Fig 2: IL-10 in vitro effects on adaptive immune cells. (a,d) Splenocytes or (b,e) purified CD4, CD8 T cells or CD19+ B cells from 28 week old NZB/W F1 animals with established autoantibodies and beginning nephritis (n = 4–9 mice) were incubated for 4 days with a-CD3/CD28 (T cell stimulation) or CpG (B cell stimulation), (a,d) in the presence or absence of neutralizing a-IL-10 or (b,e) in the presence or absence of recombinant mouse IL-10, TNF-a or IL-21. (a,b) Proliferation of CD4 T, CD8 T and B cells was examined using CFDA-SE. Depicted is one representative FACS blot showing the proliferation of CD8 T cells and gating strategy to quantify cells with 0–1, 2–4 or =5 cycles of proliferation. The mean percentages ± SEM of cells with 0–1, 2–4 or =5 cycles of proliferation from 6 (a) and 4–9 (b) independent donors are expressed as stacked bars. The p value was calculated using a Wilcoxon test with paired data analysis to determine the difference between a-IL-10-treated and untreated cells (a) or the difference between untreated cells and those treated with IL-10, IL-21 or TNF-a (b) (* p < 0.05, ** p < 0.005). (d,e) Differentiation of B cells into CD138hi plasma cells (PC) was examined by flow cytometry. The percentages of plasma cells (PC) among whole B cells (BC) are expressed as scatter blots for paired analysis; each symbol represents cells obtained from different donors. The p value was calculated using a Wilcoxon test with paired data analysis to determine the difference between a-IL-10-treated and untreated cells (d) or the difference between untreated cells and those treated with IL-10, IL-21 or TNF-a (e). (c) Splenocytes from 28 week old NZB/W F1 animals with established autoantibodies and beginning nephritis (n = 8 mice) were incubated for 48 h in the presence and absence of neutralizing a-IL-10, and the expression of Annexin V was determined by flow cytometry. Each symbol represents results obtained from different donors depicted as fold difference between a-IL-10- (+a-IL-10) and untreated (-a-IL-10) cells. Results are expressed as scatter blots with mean ± SEM. The p value was calculated using a one-sample t test. (f,g) CD138hi PC and CD138-CD19+ B cells (n = 5 mice) were sort-purified and incubated for 2 and 4 days, respectively, with CpG and in the presence or absence of recombinant IL-10. (f) The survival of PC was determined by flow cytometry and expressed as percentage of CD138hi PC after 2 days of culture/number of starts and expressed as scatter blots for paired analysis. (g) Differentiation of B cells into CD138hi plasma cells (PC) was examined by flow cytometry, and the percentages of plasma cells (PC) among whole B cells (BC) are expressed as scatter blots for paired analysis; each data symbol represents cells obtained from different donors. The p value was calculated using a Wilcoxon test with paired data analysis to determine the difference between untreated cells and those treated with IL-10. (h) Splenocytes from 28 week old NZB/W F1 animals (n = 4 mice) were stimulated for 48 h with a-CD3/CD28 and in the presence or absence of neutralizing a-IL-10. Expression of IFN-? was determined on CD4 and CD8 T cells after re-stimulation with PMA/ionomycin for an additional 4 h. Each symbol represents results obtained from different donors depicted as fold difference between a-IL-10-treated (+a-IL-10) and untreated (-a-IL-10) cells. Results are expressed as scatter blots with mean ± SEM. The p value was calculated using a one-sample t test. Depicted is also one representative FACS blot showing the expression of IFN-? on CD8 T cells in cells treated or not with a-IL-10.

Fig 3: Flow cytometry analysis of B cell differentiation in the colon of WT and Sphk2-/- mice after sub-chronic stress exposure. Representative dot plots showing the gating strategy used to distinguish lymphoid lineage cells (CD45+), B cells (CD45+B220+CD138-/low), plasmablasts (CD45+B220-CD138-/low), and plasma cells (CD45+B220-CD138high). Considering the total number of CD45+ cells as 100%, percentage of B cells (a), plasmablasts (b), and plasma cells (c) were plotted. Data are means ± SEM of 5–8 mice per group. Two-way ANOVA considering stress (S) and Sphk2-/- as independent variables followed by Tukey's post hoc; *S p < 0.05, **S p < 0.01. Sphk2: Sphingosine kinase 2; WT: wild-type; Sphk2-/-: Sphk2 knockout.

Fig 4: Loss of TET-function impairs GC maintenance and CSR upon immunization. (A) qRT-PCR analysis for TET2 and TET3 mRNA expression in FACS-sorted splenic GC B cells of the indicated genotypes 5 and 10 days post immunization with NP-CGG (n = 3 for Cg1-Cre and n = 3 for Cg1-Cre;Tet2 F/F;Tet3 F/F). (B) Mice of the indicated genotypes were immunized at the age of 8–10 weeks with NP-CGG and analysed 14 days later. Flow cytometry analysis of splenic single-cell suspensions identified the fractions of total B cells (B220+ CD19+), GC B cells (CD19+B220+ CD138- CD95hi CD38lo/-) and NP +IgG1+ GC B cells within total splenocytes. Total cell numbers for the respective cell populations are shown (n = 5 for Cg1-Cre and n = 5 for Cg1-Cre;Tet2 F/F;Tet3 F/F). (C) Representative flow cytometry plots (left) and bar graph (right; n = 5 for Cg1-Cre and n = 5 for Cg1-Cre;Tet2 F/F;Tet3 F/F) depict fractions of B cells, GC B cells and IgG1+ NP + GC B cells shown in (B). Arrows indicate gating strategy. (D) Spleen sections from the indicated genotypes 14 days post immunization with NP-CGG were analysed for Ki67 (proliferating cells) and for the ability of cells to bind PNA (GC B cells). The brown precipitate indicates positive staining. Sections were counterstained with haematoxylin. One representative mouse out of three per group is shown. Scale bars: 200 µm. (E) Representative flow cytometry plots (left) depict fractions of DZ and LZ GC B cells within total GC B cells. Scatter graph (right; n = 5 for Cg1-Cre and n = 5 for Cg1-Cre;Tet2 F/F;Tet3 F/F) shows the ratio of DZ and LZ GC B cells. (F) Scatter graph depicts the ratio of IgM+ and IgG1+ cells within GC B cells (n = 5 for Cg1-Cre and n = 5 for Cg1-Cre;Tet2 F/F;Tet3 F/F). (G) qRT-PCR analysis for Ki67 and AID mRNA in FACS-sorted naïve Cg1-cre B cells (n = 3) and GC splenic B cells (n = 3 for Cg1-Cre and n = 3 for Cg1-Cre;Tet2 F/F;Tet3 F/F) 14 days post NP-CGG immunization. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 vs Cg1-Cre control (Student's t-test).

Fig 5: Effect of intratumoral a-AED treatment in the immune cell infiltration of secondary lymphoid organs in the 4T1 tumor model. Determination of innate and adaptive immune subpopulations by flow cytometry in lymph nodes. (a) T lymphocytes CD3+. (b) CD8+ T cytotoxic lymphocytes. (c) CD4+ T helper lymphocytes. (d) CD4+/Foxp3+ T lymphocytes. (e) F4/80+ macrophages. (f) NKp46+ cells. (g) CD19+ B lymphocytes. (h) Plasmatic cells (CD138+). (i) Plasmablasts (CD138+/CD19+). Graphs represent the mean ± SD of the data from three independent experiments. Gates from 10,000 cells were collected. Statistical significance was calculated using a Kruskal–Wallis test (*, p = 0.05).