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. Author manuscript; available in PMC: 2022 Jun 23.
Published in final edited form as: Cell Rep. 2021 Aug 10;36(6):109523. doi: 10.1016/j.celrep.2021.109523

Figure 6. Perivascular BFP-CXCL10+ clusters nucleate antigen-specific T:APC interactions to boost function.

Figure 6.

Virtual 3D BFP-CXCL10+ clusters were defined by a DBSCAN-based algorithm, and Th1 cell motility was classified according to their position relative to the cluster perimeter.

(A–D) T cell motility inside and outside of the BFP-CXCL10+ clusters. (A) Mean-squared displacement (MSD) of WT (left) or CXCR3 KO (middle) OT-II Th1 cells in the OVA/CFA-immunized dermis, 30-min IV-MPM. Right panel, WT Th1 cell motility in the OVA/CFA or KLH/CFA immunized dermis. Pooled data from three independent experiments, three to six mice with 6–12 imaging volumes/group. (B) Instantaneous speed of individual Th1 cells in and out of the BFP-CXCL10+ cluster; scale bar, 50 μm. Right panels are enlarged regions from the cluster image at left. (C) Instantaneous speed of individual T cells relative to the BFP-CXCL10+ cluster. (D) Migratory paths (x–y projections) of OT-II Th1 cells relative to BFP-CXCL10+ clusters in the OVA/CFA dermis, 60-min IV-MPM. Representative data from three independent experiments, >12 imaging volumes; >30 tracks per plot.

(E and F) Average velocity (E) and arrest coefficient (F) of WT and CXCR3 KO OT-II Th1 cells described in (A).

(G) Th1:BFP-CXCL10+ cell-contact time, 60-min IV-MPM. Left, representative IV-MPM image of OT-II Th1 (green):BFP-CXCL10+ (blue) cell contacts (white), using the 3D surface overlap rendering tool in Imaris. Right, Th1:BFP-CXCL10+ cell contact duration, inside (In) and outside (Out) of BFP-CXCL10+ clusters.

(H) Th1:BFP-CXCL10+ cell-contact durations for WT and CXCR3 KO Th1 cells, 60-min IV-MPM.

(I) WTOT-II Th1:BFP-CXCL10+ cell-contact durations in the OVA/CFA (cognate antigen)- and KLH/CFA (non-cognate)-immunized dermis. Pooled data from three independent experiments, three to five mice with 6–10 imaging volumes/group.

(J and K) Frequency of CD69+ (J) and IFN-γ+ (K) WT Th1 cells in the OVA/CFA- or KLH/CFA-immunized REX3 dermis and CXCR3 KO Th1 cells in the OVA/CFA immunized dermis.

Violin plots represent the frequency distribution of the data: black dotted line, median; border-colored dotted lines, the first and third quartiles. Bars represent means ± SEM. Statistics by one-way ANOVA (E and F) or two-way ANOVA (G–K), *p < 0.05, **p < 0.01, ****p < 0.0001.