Human Mesenchymal Stem Cell–Educated Macrophages Are a Distinct High IL-6– Producing Subset that Confer Protection in Graft-versus-Host-Disease and Radiation Injury Models  Myriam N. Bouchlaka, Andrea B. Moffitt, Jaehyup Kim, John A. Kink, Debra D. Bloom, Cassandra Love, Sandeep Dave, Peiman Hematti, Christian M. Capitini  Biology of Blood and Marrow Transplantation  Volume 23, Issue 6, Pages 897-905 (June 2017) DOI: 10.1016/j.bbmt.2017.02.018 Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 MEMs express higher levels of CD206, CD163, PD-L1, PD-L2, CD39, and CD73. On day +10 of ex vivo expansion, MØ or MEMs were analyzed by flow cytometry for CD90 and CD14. (A) CD14+CD90− cells are MØs. CD14−CD90+ cells are MSCs. (B) Cell surface expression of CD206, CD163, PD-L1, and PD-L2 were determined on the CD14+CD90− population in MØ and MEM cultures by MFI (mean fluorescent intensity) of isotypes (iso), MØs, and MEMs, respectively. Numbers denote the MFIs for each group with color-matched histograms. (C) CD206, CD163, and PD-L1/L2 fold change in MFI in MEM over MØ from 6 donors. (D) Expression of CD73, CD39, and MHC II (HLA-DR) of fluorescent minus 1 (FMO) versus CD14 on MØ and MEMs. (E) MFI of iso, MØs, and MEMs for CD73, CD39 or MHC II. Numbers in plots denote MFI for each group. Bar graph statistics (mean ± SEM) by 2-way ANOVA with Bonferroni multiple comparisons. Biology of Blood and Marrow Transplantation 2017 23, 897-905DOI: (10.1016/j.bbmt.2017.02.018) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 MEMs express a unique gene expression profile that distinguishes them from MØs cultured from peripheral blood or BM. Human MØs were generated from CD14+ monocytes from PBMCs or BM cells for 1 week. MEMs were generated by incubating MØ-PBMC with MSCs at a 10:1 ratio for 3 days. On day +10 of culture, CD14+ MØs were resorted from the MØ-PBMCs, MØ-BM, and MEM cultures, and RNA was isolated for RNA-Seq. (A) Principal component analysis was applied to RNASeq gene expression of 9 samples, 3 each of MEM (red triangle), MØ-BM (green circle), and MØ-PBMC (blue cross) populations. Each symbol represents a unique sample. Samples are plotted based on their coefficients in principal component space. (B) Each column of the heatmap is a unique sample (red, MEM; green, MØ-BM; blue, MØ-PBMC). Genes shown are those with at least a 2-fold difference and false discovery rate < .15 between any of the group comparisons. (C) Gene set enrichment analysis was run comparing MEMs (n = 3) to other MØs (MØ-BM and MØ-PBMC, n = 6). Genes in each set are indicated by black lines along the bottom tracks. The enrichment score across the list is plotted in green. NES indicates normalized enrichment score; FDR, false discovery rate. (D) Log2 transformed FPKM RNAseq gene expression, averaged across the 3 replicates for each population (red, MEM; green, MØ-BM; blue, MØ-PBMC). Error bars show SEM. Biology of Blood and Marrow Transplantation 2017 23, 897-905DOI: (10.1016/j.bbmt.2017.02.018) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Decreased proinflammatory cytokine profile and increased expression of anti-inflammatory genes by MEMs. On day +10 of ex vivo expansion, CD14+ sorted MØ or CD14+ sorted MEMs were collected for reverse transcriptase-PCR. (A and B) Fold change in mRNA expression of genes normalized to GAPDH housekeeping gene. N = 3 and set up in triplicate. Mean ± SEM analyzed by 2-way ANOVA with Bonferroni's multiple comparison. ****P < .0001, ***P < .001, and *P < .05. Biology of Blood and Marrow Transplantation 2017 23, 897-905DOI: (10.1016/j.bbmt.2017.02.018) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 MEMs secrete higher IL-6 constitutively or after LPS stimulation and is dependent on direct contact with MSCs, via JAK1/JAK2, arginase, and COX-2 pathways. (A) IL-6 production by ELISA was measured in MØs exposed to media alone (MQ), in direct contact with MSCs (MEM direct), in MØ with MSCs added to the upper chamber of a transwell (MEM transwell), in MØ with rh-IL4 (MQ + IL-4) or rh-IL13 (MQ + IL-13), or in MEMs exposed to NS398 (MEM + NS398), NOR-NOHA (MEM + NOR-NOHA), or ruxolitinib (MEM + Ruxo). Each group was set up in triplicate, 3 to 8 donors. (B and C) IL-6 production by ELISA was measured in MØs sorted from MØs + media or MØs + MSCs (MEMs) and replated in fresh media with or without LPS for 2 days. Three donors and each group was set up in triplicate. (B) Human IL-6 concentration and (C) fold change in human IL-6 production by each group compared with MØ in media alone is shown. Mean ± SEM analyzed by 1-way ANOVA with Tukey's multiple comparisons. Data are representative of 3 experiments with reproducible results. n.d. indicates not detected. Biology of Blood and Marrow Transplantation 2017 23, 897-905DOI: (10.1016/j.bbmt.2017.02.018) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Treatment of xenogeneic GVHD with MEMs allow for increased survival and inhibit T cells proliferation in vitro. (A) Day +0, NSG mice received 30 × 106 PBMCs i.v. to induce a xenogeneic GVHD in the absence of total body irradiation. (A) On day +12 post-transplant, human CD45+ engraftment in spleen, blood, and BM was assessed. (B) On day +18, when mice showed clinical evidence of GVHD, mice were randomized to receive PBS, 5 × 105 MSCs, or MEMs i.v to treat GVHD and monitored for survival. Five mice per group, 1 representative experiment of 3 performed. Survival curves compared by log rank analysis, **P < .001 (C) Allogeneic CD3+ sorted CFSE-labeled T cells were cultured in triplicate with anti-CD3 and anti-CD28 antibodies either alone (T cells), with MØ (T cells + MØ), or MEM (T cells + MEM) at a 2:1 ratio for 5 days. Cells were then collected from each group and stained for CD3+ to determine the percentage of proliferating cells, which are CD3+CFSE−. Mean ± SEM calculated by 1-way ANOVA. Biology of Blood and Marrow Transplantation 2017 23, 897-905DOI: (10.1016/j.bbmt.2017.02.018) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 6 Treatment of lethal radiation with MEMs allows for increased survival and improved weight loss and clinical scores. (A-D) On day 0 NSG mice received lethal total body irradiation (3 Gy) followed by (3 hours later) PBS, 5 × 105 MØs, 5 × 105 MSCs, or 5 × 105 MEMs treatment i.v. Eight to 11 mice per group. (A) Survival curve compared by log rank analysis. (B) Median survival in days for each group.***P = .0003, **P = .0066, *P = .02, mean ± SEM by 1-way ANOVA of analysis with Bonferroni multiple comparison post-test. (C) Percent weight change compared with day 0 for each group, ***P < .0001, 2-way ANOVA with Tukey's multiple comparisons post-test. (D) Overall clinical score (weight loss, posture, activity, skin, and fur texture). On day 37: *P = .015 MEM versus PBS, *P = .011 MEM versus MSC, and ***P = .0002 MEM versus MØ, 2-way ANOVA with Tukey's multiple comparisons post-test. Data representative of 1 of 2 experiments with similar results. (E) MØ or MEM were co-cultured at 25:1 ratio with NIH-3T3-GFP+ cells prelabeled with Violet Proliferation Dye 450. On day 7 the percentage of proliferating NIH-3T3 fibroblasts was assessed by flow cytometry by gating on GFP+ VPD450− cells and compared with NIH-3T3-GFP cells cultured alone. Mean ± SEM calculated by 2-way ANOVA with Tukey's multiple comparisons post-test. Data are representative of 1 of 2 experiments with similar results. Biology of Blood and Marrow Transplantation 2017 23, 897-905DOI: (10.1016/j.bbmt.2017.02.018) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions