Optimization of in vitro expansion of macaque CD4⁺ T cells using anti-CD3 and co-stimulation for autotransfusion therapy

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/ε was able to induce T-cell proliferation and expansion in combination with antibodies against multiple co-stimulatory molecules, monkey CD3ε 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.