Tumour-infiltrating lymphocytes: from prognosis to treatment selection.

Tumour-infiltrating lymphocytes (TILs) are considered crucial in anti-tumour immunity. Accordingly, the presence of TILs contains prognostic and predictive value. In 2011, we performed a systematic review and meta-analysis on the prognostic value of TILs across cancer types. Since then, the advent of immune checkpoint blockade (ICB) has renewed interest in the analysis of TILs. In this review, we first describe how our understanding of the prognostic value of TIL has changed over the last decade. New insights on novel TIL subsets are discussed and give a broader view on the prognostic effect of TILs in cancer. Apart from prognostic value, evidence on the predictive significance of TILs in the immune therapy era are discussed, as well as new techniques, such as machine learning that strive to incorporate these predictive capacities within clinical trials.

Rapid and efficient generation of antigen-specific isogenic T cells from cryopreserved blood samples.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene editing has been leveraged for the modification of human and mouse T cells. However, limited experience is available on the application of CRISPR/Cas9 electroporation in cryopreserved T cells collected during clinical trials. To address this, we aimed to optimize a CRISPR/Cas9-mediated gene editing protocol compatible with peripheral blood mononuclear cells (PBMCs) samples routinely produced during clinical trials. PBMCs from healthy donors were used to generate knockout T-cell models for interferon-γ, Cbl proto-oncogene B (CBLB), Fas cell surface death receptor (Fas) and T-cell receptor (TCRαß) genes. The effect of CRISPR/Cas9-mediated gene editing on T cells was evaluated using apoptosis assays, cytokine bead arrays and ex vivo and in vitro stimulation assays. Our results demonstrate that CRISPR/Cas9-mediated gene editing of ex vivo T cells is efficient and does not overtly affect T-cell viability. Cytokine release and T-cell proliferation were not affected in gene-edited T cells. Interestingly, memory T cells were more susceptible to CRISPR/Cas9 gene editing than naïve T cells. Ex vivo and in vitro stimulation with antigens resulted in equivalent antigen-specific T-cell responses in gene-edited and untouched control cells, making CRISPR/Cas9-mediated gene editing compatible with clinical antigen-specific T-cell activation and expansion assays. Here, we report an optimized protocol for rapid, viable and highly efficient genetic modification in ex vivo human antigen-specific T cells, for subsequent functional evaluation and/or expansion. Our platform extends CRISPR/Cas9-mediated gene editing for use in gold-standard clinically used immune-monitoring pipelines and serves as a starting point for development of analogous approaches, such as those including transcriptional activators and/or epigenetic modifiers.