Fig. 2: Clinical heterotypic CD8+ T cell clusters.
From: Tumour-reactive heterotypic CD8 T cell clusters from clinical samples
a, Diagram of tumour sample collection and processing: fresh tumour samples were obtained, cut into small pieces, briefly enzymatically digested, stained and analysed using flow cytometry to identify tumour cells, T cells and APCs. b, Representative flow cytometry plots of a melanoma tumour digest (patient 1, P1) processed as in a. The tumour digest was stained for the tumour cell markers CD146 and NGFR, the T cell marker CD8 and APC marker CD11c. Plots were obtained from live cells gated as shown in Extended Data Fig. 2a. c, The percentage of T cell–tumour cell (T–Tum) and T cell–APC (T–APC) clusters within the total CD8+ T cell population. Each coloured point represents an individual patient (n = 21). Data are mean ± s.e.m. d, The tumour digest (patient 1) from b was visualized using imaging flow cytometry. Representative single cells (top) and heterotypic clusters with different compositions (bottom) are shown. The white arrows indicate relocalization of markers to the immunological synapse in T cell–APC clusters. e, Tumour digest (patient 6, P6) visualized using imaging flow cytometry. Representative clusters are shown. The white arrows indicate relocalization of immunological synapse markers. f, Tumour digest (patient 17, P17) visualized using imaging flow cytometry. Representative heterotypic clusters containing CD4+ T cells are shown. In d–f, numbers indicate cell identifiers. g, Multiplex immunofluorescence staining of tissue sections of patient 2 (lymph node metastasis (LN met)) and patient 6 (non-lymph node metastasis). The sections were stained for the tumour cell markers SOX10 and HMB45, T cell marker CD8 and APC marker CD11c. DAPI was included as a nuclear marker. In the top rows, merged images are shown (DAPI is not included for clarity). The white boxes indicate magnified areas. In the bottom rows, channels are separated and correspond to the second images on the top row. n = 11 patients; representative patients are shown. Scale bars, 500 μm (g, left) and 100 μm (g, right).
