Background
Intratumoral activation and expansion of tumor-specific CD8 T cells is essential for successful immunotherapy. Interleukin-10 (IL-10) is known for its anti-inflammatory function, and at higher concentrations IL-10 activates cytotoxicity and enhance proliferation of CD8 T cells.1–4 In this study, we verified the presence of IL-10RA+CD8+ T cells in the tumor microenvironment (TME) and assessed the ability of high dose rhIL-10 to induce CD8+ T cell and NK cell proliferation and cytotoxicity in vitro.
Methods
Healthy and cancerous tissue sections from various indications were stained for IL-10RA and CD8 co-expression. To study CD8 T and NK cell function, CD8 and NK cells were isolated from healthy donor PBMC. CD8 T cells were activated by anti-CD3+CD28 stimulation, and NK cells activated by rhIL-2+rhIL-15 stimulation, whereafter IL10RA expression was analysed by flow cytometry. After activation, CD8 and NK cells were treated with various doses of rhIL-10, IFN release and proliferation were measured. Activated CD8 T and NK cells were co-cultured with a prostate cancer cell line LNCaP, and imaged with Incucyte to assess functional properties, and IFN release was measured.
Results
Tissue sections from cancer indications, including brain, breast, intestine, kidney, liver, lung, ovary, pancreas, and skin, have high number of CD8+ IL10-RA+ double positive cells compared to respective healthy tissues. However, only prostate cancer reached significantly higher double positive cells. Consistent with the published data, IL10-RA expression was upregulated in activated healthy donor CD8+ T cells (52 +/- 13% IL10-RA+CD8+ cells) in comparison to non-activated CD8+ T cells (7 +/-1% IL10Ra+CD8+ cells). rhIL-10 dose-dependently induced CD8+ T cell proliferation and IFN release only in activated CD8+ T cells. In addition, GzmB was significantly elevated after 72 h of rhIL-10 treatment in CD8+ T cells. Activation of NK cells didnt increase IL10RA expression. However, the receptor levels were already detectable at baseline (25% for both non-activated and activated). Addition of rhIL-10 in co-culture settings increased IFN release in activated CD8+ T cells (figure 1A). In killing assays, addition of rhIL-10 increased cancer cell killing in activated CD8+ and NK cells (figure 1B,C and 2B,C). Overnight incubation of rhIL-10 induced GzmB and IFNg production in activated NK cells (figure 2A).
Conclusions
Followed high dose of rhIL-10 treatment, activated CD8+ T cells and NK cells released cytotoxic cytokines and killed target cancer cells efficiently. This study adds further evidence to pre-existing research of IL-10 cytokine as a potential anti-tumoral treatment.
References
Emmerich J, Mumm JB, Ivan CH, et al. IL-10 activates and expands tumor resident CD8+ T cells. Cancer Res. 2012;72:3570–3581.
Fujii S, Shimizu K, Shimizu T, et al. Interleukin-10 promotes the maintenance of antitumor CD81 T-cell effector function in situ. Blood. 2001;98:2143–2151
MacNeil IA, Suda T, Moore KW, et al. IL-10, a novel growth cofactor for mature and immature T cells. J Immunol. 1990;15:4167–73.
Mumm JB, Emmerich J, Zhang X, et al. IL-10 Elicits IFNg-Dependent Tumor Immune Surveillance. Cancer Cell. 2011;20:781–796.
Abstract 1044 Figure 1
(A) High dose of rhlL-10 (100 ng/mL) induces further IFNg release in activated CD8 T cells after 72h co-culture with cancer cells (LNCaP) measured by ELISA. (B) High dose rhlL-10 (100 ng/mL) further enhances killing of prostate cancer cells (LNCaP stained in red) in IncuCyte assay Caspase Green at 24h after co-culture. (C) Killing capacity increases over time in rh1L-10 treated CD8 T cell group measured as apoptotic tumor cells over time.
Abstract 1044 Figure 2
(A) High dose of rhIL-10 (100 ng/mL) induces further Granzyme B activation in activated NK cells measured by flow cytometry. (B) High dose rhIL-10 (100 ng/mL) further enhances killing of prostate cancer cells (LNCaP stained in red) in IncuCyte assay Caspase Green at 24h after co-culture. (C) Killing capacity increases over time in rhIL-10 treated NK cell group measured as apoptotic tumor cells over time.