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. 2022 Sep 20;41:279. doi: 10.1186/s13046-022-02481-4

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© The Author(s) 2022

Open AccessThis 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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — Characterization of immune cells infiltrating HPV-positive and HPV-negative primary tumors. A Representative 9-color multispectral images of HPV-positive and HPV-negative primary tumors. Immune markers and color code are indicated in the underlying legend. Original magnification X20. B Immune cell populations infiltrating the stromal and the intra-tumoral regions of HPV-positive and HPV-negative primary tumors. C Representative images of cell-to-cell distance analysis in HPV-positive and HPV-negative primary tumors. Cancer cells (light blue dots) within a 20 μm radius from CD8+ cells (red dots) are represented. D Nearest neighbors analysis measuring the mean distance between each tumor cell and the nearest CD8+, CD8 + CD103+ and CD8 + PD-1+ T lymphocytes (left), or each CD163+ M2-polarized macrophage (middle) or each FoxP3+ Treg cell (right) and the nearest CD8+ T lymphocytes in the stromal and intra-tumoral areas. E Count within analysis calculating the percentage of tumor cells within a radius of 20 μm from CD8+, CD8 + CD103+ and CD8 + PD-1+ T cells (left), and the percentage of CD163+ M2-polarized macrophages (middle) or FoxP3+ Treg cells (right) within a radius of 20 μm from CD8+ T lymphocytes in the stromal and intra-tumoral areas. Significantly different data are represented by *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001