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919 IL-2 Induces Selective Activation of Helios-Positive Regulatory T Cells and CD56bright NK Cells in Vitro and in Patients with Chronic Gvhd Receiving Low-Dose IL-2 TherapyClinically Relevant Abstract

Clinical Allogeneic Transplantation: Acute and Chronic GVHD, Immune Reconstitution
Program: Oral and Poster Abstracts
Type: Oral
Session: 722. Clinical Allogeneic Transplantation: Acute and Chronic GVHD, Immune Reconstitution: Biological Studies
Monday, December 7, 2015: 6:15 PM
W311EFGH, Level 3 (Orange County Convention Center)

Masahiro Hirakawa, MD, PhD1*, Tiago R Matos, MD, MSc1*, John Koreth, MBBS, DPhil1, Edouard Forcade, MD1*, Jennifer Whangbo, MD, PhD2, Kazuyuki Murase, MD, PhD1,3, Hongye Liu1*, Sarah Nikiforow, MD, PhD1, Vincent T Ho, MD4, Corey S Cutler, MD, MPH1, Philippe Armand, MD, PhD1, Edwin P. Alyea, MD1, Joseph H. Antin, MD1, Robert J. Soiffer, MD5 and Jerome Ritz, MD2

1Division of Hematologic Malignancies and Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA
2Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, MA
3Department of Medical Oncology and Hematology, Sapporo Medical University School of Medicine, Sapporo, Japan
4Dana-Farber Cancer Institute, Boston, MA
5Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA

Introduction: CD4+FoxP3+CD25+ regulatory T cells (Treg) play a central role in the maintenance of immune tolerance and prevention of chronic graft-versus-host disease (cGVHD) after allogeneic stem cell transplantation (SCT). Treg constitutively express high-affinity interleukin-2 (IL-2) receptors and murine models have established that IL-2 is a critical homeostatic regulator of Treg in vivo.  We previously reported that daily administration of low-dose IL-2 in patients with cGVHD induces selective expansion of Treg and NK cells and results in clinical improvement in approximately 50% of patients. However, the mechanisms responsible for these selective effects and the influence of IL-2 therapy on other lymphocytes have not been established due to the limited resolution of traditional cell analytic methods such as flow cytometry.

Methods: Single cell mass cytometry (CyTOF) with a panel of 33 markers was used to simultaneously examine the phenotypic and functional effects of low-dose IL-2 on lymphocyte populations in vitro and in vivo. The analytic panel included 22 cell surface markers to identify distinct T, B and NK cell subsets and 11 intracellular markers to measure functional status and activation of specific signaling pathways. viSNE, a cytometry analysis tool, was used to visualize high-dimensional cytometry data on a two-dimensional map.

Results: In unstimulated lymphocytes from healthy donors, constitutive expression of CD25 (IL-2Ra) at high levels was restricted to Treg and CD56bright NK cells. Central memory (CM) and effector memory (EM) subsets of conventional CD4 T cells (Tcon) and CM CD8 T cells expressed low levels of CD25. Within the Treg population, the highest expression of CD25 was closely associated with expression of Helios transcription factor. Helios+ Treg also express higher levels of FoxP3, HLA-DR and CD95 and lower levels of BCL2 compared to Helios- Treg. To examine responses to IL-2, we stimulated peripheral blood mononuclear cells (PBMC) from healthy donors with IL-2 for 15 min in vitro (Figure 1). At low IL-2 concentrations (1 to 10 IU/ml), pSTAT5 was preferentially activated in Treg. Notably, pSTAT5 activation was more robust in memory Treg than naïve Treg and in Helios+ Treg than Helios- Treg. In addition, we observed activation of pSTAT5 in CD56bright NK cells at low concentrations of IL-2 (10 IU/ml). Higher IL-2 concentrations (100-1000 IU/ml) were required to activate pSTAT5 in Tcon, CD8 T cells and CD56dim NK cells. At high IL-2 concentrations, pSTAT5 was activated in all Treg, NK, Tcon and CD8 subsets. To examine the response to IL-2 in vivo, we examined PBMC from 14 patients with chronic GVHD receiving daily low-dose IL-2 using the same CyTOF panel of markers. Without additional in vitro stimulation, pSTAT5 expression was increased preferentially in Helios+ Treg. Peak pSTAT5 expression occurred 1 week after starting IL-2 and decreased with continued IL-2 therapy. Similarly, increased expression of FoxP3, CD25, HLA-DR and Ki67 occurred primarily in Helios+ Treg with peak expression at 1 week. At later time points during IL-2 therapy, changes in Treg included increased expression of CD95, CTLA4, PD-1, BIM and BCL2. Although there was no activation of pSTAT5 in CD4 Tcon and CD8 T cells, expression of PD-1 increased in effector memory subsets of Tcon and CD8 T cells 1 week after starting IL-2 therapy. Selective expansion of CD56bright NK cells was also noted, with peak activation at 1 week. No other changes were noted in Tcon, CD8 T cells and B cells. All changes observed during IL-2 therapy returned to baseline levels 4 weeks after treatment was stopped. However, examination of PBMC from 8 patients who received continuous daily low-dose IL-2 therapy for approximately 1 year showed that all of the changes noted above persisted during extended therapy.

Conclusion: Comprehensive analysis of T, B and NK cells from healthy donors revealed that low concentrations of IL-2 result in selective activation of Helios+ Treg and CD56bright NK cells. Higher concentrations of IL-2 are required for activation of CD4 Tcon, CD8 T cells and CD56dim NK cells. Identical populations are activated in patients with cGVHD receiving daily low-dose IL-2 and these functional effects persist during extended IL-2 therapy. Although the function of Helios transcription factor is not well defined, Helios expression identifies those Treg most primed to respond to low concentrations of IL-2 in vitro and in vivo.

 

Disclosures: Armand: Infinity Pharmaceuticals: Consultancy ; Merck: Consultancy , Research Funding ; Bristol-Myers Squibb: Research Funding . Antin: Jazz Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees ; Gentium S.p.A.: Membership on an entity’s Board of Directors or advisory committees . Soiffer: Gentium SpA/Jazz Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees .

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