Fig 2: Fewer Tfr Cells Are Present in the GC of Cxcr5fl/flFoxp3cre-ERT2 Mice(A–C) Histogram (A), quantification (B). and dot plots (C) of CXCR5 expression on Foxp3+CD4+ Treg cells in mesenteric lymph nodes three weeks after initiating the tamoxifen diet, before immunization, in Cxcr5fl/flFoxp3cre-ERT2 and Cxcr5+/+Foxp3cre-ERT2 mice. A fluorescence minus one (FMO) control serves as a negative control, and B220+ B cells serve as a CXCR5-positive population.(D–M) Mice were immunized with NP-KLH/alum s.c., and the GC response was analyzed in draining lymph nodes 14 days after immunization.(D) Representative flow cytometry contour plots of PD-1+Bcl6+ cells within Foxp3+CD4+ cells (Tfr cells).(E and F) Quantification of the (E) percentage and (F) absolute number of Bcl6+PD-1+ Tfr cells.(G) Cryosections from iLNs were stained for Foxp3 (magenta), Ki67 (blue), CD3 (green), and IgD (orange). Scale bar, 100 µm. Representative confocal image of the GC, with Tfr cells and Tfh cells indicated by the arrows.(H) Quantification of the median number of Tfr cells, defined as CD3+Foxp3+, per 5,000 µm2 of GC area.(I) Quantification of confocal images of the median number of CD3+Foxp3+ Tfr cells per GC per mouse.(J) Quantification of the median number of Tfr cells per 10 Tfh cells, defined as Foxp3-CD3+.(K) Quantification of the median number of Treg cells, defined as CD3+Foxp3+, per 5,000 µm2 of IgD+ B cell follicle area.(L and M) CXCR4 MFI (L) and CCR7 MFI (M) (subtracting FMO control) on Bcl6+PD-1+Foxp3+CD4+ Tfr cells from mice of the indicated genotypes.Each symbol represents one mouse, the horizontal bars represent mean values, and the error bars show the SD. The p values were determined using a Mann-Whitney U test. For the quantification of confocal images, 4–10 GCs or B cell follicles were imaged per mouse. Data represent two or more independent experiments.

Fig 3: MSNs overcome resistance and prevent relapse of aPD-1-resistant tumors. (A) SEM and TEM images of spherical MSNs with diameters of 100, 65 and 45 nm. (B) SEM and TEM images of rod-like MSNs with aspect ratios of 3:1, 4:1, 8.5:1. (C–E) Tumor growth and tumor weights of B2m-sgRNA B16F10 (C), H22 (D) and CT26 (E) tumor-bearing mice treated with different MSNs treatment (top: sizes; bottom: L/Ds) prior to aPD-1. (F, G) Growth of recurrent tumors (top) and recurrence-free survival (bottom) of tumor-bearing mice after indicated treatments. (H) Growth of rechallenged tumors (top) and overall survival (bottom) of B2m-sgRNA B16F10 tumor-bearing mice after indicated treatments. Error bars represent mean±SEM (n>6 per group in C–H). P value for tumor measurement was calculated by unpaired Student’s t-test. P value for survival curve was calculated by the log-rank (Mantel-Cox) test. (*P<0.05, **p<0.01, ***p<0.001). MSNs, mesoporous silica nanoparticles; PD-1, programmed cell death protein 1; TEM, tumor microenvironment.

Fig 4: hSFRP2 mAb reduces T-cell CD38 and PD-1 protein in vivo. Splenic CD4+ and CD8+ T-cells (top) and tumor infiltrating lymphocytes (TILs; bottom) isolated from RF420 tumor-bearing mice treated with IgG1 control (red) or hSFRP2 mAb (blue) for 21 days were stained for the following immune markers labeled with fluorochromes: CD38, PD-1, TNF-a, CD103 and CD5. Dot plot graphs show mean fluorescent intensity (MFI) analyzed by FACS for each of the immune markers of interest within the CD4+/CD8+ and TIL populations. For IgG1 and hSFRP2 mAb treatments, the number of independent samples (n) was 4. CD38 was significantly decreased with hSFRP2 mAb in both splenocytes (+ p < 0.05) and TILs (+ p < 0.05). PD-1 was significantly decreased in CD8+ TILs treated with hSFRP2 mAb (+ p < 0.05). Bottom tables: median MFIs (interquartile range) for each immune marker.

Fig 5: Normal Magnitude and Output of the GC Response in Cxcr5fl/flFoxp3cre-ERT2 MiceAnalysis of the GC response 14 days after s.c. immunization with NP-KLH/alum.(A) Flow cytometry contour plots of Bcl6+Ki67+B220+ GC B cells.(B and C) Quantification of the (B) frequency and (C) absolute number of Bcl6+Ki67+ B cells.(D) Quantification of confocal images of the average GC area per mouse of the indicated genotypes.(E) Flow cytometry contour plots of Tfh cells, gated as CXCR5+PD-1+ of Foxp3-CD4+ cells.(F and G) Quantification of the (F) percentage and (G) number of CXCR5+ Tfh cells.(H) Quantification of confocal images of the average number of CD3+Foxp3- Tfh cells per GC area per mouse of the indicated genotypes.(I) Levels of anti-NP7 antibodies of the indicated isotypes in the sera.(J) Levels of anti-NP20 antibodies of the indicated isotypes in the sera.(K) Ratio of NP20/NP7 of the indicated isotypes in the sera.(L) Serum levels of IgG specific for dsDNA.Each symbol represents one mouse, the horizontal bars represent mean values, and the error bars show the SD. The p values were determined using a Mann-Whitney U test. For the quantification of confocal images, 4–10 GCs were imaged per mouse. Data represent two independent experiments.