Both PI3K p110α and p110δ Isoforms Control CD160-Mediated CLL Cell Viability and Cytokine Production
Authors: Farren Timothy W. , Liu Fengting , Agrawal Samir G. 
Published: October 2019

Abstract
Expression of CD160 on normal immune cells is primarily observed on human and murine circulating cytotoxic lymphocytes (NK cells and a small subset of T-cells). CD160 exhibits a broad specificity for major histocompatibility complex molecules. While normal B-cells do not express CD160, malignant B-cells of CLL (and HCL) are CD160+.1 Triggering of CD160 on CLL cells mediates increased viability, PI3K dependent growth signals and marked IL-6 and IL-8 secretion.2 In this study, the role of the different PI3K subunits in CD160 effector functions in CLL have been investigated in terms of cytokine/chemokine secretion and cell viability.
Unstimulated CLL cells underwent apoptosis over 5 days in culture, which was significantly inhibited by the anti-CD160 antibody, CL1-R2 (p<0.01). Similarly, the viability of purified CLL cells was enhanced by addition of a microenvironment milieu (CD19 negative fraction from BM samples) and further improved by CL1-R2 triggering of CD160 (p=0.03).
Without stimulation, CLL cells secreted minimal levels of IL-2, IL-4, IL-10, TNF-alpha, INF-gamma, CCL2 and CCL5 (as assessed by cytokine bead array (BD Biosciences). At baseline, low levels of IL-6 (median: 23 pg/ml) and IL-8 (median: 114 pg/ml) were detected. Incubating CL1-R2 with CLL cells led to a significant increase in IL-6 (median: 1431 pg/ml, p<0.01), IL-8 (median: 7945 pg/ml, p<0.01), CCL2 (monocyte chemotactic protein-1, median: 813.3 pg/ml, p=0.01) and CCL5 (median: 53.61 pg/ml, p=0.03). These effects of CD160 triggering were suppressed, in a dose-dependent manner, by the pan PI3K inhibitors LY294002 (p=0.01) and Wortmannin (p<0.01, Table 1).Table 1(above): n= 12CD160 (10μg/ml) Median pg/ml (range)CD160 (10μg/ml) and LY294002 10μM Median pg/ml (range)CD160 (10μg/ml) and LY294002 20μM Median pg/ml (range)IL-61431 (147 - 8972)108 (4.0 - 5512) p=0.003929 (3.38-5250) p=0.0019IL-87945 (950 - 9665)4394 (71 - 7744) p=0.00171958 (28 - 7024) p=0.0075CCL2813 (84 - 879)7.0 (1.3 - 81) p=0.00631.8 (0.0 - 20) p=0.0045CCL554 (42 - 106)36 (24 - 72) p=0.001821 (6.5 -27) p=0.0024
The roles of the different p110-catalytic subunits of PI3K in CD160 mediated activation were investigated. CLL cells from 5 different patients were incubated with the specific isoform-selective inhibitors PI103 (p110α), TGX-115 (p110β) and IC87114 (p110δ) at concentrations of 0.5, 1 and 10μM, in the presence of CL1-R2. CD160 induced cell viability was significantly reduced in a dose dependent manor by both PI103 (p=0.004) and IC87114 (p=0.002). Although the p110β inhibitor demonstrated a reduction in CD160 mediated cell viability it was not significant. Combining the p110α and p110δ inhibitors, a further decrease in CD160 mediated cell viability was observed (p<0.01), but not in a synergistic manner.
Upon performing cytokine and chemokine bead array assays on the same patients, the CD160 induced cytokine and chemokine production of IL-6, IL-8, CCL2 and CCL5 were suppressed by all the inhibitors at a concentration of 10 μM (Table 2). However, only p110α and p110δ subunit inhibition resulted in significant reductions, with a further decrease in both CD160 mediated cytokine and chemokine secretion when both were inhibited together (IL-6: p<0.01, IL-8: p=0.03, CCL2: p=0.02, CCL5: p=0.03), but not with a synergistic or additive effect (Table 2).Table 2(Above): n=5CD160 (10μg/ml)CD160 (10μg/ml) and PI103 (p110a)CD160 (10μg/ml) and TGX-221 (p110b)CD160 (10μg/ml) and IC87114 (p110d)CD160 (10μg/ml) and PI103+IC87114IL-6457198 p=0.05365 p=NS189 p=0.02101 p<0.01IL-848613405 p=0.044066 p=NS3143 p=0.032625 p=0.03CCL2696130 p=0.04583 p=NS111 p=0.0246 p=0.02CCL59159 p=0.0573 p=NS61 p=0.0543 p=0.03
These results demonstrate that CL1-R2 engagement of CD160 leads to: (1) a direct survival signal in CLL cells; and (2) production of IL-6, IL-8, CCL2 and CCL5. These functions are PI3K-dependent. These PI3K dependent responses signal largely through the p110α and p110δ isoform-selective catalytic subunits. From a clinical perspective, this data lends further support to targeting the p110α subunit, in addition to p110δ, as a therapeutic strategy. However, none of the inhibitors, alone or in combination, reduced cytokine secretion to basal levels, suggesting that CD160 is also signalling via other undefined pathways.
References:
1. Farren TW et al. Blood. 2011; 118 (8): 2174-2183.
2. Liu FT et al. Blood. 2010; 115 (15): 3079-3088

Disclosures:
No relevant conflicts of interest to declare.