Figure 4.
Pathogenic Tph cells and Trh cells traffic between the blood and cGVHD target tissues. Lung FFPE slides from cGVHD mice were processed to visualize lymphocyte infiltration via imaging mass cytometry (N = 4). (A) Selected lung lymphocyte infiltration area via hematoxylin-eosin stains. (B) Representative images of 12 markers are shown. (C) Merged channels image shows signals of CD4, B220, CD8, CD11c, E-cadherin, collagen, and histone. (D) Merged channels image shows signals of CD4 and B220 within lung inducible bronchus-associated lymphoid tissue (iBALT). (E) Images show signals of CD4, PSGL1, CD69, and merged channels in lung iBALT. (F) Percentage of liver (N = 23) and lung (N = 25) PSGL1loPD1hi Trh cells among total CD4+ T cells in cGVHD mice and compared with Trh cells (N = 7) from no-GVHD mice by flow cytometry on day 60 after HCT. (G) Correlations between percentage of liver Trh cells and Tph cells, and between percentage of lung Trh cells and Tph cells were analyzed. (H) Diagram of transfer experiments in which donor CD45.1+ Trh cells in the liver and lung of primary cGVHD mice were isolated on day 30 after HCT and transferred into CD45.2+ adoptive recipients of no-GVHD mice on day 14 after HCT. Fourteen days after cell transfer, CD45.1+ T cells were isolated from the adoptive recipients and analyzed (N = 8). (I) Percentages of Tph cells from blood and Trh cells from liver and lung were measured among injected CD45.1+ T cells, mean ± SEM. (J) Representative panel of CD69 and ICOS expression of CD45.1+ Tph cells in the blood and CD45.1+ Trh cells in the liver and lung compared with tissue-matched naïve CD4+ T cells. (K) Diagram of transfer experiments in which donor CD45.2+ blood Tph cells of primary cGVHD mice were enriched at day 30 after HCT and adoptively transferred into CD45.1+ cGVHD recipients at 14 days after HCT. CD45.2+ T cells were monitored 14 days later (N = 3). (L) CD45.2+ T cells in the blood, liver, and lung were identified and tested for PSGL1 versus PD1 expression as shown for 1 representative recipient (left) and calculated (right), mean ± standard deviation. (M) Representative panel of CD69 and ICOS expression of lung CD45.2+Trh cells compared with naïve T cells. P values were calculated by unpaired Student t test (F) and nonparametric Spearman correlation (G). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Pathogenic Tph cells and Trh cells traffic between the blood and cGVHD target tissues. Lung FFPE slides from cGVHD mice were processed to visualize lymphocyte infiltration via imaging mass cytometry (N = 4). (A) Selected lung lymphocyte infiltration area via hematoxylin-eosin stains. (B) Representative images of 12 markers are shown. (C) Merged channels image shows signals of CD4, B220, CD8, CD11c, E-cadherin, collagen, and histone. (D) Merged channels image shows signals of CD4 and B220 within lung inducible bronchus-associated lymphoid tissue (iBALT). (E) Images show signals of CD4, PSGL1, CD69, and merged channels in lung iBALT. (F) Percentage of liver (N = 23) and lung (N = 25) PSGL1loPD1hi Trh cells among total CD4+ T cells in cGVHD mice and compared with Trh cells (N = 7) from no-GVHD mice by flow cytometry on day 60 after HCT. (G) Correlations between percentage of liver Trh cells and Tph cells, and between percentage of lung Trh cells and Tph cells were analyzed. (H) Diagram of transfer experiments in which donor CD45.1+ Trh cells in the liver and lung of primary cGVHD mice were isolated on day 30 after HCT and transferred into CD45.2+ adoptive recipients of no-GVHD mice on day 14 after HCT. Fourteen days after cell transfer, CD45.1+ T cells were isolated from the adoptive recipients and analyzed (N = 8). (I) Percentages of Tph cells from blood and Trh cells from liver and lung were measured among injected CD45.1+ T cells, mean ± SEM. (J) Representative panel of CD69 and ICOS expression of CD45.1+ Tph cells in the blood and CD45.1+ Trh cells in the liver and lung compared with tissue-matched naïve CD4+ T cells. (K) Diagram of transfer experiments in which donor CD45.2+ blood Tph cells of primary cGVHD mice were enriched at day 30 after HCT and adoptively transferred into CD45.1+ cGVHD recipients at 14 days after HCT. CD45.2+ T cells were monitored 14 days later (N = 3). (L) CD45.2+ T cells in the blood, liver, and lung were identified and tested for PSGL1 versus PD1 expression as shown for 1 representative recipient (left) and calculated (right), mean ± standard deviation. (M) Representative panel of CD69 and ICOS expression of lung CD45.2+Trh cells compared with naïve T cells. P values were calculated by unpaired Student t test (F) and nonparametric Spearman correlation (G). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

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