Volume 16, Issue 3, Pages 412-423 (March 2014) Treatment for salivary gland hypofunction at both initial and advanced stages of Sjögren-like disease: a comparative study of bone marrow therapy versus spleen cell therapy with a 1-year monitoring period  Saeed Khalili, Denise L. Faustman, Younan Liu, Yoshinori Sumita, David Blank, Alan Peterson, Shohta Kodama, Simon D. Tran  Cytotherapy  Volume 16, Issue 3, Pages 412-423 (March 2014) DOI: 10.1016/j.jcyt.2013.10.006 Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure 1 Long-term monitoring of SFR in NOD mice treated with the two-component therapy. (A) NOD mice that were 7 weeks old and at the initial phase of SS-like (week 0). Scatter graph shows SFR of control (black circle) and CFA (black triangle) NOD mice deteriorating over the 52-week post-therapy period. NOD transplanted with spleen (white square) or bone marrow cells (white circle) maintained their SFR at levels comparable to prior disease development. Spleen cell + CFA treatment was superior to the three other NOD groups in preserving SFR (∗P < 0.05). At 52 weeks after treatment, the SFR of BM cell + CFA was statistically higher than in untreated NOD control (∗P < 0.05). (B) NOD mice that were 20 weeks old and at the advanced phase of SS-like (week 0). SFRs in bone marrow–treated and in spleen-treated NOD mice increased after therapy and reached their highest levels at week 16 after therapy (P < 0.05; n = 10 per group). At week 32 after therapy, SFRs of both cell-treated groups were significantly higher than those of CFA-treated or control NOD mice (P < 0.05). Overall, both cell therapies were most effective when given during the initial phase of SS-like; SFRs were maintained between 80–100% of pre-symptomatic NOD mice (in A). When cell therapies were given to NOD mice with advanced-phase SS-like (in B), SFRs were at best 50% of pre-symptomatic NOD mice. Note that the X-axis does not have a constant time/week interval. The “break line” on the X-axis indicates that one time interval to the next is not constant. Cytotherapy 2014 16, 412-423DOI: (10.1016/j.jcyt.2013.10.006) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure 2 Cell therapies lowered the frequency of T and B lymphocytes in foci and increased Foxp3+ Treg cells. Immunohistochemical staining of submandibular glands from NOD mice in the initial phase of SS-like at week 52 after therapy is shown. Cells positive for regulatory T cells (Foxp3+), T-helper cells (CD4), plasmacytoid dendritic cells (CD11c), T-cytotoxic cells (CD8), natural killer cells (CD49b) and B cells (CD19) appear brown in the foci. Graphs on the right represent percentages of positive cells occupying the area of the focus score. Foxp3+ Treg cells increased in bone marrow and spleen cell + CFA groups (P < 0.05), whereas CD8 and CD19 decreased when compared with control NOD (P < 0.05; n = five to six mice per group). CD4 cells decreased in the spleen-treated group (P < 0.05). No difference was observed for the percentage of CD49b and CD11c cells among the four NOD groups. Similar results were observed in foci of NOD mice treated during the advanced phase of SS-like. Cytotherapy 2014 16, 412-423DOI: (10.1016/j.jcyt.2013.10.006) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure 3 Expression of key growth factor genes involved in the pathogenesis of SS and in salivary tissue homeostasis. (A) Initial phase of SS-like. At 52-week post-treatment, BM- and spleen-treated groups had a 3- to 4-fold downregulation of inflammatory genes (TNFα, TGFβ1) when compared with the control group and a 3- to 8-fold upregulation of tissue-regenerative genes (EGF, FGF-2, IGF-IR and AQP-5) when compared with control and CFA groups (P < 0.05; n = eight to nine mice per group). (B) Advanced phase of SS-like. At 32-week post-treatment, the same trend of gene expression was observed for TNFα, TGFβ1 (2- to 2.5-fold downregulation) and EGF (10-fold upregulation) in BM- and spleen-treated groups. However, IGF-IR mRNA was 8-fold decreased in BM- and spleen-treated groups (P < 0.05). FGF-2 and AQP-5 were not statistically different among the four NOD groups. (C) EGF concentration in serum of the advanced phase of SS-like was 6–8 times higher in spleen-treated NOD versus control, CFA- or BM-treated NOD (P < 0.05). (Technical note: Unfortunately, serum from NOD mice treated during the initial phase of the disease was not preserved because we initially focused on tissue trans-differentiation.) Cytotherapy 2014 16, 412-423DOI: (10.1016/j.jcyt.2013.10.006) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure 3 Expression of key growth factor genes involved in the pathogenesis of SS and in salivary tissue homeostasis. (A) Initial phase of SS-like. At 52-week post-treatment, BM- and spleen-treated groups had a 3- to 4-fold downregulation of inflammatory genes (TNFα, TGFβ1) when compared with the control group and a 3- to 8-fold upregulation of tissue-regenerative genes (EGF, FGF-2, IGF-IR and AQP-5) when compared with control and CFA groups (P < 0.05; n = eight to nine mice per group). (B) Advanced phase of SS-like. At 32-week post-treatment, the same trend of gene expression was observed for TNFα, TGFβ1 (2- to 2.5-fold downregulation) and EGF (10-fold upregulation) in BM- and spleen-treated groups. However, IGF-IR mRNA was 8-fold decreased in BM- and spleen-treated groups (P < 0.05). FGF-2 and AQP-5 were not statistically different among the four NOD groups. (C) EGF concentration in serum of the advanced phase of SS-like was 6–8 times higher in spleen-treated NOD versus control, CFA- or BM-treated NOD (P < 0.05). (Technical note: Unfortunately, serum from NOD mice treated during the initial phase of the disease was not preserved because we initially focused on tissue trans-differentiation.) Cytotherapy 2014 16, 412-423DOI: (10.1016/j.jcyt.2013.10.006) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure 4 Detection of Y-chromosome (male) and EGFP-positive cells in salivary glands of female NOD mice: FISH and immunostaining in mouse submandibular glands. Nuclei in all tissues were stained blue with 4′,6-diamidino-2-phenylindole. (A) Male mouse salivary gland used as a positive control for detecting Y-chromosome cells (pink dot). (B) Female mouse used as a negative control. (C) NOD mouse transplanted with male bone marrow or spleen cells. No Y-chromosome–positive cell was detected. (D) Male EGFP mouse used as a positive control for detecting EGFP cells. The red signal in the cytoplasm represents EGFP-actin. (E) Non-EGFP mouse used as a negative control. (F) NOD mouse transplanted with EGFP bone marrow or spleen cells. No EGFP-positive cells were detected. (G) PCR for Y chromosome and EGFP. NOD receiving male or EGFP cell transplants were negative for Y chromosome or EGFP amplifications. (H) EGFP transgenic mouse used as donor (arrow). Cytotherapy 2014 16, 412-423DOI: (10.1016/j.jcyt.2013.10.006) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure 5 Effect of therapy on normoglycemia and body weight. Upper panel: Kaplan–Meier plot for normoglycemia in CFA (white triangle), BM (white circle) and spleen (black square) groups compared with control NOD mice (black circle) (P < 0.05). Fifty percent of control NOD developed hyperglycemia (glucose level >250 mg/dL) at week 8 after therapy. Only 10% of control NOD was normoglycemic at week 30 after therapy. However, all (100%) spleen-treated and CFA-treated NOD and 90% of BM-treated NOD had normal blood glucose levels until week 52 after treatment. Lower panel: Body weights continued to increase steadily for CFA-, BM- and spleen-treated NOD and were statistically different from controls at week 52 after treatment (P < 0.05). Cytotherapy 2014 16, 412-423DOI: (10.1016/j.jcyt.2013.10.006) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure S1 Saliva composition of NOD mice. (A) Initial phase of SS-like: Concentrations of salivary EGF, amylase and total proteins were measured for each group of NOD mice at baseline (pre-therapy, week 0) and end of the experiment (week 52 after therapy). Their concentrations did not change significantly (P > 0.05; n = 10 per group). (B) Advanced phase of SS-like: The concentrations did not change significantly (P > 0.05) from baseline to the end of the experiment (week 32 after therapy). Cytotherapy 2014 16, 412-423DOI: (10.1016/j.jcyt.2013.10.006) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure S1 Saliva composition of NOD mice. (A) Initial phase of SS-like: Concentrations of salivary EGF, amylase and total proteins were measured for each group of NOD mice at baseline (pre-therapy, week 0) and end of the experiment (week 52 after therapy). Their concentrations did not change significantly (P > 0.05; n = 10 per group). (B) Advanced phase of SS-like: The concentrations did not change significantly (P > 0.05) from baseline to the end of the experiment (week 32 after therapy). Cytotherapy 2014 16, 412-423DOI: (10.1016/j.jcyt.2013.10.006) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure S2 Concentrations of electrolytes in saliva of NOD mice. In the initial phase of SS-like, the concentrations of electrolytes (Na+, K+, Ca2+ and Cl–) were not significantly different among the four groups before versus after therapy (P > 0.05). Note that concentrations of electrolytes in saliva of the advanced phase of SS-like NOD mice are not shown because of insufficient volume of saliva for analysis. Cytotherapy 2014 16, 412-423DOI: (10.1016/j.jcyt.2013.10.006) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure S3 Number of lymphocytic aggregates (focus score) in salivary glands. (A) Chart represents the focus score distribution among the four NOD groups. The mean focus score was 3.1 for the control, 2.4 for the CFA-treated, 2.3 for the bone marrow–treated and 2.1 for the spleen-treated groups. No statistical difference was found (P = 0.596; n = eight to 10 per group). A comparable result was observed in both the initial and advanced phases of SS-like. (B) Light photomicrographs of NOD salivary glands stained with hematoxylin and eosin. The lymphocytic infiltrates in control, CFA-treated, bone marrow–treated, and spleen-treated NOD mice are shown within the yellow dash line. Cytotherapy 2014 16, 412-423DOI: (10.1016/j.jcyt.2013.10.006) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions

Figure S3 Number of lymphocytic aggregates (focus score) in salivary glands. (A) Chart represents the focus score distribution among the four NOD groups. The mean focus score was 3.1 for the control, 2.4 for the CFA-treated, 2.3 for the bone marrow–treated and 2.1 for the spleen-treated groups. No statistical difference was found (P = 0.596; n = eight to 10 per group). A comparable result was observed in both the initial and advanced phases of SS-like. (B) Light photomicrographs of NOD salivary glands stained with hematoxylin and eosin. The lymphocytic infiltrates in control, CFA-treated, bone marrow–treated, and spleen-treated NOD mice are shown within the yellow dash line. Cytotherapy 2014 16, 412-423DOI: (10.1016/j.jcyt.2013.10.006) Copyright © 2014 International Society for Cellular Therapy Terms and Conditions