The T cell differentiation landscape is shaped by tumour mutations in lung cancer.

Tumour mutational burden (TMB) predicts immunotherapy outcome in non-small cell lung cancer (NSCLC), consistent with immune recognition of tumour neoantigens. However, persistent antigen exposure is detrimental for T cell function. How TMB affects CD4 and CD8 T cell differentiation in untreated tumours, and whether this affects patient outcomes is unknown. Here we paired high-dimensional flow cytometry, exome, single-cell and bulk RNA sequencing from patients with resected, untreated NSCLC to examine these relationships. TMB was associated with compartment-wide T cell differentiation skewing, characterized by loss of TCF7-expressing progenitor-like CD4 T cells, and an increased abundance of dysfunctional CD8 and CD4 T cell subsets, with significant phenotypic and transcriptional similarity to neoantigen-reactive CD8 T cells. A gene signature of redistribution from progenitor-like to dysfunctional states associated with poor survival in lung and other cancer cohorts. Single-cell characterization of these populations informs potential strategies for therapeutic manipulation in NSCLC.

Publisher Correction: Spatial heterogeneity of the T cell receptor repertoire reflects the mutational landscape in lung cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Quantitative analysis of the T cell receptor repertoire.

The T cell receptor repertoire provides a window into the cellular adaptive immune response. In the context of cancer, determining the repertoire within a tumor can give important insights into the evolution of the T cell anti-cancer response, and has the potential to identify specific personalized biomarkers for tracking host responses during cancer therapy, including immunotherapy. We describe a protocol for amplifying, sequencing and analyzing T cell receptors which is economical, robust, sensitive and versatile. The key experimental step is the ligation of a single stranded oligonucleotide to the 3' end of the T cell receptor cDNA, which allows easy amplification of all possible rearrangements using only a single set of primers per locus, while simultaneously introducing a unique molecular identifier to label each starting cDNA molecule. After sequencing, this molecular identifier can be used to correct both sequence errors and the effects of differential PCR amplification efficiency, thus producing a more accurate measure of the true T cell receptor frequency within the sample. We describe a detailed protocol describing this method to create libraries of T cell receptors from in vitro T cell cultures, blood or tissue samples. We combine this with a computational pipeline, which incorporates sample multiplexing, T cell receptor annotation and error correction to provide accurate counts of individual T cell receptor sequences within samples. The integrated experimental and computational pipeline should be of value to researchers interested in documenting and understanding the T cell immune response to cancer, and in manipulating it for therapeutic purposes.

Spatial heterogeneity of the T cell receptor repertoire reflects the mutational landscape in lung cancer.

Somatic mutations together with immunoediting drive extensive heterogeneity within non-small-cell lung cancer (NSCLC). Herein we examine heterogeneity of the T cell antigen receptor (TCR) repertoire. The number of TCR sequences selectively expanded in tumors varies within and between tumors and correlates with the number of nonsynonymous mutations. Expanded TCRs can be subdivided into TCRs found in all tumor regions (ubiquitous) and those present in a subset of regions (regional). The number of ubiquitous and regional TCRs correlates with the number of ubiquitous and regional nonsynonymous mutations, respectively. Expanded TCRs form part of clusters of TCRs of similar sequence, suggestive of a spatially constrained antigen-driven process. CD8+ tumor-infiltrating lymphocytes harboring ubiquitous TCRs display a dysfunctional tissue-resident phenotype. Ubiquitous TCRs are preferentially detected in the blood at the time of tumor resection as compared to routine follow-up. These findings highlight a noninvasive method to identify and track relevant tumor-reactive TCRs for use in adoptive T cell immunotherapy.

Framework engineering to produce dominant T cell receptors with enhanced antigen-specific function.

TCR-gene-transfer is an efficient strategy to produce therapeutic T cells of defined antigen specificity. However, there are substantial variations in the cell surface expression levels of human TCRs, which can impair the function of engineered T cells. Here we demonstrate that substitutions of 3 amino acid residues in the framework of the TCR variable domains consistently increase the expression of human TCRs on the surface of engineered T cells.The modified TCRs mediate enhanced T cell proliferation, cytokine production and cytotoxicity, while reducing the peptide concentration required for triggering effector function up to 3000-fold. Adoptive transfer experiments in mice show that modified TCRs control tumor growth more efficiently than wild-type TCRs. Our data indicate that simple variable domain modifications at a distance from the antigen-binding loops lead to increased TCR expression and improved effector function. This finding provides a generic platform to optimize the efficacy of TCR gene therapy in humans.

Bistable switch in migration stimulating factor expression: regulation by the concerted signalling of transforming growth factor-ß1 and the extracellular matrix.

Migration stimulating factor (MSF) is an oncofetal motogenic/angiogenic cytokine constitutively expressed by epithelial and stromal cells in fetal and neoplastic tissues. Fibroblasts derived from healthy adult skin do not express MSF but can be induced to do so by treatment with transforming growth factor-ß1 (TGF-ß1). As the bioactivities of both MSF and TGF-ß1 are modulated by the extracellular matrix, we investigated whether the induction of MSF expression by TGF-ß1 is also matrix dependent. We now report that adult fibroblasts are induced to express MSF by a transient treatment with TGF-ß1 (as short as 2 hr) but only when the cells are adherent to a "wound" matrix, such as denatured type I collagen, fibrin or plastic tissue culture dishes. Unexpectedly, this induction of MSF expression persists unabated for the entire subsequent lifespan of the treated cells in the absence of further TGF-ß1 and irrespective of the substratum. Such "activated" MSF expression may, however, be persistently switched off again by a second transient exposure to TGF-ß1 but this time only when the cells are adherent to a "healthy" matrix of native type I collagen. Significantly, the constitutive expression of MSF by fetal and cancer patient fibroblasts could also be persistently switched off by this means. We conclude that TGF-ß1 may both switch on and switch off MSF expression in a manner critically determined by the nature of the matrix substratum and suggest that this may be a possible mechanism underlying the observed dual functionality of TGF-ß1 as both a tumour suppressor and tumour promoter.