs have been transplanted using the above regimen and adding either 50 (n=5), 100 (n=3), or 200 (n=3) mg/m2 MTX on day +3 followed by leucovorin rescue on days +4 and +5. Early results showed that 4 of 5 dogs experienced graft rejection in the MTX50 group, 0 of 3 dogs rejected in MTX100 (p=0.047, Monte Carlo), and all 3 dogs in MTX200 have continued to exhibit stable mixed chimerisms from +42 to +76 days after transplant. Dogs in all 3 cohorts had rapid engraftment within 1 week, with similar average peak mononuclear cell (MNC) chimerisms at comparable timepoints (83% at 5 wks, 88% at 6 wks, and 80% at 5 wks at MTX50, 100, and 200 respectively). None of the 11 dogs have shown evidence of GVHD or MTX toxicity, and all dogs cleared MTX within 48 hours. All dogs exhibited early donor tolerance on mixed lymphocyte culture assays within 2 weeks and preserved NK activity posttransplant. While these data are preliminary, adding single, progressively larger doses of MTX on day +3 to a novel, nonmyeloablative conditioning regimen appeared to improve engraftment among MHC-haploidentical canine recipients with no signs of GVHD. There was high initial donor chimerism without undue early toxicity. Further, this study will aid in our establishment of high-dose, posttransplant MTX as a way of promoting sustained donor chimerism after transplanting CD34 selected, MTX-resistant canine PBSC in the MHC-haploidentical setting. This preclinical data serve as a basis for developing nonmyeloablative transplant options for patients without HLA-identical donors.

The Role of Selective Depletion of Alloreactive Donor T Cells in Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation.

One of the main goals in allogeneic bone marrow(BM) transplantation is the abrogation of graft-versus-host disease (GVHD) with the preservation of antileukemia and antiviral activity. The Study present a selective T cell depletion strategy based on the physical separation of the alloreactive T cells, which were identified by expression of two activation-induced antigens (CD25 and CD69). T cells from C57BL/6(H-2b) mice were first activated with BALB/c (H-2d) recipient spleen cells in a 2-day mixed-lymphocyte-culture (MLC). Following this activation, this compound is selectively depleted based on expression of two activation-induced antigens CD25 and CD69 using magnetic cell sorting. The depleted cells or the untreated cells were then rechallenged respectively in a secondary MLC, with the same stimulator cells or a third-party (DBAH-2k) or tumor- specific (SP2/0, BALB/c-origin myeloma) cells. Cells proliferation were assayed at the indicated time points(1, 2, 3, 4, 5 days). These treated cells or control-cultured cells (2.0×106) mixed with 5.0×106 BM cells from C57BL/6 were transfused respectively by the trail vain into the lethally irradiated BALB/c to observe the survival time, GVHD incidence and pathological analysis. MLC assays demonstrated that this technique led to a significant decrease in alloreactivity of donor cells(29.02~64.17%), which at the same time preserved reactivity against third party cells(49.61~75.69%)and anti-tumor cells(61.14~68.62%). The mice in the group of control-coclutured were died of acute GVHD within 24days. The 7 recipient mice in the treated group were free of acute GVHD, and 3 mice were died of acute GVHD (aGVHD) within 23 days. MACS-based ex-vivo depletion of alloreactive donor T cells based on expression of two activation-induced antigens (CD25 and CD69) could inhibit anti-host responses, by contrast, anti-SP2/O and anti-third-party responses were preserved. Cotransplantation of these selected depleted cells and BM cells could reduce aGVHD.

Comparative Immunotoxicity Evaluation of Amphotericin B and ABELCET®, an Amphotericin B Lipid Complex (ABLC)

ABELCET, an amphotericin B lipid complex formulation (ABLC) and an aqueous, non-lipid-containing formulation with sodium deoxycholate (AmBd), were evaluated for their potential to induce immunotoxicity in B6C3F1 female mice. ABLC was administered intravenously at doses of 1, 3, and 10 mg/kg daily for 28 days, while AmBd at 1 mg/kg was administered by the same route and duration. The effect of ABLC and AmBd on clinical signs, body weight, and spleen weight was determined. Peritoneal macrophage function was measured by phagocytosis of 51Cr-labeled chicken red blood cells and generation of hydrogen peroxide during respiratory burst. The ability of natural killer cells to lyse radiolabeled tumor target cells was evaluated in a short-term chromium-release assay. The ability of splenic T and B cells to undergo blastogenesis and of splenic T cells to recognize alloantigens present on foreign cells was assessed in a splenic lymphocyte assay and the ability of mice to generate antibody-forming cells following immunization with sheep red blood cells was measured. Neither ABLC nor AmBd affected the metabolic or functional activity of murine phagocytic cells. These agents also did not cause any biologically significant or dose-related changes in B- or T-cell responses to mitogens, T-cell responses to allogeneic cells in the mixed lymphocyte culture assay, or natural killer cell function. The ability to generate a primary antibody response to a T cell-dependent antigen was also unimpaired. Based on the results of this study, it was concluded that neither ABLC at dose up to 10 mg/kg nor AmBd at dose up to 1.0 mg/kg produce biologically significant immunologic changes in B6C3F1 mice.

Effect of interleukin-15 on alloreactivity in umbilical cord blood

Interleukin(IL)-15 is a promising immunotherapeutic agent for immune reconstitution following stem cell transplantation. To investigate whether IL-15 would aggravate graft-versus-host disease (GVHD) in the setting of unrelated umbilical cord blood (CB) transplantation, we examined the effect of IL-15 on activation marker expression, proliferation and cytokine production of CB in a one-way mixed lymphocyte culture (MLC) assay. We found that IL-15 differentially enhanced CD69 and CD25 expression on CB T cells following allo-stimulation. The maximum degree of allo-specific CB proliferation was achieved on Day 6. IL-15 down-regulated the CB alloreactive proliferative response on Days 4, 6, and 8, with preferentially enhanced autologous proliferation. Exogenous IL-15 further enhanced CB TNF-α and IL-10 production in both autologous and allogeneic MLC 6 days after allopriming. Thus, IL-15 was effective in enhancing activation marker expression and cytokine production during CB alloreactivity, but failed to enhance allospecific proliferation. Further studies would be needed to study the role of IL-15 on GVHD in the setting of CB transplantation.

Identification of non-phosphate-containing small molecular weight inhibitors of the tyrosine kinase p56 Lck SH2 domain via in silico screening against the pY + 3 binding site.

The protein p56 lymphoid T cell tyrosine kinase (Lck) is predominantly expressed in T lymphocytes where it plays a critical role in T-cell-mediated immune response. Lck participates in phosphotyrosine-dependent protein-protein interactions through its modular binding unit, the Src homology-2 (SH2) domain. Accordingly, virtual screening methods combined with experimental assays were used to identify small molecular weight nonpeptidic compounds that block Lck SH2 domain-dependent interactions. Virtual screening included scoring normalization procedures and postdocking structural clustering that is shown to facilitate the selection of active compounds. By targeting the well-defined hydrophobic binding pocket known to impart specificity on Lck-protein interactions (i.e., pY + 3 site), inhibitors of the Lck SH2 domain were discovered that omit the phosphotyrosine (pY) or related moieties. The 34 out of 196 computationally selected compounds were shown to inhibit Lck SH2 domain association with phosphorylated immunoreceptor tyrosine based activation motifs peptide. Twenty-four of the active compounds were further tested for their ability to modulate biological function. Thirteen of these compounds showed inhibitory activity in mixed lymphocyte culture assay. Fluorescence titration experiments on four of these active compounds further verified their binding to the SH2 domain. Because of their simple chemical structures, these small organic compounds have the potential to act as lead compounds for the development of novel immunosuppressant drugs.

Embryonic stem cells as an alternate marrow donor source: engraftment without graft-versus-host disease.

A single embryonic stem cell (ESC) line can be repetitively cryopreserved, thawed, expanded, and differentiated into various cellular components serving as a potentially renewable and well-characterized stem cell source. Therefore, we determined whether ESCs could be used to reconstitute marrow and blood in major histocompatibility complex (MHC)-mismatched mice. To induce differentiation toward hematopoietic stem cells (HSCs) in vitro, ESCs were cultured in methylcellulose with stem cell factor, interleukin (IL)-3, and IL-6. ESC-derived, cytokine-induced HSCs (c-kit+/CD45+) were isolated by flow cytometry and injected either intra bone marrow or intravenously into lethally irradiated MHC-mismatched recipient mice. From 2 wk to 6 mo after injection, the peripheral blood demonstrated increasing ESC-derived mononuclear cells that included donor-derived T and B lymphocytes, monocytes, and granulocytes without clinical or histologic evidence of graft-versus-host disease (GVHD). Mixed lymphocyte culture assays demonstrated T cell tolerance to both recipient and donor but intact third party proliferative responses and interferon γ production. ESCs might be used as a renewable alternate marrow donor source that reconstitutes hematopoiesis with intact immune responsiveness without GVHD despite crossing MHC barriers.