T cells play a key role in tumour surveillance, both identifying and eliminating transformed cells.
1. Tumour-Specific T Cell Responses
As a part of the body’s immunosurveillance mechanism, all nucleated cells display major histocompatibility (MHC) class I molecules, enabling the routine presentation of cellular peptide fragments on their plasma membrane. Internal proteins are broken up by proteasomes to form peptides that are then transported by transporter associated with antigen processing (TAP) to MHC I molecules in the lumen of the endoplasmic reticulum. The peptide–MHC I complexes generated via these antigen processing and presentation pathways can be recognized by T cell receptors (TCRs) on Cytotoxic T lymphocytes (CTLs). Tumour-specific peptides are now known to exist, and to be capable of triggering spontaneous T cell responses in patients [1][2][3][4][5]. Upon detection of tumour-specific, ‘foreign’ antigens, CTLs can become activated leading to the destruction of tumour cells presenting target antigens. However, tumour cells may evade this pathway of immune-mediated destruction through multiple mechanisms.
As a part of the body’s immunosurveillance mechanism, all nucleated cells display major histocompatibility (MHC) class I molecules, enabling the routine presentation of cellular peptide fragments on their plasma membrane. Internal proteins are broken up by proteasomes to form peptides that are then transported by transporter associated with antigen processing (TAP) to MHC I molecules in the lumen of the endoplasmic reticulum. The peptide–MHC I complexes generated via these antigen processing and presentation pathways can be recognized by T cell receptors (TCRs) on Cytotoxic T lymphocytes (CTLs). Tumour-specific peptides are now known to exist, and to be capable of triggering spontaneous T cell responses in patients [1,2,3,4,5]. Upon detection of tumour-specific, ‘foreign’ antigens, CTLs can become activated leading to the destruction of tumour cells presenting target antigens. However, tumour cells may evade this pathway of immune-mediated destruction through multiple mechanisms.
The microenvironment within solid tumours is often hostile to tumour-specific T cells, negatively affecting their function and survival. Metabolic alterations to tumour and stromal cells result in the overexpression of glucose transporters and key glycolytic enzymes, altered growth factor signalling, hypoxic conditions, increases in acidity, and abnormal angiogenesis. Furthermore, an abundance of suppressive immune cells, including regulatory T cells (Tregs), myeloid-derived suppressor cells, and tumour-associated macrophages, infiltrate into the tumour and deliver a host of negative signals to tumour-specific T cells and antigen-presenting cells including dendritic cells. As these features of the tumour microenvironment have been reviewed extensively elsewhere (see Refs [6][7][8][9][10][11]), we will focus here on an examination of alterations to antigen processing and presentation pathways [12][13][14], factors influencing T cell infiltration, and strategies designed to improve tumour-targeting by T cells in the sections below.
The microenvironment within solid tumours is often hostile to tumour-specific T cells, negatively affecting their function and survival. Metabolic alterations to tumour and stromal cells result in the overexpression of glucose transporters and key glycolytic enzymes, altered growth factor signalling, hypoxic conditions, increases in acidity, and abnormal angiogenesis. Furthermore, an abundance of suppressive immune cells, including regulatory T cells (Tregs), myeloid-derived suppressor cells, and tumour-associated macrophages, infiltrate into the tumour and deliver a host of negative signals to tumour-specific T cells and antigen-presenting cells including dendritic cells. As these features of the tumour microenvironment have been reviewed extensively elsewhere (see Refs [6,7,8,9,10,11]), we will focus here on an examination of alterations to antigen processing and presentation pathways [12,13,14], factors influencing T cell infiltration, and strategies designed to improve tumour-targeting by T cells in the sections below.
1.1. Antigen Processing and Presentation in Tumours
Interference with the expression level of single or multiple components of antigen processing and presentation pathways are common mechanisms used by malignant cells to reduce visibility to CTL. Examples include alterations in the expression of proteasome subunits involved in the processing of endogenous antigens, downregulation, loss, or imbalance in the expression of enzymes such as endoplasmic reticulum aminopeptidase (ERAP)1 and ERAP involved in the loading of peptides into MHC I molecules, the loss of IFN-γ-signaling components resulting in the prevention of MHC I upregulation, and defects in MHC I expression itself, all of which result in changes to the repertoire of antigenic peptides presented to CTL [15][16]. The down-regulation or total loss of cell surface MHC I in particular has been reported in many studies on a wide array of tumours of different origin, revealing tight associations with the level of tumour-infiltrating lymphocytes, disease progression, and overall survival [15][17][18]. Based upon the functional ability of tumours to recover or up-regulate MHC I expression following cytokine treatment, Garrido et al. reported that the alteration of MHC I can be classified into two categories: (1) Irreversible genetic defects, and (2) Reversible epigenetic modifications (
Interference with the expression level of single or multiple components of antigen processing and presentation pathways are common mechanisms used by malignant cells to reduce visibility to CTL. Examples include alterations in the expression of proteasome subunits involved in the processing of endogenous antigens, downregulation, loss, or imbalance in the expression of enzymes such as endoplasmic reticulum aminopeptidase (ERAP)1 and ERAP involved in the loading of peptides into MHC I molecules, the loss of IFN-γ-signaling components resulting in the prevention of MHC I upregulation, and defects in MHC I expression itself, all of which result in changes to the repertoire of antigenic peptides presented to CTL [15,16]. The down-regulation or total loss of cell surface MHC I in particular has been reported in many studies on a wide array of tumours of different origin, revealing tight associations with the level of tumour-infiltrating lymphocytes, disease progression, and overall survival [15,17,18]. Based upon the functional ability of tumours to recover or up-regulate MHC I expression following cytokine treatment, Garrido et al. reported that the alteration of MHC I can be classified into two categories: (1) Irreversible genetic defects, and (2) Reversible epigenetic modifications ( ) [19]. For irreversible genetic defects (“hard” lesions), the most common aetiologies are loss of heterozygosity in chromosomes 6 and 15 harbouring MHC class I genes or the β2-microglobulin (β2m) gene, respectively [19][20][21][22]. In contrast, reversible epigenetic modifications (“soft” lesions) mainly concern deficiencies in the regulation of genes including β2m, MHC I heavy chain and components of the antigen-processing machinery (APM) and can be compensated for through the administration of therapeutic drugs, such as IFN-γ [19][23]. This framework is supported by evidence from both clinical and experimental animal studies [19][24][25]. Hence, it is important to carefully define the molecular mechanisms responsible for a particular alteration in MHC I phenotype prior to designing specific ways to restore in situ tumour MHC I expression.
) [19]. For irreversible genetic defects (“hard” lesions), the most common aetiologies are loss of heterozygosity in chromosomes 6 and 15 harbouring MHC class I genes or the β2-microglobulin (β2m) gene, respectively [19,20,21,22]. In contrast, reversible epigenetic modifications (“soft” lesions) mainly concern deficiencies in the regulation of genes including β2m, MHC I heavy chain and components of the antigen-processing machinery (APM) and can be compensated for through the administration of therapeutic drugs, such as IFN-γ [19,23]. This framework is supported by evidence from both clinical and experimental animal studies [19,24,25]. Hence, it is important to carefully define the molecular mechanisms responsible for a particular alteration in MHC I phenotype prior to designing specific ways to restore in situ tumour MHC I expression.
Figure 1.
MHC I aberrations in cancer. Tumours can be categorised into “Hard” or “Soft” lesions based on the nature of MHC I defects [19]. Mutations, deletions, or loss of heterozygosity result in irreversible structural defects in the MHC I pathway and can be categorised as “Hard” lesions. Downregulation or inhibition of gene expression result in reversible regulatory defects in the MHC I pathway and can be categorised as “Soft” lesions. Therapeutic approaches differ according to lesion type.
1.2. Inducing MHC I Expression in Cancer
While defects in peptide-MHC I expression in tumours have been examined extensively, strategies to counteract these defects remain scarce and challenging. The majority of MHC I defects in human cancers such as melanoma, gastric, colorectal, and cervical carcinoma, belong to the category of “soft” lesions [26][27]. The repression of gene transcription, such as the lack of MHC I and APM gene expression in tumour cells, can be recovered by histone deacetylation inhibitors and DNA methyltransferase inhibitors, and/or by activation of the interferon (IFN) signalling pathway and stabilization of the NFkB pathway [16][28][29][30][31][32]. Interestingly, combinatory treatment with IFN-γ, or knockout of known inhibitors of NFkB signalling can further exaggerate the effect of histone deacetylation and DNA methyltransferase inhibitors [30][33]. Additionally, combination treatment of these epigenetic modulators is known to enhance MHC I expression in breast cancer patients [33]. These findings indicate the potential of appropriate combination therapies to increase anti-tumour immunity when treating “soft” lesions.
While defects in peptide-MHC I expression in tumours have been examined extensively, strategies to counteract these defects remain scarce and challenging. The majority of MHC I defects in human cancers such as melanoma, gastric, colorectal, and cervical carcinoma, belong to the category of “soft” lesions [26,27]. The repression of gene transcription, such as the lack of MHC I and APM gene expression in tumour cells, can be recovered by histone deacetylation inhibitors and DNA methyltransferase inhibitors, and/or by activation of the interferon (IFN) signalling pathway and stabilization of the NFkB pathway [16,28,29,30,31,32]. Interestingly, combinatory treatment with IFN-γ, or knockout of known inhibitors of NFkB signalling can further exaggerate the effect of histone deacetylation and DNA methyltransferase inhibitors [30,33]. Additionally, combination treatment of these epigenetic modulators is known to enhance MHC I expression in breast cancer patients [33]. These findings indicate the potential of appropriate combination therapies to increase anti-tumour immunity when treating “soft” lesions.
Concurrently, “hard” MHC class I genetic aberrations exist in 30–40% of human cancers [20][34] correlating with a poor response to cytokine treatments, and acquired resistance to immune checkpoint (anti-PD-1) inhibitors [35]. Alternative approaches have therefore been investigated, such as replacement of defective genes to solve structural alterations in MHC I and APM. The application of viral vectors encoding peptide transporter TAP or β2m genes has been shown to be capable of up-regulating surface expression of MHC I in APM-deficient tumour cells and restoring recognition by antigen-specific CTL [36][37][38][39]. In mouse models of prostate and lung cancer, this gene therapy approach augmented T cell activation and increased T cell numbers within the tumour microenvironment thereby delaying tumour growth and prolonging survival [36][39]. However, this approach is constrained by the local delivery of the gene of interest, which presents particular challenges for tumours situated in internal organs.
Concurrently, “hard” MHC class I genetic aberrations exist in 30–40% of human cancers [20,34] correlating with a poor response to cytokine treatments, and acquired resistance to immune checkpoint (anti-PD-1) inhibitors [35]. Alternative approaches have therefore been investigated, such as replacement of defective genes to solve structural alterations in MHC I and APM. The application of viral vectors encoding peptide transporter TAP or β2m genes has been shown to be capable of up-regulating surface expression of MHC I in APM-deficient tumour cells and restoring recognition by antigen-specific CTL [36,37,38,39]. In mouse models of prostate and lung cancer, this gene therapy approach augmented T cell activation and increased T cell numbers within the tumour microenvironment thereby delaying tumour growth and prolonging survival [36,39]. However, this approach is constrained by the local delivery of the gene of interest, which presents particular challenges for tumours situated in internal organs.
1.3. Circumventing MHC I Down-Regulation
1.3.1. TEIPP Antigens
Subsets of CD8
+ T cells identified by van Hall et al., may be able to recognise ‘hard’ lesions with impaired TAP, tapasin, or proteasome function [40][41][42]. These T cells appear to recognise an alternative peptide repertoire that emerges in residual MHC I as a result of stimulating alternative processing pathways. These alternative peptides are also known as “T cell epitopes associated with impaired peptide processing” (TEIPP). TEIPP antigens have been shown to be exploitable in mouse models of TAP-deficient RMA-S or MCA tumours through vaccination with molecularly identified TEIPP peptides, cellular vaccination with TAP-deficient dendritic cells presenting TEIPP antigens, and adoptive transfer of in vitro expanded TEIPP-specific CTLs [40][43][44]. Vaccine-mediated priming of TEIPP-specific CTLs resulted in efficient homing to the tumour site and subsequently protection against outgrowth of APM-deficient tumours in mice [45]. The potential for clinical translation of these TEIPP antigens has been demonstrated by several studies including the identification of 16 novel human TEIPP peptides by Marijt et al. [46], and the success by Durgeau et al. utilizing a vaccination strategy of pooled peptides (TEIPP included) in a humanized mouse model to control outgrowth of TAP-deficient lung carcinoma cells [47]. These studies shed new light on the clinical exploration of TEIPP antigens and TEIPP-specific CTLs, which could be employed to mitigate the effects of APM-loss in cancer, and show potential for combination with currently available tumour antigens to improve cancer vaccine efficacy.
T cells identified by van Hall et al., may be able to recognise ‘hard’ lesions with impaired TAP, tapasin, or proteasome function [40,41,42]. These T cells appear to recognise an alternative peptide repertoire that emerges in residual MHC I as a result of stimulating alternative processing pathways. These alternative peptides are also known as “T cell epitopes associated with impaired peptide processing” (TEIPP). TEIPP antigens have been shown to be exploitable in mouse models of TAP-deficient RMA-S or MCA tumours through vaccination with molecularly identified TEIPP peptides, cellular vaccination with TAP-deficient dendritic cells presenting TEIPP antigens, and adoptive transfer of in vitro expanded TEIPP-specific CTLs [40,43,44]. Vaccine-mediated priming of TEIPP-specific CTLs resulted in efficient homing to the tumour site and subsequently protection against outgrowth of APM-deficient tumours in mice [45]. The potential for clinical translation of these TEIPP antigens has been demonstrated by several studies including the identification of 16 novel human TEIPP peptides by Marijt et al. [46], and the success by Durgeau et al. utilizing a vaccination strategy of pooled peptides (TEIPP included) in a humanized mouse model to control outgrowth of TAP-deficient lung carcinoma cells [47]. These studies shed new light on the clinical exploration of TEIPP antigens and TEIPP-specific CTLs, which could be employed to mitigate the effects of APM-loss in cancer, and show potential for combination with currently available tumour antigens to improve cancer vaccine efficacy.
1.3.2. CAR T Cells
Another way to overcome the problem of MHC I down-regulation on tumour cells is through the use of chimeric antigen receptor T cells (CAR T cells) [48]. These T cells do not rely on peptide-MHC I complex recognition to kill target cells, but instead have been engineered to express antibody receptors that recognize tumour-associated surface proteins. The adoptive transfer of CAR T cells is a promising anti-tumour therapeutic, which has led to high complete response rates in patients with haematological malignancies [49][50][51]. There have been hundreds of clinical trials involving CAR T cells worldwide since the first two US Food and Drug Administration (FDA)-approved CAR T therapies targeting CD19 on B cell malignancies [8][11]. CD19 continues to be the most popular antigen target [10], though patient relapses have been shown in multiple trials due to the emergence of CD19-negative leukaemia cells, a phenomenon known as antigen escape [52][53]. This may explain the low long-term survival rates versus the high initial remission rates in all patients after CD19 CAR T treatments. Further challenges remain for CAR T cell therapy including treatment-associated toxicities [52][54]. Targeting solid tumours with CAR T cells poses additional obstacles including the lack of tumour-specific antigens (ideally antigens must be highly expressed on most tumour cells but absent on normal tissues), the inefficient infiltration of CAR T cells into solid tumour masses, and the immunosuppressive effects of the tumour microenvironment.
Another way to overcome the problem of MHC I down-regulation on tumour cells is through the use of chimeric antigen receptor T cells (CAR T cells) [48]. These T cells do not rely on peptide-MHC I complex recognition to kill target cells, but instead have been engineered to express antibody receptors that recognize tumour-associated surface proteins. The adoptive transfer of CAR T cells is a promising anti-tumour therapeutic, which has led to high complete response rates in patients with haematological malignancies [49,50,51]. There have been hundreds of clinical trials involving CAR T cells worldwide since the first two US Food and Drug Administration (FDA)-approved CAR T therapies targeting CD19 on B cell malignancies [8,11]. CD19 continues to be the most popular antigen target [10], though patient relapses have been shown in multiple trials due to the emergence of CD19-negative leukaemia cells, a phenomenon known as antigen escape [52,53]. This may explain the low long-term survival rates versus the high initial remission rates in all patients after CD19 CAR T treatments. Further challenges remain for CAR T cell therapy including treatment-associated toxicities [52,54]. Targeting solid tumours with CAR T cells poses additional obstacles including the lack of tumour-specific antigens (ideally antigens must be highly expressed on most tumour cells but absent on normal tissues), the inefficient infiltration of CAR T cells into solid tumour masses, and the immunosuppressive effects of the tumour microenvironment.
Bispecific CAR T cells that recognize CD19 and an additional antigen, have the potential to offset antigen-loss-associated tumour escape and improve long-term survival rates and are the subject of much interest in the field. In the last 5 years in particular, preclinical animal models have emerged to test the viability of CARs with dual targets, such as CD19/HER2 [55], CD19/CD20 [56], and CD19/CD22 [57], and have shown promise. Grada et al. were amongst the first to construct a single-chain CAR with two distinct antigen recognition domains presented in tandem: CD19 and the human epidermal growth factor receptor 2 (HER2/neu) [55]. This tandem CAR (TanCAR) responded to either CD19 or HER2 and was able to trigger T cell activation, which was confirmed in a xenograft mouse model utilising Daoy.TET.CD19 tumour cells. The Daoy.TET.CD19 cells expressed HER2 constitutively and CD19 only when cultured with doxycycline. The adoptive transfer of TanCAR T cells alone, in the absence of doxycycline, resulted in significantly delayed tumour progression, compared to the group without transferred T cells. The tumour growth was further delayed by induction of CD19 with doxycycline. Hence, simultaneous recognition of two antigens enhanced in vivo anti-tumour activity by these bispecific CAR T cells. However, as HER2 and CD19 are not typically expressed on the same cell, tumour cells can still escape TanCAR detection by eliminating CD19 expression. To generate a bispecific CAR T cell that can control both CD19 +
B-cell lymphoma and CD19
−
loss variants with equal efficiency, Zah et al. determined the structural parameters for dual-antigen recognition and created CD19-OR-CD20 CAR, an “OR-gate CAR”, to trigger robust T cell function by either antigen expression [56]. The in vivo functionality of CD19-OR-CD20 CAR has been determined in a NOD SCID Gamma mouse model utilising Raji tumour cells [56]. While CD19 CAR T cells could control the tumour growth of wild-type Raji cells but not mixed populations of wild-type Raji cells and CD19 −
loss variant cells, OR-gate CAR T cells could efficiently target both. Their work presented the potential of CD19-OR-CD20 CAR as a powerful safeguard against antigen escape in CAR T treatments for wild-type and CD19
−
loss variant B-cell lymphomas.
Another bispecific CAR, the CD19/CD22 bivalent CAR, was generated by Qin et al. and tested in a “spike-in” leukaemia relapse model utilising NALM6 cells and CD19
−
/CD22
−
loss variants edited by CRISPR-Cas9 technology [57]. The inoculum in this B-ALL xenograft model was a mixture of 1% CD19 −
or CD22
−
B-ALL with 99% wild-type cells (CD19
+
/CD22
+
), which mimics relapse from a small pre-existent clone as observed in clinical trials. Adoptive transfer of T cells expressing CD19/CD22 bivalent CAR eradicated CD19
+
/CD22
−
B-ALL xenografts and significantly inhibited the outgrowth of CD19
−
/CD22
+
B-ALL populations. The potency of this bivalent CAR to reduce the risk of antigen escape was further investigated in the patient-derived xenografts: HMB15 (CD19
+
/CD22
+
) and HMB28 (CD19
−
/CD22
+
). Encouragingly, HMB28 was resistant to CD19 CAR but cleared by the bivalent CAR, while HMB15 was eradicated by either CAR. These clinically relevant models of CD19 CAR resistance have helped to confirm proof-of-concept in vivo activity of bispecific CARs and their potential applicability as adoptive T cell transfer therapies for cancer treatments.
It will be interesting to further utilize the design principles of bispecific CARs highlighted in the studies above to construct novel CARs targeting additional antigen pairs. Examinations of promising alternative targets are underway, such as CD30 in refractory Hodgkin’s lymphoma, BCMA in multiple myeloma, and CD33, CD123, and FLT3 in AML [52]. It remains possible that minor modifications to individual CAR components could further increase the functionality of TanCAR, OR-gate CAR, or bivalent CAR. Several studies have shown the importance of single-chain variable fragment framework regions, costimulatory signals and extracellular spacer length in enabling robust T cell-mediated responses, which provides critical clues for the optimization of bispecific CAR targeting strategies [58][59][60].
It will be interesting to further utilize the design principles of bispecific CARs highlighted in the studies above to construct novel CARs targeting additional antigen pairs. Examinations of promising alternative targets are underway, such as CD30 in refractory Hodgkin’s lymphoma, BCMA in multiple myeloma, and CD33, CD123, and FLT3 in AML [52]. It remains possible that minor modifications to individual CAR components could further increase the functionality of TanCAR, OR-gate CAR, or bivalent CAR. Several studies have shown the importance of single-chain variable fragment framework regions, costimulatory signals and extracellular spacer length in enabling robust T cell-mediated responses, which provides critical clues for the optimization of bispecific CAR targeting strategies [58,59,60].
A current challenge for CAR T cell therapy is to overcome barriers to their persistence following adoptive transfer. Emerging research indicates that cell-intrinsic mechanisms affect CAR T cell exhaustion, activation-induced cell death, and lymphocyte contraction, suggesting that the production of CAR T cells from less differentiated T cells (i.e., naïve, central memory or stem like T cells) is associated with superior functional properties (reviewed in Ref. [61]). A more comprehensive understanding of CAR signalling, improving CAR trafficking and survival, and scrupulously studying their efficacy for tumour treatments in related preclinical animal models will continually facilitate the development of more effective CAR T therapeutics [48].
1.3.3. Gamma-Delta T Cells
An alternative to CAR T cell transfer for the treatment of tumours with down-regulated or defective MHC I expression is the adoptive transfer of γδ T cells. Like conventional αβ T cells, γδ T cells demonstrate cytotoxic activity via the granzyme-perforin axis, release cytokines such as TNF-α, IFN-γ, and IL-17, and can kill a wide array of tumour types [62]. However, unlike conventional αβ T cells, γδ T cells are activated in an MHC-independent manner and are capable of recognising a broad range of stress-induced molecules on cancer cells [63]. The adoptive transfer of γδ T cells into cancer patients is known to be safe and associated with improved overall survival [64], although overall treatment efficacy is highly variable, most likely as a result of the exploration of greatly varied γδ T cell expansion and delivery protocols [62]. While there is much to be learned regarding the optimal methodology for isolating and expanding suitable numbers of γδ T cells for clinical use, the early indications are that γδ T cells may present great potential as an off-the-shelf cancer therapy.
An alternative to CAR T cell transfer for the treatment of tumours with down-regulated or defective MHC I expression is the adoptive transfer of γδ T cells. Like conventional αβ T cells, γδ T cells demonstrate cytotoxic activity via the granzyme-perforin axis, release cytokines such as TNF-α, IFN-γ, and IL-17, and can kill a wide array of tumour types [62]. However, unlike conventional αβ T cells, γδ T cells are activated in an MHC-independent manner and are capable of recognising a broad range of stress-induced molecules on cancer cells [63]. The adoptive transfer of γδ T cells into cancer patients is known to be safe and associated with improved overall survival [64], although overall treatment efficacy is highly variable, most likely as a result of the exploration of greatly varied γδ T cell expansion and delivery protocols [62]. While there is much to be learned regarding the optimal methodology for isolating and expanding suitable numbers of γδ T cells for clinical use, the early indications are that γδ T cells may present great potential as an off-the-shelf cancer therapy.
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