Low-Dose Interleukin-2 Therapy Enhances Cytotoxicity of CD56^bright NK Cells in Patients with Chronic Gvhd

Introduction: Natural killer (NK) cells play an important role in defense against infections and cancer. Two major subsets of mature NK cells have been described. CD56^bright CD16^- NK cells, which normally represent only 5-10% of circulating NK cells, are thought to exhibit less cytolytic activity and greater immune regulatory functions than CD56^dim CD16⁺ NK cells. However, recent studies have reported that cytolytic activity of CD56^bright NK cells increases when these cells are stimulated with IL-15or the combination of IL-12, IL-15 and IL-18. Previous studies from our center have shown that daily administration of low-dose IL-2 in patients with chronic graft-versus-host disease (cGVHD) induces selective expansion of CD4⁺FoxP3⁺Helios⁺ regulatory T cells and CD56^bright NK cells and improves clinical manifestations of cGVHD. The function of CD56^bright NK cells expanded by low-dose IL-2 has not previously been studied.

Methods: Single cell mass cytometry (CyTOF) with a panel of 35 metal tagged antibodies was performed on cryopreserved peripheral blood mononuclear cells (PBMC) from 10 adult patients with active cGVHD receiving daily low-dose IL-2 therapy. Patients in this clinical trial received extracorporeal photopheresis (ECP) for 8 weeks prior to starting daily low dose IL-2. ECP therapy (twice weekly) was continued when patients began low-dose IL-2 (1x10⁶ IU/M²/day x 8 weeks). The analytic panel included 26 cell surface markers to identify distinct lymphocyte subsets and 9 intracellular markers to measure functional status and activation of specific signaling pathways. viSNE was used to visualize of high-dimensional data on a two-dimensional map and quantify single cell mass cytometry data. NK cytolytic activity was measured in flow cytometry-based cytotoxicity assays. CD56^bright and CD56^dim NK cells from 8 adult patients were purified from cryopreserved PBMC by cell sorting and incubated with labeled K562 cells for 4 hours followed by staining with 7-AAD and Annexin-V. E:T ratio of 1:1 was used for incubation with K562 targets.

Results: No changes in extracellular or intracellular NK cell markers or quantitative changes in NK cells were observed during the initial 8 week ECP treatment period. Selective expansion of CD56^bright NK cells was noted after 1 week of IL-2 therapy (9W) and continued during 8 weeks of daily IL-2 therapy. Increased expression of NKp30, Nkp46, NKG2D, HLA-DR and Ki67 occurred in expanded CD56^bright NK cells with peak expression at 1 week after starting IL-2 (9W). At later time points during IL-2 therapy, expression of NKG2D, HLA-DR and Ki67 returned to baseline (Figure 1A). Expression of CD56, CD122 and NKG2A continued to increase during IL-2 treatment. In contrast, expression of CD25 by expanded CD56^bright NK cells decreased during IL-2 treatment. Expression of phosphorylated signaling proteins did not change in any NK cell subset during IL-2 treatment.　Cytolytic activity was measured in CD56^bright and CD56^dim NK cell subsets at different times during ECP and IL-2 therapy. After thawing, flow-sorted NK cell subsets were cultured for 16-20 hours with IL-2 (100 IU/ml). Cells were then washed and incubated with tumor targets (K562) for 4 hours and % killing was assessed by flow cytometry. Compared to pre IL-2 treatment, cytolytic activity of CD56^bright NK cells increased during IL-2 treatment while cytotoxicity of CD56^dim NK cells did not change. Notably, cytotoxicity of CD56^bright NK cells became significantly higher than CD56^dim NK cells during IL-2 therapy (Figure 1B).

Conclusion: Single cell mass cytometry revealed that daily low dose IL-2 therapy induces selective expansion, activation and increased expression of activating NK receptors in CD56^bright NK cells. CD56^dim NK cells were not affected by IL-2 therapy. In vitro assays revealed that cytolytic activity of CD56^bright NK cells increased during IL-2 treatment and exceeded the cytotoxicity of CD56^dim NK cells. CD56^bright NK cells, traditionally considered to be minimally tumor-responsive, are effectively stimulated by daily low dose IL-2 exposure to enable potent cytotoxicity in response to tumor targets. In patients receiving low-dose IL-2 after allogeneic HSCT, expanded CD56^bright NK cells may contribute to graft versus leukemia (GVL) and help prevent relapse after transplant.

View largeDownload slide

View largeDownload slide

Close modal