[Study of reducing graft-versus-host disease by in vitro blockade of CD40-CD40 ligand co-stimulatory pathway in allogeneic bone marrow transplantation mouse model].
OBJECTIVE
To investigate the effect and its mechanism of reducing graft-versus-host disease (GVHD) by in vitro blockade of CD(40)-CD(40)L pathway in vitro, the donor T lymphocytes cultured in vitro with anti-CD(40)L mAb were transfused in bone marrow transplantation (BMT) GVHD mouse model.


METHODS
C57BL/6(H-2b) spleen T cells were isolated as responder cells, and BALB/c(H-2d) spleen cells as stimulator cells. They were cocultured with or without Anti-CD(40)L mAb as anti-CD(40)L mAb group and control group, respectively. At day 5, the mixed lymphocyte response (MLR)-culture cells mixed with bone marrow cells and transfused respectively into the TBI conditioned recipient mice. The mice were divided into two groups: group A, bone marrow cells (2 x 10(6)) and spleen T lymphocytes (2 x 10(6)) from MLR control group; group B, bone marrow cells (2 x 10(6)) and spleen T lymphocytes (2 x 10(6)) from MLR anti-CD(40)L mAb group. The GVHD incidence and hematopoietic reconstitution were observed. Peripheral blood sera and spleen cells of the recipients mice were harvested at scheduled time points for the measurement of cytokines and T cell immunophenotyping with flow cytometry.


RESULTS
The incidence of GVHD in group A was 100% (10/10), and in group B was 20% (2/10). The percentage of H-2D(b) positive cells in group B (n = 8) was (93.54 +/- 2.32)% at day 40 after transplantation. The levels of cytokines in serum from group B were significantly lower than those from group A (P < 0.05). The expressions of CD(4)(+), CD(8)(+), CD(4)(+)CD(25)(+), CD(8)(+)CD(25)(+), CD(4)(+)CD(69)(+), CD(8)(+)CD(69)(+) and CD(4)(+)CD(40)L(+) were lower in group B than in group A (P < 0.05). The expressions of CD(8)(+)CD(40)L(+) and CD(4)(+)CD(45)RA(+) were similar in the two groups (P > 0.05).


CONCLUSIONS
Blockade of CD(40)-CD(40)L interaction in vitro could induce immune tolerance in vivo, reduce aGVHD in aGVHD mice model and form chimerism, which was mediated by inhibiting the Th1 and Th2 cytokines production, inducing tolerance of CD(4)(+) and CD(8)(+) cells to alloantigens. The obstruction of T cells activation after tolerance happened mainly at the early and mature phase of T cells activation. These provided the experimental basis for the use of anti-CD(40)L mAb in the clinical transplantation to prevent aGVHD.
Huang
Shao-liang
SL
Department of Pediatrics, the Second Affiliated Hospital, Sun Yat-sen University, Guangzhou 510120, China.
Chen
Chun
C
Duan
Lian-ning
LN
Li
Hao-wei
HW
Wen
Guan-mei
GM
Li
Lin
L
Zhan
Mei-yi
MY
Wei
Jing
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D016026:Bone Marrow Transplantation / Q000009:adverse effects*; D019013:CD40 Antigens / Q000502:physiology*; D023201:CD40 Ligand / Q000276:immunology / Q000502:physiology*; D006086:Graft vs Host Disease / Q000517:prevention & control*; D016753:Interleukin-10 / Q000097:blood; D015847:Interleukin-4 / Q000097:blood; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL
D000911:Antibodies, Monoclonal; D019013:CD40 Antigens; D016753:Interleukin-10; D023201:CD40 Ligand; D015847:Interleukin-4