Selective expansion of memory CD4⁺ T cells by mitogenic human CD28 generates inflammatory cytokines and regulatory T cells

Costimulatory signals are important for development of effector and regulatory T cells. In this case, CD28 signaling is usually considered inert in the absence of signaling through the TCR. By contrast, mitogenic rat CD28 mAb reportedly expand regulatory T cells without TCR stimulation. We found that a commercially available human CD28 mAb (ANC28) stimulated PBMC without TCR co-ligation or cross-linking; ANC28 selectively expanded CD4⁺CD25⁺FOXP3^- (Teff) and CD4⁺CD25⁺FOXP3⁺ (Treg) cells. ANC28 stimulated the CD45RO⁺ CD4⁺ (memory) population, whereas CD45RA⁺CD4⁺ (naive) cells did not respond. ANC28 also induced inflammatory cytokines. Treg induced by ANC28 retain the Treg phenotype longer than costimulated Treg. Treg induced by ANC28 suppressed CD25^- T cells through a contact-dependent mechanism. Purity influenced the response of CD4⁺CD25⁺ cells because bead-purified CD4⁺CD25⁺ cells (85-90% pure) responded strongly to ANC28, whereas 98% pure FACS-sorted CD4⁺CD25^bright (Treg) did not respond. Purified CD4⁺CD25^int cells responded similarly to the bead-purified CD4⁺CD25⁺ cells. Thus, pre-activated CD4⁺ T cells (CD25^int) respond to ANC28 rather than Treg (CD25^bright). The ability of ANC28 to expand both effectors producing inflammatory cytokines aswell as suppressive regulatory T cells might be useful for ex vivo expansion of therapeutic T cells.