Matrix Metalloproteinase-9 in Monocytic Myeloid-Derived Suppressor Cells Correlate with Early Infections and Clinical Outcomes in Allogeneic Hematopoietic Stem Cell Transplantation  Sung-Eun Lee, Ji-Young Lim, Tae Woo Kim, Young-Woo Jeon, Jae-Ho Yoon, Byung-Sik Cho, Ki-Seong Eom, Yoo-Jin Kim, Hee-Je Kim, Seok Lee, Seok-Goo Cho, Dong-Wook Kim, Jong Wook Lee, Woo-Sung Min, Dong-Mi Shin, Eun Young Choi, Chang-Ki Min  Biology of Blood and Marrow Transplantation  Volume 24, Issue 1, Pages 32-42 (January 2018) DOI: 10.1016/j.bbmt.2017.08.017 Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Transplantation outcomes based on the recovery of G-MDSCs and M-MDSCs at engraftment. The 130 patients were grouped (low versus high groups) according to the median values of the frequency of G-MDSCs and M-MDSCs. (A to F) Transplantation outcomes according to G-MDSC frequency. Shown are the cumulative incidence of (A) acute GVHD (grade ≥II), (B) infections within 100 days, (C) infections by 1 year and 2 years, (D) TRM by 1 year, (E) relapse, and (F) probability of EFS between the low and high G-MDSC groups. (G to L) Transplantation outcomes according to M-MDSC frequency. Shown are the cumulative incidence of (G) acute GVHD (grade ≥II), (H) infections within 100 days, (I) infections by 1 year and 2 years, (J) TRM by 1 year, (K) relapse, and (L) probability of EFS between the low and high M-MDSC groups. Biology of Blood and Marrow Transplantation 2018 24, 32-42DOI: (10.1016/j.bbmt.2017.08.017) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Relationship between MDSC expansion and mucositis before engraftment. (A) Correlation between the recovery of MDSCs and mucositis (grade ≥II) before engraftment. (B) Serum levels of C-reactive protein at day 7 post-transplantation correlated with the occurrence of mucositis. Data are presented as mean ± SEM. Biology of Blood and Marrow Transplantation 2018 24, 32-42DOI: (10.1016/j.bbmt.2017.08.017) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Transcriptome profiling analysis of isolated G-MDSCs and M-MDSCs. Genes differentially expressed between G-MDSC and M-MDSC populations (n = 3, respectively) were identified with a threshold of a 2-fold change and P <.05. (A) Hierarchical clustering analysis and (B) principal component analysis showing distinct patterns in the transcriptomes from the 2 populations. (C) Gene Set Enrichment Analysis identifying the biological functional category of cell movement as the most significant category (false discovery rate q value = .0087). (D) The top 25 genes in cell movement shown in a heat map. Transcript levels of genes labeled with * were determined by qRT-PCR. Biology of Blood and Marrow Transplantation 2018 24, 32-42DOI: (10.1016/j.bbmt.2017.08.017) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Comparison of transcript levels of MMP-9 and its upstream genes in G-MDSCs and M-MDSCs. To compare the expression of MMP-9 and its upstream genes between G-MDSCs and M-MDSCs, qRT-PCR analyses were performed in each subset of MDSCs isolated from PBMCs of 34 patients at engraftment. (A) Expression levels of MMP-9 in M-MDSCs and G-MDSCs measured in RNA extracts (n = 34; left), cell lysates (n = 54; middle), and culture supernatants (n = 46; right). Transcript levels of (B) ARRB2, (C) F2RL1, and (D) GNA12 were measured in 2 isolated subsets of MDSCs (n = 34). Biology of Blood and Marrow Transplantation 2018 24, 32-42DOI: (10.1016/j.bbmt.2017.08.017) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 MDSC-induced T cell suppression via MMP-9. (A) The microscopic findings of G-MDSCs and M-MDSCs after their isolation from patient samples obtained at engraftment. (B) T cell suppression mediated by G-MDSCs and M-MDSCs tested at a 1:.2, 1:1, and 1:5 T cell to MDSC ratios. (C) Effect of MMP-9 blockade on T cell proliferation. The proliferation of both CD4+ (top) and CD8+ (bottom) T cells was compared after co-culturing with purified M-MDSCs or G-MDSCs in the presence of 5 nM or 100 nM MMP-9 inhibitor, respectively. The left figures are representative of duplicate experiments, and the individual data from 2 independent experiments are presented in the right figure. The data are presented as mean ± SEM. *P < .05; **P < .01; ***P < .001. Biology of Blood and Marrow Transplantation 2018 24, 32-42DOI: (10.1016/j.bbmt.2017.08.017) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 6 Predictive effect of serum MMP-9 level on transplantation outcomes. The effects of serum MMP-9 level on the cumulative incidences of (A) acute GVHD (grade ≥II), (B) infections within 100 days, (C) TRM by 1 year, and (D) EFS were evaluated in 106 patients with available serum samples. Patients were grouped on the basis of high versus low MMP-9 level with the use of the identical cutoff after log transformation (i.e., log-10 MMP-9 < 3.9 versus ≥3.9). (E and F) In an independent cohort of 78 patients, a consistent effect of serum level of MMP-9 on the cumulative incidence of (E) acute GVHD (grade ≥II) and (F) infections within 100 days is shown. Biology of Blood and Marrow Transplantation 2018 24, 32-42DOI: (10.1016/j.bbmt.2017.08.017) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions