Marked in Vivo Donor Regulatory T Cell Expansion via Interleukin-2 and TL1A-Ig Stimulation Ameliorates Graft-versus-Host Disease but Preserves Graft-versus- Leukemia in Recipients after Hematopoietic Stem Cell Transplantation  Dietlinde Wolf, Henry Barreras, Cameron S. Bader, Sabrina Copsel, Casey O. Lightbourn, Brent J. Pfeiffer, Norman H. Altman, Eckhard R. Podack, Krishna V. Komanduri, Robert B. Levy  Biology of Blood and Marrow Transplantation  Volume 23, Issue 5, Pages 757-766 (May 2017) DOI: 10.1016/j.bbmt.2017.02.013 Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Synergistic effect of TL1A-Ig and IL-2 on Treg expansion in vivo. (A-E) Combined administration of TL1A-Ig and recombinant murine interleukin (rmiL-2) in vivo expands Tregs to much higher levels than single agents alone (n = 6, P values are shown). TL1A-Ig (50 µg) was administered i.p. on days 1 to 4; rmIL-2 (1.5 µg) bound to α-IL-2 mAb (clone JES6-5H4; 8 µg) on days 4 and 6. Mice were sacrificed on day 7. Treg expansion is shown as frequency (%) of total CD4+ cells in spleen (A), LN (B), and PB (C) and total cell numbers in spleen (D) and LN (E). (F) Example dot plot of Treg expansion with TL1A-Ig + IL-2 in spleen on day 7. (G-H) Total cell numbers in spleen (G) and LN (H) post expansion on day 7 (n = 6, P = ns if not shown). Consistent with an expansion of Tregs, increased numbers of spleen and lymph node cells (G,H) with elevated frequencies of CD4+ and diminished frequencies of CD8+ T cells, respectively were detected (I,J). (I-L) The frequency (%) of CD4 (I), CD8 (J), B (K) and NK1.1 cells (L) out of the lymphocyte compartment after Treg expansion in LN on day 7 is shown (n = 6, P = ns if not shown). (M) Kinetics of Treg expansion indicate return to normal levels 2 to 3 weeks after final injection. (N) Kinetics of CD4, CD8 expression following final injection. Biology of Blood and Marrow Transplantation 2017 23, 757-766DOI: (10.1016/j.bbmt.2017.02.013) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 TL1A-Ig/IL-2–expanded Treg activation and function: amelioration of GVHD after MHC-mismatched and MHC-matched allogeneic hematopoietic stem cell transplantation. Tregs were expanded with TL1A-Ig/IL-2 (single or combined) as in Figure 1. Mice were sacrificed at different time points after the last IL-2 injection. (A) pSTAT5 staining (flow cytometry) showing the heightened activation status of splenic Tregs compared to Tcon CD4 and CD8 cells, 1 and 24 hours after the last IL-2 injection (combined TL1A-Ig/IL-2). (B) Elevated pSTAT5 expression shown by flow cytometry in the spleens of IL-2 and TL1A-Ig/IL-2 but not TL1A-Ig only–treated B6-FoxP3rfp animals 1 day after the last IL-2 injection. (C) Decreased methylation of FoxP3 CpG sites in the CNS2 (Treg-specific demethylation CNS2) region of TL1A-Ig/IL-2–expanded and unexpanded Tregs versus FoxP3− CD4 T (Tcon) cells. (D) Tregs expanded with single or combined treatment show comparable suppressive activity in a suppression assay. (E, F) Donor B6-FoxP3rfp mice were either left untreated or were treated with TL1A-Ig/IL-2 (TL1A: 50 µg on days −6 to −3; recombinant murine interleukin-2 (rmiL-2): 1.5 µg bound to α-IL-2 mAb [clone JES6-5H4] 8 µg on days −3 and −1) or 4C12 (.5 mg/kg on day −4). Treg expansion was assessed on day 0 at the time of BMT (E). (F) A HSCT utilizing a B6 → BALB/c donor/recipient mouse model involving a complete MHC mismatch was performed on day 0. Lethally irradiated (8.5 Gy) BALB/c mice received 5 × 106 TCD B6-CD45.1 BM cells and spleen cells from expanded (TL1A-Ig/IL-2 or 4C12) or untreated B6-FoxP3rfp donor mice adjusted to contain 1.2 × 106 total T cells. Clinical scoring and survival are presented (F). HSCT with cells from TL1A-Ig/IL-2–expanded donors demonstrates more efficient amelioration of GVHD compared to 4C12 (n = 5). (G) A HSCT utilizing a B10.D2 → BALB/c donor/recipient mouse model across a MHC-matched, minor histocompatibility antigen mismatch was performed using 8 × 106 non-TCD BM cells + 25 × 106 spleen cells from either untreated or TL1A-Ig/IL-2–expanded B10.D2 donor mice. Percentage of initial weight and clinical score are presented (G). Amelioration of GVHD shown was readily detected using TL1A-Ig/IL-2 Treg–expanded donor cells (n = 7). P values are shown. (H) Massons Trichrome (collagen) and H&E staining from recipient skin 4 to 5 weeks post-HSCT as described in G. In recipients of Treg-expanded donor cells (TL1A-Ig + IL2) note the thin epidermis, scant collagen staining (blue) in the dermis and absence of inflammatory cells whereas recipients of nonexpanded donor cells (GVHD control) exhibited a thickened epidermis, extensive collagen deposition in the dermis and a modest infiltration of chronic inflammatory cells. Magnification = 25×. (This figure is available in color online at www.bbmt.org.) Biology of Blood and Marrow Transplantation 2017 23, 757-766DOI: (10.1016/j.bbmt.2017.02.013) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Expansion of Tregs in certain hematopoietic compartments is accompanied by a decrease in T and B cell immune responsiveness. (A-E) Using standard protocol of treatment (TL1A-Ig/IL-2), the frequency of Tregs is increased in blood, spleen, LN, and gastrointestinal tract (colon) but barely bone marrow (n = 13, except colon: n = 3). (F,G) Expanded Tregs are highly proliferative indicated by expression of Ki67 in spleen and LN. One example is shown out of 3 independent experiments. In each experiment the elevation of Ki67 expression in expanded Tregs was ~10 fold greater than that in Tregs from control animals. (H,I) Increased cytokine production (granzyme B, IL-10) by Tregs from expanded mice after phorbol 12-myristate 13-acetate (1 ng/mL) + ionomycin (1 µM) stimulation for 5 hours in the presence of monensin indicate higher levels in splenic versus LN-derived Tregs. These increases were observed in each of 3 independent experiments performed ranging from 7 × for granzyme B between the control versus expanded Tregs in the spleen, ~4 to 5 times for granzyme B between the control versus expanded Tregs in the LN and 3 to 5 times for IL-10 between the spleen versus LN for expanded Tregs. (J) Higher pSTAT5 expression (assessed by flow cytometry) in spleen versus LN 1 hour after final IL-2 injection. For (H-J) 1 example is shown out of 3 independent experiments. (K) Increased expression of killer cell lectin-like receptor subfamily G member 1 (KLRG1) on Tregs after expansion with higher expression on spleen compared to LN (n = 5). (L,M) Treg expansion leads to a suppressive environment (T and B cells) in spleen and LN with increased suppression in the splenic compartment compared to LN despite similar Treg numbers (1.9 × 104 versus 1.8 × 104 Tregs in spleen versus LN cultures respectively) (n = 3). These cultures contained cell suspensions of spleen or lymph node cells obtained from mice which underwent TL1A-Ig + IL-2–induced expansion or normal, unexpanded mice. The cultures were then stimulated with either anti-CD3mAb or LPS for 72 hours (see Methods). (N) An in vitro suppression assay with sorted Tregs from control (normal) mice demonstrates more effective suppression by splenic versus LN derived CD4+FoxP3+ cells (n = 3). Biology of Blood and Marrow Transplantation 2017 23, 757-766DOI: (10.1016/j.bbmt.2017.02.013) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Hematopoietic stem cell transplantation (HSCT) with cells from TL1A-Ig/IL-2–expanded donors indicates more efficient amelioration of GVHD by spleen cells compared with LN cells. A B6 → BALB/c donor/recipient mouse model involving a complete MHC mismatch was utilized. Lethally irradiated mice received 5 × 106 TCD B6-CD45.1 BM cells and either spleen or LN cells from TL1A-Ig/IL-2–expanded or untreated B6-FoxP3rfp mice adjusted to contain 1.2 × 106 total T cells. (A,B) HSCT with cells from TL1A-Ig/IL-2–expanded donors indicates more efficient amelioration of GVHD by spleen cells compared to LN cells (n = 10). (A) Clinical GVHD score. (B) Overall survival through 9 to 10 weeks post-HSCT. Median survival: A > 100 days; B = 31 days; C > 100 days; D = 68 days. P values are shown. (C-F) Bleed day 31. (C) Frequency of CD4+, CD8+ and CD19+ cells out of total lymphocytes. (D) Frequency of Treg cells out of donor T CD4+ cells. Group A is not included in this graph because recipients did not receive donor T cells. (E) Cell origin/engraftment (BM donor, LN donor, or recipient) of CD4+ and CD8+ cells. (F) Naïve/memory compartment of CD4+ and CD8+ cells. Biology of Blood and Marrow Transplantation 2017 23, 757-766DOI: (10.1016/j.bbmt.2017.02.013) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Treg-expanded donor cells mediate GVL responses as effective as normal donors. HSCT with splenocytes from unexpanded or TL1A-Ig/IL-2–expanded B6-FoxP3rfp donors was performed as described in Figure 4. Some groups also received 1 × 105 A20luc/YFP cells i.v. at the time of HSCT. Donor Treg expansion leads to an efficient amelioration of GVHD while retaining the GVL effect (n = 8) shown by (A) % initial weight, (B) clinical GVHD score, and (C) survival. Statistical analyses (see Methods) was performed on day 35. The GVL effect was identified by bioluminescence imaging (D) and quantified by measurement of the total photon flux from each recipient group (E). Treg-expanded donors (D, right panel) mediate GVL responses as effective as normal donors (D, middle panel). Positive (BM + A20) tumor growth control (D, left panel). Color bar represents signal intensity code over body surface area. (This figure is available in color online at www.bbmt.org.) Biology of Blood and Marrow Transplantation 2017 23, 757-766DOI: (10.1016/j.bbmt.2017.02.013) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions