• IgM in cultures;
  • immune response;
  • mitogenesis;
  • regulatory T cells;
  • somatic LDH;
  • sperm LDH-C4

PROBLEM: Sperm-specific lactate dehydrogenase-C4 (LDH-C 4) is an autoantigen that produces experimentally induced autoimmune orchitis in testes. In the present study, immunological functions of B and T cells have been examined and compared after immunization with sperm-specific LDH and the LDH from somatic cells. METHODS: Three sets of experiments were performed. In the first set, effects of Balb/C LDH isozymes at 10−3−1 L μg/well were investigated: (i) by mixed lymphocyte cultures (MLC) using C-57 Bl/6 female cells as responders and AKR lymphocytes (irradiated) as stimulators, (ii) for regulatory T cell activity in MLC co-cultured along with Con-A-induced AKR lymphoblasts and (iii) for modulation of lymphocyte activation by PHA in vitro. In the second set of experiments, female mice (C-57 Bl/6) were distributed in six groups for various treatments: i) saline (as vehicle), ii) adjuvant, iii) LDH-B4 (20×3 μg), iv) LDH-B4 (40×3 μg), v) LDH-C4 (20×3 μg), and v) LDH-C4 (40×3 μg). Mice were hyperimmunized with -B4 or -C4 (Balb/c) with a primary dose of 20 or 40 μg of protein per mouse, emulsified in Freund's complete adjuvant (FCA) and two identical doses in Freund's incomplete adjuvant (s.c.) within 22 days. Saline (group i) or adjuvant treated dams (group ii) served as controls. One week after the second booster, sera were tested for IgG response and lymphocytes harvested for polyclonal activation in vitro using LPS and Con-A as mitogens. In the third set of experiments, female Balb/c mice were divided into six groups as in the second experiment and immunized with a single primary dose of isogenic LDH-B4 or LDH-C4 at 20 or 40 μg of protein in FCA. On day 5, after sensitization with LDH, lymphocytes were evaluated for mitogenesis and for IgM production in vitro using LPS and Con-A as mitogens. RESULTS: i) Primary MLC(s) were non-specifically suppressed in the presence of 10−3–l μg allogenic LDH-C4 or -B4, although LDH-C4 tended to abolish MLC completely. But MLC co-cultured with blast cells was suppressed by LDH-C4 alone, indicating that sperm LDH suppresses induced formation of regulatory T cells. ii) FCA primed lymphocytes in situ were significantly inhibited for Con-A stimulation in vitro. Since LPS stimulation remained unaffected, it appeared that FCA is immunosuppressive for T cell proliferation alone. iii) Cells primed with LDH increased mitogenic activity of LPS several fold, although LDH-C4 was less effective than LDH-B4 in sensitization of B lymphocytes. iv) However, effect of Con-A in mitogenesis was dose-dependent, viz. cells primed at 20×3 μg of each isozyme overcame the immunosuppressive nature of FCA by bringing back the SI (×25) equivalent to saline primed cells, while pre-treatment of cells with 40×3 μg LDH-C4 abolished SI completely, indicating that -C4 primed cells were immunologically suppressed for Con-A stimulation. Such a response was markedly visible when allogenic LDH-C4 was used for hyperimmunization; lymphocytes challenged with somatic LDH under similar conditions did not react. Loss of T cell functions by LDH-C4 was confirmed in the presence of PHA in primary cultures. v) For antibody responses, although sperm LDH was highly reactive and dose-dependent, somatic LDH was also immunogenic for IgG production in serum to a lesser degree. Besides, IgM antibody was also discernible by two isozymes in LPS-induced cultures. Significantly, -C4 primed cells at the higher dose, in comparison with the lower dose, were less responsive for IgM production. CONCLUSIONS: It is concluded that LDH(s) from sperm and somatic cells share functionally related antigenic epitopes that can generate/modify immune responses in vivo and in vitro with qualitative differences. However, immunosuppressive determinant of LDH-C4 is cell specific and dose selective.