Abstract:Background Our laboratory has previously shown that adoptive transfer of in vitro‐expanded autologous purified polyclonal CD4+ T cells using anti‐CD3/CD28‐coated beads induced antiviral responses capable of controlling SIV replication in vivo.Methods As CD4+ T cells comprise several phenotypic and functional lineages, studies were carried out to optimize the in vitro culture conditions for maximal CD4+ T‐cell expansion, survival and delineate the phenotype of these expanded CD4+ T cells to be linked to maximal clinical benefit.Results and Conclusions The results showed that whereas anti‐monkey CD3γ/ɛ was able to induce T‐cell proliferation and expansion in combination with antibodies against multiple co‐stimulatory molecules, monkey CD3ɛ cross reacting antibodies failed to induce proliferation of macaque CD4+ T cells. Among co‐stimulatory signals, anti‐CD28 stimulation was consistently superior to anti‐4‐1BB, CD27 or ICOS while the use of anti‐CD154 failed to deliver a detectable proliferation signal. Increasing the relative anti‐CD28 co‐stimulatory signal relative to anti‐CD3 provided a modest enhancement of expansion. Additional strategies for optimization included attempts to neutralize free radicals, enhancement of glucose uptake by T cells or addition of T‐cell stimulatory cytokines. However, none of these strategies provided any detectable proliferative advantage. Addition of 10 autologous irradiated feeder cells/expanding T cell provided some enhancement of expansion; however, given the high numbers of T cell needed, this approach was deemed impractical and costly, and lower ratios of feeder to expanding T cells failed to provide such benefit. The most critical parameter for efficient expansion of purified CD4+ T cells from multiple monkeys was the optimization of space and culture conditions at culture inception. Finally, anti‐CD3/28‐expanded CD4+ T cells uniformly exhibited a central memory phenotype, absence of CCR5 expression, marked CXCR4 expression in vitro, low levels of caspase 3 but also of Bcl‐2 expression.