Figure 2
Figure 2. Treg infusions treat cGVHD in a CXCR5-dependent manner. B10.BR mice were transplanted with either T-cell–depleted (TCD), B6 BM (BM Only), or TCD B6 BM with purified B6 Tcon (cGVHD) as in Figure 1A-F. Some groups of mice given BM and Tcon were also given either WT or CXCR5KO Tregs on day 0 (prophylactic) or day 28 (therapeutic). (A-C) PFTs assessing (A) airway resistance, (B) total lung elastance, and (C) total lung compliance were performed on day 56 after transplant. PFTs demonstrate reversal of PFTs characteristic of cGVHD-associated BOS in all mice given WT Tregs, and CXCR5KO Tregs on day 0, but not mice given CXCR5KO Tregs on day 28. (D-F) Frequency of (D) Tfr and (E) Tfh out of total splenic CD4+ T cells, along with frequency of (F) GC B cells (CD19+GL7hiFAShi) out of total splenic B cells were measured by flow cytometry on day 56 after transplant. (D) Tfr frequency was found to be higher, but (E) Tfh and (F) GC B-cell frequency lower in mice given WT Tregs and CXCR5KO Tregs on day 0, but not mice given CXCR5KO Tregs on day 28. (G) Representative images (original magnification ×200) of frozen spleen tissues stained with Cy3-peanut agglutinin (red) to delineate GCs. (H) Quantification of GC area from frozen spleen tissues stained with Cy3-peanut agglutinin as in (G) from 5 spleens/group. WT CXCR5KO Tregs given on day 0 reduce GC area to levels near that of BM-only mice. CXCR5KO Tregs given on day 28 reduce GC area slightly, but not significantly, compared with cGVHD mice, and do not decrease GC area to the same degree as WT Tregs. (I) Histopathology scoring of the liver based on H&E-stained cryopreserved sections of organs harvested on day 56 after transplant. Scores are not significantly different between mice given CXCR5KO Tregs on day 28 and cGVHD mice. (J) Histopathology scoring of lung based on H&E staining as in (I). Scores are not significantly different between mice given CXCR5KO Tregs on day 28 and cGVHD mice. (A-J) Representative data from 2 independent experiments; n = 5-8 mice/group/experiment. Bar graphs show mean ± SEM. Multiple comparisons made using one-way ANOVA with Tukey’s posttest. Significance: *P < .05; **P < .01; ***P < .001; ****P < .0001.

Treg infusions treat cGVHD in a CXCR5-dependent manner. B10.BR mice were transplanted with either T-cell–depleted (TCD), B6 BM (BM Only), or TCD B6 BM with purified B6 Tcon (cGVHD) as in Figure 1A-F. Some groups of mice given BM and Tcon were also given either WT or CXCR5KO Tregs on day 0 (prophylactic) or day 28 (therapeutic). (A-C) PFTs assessing (A) airway resistance, (B) total lung elastance, and (C) total lung compliance were performed on day 56 after transplant. PFTs demonstrate reversal of PFTs characteristic of cGVHD-associated BOS in all mice given WT Tregs, and CXCR5KO Tregs on day 0, but not mice given CXCR5KO Tregs on day 28. (D-F) Frequency of (D) Tfr and (E) Tfh out of total splenic CD4+ T cells, along with frequency of (F) GC B cells (CD19+GL7hiFAShi) out of total splenic B cells were measured by flow cytometry on day 56 after transplant. (D) Tfr frequency was found to be higher, but (E) Tfh and (F) GC B-cell frequency lower in mice given WT Tregs and CXCR5KO Tregs on day 0, but not mice given CXCR5KO Tregs on day 28. (G) Representative images (original magnification ×200) of frozen spleen tissues stained with Cy3-peanut agglutinin (red) to delineate GCs. (H) Quantification of GC area from frozen spleen tissues stained with Cy3-peanut agglutinin as in (G) from 5 spleens/group. WT CXCR5KO Tregs given on day 0 reduce GC area to levels near that of BM-only mice. CXCR5KO Tregs given on day 28 reduce GC area slightly, but not significantly, compared with cGVHD mice, and do not decrease GC area to the same degree as WT Tregs. (I) Histopathology scoring of the liver based on H&E-stained cryopreserved sections of organs harvested on day 56 after transplant. Scores are not significantly different between mice given CXCR5KO Tregs on day 28 and cGVHD mice. (J) Histopathology scoring of lung based on H&E staining as in (I). Scores are not significantly different between mice given CXCR5KO Tregs on day 28 and cGVHD mice. (A-J) Representative data from 2 independent experiments; n = 5-8 mice/group/experiment. Bar graphs show mean ± SEM. Multiple comparisons made using one-way ANOVA with Tukey’s posttest. Significance: *P < .05; **P < .01; ***P < .001; ****P < .0001.

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