1. Mesenchymal Stromal Cell (MSC)-Derived Combination of CXCL5 and Anti-CCL24 Is Synergistic and Superior to MSC and Cyclosporine for the Treatment of Graft-versus- Host Disease 
Xiubo Fan, Dianyang Guo, Alice M.S. Cheung, Zhi Yong Poon, Chui Sun Yap, Shane Ee Goh, Dianyan Guo, Huihua Li, Sudipto Bari, Shang Li, Kiat Hon Lim, William Ying Khee Hwang 
Biology of Blood and Marrow Transplantation 
Volume 24, Issue 10, Pages (October 2018)
DOI: /j.bbmt
Copyright © 2018 The American Society for Blood and Marrow Transplantation Terms and Conditions

2. Figure 1
BM-MSCs mediate immunosuppression mainly through soluble factors. (A) Setup of wells with/without transwell inserts and BM-MSC-conditioned media. (B) Reduction in cell-mediated cytotoxicity is inversely correlated with the physical distance between MSCs and MLs, and the maximal reduction occurs when BM-MSCs are in direct physical contact with MLs. All results are expressed as mean ± standard deviation. For 8 comparisons, the P value was adjusted with Bonferroni's correction (*P< .0063; **P < .0013; ***P< .00013).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2018 The American Society for Blood and Marrow Transplantation Terms and Conditions

3. Figure 2
Sequential FFD screening of potential BM-MSC-mediated immunosuppressive factors. (A) Condition-6 (C6), consisting of 7 factors (CCL20, CXCL6, OPG, IL-10, and CXCL5 as well as anti-CCL1 and anti-CCL24 antibodies), was identified as the optimal combination in the first FFD experiment with 12 factors (212; Table 2; results are expressed as mean ± standard deviation, n = 1). (B) These 7 factors were subjected to a second FFD analysis (Supplementary Table S1; results are expressed as mean ± standard error [SE], n = 3). The optimal combination C21 consisted of 4 factors: CXCL5, OPG protein, and anti-CCL1 and anti-CCL24 antibodies. (C) These 4 factors were subjected to the third (full) FD analysis with 4 factors (Supplementary Table S2; results are expressed as mean ± SE, n = 2). C32 (CXCL5 and anti-CCL24 antibody) was identified as the optimal combination with the least factors. (D) The immunosuppressive effect of MSCs was abrogated by addition of 2FC flip-over (2FC-F) with 5 µg/mL of anti-CXCL5 and 100 ng/mL of CCL24. Results are expressed as mean ± SE, n = 3 (*P < .0167; **P < .0033; ***P <  for multiple comparisons). (E) The 2FC treatment could suppress GVH but preserve graft-versus-leukemia effects. Results are expressed as mean ± SE, n = 2 (*P < .05).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2018 The American Society for Blood and Marrow Transplantation Terms and Conditions

4. Figure 3
Clinical relevance of administrating 2FC and its toxicities. (A) The concentration changes of human CCL24 and CXCL5 as well as (B) clinical scores of animals in NSG mice with moderate GVHD (n = 3 mice per group). (C-F) It was deemed safe to use the 2FC to treat diseases because there was no observed body weight loss or liver and kidney toxicities (3 ICR mice per group, *P < .05).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2018 The American Society for Blood and Marrow Transplantation Terms and Conditions

5. Figure 4
2FC treatment demonstrates consistent immunosuppressive capacity no matter the severity of GVHD. The 2FC (CXCL5 and anti-CCL24 antibody) was as effective as BM-MSCs and CsA, in improving (A) survival and (E) clinical scores of animals with moderate GVHD (ndPBS = 28 mice, n2FC = 21 mice, nMSC = 21 mice, nCsA = 24 mice in 6 repeats). (B and F) It remains effective in animals with severe GVHD, whereas MSCs and CsA are less effective (ndPBS = 30 mice, n2FC = 21 mice, nMSC = 24 mice, nCsA = 23 mice in 5 repeats). Used individually, CXCL5 and anti-CCL24 antibody are as effective as the 2FC in improving (C and G) moderate GVHD (nCXCL5 = 23 mice, nanti-CCL24= 23 mice in 5 repeats) but much less effective in (D and H) severe GVHD (nCXCL5 = 23 mice, nanti-CCL24= 18 mice in 5 repeats). (I) This result is concordant with histologic observation after hematoxylin and eosin staining. With moderate GVHD, sections of skin, intestine, and kidney from 1 representative mouse were examined at day-30 post-transplantation (scale bar, 100 µm). This representative mouse was chosen from the 1 representing the average clinical scoring of each group (5 mice per group). The single-headed arrow indicates lymphocyte infiltration; double-headed arrow, the length of villus; CI, clinical index; HI, histologic index. For clinical score, results are expressed as mean ± SE. The overall trend difference in clinical score between different treatments was compared by means of generalized estimating equation model (*P < .0167; **P < .0033; ***P <  for multiple comparisons). For survival curve, the significance was defined as *P < .05; **P < .01; ***P < .001.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2018 The American Society for Blood and Marrow Transplantation Terms and Conditions

6. Figure 5
The immunosuppression can be improved by extended treatment. Extended treatment significantly improved (A) mice survival and (B) clinical score of animals with severe GVHD (ndPBS = 35 mice, n2FC-4 injections= 20 mice, n2FC-8 injections= 15 mice in 4 repeats). For clinical score, results are expressed as mean ± SE. The overall trend difference in clinical score between different treatments was compared by means of generalized estimating equation model. The significance was defined as *P < .025; **P < .005; ***P < .0005 for multiple comparisons.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2018 The American Society for Blood and Marrow Transplantation Terms and Conditions

7. Figure 6
The in vivo target cells of 2FC treatment. The 2FC treatment not only reduced (A) the proliferation of human Th 1 and Th 17 cells but also reduced CTLs in the circulation and (B) NK cells in the spleen (at day 40 post-transplantation). It increased (C) mouse immunosuppressive neutrophils (Ly6G+CD11b+ cells at day-25 post-transplantation) but did not affect (D) human immunosuppressive neutrophils (CD16briCD62Ldim cells at day-25 post-transplantation) and the reconstitution of (E) human hematopoietic stem/progenitor cells in the BM (CD34+ cells at day 40 post-transplantation) in NSG mice with moderate GVHD. Results are expressed as mean ± SE (n = 5) in A and geometric mean with 95% confidence intervals in B through E. (F) Circulating levels of human proinflammatory cytokines IFN-γ, IL-6, IL-8, IL-17A, MIP-1β, and MCP-1 were reduced by 2FC, MSCs, and CsA treatment. Results are expressed as mean ± SE (n = 3). For A and F the overall trend difference between different treatments was compared by means of generalized estimating equation model (* P < .0167; ** P < .0033, for multiple comparisons). For B through E significance was defined as *P < .05; **P < .01; ***P < .001.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2018 The American Society for Blood and Marrow Transplantation Terms and Conditions