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1046 Targeting novel pathways in chronically activated T Cells prevents functional exhaustion
  1. Justyna Rzepecka,
  2. Andrew Hall,
  3. Mark Barbour,
  4. Emma Mallon,
  5. Marija Bedaj,
  6. Lucia Janicova,
  7. Xabier Cortes-Lavaud,
  8. Frances Acklam,
  9. Joanne Hay,
  10. Hayley Gooding,
  11. Darryl Turner,
  12. Preeti Singh and
  13. Aaron Crawford
  1. Concept Life Sciences, Edinburgh, UK


Background In cancer, persistent antigenic challenge leads to T cells acquiring a hyporesponsive cell state, also referred to as T cell exhaustion. We have previously demonstrated that human CD3+ T cells repeatedly stimulated in vitro, via their TCR, develop phenotypical characteristics of exhausted T cell found in vivo, including increased expression of inhibitory receptors PD-1, TIM-3 and LAG-3 and diminished responsiveness to dendritic cell activation and cancer cell cytotoxicity. We showed that PD-1 blockade with nivolumab and treatment with an IKZF3 inhibitor, lenalidomide, reinvigorated the exhausted T cells. We next wished to evaluate if blocking of IKZF3 during the development of T cell exhaustion could protect them from acquiring the exhausted phenotype.

Methods Human CD3+ T cells, isolated from healthy PBMC donors, were repeatedly stimulated with anti-CD3/anti-CD28 coated beads to mimic chronic antigenic challenge. The stimulations were conducted in the presence or absence of lenalidomide. T cells, both CD4+ and CD8+, were assessed for changes in expression of inhibitory receptors and cytokine release was quantified. Following the final round of TCR stimulation, T cells were co-cultured with allogeneic dendritic cells to determine if their functionality has been altered by lenalidomide treatment. T cell proliferation and IFN-g release were assessed as well as changes in inhibitory receptor expression at the end of the co-culture.

Results We demonstrated that lenalidomide had no impact on T cell viability or CD4+/CD8+ ratio in repeatedly stimulated cultures. Lenalidomide did however affect inhibitory receptor expression and impacted cytokine secretion from chronically stimulated T cells. Lenalidomide led to increased PD-1 expression on CD8+ T cells and LAG-3 expression on CD4+ T cells whereas TIM-3 expression was downregulated on both T cells subsets in the presence of compound. Lenalidomide enhanced production of cytokines in repeatedly stimulated T cells. To assess if lenalidomide-driven changes had any impact on T cell functionality, we cultured lenalidomide-treated and untreated repeatedly activated T cells with allogeneic dendritic cells. Whilst repeatedly stimulated T cells not exposed to lenalidomide showed diminished ability to proliferate and secrete IFN-g, consistent with their exhausted cell state, lenalidomide treated T cells displayed increased IFN-g secretion suggesting that lenalidomide protected repeatedly stimulated T cells from acquiring an exhausted phenotype.

Conclusions The in vitro assays described herein offer the opportunity to investigate the effect of candidate therapeutics, including combination therapies, on exhausted T cell development and function.

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