Blocking ICOS-B7h Signal Can Inhibit the Function of Allogene T Lymphocytes and Decrease the Incidence and Severity of Acute Graft Versus Host Disease in Mouse Model

ICOS, a CD28 family member expressed on activated T cells, plays important roles in T cell activation and effector function. Here we report our results of biological activity of ICOS signal on allogeneic T lymphocytes and its effect on acute graft-versus-host disease in mouse model by blocking ICOS-B7h signal with ICOS-Ig fusion protein. Human ICOSIg fusion protein was harvested and purified from supernatant of CHO cells transfected with pSecTag2/Hygro A-ICOS-Ig in our lab. Spleen CD4⁺ cells from C57BL/6 mouse were stimulated with dendritic cells from BALB/C mouse, with different doses of ICOS-Ig or human-Ig (h-Ig) as controls. Allogeneic aGVHD model was established with lethally irradiated BALB/c recipients receiving allogeneic BM and spleen T cells from C57BL/6 mouse with 100ug ICOS-Ig or h-Ig intropenetoneally 4 times at day 0, day +2, +4 and +6 of transplantation.

RESULTS:

1. ICOS-Ig (10ug/mL) significantly inhibited proliferation of CD4⁺T cells ( P<0.01), decreased the level of TNF-α and elevated level of IL-4 in the supernatants of CD4⁺ T cells in response to allogeneic mature DCs but had no effect on IFN-γ production; ICOS-Ig blockade elevated apoptosis of splenic CD4⁺ T cells while had no effect on T cell activation (CD25 expression).

2. ICOS-Ig blockade significantly attenuated the lethal GVHD that occurred in control recipient mice. The average survival time was 13.25±5.87 days for mice in h-Ig group, while 21.42±3.02 days for animals in ICOS-Ig group(p=0.0217). Pathologic evaluation revealed that the liver and intestine of animals in ICOS-Ig group has less lymphocyte infiltration and less architectural disruption than those in control h-Ig group;

3. In vivo, ICOS-Ig had no effect on allogeneic T cells division (h-Ig :98.40±1.32, ICOS-Ig: 97.69±2.19 by FACS analysis of CFSE labeled lymphocyte at day 3 of transplantation) and no effect on the proportion of CD4⁺/CD8⁺ (h-Ig: 26.35±0.07, ICOS-Ig: 22.12±0.21), but increased apoptosis of allogeneic CD8⁺ T cells in GVHD model by FACS analysis of Annexin-V staining lymphocytes at day 10 of transplantation (h-Ig: 20.44±3.83, ICOS-Ig: 22.87±6.94 in CD4⁺ T cells; h-Ig: 18.73±7.43, ICOS-Ig: 24.03±5.4 in CD8⁺ T cells).

Spleen T cells from mice after transplantation were stimulated by ConA ex vivo, ICOS-Ig group proliferated less than control h-Ig group through cell counting with CCK-8 (h-Ig: 0.86±0.04,ICOS-Ig: 0.69±0.12,P<0.05). (4) ICOS-Ig significantly reduced the secretion of IFN-γ and elevated IL-4 in the serum of recipient mouse. The IFN-γ (pg/mL) detected were 562.27±49.97 in h-Ig group, 49.79±2.81 in ICOS-Ig group; and the IL-4 (pg/mL) detected were 38.819±27.56 in h-Ig group,456.03±69.63 in ICOS-Ig group. (p<0.05). (5)ICOS-Ig significantly reduced the secretion of T-bet and elevated GATA-3 in the spleens of recipient mouse. The T-bet/GATA-3 detected were 1.87±0.65 in h-Ig group, 0.56±0.03 in ICOS-Ig (p=0.03).

CONCLUSION:

1. The ICOS-Ig fusion protein had bioactivity of inhibition of T cell proliferation and alternated the polarization of T helper cells; It promoted the apoptosis of allo-reactive T cells from donor animals but had no effect on the activation of allo-reactive CD4⁺T cells;

2. ICOS-Ig blockade can prevent aGVHD through attenuating the function of the allo-reactive T cells, elevating apoptosis of allo-reactive T cells and alternating the polarization of T helper cells.