IL-2 Dependent Modulation of CD8+ T Cells Using E3 Ubiquitin Ligase Cbl-b Deficient CD4+ T Cells
SeongJun Han1,2, Douglas Chung2, Michael St.Paul1,2, Alisha Elford2, Pamela Ohashi1,2,•
1Department of Immunology, University of Toronto, Canada, 2Princess Margaret Hospital, University Health Network, Canada •Corresponding author
Adoptive T cell therapy (ACT) has been explored for the treatment of multiple malignancies including melanoma. However, ACT can be limited by lack of IL-2 and functional CD4+ T cells, and the presence of regulatory T cells (Treg) within the tumor environment. To address the challenge, adoptive transfer of CD4+ T cells has been previously studied to restore robust CD8+ T cell response. Lymphocytes deficient in Cbl-b, an E3 ubiquitin ligase, display a hyper-inflammatory phenotype and resistance against immunosuppression, serving as a potential target for improving ACT. We investigated thecellular consequences of Cbl-b deficiency in CD4+ T cells, and its potential in mediating robust CD8+ T cell responses and Treg cell-resistance. CD8+ or CD4+ T cells from B6 and Cbl-b-/- splenocytes were stimulated in different combinations for surface immunophenotyping and cytokine profiling. Treg suppression assays and cytokine screening were performed to identify the role of Cbl-b-mediated cytokine regulation in Treg cell-resistance. Lastly, Treg suppression assays were performed using exogenous IL-2, anti-IL-2/CD25 antibodies, as well as with IL-2R-/- T cells. We demonstrate that Cbl-b-/- CD4+ T cells display enhanced Th1/17 differentiation, secreting high level of IL-2, IFN and IL-17A, as previously explored. The pro-inflammatory cytokines secreted by Cbl-b-/- CD4+ T cells, specifically IL-2, induced significantly elevated surface activation markers on CD8+ T cells and resulted in reduced Treg suppression specifically through IL-2R. Abrogation of IL-2 signaling reversed the resistance of CD4+ and CD8+ T cells to Treg suppression. Overall, we demonstrate that hypersecretion of IL-2 by Cbl-b-/- CD4+ T cells is an important mechanism to 1) boost pro-inflammatory CD8+ T cell function and 2) enable CD4+ or CD8+ T cells to counter IL-2 deprivation by Tregs.
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