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. 2020 May 19;52(5):794–807.e7. doi: 10.1016/j.immuni.2020.03.010

Figure 5.

Figure 5

LFA-1 and VLA-4 Promote T Cell Migration during Entry

(A) Flow-cytometric detection of GFP+ T cells labeled 5 min in vivo with anti-CD3-PE 2.5 h after being transferred into hosts injected with PBS (control) or blocking antibodies against integrin subunits α4, αL, or α4+αL, as indicated. Mice were treated with the above antibodies 2 h prior to T cell transfer. Red numbers show frequency of unlabeled cells. Right, summary showing one out of three representative experiments. Error bars represent SD.

(B) Immunohistochemistry on spleen sections from mice treated as in (A), stained with anti-GFP (transferred T cells, brown) and anti-CD169 (to highlight the MZ macrophages, blue). Data are representative of four independent experiments.

(C–F) TPLSM of hCD2-DsRed mice transferred with GFP+ T cells following combined (α4 and αL) integrin blockade.

(C) Schematic of experimental design.

(D) 63-μm z-projection view showing endogenous (red) and transferred (green) T cells. Grey line highlights PT-tracks connected to a T zone. Left, trajectories of transferred T cells with cyan lines indicating cell moving toward the T zone and purple lines showing cells moving away from it.

(E) Summary of frequencies of cells migrating toward or away from the T zones. Each circle represents the average frequency measured in one mouse (n ≥ 4).

(F) Mean velocities and straightness of migration path of transferred GFP+ T cells. Data were pooled from 10 videos imaged in four mice.

(G) Top, schematic of experimental design. Bottom left, 72-μm z-projection view of TPLSM intravital imaging of mice imaged 2 h after GFP+ T cells transfer. Bottom right, mean velocities and straightness of migration path of the transferred cells. Data in (G) are representative of one of three independent experiments performed. Error bars represent SD. See also Video S7.

(H) Similar analysis as in (A), performed 24 h after T cell transfer. Shows one of three representative experiments. Error bars represent SD. ∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01.