Extended Survival in CLL Patients Who Discontinued Idelalisib Because of Colitis Correlates with Higher Th17 Levels in Blood and Colon Tissue
Authors: Aslam Rukhsana , Barrientos Jacqueline C , Ferrer Gerardo , Chiu Pui Yan , Rai Kanti R , Chiorazzi Nicholas , Sherry Barbara 
Published: October 2019

Abstract
Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Western countries. Despite significant clinical and molecular advances, CLL remains an incurable disease. In CLL there is ongoing communication between leukemic B cells and non-leukemic cells in the tissue microenvironment. Idelalisib, a PI3Kδ inhibitor, is efficacious in CLL although side effects like inflammatory reactions in different tissues (e.g., colitis, transaminitis and pneumonitis) and progression of disease can lead to discontinuation of the drug. We previously presented an intriguing finding that a cohort of patients who discontinued idelalisib therapy due to colitis had extended survival compared to other patients who discontinued drug but did not have colitis. Of note, the colon tissues from patients with ulcerative colitis (UC) often contain elevated levels of Th17 cells and decreased levels of Treg (Foxp3+) cells. Moreover, Th17 cells from humans and mice have been shown to promote autoimmune B-cell maturation. In this regard, we have previously published that circulating Th17 levels are elevated in CLL patients and Th17 numbers correlate with extended survival. For these reasons, we have now quantified the numbers of Th17 and Treg cells in the blood and colon tissues of a cohort of CLL patients with drug-associated colitis and extended survival and have investigated the effects of in vitro exposure to idelalisib on Th17 generation.
Circulating Th17A levels were quantified in a cohort of patients (n=11) before starting idelalisib-treatment and at the time the drug was discontinued due to colitis by flow cytometric analysis of intracellular IL-17A in CD4+ T cells. In addition, colon tissues from those CLL patients who developed colitis during idelalisib treatment taken at the time of drug discontinuation and patients with UC were examined to quantify the infiltration of T (CD3+) cells, Th17 (IL17A+) cells and Treg (Foxp3+) cells by immunohistochemistry (IHC). As negative control tissues (CT) for these studies we used colectomy samples from patients with inactive diverticulosis.
FACS analysis of peripheral blood mononuclear cells from the CLL patients treated with idelalisib and having extended survival showed significantly higher circulating Th17A levels (P < 0.01) at the time of drug discontinuation compared to pre-treatment levels. In addition, IHC analyses on tissues from a subset of these same patients indicated that the ratio of % IL-17A+ to CD3+ cells was significantly higher in CLL than in CT (P = 0.0002). It was also significantly higher in CLL than UC (P = 0.001), even though the average number of CD3+ cells in UC was higher than in CLL (P = 0.0001). We also determined the ratio of % FoxP3+ to CD3+ cells in the colon tissues of 12 CT, 16 UC and 6 CLL patients from the cohort studied above. This revealed the average percentage of FoxP3+ cells within the total number of infiltrating T cells was significantly higher in CLL than CT (P = <0.0001), but not significantly different from UC patients (P = 0.90). When analyzing solely the CLL samples, there was a reciprocal relationship between Th17A+ cells and FoxP3+ cells, with the former being higher and the latter lower.
To examine the effect of idelalisib on Th17-cell generation, naïve CD4+ CLL T cells from 6 patients (3 M-CLL + 3 U-CLL) were activated in vitro by anti-CD3/28 ligation plus IL-2 in the presence or absence of idelalisib. After 7 days, T helper subset profiling was performed by intracellular cytokine staining. Significantly higher percentages of Th17A cells and significantly lower percentages of Th1 cells (IFNg+) (P= 0.006) were found in cultures containing idelalisib. The percentages of Tregs were unchanged in the same cultures. These findings suggest that the higher numbers of Th17s in blood and tissue of patients after idelalisib treatment may reflect the action of idelalisib on Th17 generation.
In conclusion, CLL patients who discontinued idelalisib due to colitis and yet had extended survival have increased circulating and tissue-resident Th17 cells and decreased levels of Treg cells. These findings suggest that an immune imbalance between Th17 and Treg cell numbers and function promotes colitis and at the same time favors improved clinical outcome. This is consistent with the known action of Th17 cells enhancing autoimmune B-cell responses.

Disclosures
Barrientos: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rai:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.