Imbalance in T Cell Subsets Triggers the Autoimmune Toxicity of PI3K Inhibitors in CLL

Abstract

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.

A T cell inflammatory phenotype is associated with autoimmune toxicity of the PI3K inhibitor duvelisib in chronic lymphocytic leukemia

“…Interestingly, a subset of these CD8 T cells also demonstrate Th17-type differentiation. We also find that the cytokines most differentially expressed with toxicity are those associated with Th17 response, consistent with our prior observations with idelalisib 22 and suggesting that Th17 activation is a significant contributor to PI3Kδ inhibitor-related toxicity.…”

“…We have previously demonstrated that increased activation of the Th17 pathway is associated with the autoimmune toxicity of idelalisib treatment in previously untreated patients 19 . Given those findings and the interesting above observation that a significantly increased CD8 cluster in this cohort shows Th17 differentiation specifically in patients with toxicity, we further evaluated the impact of duvelisib on Th17 subsets.…”

Idelalisib reduces regulatory T cells and activates T helper 17 cell differentiation in relapsed refractory patients with chronic lymphocytic leukaemia

“…CD161 is a lectin‐like receptor associated with tissue homing and found on CD8 and interleukin 17‐expressing T cells, 8,9 while RORγt is a transcription factor associated with Th17 differentiation. Hence, both these clusters suggest increased Th17 activity at baseline in patients who go on to develop toxicity, consistent with our findings also in previously untreated patients with CLL receiving PI3Kδ inhibitors 10 . Both clusters also increase at the time of toxicity (Figure S2A,B and S3A).…”

“…Hence, both these clusters suggest increased Th17 activity at baseline in patients who go on to develop toxicity, consistent with our findings also in previously untreated patients with CLL receiving PI3Kδ inhibitors. 10 Both clusters also increase at the time of toxicity (Figure S2A,B and S3A). Clusters nine and 12, that show enrichment at the later toxicity timepoint but not at baseline, are also both activated CD8 T cells (Figures S1C,D, S2C,D, and S3B).…”

Promises and pitfalls of targeted agents in chronic lymphocytic leukemia

“…Immune phenomena, particularly pneumonitis, colitis, rash and transaminitis, were the most common reasons for cessation [ 175 ] . Although poorly understood, these events may be related to inhibition of PI3K p100d in regulatory T cells, leading to enhanced CD8+ T cell activity [ 197 , 198 ] and Th17 phenotype [ 199 ] . In one young TN cohort treated with idelalisib and ofatumumab, Grade ≥ III immune mediated hepatotoxicity occurred in 52% of patients, and 55% of patients discontinued therapy at a median of eight months due to toxicity [ 201 , 206 ] .…”