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. 2023 Jun 14;618(7967):1033–1040. doi: 10.1038/s41586-023-06199-x

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a,d, Experimental protocols to assess the distribution of adoptively transferred T cells (left) and graphics depicting the invasive tumour margin (right) of CRISPR-ctrl (a) or Jak1-KO (d) tumours. b,e, Arrest coefficient and mean speed of adoptively transferred Venus⁺ Pmel-1 CD8⁺ (red) and eGFP⁺ TRP-1 CD4⁺ T cells (green) in the stromal (S) and tumoural (T) compartment at the invasive margin (bars indicate the median) of CRISPR-ctrl (b) or Jak1-KO (e) tumours (75–842 cells examined from three independent experiments; ****P < 0.0001, ***P = 0.0007, **P = 0.0068, CD4⁺ versus CD8⁺ in stroma *P = 0.0204, CD4⁺ in stroma versus CD8⁺ in tumour *P = 0.0107 using a Kruskal–Wallis test with Dunn’s multiple comparison test). c,f, Representative intravital microscopic images (scale bars, 100 µm) and insets exemplifying 450 s motion tracks of Venus⁺ Pmel-1 CD8⁺ and eGFP⁺ TRP-1 CD4⁺ T cells at the stromal (S) and tumoural (T) area of the invasive tumour margin of CRISPR-ctrl (c) or Jak1-KO (f) melanomas. g, Experimental protocol to assess antigen-dependent interactions between eGFP⁺ TRP-1 CD4⁺ T cells and CD11c⁺ immune cells. h, Arrest coefficient, mean speed and relative contact duration between eGFP⁺ TRP-1 CD4⁺ T cells and CD11c-Venus cells (the bars indicate the median; 43–132 cells examined from three independent experiments; ****P < 0.0001, **P = 0.0022 with a Mann–Whitney U-test). i, Representative motion tracks of eGFP⁺ TRP-1 CD4⁺ T cells interacting with CD11c-Venus cells in CRISPR-ctrl and Trp1-KO melanomas. Scale bars, 20 µm. j, Experimental protocol to assess the impact of MHC-II blockade on antigen-dependent interactions between eGFP⁺ TRP-1 CD4⁺ T cells and CD11c⁺ immune cells. k, Arrest coefficient, mean speed and relative contact duration between eGFP⁺ TRP-1 CD4⁺ T cells and CD11c-Venus cells (the bars indicate the median; 203–273 cells examined from three independent experiments; ****P < 0.0001, ***P = 0.0003, with a Mann–Whitney U-test). l, Representative motion tracks of eGFP⁺ TRP-1 CD4⁺ T cells interacting with CD11c-Venus cells in CRISPR-ctrl Tyr-KO tumours with and without MHC-II blockade. Scale bars, 20 µm. Data were pooled from at least three independent mice and groups statistically compared using a one-way ANOVA with Tukey post hoc.

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