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Purpose <p>Emerging evidence indicates that granzyme K expressing tumor infiltrating CD8 + T-cells play a critical role in mediating anti-tumor T-cell immunity and immunotherapy treatment response. However, precise characterization and cell surface markers for the viable isolation of these cells from tumor samples are lacking.</p> Methods <p>We constructed a tumor-infiltrating CD8 + T cell subpopulation atlas (<i>n</i> = 286,827 cells) by integrating single-cell RNA sequencing datasets from two immunotherapy-treated patient cohorts with aerodigestive tract malignancies. This unified dataset facilitated detailed mapping of transcriptional trajectories and stringent identification of distinctive cell surface markers across major CD8 + tumor-infiltrating lymphocyte (TIL) compartments.</p> Results <p>Analysis of the CD3 + CD8+ TIL transcriptional landscape revealed three predominant populations across all tumor types and treatment conditions: stem-like cells, dysfunctional effector cells, and GZMK+ effector cells. Notably, the GZMK+ effector population displayed a distinctive transcriptional profile lacking positive enrichment of pre-defined gene sets, suggesting a previously poorly characterized subpopulation without established cell surface markers. We performed high-dimensional flow cytometry on primary human tumor samples to validate candidate CD8 + TIL subpopulations. Through validation in primary human tumors, we established that cell surface KLRG1 reliably identifies and enables viable isolation of the GZMK+ effector TIL population. Concurrently, we demonstrated that dysfunctional and stem-like populations can be effectively isolated as KLRG1-CD39 + and KLRG1-CD39- CD55 + subsets, respectively. Importantly, accurate enrichment occurs despite decreased starting cell type proportion.</p> Conclusions <p>Our characterization of CD8 + TIL subpopulations and validation of their distinguishing surface markers establishes a robust platform for isolation and protein-level granzyme assessment of these key cells in patient samples.</p>

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KLRG1 defines a distinct tumor-infiltrating granzyme K+ CD8 + T cell population

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月19日

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  • Jerin Thomas,
  • Sophia Kennedy,
  • Stella Darcy,
  • Henrique Malaco,
  • Nyasha Chambwe,
  • Dev Kamdar,
  • Lucio Periera,
  • Andrew Salama,
  • Danielle Scarola,
  • Brett Miles,
  • Douglas Frank,
  • Nagashree Seetharamu,
  • Rajarsi Mandal

摘要

Purpose

Emerging evidence indicates that granzyme K expressing tumor infiltrating CD8 + T-cells play a critical role in mediating anti-tumor T-cell immunity and immunotherapy treatment response. However, precise characterization and cell surface markers for the viable isolation of these cells from tumor samples are lacking.

Methods

We constructed a tumor-infiltrating CD8 + T cell subpopulation atlas (n = 286,827 cells) by integrating single-cell RNA sequencing datasets from two immunotherapy-treated patient cohorts with aerodigestive tract malignancies. This unified dataset facilitated detailed mapping of transcriptional trajectories and stringent identification of distinctive cell surface markers across major CD8 + tumor-infiltrating lymphocyte (TIL) compartments.

Results

Analysis of the CD3 + CD8+ TIL transcriptional landscape revealed three predominant populations across all tumor types and treatment conditions: stem-like cells, dysfunctional effector cells, and GZMK+ effector cells. Notably, the GZMK+ effector population displayed a distinctive transcriptional profile lacking positive enrichment of pre-defined gene sets, suggesting a previously poorly characterized subpopulation without established cell surface markers. We performed high-dimensional flow cytometry on primary human tumor samples to validate candidate CD8 + TIL subpopulations. Through validation in primary human tumors, we established that cell surface KLRG1 reliably identifies and enables viable isolation of the GZMK+ effector TIL population. Concurrently, we demonstrated that dysfunctional and stem-like populations can be effectively isolated as KLRG1-CD39 + and KLRG1-CD39- CD55 + subsets, respectively. Importantly, accurate enrichment occurs despite decreased starting cell type proportion.

Conclusions

Our characterization of CD8 + TIL subpopulations and validation of their distinguishing surface markers establishes a robust platform for isolation and protein-level granzyme assessment of these key cells in patient samples.