Two-Round Mixed Lymphocyte Reaction for Evaluation of the Functional Activities of Anti-PD-1 and Immunomodulators

Immune checkpoint inhibitors (ICIs), such as anti-PD-1 and anti-PD-L1 Abs, have shown efficacy for the treatment of various cancers. Although research has actively sought to develop new ICIs and immunomodulators, no efficient in vitro assay system is available to evaluate their functional activities. In the present study, we established a two-round MLR with human PBMCs for evaluation of the T cell-activating capacity of anti-PD-1 and other immunomodulators. We initially performed conventional MLR for this purpose. However, anti-PD-1 blocking Abs could not increase the proliferation of allo-reactive T cells in conventional MLR because PD-L1+ and PD-L2+ cells disappeared gradually during MLR. Therefore, we re-applied the same stimulator PBMCs to the allo-stimulated responder cells as a second-round MLR on day 6 when anti-PD-1 or immunomodulators were also added. In this two-round MLR, the proliferation of allo-reactive T cells was enhanced by anti-PD-1 in a dose-dependent manner or by immunomodulators, such as lenalidomide and galunisertib, a TGF-β receptor-1 inhibitor. Proliferation was further increased by the combination of immunomodulators with anti-PD-1. Here, we established a modified two-round MLR method with human PBMCs for evaluation of the functional activities of anti-PD-1 and immunomodulators.

Pretreatment of IL-2 influences the proliferation and polarization of mice splenic lymphocyte following mobilization with G-CSF / 中华微生物学和免疫学杂志

Objective To explore the influence of IL-2 pretreatment on splenic lymphocyte following mobilization with G-CSF, which may provide a new approach to attenuate acute graft-versus-host disease (GVHD). Methods Splenic cells and na(i)ve CD4+T cells from C57BL/6N mice receiving G-CSF-mobilized were pretreated with IL-2(50 U/ml) , and cocultured with the allogeneic antigens from BALB/c mice. The proliferation responses and the polarization of T cells were determined. C57BL/6N mice were randomly divided into 4 groups: G-CSF + IL-2 group, G-CSF group, IL-2 group , control group. Results Compared with the control group, IL-4 increased obviously while IFN-γ decreased significantly in the group of G-CSF + IL-2. The proliferation responses were also suppressed in vitro. Conclusion The proliferation responses of splenic cells and naive CD4 + T cells from C57BI/6N mice receiving G-CSF-mobilized to the allogeneic antigens were significantly abrogated by the pretreatment with IL-2, T cells were polarized toward the production of type-2 cytokines. The combination of G-CSF and IL-2 is potentially synergetic in the induction of T lymphocyte immune tolerance.

Clinical Grade of Gerneration of Dendritic Cells for Immunotherapy / 华中科技大学学报（医学）（英德文版）

In order to develop a protocol for clinical grade generation of dendritic cells (DCs) for cancer immumotherapy, aphereses were performed with the continuous flow cell separator and materials were derived from 10 leukemia patients that had achieved complete remission. Peripheral blood monocytes were cultured in vitro with GM-CSF, IL-4 for 6 days, then TNF-α (the TNF-α group) or TNF-α, IL-1β, IL-6, PGE2 (the cytokine mixture group) were added to promote maturation. Cell number was counted by hematology analyzer, and phenotype study (CD1a, CD14, CD83) was carried out by flow cytometry, and the function of DCs was examined by mixed lymphocyte reaction. The results showed that (0.70±0.13)×107/mL (the TNF-α group) and (0.79±0.04)×107/mL (the cytokine mixture group) DCs were generated respectively in peripheral blood obtained by leucapheresis. The phenotypes were as follows: CD1a+ (74.65±4.45)%, CD83+(39.50±4.16)%, CD14+(2.90±1.76)% in TNF-α group, and CD1a+ (81.86±5.87)%, CD83+ (81.65±6.36)%, CD14+ (2.46±1.68)% in the cytokine mixture group. It was concluded that leucapheresis may be a feasible way to provide large number of peripheral blood monocytes for DC generation, and combined administration of TNF-α, IL-1β,IL-6, and PGE2 may greatly promote maturity.

Effects of Trifoliumpratense Leguminosae extract on mouse allogenetic skin transplantation / 中华中医药杂志

Objective: To observe the effect of Trifoliumpratense Leguminosae extract (TLE) on mouse allogenetic skin transplantation. Methods: Recipient BALB/c was divided into physiologic saline (PS) group and TLE group, full-thickness skins were transplanted through back to back method from donor C57BL/6. The allogenetic transplanted skin growth condition was observed. The proliferation of lymphocytes of recipient mice in vitro were detected by CFDA-SE stain and mixed lymphocyte reaction respectively. Results: The allogenetic transplanted skin injected with TLE 25g/kg per day by vena caudalis growed better than that in PS group. The proliferation of lymphocyte in TLE group was smaller than that in PS group. Conclusion: TLE maybe participate in the regulation of mouse immune system and induce its tolerance to the allogenetic transplanted skin.

Mechanism of Cultured Mouse Mesenchymal Stem Cell-Induced Suppression of Allogeneic Lymphocytes Proliferation / 대한혈액학회지

BACKGROUND: Mesenchymal stem cells (MSC) can be defined by their extensive in vitro self renewal capacity and multi-lineage differentiation potentiality. These cells possess in vitro immunosuppressive properties that appear not to be major histocompatibility complex (MHC) restricted. This study evaluated the immune suppressive effect of mouse MSC on mixed lymphocyte reaction (MLR), and the mechanisms were investigated. METHODS: MSC 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. RESULTS: The BALB/c MSC constitutively expressed MHC class I and CD54 (ICAM-1) antigens but were negative for MHC class II, CD40, CD80 (B7-1) and CD106 (VCAM-1) antigens. MSC 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 MSC but independent of MHC. C57BL/6 T lymphocytes proliferation was still inhibited when BALB/c MSC were added in culture 3 days after starting of MLR. When MSC 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 MSC and C57BL/6 T cells. When C57BL/6 T lymphocytes were cultured with MSC, 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 to 66.2+/-7.46%. CONCLUSIONS: We concluded that the cell cycle of responder T lymphocytes in MLR is arrested at G0 phase by MSC.

Effective activation of CD11c and anti-tumor immune in immunized mice with recombinant E.coli. LLO/OVA / 第三军医大学学报

Objective To investigate the anti-tumor pathway and the effect of recombinant E.coli. LLO/OVA in C57BL/6 mice. Methods Magnetic sorting of splenic CD11c, CD4+ and CD8+ T cell from immunized mice with E.coli. LLO/OVA and E.coli. OVA vaccination were performed. In addition, the lymphocytes mixed reaction and IL-2 and IFN-? in the supernatant were detected. The percentage of OVA257-264 SIINFEKL specific CD8+ T cell were checked by flow cytometry. B16-OVA melanoma protection and inhibition response in E.coli. vaccinated mice were compared. Results Compared to E.coli. OVA, E.coli. LLO/OVA-vaccinated splenic CD11c cells stimulated proliferation of autogenic CD4+ T cells and IL-2 production of these cells. CD11c cells induced autogenic CD8+T cells proliferation and IFN-? secretion. A great number of OVA257-264 SIINFEKL-specific splenic CD8+ T cells were induced and the number of lung tumor nodules was significantly reduced in E.coli. LLO/OVA vaccinated mice. Conclusion Recombinant LLO, as an effective adjuvant of E.coli. OVA, can induce murine splenic CD11c cells activation and play potential roles on CD4+ and CD8+ T cells proliferation and IL-2 and IFN-? secretion of these cells, induce more OVA-specific CD8+ T cells and stimulate stronger tumor inhibition in vivo of vaccinated C57BL/6 mice.

Effects of HLA-derived peptide on proliferation and heme oxygenase-1 activity of rat spleen cells / 第三军医大学学报

Objective To investigate the immunosuppression function of a novel HLA derived peptide, RDP1258, and its mechanisms. Methods A peptide derived from HLA, RDP1258, was chemically synthesized by artificial solid phase synthesis. Effects of the peptide on alloreactive cytotoxic activity of rat spleen cells and heme oxygenase 1 (HO 1) activity were observed using 3H TdR method and enzyme chemistry method, respectively. Results The synthetic HLA derived peptide could obviously inhibit the proliferation of rat spleen cells and mixed lymphocyte reaction, and reduce HO activity in a dose dependent manner in vitro . Conclusion RDP1258 can significantly inhibit the proliferation of rat spleen cells induced by mitogen and isoantigen possibly by means of affecting HO 1 activity.