Most studies use IFN-gamma as the readout of T-cells responding to wildtype and mutant epitopes provided from cancer cells, yet Th2 responses, with the signature cytokines IL-4, IL-5 and IL-13 may also be present, either as an ‘original’ Th2 response or as a result of partial agonist peptides, imposed by the mutational event (see below) that may turn Th1 T-cells into Th2 cytokine-producing T-cells [29]. Th2-type T-cell responses may not per se signify an unproductive and potentially ‘tolerizing’ immune response; more recent reports indicate that Th2-type immune responses may also be able to mediate clinically relevant anti-cancer immune reactivities [30]. In a preclinical model, antigen-specific Th2 cells eradicated myelomas without the help of CD8 T-cells, leading to massive inflammation at the tumor site [30]. Th2-mediated tumour destruction has been shown to be associated with IL-1, TNF-alpha (Th1) and Th2 cytokine (IL-4, IL-5, IL-13) production in situ, while passively transferred Th2 cells were able to confer long-lasting cellular anti-cancer directed immune responses. CD8-independent and antigen-specific T-cells in Th2-mediated immune responses were shown to be eotaxin- and STAT6-dependent [31–36]. In general, Th2 infiltrates in human cancers have not been studied extensively and some studies even suggested a better outcome with Th2-type cytokines [36]. The nature of Th2 responses in recognising mutant epitopes is not well explored at this time. The more detailed association of CD4 Th2 responses may also benefit from closer association of T-cells with the restricting MHC class II elements. For instance, previous studies reported Th1/Th2 CD4+ T-cell responses against NY-ESO-1 in DPB1*0401/0402-positive patients with ovarian cancer [37]. Much more information is available concerning the nature of the cellular immune response directed against peptides presented by the rather less variant (as compared to HLA-DR) HLA-DP molecules from infectious pathogens, e.g. Hepatitis B or MHC class II molecules that pre-dispose humans to certain autoimmune diseases (e.g. gluten-associated colitis) [37–45]. The impact of variant epitopes in association with certain MHC alleles that are associated with certain cytokine production patterns (IL-17, Th1, Th2) is unexplored up to now. Table 4.1 provides an overview of wildtype and mutant target epitopes recognised in TIL from patients with glioma, demonstrating that Th2 responses exist in the TCR repertoire from individual patients directed against mutant epitopes.

Increasing immune effector functions by recruiting T-cells that recognise mutant epitope sequences is a clinically attractive attempt to improve and broaden the TCR repertoire directed against mutations that exclusively exist in malignancies and would therefore represent the ‘ideal’ cancer-associated antigen – a ‘biological scalpel’ that would only target cancer cells and not harm non-transformed cells. One method is to modify peptides at residues that do not interact with the nominal MHC restricting molecule, yet with the TCR contact residues: these variants are called ‘heteroclitic’ analogues and are able to trigger the nominal TCR with differential T-cell effector functions, e.g. cytotoxicity, quality and quantity of cytokine production, as well as proliferation [79]. This approach has been used to induce T-cells that react to wildtype peptides, e.g. to p53, yet are elicited with a variant peptide. If single mutations occur in epitopes, it could yield peptides that are naturally processed and presented by tumour cells to TCRs. What could potentially happen if a T-cell response, directed against a wildtype target, will also be able to able to react to the corresponding mutant target epitope, presented by the identical, nominal HLA-restricting element?. The following scenarios may occur, which have been described already in the early 1990s from several groups: In general, a single ligand specificity for each individual TCR appears to be rare. In contrast, the TCR recognition has been shown to be flexible, induced by altered peptide ligands, grouped into antagonists, partial agonists and superagonists. Mutations within peptide targets can induce differential phosphorylation of the TCR/CD3 complex with a differential downstream signalling pathway configuration [80]. Mutations may therefore—in case the wildtype peptide is also recognized—lead to abrogation of T-cell recognition simply because the ligand is not processed and presented. Alternatively, the T-cell ligand may well be processed and presented, but the T-cell signal may be abrogated potentially due to cellular anergy [29]. Partial agonists, i.e. by inducing a single amino acid residue, will still be able to stimulate the T-cells directed against the wildtype peptide, even across a similar dose range of the nominal epitopes: as the T-cells with the wildtype TCR ligand react with proliferation, cytokine production and cytotoxicity, peptide variants may either induce cytotoxicity and/or cytokine production, in the absence of T-cell proliferation [81, 82]. Of note, a similar observation may be true for TCRs directed against the mutant epitope that would react with a qualitatively and quantitatively differential T-cell reactivity pattern. Single amino acid exchanges may also turn T-cell clones from a Th1 into a Th2 cytokine production pattern, or lead to T-cell clones with abrogated cytokine production, yet strong cytotoxic T-cell responses as shown for viral pathogens [81–83]. Mutations in nominal targets, associated with differential signalling events, may also be crucial for the differentiation status of T-cells reacting to wildtype as well as to mutant targets, first described in preclinical models of thymocyte differentiation and maturation [84, 85]. Differential triggering of the nominal TCR is associated with T-cell maturation and differentiation—a quality that is important for long-term immune memory, access to (tumor) tissue as well as for clinical efficacy of T-cell therapy. The passive transfer of immune cells directed to TAAs has been shown to be clinically relevant as the transfer of the T-cell product leads to the generation of central memory T-cells [86]. The biochemical signals that govern T-cell memory rely not only on the cytokine environment, yet also on the signal strength delivered by the TCRζ chain complex; the quality and quantity of T-cell responses including T-cell memory is strikingly shaped by the strength of the MHC/peptide–TCR interaction [87], which may in part be relevant for CD8+ T-cells. A decreasing potential model has been proposed, gauging the signal strength delivered by the target epitope to the corresponding TCR that is dictating whether the T-cell most likely enters the T-cell memory pool [88, 89]. This ‘signal strength’ model will need to take into account the locally produced cytokines and pro-inflammatory signals associated with moving T-cells into the diversity of the memory T-cell pool. In general, weaker TCR signals are sufficient in order to move T-cells into a memory T-cell program [90, 91]. Not mutually exclusive, the length of the TCR signalling (i.e. shortening the TCR stimulation) will also decide whether T-cells enter the memory T-cell pool [92, 93]. The observation that point mutations within peptides affect the contact with the nominal TCR also impairs CD8+ T-cell memory development, mediated in part by TCR-dependent NFκB signalling [94]. This may partly explain why T-cell clones targeting the identical (mutant) tumor epitope exist in heterogeneous differentiation states (see Fig. 4.2).