Background The adoptive cell transfer (ACT) of tumor-infiltrating T lymphocytes (TILs) has shown remarkable results in patients with different cancer types. The antitumor effect of this therapy is mainly attributed to a small fraction of tumor-reactive T lymphocytes (TRLs) that recognize mutated peptides as well as overexpressed self-antigens. Therefore, the enrichment and expansion of TRLs constitutes a promising immunotherapy approach. However, the specific targeting of individual mutated antigens represents a daunting challenge for widespread therapeutic application. Alternatively, we hypothesize that TRLs could be identified and enriched by a surface marker (or combination thereof) in an antigen-independent manner as a result of the chronic antigen exposure and other factors present in the tumor microenvironment (TME).

Materials and Methods We screened T cell activation and exhaustion markers, among others, on different tumor tissues using the MACSima™ Imaging Platform, an instrument for the highly multiplexed immunofluorescence imaging technology MICS (Multiparameter Imaging Cell Screen), enabling investigation of hundreds of markers on a single section. Moreover, flow cytometry and single-cell RNA sequencing analyses of T cells from tumor digests were performed to complement the characterization of TILs.

Results The MICS results highlighted the complexity of the TME, mainly composed of tumor cells, fibroblasts and endothelial vessels. In some cases, an extensive immune infiltrate consisted of T cells, plasma cells, some B cells and distinct myeloid cells was observed. Particularly, CD8 T cells from different tumor areas exhibited a tissue-resident memory phenotype with the expression of CD69, CD45RO or CD103. Activated/exhausted CD8 T cells were homogenously found across the imaged tumor areas. However, there was a tendency to find them in close proximity to tumor cells, especially for CD8 subsets expressing CD39 and other relevant markers, which may suggest the identification of tumor-reactive CD8 T cell populations. Flow cytometry data revealed the presence of similar T cell phenotypes in the patient´s TILs from tumor digests.

Conclusions This imaging technology offers the possibility to study multiple parameters—including the localization—of relevant cells in the TME such as T cells. The phenotypic and functional characterization of different T cell subsets will allow the further investigation of their anti-tumor reactivity. Ultimately, the enrichment and expansion of the identified tumor-reactive T cell population hold great promises to improve the efficiency of T cell therapy against cancer.

Disclosure Information E. Criado-Moronati: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. A. Gosselink: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. J. Kollet: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. A. Dzionek: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. B. Heemskerk: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG.