Session: 722. Clinical Allogeneic Transplantation: Acute and Chronic GVHD, Immune Reconstitution: GVHD Treatment and Prevention Strategies
Hematology Disease Topics & Pathways:
Adult, Diseases, Biological, Therapies, GVHD, Biological Processes, Technology and Procedures, cell expansion, Immune Disorders, Study Population, immunotherapy, Clinically relevant, NK cells, imaging, flow cytometry, immune mechanism
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. CD56bright and CD56dim 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 CD56bright 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 CD56bright 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 CD56bright 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 CD56bright and CD56dim 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 CD56bright NK cells increased during IL-2 treatment while cytotoxicity of CD56dim NK cells did not change. Notably, cytotoxicity of CD56bright NK cells became significantly higher than CD56dim 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 CD56bright NK cells. CD56dim NK cells were not affected by IL-2 therapy. In vitro assays revealed that cytolytic activity of CD56bright NK cells increased during IL-2 treatment and exceeded the cytotoxicity of CD56dim NK cells. CD56bright 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 CD56bright NK cells may contribute to graft versus leukemia (GVL) and help prevent relapse after transplant.
Disclosures: Koreth: equillium: Consultancy; amgen: Consultancy; prometheus: Research Funding; fortress biotech: Consultancy; miltenyi: Research Funding; BMS: Research Funding; Millennium: Research Funding. Nikiforow: Kite Pharma: Consultancy. Ho: Jazz Pharmaceuticals: Consultancy. Antin: Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Soiffer: Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.
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