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Abstract
Background Exhausted T cells (Tex) represent a distinct cell state, with a unique transcriptional and chromatin accessibility profile compared to their functional effector counterparts. Terminally exhausted T cells are epigenetically committed and non-responsive to checkpoint blockade therapies.1,2 With the aim to identify epigenetic factors which contribute to T cell exhaustion, we carried out a CRISPR screen in an in vitro T cell exhaustion system.
Methods In vitro exhausted T cells were generated by repeated stimulation of human T cells (isolated from healthy donor leukopaks) with anti-CD3 and anti-CD28 antibodies over a period of 10 days. In vitro Tex were characterized by flow cytometry, cytokine secretion, RNA Seq and ATAC Seq. An arrayed CRISPR screen was carried out by Horizon Discovery in this Tex model targeting 829 genes (focused on the human epigenome) for KO in T cells from 3 donors (3 biological replicates). IFNγ, IL-2, TNFα and cell titer glo (viability readout) were used to assess functional ability of KO cells after the final stimulation.
Results We first established an in vitro T cell exhaustion system and showed that it recapitulated phenotypic, functional, transcriptomic and epigenetic features of exhausted T cells (figure 1). An epigenome focused CRISPR screen was then performed in the Tex model to evaluate the role of different epigenetic factors in T cell exhaustion (figure 2). Hits were identified as genes which when knocked out were able to increase cytokine secretion across at least 2 donors. From the screen, we identified previously established3,4 and novel regulators of T cell exhaustion (figure 3). We validated the role of ikaros, a top hit from the screen, in T cell exhaustion using functional cytokine and T cell mediated tumor killing assays (figure 4).
Conclusions A positive control (CBLB) used in the screen and other previously reported factors were hits in the screen. Several novel regulators of T cell exhaustion were also identified. Hits from the screen could potentially be targeted to reinvigorate exhausted T cells and expand responses to check point therapies. We identified a novel role for ikaros in controlling T cell exhaustion, perhaps supporting a possibility in treating solid tumors. The therapeutic target of IMiDs are the Ikaros family of transcription factors which are approved drugs for the treatment of multiple myeloma and other hematological malignancies.
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In vitro exhausted T cells show phenotypic and epigenetic hallmarks of exhaustion
Overview of the screen
Top hits from the screen included known regulators of T cell exhaustion as well as novel regulators
Role of a top hit (IKZF1/Ikaros1) in T cell exhaustion validated using. A) rescue of cytokine secretion and B) tumor cell killing