The efficacy of checkpoint blockade immunotherapies in colorectal cancer is currently restricted to a minority of patients diagnosed with mismatch repair-deficient tumors having high mutation burden. However, this observation does not exclude the existence of neoantigen-specific T cells in colorectal cancers with low mutation burden and the exploitation of their anti-cancer potential for immunotherapy. Therefore, we investigated whether autologous neoantigen-specific T cell responses could also be observed in patients diagnosed with mismatch repair-proficient colorectal cancers.
Whole-exome and transcriptome sequencing were performed on cancer and normal tissues from seven colorectal cancer patients diagnosed with mismatch repair-proficient tumors to detect putative neoantigens. Corresponding neo-epitopes were synthesized and tested for recognition by in vitro expanded T cells that were isolated from tumor tissues (tumor-infiltrating lymphocytes) and from peripheral mononuclear blood cells stimulated with tumor material.
Neoantigen-specific T cell reactivity was detected to several neo-epitopes in the tumor-infiltrating lymphocytes of three patients while their respective cancers expressed 15, 21, and 30 non-synonymous variants. Cell sorting of tumor-infiltrating lymphocytes based on the co-expression of CD39 and CD103 pinpointed the presence of neoantigen-specific T cells in the CD39+CD103+ T cell subset. Strikingly, the tumors containing neoantigen-reactive TIL were classified as consensus molecular subtype 4 (CMS4), which is associated with TGF-β pathway activation and worse clinical outcome.
We have detected neoantigen-targeted reactivity by autologous T cells in mismatch repair-proficient colorectal cancers of the CMS4 subtype. These findings warrant the development of specific immunotherapeutic strategies that selectively boost the activity of neoantigen-specific T cells and target the TGF-β pathway to reinforce T cell reactivity in this patient group.

Animal models provide an essential tool to study the efficacy of CAR-T cell treatments. Most of the current works test human CAR-T cells in immunodeficient animals, typically NOD Scid Gamma (NSG) mice transplanted with human tumors. Despite the limitations of this model, including the difficulty to study the interaction between CAR-T cells and the human innate system and to assess the toxicity of this therapy, NSG are extensively used for adoptive T cell transfer studies. In this chapter, we will describe the protocols to test CAR-T cells in NSG animals with solid tumors. We first describe the implantation of human xenograft tumors in NSG animals, followed by CAR-T cell administration and assessment of antitumor responses. We will also review the protocols to analyze T cell persistence in the blood of treated animals. Finally, we will focus on the analysis of the tumors at the end point of the experiment, including the percentage, phenotype, and function of tumor infiltrating T cells, and loss of antigen expression.

CARs are synthetic receptors designed to drive antigen-specific activation upon binding of the scFv to its cognate antigen. However, CARs can also elicit different levels of ligand-independent constitutive signaling, also known as tonic signaling. Chronic T cell activation is observed in certain combinations of scFv, hinge, and costimulatory domains and may be increased due to high levels of CAR expression. Tonic signaling can be identified during primary T cell expansion due to differences in the phenotype and growth of CAR-T cells compared to control T cells. CARs displaying tonic signaling are associated with accelerated T cell differentiation and exhaustion and impaired antitumor effects. Selecting CARs which configuration does not induce tonic signaling is important to enhance antigen-specific T cell responses. In this chapter, we describe in detail different protocols to identify tonic signaling driven by CARs during primary T cell ex vivo expansion.

T cell function is dictated by a delicate balance of stimuli that shapes T-cell phenotype, the latter characterizable using a number of immunogenic assays. Thanks to advancements in next-generation sequencing technology, and the intersection between genetics, engineering, computer science, biostatistics, and immunology, it is now possible to profile immune cells residing in any organ or disease site at single cell resolution. Herein we review the most common approaches available to describe T cell activation, T cell molecular heterogeneity, and T cell function. We present informatic tools useful to both seasoned bioinformaticians and novices alike, providing a comprehensive overview of the in silico methods used to study T cell biology. With the goal of making this manuscript useful to a broad range of readers, we focus only on freely available tools and algorithms, and we describe the concepts using simple and direct language. We hope this work will serve to assist and inspire researchers interested in T cell biology.