Journal: Blood Advances
Article Title: Modulating endothelial cells with EGFL7 to diminish aGVHD after allogeneic bone marrow transplantation in mice
doi: 10.1182/bloodadvances.2021005498
Figure Lengend Snippet: Effect of EGFL7 on immune reconstitution and graft-versus-leukemia effect. (A-C) Splenocytes and thymocytes were collected from PBS-treated mice as they reached their endpoints (day +28 to 35) and from EGFL7-treated mice (day +30 to 35 post-BMT). (A) Absolute counts of TCR + , CD4 + , and CD8 + lymphocytes in the spleen of Syn, GVHD + rEGFL7, and GVHD + PBS treated (B6→B6D2F1) mice. Data were pooled from 3 experiments with 4 to 7 mice per group. Gating was performed on CD45.1 + and CD45.2 + cells. (B) Absolute counts of BM-derived thymocytes (CD45.2 + ) in Syn, GVHD + rEGFL7, and GVHD + PBS treated (B6→B6D2F1) mice. Data were pooled from 3 experiments with 4 to 8 mice per group. Results show mean ± SEM. Statistical analysis compared GVHD + rEGFL7 vs GVHD + PBS treated (B6→B6D2F1) mice, and P values were determined by a Mann-Whitney U test. (C) Dot-plot analysis of thymocytes (derived from the BM cells [CD45.2 + CD45.1 − ]), based on the expression of CD4, CD8, and CD45.2 antigens, in the thymus of SYN control, GVHD + rEGFL7, and GVHD + PBS mice. These data are representative of 3 or more experiments with 6 to 8 mice per group. Histogram showed mean ± SEM. Statistical analysis compared GVHD + rEGFL7 vs GVHD + PBS treated (B6→B6D2F1) mice, and P values were determined by a Mann-Whitney U test. Transplant for GVL was performed in BALB/c mice as described in “Methods.” (D-E) Whole-body bioluminescent signal intensity of recipient mice (n = 4-5 per cohort). Mice were imaged on indicated days. Average radiance expressed as mean ± SEM. One representative transplant experiment of 2 is shown. (F) Splenocytes were isolated at the time when mice reached their endpoints, days 28 to 33 post-BMT for PBS vehicle and days 30 to 35 post-BMT in rEGFL7-treated mice. Percentage GFP positivity representing P815 leukemic cell infiltration in the spleen. Each dot represents a single mouse. (G) Representative flow cytometric contour plots. Allo. spl., allogeneic splenocytes; GFP, green fluorescent protein; Min, minimum; Max, maximum; SSC-A, side-scatter-area; SYN, syngeneic control; TCD-BM, T cell depleted bone marrow. *** P < .001.
Article Snippet: Recombinant human EGFL7 (rEGFL7) was purchased from Peprotech (Rocky Hill, NJ).
Techniques: Derivative Assay, MANN-WHITNEY, Expressing, Control, Isolation
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![Effect of <t>EGFL7</t> on GVHD severity in B6→B6D2F1 mice. (A) Weights of animals that had undergone transplantation were measured daily and averaged for the group (green circles: syngeneic T cells [no GVHD]; red triangles: allogeneic treated with PBS; blue squares: allogeneic treated with <t>rEGFL7).</t> Daily EGFL7 treatment was initiated at day +21 post-BMT. Data were pooled from 3 experiments with 6 to 13 mice per group. (B) Survival curve of transplanted mice (dotted green line: syngeneic control [SYN], blue line: allogeneic treated with rEGFL7, and thin dotted red line: allogeneic treated with PBS). Data were pooled from 3 experiments with 6 to 13 mice per group. (C) Clinical scores of GVHD + PBS, GVHD + rEGFL7, and syngeneic mice at day +28 post-BMT. (D) Histopathology of the gut 28 days after allo-HSCT. Left: magnification ×200 and right: magnification ×400. (E) Gastrointestinal (GI) histopathology score of the gut of GVHD + rEGFL7 and GVHD + PBS-treated mice. Results show mean ± standard error of the mean (SEM). (F) Immunofluorescence analysis of the intestine from PBS or rEGFL7-treated mice transplanted with allogenic splenocytes that were stained for immunofluorescence. Cells were stained with CD31 (secondary antibody: donkey anti-goat alexa fluor 488), CD45 (secondary antibody: donkey anti-rabbit alexa fluor 647), and Ki-67 antibodies (secondary antibody donkey anti-rat alexa fluor 594). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Magnification ×400. (G) Cells were stained with CD3 (secondary antibody donkey anti-goat alexa fluor 488), CD4 (secondary antibody donkey anti-rabbit alexa fluor 647), and CD8 antibodies (secondary antibody donkey anti-rat alexa fluor 594). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Magnification ×400. Data show 1 representative sample from each experimental group, which consists of 3 to 4 mice per group.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6300/pmc09006300/pmc09006300__advancesADV2021005498f1.jpg)
![Effect of EGFL7 on immune reconstitution and graft-versus-leukemia effect. (A-C) Splenocytes and thymocytes were collected from PBS-treated mice as they reached their endpoints (day +28 to 35) and from EGFL7-treated mice (day +30 to 35 post-BMT). (A) Absolute counts of TCR + , CD4 + , and CD8 + lymphocytes in the spleen of Syn, GVHD + rEGFL7, and GVHD + PBS treated (B6→B6D2F1) mice. Data were pooled from 3 experiments with 4 to 7 mice per group. Gating was performed on CD45.1 + and CD45.2 + cells. (B) Absolute counts of BM-derived thymocytes (CD45.2 + ) in Syn, GVHD + rEGFL7, and GVHD + PBS treated (B6→B6D2F1) mice. Data were pooled from 3 experiments with 4 to 8 mice per group. Results show mean ± SEM. Statistical analysis compared GVHD + rEGFL7 vs GVHD + PBS treated (B6→B6D2F1) mice, and P values were determined by a Mann-Whitney U test. (C) Dot-plot analysis of thymocytes (derived from the BM cells [CD45.2 + CD45.1 − ]), based on the expression of CD4, CD8, and CD45.2 antigens, in the thymus of SYN control, GVHD + rEGFL7, and GVHD + PBS mice. These data are representative of 3 or more experiments with 6 to 8 mice per group. Histogram showed mean ± SEM. Statistical analysis compared GVHD + rEGFL7 vs GVHD + PBS treated (B6→B6D2F1) mice, and P values were determined by a Mann-Whitney U test. Transplant for GVL was performed in BALB/c mice as described in “Methods.” (D-E) Whole-body bioluminescent signal intensity of recipient mice (n = 4-5 per cohort). Mice were imaged on indicated days. Average radiance expressed as mean ± SEM. One representative transplant experiment of 2 is shown. (F) Splenocytes were isolated at the time when mice reached their endpoints, days 28 to 33 post-BMT for PBS vehicle and days 30 to 35 post-BMT in rEGFL7-treated mice. Percentage GFP positivity representing P815 leukemic cell infiltration in the spleen. Each dot represents a single mouse. (G) Representative flow cytometric contour plots. Allo. spl., allogeneic splenocytes; GFP, green fluorescent protein; Min, minimum; Max, maximum; SSC-A, side-scatter-area; SYN, syngeneic control; TCD-BM, T cell depleted bone marrow. *** P < .001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6300/pmc09006300/pmc09006300__advancesADV2021005498f2.jpg)
