Culture expanded primary chondrocytes have potent immunomodulatory properties and do not induce an allogeneic immune response  P. Lohan, O. Treacy, K. Lynch, F. Barry, M. Murphy, M.D. Griffin, T. Ritter, A.E. Ryan  Osteoarthritis and Cartilage  Volume 24, Issue 3, Pages 521-533 (March 2016) DOI: 10.1016/j.joca.2015.10.005 Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Culture expanded primary chondrocytes maintain their chondrogenic phenotype over time in vitro. CEPC were isolated from DA rat knee and hip articular cartilage and (A) cultured in monolayer. (B) Growth curve of CEPC-N and CEPC-H over passage in monolayer culture. (C) Relative expression of CD29, CD44, CD45, CD73 and CD90 on MSC, CEPC-N and CEPC-H relative to isotype control staining on the same cells. (D) Collagen II (E) Collagen I and (F) Collagen X mRNA expression in MSC, freshly isolated DA cartilage, P1, 2 and 4 CEPC-N and P1, 2 and 4 CEPC-H. Cell surface expression histograms are a representative example from one of three independent experiments. Cell surface expression graph is representative of three independent experiments. Error bars represent mean ±95% CI of three to four independent experiments (n = 3 independent donors; three cultures with three technical replicates). Statistical analysis was performed using one way ANOVA with Tukey's post-test. Growth curve and qPCR data is representative of one of three CEPC donors with 2 technical replicates per experiment. Osteoarthritis and Cartilage 2016 24, 521-533DOI: (10.1016/j.joca.2015.10.005) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Culture-expanded primary chondrocytes do not elicit allogeneic T cell proliferation, activation or cytotoxic activity in vitro. (A) Immunogenicity assays were performed by co-culture of DA rat derived MSC, CEPC-N or CEPC-H at a ratio of 1:50 with LEW rat derived CFSE stained mixed lymphocytes for 5 days in standard culture conditions. LEW T cell proliferation was measured by CFSE dilution, (B) Representative histograms of CD4+ T cell proliferation after co-culture with MSC, CEPC-N or CEPC-H (top three panels, left to right), unstimulated T cells and anti-CD3/anti-CD28 stimulated T cells (bottom 2 panels, left to right). (C) Proliferation of LEW CD4+ T cells in co-culture with DA MSC, CEPC-N and CEPC-H. (D) Proliferation of LEW CD4− T cells in co-culture with DA MSC, CEPC-N and CEPC-H. (E) Expression of CD25 on CD4+ LEW T cells in co-cultures with DA MSC, CEPC-N or CEPC-H. (F) Expression of CD25 on CD8+ LEW T cells in co-cultures with DA MSC, CEPC-N or CEPC-H. LEW lymphocytes from co-cultures with DA MSC, CEPC-N and CEPC-H were stained for expression of intra-cellular Granzyme B. (G) Representative histograms of Granzyme B expression in CD8+ T cells from these co-cultures as measured by flow cytometry. (H) Percentage of Granzyme B expressing LEW CD8+ T cells in co-cultures with DA MSC, CEPC-N and CEPC-H. (I) Secretion of IFN-γ was measured in the supernatants of co-culture experiments by ELISA. Histograms and dot plots are representative of three independent experiments. Error bars represent mean ± 95% CI of three to five independent experiments (n = 3–5 independent donors; three cultures with three technical replicates). Statistical analysis was performed using one way ANOVA with Tukey's post-test*P ≥ 0.01 and ≤0.05, **P < 0.001. Osteoarthritis and Cartilage 2016 24, 521-533DOI: (10.1016/j.joca.2015.10.005) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Oxygen tension and inflammatory cytokines alter CEPC cell surface profile, but not their immunogenicity. CEPC-N and CEPC-H were treated with 100 U/ml IFN-γ, IL-1β and TNF-α either alone or in combination with each other for 48 h. Cell surface marker expression of MHC-I was measured by flow cytometry. (A) Representative histograms of MHC-I expression on CEPC-N and CEPC-H which were not treated with any cytokines (UTR) (left two panels) or treated with a combination of IFN-γ and TNF-α for 48 h compared to the same cells stained with an isotype control. (B) Relative fluorescence of MHC-I compared to isotype control on CEPC-N (white bars) and CEPC-H (grey bars) treated with cytokines alone or in combination. Cytokine treated DA (C&E) CEPC-N or (D&F) CEPC-H were co-cultured with CFSE stained LEW lymphocytes for 5 days [Fig. 2(A)]. LEW CD4+ T cell proliferation was measured after co-culture with (C) CEPC-N or (D) CEPC-H. Expression of CD25 was measured by flow cytometry on CD4+ T cells after co-culture with (E) CEPC-N or (F) CEPC-H. Error bars represent mean ±95% CI of three to four independent experiments (n = 3–4 independent donors; three cultures with three technical replicates). Statistical analysis was performed using one way ANOVA with Tukey's post test, MHC-I expression graph: $P < 0.05 compared to same treatment on CEPC-N, *P ≥ 0.01 and ≤0.05, **P < 0.001 compared to IFN-γ and TNF-α treated CEPC-N. Other graphs: **P < 0.001 compared to anti-CD3/anti-CD28 stimulated lymphocytes. Osteoarthritis and Cartilage 2016 24, 521-533DOI: (10.1016/j.joca.2015.10.005) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 CEPC exert immunomodulatory effects on activated T cells. (A) The immunosuppressive ability of CEPC was determined by co-culturing DA rat derived MSC, CEPC-N or CEPC-H with LEW rat derived CFSE stained lymphocytes for 4 days in the presence of anti-CD3/anti-CD28 stimulation. (B) T cell proliferation was measured by CFSE dilution by flow cytometry. (C) Proliferation of CD4+ T cells from co-cultures was measured after 4 days by flow cytometry. (D) Proliferation of CD4− T cells from co-cultures was measured after 4 days by flow cytometry. (E) Nitric oxide levels in the supernatants of DA MSC, CEPC-N or CEPC-H co-cultures were measured by Griess assay. (F) PGE2 levels in the supernatants of DA MSC, CEPC-N or CEPC-H co-cultures were measured by ELISA. (G) The immunosuppressive ability of DA rat derived MSC, CEPC-N or CEPC-H without contact was determined by culturing the MSC/CEPC in a 24 well plate with anti-CD3/anti-CD28 stimulated LEW rat derived CFSE stained lymphocytes in a transwell for 4 days. (H) CD4+ T cell proliferation was measured and is presented as the percentage of T cells which had proliferated greater than four generations. Histogram is a representative plot of CD4+ T cells from three independent experiments. Error bars represent mean ±95% of three independent experiments (n = 3 independent donors; three cultures with three technical replicates). Statistical analysis was performed using One way ANOVA with Tukey's post test, *P ≥ 0.01 and ≤0.05, **P < 0.001 compared to anti-CD3/anti-CD28 stimulated T cells. Osteoarthritis and Cartilage 2016 24, 521-533DOI: (10.1016/j.joca.2015.10.005) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 CEPC exert immunomodulatory effects on activated T cells through the release of nitric oxide. Immunosuppression co-culture assays were set up as outlined in Fig. 4(A). 100 μM of the iNOS inhibitor SMT was added to co-cultures to assess the effect of inhibiting nitric oxide on the immunosuppressive ability of CEPC. (A) Proliferation of LEW CD4+ T cells co-cultured with MSC, CEPC-N or CEPC-H with (black bars) or without (white bars) SMT. (B) Nitric oxide levels, as measured by Griess assay, from co-cultures in A. To assess the effect of inhibition of PGE2 secretion by CEPC on their immunosuppressive ability immunosuppression co-culture assays were set up as outlined in Fig. 4(A) in the presence or absence of 10 μM of the Cox-2 inhibitor NS-398. (C) Proliferation of LEW CD4+ T cells co-cultured with MSC, CEPC-N or CEPC-H with (black bars) or without (white bars) NS-398. (D) PGE2 levels, as measured by ELISA, from co-cultures in C. PGE2 levels in the supernatants of SMT and NS-398 co-cultures of (E) CEPC-N and (F) CEPC-H. Error bars represent mean ±95% CI of three independent experiments (n = 3 independent donors; three cultures with three technical replicates). Statistical analysis was performed using Two way ANOVA with Tukey's post test in A, B, C and D, *P ≥ 0.01 and ≤0.05, **P < 0.001 compared to anti-CD3/anti-CD28 stimulated T cells. Statistical analysis was performed using One way ANOVA with Tukey's post test E and F, *P ≥ 0.01 and ≤0.05, **P < 0.001 compared to CEPC-N/CEPC-H without inhibitor. Osteoarthritis and Cartilage 2016 24, 521-533DOI: (10.1016/j.joca.2015.10.005) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Allogeneic chondrocytes can modulate inflammatory macrophage activity. (A) LEW rat bone marrow derived macrophages were generated by culturing in 15% L-929 cell conditioned medium for 6 days. (B) Macrophages were assessed for cell surface CD45, CD11b/c, CD163, MHC-II and CD86 by flow cytometry. (C) DA rat derived MSC, CEPC-N or CEPC-H were co-cultured with LPS and IFN-γ pre-stimulated LEW macrophages at a ratio of 1:5 for 24 or 48 h. (D) Macrophages were MACS sorted from co-cultures after 24 h, RNA isolated and qPCR performed for IL-10 expression. (E) MHC-II expression was measured on macrophages from co-cultures after 48 h co-culture with MSC, CEPC-N or CEPC-H. (F) TNF-α secretion into the co-culture supernatant was measured after 48 h by ELISA. Histograms are representative of three independent experiments. Error bars represent mean ±95% CI of three-four independent experiments (n = 3–4 independent donors; three cultures with three technical replicates). Statistical analysis was performed using One way ANOVA with Tukey's post test, *P ≥ 0.01 and ≤0.05, **P < 0.001. Osteoarthritis and Cartilage 2016 24, 521-533DOI: (10.1016/j.joca.2015.10.005) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. S1 CEPC maintain their chondrogenic phenotype in vitro. (A) MSC, CEPC-N and CEPC-H were isolated from male DA rats. To test the immunogenicity and allogeneic immunomodulatory capacity of CEPC lymphocytes and macrophages were isolated from the fully allogeneic male LEW rats. (B) CEPC were isolated from the articular cartilage of the knee and hip. Cartilage was scraped from the bones in chips, these chips were digested with protease and collagenase and cultured in 19% (CEPC-N) or 2% (CEPC-H) O2. (C) CEPC-N and (D) CEPC-H were placed in 3D alginate culture for 21 days and stained with Safranin-O. (E) DMMB and pico-green assay used to calculate GAG:DNA ratio of CEPC-N after 21 day alginate culture. Error bars represent mean ± 95% CI of three independent experiments (n = 3 independent donors; three cultures with three technical replicates). Statistical analysis was performed using Unpaired Student's t test, *P < 0.05. Osteoarthritis and Cartilage 2016 24, 521-533DOI: (10.1016/j.joca.2015.10.005) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. S2 Cytokine treatment or oxygen tension have no effect on cell surface expression of MHC-II, CD80 or CD86 on CEPC. CEPC-N and CEPC-H were treated with 100 U/ml IFN-γ, IL-1β and TNF-α either alone or in combination with each other for 48 h. Cell surface marker expression of MHC-II, CD80 and CD86 were measured by flow cytometry. (A) MHC-II expression on CEPC-N. (B) CD80 expression on CEPC-N. (C) CD86 expression on CEPC-N. (D) MHC-II expression on CEPC-H. (E) CD80 expression on CEPC-H. (F) CD86 expression on CEPC-H. Data presented as relative fluorescence compared to isotype control stained cells. Statistical analysis was performed using one way ANOVA with Tukey's post-test (n = 3 independent donors; three cultures with three technical replicates). Osteoarthritis and Cartilage 2016 24, 521-533DOI: (10.1016/j.joca.2015.10.005) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. S3 Cytokine treatment or inactivation has no effect on CEPC immunogenicity. Cytokine treated DA (A&C) CEPC-N or (B&D) CEPC-H were co-cultured with CFSE stained LEW lymphocytes for 5 days [Fig. 2(A)]. LEW CD4− T cell proliferation was measured after co-culture with (A) CEPC-N or (B) CEPC-H. Expression of CD25 was measured by flow cytometry on CD8+ T cells after co-culture with (C) CEPC-N or (D) CEPC-H. CEPC-N and CEPC-H were treated with UV radiation for 10 min or 4% PFA for 10 min prior to co-culture with LEW lymphocytes. (E) CD4+ T cell proliferation after co-culture with untreated, UV treated or PFA treated CEPC-N and CEPC-H and UV treated or untreated DA splenocytes. (F) CD4− T cell proliferation after co-culture with untreated, UV treated or PFA treated CEPC-N and CEPC-H and UV treated or untreated DA splenocytes. (G) Percentage of CD4+ LEW T cells expressing CD25 after co-culture with DA untreated, UV treated or PFA treated CEPC-N of CEPC-H. (H) Percentage of CD4− LEW T cells expressing CD25 after co-culture with DA untreated, UV treated or PFA treated CEPC-N of CEPC-H. Error bars represent mean ± 95% CI from three-four independent experiments (n = 3–4 independent donors; three cultures with three technical replicates). Statistical analysis was performed using One way ANOVA with Tukey's post test, *P ≥ 0.01 and ≤0.001, **P < 0.001, compared to anti-CD3/anti-CD28 stimulated lymphocytes, $P < 0.05, $$P < 0.01, $$$<0.001 compared to UV treated DA splenocytes, +++P < 0.001 compared to DA splenocytes, #P < 0.05 compared to MSC. Osteoarthritis and Cartilage 2016 24, 521-533DOI: (10.1016/j.joca.2015.10.005) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. S4 CEPC exert immunomodulatory effects on activated T cells through the release of nitric oxide. Immunosuppression co-culture assays were set up as outlined in Fig. 4(A). 100 μM of the iNOS inhibitor SMT was added to co-cultures to assess the effect of inhibiting nitric oxide on the immunosuppressive ability of CEPC. (A) Proliferation of LEW CD4− T cells co-cultured with MSC, CEPC-N or CEPC-H with (black bars) or without (white bars) SMT. (B) Proliferation of LEW CD4− T cells co-cultured with MSC, CEPC-N or CEPC-H with (black bars) or without (white bars) NS-398. Error bars represent mean ± 95% CI from three independent experiments (n = 3 independent donors; three cultures with three technical replicates). Statistical analysis was performed using Two way ANOVA with Tukey's post test, **P < 0.001 compared to anti-CD3/anti-CD28 stimulated T cells. Osteoarthritis and Cartilage 2016 24, 521-533DOI: (10.1016/j.joca.2015.10.005) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. S5 CEPC do not cause an increase in MMP production in in vitro co-cultures. DA rat derived MSC or CEPC-H were co-cultured with LPS and IFN-γ pre-stimulated LEW macrophages at a ratio of 1:5 for 48 h as outlined in Fig. 6(C). Supernatants from these co-cultures were concentrated and underwent zymographic analysis. (A) Relative quantification of inactive MMP-9 from co-culture supernatants. (B) Relative quantification of active MMP-9 from co-culture supernatants. (C) Relative quantification of inactive MMP-2 from co-culture supernatants. (D) Relative quantification of active MMP-2 from co-culture supernatants. Band density was quantified relative to stimulated macrophages. Error bars represent mean ± 95% CI from three independent experiments (n = 3 independent donors; three cultures with three technical replicates). Statistical analysis was performed using One way ANOVA with Tukey's post test. Osteoarthritis and Cartilage 2016 24, 521-533DOI: (10.1016/j.joca.2015.10.005) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions