The involvement of STAT proteins in cytokine-mediated regulation of CD28 surface expression on antigen-specific CD8+ T cells expanded for adoptive cell therapy

Abstract

Metastatic melanoma is an aggressive cancer responsive to adoptive cell therapy (ACT) with tumor&ndashreactive CD8 T lymphocytes. ACT efficacy correlates with persistence and proliferation of infused T cells; literature suggests strategies to inhibit terminal differentiation of tumor&ndashreactive CD8 T cells during expansion increases their anti&ndashtumor effect. IL&ndash21 suppresses effector T cell differentiation and promotes memory qualities, such as CD62L and CD28 expression, in human antigen&ndashspecific CD8 T cells. Compared to common gamma chain cytokines IL&ndash2, IL&ndash7 and IL&ndash15, IL&ndash21 is unique in its ability to support CD28 surface expression on activated CD8 T cells. Although the role of CD28 in T cell&ndashmediated immunity is well understood, mechanisms that regulate surface CD28 expression are not well defined. We investigated the ability of cytokines to support CD28 surface expression on human CD8 T cells via STAT protein activation. We found IL&ndash21 promotes sustained STAT3 activation; IL&ndash21 mediated STAT3 activation is required for expansion of MART&ndash1&ndashspecific CD8 T cells characterized by CD28 surface expression. Using chromatin immunoprecipitation (ChIP) analysis, we found IL&ndash21&ndashactivated STAT3 associates with a consensus STAT binding sequence within the human CD28 promoter, supporting a role for STAT3 as a CD28 transcriptional activator. In contrast to IL&ndash21, conventional STAT3&ndashactivating cytokine IL&ndash6 was less efficient at sustaining STAT3 activation, and supported only low levels of CD28 surface expression. In contrast to IL&ndash21, IL&ndash15 actively inhibits CD28 surface expression on activated CD8 T cells. ChIP analysis demonstrated IL&ndash15&ndashmediated STAT5 associates with the CD28 promoter at the same site as STAT3, supporting a role for STAT5 as a CD28 transcriptional inhibitor. Our observations suggest antagonism between STAT3 and STAT5 for transcriptional modulation of CD28 and support IL&ndash21 as the ideal cytokine with which to modulate antigen&ndashspecific CD8 T cells expanded for ACT, based upon the unique ability of IL&ndash21 to support CD28 surface expression.