Effect of New Immunosuppressant-Rapamycin on One-way Mixed Lymphocyte Culture

Objective: Observing human to mouse one-way mixed lymphocyte culture(xMLC) and the effect of new immunosuppressant-Rapamycin on XMLC. Methods: Mouse splenic lymphocyte were collected and treated by mitomycin as activating cell; Human Peripheral blood lymphocytes(hPBL)were separated and gathered as reacting cell; Mouse splenic lymphocyte and hPBL wee mixed to incubate for 1 week; The researchers designed control、RPM groups,and experiment(drugs)grup have different concentration. Results: HPBL in the experiment groups (mixed mouse lymphocyte)proliferated obviously,the amount of3H-TdR in corporation increased evidently(P<0 05,The mean percentage of CD 4, CD 8, LgG, LgM positive cells rose markedly. HPBL in the experiment groups less proliferated,the amount of 3H-TdR incorporation declined,RPM's ic50(50%inhibition concentration)approximately in 1.5 nmol/L; the mean percentage of CD 4, CD 8, IgG, IgM positive cells fell obviously. Conclusion: The human to mouse one-way MLC has obvious lymphocyte proliferation. New immunosuppressants-Rapamycin have powerful effete on XMLC.

Anti-CD132 Monoclonal Antibodies Inducing T Cells Apoptosis after Alloantigen Stimulation and Its Possible Clinical Applications

Aim To investigate the mechanism of anti-CD132 monoclonal antibodies (mAbs)inhibiting T cells proliferation in vitro, and their potential values for clinical use. MethodsBALB/c and C57BL/6 mice splenocytes were harvested for two-ways mixed lymphocyte culture (MLC).Anti-CD132 mAbs (final concentration 100 mg·L^(-1)) were added in MLC on day 0 (group 1) or day 3(group 2). Fluorescence activated cell sorting (FACS) was used to measure the proliferation(carboxy-fluorescein dia cetate, succinimidyl ester, CFSE), apoptosis of T cells (PE-CD3,FTTC-annexin-v), and cell cycle (pro-pidium iodide stain) . The expression of survivin in T cellswas detected by immunochemical stai-ning. Re-sults Multi-generation CFSE-labeled splenocytes werefound dividing and their fluorescent strength decreased in MLC. There was no noticeable change influorescent intensity in group 1 and group 2. On day 3, apoptosis induced by anti-CD132 mAbs wasdetected in part of T cells, but was not detected in the former two days in group 1. In group 2, thenumber of cells in M phase (activated T cells) decreased and apoptot-ic cells increased on day 4.The phenomena were not observed in control group (P < 0.01). Expression of survivin in T cells wasdetected in control group but not in groups 1 and 2. Conclusion Blockade of CD132 signaling pathwayinhibits T cell proliferation in vitro by means of inducing activated alloreactive T cell apoptosisbut not the resting T cells. Anti-CD132 mAbs may be candidates for clinical applications.

Transplantation of human hepatocytes into tolerized genetically immunocompetent rats

AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human hepatocytes was established by injection of primary human hepatocytes or Huh7 human hepatoma cells into the peritoneal cavities of fetal rats. Corresponding cells were subsequently transplanted into newborn rats via intrasplenic injection within 24h after birth. RESULTS: Mixed lymphocyte assays showed that spleen cells from non-tolerized rats were stimulated to proliferate when exposed to human hepatocytes, while cells from tolerized rats were not. Injections made between 15 d and 17 d of gestation produced optimal tolerization. Transplanted human hepatocytes in rat livers were visualized by immunohistochemical staining of human albumin. By dot blotting of genomic DNA in livers of tolerized rats 16 weeks after hepatocyte transplantation, it was found that approximately 2.5 X 10(5) human hepatocytes survived per rat liver. Human albumin mRNA was detected in rat livers by RT-PCR for 15 wk, and human albumin protein was also detectable in rat serum. CONCLUSION: Tolerization of an immuno-competent rat can permit transplantation, and survival of functional human hepatocytes.

The Inhibitory Effects of Mouse ICOS-Ig Gene-Modified Mouse Dendritic Cells on T Cells

The main approach to reduce graft rejection has been focused on the development of immunosuppressive agents at present.Although these strategies have reportedly reduced graft rejection,there has been a reciprocal increase in more severe immunosuppression and lethal infections,as well as severe side effects.Blockade of costimulatory T cell response has been proved as one of useful strategies to reduce graft rejection.Furthermore, it has been shown that infusion of dendritic cells(DCs)with a potent negative regulatory ability for T cells could prolong allograft survival.In this study mouse DCs(mDCs)were transfected with the recombinant plasmid pcDNA3.0 containing mouse inducible costimulator-Ig(mICOS-Ig) cDNA by electroporation.The transient expression of mICOS-Ig in mDC could be detected by ELISA and SDS-PAGE.Mouse ICOS-Ig fusion protein expressed in mDC and mICOS-Ig gene-modified mDC could inhibit lymphocyte proliferation in mixed lymphocyte culture(MLC)in vitro.Furthermore,mICOS-Ig gene-modified mDC could inhibit lymphocyte proliferation in recipient mice.These results suggested that mICOS-Ig gene-modified mDC exerted inhibitory effects on T cells,and might be suitable for treatment or prevention of graft rejection and immunopathologic diseases.Cellular & Molecular Immunology.2004;1(2):153-157.