Immunomagnetic Depletion of CD25-Expressing T Cells from Donor Blood Removes a Mixed Population of Regulatory and Effector T Cells - Implications for Graft Engineering.

CD4+CD25+FOXP3+ regulatory T cells (T_R) control proliferative CD4 and CD8 T cell responses to self and foreign antigens such as cytomegalovirus (CMV) and tumor-specific antigens. Thus, depletion of CD25-expressing cells from a resting population of T cells prior to antigen stimulation could boost the generation of antigen-specific T cells for adoptive transfer to treat viral infection or tumors. We depleted peripheral blood mononuclear cells (PBMC) from nine CMV seropositive donors using Miltenyi CD25 microbeads (20 μl/10⁷ cells). CD25-depleted or unmanipulated PBMC were stimulated with CMV pp65-expressing antigen presenting cells for 10–14 days with low dose IL-2, and intracellular interferon-gamma (IFNγ) production by flow cytometry was compared between CD25-depleted and -undepleted cultures. An absolute increase in antigen-specific CD4+ and CD8+ T cells was seen after CD25 depletion in 4/8 and 5/8 cultures respectively. However, in other cultures there was a decrease or no change in IFNγ+ CD4+ T cells in CD25-depleted cultures, suggesting that the pp65-specific precursor cells had also been removed. We then used 4 μl beads per 10⁷ PBMC to selectively remove only the CD25^bright (predominantly T^reg) population in nine donors and confirmed by flow cytometry that only CD25^bright cells had been removed from the starting population. However, real-time quantitative PCR (Q-PCR) showed that even though the CD25+ fraction was enriched in FOXP3-expressing cells, a substantial proportion of the CD25-depleted PBMC still expressed FOXP3. Flow cytometric analysis of FOXP3 expression by CD25+ and CD25-negative CD4+ T cells showed that a substantial proportion of CD25- cells expressed FOXP3, confirming that CD25 is not a suitable single marker for depletion of T^regs. Again no reproducible augmentation of antigen-specific T cell responses was observed: one and five donors showing an increase in CD4 and CD8+ antigen-specific T cells, respectively, while the remainder showed a decrease or no change in CD4+ and CD8+ IFNγ-producing cells. These results suggest that removal of CD25+ cells from PBMC using CD25 microbeads removes both T^reg and pp65-specific effector CD4+ and CD8+ T cells. Further, since FOXP3 is induced in responder cells as confirmed by FOXP3 Q-PCR, depletion of T^reg at the start of the cultures may only transiently alleviate the negative regulation of the antigen response. Thus, CD25 depletion using microbeads is not a reliable method to boost antigen specific T cell expansions because of the inadvertent removal of a portion of the memory response to the antigen. Since it recently has been demonstrated that Treg act as an IL-2 sink, the addition of this cytokine should functionally silence the Tregs while preserving the inflammatory response.