CD8+ T Cells from Patients with CLL Show Evidence of Granzyme B and Perforin Cytotoxic Capacity, Which Is Enhanced upon Exposure to Exogenous IL2 and/or IL21

Objective: To identify mechanisms underlying impaired cytotoxic T cell (CTL) activity in CLL, levels of the cytotoxicity associated molecules, CD107a, granzyme B (GZB) and perforin (PRF) were compared in CD8⁺ T cells from CLL patients and age-matched healthy controls (HC), before or after culture with IL2, 21, or both, +/- T-cell receptor (TCR) stimulation.

Methods: All studies used CD8⁺ cells present in CD3⁺- enriched PBMCs. For ex vivo analyses, CD8⁺ cells from 20 untreated CLL patients (U-CLL = 11, M-CLL = 9) and 23 HCs were examined for surface CD107a and intracellular GZB (iGzB) and PRF (iPRF) by flow cytometry. For in vitro experiments, CD3-enriched PBMCs from 11 CLL (U-CLL = 6, M-CLL = 5) and 16 HC were cultured for 24h in medium alone or medium supplemented with cytokines +/- anti-CD3+CD28 mAbs and then levels of surface CD107a and intracellular and released GZB and PRF were quantified by flow cytometry or ELISA.

Results: HC and CLL expressed comparable percentages and densities of CD107a⁺CD8⁺ cells ex vivo. However, CLL patients had a higher percentage of CD8⁺ cells containing iGZB than HC (P < 0.0001); this difference was also seen in U-CLL and M-CLL (P < 0.001, < 0.05, respectively). Moreover, the amount of iGZB within CD8⁺ cells, defined by mean fluoresence intensity, was higher in CLL than HC (P < 0.05). The percentage of CD8⁺ cells expressing PRF was comparable for HC and CLL, although U-CLL had higher levels than HC (P < 0.05). iPRF amounts were similar in CLL and HC.

After 24h culture with IL2, 21, or 2+21, a higher percentage of CLL CD8s expressed CD107a than HC (P < 0.01), but cells from both HC and CLL secreted higher levels of GZB (HC: P < 0.05, < 0.05, < 0.001; CLL: ns, < 0.05, < 0.0001) than when cultured in medium alone. Also, addition of the cytokines increased PRF levels for HCs (P < 0.01 each) and CLL (P < 0.05, < 0.001, < 0.0001), compared to medium alone. When examining the CLL subtypes, U-CLL CD8s cultured with IL21 or 2+21 released more GZB (P < 0.05, < 0.0001), whereas M-CLL did the same only in IL2+21 cultures (P < 0.05). U-CLL CD8s showed a similar pattern for PRF release with IL21 or 2+21 (P < 0.01, < 0.5), while M-CLL released more PRF in response to all cytokines (P < 0.05, < 0.01, < 0.01). Furthermore, while treatment with IL2, 21 and 2+21 increased HC iPRF levels in CD8⁺ cells (P < 0.001, < 0.05, < 0.001), only IL21 and 2+21 increased iPRF in CLL (P < 0.001, < 0.01).

TCR stimulation of HC and CLL in the absence of exogenous cytokines led to an increase in the percentage (P < 0.0001, < 0.05) and density (P < 0.0001, < 0.001) of CD107a expressing CD8⁺ cells. For CLL, this effect was driven by U-CLL, as only U-CLL cells showed an increase in the percentage and density of CD107a (both P < 0.01). TCR engagement also led to increased GZB and PRF release by HC (P < 0.0001 and 0.0001) and CLL (P < 0.0001 and 0.01) CD8⁺ cells. Notably, the GZB increase was greater for HC than CLL (P <0.01), with M-CLL driving the response by releasing less GZB than HC (P < 0.05).

Upon combining TCR stimulation with cytokine supplementation, HC and CLL cells exhibited a progressively increased release of GZB (HC: ns, P < 0.05, < 0.01; CLL; P < 0.05 , < 0.001, < 0.001) and PRF (HC: ns, P < 0.01, < 0.01; CLL; P < 0.05, < 0.01, < 0.01). Furthermore, whereas all cytokines increased GZB release in U-CLL cells (P < 0.05), M-CLL cells only responded to IL21 and 2+21 (P < 0.01 , < 0.05). IL21 and 2+21 also increased PRF release in U-CLL (P < 0.05). Finally, only the combination of IL2+21 increased PRF levels in HC(P < 0.05), whereas in CLL IL21 and 2+21 did (both P < 0.001).

Conclusions: Despite the known defect in CTL function in CLL, more CLL CD8⁺ cells express GZB and at greater cellular density than HC in vivo. Moreover, upon culture with IL2, 21, or 2+21, CLL CD8s display more surface CD107a, a marker of degranulation and cytotoxic capacity, than HC, while producing and releasing comparable amounts of GZB and PRF. Conversely, TCR activation of CLL CD8s leads to the release of less GZB than HC. Finally, when exposed to cytokines along with TCR engagement, CLL and HC cells release comparable levels of GZB and PRF. Of note, while IL21 and 2+21 supplementation enhanced GZB release in U-CLL and M-CLL, only U-CLL responded to IL2 supplementation alone. Together these data suggest that CLL CD8s have at least adequate cytotoxic capabilities in vivo and these increase with the addition of IL2 and/or 21. Thus, IL2 and/or 21 could potentially synergize to permit adequate effector CTL function. This is currently under investigation.

Barrientos:Merck, Oncternal, Pharmacyclics/Abbvie: Research Funding; Beigene, AstraZeneca, Pharmacyclics/Abbvie: Consultancy. Rhodes:Abbive: Consultancy; Beigene: Consultancy; Genentech: Consultancy; Genmab: Consultancy; Janssen: Consultancy, Research Funding; Morphosys: Consultancy; Pharmacyclics: Consultancy, Research Funding; SeaGen: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy; Oncternal: Research Funding; Velosbios: Research Funding; Loxo Oncology: Research Funding; Epizyme: Research Funding. Allen:BMS: Current equity holder in publicly-traded company; C4 Therapeutics: Current equity holder in private company; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees; Alexion: Consultancy, Research Funding.