A Practical Approach to T-Cell Receptor Cloning and Expression

Although cloning and expression of T-cell Receptors (TcRs) has been performed for almost two decades, these procedures are still challenging. For example, the use of T-cell clones that have undergone limited expansion as starting material to limit the loss of interesting TcRs, must be weighed against the introduction of mutations by excess PCR cycles. The recent interest in using specific TcRs for cancer immunotherapy has, however, increased the demand for practical and robust methods to rapidly clone and express TcRs. Two main technologies for TcR cloning have emerged; the use of a set of primers specifically annealing to all known TcR variable domains, and 5′-RACE amplification. We here present an improved 5′-RACE protocol that represents a fast and reliable way to identify a TcR from 105 cells only, making TcR cloning feasible without a priori knowledge of the variable domain sequence. We further present a detailed procedure for the subcloning of TcRα and β chains into an expression system. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TcR transgene into different expression systems. The presented comprehensive method can be performed in any laboratory with standard equipment and with a limited amount of starting material. We finally exemplify the straightforwardness and reliability of our procedure by cloning and expressing several MART-1-specific TcRs and demonstrating their functionality

Tryptophan depletion results in tryptophan-to-phenylalanine substitutants

Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme1–4. Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides ‘substitutants’ to distinguish them from genetically encoded mutants. Using large-scale proteomics analyses, we demonstrate W>F substitutants to be highly abundant in multiple cancer types. W>F substitutants were enriched in tumours relative to matching adjacent normal tissues, and were associated with increased IDO1 expression, oncogenic signalling and the tumour-immune microenvironment. Functionally, W>F substitutants can impair protein activity, but also expand the landscape of antigens presented at the cell surface to activate T cell responses. Thus, substitutants are generated by an alternative decoding mechanism with potential effects on gene function and tumour immunoreactivity

The Potential of Donor T-Cell Repertoires in Neoantigen-Targeted Cancer Immunotherapy

T cells can recognize peptides encoded by mutated genes, but analysis of tumor-infiltrating lymphocytes suggests that very few neoantigens spontaneously elicit T-cell responses. This may be an important reason why immune checkpoint inhibitors are mainly effective in tumors with a high mutational burden. Reasons for clinically insufficient responses to neoantigens might be inefficient priming, inhibition, or deletion of the cognate T cells. Responses can be dramatically improved by cancer immunotherapy such as checkpoint inhibition, but often with temporary effects. By contrast, T cells from human leukocyte antigen (HLA)-matched donors can cure diseases such as chronic myeloid leukemia. The therapeutic effect is mediated by donor T cells recognizing polymorphic peptides for which the donor and patient are disparate, presented on self-HLA. Donor T-cell repertoires are unbiased by the immunosuppressive environment of the tumor. A recent study demonstrated that T cells from healthy individuals are able to respond to neoantigens that are ignored by tumor-infiltrating T cells of melanoma patients. In this review, we discuss possible reasons why neoantigens escape host T cells and how these limitations may be overcome by utilization of donor-derived T-cell repertoires to facilitate rational design of neoantigen-targeted immunotherapy

Advances in immune therapies in hematological malignancies

Immunotherapy in cancer takes advantage of the exquisite specificity, potency, and flexibility of the immune system to eliminate alien tumor cells. It involves strategies to activate the entire immune defense, by unlocking mechanisms developed by tumor cells to escape from surrounding immune cells, as well as engineered antibody and cellular therapies. What is important to note is that these are therapeutics with curative potential. The earliest example of immune therapy is allogeneic stem cell transplantation, introduced in 1957, which is still an important modality in hematology, most notably in myeloid malignancies. In this review, we discuss developmental trends of immunotherapy in hematological malignancies, focusing on some of the strategies that we believe will have the most impact on future clinical practice in this field. In particular, we delineate novel developments for therapies that have already been introduced into the clinic, such as immune checkpoint inhibition and chimeric antigen receptor T-cell therapies. Finally, we discuss the therapeutic potential of emerging strategies based on T-cell receptors and adoptive transfer of allogeneic natural killer cells

Soluble T-Cell Receptors Produced in Human Cells for Targeted Delivery

<div><p>Recently, technology has become available to generate soluble T-cell receptors (sTCRs) that contain the antigen recognition part. In contrast to antibodies, sTCRs recognize intracellular in addition to extracellular epitopes, potentially increasing the number of applications as reagents for target detection and immunotherapy. Moreover, recent data show that they can be used for identification of their natural peptide ligands in disease. Here we describe a new and simplified expression method for sTCRs in human cells and show that these sTCRs can be used for antigen-specific labeling and elimination of human target cells. Four different TCRs were solubilized by expression of constructs encoding the TCR alpha (α) and beta (β) chains lacking the transmembrane and intracellular domains, linked by a ribosomal skipping 2A sequence that facilitates equimolar production of the chains. Cell supernatants containing sTCRs labeled target cells directly in a peptide (p)-human leukocyte antigen (HLA)-specific manner. We demonstrated that a MART-1p/HLA-A*02:01-specific sTCR fused to a fluorescent protein, or multimerized onto magnetic nanoparticles, could be internalized. Moreover, we showed that this sTCR and two sTCRs recognizing CD20p/HLA-A*02:01 could mediate selective elimination of target cells expressing the relevant pHLA complex when tetramerized to streptavidin-conjugated toxin, demonstrating the potential for specific delivery of cargo. This simple and efficient method can be utilized to generate a wide range of minimally modified sTCRs from the naturally occurring TCR repertoire for antigen-specific detection and targeting.</p></div

DMF5 sTCR is internalized upon specific ligand binding.

<p>(a) HeLa cells transfected with either SCT-M1-mCherry or SCT-CD20-mCherry (red) were incubated with His-tagged DMF5 sTCR labeled with an anti-His antibody and visualized using anti-mouse AF488 (green). Co-localization of the sTCR and SCT-M1 is shown in yellow (arrow). (b) Sup-T1 cells expressing SCT-M1 were incubated with DMF5 supernatants containing monomeric sTCR mCherry-His at 37°C for 30 minutes. The cells were subsequently put on ice to block endocytosis and stained with anti-His-AF647 (His). The nucleus was visualized by Hoechst stain (blue). (c) Sup-T1 cells expressing SCT-M1 were incubated with biotinylated DMF5 sTCR-mCherry bound to SA-Miltenyi nanobeads at 37°C.</p