Regulation Of Immune Functions by Sperm‐Specific LDH and its Differences with Somatic Isozyme in Primary and Secondary Lymphocyte Cultures
Authors: GUPTA G.S. , CHATURVEDI G. 
Published: December 2003

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.