Imbalance in T Cell Subsets Triggers the Autoimmune Toxicity of PI3K Inhibitors in CLL
Authors: Gadi Deepti , Kasar Siddha , Griffith Alec , Chiu Pui Yan , Tyekucheva Svitlana , Rai Vanessa , Vartanov Alexander R. , Fernandes Stacey M. , Pan Yang , Lederer James A , Sherry Barbara , Brown Jennifer R. 
Published: November 2019

Idelalisib and duvelisib are FDA approved oral inhibitors of PI3K delta and PI3K delta/gamma, respectively. Previously, we reported a decrease in regulatory T cells (Tregs) associated with autoimmune hepatotoxicity in untreated CLL patients receiving idelalisib-ofatumumab. We hypothesized that a Th17 inflammatory phenotype may be involved. To directly assess Th17s, we performed IL-17A and IL-17F intracellular staining in CD4 and CD8 T cells from this cohort (no / low toxicity group (grade 0-2 AEs) n=4; high toxicity group (grade 3+ by CTCAE) n=6), and found that the percentage of CD8 cells positive for IL-17F was significantly higher at cycle 2 in patients with high tox compared to patients with low tox (p = 0.0095). Interestingly, pre-treatment, patients who developed high tox had higher IL-17F expression in CD8s compared to CD4s compared to patients with low or no tox. The ratio of IL-17F staining in CD8 to CD4 T cells was significantly higher in patients who developed high tox compared to patients with low tox at the pre-treatment timepoint (p = 0.0381). Furthermore, IL-17A and IL-17F in CD8 T cells were also higher at baseline among mutated IGHV patients (p = 0.1048 and p = 0.0095 respectively), who were also at much greater risk of autoimmune toxicity. These data implicate the Th17 pathway in the autoimmune toxicity of idelalisib. Additionally, single cell mass cytometry (CyTOF) on relapsed/refractory patients treated with idelalisib (n=10) and duvelisib (n=2) showed that the percentage of Tregs decreases with treatment (p = 0.0405 for idelalisib), consistent with our data from the previously untreated idelalisib cohort.
To understand this decrease in Tregs, we tested whether PI3K inhibition skews the differentiation of T cell subsets. Naïve CD4 T cells from healthy individuals were isolated and differentiated toward a Treg or Th17 phenotype in the presence of 10 µM idelalisib, duvelisib, or DMSO. Treg differentiation was determined by measuring intracellular FOXP3 by flow cytometry and Th17 differentiation was evaluated by RORγT intracellular staining. Idelalisib treatment significantly decreased Treg differentiation from a median of 53.8% with DMSO to 43.4% (n=4; p = 0.0002), and increased Th17 differentiation from 24.7% with DMSO to 37.2% (n=5; p = 0.0005). Similarly, with duvelisib, Treg differentiation decreased significantly from a median of 53.8% with DMSO to 47.1% (n=4; p = 0.0005), and Th17 differentiation increased from 24.7% with DMSO to 36.9% (n=5; p = 0.02). These results indicate that both idelalisib and duvelisib can skew T cell differentiation toward a more inflammatory phenotype in vitro. Additionally, the effects of idelalisib and duvelisib on proliferation of CD3 cells from healthy PBMCs were evaluated with 3 days in-vitro treatment using the cell division tracker dye CellTrace Violet. The proliferation index was calculated using the ModFit algorithm to quantify the increase in cell number. The proliferation index of CD3 cells in the presence of 1 µM idelalisib and 1 µM duvelisib decreased by a median of 19.3% (n=4; p = 0.0031) and 28.6% (n=4; p = 0.0028) respectively, compared to DMSO; duvelisib led to greater inhibition of T cell proliferation in vitro than idelalisib (n=4; p = 0.0241) which may impact on risk of irAEs.
To further identify predictors of toxicity with PI3Ki therapy and to characterize differences between idelalisib and duvelisib, we are analyzing CyTOF data on a cohort of 13 patients treated on a DFCI upfront trial with 7 days duvelisib lead-in, then FCR. With one week of duvelisib monotherapy we found an increase in ICOS expression on CD8 T cells in the low tox group (n=5; p = 0.0495) but not in the high tox group (n=8; p = 0.2245). Furthermore, cluster analysis of the CyTOF data by RPhenograph and FlowSOM demonstrated significant changes associated with high tox in 3 cell population clusters showing markers indicative of Tregs.
In conclusion, frontline idelalisib treatment appears to increase a Th17 phenotype while decreasing Tregs, which we showed in vitro to be likely related to the drug altering naïve CD4 T cell differentiation. We hypothesize that an imbalance of Tregs and Th17 T cells is contributing to irAEs in some idelalisib treated patients. Preliminary data with duvelisib suggest alternative mechanisms which we are still analyzing, along with performing cytokine analysis and immunohistochemistry on patient biopsies to further validate these findings.

Disclosures
Pan: Gilead Sciences: Employment, Equity Ownership. Brown:Teva: Honoraria; Verastem: Consultancy, Research Funding; Sun Pharmaceuticals: Research Funding; TG Therapeutics: Consultancy; Pfizer: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; Octapharma: Consultancy; Morphosys: Other: Data safety monitoring board; Juno/Celgene: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; Loxo: Consultancy, Research Funding; Novartis: Consultancy; Gilead: Consultancy, Research Funding; Dynamo Therapeutics: Consultancy; Genentech/Roche: Consultancy; BeiGene: Consultancy; Catapult Therapeutics: Consultancy; Acerta Pharma: Consultancy; AstraZeneca: Consultancy; AbbVie: Consultancy; Invectys: Other: Data safety monitoring board; Janssen: Honoraria.