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606 Low-Dose Interleukin-2 Therapy Enhances Cytotoxicity of CD56bright NK Cells in Patients with Chronic Gvhd

Program: Oral and Poster Abstracts
Type: Oral
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
Monday, December 3, 2018: 8:15 AM
Grand Hall A (Manchester Grand Hyatt San Diego)

Tomohiro Kubo, MD, PhD1,2*, Rizwan Romee, MD1,2,3, John Koreth, MBBS, DPhil1,2,3, Roger Belizaire, MD, PhD2,3, Yusuke Kamihara, MD, PhD1,2*, Hongye Liu, PhD1,2*, Takeru Asano, MD, PhD1,2*, Jennifer Whangbo, MD, PhD1,2,4, Masahiro Hirakawa, MD, PhD1,2*, Sarah Nikiforow, MD, PhD1,2,3, Mahasweta Gooptu, MD1,2,3, Vincent T. Ho, MD1,2,3*, Corey S. Cutler, MD, MPH1,2,3*, Edwin P. Alyea III, MD1,2,3, Joseph H. Antin, MD1,2,3, Robert J. Soiffer, MD1,2,3 and Jerome Ritz, MD, PhD1,2,3

1Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, MA
2Harvard Medical School, Boston, MA
3Brigham & Women's Hospital, Boston, MA
4Boston Children’s Hospital, Boston, MA

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. CD56bright 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 CD56dim CD16+ NK cells. However, recent studies have reported that cytolytic activity of CD56bright NK cells increases when these cells are stimulated with IL-15 or 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 CD56bright NK cells and improves clinical manifestations of cGVHD. The function of CD56bright 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. 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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