Lymphocytes In Mixed Lymphocyte Reaction Research Articles

Immunosuppressive Diversity of Umbilical Cord-Derived Mesenchymal Stromal Cells

Recently, umbilical cord (UC) has become attracted source of mesenchymal stromal cells (MSCs) for immunotherapy such as treatment of severe acute graft versus host disease, because of abundant sources and ease of collection without invasive process. In previous reports on bone marrow-derived MSCs, there is diversity of immunosuppressive mechanisms of MSCs, which have not yet fully clarified in UC-MSCs. Here we studied the immunosuppressive properties of UC-MSCs on the activated immune system in vitro. Methods; UC was collected after obtaining informed consent from the mother. UC-MSCs were obtained from frozen-thawed UC tissue with explant method and expanded in culture medium. Mixed lymphocyte reaction (MLR) consisted with mononuclear cells (MNCs) stained with CFSE and allogeneic dendritic cell (DC) line (PMDC05) co-cultured with or without UC-MSCs was carried out, and the proliferation of CD4 and CD8-positive lymphocytes were analyzed by flowcytometry followed by FlowJo software. Migratory activities of UC-MSCs toward activated lymphocytes in MLR were evaluated by counting the migrating cells stained with DAPI through the insert filter under the microscope. The percentage of CD4+CD25+FOXP3+ regulatory T cells (Treg) before and after co-culture with UC-MSCs was analyzed by flowcytometry. Monocyte phenotypes co-cultured with UC-MSCs directly or separately by insert filter were analyzed with anti-CD14, CD206, and CD168 antibodies. Results: The UC-MSCs were positive for CD105, CD73, CD90, and negative for CD45 and HLA-DR. HLA-DR, CD80, CD86, and CD40 were negative even in the high concentration of IFN-γ, while BM-MSCs became positive for HLA-DR. PD-L2 was constitutively expressed in UC-MSC, while PD-L1 was induced upon the addition of IFN-γ. In MLR, responder T cell proliferation triggered by allogeneic DC was inhibited efficiently by 3rd party derived UC-MSCs. Mean+/- SD distribution index of proliferation in CD4- and CD8- positive cells co-cultured with UC-MSCs showed 7.6+/-4.4% and 8.8+/-3.9% compared to those without UC-MSCs (n=4, as different donor derived MSCs). This inhibition was attenuated by the addition of 1-Methyl-dl-tryptophan, Indoleamine 2, 3-dioxygenase (IDO-1) inhibitor, in a dose-dependent manner. The supernatant of MLR showed the increase of IFN-γ and TNF-α, which were also suppressed in MLR co-cultured with UC-MSCs. UC-MSCs migrated toward activated lymphocytes in MLR compared with those of non-activated lymphocytes significantly, which was inhibited by the addition of AG1296, known as platelet-derived growth factor inhibitor; and PPP as insulin-like growth factor (P<0.05). Cytokine beads array indicated the increase of RANTES, IL-8, and MIP-1. Regulatory T cells in CD4-positive T cells were significantly increased in the MNCs co-cultured with UC-MSCs. Interestingly, CD206-positive M2 monocytes were significantly increased in the MNCs cu-cultured with UC-MSC. Mean +/- SD percentage of CD206+CD80- M2 cells indicated 28.9+/-15.3% in MNCs and 83.3+/-11.2% in MNCs cu-cultured with UC-MSC (n=4, P<0.05). On the other hand, CD206-CD80+ M1 monocytes significantly increased in addition of LPS, which were inhibited by the co-culture with UC-MSCs (P<0.05). Furthermore IFN-γ and TNF-α secreted in the MNCs with LPS were suppressed by the co-culture of UC-MSCs. Discussions and Conclusion: These results demonstrated that UC-MSCs have the diverse immunosuppressive potency through migration toward the activated lymphocytes, suppressing activated T cells proliferation, regulatory T cells induction, and transition of monocyte phenotype. And UC-MSCs are the feasible and abundant source for immunotherapy. Disclosures Nagamura-Inoue: AMED: Research Funding. Nagamura:AMED: Research Funding. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding.

Characteristic Expression of Major Histocompatibility Complex and Immune Privilege Genes in Human Pluripotent Stem Cells and Their Derivatives

Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have been regarded as useful sources for cell-based transplantation therapy. However, immunogenicity of the cells remains the major determinant for successful clinical application. We report the examination of several hESC lines (NTU1 and H9), hiPSC lines, and their derivatives (including stem cell-derived hepatocytes) for the expression of major histocompatibility complex (MHC), natural killer (NK) cell receptor (NKp30, NKp44, NKp46) ligand, immune-related genes, human leukocyte antigen (HLA) haplotyping, and the effects in functional mixed lymphocyte reaction (MLR). Flow cytometry showed lower levels (percentages and fluorescence intensities) of MHC class I (MHC-I) molecules, β2-microglobulin, and HLA-E in undifferentiated stem cells. The levels were increased after cotreatment with interferon-γ and/or in vitro differentiation. Antigen-presenting cell markers (CD11c, CD80, and CD86) and MHC-II (HLA-DP, -DQ, and -DR) remained low throughout the treatments. Recognition of stem cells/derivatives by NK lysis receptors were lower or absent. Activation of responder lymphocytes was significantly lower by undifferentiated stem cells than by allogeneic lymphocytes in MLR, but differentiated NTU1 hESCs induced a cell number-dependent lymphocyte proliferation comparable with that by allogeneic lymphocytes. Interestingly, activation of lymphocytes by differentiated hiPSCs or H9 cells became blunted at higher cell numbers. Real-time reverse transcriptase PCR (RT-PCR) showed significant differential expression of immune privilege genes (TGF-β2, Arginase 2, Indole 1, GATA3, POMC, VIP, CALCA, CALCB, IL-1RN, CD95L, CR1L, Serpine 1, HMOX1, IL6, LGALS3, HEBP1, THBS1, CD59, and LGALS1) in pluripotent stem cells/derivatives when compared to somatic cells. It was concluded that pluripotent stem cells/derivatives are predicted to be immunogenic, though evidence suggests some level of potential immune privilege. In addition, differential immunogenicity may exist between different pluripotent stem cell lines and their derivatives.

S48 Air Pollution Particulate Matter Promotes Dc Maturation And Enhances Their Stimulation Of Cd8 Lymphocyte Responses

Background High levels of ambient urban particulate matter (UPM), a component of air pollution, are associated with respiratory tract infections and exacerbations of airways diseases. Dendritic cells (DCs) exposed to inhaled UPM orchestrate the resulting immune response. We have previously shown that UPM-stimulation of DCs results in enhanced proliferation of naive CD4 lymphocytes but decreased priming of IFNγ-producing CD4 lymphocytes. These CD4 lymphocytes are important in anti-viral immune responses; however, Tc1 CD8 lymphocytes have more direct anti-viral action. In this research we have studied the effect of UPM on DC priming of CD8 lymphocytes. Methods CD1c peripheral blood DCs were isolated, cultured in the presence/absence of UPM stimulation, with GM-CSF or in medium alone. DC expression of CD83, CCR7, CD40 and MHC Class I were measured by flow-cytometry at 24 h. Pre-treated DCs were also cultured with naive CD8 lymphocytes in an allogeneic mixed-lymphocyte reaction (MLR). Lymphocyte proliferation (flow-cytometric measurement of CFSE) and cytokine production (multiplex bead array) were assessed at day 5. The proportion of lymphocytes primed to produce IFNγ was measured at day 7 (intracellular staining). Results UPM-stimulation increased DC expression of the maturation marker CD83 (p = 0.0038) and chemokine receptor CCR7 (p = 0.0018). It had no effect on CD40 or MHC Class I expression. UPM-stimulation of DCs also significantly increased CD8 lymphocyte proliferation (p = 0.020), and the production of IFNγ, TNFα and IL-13 by CD8 lymphocytes in MLR at day 5 (all p Conclusion No evidence of an impaired Tc1 response was seen with UPM-stimulated DCs, in contrast to our previous findings with CD4 T lymphocytes. This may be because CD8 lymphocytes are more primed to respond and produce cytokines at baseline. However, UPM-treatment of DCs did significantly increase DC expression of CCR7, which directs DCs to lymph nodes, and increased the priming of Tc1 and Tc2 responses in the absence of any other stimulation. Inhalation of UPM may give rise to pathological CD8 responses to otherwise innocuous novel antigens.

Effects of simvastatin on the function of dendritic cells in patients with rheumatic arthritis

The present study examined the functional profile of dendritic cells (DCs) in patients with rheumatoid arthritis (RA) and the effects of simvastatin on the function of DCs. A total of 40 patients who was recently diagnosed as having RA were equally assigned to two groups: the routine treatment group (group R) and the routine treatment plus simvastatin group (group R+S). Twenty healthy individuals served as control. The peripheral blood mononuclear cells (PBMCs) were isolated before and 4 weeks after the treatment and then cultured with interleukin-4 (IL-4) and granulocyte-macrophage colony stimulatory factor (GM-CSF) to prepare mature DCs. The expression of co-stimulating factor CD86 on the surface of DCs was assessed by flow cytometry. And the stimulating capacity of DCs was measured by mixed lymphocyte reaction (MLR). The contents of cytokines in culture supernatants of DCs in MLR were detected by ELISA. Blood lipids and high-sensitivity C-reactive protein (hs-CRP) were detected. The relationship between the expression of CD86 and the blood CRP level was also investigated. The results showed that, as compared with the control group, the CD86 expression and the level of cytokines secreted by DCs were significantly increased in RA patients and greater stimulating capacity of DCs in MLR was demonstrated in RA patients. T lymphocytes in MLR secreted higher levels of proinflammatory cytokines (IL-2, IL-17, TNF-α and INF-γ) and lower level of anti-inflammation cytokine (IL-10). The function of DCs was markedly weakened and the level of hs-CRP and low-density lipoprotein was substantially lowered in group R+S in comparison to group R. The CD86 expression was positively correlated with hs-CRP. It was concluded that DCs in RA are highly activated and DC-initiated immune reaction may play an important role in the pathogenesis of RA. Simvastatin administration can significantly inhibit the DCs function and reduce the level of hs-CRP, indicating the suppression on inflammatory reaction may be one of the mechanisms by which simvastatin exerts its effect in treating RA.

Differential effect of IFN-τ on proliferation and distribution of lymphocyte subsets in one-way mixed lymphocyte reaction in cows and heifers

IFN-τ is a signaling protein secreted by the bovine conceptus during the peri-implantation period and responsible for pregnancy recognition. Its main role is the prevention of pulsatile release of luteolytic PGF2α, but it also exerts immunomodulatory activities characteristic for other type I interferons. The aim of the study was to examine the effect of IFN-τ on the proliferation and distribution of peripheral blood lymphocyte subsets during one-way mixed lymphocyte reaction (MLR) in cows and heifers. IFN-τ inhibited the proliferative response of lymphocytes in MLR both in cows and heifers in a dose-dependent manner, but cow lymphocytes were less susceptible than those ones from heifers. It was also showed that IFN-τ differentially changed lymphocyte subsets distribution in MLR in cows and heifers. In cows, the relative percentage of CD8 + cells after MRL in the presence of IFN-τ was significantly lower than in heifers. Differential effect of rIFN-τ on proliferation and lymphocyte subsets distribution in a one-way MRL in cows and heifers indicated that the age of the mother is an important factor in immunomodulatory effect towards developing bovine embryo.

The Arrest of Cell Cycle Progression in Mixed Lymphocyte Reaction by Cultured Mouse Mesenchymal Stem Cell.

Mesenchymal stem cells (MSCs) possess in vitro immunosuppressive properties that appear not to be major histocompatibility complex (MHC)restricted. This study evaluated the immune suppressive effect of mouse MSCs on mixed lymphocyte reaction (MLR), and the mechanisms were investigated. MSCs were obtained from BALB/c bone marrow and cultured in low-glucose DMEM media. The expression of surface antigens and cell cycle were analyzed by flow cytometry. The MSC-induced suppression was assessed by MLR and transwell culture. The BALB/c MSCs constitutively expressed MHC class I and CD54 (ICAM-1) antigens but were negative for MHC class II, CD40, CD80 (B7–1) and CD86 (B7–2) antigens. MSCs suppressed allogeneic C57BL/6 T lymphocytes proliferation by adding them to MLR in which C3H spleen cells were used as a stimulator. This inhibition was dependent on the dose of BALB/c MSCs but independent of MHC. C57BL/6 T lymphocytes proliferation was still inhibited when BALB/c MSCs were added in culture 3 days after starting of MLR. When MSCs were separated from C57BL/6 T cells by using the transwell membrane, the suppression of immune response wasn't observed, which suggested that the suppressive effect was dependent on cell-cell contact between BALB/c MSCs and C57BL/6 T cells. When C57BL/6 T lymphocytes were cultured with MSCs, the percentage of C57BL/6 T cells in G0 phase increased from 51.8±7.66% to 77.2±7.39% compared with the case that only C57BL/6 T cells were cultured. When the C57BL/6 T cells were cultured with C3H spleen cells, most of C57BL/6 T cells were in G2/M (96.38±3.33%). But by the addition of MSC to MLR, the percentage of T cells in G2/M decreased to 33.0±9.66% while that of T cells in G0 increased from 2.0±0.71% to 66.2±7.46%. We concluded that the cell cycle of responder T lymphocytes in MLR is arrested at G0 phase by MSCs. [Display omitted]

Decreased Inducible Expression of CD80 and CD86 in Human Monocytes After Ultraviolet-B Irradiation: Its Involvement in Inactivation of Allogenecity

Ultraviolet-B (UV-B) irradiation of antigen presenting cells (APCs) modifies their allogenecity, resulting in inhibition of the proliferative response of T cells in mixed lymphocyte reaction (MLR). Costimulation by the CD28 ligand CD80 (B7/B7-1) and CD86 (B70/B7-2) plays an important role during T-cell proliferation by augmenting synthesis of interleukin-2 (IL-2) and other cytokines. In this study, we demonstrated induced expression of both CD80 and CD86 during allogeneic MLR, though human freshly isolated monocytes express CD86 constitutively with a much lower level of CD80. A monoclonal antibody (MoAb) against CD86, but not CD80, efficiently inhibited allogeneic T-cell proliferative responses stimulated with highly purified monocytes. UV-B exposure (0 to 1,000 J/m2) of monocytes inhibited the proliferation of T lymphocytes in MLR in a dose-dependent manner. Flow cytometric analysis showed that UV-B exposure of monocytes impaired the constitutive expression of CD54 (intercellular adhesion molecule-1) by 24 hours after irradiation, but the effect on CD86 was relatively less. The surface expression of CD80, CD86, CD54, and HLA-DR on monocytes was further augmented by interferon (IFN)-gamma; this cytokine-induced expression was dose-dependently reduced by UV-B irradiation. Similarly, the upregulation of these molecules following allogeneic MLR was downregulated by UV-B irradiation. UV-B irradiation of monocytes inhibited the expression of IL-2 mRNA in monocyte-stimulated allogeneic MLR. In contrast, the addition of anti-CD28 MoAb at the onset of MLR prevented, at least partially, the reduction of IL-2 mRNA. These results strongly suggest that the impairment of inducible expression of CD86 and CD80 may contribute to the reduced MLR response following exposure of monocytes of UV-B.

Effect of ultraviolet-B-irradiated donor-specific blood transfusions and peritransplant immunosuppression with cyclosporine on rat cardiac allograft survival.

We have previously demonstrated that pretreatment of ACI recipients with ultraviolet-irradiated donor-specific blood transfusion (UV-DST) leads to permanent cardiac allograft survival without further host immunosuppression (ACI rats are weak responders to Lewis lymphocytes in mixed-lymphocyte reaction). This study examines the effect of UV-DST and the timing of transfusions on ACI cardiac allograft survival in Lewis recipients with and without the addition of peritransplant cyclosporine (CsA) (20 mg/kg i.m.) given on days 0, +1, and +2 in relation to the time of transplantation. The mean survival time (MST) of ACI cardiac allografts in Lewis recipients was significantly increased to 33.6 +/- 5.7 days (P less than 0.001) by CsA treatment alone as compared to 6.5 +/- 0.5 days survival in control. When DST was given on day -3 combined with CsA, graft survival was increased to 42.0 +/- 9.3 days (P less than 0.01), as compared to 5.8 +/- 1.3 days when DST alone was used. When DST was irradiated with ultraviolet B (UV-DST) and administered on day -3 combined with peritransplant CsA, the MST was increased to 68.83 +/- 16.1 days as compared to an MST of 10.0 +/- 1.0 days in controls treated with UV-DST alone. When UV-DST was given on day -7 and combined with peritransplant CsA immunosuppression, the results were similar. However, when UV-DST was peritransplant CsA course, 4 of 6 recipients maintained their ACI heart allografts indefinitely (greater than 300 days) in contrast to the effect of UV-DST alone (MST of 13.5 days). Third-party (W/F) UV-irradiated blood transfusions were ineffective in prolonging ACI cardiac allografts in Lewis rats, regardless of whether the transfusions were given alone or in combination with peritransplant immunosuppression with CsA. In conclusion, these results demonstrate that UV-DST combined with a brief peritransplant immunosuppression with CsA induces prolonged heart allograft survival in a histoincompatible, strong responder host, and that such effect is donor specific. The use of UV-DST combined with peritransplant CsA immunosuppression offers a promising approach to achieving organ transplant unresponsiveness, and decreased sensitization to the donor blood elements, which eventually may have important clinical implications.

Suppression of cytolytic T-cell activity by staphylococcal enterotoxin B-induced suppressor cells: role of interleukin 2.

We have previously shown that staphylococcal enterotoxin B (SEB) is a potent inducer of suppressor T cells which function to inhibit antibody production in vitro. In the present paper we extend these studies and show that the SEB-induced suppressor cells also inhibit the development of cytotoxic lymphocytes in mixed-lymphocyte reaction (MLR) cultures. Since further analysis also showed that the level of interleukin 2 (IL-2) in cultures of SEB-primed cells was significantly reduced, experiments were carried out to determine the role of IL-2 in the inhibition of cytotoxic cell activity. Attempts to neutralize the suppression by supplementing MLR cocultures with delectinated supernatants from concanavalin A (Con A)-stimulated rat splenocytes were not successful. In addition, MLR cocultures supplemented on Day 0 with 50 units of affinity-purified IL-2 were also suppressed. Further analysis showed that the IL-2 activity in the supplemented MLR cocultures containing suppressor cells were significantly reduced by Day 3. However, repeated supplementation of the MLR cocultures with exogenous IL-2 was successful in achieving (and maintaining) "normal" levels of IL-2. The cytotoxic cell activity in these MLR cocultures remained suppressed. These results suggest that the inhibition of cytotoxic cell activity by SEB-induced suppressor cells is independent of IL-2 levels in the MLR coculture.