T cell alloreactivity induced by normal G-CSF-mobilized CD34⁺ blood cells

Abstract

In this study, the hypothesis that a subset of granulocyte colony-stimulating factor (G-CSF)-mobilized CD34⁺ blood cells may actively induce an allogeneic T cell response in vitro was tested. Circulating CD34⁺ cells were purified to ⩾98% by high gradient magnetic separation and then analyzed for the coexpression of HLA-DR, the common β-chain of the leukointegrin family CD18 and costimulatory molecules CD80 (B7–1) and CD86 (B7–2). These antigens were expressed on average on: 94.9 ± 2.5%, 64.4 ± 15.4%, 0% and 1.9 ± 1.2% CD34⁺ blood cells, respectively. Irradiated CD34⁺ cells induced a high proliferative response of allogeneic, but not autologous, purified CD4⁺ and CD8⁺ T cells in primary mixed leukocyte culture (MLC). An average three-fold lower CD4⁺ and CD8⁺ T cell response was induced by mononuclear cells from G-CSF-treated donors. A lower frequency of allostimulating cells among mononuclear cells rather than among CD34⁺ cells in the apheresis was documented by limiting dilution assay (LDA). As previously observed with marrow, sorted CD34⁺/CD18⁺ cells induced the proliferation of allogeneic T cells in MLC, while CD34⁺/CD18⁻ cells, which were >94% HLA-DR⁺ and contained both committed (CFU-C) and early (LTC-IC) hematopoietic progenitors, stimulated allogeneic T cells poorly. Three-color staining cytofluorimetry indicated that expression of CD80 and CD86 were upregulated in 6.9 ± 4.9 and 10.7 ± 2.6% CD34⁺ blood cells respectively, after 24–30 h of culture with autologous or allogeneic mononuclear cells, or with CD4⁺, or CD8⁺ T cells, but not with medium alone. Moreover, the upregulation of CD86 was observed on CD34⁺/CD18⁺ rather than on CD34⁺/CD18⁻ cells after 30 h in MLC. Blocking experiments demonstrated that preincubation of stimulator and responder cells with anti-CD80 plus anti-CD86 monoclonal antibodies induced a 84 ± 8% inhibition of CD34⁺ cell allostimulating activity after 6 days in primary MLC. These results suggest that G-CSF-mobilized CD34⁺ hematopoietic progenitors with alloantigen presenting function express CD18 and may upregulate CD80 and CD86 upon interaction with T cells. Since activation of B7 costimulatory molecules represents an active costimulatory pathway on G-CSF-mobilized CD34⁺ cells, the blockade of these molecules or, alternatively, the use of selected non-immunogenic CD34⁺/CD18⁻ blood stem cells may represent a new strategy for reducing graft rejection and overcoming HLA barriers in allogeneic stem cell transplantation.