Allogeneic T-cell Proliferation Research Articles

ANALYSIS OF THE RESPONSE OF HUMAN NEONATAL DENDRITICS CELLS AND MACROPHAGES AGAINST ROTAVIRUS (137.26)

Abstract Rotavirus (RV) is the most common cause of infectious diarrhea. Dendritic cells (DCs) and macrophages play a crucial role in the immune response against viral infections; therefore it is important to dissect the interaction of RV with these cells. Here we analyzed the interaction of RV with neonatal DCs and macrophages in an in vitro model. Cord blood-derived mononuclear cells were culture in vitro for 6 days in the presence of either hGM-CSF/IL-4 or hM-CSF to generated enriched DCs and macrophages, respectively. These cells were cultured for 24 hrs with rotavirus and the levels of the activation markers CD40, CD86 and MHC class-II, and the intracellular cytokines IL-6, IL-12 and IL-10 were analyzed by flow cytometry. TGF-β was evaluated by ELISA. Finally, the induction of allogenic T-celll proliferation was analyzed. We found that RV could induce the activation of the neonatal DCs and macrophages since all the activation markers analyzed were up regulated, albeit at different levels. As result of this activation, the neonatal DCs and macrophages produced high levels of the regulatory cytokines, but moderate levels of pro-inflammatory cytokines. Finally, the activated DCs and macrophages were able to induce a strong allogenic T-cell proliferation. These results showed that neonatal DCs and macrophages could be activated by RV and that this activation was sufficient to induce the priming of allogenic T-cells. Supported by grant SEP-CONACYT (80149) and DGAPA, UNAM.

Interferon-α and Interleukin-6 in SLE serum induce the differentiation and maturation of dendritic cells derived from CD34+ hematopoietic precursor cells

The objective of this study was to investigate the effects of the cytokines IFN-alpha, IL-6 and IL-10 present in the serum of systemic lupus erythematosus (SLE) patients on the differentiation and maturation of DCs derived from CD34(+) hematopoietic precursor cells (HPCs). CD34(+) HPCs cultured in SLE serum containing elevated levels of IFN-alpha or IL-6 differentiated into DCs showing an increased expression of HLA-DR, CD80 and CD86, decreased IL-12 secretion along with increased IL-10 production, and an increased ability to stimulate allogenic T-cell proliferation, compared with DCs cultured in normal serum. DCs cultured with high levels of IFN-alpha increased the proportion of both CD3(+)CD8(-)IFN-gamma(+) and CD3(+)CD8(+)IFN-gamma(+) T-cell subsets, and increased the production of IFN-gamma in the allogenic MLR. DCs cultured with high levels of IL-6 decreased the proportion of both IFN-gamma(+) T-cell subsets, and decreased the secretion of IFN-gamma, but increased the production of IL-10. The IL-10 present in SLE serum did not significantly alter the phenotype or function of the DCs. IFN-alpha and IL-6 present in SLE serum induce CD34(+) HPCs to differentiate into DCs with different regulatory effects on T-cell differentiation, which might be involved in the initiation and maintenance of SLE.

Dendritic cells transduced with Rsf-1/HBXAP gene generate specific cytotoxic T lymphocytes against ovarian cancer in vitro

Recently, some studies have indicated that Rsf-1/HBXAP plays a role in chromatin remodeling and transcriptional regulation that may contribute to tumorigenesis in ovarian cancer. The present study demonstrates that using dendritic cells (DCs) from human cord blood CD34 + cells transduced with Rsf-1/HBXAP DNA plasmids by nucleofection generate specific cytotoxic T lymphocytes (CTL) against ovarian cancer in vitro. After transfection, DCs were analyzed for Rsf-1/HBXAP mRNA expression by RT-PCR and protein expression by Western blot. Then the DC phenotypes, T-cell stimulatory capacity, endocytic activity and migration capacity were explored by flow cytometry analysis, allogeneic mixed lymphocyte reaction, endocytosis and transwell chemotaxis assay, respectively. After transfection, Rsf-1/HBXAP expression was detected at mRNA and protein levels. Allogeneic T-cell proliferation induced by transfected DCs was obviously higher than non-transfected DCs, but the endocytosis capacity and migratory ability were not different. Rsf-1/HBXAP gene-transduced DCs could induce antigen-specific CTL and generate a very potent cytotoxicity to OVCAR3 cells. These data suggest that Rsf-1/HBXAP gene-transduced DCs may be a potential adjuvant immunotherapy for ovarian cancer in clinical applications.

Donor‐strain‐derived immature dendritic cell pre‐treatment induced hyporesponsiveness against allogeneic antigens

The maturation of antigen-presenting dendritic cells (DCs) serves as an important determinant for the regulation of immunity, and overall immune response. We hypothesized that a reduced immune response to donor alloantigens and improved allograft survival could be induced by pre-treating recipients with bone-marrow-derived donor-strain fixed immature DCs (FIDCs). Donor-strain-derived mature and immature DCs were fixed before grafting to ensure that they possessed a stable immunogenic phenotype. The fixed mature DCs effectively induced allogeneic T-cell proliferation in recipients, whereas FIDCs were unable to elicit an allogeneic T-cell response. T cells that had previously been exposed to FIDCs maintained naïve phenotypes and were unable to extensively divide after injection into lethally irradiated donor-strain mice. The pre-treatment of recipients with donor-strain FIDCs markedly prolonged the survival of islet as well as skin allografts. However, T-cell hyporesponsiveness induced by FIDC injection was abrogated by the depletion of CD4(+) CD25(+) T cells. Consequently, FIDC-induced T-cell hyporesponsiveness could reflect anergy rather than specific deletion. Our findings suggest that FIDCs of donor strain could be used to induce long-term graft survival.

Highlights of This Issue

Chemotherapy is used to treat metastatic/recurrent nasopharyngeal carcinoma (NPC), but there is a need for reliable biomarkers to predict treatment outcome early. Wang and colleagues explored the role of Epstein-Barr virus (EBV) clearance rate by quantifying serial changes of circulating EBV DNA concentration in NPC patients undergoing chemotherapy. They found that the rate of EBV clearance within the first month of treatment reflects chemosensitivity and predicts tumor response and patient survival. These results suggest that clearance of EBV DNA could be used as an early tumor response marker, and NPC patients with a slow plasma EBV DNA clearance rate may be considered for a chemotherapy regimen change.Cancer stem cell (CSC)–targeted therapeutic interventions, such as immunotherapy, hold promise for treatment of human cancers. Di Tomaso and colleagues performed an immunobiological characterization of a large panel of glioblastoma multiforme (GBM) CSC lines and paired non-CSC lines cultured in FBS. They found that T-cell responses against CSCs pretreated with IFN-γ could be induced in vitro. Notably, CSCs, but not autologous FBS-cultured cells, could inhibit allogeneic T-cell proliferation. These results could help to identify GBM patients with CSCs that can be targeted by immunotherapy.Mechanism-driven drug discovery relies on a thorough validation of molecular targets whose inhibition selectively kills cancer cells with a minimal effect on normal cells. In this study, Jia and colleagues identified and validated the SCF E3 ubiquitin ligase component SAG as a novel anticancer and radiosensitizing target. They found that SAG is overexpressed in a number of human primary cancers, and that silencing SAG with siRNA selectively kills cancer cells and sensitizes radioresistant cancer cells. Thus, development of small molecule inhibitors or RNAi-based therapy targeting SAG is a promising cancer treatment strategy.GMX1777 is a novel inhibitor of nicotinamide phosphoribosyl-transferase, an enzyme involved in NAD biosynthesis. Kato and colleagues used in vitro and in vivo models of head and neck cancer (HNC) to show that GMX1777 can enhance radiation efficacy via interference with DNA repair and tumor angiogenesis. Further, FDG-PET imaging corroborated the metabolic shutdown in these tumors. This study illustrates a potential therapeutic opportunity in targeting tumor metabolism, and suggests that GMX1777 plus radiotherapy warrants further clinical evaluation in patients with HNC.

Evidence for the Involvement of Galectin-3 in Mesenchymal Stem Cell Suppression of Allogeneic T-Cell Proliferation

Human bone marrow-derived mesenchymal stem cells (MSC) are multipotent non-hematopoietic progenitors that have regulatory activity on immune cells. NOD- and Toll-like receptors (NLR, TLR) have several roles in immunity, including those relevant to pathogen recognition and shaping the course of immune responses by controlling gene expression. We have shown that these innate immune receptors are expressed by hematopoietic CD34+ progenitors and MSC. To uncover genes critical in MSC function, first we have used microarray to screen for potential transcripts whose levels are altered in response to NOD-1 and TLR-2 activation, and second we validated some candidate genes using real-time RT-PCR, Western blots and cellular assays. Amongst the altered genes, galectin-3 was upregulated at both mRNA and protein levels in response to TLR-2 activation. Interestingly, MSC secreted galectin-3, a protein known to modulate T-cell proliferation, gene expression, cell adhesion and migration. Knockdown of galectin-3 in MSC using small interfering RNA (siRNA) reduced the immunosuppressive effect of MSC on mixed lymphocyte cultures when compared to cells treated with an irrelevant siRNA (P < 0.05). Collectively, the data emphasize a new role of galectin-3 in the immunomodulatory function of MSC and indicate that NOD signalling pathway is also functional in these cells.

Immunomodulatory effects of vitamin D: implications for GVHD

GVHD remains a major source of morbidity and mortality after allogeneic BMT. GVHD is mediated by alloreactive T cells derived from the hematopoietic graft that target host tissues. Pre-clinical models have shown that presentation of alloantigens by host DCs results in the activation of donor-derived T cells that mediate GVHD. Strategies that interfere with the Ag-presenting capacity of DCs after allogeneic transplantation may decrease the risk of developing GVHD. Vitamin D is a hormone essential for calcium metabolism that shows immunomodulatory properties. We showed that correction of vitamin D deficiency appeared to mitigate manifestations of GVHD. In pre-clinical studies, we have shown that vitamin D inhibits DC maturation, polarizes T-cell populations toward the expression of Th2 as compared with Th1 cytokines, and blunts allogeneic T-cell proliferation in response to DC stimulation. Exposure to vitamin D resulted in increased expression of IDO, an enzyme responsible for tryptophan metabolism that is upregulated in tolerizing DCs. These data suggest that exposure to vitamin D results in immature DC populations that bias toward tolerizing rather than stimulatory T-cell populations. Vitamin D may therefore have a role in the prevention of GVHD.

Patients with oral squamous cell carcinoma are characterized by increased frequency of suppressive regulatory T cells in the blood and tumor microenvironment.

Oral squamous cell carcinoma (OSCC) is a cancerous lesion with high incidence worldwide. The immunoregulatory events leading to OSCC persistence remain to be elucidated. Our hypothesis is that regulatory T cells (Tregs) are important to obstruct antitumor immune responses in patients with OSCC. In the present study, we investigated the frequency, phenotype, and activity of Tregs from blood and lesions of patients with OSCC. Our data showed that >80% of CD4(+)CD25(+) T cells isolated from PBMC and tumor sites express FoxP3. Also, these cells express surface Treg markers, such as GITR, CD45RO, CD69, LAP, CTLA-4, CCR4, and IL-10. Purified CD4(+)CD25(+) T cells exhibited stronger suppressive activity inhibiting allogeneic T-cell proliferation and IFN-gamma production when compared with CD4(+)CD25(+) T cells isolated from healthy individuals. Interestingly, approximately 25% of CD4(+)CD25(-) T cells of PBMC from patients also expressed FoxP3 and, although these cells weakly suppress allogeneic T cells proliferative response, they inhibited IFN-gamma and induced IL-10 and TGF-beta secretion in these co-cultures. Thus, our data show that Treg cells are present in OSCC lesions and PBMC, and these cells appear to suppress immune responses both systemically and in the tumor microenvironment.

Indoleamine 2,3-Dioxygenase Is Expressed and Functionally Active in Human Dermal Dendritic Cells, but Not in Epidermal Langherans Cells.

Abstract 278Indoleamine 2,3-dioxigenase (IDO) is the rate-limiting enzyme in tryptophan catabolism along the kynurenine pathway. IDO expression by different cell subsets inhibits T-cell activation, proliferation and survival and induces regulatory T cells (Tregs). Although human monocyte-derived dendritic cells have been shown to express IDO, little is known about its expression in other subsets of human DCs, including those generated from CD34+ hematopoietic progenitors (CD34+-derived DCs). In particular, no data are currently available for IDO expression in CD34+-derived DC subsets, such as dermal DCs and epidermal Langherans cells (LCs), which are physiologically resident DCs in the skin and act as the main sentinels against pathogens. In the present study, we performed a full characterization of IDO expression and function by human DCs generated from CD34+ cells. CD34+-derived DCs were generated from purified CD34+ cells after 7 days of culture with GM-CSF and TNF-alpha. In some experiments, day 7-DCs were sorted into different subsets and tested for IDO expression and function. Alternatively, day 7-DCs were cultured with GM-CSF and IL-4 and then matured with a cytokine cocktail of maturation stimuli (IL-1beta, TNF-alpha, IL-6, PGE2). After culture, DCs were analyzed for IDO expression by real-time PCR and western immunoblot, kynurenine production, inhibition of allogenic proliferation and Tregs induction. CD34+ cells did not express IDO mRNA, without significant differences among different progenitor cell sources (cord blood, mobilized peripheral blood, bone marrow). During DC differentiation, IDO mRNA expression was observed at day 7, but not at day 14. At day 14, maturation stimuli induced a marked up-regulation of IDO mRNA and protein, which resulted in increased kynurenine production and inhibition of T-cell proliferation. Flow cytometry analysis of day-7 cells revealed a double population, which comprised CD14+CD1a− dermal DCs and CD14−CD1a+ LCs. Interestingly, IDO mRNA and protein were observed only in dermal DCs, but not in LCs. IDO expression by dermal DCs resulted in increased production of kynurenine and in reduced allostimulatory capacity of T-cell proliferation. Dermal DCs were shown to induce a population of CD4+CD25+Foxp3+ which acted as Tregs by inhibiting allogeneic T-cell proliferation. This effect was abrogated by the addition of the IDO inhibitor 1-methyl tryptophan. Interestingly, dermal DCs and LCs showed a differential expression pattern of chemokine receptors. In particular, while both LCs and dermal DC expressed CCR7, CXCR4 expression segregated only in IDO-expressing dermal DCs. These data may correlate to different trafficking features of CD34+-derived DC subsets and may be associated to IDO expression. In conclusion, DC differentiation of CD34+ cells results in the expression of a functionally active IDO protein in dermal DCs, but not in LCs. Given the role of IDO in regulating immune tolerance, our data may suggest that within the complex skin microenvironment dermal DCs are intrinsically committed to function as regulatory DCs, whereas LCs are devoted to act as activating DCs. These data have implications for a better understanding of the development of the immune response during inflammation/infection. Disclosures:No relevant conflicts of interest to declare.

Ovarian Cancer Cells Induce Peripheral Mature Dendritic Cells to Differentiate Into Macrophagelike Cells In Vitro

Precursors of dendritic cells (DCs) are able to differentiate into macrophages induced by some tumor-associated molecules; however, whether peripheral mature DCs could differentiate into macrophages remains unknown. This study was designed to find out whether ovarian cancer cells could induce peripheral mature DCs to differentiate into macrophages. Mature DCs were cultured from monocytes with granulocyte-macrophage colony-stimulating factor and interleukin 4 (IL-4) for 6 days and lipopolysaccharide for another 24 hours and then were cocultured for 48 hours with ovarian cancer ascites or cell-free supernatants of SKOV3 and CAOV3 cell lines. In some experiments, mature DCs were cultured in the absence or presence of IL-10 or leukemia inhibitory factor (LIF) for the same time. In neutralization experiments, neutralizing monoclonal antibodies to IL-10 or LIF were added to the cultures. Cell phenotypes and phagocytosis were analyzed using flow cytometry; allogeneic T-cell proliferation assay was used to examine stimulatory activity of cells. Mature DCs cocultured with ovarian cancer ascites or supernatants of SKOV3 and CAOV3 differentiated into a group of macrophagelike cells that exhibited increased expression of surface marker CD14+CD1a-, decreased expression of CD83, poorer T-cell costimulatory properties, and greater endocytosis of fluorescein isothiocyanate-dextran in vitro. Interleukin 10 but not LIF mediated this differentiation pathway.

Myeloid Dendritic Cells from Human Cutaneous Squamous Cell Carcinoma Are Poor Stimulators of T-Cell Proliferation

To determine the phenotype and function of myeloid dendritic cells (DCs) from human cutaneous squamous-cell carcinoma (SCC), we studied their surface marker expression and allo-stimulatory potential ex vivo. There were abundant CD11c(+) myeloid DCs, as well as TNF and inducible nitric oxide synthase (iNOS)-producing DCs, in and around SCC tumor nests. Although myeloid DCs from SCC, adjacent non-tumor-bearing skin, and normal skin, were phenotypically similar by flow cytometry, and there was a pronounced genomic signature of mature DCs in SCC, they showed different T-cell stimulatory potential in an allogeneic mixed leukocyte reaction. Myeloid DCs from SCC were less potent stimulators of allogeneic T-cell proliferation than DCs from non-tumor-bearing skin. Culture with a DC-maturing cytokine cocktail (IL-1beta, IL-6, TNF-alpha, and PGE(2)) enhanced stimulatory potential in DCs from non-tumor-bearing skin, whereas SCC-associated DCs remained poor stimulators of T-cell proliferation. The microenvironment associated with SCC showed expression of TGF-beta, IL-10, and VEGF-A, factors capable of suppressing the DC function. These findings indicate that CD11c(+)/HLA-DR(hi) DCs from SCC are mature, but are not potent stimulators of T-cell proliferation compared with phenotypically similar DCs isolated from non-tumor-bearing skin. Identification of mechanisms responsible for suppression of tumor-associated DCs may provide insight into the evasion of immunosurveillance by SCC.

Immunophenotypical, morphologic, and functional characterization of maturation‐associated plasmacytoid dendritic cell subsets in normal adult human bone marrow

Information about maturation of plasmacytoid dendritic cell precursors (pre-pDCs) in normal bone marrow (BM) remains limited. Immunophenotypical, morphologic, and functional changes associated with maturation of pre-pDCs were analyzed in adult normal human BM (n = 45). Three pre-pDC maturation stages, with an increasingly higher degree of maturity, were systematically identified: CD34++/HLA-DR++/+++/CD123++/CD45+/++ (Stage I), CD34+/HLA-DR+/++/CD123++/+++/CD45+/++ (Stage II), and CD34-/HLA-DR++/CD123++/+++/CD45++ (Stage III) cells. Lymphoid- and early myeloid-associated molecules, as well as CD86, were coexpressed in Stage I pre-pDCs, being down regulated afterward. CD304 and CD85j appeared in Stage I, progressively increasing their levels thereafter. Interestingly, cutaneous lymphocyte-associated antigen was heterogeneously expressed throughout the maturation, particularly in Stage I pre-pDCs. The morphologic appearance of Stage I pre-pDCs was consistent with their undifferentiated stage, while Stage II/III cells showed morphologic features of more differentiated cells. From the functional point of view, only Stage II and Stage III pre-pDCs were capable of inducing allogeneic T-cell proliferation, the later subset also showing interferon-g secretion; in contrast, Stage I pre-pDCs showed the highest endocytic ability. In summary, three maturation stages of pre-pDCs can be identified in adult normal BM, which show unique phenotypical, morphologic, and functional characteristics; these results provide a frame of reference for a better understanding of pDC malignancies.

Indoleamine 2,3-dioxygenase–expressing mature human monocyte-derived dendritic cells expand potent autologous regulatory T cells

AbstractA comprehensive understanding of the complex, autologous cellular interactions and regulatory mechanisms that occur during normal dendritic cell (DC)–stimulated immune responses is critical to optimizing DC-based immunotherapy. We have found that mature, immunogenic human monocyte-derived DCs (moDCs) up-regulate the immune-inhibitory enzyme, indoleamine 2,3-dioxygenase (IDO). Under stringent autologous culture conditions without exogenous cytokines, mature moDCs expand regulatory T cells (Tregs) by an IDO-dependent mechanism. The priming of resting T cells with autologous, IDO-expressing, mature moDCs results in up to 10-fold expansion of CD4+CD25brightFoxp3+CD127neg Tregs. Treg expansion requires moDC contact, CD80/CD86 ligation, and endogenous interleukin-2. Cytofluorographically sorted CD4+ CD25brightFoxp3+ Tregs inhibit as much as 80% to 90% of DC-stimulated autologous and allogeneic T-cell proliferation, in a dose-dependent manner at Treg:T-cell ratios of 1:1, 1:5, and as low as 1:25. CD4+CD25brightFoxp3+ Tregs also suppress the generation of cytotoxic T lymphocytes specific for the Wilms tumor antigen 1, resulting in more than an 80% decrease in specific target cell lysis. Suppression by Tregs is both contact-dependent and transforming growth factor-β–mediated. Although mature moDCs can generate Tregs by this IDO-dependent mechanism to limit otherwise unrestrained immune responses, inhibition of this counter-regulatory pathway should also prove useful in sustaining responses stimulated by DC-based immunotherapy.

Multipotent adult progenitor cells can suppress graft-versus-host disease via prostaglandin E2 synthesis and only if localized to sites of allopriming

AbstractMultipotent adult progenitor cells (MAPCs) are nonhematopoietic stem cells capable of giving rise to a broad range of tissue cells. As such, MAPCs hold promise for tissue injury repair after transplant. In vitro, MAPCs potently suppressed allogeneic T-cell activation and proliferation in a dose-dependent, cell contact–independent, and T-regulatory cell–independent manner. Suppression occurred primarily through prostaglandin E2 synthesis in MAPCs, which resulted in decreased proinflammatory cytokine production. When given systemically, MAPCs did not home to sites of allopriming and did not suppress graft-versus-host disease (GVHD). To ensure that MAPCs would colocalize with donor T cells, MAPCs were injected directly into the spleen at bone marrow transplantation. MAPCs limited donor T-cell proliferation and GVHD-induced injury via prostaglandin E2 synthesis in vivo. Moreover, MAPCs altered the balance away from positive and toward inhibitory costimulatory pathway expression in splenic T cells and antigen-presenting cells. These findings are the first to describe the immunosuppressive capacity and mechanism of MAPC-induced suppression of T-cell alloresponses and illustrate the requirement for MAPC colocalization to sites of initial donor T-cell activation for GVHD inhibition. Such data have implications for the use of allogeneic MAPCs and possibly other immunomodulatory nonhematopoietic stem cells for preventing GVHD in the clinic.

Differential expression of CD97 on human lymphocyte subsets and limited effect of CD97 antibodies on allogeneic T-cell stimulation

CD97 is a large heptahelical EGF-TM7 receptor broadly expressed on hematopoietic cells as three isoforms with respectively three, four, or five epidermal growth factor (EGF)-like domains. We here describe the expression characteristics of CD97 on human lymphocyte subsets. We found CD97 to be present on all lymphocytes in blood and lymphoid tissue. Expression of CD97 on B cells was lower compared to T and NK cells and did not differ between B-cell subsets. In CD4 + T cells, CD97 expression was higher on memory cells compared to naive cells. In CD8 + T and NK cells, we found a downregulation of CD97 on cytolytic effector cells. Stimulation through CD3 and CD28 resulted in a rapid upregulation of CD97 in all T-cell subsets within 2–4 h. A link between CD97 expression and lymphocyte proliferation was established in NK cells, which markedly upregulated CD97 in response to IL-2 and IL-15. Mixed lymphocyte cultures revealed a limited ability of the stalk region-specific monoclonal antibody CLB-CD97/3 to inhibit CD8 + and CD4 + allogeneic T-cell proliferation.

Genetically Engineered Lactococcus lactis Secreting Murine IL‐10 Modulates the Functions of Bone Marrow‐Derived Dendritic Cells in the Presence of LPS

Oral delivery of IL-10 by genetically modified Lactococcus lactis (LL-pTmIL10) has been shown to efficiently reduce intestinal inflammation in mice with chronic colitis, but the mechanisms involved have not been elucidated. It has been suggested that IL-10 controls intestinal inflammation by inhibiting microbe-induced activation of dendritic cells. We therefore investigated whether LL-pTmIL10 can modulate the functions of bone marrow-derived dendritic cells (BM-DC) responding to LPS. Incubation of these cells with LL-pTmIL10 or with the control strain LL-pTREX reduced their ability to activate allogeneic T-cell proliferation. However, in contrast to LL-pTREX, LL-pTmIL10 inhibited the LPS-stimulated secretion of MCP-1 by BM-DC and reduced the synergistic up-regulation of IL-12/IL-23p40. In addition, LL-pTmIL10 treatment of LPS-stimulated BM-DC significantly inhibited their capacity to induce strong secretion of IL-17 by CD4+ T cells. Our data suggest that the beneficial effects of LL-pTmIL10 treatment during chronic colitis might involve inhibition of CD4+ Th17 cells and a reduced accumulation of these cells as well as other immune cells at the site of inflammation.

Ovarian carcinoma cells influence differentiation of Lin−CD45RA− dendritic cell precursors into two mature subtypes in vitro

Objective Decreased number and impaired function of dendritic cells (DCs) have been found in ovarian carcinoma microenvironment. The study was designed to detect if this phenomenon was associated with abnormal DC differentiation influenced by ovarian carcinoma cells. Materials and methods FLT-3L and SCF were used for expanding DC precursors from CD34+ progenitors. GM-CSF and TNF-α were used to induce mature DCs. Supernatants of cultured ovarian carcinoma cell line SKOV3 were added, in order to study their influence on the differentiation and maturation of Lin−CD45RA− DC precursors. Flow cytometry was used to analyze cell subtypes and molecular surface markers. Allogeneic T-cell proliferation assay was used to exam stimulatory activity of DCs. IL-12 secretion was tested by ELISA. Results Lin−CD45RA− DC precursors cultured with GM-CSF and TNF-α generated HLA-DR+CD11C+CD123− myeloid DCs (mDCs) and HLA-DR+CD11C−CD123+ plasmacytoid DCs (pDCs) in vitro. The supernatants from ovarian carcinoma cell line SKOV3 (SKOV3-supernatants) increased pDCs and decreased mDCs compared with pure medium or supernatants of normal ovarian surface epithelial (OSE) cells. There were no significantly different expressions of HLA-DR and CD80 by DCs between with and without SKOV3-supernatants. But DCs treated with SKOV3-supernatants were shown to have impaired immune activity to stimulate proliferation of allogeneic CD3+ T cells and secrete IL-12. Conclusion Ovarian carcinoma cells influence differentiation of Lin−CD45RA− DC precursors into subtypes of mature DCs in vitro. This resulted in fewer mDCs, increased number of pDCs, and impairment of mature DCs immune activity.

Gene Transfer Using Recombinant Simian Virus 40 Viral Vectors Into Mice Bone Marrow Progenitor Cells Depressed the Immunogenicity of Keratinocyte Stem Cells

Hematopoietic stem cell (HSC) gene transfer has been attempted almost entirely ex vivo and has been limited by loss of self-renewal capacity and transplantation-related defects in homing and engraftment. Herein we have attempted to overcome these limitations by injecting vectors directly into the bone marrow (BM) to transduce HSCs in their native environment. Simian virus 40 (SV40)-derived gene delivery vectors were used because they efficiently transduce resting CD34+ cells. Neonatal C57BL/6 (H-2b) mice (3 days old) received SV(Nef-FLAG), carrying FLAG marker epitope directly into both femoral marrow cavities. Keratinocyte stem cells (KSCs) were purified at 7 and 14 days after SV40 injection. The KSCs from 10-day-old C57BL/6 mice were designated as controls. Flow cytometric (FCM) analyses indicated that KSCs from transgenic mice showed strong down-regulation of surface immunological molecules CD40, CD80, CD86, and human major histocompatibility complex class I chain-related antigen A (MICA). Mixed lymphocyte reaction (MLR) assays showed that transgenic KSCs depressed allogeneic T-cell proliferation. Immunofluorescence showed transgenic KSCs expressed FLAG for the entire study as well as high levels of transforming growth factor (TGF)-β and BCL-2. Thus, direct intramarrow administration of recombinant SV40 yielded efficient gene transfer to mice BM progenitor cells. KSCs with low immunogenicity may be obtained for further investigations of skin transplantation immunity.

Stromal-Derived Factor-1 Up-regulated the Expression of MIC on Mouse Keratinocyte Stem Cells

Although previous studies have demonstrated that stromal-derived factor-1 (SDF-1) played a key role in chronic graft dysfunction (CGD), the precise mechanisms underlying this process are not clear. In this study, SDF-1 was injected into keratinocyte stem cells (KSCs) which were isolated and purified from neonatal C57BL/6 (H-2b) mice. Adenylyl cyclase (AC) activity of KSCs was measured and expressions of the human major histocompatibility complex (MHC) class I chain-related antigens A and B (MICA, MICB) detected by immunofluorescence. Cultured KSCs were negative for IA/IE MHC class II molecules by immunofluorescence, indicating the absence of any contamination with Langerhans cells and certifying the purity of KSCs. Over a 7-day culture period, SDF-1 up-regulated AC activity to 2.783 ± 0.799, which was higher than that of the control group (1.290 ± 0.476; P < .01). Immunostaining showed that KSCs expressed increased amounts of MICA protein (0.790 ± 0.134 versus 0.200 ± 0.022; P < .01) and MICB protein (0.610 ± 0.832 versus 0.230 ± 0.016; P < .01). Mixed lymphocyte reaction assays showed that KSCs cultured with SDF-1 injection for 7 days stimulated allogeneic T-cell proliferation. The data indicated that SDF-1 may accelerate the ultimate rejection of allogeneic keratinocytes by enhancing MIC through the AC signal pathway.

Functional IDO Is Expressed on CD34+- and Monocyte-Derived Dendritic Cells According to Differentiation Status.

Indoleamine 2,3-dioxigenase (IDO) is the rate-limiting enzyme in tryptophan catabolism along the kynurenine pathway. IDO expression by different cell subsets inhibits T-cell activation, proliferation and survival and induces regulatory T cells (Tregs). Although human monocyte-derived dendritic cells (Mo-DCs) have been shown to express IDO, little is known about the expression of IDO in other subsets of human DCs, including those generated from CD34+ hematopoietic progenitors (CD34+-derived DCs) In the present study, we performed a full characterization of IDO expression and function by human dendritic cells including Mo-DCs and CD34+-derived DCs. Mo-DCs were generated from purified CD14+ monocytes after culture with GM-CSF and IL-4 and then matured with CD40L, LPS alone, LPS plus IFN-gamma and a cytokine cocktail (IL-1beta, TNF-alpha, IL-6, PGE2). CD34+-derived DCs were generated from purified CD34+ cells after 7 days of culture with GM-CSF and TNF-alpha, followed by 7 day-treatment with GM-CSF and IL-4 and then matured with the same stimuli used for MoDCs. After culture, DCs were analyzed for IDO expression by real-time PCR and western immunoblot, kynurenine production, inhibition of allogenic proliferation and Tregs induction. Monocytes lack IDO expression. Immature Mo-DCs have little if any expression of IDO. During maturation, the cytokine cocktail is the most effective in up-regulating IDO, both at mRNA and protein level, which is paralleled with higher kynurenine production and inhibition of allogeneic T cell proliferation. PGE2 has a crucial role in inducing IDO expression, while IL6 has an opposite effect. Mature Mo-DCs are shown to generate a population of CD4+CD25+FOXP3+ Tregs which are capable of suppressing allogeneic T-cell proliferation. This inhibitory effect is abrogated by the addition of the IDO inhibitor 1-methyl tryptophan (1-MT). CD34+ cells lack IDO mRNA expression. During DC differentiation, IDO expression is observed at day 7, but not at day 14. Flow cytometry analysis of day 7 cells reveal a population of CD34−, CD14+, CD1a+/− cells. Similarly to Mo-DCs, maturation stimuli induce a marked up-regulation of IDO mRNA. Similar results are observed when IDO protein is measured. Production of kynurenine, inhibition of allogeneic T cell proliferation and generation of Tregs from normal CD3+ T-cells are also paralleled with IDO expression. In conclusion, in human DCs the maturation status is associated with a differential expression of IDO both in Mo-DCs and CD34+-derived DCs. Differentiation of CD34+ cells into DCs results in a transient and early expression of IDO at precursor level. Given its functional capacity to inhibit T-cell-mediated immune response, the expression of IDO along DC differentiation may indicate a protective role of the pool of precursor cells which are committed to DC lineage.