Soluble HLA-G protein secreted by allo-specific CD4+ T cells suppresses the allo-proliferative response: a CD4+ T cell regulatory mechanism

Abstract

We recently reported that the nonclassical HLA class I molecule HLA-G was expressed in the endomyocardial biopsies and sera of 16% of heart transplant patients studied. The aim of the present report is to identify cells that may be responsible for HLA-G protein expression during the allogeneic reaction. Carrying out mixed lymphocyte cultures in which the responder cell population was depleted either in CD4(+) or CD8(+) T cells, we found that soluble HLA-G5 protein but not the membrane-bound HLA-G isoform was secreted by allo-specific CD4(+) T cells from the responder population, which suppressed the allogeneic proliferative T cell response. This inhibition may be reversed by adding the anti-HLA-G 87G antibody to a mixed lymphocyte culture. That may indicate a previously uncharacterized regulatory mechanism of CD4(+) T cell proliferative response.

Figure 1

Immunoprecipitation followed by Western blot analysis of cell culture supernatants after 10 days of MLR revealed the presence of soluble HLA-G5 in three MLR combinations (MLR1, MLR2, and MLR3). Immunoprecipitation and Western blot analysis of soluble HLA-G were carried out by using the PAG5-6 Ab and 4H84 mAb, respectively. In these HLA-G5-positive combinations, when the responder cells were CD4⁺-depleted (MLR1 CD4⁻CD8⁺, MLR2 CD4⁻CD8⁺, and MLR3 CD4⁻CD8⁺), HLA-G5 could not be detected. In contrast, when responder cells were CD8⁺-depleted (MLR1 CD8⁻CD4⁺, MLR2 CD8⁻CD4⁺, and MLR3 CD8⁻CD4⁺), HLA-G5 could be observed. M8-HLA-G5 and M8-pcDNA transfectant cells were used as positive and negative controls, respectively.

Figure 2

Detection of HLA-G5 by immunoprecipitation followed by Western blot analysis of cell culture supernatants on various days after MLR. Stimulator cells were used against total peripheral blood mononuclear responder cells in the three HLA-G-positive combinations (MLR1, MLR2, and MLR3). (A) Days 6 and 10 of primary MLR. (B) Days 12 and 15 of secondary MLR. M8-HLA-G5 transfectant cells were used as the positive control, and M8-pcDNA was used as the negative control.

Figure 3

Flow cytometry and immunocytochemical analysis after 10 days of MLR by using the 87G and 4H84 anti-HLA-G mAbs. (A) Flow cytometry profiles were all negative with anti-HLA-G mAbs, whereas HLA-G1-transfected cells were positively stained with 87G. (B) Immunocytochemical analysis was carried out without permeabilization. In this HLA-G-positive combination, the PBMCs were negatively stained; after permeabilization, the responder cells correspond to all of the PBMCs (C) and CD8⁺-depleted PBMCs (D) were positively stained.

Figure 4

Western blot analysis of responder cell lysates after MLR. Stimulator cells were used against total peripheral blood mononuclear responder cells in the three HLA-G-positive combinations (MLR1, MLR2, and MLR3). (A) Days 6 and 10 of primary MLR. (B) Days 12 and 15 of secondary MLR. M8-HLA-G5 and M8-HLA-G1 transfectant cells were used as positive controls, and M8-pcDNA was used as the negative control.

Figure 5

Effect of HLA-G5 production on T cell proliferation when (i) the responder cells were CD8⁺-depleted (CD4⁺-enriched) and (ii) the anti-HLA-G5 mAb was used in HLA-G-positive and -negative combinations. The data are expressed as the mean of thymidine incorporation (cpm) in triplicate wells.