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. 2023 Oct;43(10):1143-1163.
doi: 10.1002/cac2.12480. Epub 2023 Sep 1.

Intratumoral CD103+ CD8+ T cells predict response to neoadjuvant chemoimmunotherapy in advanced head and neck squamous cell carcinoma

Siqi Ren  1   2 , Tianjun Lan  1   2 , Fan Wu  1   2 , Suling Chen  1   2 , Xue Jiang  2 , Chuying Huo  3 , Zitian Li  4 , Shule Xie  1   2 , Donghui Wu  5 , Ruixin Wang  1   2 , Yanyan Li  1   2 , Lin Qiu  1   2 , Guoxin Huang  1   2 , Shurui Li  2 , Xiaojuan Wang  2 , Meifeng Cen  2 , Tingting Cai  1 , Zhaoyu Lin  1   2 , Jinsong Li  1   2 , Bowen Li  1   2
Affiliations

Intratumoral CD103+ CD8+ T cells predict response to neoadjuvant chemoimmunotherapy in advanced head and neck squamous cell carcinoma

Siqi Ren et al. Cancer Commun (Lond). 2023 Oct.

Abstract

Background: Immune cell heterogenicity is known to determine the therapeutic response to cancer progression. Neoadjuvant chemoimmunotherapy (NACI) has shown clinical benefits in some patients with advanced head and neck squamous cell carcinoma (HNSCC), but the underlying mechanism behind this clinical response is unknown. The efficacy of NACI needs to be potentiated by identifying accurate biomarkers to predict clinical responses. Here, we attempted to identify molecules predicting NACI response in advanced HNSCC.

Methods: We performed combined single-cell RNA sequencing (scRNA-seq) and multiplex immunofluorescence (mIHC) staining with tumor samples derived from NACI-treated HNSCC patients to identify a new tumor-infiltrating cell (TIL) subtype, CD103+ CD8+ TILs, associated with clinical response, while both in vitro and in vivo assays were carried out to determine its antitumor efficiency. The regulatory mechanism of the CD103+ CD8+ TILs population was examined by performing cell-cell interaction analysis of the scRNA-seq data and spatial analysis of the mIHC images.

Results: We established intratumoral CD103+ CD8+ TILs density as a determinant of NACI efficacy in cancers. Our scRNA-seq results indicated that the population of CD103+ CD8+ TILs was dramatically increased in the responders of NACI-treated HNSCC patients, while mIHC analysis confirmed the correlation between intratumoral CD103+ CD8+ TILs density and NACI efficacy in HNSCC patients. Further receiver operating characteristic curve analysis defined this TIL subset as a potent marker to predict patient response to NACI. Functional assays showed that CD103+ CD8+ TILs were tumor-reactive T cells, while programmed cell death protein-1 (PD-1) blockade enhanced CD103+ CD8+ TILs cytotoxicity against tumor growth in vivo. Mechanistically, targeting the triggering receptor expressed on myeloid cells 2-positive (TREM2+ ) macrophages might enhance the population of CD103+ CD8+ TILs and facilitate antitumor immunity during NACI treatment.

Conclusions: Our study highlights the impact of intratumoral CD103+ CD8+ TILs density on NACI efficacy in different cancers, while the efforts to elevate its population warrant further clinical investigation.

Keywords: CD103; CD8; head and neck squamous cell carcinoma; neoadjuvant chemoimmunotherapy; predictive marker; tumor-infiltrating lymphocyte.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

FIGURE 1
FIGURE 1
Immune cell profile of advanced HNSCC at single‐cell atlas. (A) Schematic overview of the experimental design and analytical workflow. We performed combined scRNA‐seq and mIHC staining with tumor samples derived from NACI‐treated HNSCC patients to identify CD103+CD8+ TILs, associated with clinical response. (B) A total of 17 mega‐clusters in advanced HNSCC. The clusters are visualized and labeled by cell type via UMAP visualization. The distributions of cell clusters between the responders and non‐responders are visualized by UMAP visualization. The distributions of cell clusters from 8 tumor samples are visualized respectively by UMAP visualization. (C) All cell type clusters are visualized via UMAP visualization in the responders versus non‐responders. Boxplots showing the alterations of cell clusters between the responders and non‐responders. (D) UMAP visualization of T and NK cell subclusters. The clusters are visualized and labeled by cell type. The bar graph represents the proportion of these subclusters in responders and non‐responders. (E) The bar graph represents the proportion of CD103+CD8+ T cells in the T cell expression between the responders and non‐responders. (F) Heatmap depicting the cell‐type‐specific markers in the T and NK cell subclusters of scRNA‐seq analysis. Abbreviations: HNSCC, head and neck squamous cell carcinoma; scRNA‐seq, single‐cell RNA sequencing; NK, natural killer; GZMK, Granzyme K; LEF1, lymphoid enhancer binding factor 1; ISG, interferon stimulated exonuclease gene; TNFRSF4, TNF receptor superfamily member 4; CXCL13, C‐X‐C motif chemokine ligand 13; FCGR3A, Fc gamma receptor IIIa; TRDC, T cell receptor delta constant; MKI67, Marker of proliferation Ki‐67; TRDV2, T cell receptor delta variable 2.
FIGURE 2
FIGURE 2
The molecular characterization of CD103+CD8+ TILs. (A) GO analysis of gene set from the CD8+ TILs subtypes. (B) KEGG analysis of gene set from CD8+ TILs subtypes. (C) PID analysis of gene set from the CD8+ TILs subtypes. (D) Heatmap depicting the cell‐type‐specific markers in the CD8+ TILs subtypes. (E) FACS analysis of gene expression in CD103+CD8+ T cells and CD103−CD8+ T cells from advanced HNSCC tissues (n = 4). (F) FACS analysis of CD103+CD8+ T cell population in PBMC (B), responders (R) and non‐responders (NR) tumor tissues, respectively (n = 10). Horizontal bar indicates the median. Significance was determined with an unpaired 2‐tailed student's t‐test or one‐way ANOVA with Bonferroni post hoc test. Abbreviations: TIL, tumor‐infiltrating cell; HNSCC, head and neck squamous cell carcinoma; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PID, Pathway Interaction Database; IL‐2, Interleukin 2; TNF‐α, tumor necrosis factor; GZMB, Granzyme B; CXCL13, C‐X‐C motif chemokine ligand 13; PD‐1, programmed cell death protein‐1; CCR7, C‐C Motif Chemokine Receptor 7; PBMC, peripheral blood mononuclear cell; FACS, flow cytometry.
FIGURE 3
FIGURE 3
The increased CD103+CD8+ TIL density is associated with better therapeutic response in NACI‐treated HNSCC patients. (A) mIHC image of DAPI, Cytokeratin (CK), CD103, and CD8 staining of advanced HNSCC tissues in the responders and non‐responders. Scale bar, 100 μm. (B) Density of intratumoral CD103+CD8+ TILs and CD103−CD8+ TILs in tumors was assessed in the MPR group (n = 22) and IPR group (n = 17) patients to NACI via mIHC assay. Density of intratumoral CD103+CD8+ TILs and CD103−CD8+ TILs in tumors was assessed in the responders (R; n = 19) and non‐responders (NR; n = 20) patients to NACI. Image shows CD103+CD8+ (yellow), CD103−CD8+ (red), and CD8+ (red) cells in advance HNSCC (lower panel). Scale bar, 10 μm. (C) MRI showing the tumor size in responders and non‐responders before and after NACI, respectively. (D) mIHC image shows the density of CD103+CD8+ TILs in tumors from responders and non‐responders before and after NACI. Scale bar, 200 μm. (E) The temporal alterations of the density of CD103+CD8+ TILs and CD103−CD8+ TILs from pre‐ and post‐NACI in the responders (n = 19) and non‐responders (n = 20) via mIHC assay. (F) Distribution of CD103+CD8+ TILs between different clinical characteristics in patients with advanced HNSCC. Horizontal lines indicate the mean ± SEM. Significance was determined with a 2‐tailed Mann‐Whitney U test. Pearson's chi‐square test was used for studying the distribution of intraepithelial CD103+CD8+ TILs across different subgroups. Abbreviations: TIL, tumor‐infiltrating cell; HNSCC, head and neck squamous cell carcinoma; mIHC, multiplex immunofluorescent; MRI, Magnetic resonance imaging; MPR, major pathologic response; IPR, incomplete pathologic response; R, responders; NR, non‐responders; SEM, standard error of the mean; NACI, neoadjuvant chemoimmunotherapy; MRI, magnetic resonance imaging.
FIGURE 4
FIGURE 4
Intraepithelial CD103+CD8+ TIL density has a strong predictive value for therapeutic response in HNSCC patients treated with NACI. (A) Overview of the automated image analysis pipeline via inform analysis (upper panel). The spatial distribution of CD103+CD8+ TILs in the responders and non‐responders (lower panel). Digital markup image shows tumor (red) and stromal (green) zones of the tumor section defined by cytokeratin staining. Digital markup image shows CD103+CD8+ TILs (red). Scale bar, 100 μm. (B) Density of intraepithelial and stromal CD103+CD8+ TILs in tumors were assessed in the MPR group (n = 22) and IPR group (n = 17) patients to NACI via inform analysis. Density of intraepithelial and stromal CD103+CD8+ TILs in tumors were assessed in the responders (R; n = 19) and non‐responders (NR; n = 20) with NACI. (C) HALO® spatial analyses demonstrate the spatial distribution of CD103+CD8+ TILs in the responders and non‐responders. Digital markup image shows tumor (red) and stromal (green) zones of the tumor section defined by cytokeratin staining. Digital markup image shows the density of intratumoral CD103+CD8+ TILs (blue), intraepithelial CD103+CD8+ TILs (red), and stromal CD103+CD8+ TILs (green). Scale bar, 1 mm, 200 μm. Proximity analysis histograms of CD103+CD8+ TILs within an 800 μm radius by progressive segments of 200 μm bands in advanced HNSCC tissues. (D) Temporal alterations in the density of intraepithelial CD103+CD8+ TILs from pre‐ and post‐NACI in the responders (n = 19) and non‐responders via inform analysis (n = 20). (E) Predictive value of intratumoral CD103+CD8+ TILs, intraepithelial CD103+CD8+ TILs, and stromal CD103+CD8+ TILs via ROC analysis. (F) Distribution of intraepithelial CD103+CD8+ TILs between different clinical characteristics in patients with advanced HNSCC. Horizontal lines indicate the mean ± SEM. Significance was determined with a 2‐tailed Mann‐Whitney U test. Pearson's chi‐square test was used for studying the distribution of intraepithelial CD103+CD8+ TILs across different subgroups. Abbreviations: TIL, tumor‐infiltrating cell; HNSCC, head and neck squamous cell carcinoma; MPR, major pathologic response; IPR, incomplete pathologic response; ROC, Receiver Operating Characteristic; R, responders; NR, non‐responders; SEM, standard error of the mean; NACI, neoadjuvant chemoimmunotherapy.
FIGURE 5
FIGURE 5
Comparison and association of CPS with CD103+CD8+ TIL level. (A) The waterfall plot depicting the correlation between the density of CD103+CD8+ TILs and clinicopathological features of individual patients who NACI (n = 39). The black horizontal line represents 90% pathological response. (B) CPS was assessed in the responders (R; n = 19) and non‐responders (NR; n = 20) with NACI. (C) Correlation between CPS and radiographic response (n = 39). (D) Correlation between intraepithelial CD103+CD8+ TILs and CPS (n = 39). (E) Intraepithelial CD103+CD8+ TILs were assessed in the responders (R; n = 9) and non‐responders (NR; n = 11) with NACI from CPS < 20 groups (left panel). Intraepithelial CD103+CD8+ TILs were assessed in the responders (R; n = 10) and non‐responders (NR; n = 9) with NACI from CPS ≥ 20 groups (right panel). (F) Predictive value of intratumoral CD103+CD8+ TIL level, intraepithelial CD103+CD8+ TIL level, and CPS via ROC analysis. (G) Predictive value of CPS was compared with intraepithelial CD103+CD8+ TIL level and elevated when combined with intraepithelial CD103+CD8+ TIL level (left panel). Decision curve analysis comparison of CPS, intraepithelial CD103+CD8+ TIL level, and CPS combined with intraepithelial CD103+CD8+ TIL level. Horizontal lines indicate the mean ± SEM. Significance was determined with a 2‐tailed Mann‐Whitney U test. Spearman correlation analyses were used to study the linear association. Abbreviations: TIL, tumor‐infiltrating cell; HNSCC, head and neck squamous cell carcinoma; MPR, major pathologic response; IPR, incomplete pathologic response; ROC, receiver operating characteristic; R, responders; NR, non‐responders; CPS, combined positive score; SEM, standard error of the mean; NACI, neoadjuvant chemoimmunotherapy.
FIGURE 6
FIGURE 6
Correlation between CD103+CD8+ TILs and TREM2+ macrophages. (A) Correlative network showing interactions of cell subsets identified in advanced HNSCC tissues based on scRNA‐seq. The arrow width indicates the degree of correlation between two clusters. (B) Bar plot shows the relative proportion of interaction strength for each signaling pathway between responders and non‐responders. The pathways labeled blue are enriched in responders, and labeled red are enriched in non‐responders. R: responders; NR: non‐responders. (C) mIHC staining of TREM2+ macrophages marker CD68 (pink) and TREM2 (yellow), and CD103+CD8+ TILs marker CD103 (green) and CD8 (red), and DAPI staining (blue) in the advanced HNSCC with NACI. Scale bars, 100 μm. Quantification of CD103+CD8+ TILs at a distance of 250 μm or 500 μm from TREM2+ macrophages. (D) Proximity line series between CD103+CD8+ TILs and TREM2+ macrophages. (E) Proximity analysis histograms of CD103+CD8+ TILs to TREM2+ macrophages within a 400 μm radius by progressive segments of 20 μm bands. Abbreviations: TIL, tumor‐infiltrating cell; HNSCC, head and neck squamous cell carcinoma; mIHC, multiplex immunofluorescent; TREM2, triggering receptor expressed on myeloid cells 2; R, responders; NR, non‐responders; NACI, neoadjuvant chemoimmunotherapy; scRNA‐seq, single‐cell RNA sequencing.
FIGURE 7
FIGURE 7
Schematic diagram represents the role of CD8+CD103+ TILs in regulating NACI efficacy. For advanced HNSCC patients treated with NACI, the responders exhibit a “hot tumor” (immune‐inflamed) phenotype, in which the density of intratumoral CD103+CD8+ TILs were increased, and these cells were mostly found in the cancer nest. In contrast, the non‐responders may have a “cold tumor” (immune‐desert or immune‐excluded) phenotype, in which the cell number of intratumoral CD103+CD8+ TILs were lower, and these cells were frequently located in the stroma. Abbreviations: TIL, tumor‐infiltrating cell; HNSCC, head and neck squamous cell carcinoma; NACI, neoadjuvant chemoimmunotherapy; NACI, neoadjuvant chemoimmunotherapy.
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References

    1. Vos JL, Elbers JBW, Krijgsman O, Traets JJH, Qiao X, van der Leun AM, et al. Neoadjuvant immunotherapy with nivolumab and ipilimumab induces major pathological responses in patients with head and neck squamous cell carcinoma. Nat Commun. 2021;12(1):7348. - PMC - PubMed
    1. Qiu H, Cao S, Xu R. Cancer incidence, mortality, and burden in China: a time‐trend analysis and comparison with the United States and United Kingdom based on the global epidemiological data released in 2020. Cancer Commun (Lond). 2021;41(10):1037–48. - PMC - PubMed
    1. Zhang J, Huang D, Saw PE, Song E. Turning cold tumors hot: from molecular mechanisms to clinical applications. Trends Immunol. 2022;43(7):523–45. - PubMed
    1. Wang Y, Wang M, Wu HX, Xu RH. Advancing to the era of cancer immunotherapy. Cancer Commun (Lond). 2021;41(9):803–29. - PMC - PubMed
    1. Uppaluri R, Campbell KM, Egloff AM, Zolkind P, Skidmore ZL, Nussenbaum B, et al. Neoadjuvant and Adjuvant Pembrolizumab in Resectable Locally Advanced, Human Papillomavirus–Unrelated Head and Neck Cancer: A Multicenter, Phase II Trial. Clin Cancer Res. 2020;26(19):5140–52. - PMC - PubMed

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