scholarly journals D-mannose ameliorates autoimmune phenotypes in mouse models of lupus

2020 ◽  
Author(s):  
Haiting Wang ◽  
Xiangyu Teng ◽  
Georges Abboud ◽  
Wei Li ◽  
Shuang Ye ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell Proliferation ◽  
Autoantibody Production ◽  
Models Of Lupus

Abstract Background: Systemic lupus erythematosus is a disorder of immune regulation characterized by overproduction of autoantibodies. D-mannose is a C-2 epimer of glucose that exhibits immunoregulatory effects in models of autoimmune diseases, such as type 1 diabetes, induced rheumatoid arthritis, and airway inflammation. This study was conducted to evaluate the efficacy of D-mannose treatment in mouse models of lupus.Methods: The effect of D-Mannose was evaluated by flow cytometry on the in vitro activation of C57BL/6 (B6) murine bone marrow derived dendritic cells and their ability to induce antigen specific CD4+ T cell proliferation and activation. The effect of D-mannose administration in vivo on the frequency of Foxp3+ regulatory T cells in B6 mice was assessed by flow cytometry. D-mannose was administered to two models of lupus: the chronic graft-versus-host disease (cGVHD) induced model and the B6.lpr spontaneous model. Autoantibody production was measured by ELISA and immune activation by flow cytometry. Results were compared by two-tailed statistics: unpaired or paired t tests, or Mann-Whitney U tests depending on whether the data was normally distributed.Results: D-mannose inhibited the maturation of bone marrow dendritic cells and their induction of antigen-specific T cell proliferation and activation in vitro. In vivo, D-mannose increased the frequency of Foxp3+ regulatory T cells in unmanipulated control mice. In the cGVHD model of induced lupus, D-mannose treatment decreased autoantibody production, with a concomitant reduction of the frequency of effector memory and follicular helper T cells as well as germinal center B cells and plasma cells. These results were partially validated in the B6.lpr model of spontaneous lupus. Conclusion: Overall, our results suggest that D-mannose ameliorates autoimmune activation in models of lupus, at least partially due to its expansion of Treg cells, the induction of immature conventional dendritic cells and the downregulation of effector T cells activation. D-Mannose showed however a weaker immunomodulatory effect in lupus than in other autoimmune diseases.

scholarly journals D-mannose ameliorates autoimmune phenotypes in mouse models of lupus

2020 ◽  
Author(s):  
Haiting Wang ◽  
Xiangyu Teng ◽  
Georges Abboud ◽  
Wei Li ◽  
Shuang Ye ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell Proliferation ◽  
Autoantibody Production ◽  
Models Of Lupus

Abstract Background Systemic lupus erythematosus is a disorder of immune regulation characterized by overproduction of autoantibodies. D-mannose is a C-2 epimer of glucose that exhibits immunoregulatory effects in models of autoimmune diseases, such as type 1 diabetes, induced rheumatoid arthritis, and airway inflammation. This study was conducted to evaluate the efficacy of D-mannose treatment in mouse models of lupus. Methods The effect of D-Mannose was evaluated by flow cytometry on the in vitro activation of C57BL/6 (B6) murine bone marrow derived dendritic cells and their ability to induce antigen specific CD4+ T cell proliferation and activation. The effect of D-mannose administration in vivo on the frequency of Foxp3+ regulatory T cells in B6 mice was assessed by flow cytometry. D-mannose was administered to two models of lupus: the chronic graft-versus-host disease (cGVHD) induced model and the B6.lpr spontaneous model. Autoantibody production was measured by ELISA and immune activation by flow cytometry. Results were compared by two-tailed statistics: unpaired or paired t tests, or Mann-Whitney U tests depending on whether the data was normally distributed. Results D-mannose inhibited the maturation of bone marrow dendritic cells and their induction of antigen-specific T cell proliferation and activation in vitro. In vivo, D-mannose increased the frequency of Foxp3+ regulatory T cells in unmanipulated control mice. In the cGVHD model of induced lupus, D-mannose treatment decreased autoantibody production, with a concomitant reduction of the frequency of effector memory and follicular helper T cells as well as germinal center B cells and plasma cells. These results were partially validated in the B6.lpr model of spontaneous lupus. Conclusion Overall, our results suggest that D-mannose ameliorates autoimmune activation in models of lupus, at least partially due to its expansion of Treg cells, the induction of immature conventional dendritic cells and the downregulation of effector T cells activation. D-Mannose showed however a weaker immunomodulatory effect in lupus than in other autoimmune diseases.

scholarly journals Tasquinimod Targets Immunosuppressive Myeloid Cells, Increases Osteogenesis and Has Direct Anti-Myeloma Effects By Inhibiting c-Myc Expression in Vitro and In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1594-1594
Author(s):  
Rong Fan ◽  
Hatice Satilmis ◽  
Niels Vandewalle ◽  
Elke De Bruyne ◽  
Eline Menu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
Cell Lines ◽  
Interferon Gamma ◽  
Myeloid Cells ◽  
T Cell Proliferation

Abstract Introduction Immunotherapy has revolutionized cancer treatment and significantly affected the management of Multiple Myeloma (MM) patients. Unfortunately, these immunotherapeutic approaches are hampered by the presence of a suppressive bone marrow microenvironment including myeloid derived suppressor cells and tumor associated macrophages. Tasquinimod (TasQ), an immunomodulatory compound, is currently in phase Ib/IIa for relapsed/refractory MM patients (NCT04405167). TasQ blocks the interaction between S100A9 and its receptors, which is associated with reduced MDSC accumulation. In this study, we investigated TasQ-mediated direct and indirect effects on MM cell growth, bone disease and immunomodulation in vitro and in vivo using human myeloma cell lines and the immunocompetent 5TMM models. Material and methods In vitro, murine (5T33vt, 5TGM1) and human (JJN3, LP1, OPM2, and RPMI8226) MM cell lines were cultured at different concentrations of TasQ. Cell proliferation was assessed by BrdU staining using flow cytometry. C-Myc and pSTAT3 expression were analyzed by western blot. In vitro T cell proliferation experiments were performed using MACS-sorted CD11b + cells and CFSE-labeled T cells from naïve mice. Cells were cocultured for 72h in the presence of MM conditioned medium (5T33MMvt CM) with CD3/CD28 microbeads, followed by flow cytometry to assess T cell proliferation. For in vivo experiments, we used the 5T33 (aggressive) and 5TGM1 (moderate) MM models. On the second day after tumor cell injection, the mice were randomly assigned to the treatment group and the control group. The treatment group received 30 mg/kg of TasQ in drinking water for 35 days (5TGM1) and 21 days (5T33). Anti-tumor and immunomodulating effects were analyzed by flow cytometry (e.g. tumor cells, myeloid subsets, CD4/CD8 + T cells), qRT-PCR, western blot and serum ELISA (interferon-gamma). Effects on osteogenesis in the 5TGM1 model was investigated by Micro-CT. Statistical differences were assessed by Mann-Whitney U test and One-way ANOVA with p<0.05 considered as statistically significant. Results TasQ-treatment of murine and human myeloma cell lines (HMCL), at concentrations of 10-25uM, significantly reduced MM cell proliferation after 24h and 48h in vitro (n=3, p<0.05). In addition, a downregulation in c-Myc expression could be observed 6h after treatment of human MM cell lines (n=3). In vitro, TasQ significantly increased T cell proliferation in co-culture experiments with T cells and myeloid cells in 5T33MMvt CM (n=3, p<0.05). Using the immunocompetent 5TGM1 and 5T33MM model, we investigated direct and indirect anti-tumor effects of TasQ. We found that TasQ significantly reduced tumor load in the bone marrow of 5TGM1 (n=10/group, p=0.0012) and 5T33MM mice (n=10/group, p=0.0106) compared to vehicle-treated control mice. Using flow cytometry, we could not observe a difference in the percentage of CD4 + and CD8 + T cells. However, a significant upregulation in serum interferon-gamma could be observed in the 5T33MM mice (p=0.0284). While the percentage of CD11b + cells in the TasQ-treated group was significantly increased (p<0.05), the percentage of monocytic myeloid cells (CD11b +Ly6G -) was significantly reduced in both models (p<0.05). qRT-PCR results showed that the expression of IL-10 was downregulated in purified CD11b + myeloid cells (p<0.05). Consistent with the in vitro data, we observed a decrease in the protein expression of c-Myc in purified MM cells obtained from TasQ-treated mice compared to control mice. Micro-CT analysis of femurs demonstrated a significant increase in the percentage BV/TV (ratio of bone material volume over tissue volume) and trabeculae number (p<0.0001) in TasQ-treated 5TGM1 mice compared to untreated mice. Conclusion TasQ has pleiotropic effects on the MM cells and its surrounding bone marrow microenvironment. It affects MM cell growth by decreasing c-Myc expression. In addition, TasQ targets the immunosuppressive monocytic myeloid cell population and increases serum interferon-gamma levels, indicative for immune cell activation. Moreover, it stimulates osteogenesis in vivo. Taken together, all these data provide evidence for the therapeutic benefits of TasQ as an anti-MM therapy for patients. Disclosures Törngren: Active Biotech: Current Employment. Eriksson: Active Biotech: Current Employment. De Veirman: Active Biotech AB: Research Funding.

Imatinib mesylate inhibits T-cell proliferation in vitro and delayed-type hypersensitivity in vivo

Blood ◽  
2004 ◽  
Vol 104 (4) ◽  
pp. 1094-1099 ◽  
Author(s):  
Allan B. Dietz ◽  
Lina Souan ◽  
Gaylord J. Knutson ◽  
Peggy A. Bulur ◽  
Mark R. Litzow ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Imatinib Mesylate ◽  
Cyclin D3 ◽  
Delayed Type Hypersensitivity ◽  
T Cell Proliferation

Abstract Imatinib mesylate (STI571, imatinib) inhibited DNA synthesis in primary human T cells stimulated with allogeneic mature dendritic cells or phytohemagglutinin (PHA) but did not induce apoptosis. The values for the concentration that inhibits 50% (IC50) of T-cell proliferation stimulated by dendritic cells and PHA were 3.9 μM and 2.9 μM, respectively, that is, within the concentration range found in patients treated with imatinib mesylate. Interestingly, imatinib mesylate did not inhibit expression of T-cell activation markers CD25 and CD69, although it reduced the levels of activated nuclear factor-κB (NF-κB) and changed phosphorylation or protein levels of Lck, ERK1/2, retinoblastoma protein, and cyclin D3. When T cells were washed free of imatinib mesylate, they proliferated in response to PHA, demonstrating that inhibition is reversible. Treatment with imatinib mesylate led to accumulation of the cells in G0/G1 phase of the cell cycle. The in vitro observations were confirmed in vivo in a murine model of delayed-type hypersensitivity (DTH). In mice treated with imatinib mesylate, DTH was reduced in comparison to sham-injected controls. However, the number of splenic T cells was not reduced showing that, similarly to in vitro observations, imatinib mesylate inhibited T-cell response, but did not cause apoptosis. These findings indicate that long-term administration of high-dose imatinib mesylate might affect immunity.

scholarly journals D-mannose ameliorates autoimmune phenotypes in mouse models of lupus

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Haiting Wang ◽  
Xiangyu Teng ◽  
Georges Abboud ◽  
Wei Li ◽  
Shuang Ye ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Autoimmune Diseases ◽  
Mouse Models ◽  
Effector Memory ◽  
T Cell Proliferation ◽  
Autoantibody Production ◽  
Models Of Lupus

Abstract Background Systemic lupus erythematosus is an autoimmune disease characterized by an overproduction of autoantibodies resulting from dysregulation in multiple immune cell types. D-mannose is a C− 2 epimer of glucose that exhibits immunoregulatory effects in models of autoimmune diseases, such as type 1 diabetes, induced rheumatoid arthritis, and airway inflammation. This study was conducted to evaluate the efficacy of D-mannose treatment in mouse models of lupus. Results Firstly, the effect of D-Mannose was evaluated by flow cytometry on the in vitro activation of non-autoimmune C57BL/6 (B6) bone marrow-derived dendritic cells (BMDCs) and their ability to induce antigen-specific CD4+ T cell proliferation and activation. D-mannose inhibited the maturation of BMDCs and their induction of antigen-specific T cell proliferation and activation. In vivo, D-mannose increased the frequency of Foxp3+ regulatory T cells in unmanipulated B6 mice. To assess the effect of D-mannose in mouse models of lupus, we used the graft-versus-host disease (cGVHD) induced model and the B6.lpr spontaneous model. In the cGVHD model, D-mannose treatment decreased autoantibody production, with a concomitant reduction of the frequency of effector memory and follicular helper T cells as well as germinal center B cells and plasma cells. These results were partially validated in the B6.lpr model of spontaneous lupus. Conclusion Overall, our results suggest that D-mannose ameliorates autoimmune activation in models of lupus, at least partially due to its expansion of Treg cells, the induction of immature conventional dendritic cells and the downregulation of effector T cells activation. D-Mannose showed however a weaker immunomodulatory effect in lupus than in other autoimmune diseases.

Monocytic Myeloid-Derived Suppress Cells Restore Adipogenic Bone Marrow Microenvironment in Aplastic Anemia By Inhibiting Intra-BM CD8+ T Cells Proliferation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1211-1211
Author(s):  
Ying Qu ◽  
Zhengxu Sun ◽  
Yan Yuan ◽  
Fen Wang ◽  
Kunpeng Wu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Proliferation ◽  
Immune Balance ◽  
Ifn Γ

Aplastic anemia (AA) is a hematopoietic disorder resulted from immune-related hypocellular hematopoiesis in bone marrow (BM). It has been clearly addressed that the activated T cells contribute to the exhaustion of hematopoietic progenitors and hypo-hematopoiesis. The adipogenic BM is one of the characteristics to make AA diagnosis. However, little is known about the relationship of intra-BM immune imbalance and hematopoietic microenvironment abnormity in this disease entity. Functional hematopoiesis relies on not only abundant hematopoietic stem cells (HSCs) but also the balanced supportive hematopoietic niche. Intra-BM immune balance, at either cellular or cytokine level, is one of the key footstones to maintain hematopoietic microenvironment. Various intra-BM immune cellular components play both sides of one coin. Among them, myeloid-derived suppressive cells (MDSCs) are heterogeneous myeloid progenitor cells characterized by the negative immune response in cancers and other inflammatory diseases. In BM aspiration and biopsy samples from the patients who were diagnosed as AA in our study, massive activated lymphocytes infiltration and adipocytes accumulation were observed. Interestingly, the absolute numbers of immune modulatory MDSCs either in AA patients' PB or in BM of immune-related AA mice were reduced, indicating a potential link between polarized BM adipo-osteogenic microenvironment and immune disorder under AA circumstance. We thus adopted AA mice model to look into the embedded details both in vivo and in vitro. We clarified that BM components were more vulnerable to the attack of CD8+ T cells than that of CD4+ T cells. Taking into the fact that BM adipocytes are more abundant either in AA patients or in AA mice models, we differentiated mesenchymal stromal cells (MSCs), the major BM stroma cells, into osteoblastic or adipogenic lineages to mimic the osteo-adipogenic differentiation in BM microenvironment. Interestingly, CD8+ T cells and interferon-γ(IFN-γ) exerted dramatically adipocytic stimulation on BM-MSCs either in vitro or in vivo, by determination of increasing expression of adipogenetic genes including Ap2, Perilipin, Pparg and Cebpα, as well as staining of Oil Red O and perilipin. To dissect intra-BM cellular immune balance, MDSCs were isolated as representative immune regulating population to investigate their function on osteo-adipogenic balance. Interestingly, not CD11b+Ly6G+Ly6C-granulocytic-MDSCs (gMDSCs) but CD11b+Ly6G-Ly6C+monocytic-MDSCs (mMDSCs) inhibited both T cell proliferation and IFN-γ production. Addition of L-NMMA, the antagonist of iNOS pathway in mMDSCs-containing system restored T cell proliferative curve and cell numbers, whereas Nor-NOHA, the antagonist of Arg-1 pathway didn't abrogate mMDSCs' immune-regulation properties, indicating that mMDSCs inhibited T cell proliferation via iNOS pathway. We then performed single dose or multi-dose injection of mMDSCs in AA mice to see whether mMDSCs are able to reconstitute the impacted hematopoiesis. Single injection of mMDSCs was able to prevent from CTL infiltration in a very short term. However, multi-injection of mMDSCs showed significant benefit in overall survival rate compared to AA mice. We further detected the function of mMDSCs on polarized BM-MSCs adipo-osteogenic differentiation potential. To detect sequential BM adipogenetic progression in AA microenvironment, we performed in vivo fluorescent microscopy on AP2 (Fabp4)-Cre×mT/mG reporting mice at different transfusion time points of T cells and mMDSCs. GFP-expressing AP2+ adipocytes accumulated adjacently to perivascular niches whose boarders were labelled by Dextran-CY5 in a time-dependent manner after T cell infusion. Monocytic MDSCs transfused AA mice showed decreased GFP+ adipocytes which was coincident with our in vitro findings. In conclusion, intra-BM immune balance is one of the environmental factors seesawing by activating and suppressive ends to support functional hematopoiesis. Adoptive transfusion of mMDSCs, the immune-suppressive population might be a novel immune-regulating strategy to treat AA, relying on not only restoring the intra-BM immune balance but also improving stroma's multi-differentiating microenvironment. Figure Disclosures No relevant conflicts of interest to declare.

Myeloid Suppressive Cells Mobilized by GM-CSF in Non-Tumor Bearing Mice Are Dependent On Interferon Gamma for Function

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 832-832
Author(s):  
Mark A. Schroeder ◽  
Julie Ritchey ◽  
John F. DiPersio
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Proliferation ◽  
Suppressive Function ◽  
Gm Csf ◽  
Tumor Bearing

Abstract Abstract 832 Myeloid-derived-suppressor cells (MDSCs) are enriched in tumors, and exist to a lesser extent in the blood, spleen and bone marrow of tumor bearing mice. Monocytic MDSCs (monoMDSCs) suppress CD8+ T-cells via expression of Arginase 1 (ARG1) and inducible nitric oxide synthase (iNOS). Tumor derived factors are critical to the maintenance of MDSCs and preventing differentiation to mature macrophages and dendritic cells. GM-CSF is a hematopoietic cytokine that can be secreted by tumors and promotes MDSC generation. Cells phenotypically similar to MDSCs can be isolated from blood of normal individuals but lack suppressive function. Hematopoietic peripheral blood stem cell mobilization with G-CSF and GM-CSF enriches for cells phenotypically similar to MDSCs. There is limited data on the role and function of these cells isolated from non-tumor bearing, normal individuals. Recent evidence suggests that graft-versus-host disease (GvHD) can be abrogated in mice by ex vivo expanded, bone marrow derived, MDSCs generated in the presence of GM-CSF, G-CSF and IL-13 (Highfill et al. Blood 2010 116:5738). It remains to be shown whether phenotypic MDSCs identified in non-tumor bearing mice are capable of immune suppression; and, the mechanism by which an immature myeloid cell becomes a functional MDSC remains unknown. We have observed an increase (up to 8 fold) in a population of cells phenotypically resembling monoMDSCs (CD11b+/Ly6C+/Ly6G-) in the spleens and blood of mice mobilized with pegylated-murine-GM-CSF (peg-mGM-CSF). We hypothesized that this population of cells would have suppressive function similar to MDSCs in vitro and in vivo, and may have the potential to abrogate graft-versus-host disease (GvHD). To investigate the function of MDSCs found in spleens of C57/Bl6 (B6) mice treated with peg-mGM-CSF we performed CFSE based anti-CD3/CD28 antibody stimulated T-cell proliferation assays, mixed leukocyte reactions and transwell assays. We observed that CD11b+Ly6C+Ly6G- cells isolated from spleens of mice treated with peg-mGM-CSF have potent suppressive function in vitro that is contact dependent and abrogated by blocking ARG1 or iNOS. This suppressive effect was lost in APC stimulated MLRs using B6 T-cells and Balb/C stimulators (confirmed in two separate experiments). Furthermore, the in vivo potential of these putative MDSCs to abrogate murine GvHD was investigated using a B6 to Balb/C donor leukocyte infusion GvHD model. Adoptive transfer of purified splenic CD11b+Ly6C+Ly6G- cells from peg-mGM-CSF mobilized B6 donors along with an equivalent number of congenic T-cells failed to abrogate GvHD. We investigated timing of MDSC infusion in the B6 to Balb/C GvHD model and found no improvement in weight loss, GvHD score or survival in mice receiving 5×105 monoMDSCs IV on day 1, 6 or 10 after transplant compared to T-cells alone control (n = 5 – 10/group, Log rank, p= NS). To address in vivo function further in a bioluminescent imaging (BLI) tumor model. Balb/C recipients were injected SC with A20 cells mixed +/− monoMDSCs at a 1:10 ratio after lethal irradiation and T-cell deplete bone marrow on day 0. Donor T-cells were infused at day +11. The rate of tumor growth measured by photon flux was the same between subcutaneous tumors either with or without monoMDSCs. (two separate experiments, 5 mice/group). This in vivo data suggested that a critical factor present in vitro might be lacking or insufficient in vivo. To investigate the critical factor(s) present in vitro we performed T-cell proliferation assays in the presence of blocking antibodies against IFNy, TNFalpha, IL-10, GM-CSF and CD154. Only neutralization of IFNy resulted in negation of the suppressive effects of these cells. To investigate the source of IFNy production we used transgenic IFNy knockout mice as T-cell and MDSC donors. Proliferation of IFNy deficient T-cells was suppressed efficiently by wild-type (WT) MDSCs, and, neutralizing IFNy using a blocking antibody negated suppression. This suggested IFNy production by a cell within the putative MDSC sorted population might be critical for MDSC function. IFNy deficient peg-mGM-CSF mobilized CD11b+Ly6C+Ly6G- spleen cells failed to suppress WT or IFNy deficient T-cell proliferation. These results suggest a critical role for IFNy production by CD11b+Ly6C+Ly6G- myeloid cells in maintaining their suppressive phenotype in vitro and perhaps in vivo. Disclosures: No relevant conflicts of interest to declare.

Pharmacological Inhibition Of Semi-Direct Alloantigen Presentation and The Generation Of Cross-Dressed Antigen Presenting Cells: A Novel approach To Limit Graft Versus Host Disease Following Allogeneic Hematopoetic Stem Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 294-294
Author(s):  
Sravanti Rangaraju ◽  
Junghwa Choi ◽  
Cynthia R. Giver ◽  
Edmund K. Waller
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Proliferation ◽  
Mhc Ii ◽  
Direct Pathway ◽  
Antigen Presenting

Abstract Background Graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplant (allo-HSCT) is caused by CD4+ and CD8+ donor T cells directed against mismatched recipient antigens, presented in the context of donor MHC-II (indirect pathway) and recipient MHC-I (direct pathway). Recently, the presence of 'cross-dressed' CD11c+ antigen presenting cells (APCs) expressing both donor and recipient type MHC-I molecules has been demonstrated in animal organ and HSCT transplant models supporting 'semi-direct' pathway of allo-activation (Wang et al, Blood. 2011).These APCs can efficiently present allo-antigens to both CD4+ and CD8+ T cells and activate immune responses that could lead to allograft rejection or GVHD. Exchange of membrane fragments and associated proteins between cells, termed trogocytosis, generates cross-dressed APCs.We sought to test whether cross-dressed APCs facilitate antigen presentation to donor T cells and initiate GVHD following allo-HSCT. Further, we tested an array of drugs as inhibitors of trogocytosis, to interrupt the semi-direct pathway of allo-antigen presentation. Methods In vivo experiments used a B6(H2Kb) ˆ B10.BR(H2Kk) murine transplant model. Spleens of transplanted mice were analyzed on days 10, 15, 20 post-transplant for presence of cross dressed CD11c+cells, and their expression of CD80, CD86 and MHC-II by flow cytometry. Cross dressed donor CD11c+ FACS sorted cells from recipient spleens were co-cultured with CFSE labeled donor type T-cells for 6 days, and T-cell proliferation was measured as dilution of CFSE by flow cytometry. In vitro experiments used primary MLR consisting of CFSE labeled B6 bone marrow cells co-cultured with PKH26 (membrane dye) labeled B10.BR splenocytes. B6 antigen presenting cells were analyzed by flow cytometry for the presence of CFSE+PKH26+ double positive cells generated by trogocytosis. Pharmacological inhibitors of cytoskeleton function were added to the primary MLR and their effect on trogocytosis as well as T cell proliferation was assessed. Results Cross-dressed donor CD11c+ APCs were generated in vivo following allo-HSCT (Figure 1). Recipient spleens showed that 50%, 28.6% (p=0.01) and 12% (p=0.02) of donor type CD11c+ cells were cross dressed on days 10, 15 and 20 respectively post transplant (n=5). These cross dressed APCs expressed higher levels of co-stimulatory molecules CD80 (p<0.001) and CD86 (p<0.001), and MHC-II compared to non-cross-dressed donor CD11c+ cells (Figure 2). Sorted cross dressed CD11c+ cells from recipient mice were able to induce in vitro proliferation of co-geneic CD8 T-cells, while their non-crossdressed counterparts did not. We demonstrated that cross-dressed CD11c+ cells were generated in vitro, by exchange of plasma membrane fragments and could be inhibited in vitro by low doses of paclitaxel and VIP antagonist (Figure 3), while preserving cell viability. Further more, bone marrow treated with 0.05uM of paclitaxel, caused significantly decreased T cell proliferation in primary MLR compared to non drug treated bone marrow. Discussion The high frequencies of cross-dressed donor CD11c+ APCs following allo-HSCT suggests that semi-direct allo-antigen presentation may play a key role in the initiation of GVHD, while the decreasing trend could reflect replacement of host cells by donor hematopoetic cells. Reducing the generation of cross-dressed APCs by pharmacological inhibition of trogocytosis is a novel approach to reduce GVHD post allo-HSCT, targeting the semi-direct pathway of allo-antigen presentaion. Our data shows that very low doses of paclitaxel, a microtubule inhibitor and VIPHyb, an antagonist of Vasoactive Intestinal Peptide signaling, can reduce semi-direct presentaion of allo-antigen to Tcells and reduce alloreactivity without direct cytotoxic effect. Disclosures: No relevant conflicts of interest to declare.

ATG5 Dependent Autophagy Uncouples T Cell Functions and Modulates Experimental Graft-Versus-Host Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 149-149
Author(s):  
Katherine Oravecz-Wilson ◽  
Corinne Rossi ◽  
Chen Liu ◽  
Tomomi Toubai ◽  
Hiroya Tamaki ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
Apoptotic Proteins

Abstract ATG5 is a key protein that regulates autophagy, a vital cellular process whose role in various immune cells is poorly understood. A recent report showed that the deficiency of autophagy gene Atg16l1 in host DCs increased graft-vs-host disease (GVHD). Nevertheless, the direct role of autophagy in regulating T cell alloreactivity after bone marrow transplant (BMT) is unknown. In order to investigate the role of autophagy in T cells, we first analyzed the changes in autophagosome marker LC3 upon WT T cell activation. TCR stimulation with anti-CD3/CD28, increased cytosolic LC3-I and its membrane-bound LC3-II form. Interestingly, we found that the upregulation of LC3 was predominant in dividing cells, which lead us to hypothesize that autophagy is essential for T cell proliferation. Therefore we next explored if the deficiency in autophagy impaired T cell proliferation utilizing B6 T cell-specific ATG5 knockout (ATG5 KO T cell) mouse and hydroxychloroquine (CQ, a known inhibitor of autophagy). As hypothesized, when compared with WT controls, both CQ treated WT T cells and the ATG5 KO T cells, in vitro, demonstrated a significant decrease in proliferation as demonstrated by 3H-thymidine incorporation and CFSE staining (p<0.0001) and were associated with a failure to upregulate LC3. These effects were observed after anti-CD3/CD28 TCR stimulation as well as following allogenic stimulation in a mixed lymphocyte reaction (MLR) with bone marrow-derived dendritic cells (DCs) from BALB/c mice. The reduction in T cell proliferation was accompanied by a significant increase in apoptosis (p<0.0001). However it was not associated with a decrease in T-helper (TH) signature cytokines (IFNγ, IL-2, IL-17, IL-4) suggesting no impact on T cell differentiation. Furthermore ATG5 deficiency also did not alter T cell activation as determined by upregulation of NFAT and ZAP70. Thus lack of autophagy lead to the decrease survival of T cell along with decreased proliferation after TCR stimulation but did not affect TH differentiation and T cell activation. Given the in vitro observations, we hypothesized that ATG5 KO T cells would also induce less GVHD following allogenic bone marrow transplantation (BMT). Utilizing clinically relevant MHC-mismatched B6 → BALB/c BMT model, we lethally irradiated (800cGy) WT-BALB/c mice and transplanted 5x106 T cell-depleted bone marrow from WT-B6 mice along with 0.5x106 splenic T cells purified from ATG5 KO or WT- B6 mice. WT-BALB/c TCD BM and T cells were used for syngeneic controls. Consistent with in vitro results, ATG5 KO T cells showed decreased proliferation in vivo but showed no difference in Th1/Th17 differentiation. Allogenic animals transplanted with ATG5 KO T cells also showed a significantly improved survival (p=0.001) and reduced GVHD severity (p=0.03). Phenotypic analyses prior to BMT showed that ATG5 KO T cells show decreased CD62L and an increased expression of CD44. Because naïve (CD62L+CD44-) T cells are critical for GVHD, we next explored if the observed improvement in GVHD could be due to a decreased population of these naïve T cells in the transplant inoculum of ATG5 KO animals. Therefore, using the same BMT model and design, we transplanted WT-BALB/c mice 0.5x106 isolated splenic CD62L+ T cells only from either ATG5 KO or WT-B6 animals and observed that GVHD mortality was reduced in the allo-recipients of ATG5 KO T cells compared with WT T cells (p=0.005). To determine the potential molecular mechanism, we next hypothesized that upon activation ATG KO T cells may show alterations in pro and anti-apoptotic proteins. We observed that ATG5 KO failed to increase Bcl-2 level and showed a decrease in Bcl-XL level upon TCR stimulation compared to WT T cells. In contrast to the relative lack of anti-apoptotic proteins, they displayed similar levels of the pro-apoptotic proteins BIM, Bak and Bax. These results suggest that an imbalance between pro- and anti-apoptotic factors is likely a cause for the reduced T cell expansion by ATG5 KO T cells. Our results collectively demonstrate that inhibition of autophagy decreases T cell expansion but not its differentiation in vitro and in vivo. Furthermore contrary to the aggravation of GVHD when autophagy is targeted in host DCs, it mitigated GVHD when targeted in donor T cells. Thus the net impact of manipulating autophagy after allogeneic BMT on GVHD is dependent on which immune cell subsets are being targeted. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Pulmonary Immunity to Viral Infection: Adenovirus Infection of Lung Dendritic Cells Renders T Cells Nonresponsive to Interleukin-2

2006 ◽  
Vol 80 (4) ◽  
pp. 1826-1836 ◽  
Author(s):  
Allison T. Thiele ◽  
Tina L. Sumpter ◽  
Joanna A. Walker ◽  
Qi Xu ◽  
Cheong-Hee Chang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Interleukin 2 ◽  
In Vivo Studies ◽  
T Cell Proliferation ◽  
Interleukin 12

ABSTRACT Adenovirus (Ad) infection has been identified as predisposing hosts to the development of pulmonary disease through unknown mechanisms. Lung dendritic cells (DCs) are vital for initiating pulmonary immune responses; however, the effects of Ad infection on primary lung DC have not been studied. In contrast to the effects on bone marrow- and monocyte-derived DCs, the current study shows that Ad infection of murine BALB/c lung DCs in vitro and in vivo suppresses DC-induced T-cell proliferation. The effect of Ad on DCs was not due to a downregulation of major histocompatibility complex or costimulatory molecules. Analysis of the production of interleukin-12 (IL-12), alpha interferon (IFN-α), and IFN-γ by the Ad-infected DCs shows no significant differences over noninfected control lung DCs. Ad-induced suppression was not due to a deficiency of IL-2 or other DC-secreted factors and was dependent on viral protein synthesis, as UV irradiation of Ad abrogated the suppressive effect. Results suggest that Ad-infected DCs induce T cells to be nonresponsive to IL-2 during primary coculture, as the addition of IL-2 in secondary cultures recovered T-cell proliferation. In vivo studies supported in vitro results showing that Ad infection resulted in lung T cells with decreased proliferative ability. This study demonstrates that Ad infection induces local immunoincompetence by altering DC-T-cell interactions.

Bone marrow-derived mesenchymal stem cells inhibit T follicular helper cell in lupus-prone mice

Lupus ◽  
2017 ◽  
Vol 27 (1) ◽  
pp. 49-59 ◽  
Author(s):  
X Yang ◽  
J Yang ◽  
X Li ◽  
W Ma ◽  
H Zou
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Lupus Nephritis ◽  
Bone Marrow Cells ◽  
Tfh Cells ◽  
Follicular Helper

Background The objective of this paper is to analyze the role of bone marrow-derived mesenchymal stem cells (BM-MSCs) on the differentiation of T follicular helper (Tfh) cells in lupus-prone mice. Methods Bone marrow cells were isolated from C57BL/6 (B6) mice and cultured in vitro, and surface markers were identified by flow cytometry. Naïve CD4+ T cells, splenocytes and Tfh cells were isolated from B6 mice spleens and co-cultured with BM-MSCs. The proliferation and the differentiation of CD4+ T cells and Tfh cells were analyzed by flow cytometry. Lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice were treated via intravenous injection with expanded BM-MSCs, the differentiation of Tfh cells was detected, and the relief of lupus nephritis was analyzed. Results MSCs could be successfully induced from bone marrow cells, and cultured BM-MSCs could inhibit T cell proliferation dose-dependently. BM-MSCs could prevent Tfh cell development from naïve CD4+ T cells and splenocytes. BM-MSCs could inhibit IL-21 gene expression and cytokine production and inhibit isolated Tfh cells and STAT3 phosphorylation. In vivo study proved that BM-MSCs intravenous injection could effectively inhibit Tfh cell expansion and IL-21 production, alleviate lupus nephritis, and prolong the survival rate of lupus-prone mice. Conclusions BM-MSCs could effectively inhibit the differentiation of Tfh cells both in vitro and in vivo. BM-MSC treatment could relieve lupus nephritis, which indicates that BM-MSCs might be a promising therapeutic method for the treatment of SLE.

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