Fig 2: Plasmodium‐specific memory CD4+ T cells induced under IL‐27 neutralization exhibit enhanced cytokine productionCytokine production was analyzed for PbT‐II cells transferred to B6 mice infected with Pcc and were treated with control (IgG, blue) or anti‐IL‐27 mAb (α‐IL‐27, red) between −1 and 7 days after infection. A, BSplenocytes from IgG and α‐IL‐27‐treated mice collected 7 and 28 days after infection were stimulated with PMA and ionomycin for 4 h (A), and splenic CD4+ T cells were cultured with PbT‐II peptide‐pulsed dendritic cells for 4 h (B), stained for surface markers, fixed, permeabilized, and stained for IFN‐γ, TNF, IL‐10, and IL‐2. Representative flow cytometry profiles (left) of cytokine expression of PbT‐II cells (CD45.1+CD45.2−TCRβ+CD4+) and frequencies of cytokine‐producing PbT‐II cells (right) are shown for control (blue) and anti‐IL‐27 mAb (red)‐treated mice at day 7 (n = 4 mice/group) and day 28 (n = 5 mice/group) days pi. Representative data of two independent experiments are shown.CProportions of IFN‐γ‐ or TNF‐producing cells in response to PbT‐II peptide within indicated subpopulations of control (n = 4) and anti‐IL‐27 mAb‐treated (n = 3) mice.D, ESplenic cells were prepared 28 days after Pcc infection and stained for TCRβ, CD4, CD45.1, CD127, and KLRG1, fixed, permeabilized, and stained for Ki67 to analyze expression in PbT‐II cell subpopulations.DRepresentative histogram plots (left) and summary graphs (right) of Ki67 expression in PbT‐II cells, and isotype control of IgG group (gray) at 28 days pi (n = 4 mice/each group). Data are representative of three independent experiments.ERepresentative histograms (left) and summary graph (right) of median fluorescence intensity (MFI) levels of Ki67 expression of total PbT‐II cells from IgG, and CD127+KLRG1− (orange), CD127−KLRG1+ (red), CD127−KLRG1− (green) PbT‐II subpopulations from anti‐IL‐27 mAb‐treated mice (n = 3 mice/each group). Data are representative of three independent experiments. Data information: Statistical significance was assessed by Student's t test for comparing IgG and anti‐IL‐27 mAb‐treated mouse groups in (A), (B), and (D), and per subpopulation in (C), and one‐way ANOVA followed by Tukey's multiple comparison test in (E). P values (< 0.05) are shown. Error bars represent SD in all graphs. Source data are available online for this figure.

Fig 3: Effects of inflammation on LRRK2 activity in WT and Lrrk2-R1441C mutant mice.(A) Cellular composition of mouse spleen (n = 4 mice) 24 h after i.p. injection of anti-CD3 (αCD3, red) or PBS (blue). Frequencies of indicated cell types as a percent of single live CD45+ cells is shown. Statistical analysis was not applied to T cells, since strong reduction in the number of T cells is most likely due to incomplete identification of T cells resulting from anti-CD3 induced internalisation of the TCR. (B) Distinction between mature and immature neutrophils by Ly6G level from cells in A. Note a strong expansion of the CD11bhi/Ly6Gint immature neutrophil subset in anti-CD3 ab injected sample. (C) EGFP-LRRK2 fluorescence (for EGFP-Lrrk2-KI sample) or autofluorescence measured in the same channel (for matching WT control) in B cells among splenocytes from WT (left panel) or EGFP-Lrrk2-KI (right panel) mice 24 h after anti-CD3 (red) or PBS (blue) injection, plotted on a linear scale against FSC-H. (D, E) WT mice were i.p. injected with either 1 mg/mouse Zymosan A (red, n = 4) or vehicle (PBS, blue, n = 4). 18 h later the total numbers of neutrophils, B cells and macrophages (D) and their LRRK2 activity (E) were measured in peritoneal lavage (left) and spleens (right) and displayed as in A. (F). Body weights before and 24 h after i.p. injection of anti-CD3 antibody (αCD3, red, n = 3 and 4) or equal volume of PBS (PBS, blue, n = 3 and 4) were measured for wild-type (WT) and Lrrk2-R1441C knock-in (R1441C) mice, respectively, and % weight loss was calculated. (G) Relative expression of Tnf, Ifng, Usp18 and S100a8 mRNA was measured by qPCR in ileums isolated from Lrrk2-R1441C mice (R1441C) and their wild-type littermates (WT) 24 h after anti-CD3 (red, n = 4 and 3) or PBS (blue, n = 4 and 3) injection. Graphs show fold changes relative to PBS-injected WT mice, with Tbp as a reference gene. (H). Frequencies of indicated cell types among live single CD45+ splenocytes obtained from WT or R1441C mice 24 h after anti-CD3 (grey, n = 3, or red, n = 4) or PBS (purple, n = 3, or blue, n = 4) injection are displayed. Note that there was no statistically significant difference between two genotypes. Data information: (A, E, D–H): Dots depict values in individual mice, bars show means, and error bars represent SEM. Statistical significance calculated by one-way ANOVA (A), 2-way ANOVA (F–H) or 2-tailed T test (E) is shown as P values or ns = not significant.

Fig 4: IL-4 induces LRRK2 expression and activity in splenic B cells.(A) EGFP-LRRK2 fluorescence was measured in B cells isolated from EGFP-Lrrk2-KI spleens (n = 3 mice) and incubated for 40 h with conditioned media from splenocytes as described in Fig. 4E, supplemented where indicated with 10 µg/ml of neutralising antibodies. The autofluorescence of identically treated B cells from wild-type mice (n = 2) was subtracted as a background. (B) B cells isolated from EGFP-Lrrk2-KI spleens (n = 3) were cultured for 40 h in media containing 5 ng/ml IL-7 with or without 10 ng/ml IFNγ, 100 ng/ml TNF or 10 ng/ml IL-4 as indicated, and EGFP fluorescence above background measured. (C) LRRK2-dependent pRab10 gMFI (LRRK2 activity) in B cells isolated from n = 3 WT mice treated as in (B) was measured. (D) Relative mRNA levels of Lrrk2 measured by qPCR in WT B cells (n = 4) from 24 h control (blue) or IL-4 stimulated (red) cultures. Bars are means of fold change relative to a control group, with Tbp used as a reference gene. (E) Graph shows the estimated copy number of LRRK2 protein per cell in lymph node naive B cells (n = 3 mice) or lymph node B cells stimulated for 40 h with 10 ng/ml IL-4 (data from 4 mice). (F) B cells isolated from EGFP-Lrrk2-KI and WT mice (4 mice per group) and cultured for 40 h with (red) or without (blue) 10 ng/ml IL-4 as in (B) were co-stained with markers for splenic B-cell subsets, and subset frequencies (left, both phenotypes pooled together) and EGFP-LRRK2 expression above background (right) were measured in T1, T2, and Fo B-cell subsets gated as in Appendix Fig. S3. (G) Immunoblot of WT splenic B cells (n = 3 mice) purified using positive selection and cultured for 24 h with 10 ng/ml IL-4 (IL-4) or without (cntr). Lysates from equivalent cell numbers were loaded and blotted for total LRRK2, pS935-LRRK2, pY641 STAT6, total Rab10 and pT73 Rab10. β-actin was used as a loading control. Quantifications of the total LRRK2 protein levels, LRRK2 activity towards Rab10, pS935-LRRK2 and pSTAT6 are shown. The experiment was repeated three times. (H) GFP fluorescence imaging in live B cells isolated from EGFP-Lrrk2-KI or WT littermate murine spleens that were cultured for 1 day with (IL-4) or without (cntr) 10 ng/ml IL-4. WT B cells shown in the left panels were additionally imaged using a transmitted light detector (T-PMT, second panels) to confirm the presence of the cells in the field of view. Scale bar = 2 µm. Images representative of three mice from three independent experiments. Data information: in (A–G), dots show values from individual mice, with means and SEM shown as bars and error bars. Statistical significance calculated by two-tailed paired t test (A–D, F, G) or two-tailed unpaired t test (E) is shown by the P value, ns not significant. Source data are available online for this figure.

Fig 5: OVV-αCD47nb enhances immune infiltration and reprograms tumor-associated macrophages(A) BALB/c mice bearing subcutaneous NS-1 tumors received intratumoral injections of PBS, OVV, or OVV-αCD47nb (2 × 107 PFU/mouse). Tumors were harvested within 24 h after the final injection for single-cell suspension preparation and flow cytometric analysis of CD8+ T cell proportions. Parallel experiments assessed TNF production, CD107a expression, and LAG-3 surface levels on tumor-infiltrating CD8+ T cells.(B) Representative diagram of flow cytometric analysis of CD8+ T cells.(C) Representative diagram of flow cytometric analysis of the expression of CD69 on CD8+ T cells.(D) Representative diagram of flow cytometric analysis of the expression of CD107a on CD8+ T cells.(E) Representative diagram of flow cytometric analysis of secreting granzyme B in CD8+ T in tumors.(F) Representative diagram of flow cytometric analysis of producing TNF-α in CD8+ T in tumors.(G) Representative diagram of flow cytometric analysis of CD8+ T cell surface depletion indices LAG-3 in tumors.(H) Representative diagram of flow cytometric analysis of CD8+ T cell surface depletion indices CTLA-4 in tumors.(I) Flow cytometric analysis of the proportion of F4/80+ CD11b+ macrophages.(J) Flow cytometric analysis of the proportion of CD86+ M1 macrophages.(K) Flow cytometric analysis of the proportion of CD206+ M2 macrophages.(L) Detection of CD8+ T and CD68+ macrophages infiltration in NS-1 tumors by immunohistochemistry. Scale bars represent 100 μm. Data represent the mean ± standard deviation (SD) of ≥three independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparisons post-test. ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. LAG-3, lymphocyte-activation gene 3; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; TAMs, tumor-associated macrophages.