Human CD4⁺ T Cells Help CD8⁺ T Cells Proliferate Ex Vivo by Secreting Both IL-2/IL-21 and Upregulating IL-21R

Abstract 4284

While adoptive T cell therapy is a promising treatment modality for cancer, the optimal approach to generate T cell grafts ex vivo is currently unknown. CD4⁺ T cells help generate effective immune responses by sustaining CD8⁺ T cell proliferation, preventing exhaustion, and establishing long-lived functional memory. Incorporation of CD4⁺ T cell help to expand CD8⁺ T cells may provide a novel strategy to generate CTL grafts for adoptive therapy.

In mouse models, common γ-chain receptor cytokines and CD40/CD40L can mediate CD4⁺ T cell help. However, CD4⁺ T cell help in humans has yet to be fully defined. We therefore developed an in vitro model for human CD4⁺ T cell help, which utilizes a novel artificial APC, aAPC/mOKT3. K562-based aAPC/mOKT3 expresses a membranous form of anti-CD3 mAb, CD54, CD58, CD80, and CD83 and stimulates CD3⁺ T cells regardless of HLA haplotype or antigen specificity. Using aAPC/mOKT3, we stimulated CD8⁺ T cells in the presence or absence of CD4⁺ T cells and found that CD8⁺ T cells expanded better when coincubated with CD4⁺ T cells, suggesting the presence of CD4⁺ T cell help. Coculture experiments using transwell plates suggested that the observed CD4⁺ T cell help of CD8⁺ T cell expansion involved both soluble factors and cell-cell contact.

To identify molecules mediating the observed CD4⁺ T cell help, supernatants of CD4⁺/CD8⁺ T cell mixed and separate cultures were measured for a panel of soluble factors. IL-2 and IL-21 were detected at lower levels in mixed cultures, consistent with more consumption or less production of these cytokines. Blockade of either IL-2 or IL-21 in CD4⁺/CD8⁺ T cell mixed cultures resulted in a reduction of CD8⁺ T cell expansion, indicating that, for both cytokines, more consumption rather than less production occurred and that IL-2 and IL-21 may serve as mediators of CD4⁺ T cell help. However, the addition of IL-21 to CD8⁺ T cells stimulated with aAPC/mOKT3 in the presence of IL-2 did not improve CD8⁺ T cell expansion, suggesting that IL-2 plus IL-21 cannot solely replace CD4⁺ T cell help. We found that the presence of CD4⁺ T cells upregulated the expression of IL-21R on CD8⁺ T cells. When we introduced IL-21R on CD8⁺ T cells and stimulated with aAPC/mOKT3 in the presence of IL-2 and IL-21, CD8⁺ T cell proliferation was restored. These results suggest that CD4⁺ T cells help CD8⁺ T cells proliferate ex vivo by secreting both IL-2/IL-21 and upregulating IL-21R.

When peripheral CD3⁺ T cells from normal donors were stimulated with aAPC/mOKT3, the number of both CD4⁺ and CD8⁺ T cells increased. However, in contrast to other pan T cell expansion systems, aAPC/mOKT3 preferentially expanded CD8⁺ T cells. No obvious skewing in the Vβ usage of both CD4⁺ and CD8⁺ T cell populations was revealed by TCR Vβ repertoire analysis, supporting “unbiased” T cell expansion by aAPC/mOKT3. Moreover, HLA-restricted antigen-specific CD8⁺ CTL with high functional avidity could be generated from CD3⁺ T cells initially expanded for 4 weeks using aAPC/mOKT3.

Using aAPC/mOKT3, tumor-infiltrating lymphocytes (TIL) were successfully expanded without adding soluble mAb or allogeneic feeder cells. As in peripheral T cell cultures, CD8⁺ T cells predominantly expanded in all cultures, including those that initially contained a minimal percentage of CD8⁺ T cells. Importantly, Foxp3⁺ Treg cells did not proliferate. Expanded T cells highly expressed CD27 and CD28, which are associated with T cell survival and persistence in vivo. They also secreted high levels of IFN-γ and IL-2, lower amounts of IL-4, and no IL-10. These results demonstrate that the aAPC/mOKT3-based system can expand functional CD8⁺ TIL in the presence of autologous CD4⁺ T cells.

In conclusion, we have determined that CD4⁺ T cell-dependent CD8⁺ T cell expansion required both soluble factors secreted by and cell contact with CD4⁺ T cells. Among the soluble factors secreted by CD4⁺ T cells, IL-2 and IL-21 were necessary. Furthermore, upregulation of IL-21R on CD8⁺ T cells by CD4⁺ T cells was critical for an optimized response to IL-21. Thus, in humans, CD4⁺ T cells help CD8⁺ T cells proliferate by secreting IL-2/IL-21 and upregulating IL-21R. Our aAPC enabled expansion of CD8⁺ TIL in the presence of CD4⁺ T cell help without using soluble mAb or allogeneic feeder cells. Taken together, these results demonstrate the indispensable role of CD4⁺ T cell help on expanding CD8⁺ T cells and suggest a novel strategy to generate anti-tumor T cells ex vivo for adoptive therapy.