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In vivo dynamics of T cells and their interactions with dendritic cells in mouse cutaneous graft-versus-host disease

Sarah Morin-Zorman, Christian Wysocki, Jieqing Zhu, Hongmei Li, Sylvain Zorman, Catherine Matte-Martone, Edwina Kisanga, Jennifer McNiff, Dhanpat Jain, David Gonzalez, David M. Rothstein, Fadi G. Lakkis, Ann Haberman and Warren D. Shlomchik

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    CD4 cell dynamics. Irradiated 129 mice were reconstituted with B6 CD11c-YFP BM, B6 RFP+ CD4 cells, and unlabeled B6 CD8 cells. Ears were imaged ∼28 days after transplantation. (A) Image from a representative video (supplemental Video 1). Mean speeds and displacements for tracked cells are shown in panel B. Each symbol is a unique tracked CD4 cell. (C) Representative tracking from a single video with the starting point of each CD4 cell at the origin. Color denotes time. (D) The mean speed, distance to the YFP surface, and displacement were calculated for each cell in 6 videos (color coded). (E) Average CD4 cell behaviors from 6 videos, represented in a 100-circle plot. “Stable” indicates an instantaneous velocity of less than 5 μm/minute. “In contact” indicates a distance 2 μm or less from the DC surface. “Black space” indicates a distance more than 2 μm from DC surface.

  • Figure 2.

    CD8 cell dynamics. Irradiated 129 mice were reconstituted with B6 CD11c-YFP BM, B6 RFP+ CD8 cells, and unlabeled B6 CD4 cells. Ears were imaged ∼28 days after transplantation. (A) Image from a representative video (supplemental Video 2). Mean speeds and displacements for tracked cells are shown in panel B. Each symbol is a unique tracked CD8 cell. (C) Representative tracking from a single video with the starting point of each CD8 cell at the origin. Color denotes time. (D) Mean speed, distance to the YFP surface, and displacement were calculated for each cell in 3 videos (color coded). (E) Average CD8 cell behaviors from 3 videos, represented in a 100-circle plot.

  • Figure 3.

    CD8 cells make TCR:MHCI-dependent interactions with CD11c+ donor hematopoietic cells. (A) Experimental design. Irradiated 129 mice were reconstituted with B6 RFP+ CD8 cells, unlabeled B6 CD4 cells, and 1:1 mix of CD11c-YFP/β2M−/− BM and wt B6 BM or CD11c-YFP BM and B6 β2M−/− BM. (B) Representative images from videos with wt (left column) or β2M−/− (right column) CD11c-YFP BM. The upper row depicts original videos; the lower row has been rendered to show the YFP+ surface (blue) with red spheres fit into CD8 cells (supplemental Videos 3-6). (C) Cumulative interaction times between CD8 cells and the YFP+ surface of CD8 cells that ever contact the YFP surface. P = .0001 comparing contacts with wt and β2M−/− YFP+ cells in panel B. (D) Percentage of CD8 cells that ever have contacted the YFP+ surface that stayed in continuous contact for at least 30 minutes. P = .0064.

  • Figure 4.

    muLangerin-CD11c+ cells make stable interactions with CD8 cells. Irradiated 129 mice were reconstituted with B6 F1(muLangerin-DTRxCD11c-YFP) BM, RFP+ CD8 cells and unlabeled CD4 cells. Approximately 28 days after bone marrow transplantation, mice were treated with PBS or with DT. Mice were imaged 48 hours later. (A) Representative flow cytometry of digested skin, which demonstrates the depletion of CD103+MHCII+CD11c+ cells after DT treatment. (B) Still images from representative videos in PBS (left panel; supplemental Video 7) or DT (right panel; supplemental Video 8). The percentage of CD8 cells in continuous contact with YFP+ cells for at least 30’ or 40’ are shown in panel C. P = .26 comparing PBS and DT-treated groups. (D) Cumulative contact durations between CD8 cells and CD11c+ cells (of CD8 cells that ever contact the DC surface). P = .54.

  • Figure 5.

    CD4 stability is dependent on TCR:MHCII contacts. Irradiated 129 mice were reconstituted with B6 RFP+ CD4 cells, unlabeled B6 CD8 cells, and 1:1 mix of CD11c-YFP/MHCII−/− BM and wt B6 BM or CD11c-YFP BM and B6 MHCII−/− BM. Shown in panel A are cumulative interaction times between CD4 cells and the YFP+ surface (of CD4 cells that ever contact the YFP surface). P = .67 comparing interactions between CD4 cells and wt or MHCII−/− DCs. (B-C) In vivo treatment with an anti-MHCII mAb (Y3P) increased CD4 cell speeds. (B) Experimental design. (C) Representative images from videos of an ear pre- and posttreatment with Y3P or isotype (supplemental Videos 9-12). (D) Mean CD4 speeds pre- and posttreatment with Y3P (upper panel) or isotype (lower panel). P ≤ .005 comparing speeds before and after Y3P for each mouse. P ≥ .09 comparing speeds before and after isotype antibody treatment of each mouse.

  • Figure 6.

    Acute depletion of DCs does not increase CD8 T-cell motility. Irradiated 129 mice were reconstituted with BM from B6 F1(CD11c-YFPxCD11c-DTR) mice, wt B6 CD4 cells and B6 RFP+ CD8 cells. Approximately 28 days after bone marrow transplantation, mice were treated with PBS or with DT 48 and 24 hours before imaging. (A) Still images from representative videos before DT (left panel; supplemental Video 13) or after DT (right panel; supplemental Video 14). CD8+ T-cell mean speeds (B), displacements (C), and sphericity (D) before and after DT treatment. *P < .0001.

  • Figure 7.

    OT-1 effector motility in GVHD skin lesions. One hundred twenty-nine mice were irradiated and transplanted with B6 CD11c-YFP BM and CFP+ B6 CD4 and CD8 cells. On day 28, mice were injected with DsRed+ OT-1 effectors and imaged by 2PIM the following day. Each column shows images from a single mouse. In images in the second row, the YFP channel is masked to better visualize the dsRed+ OT-1 (supplemental Videos 15-20).