The Promise of Cd T Cells and the Cd T Cell Receptor for Cancer Immunotherapy

REVIEW

The promise of cd T cells and the cd T cell receptor for cancer immunotherapy

Mateusz Legut, David K Cole and Andrew K Sewell cd T cells form an important part of adaptive immune responses against infections and malignant transformation. The molecular targets of human cd T cell receptors (TCRs) remain largely unknown, but recent studies have confirmed the recognition of phosphorylated prenyl metabolites, lipids in complex with CD1 molecules and markers of cellular stress. All of these molecules are upregulated on various cancer types, highlighting the potential importance of the cd T cell compartment in cancer immunosurveillance and paving the way for the use of cd TCRs in cancer therapy. Ligand recognition by the cd TCR often requires accessory/co-stimulatory stress molecules on both T cells and target cells; this cellular stress context therefore provides a failsafe against harmful self-reactivity. Unlike ab T cells, cd T cells recognise their targets irrespective of HLA haplotype and therefore offer exciting possibilities for off-the-shelf, pan-population cancer immunotherapies. Here, we present a review of known ligands of human cd T cells and discuss the promise of harnessing these cells for cancer treatment. Cellular & Molecular Immunology (2015) 12, 656–668; doi:10.1038/cmi.2015.28; published online 13 April 2015

Keywords: cancer; cd T cells; immunotherapy; phosphoantigens; T cell receptor

INTRODUCTION humans.4 Some Vd segments can be used to generate both the a The adaptive immune compartment comprises of three dis- and d chains of the TCR, as they are located within the tcra tinct cell subsets, namely B cells, T cells expressing an ab T cell locus.5,6 The number of V segments that can be used for cd T receptor (TCR) and T cells expressing a cd TCR. These cells cells is much smaller than that for ab T cells (46 Va and 48 Vb generate their defining B cell receptors (BCRs), or TCRs using segments). However, the potential diversity of cd TCR sur- somatic V(D)J recombination which enables them to recognise passes that of ab TCR, owing to extensive N-region nucleotide a vast spectrum of antigens. Despite the fact that gene segments additions and presence of distinct D segments (present only in used in rearrangement leading to ab and cd TCRs were dis- tcrd but not tcrg locus) which can be used simultaneously and covered almost at the same time,1 the elucidation of ab T read in all three frames. This junctional variability results in the cell biology progressed rapidly, while the complexities of cd generation of hyperdiversity focused on the complementarity T cells have been slower to emerge. Nevertheless, the tripartite determining region (CDR)3 loops which are crucial for antigen organisation of the lymphocytic immune system appears to recognition.7 Furthermore, the length of the CDR3s of both a be a fundamental requirement for efficient function, as this and b chains is constrained, due to the requirement to make a organisation is present both in jawed and jawless vertebrates, well-defined contact with peptide-MHC complexes, while even though the manner of generating the defining receptors CDR3 in the d chain is usually more variable and longer than completely differs between these lineages.2 The unique func- its c counterpart.8 With regard to CDR3 length, the cd TCR tions of cd T cells, particularly in terms of antigen recognition resembles the BCR more than ab TCR. This greater variability and kinetics of response, provide further evidence that cd T of cd TCRs may translate into recognition of both proteins and cells represent an important and non-redundant lymphocyte smaller molecules. The CDRs form loops in the cd TCR struc- subset. ture to provide a highly variable antigen-binding domain at the V(D)J recombination of the c and d chain genes is shown in membrane-distal end of the molecule (Figure 2). Figure 1 (IMGT nomenclature3). There are 4–6 (depending on After arising from a common progenitor in the thymus, the haplotype) functional variable (V)c, and 8 Vd gene segments in maturation pathways of cd and ab T cells diverge. Notably, the

Division of Infection and Immunity and Systems Immunity University Research Institute, Cardiff University School of Medicine, Cardiff, UK Correspondence: Andrew K. Sewell, Henry Wellcome Building, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, UK. E-mail: [email protected] Received: 24 January 2015; accepted: 1 March 2015 cd T cells for cancer immunotherapy M Legut et al. 657

V-J recombination at tcrg locus V2 V3 V4 V5 V8 V9 JP1 JP J1 C1 JP2 J2 C2

5′ 3′

V-J rearrangement

5′ 3′

splicing

TCR-g chain transcript

V-D-J recombination at tcra/tcrd locus DV4/AV14 DV6/AV23 DV1 DV5/AV29 DV7/AV36 DV8/AV38.2 DV1 D1 D2 D3 J1 J2 J4 J3 C DV3

5′ 3′

D-J rearrangement

5′ 3′

V-D-J rearrangement

5′ 3′

splicing

TCR-d chain transcript

Figure 1 V(D)J recombination at the tcrg (upper panel) and tcra/tcrd (lower panel) locus. Only the functional gene segments are shown. The TCR-c chain is produced using only a single V-J recombination, with P/N additions occurring at the V-J junction. The TCR-d chain is produced using V-D-J recombinations that can involve either 2 or 3 D segments, leading to the creation of up to 4 N diversity regions. For the clarity of the figure, only the gene segments that can be used in TCR-d chain production are presented (lower panel). The organisation of loci tcrg and tcra/tcrd was adapted from IMGT database.3 development of cd TCR1 thymocytes does not require the subset of mouse cd Tcells.13 The identity of other ligands expression of Aire,9 a transcriptional regulator crucial for the required for positive selection of cd remains to be elucidated. negative selection of autoreactive ab T cells. The mechanism by Strong cd TCR-mediated interactions in the thymus have been which T cells become committed to the ab or cd lineage is not shown to result in upregulation of CD73, the earliest identified yet fully understood as thymocytes rearrange b, c and d genes at marker of cd lineage commitment.14 CD73 is expressed by the the same time which can lead to simultaneous expression of vast majority of cd T cells in the periphery, supporting the the cd TCR and pre-TCR (invariant Ta paired with TCR-b).10 notion that recognition of the ligand in the thymus is a common However, recent evidence suggests that thymocytes adopt the occurrence in cd T cell development. Another striking difference cd T cell lineage after receiving a strong signal via cd TCR, in development between ab and cd T cells is the acquisition of which can be additively enforced by additional signalling via effector functions. Conventional ab T cells acquire their effector pre-TCR – thus enabling weak ligands to drive cd T cell lineage phenotype, in terms of produced cytokines, upon interactions commitment as well.11 If cells fail to receive this survival signal with their targets in the periphery, while cd T cell functions, like they silence the cd TCR and undergo TCR-a rearrangement.12 their anatomical location, appear to be pre-determined in the This signal strength model implies that cd T cells need to thymus by the chain usage of their TCR.15 encounter a cognate ligand in the thymus. However, to date In humans, cd T cells constitute 0.5–10% of T cells in peri- only one molecule, namely Skint-1, has been described as a pheral blood but are substantially enriched in epithelial tissues thymically expressed ligand necessary for development of a (e.g. in skin, lungs, intestine). The majority of peripheral blood

Cellular & Molecular Immunology cd T cells for cancer immunotherapy M Legut et al. 658

TCR protein structure TCR gene structure

V DDJ C

P/N additions CDR1d CDR2d CDR3d

V-encoded D-J-encoded (germline) + P/N additions (hypervariable)

VJC

P/N additions CDR1g CDR2g CDR3g

Variable domainVariable Constant domain V-encoded J-encoded (germline) + P/N additions CDR2d (hypervariable) CDR3d CDR3g CDR2g CDR1d CDR1g

Antigen binding domain

Figure 2 9C2 cd TCR protein structure (left panel) and c and d chain mRNA architecture (right panel). The CDR loops are colour-coded. PDB ID: 4LHU.37

cd T cells express the Vd2 chain, while the tissue-resident cd T and eukaryotic cells, using non-mevalonate and mevalonate cells are mainly Vd1pos or Vd3pos. The precise reasons for this biosynthetic pathways, respectively. The accumulation of tissue specificity and the mechanisms that underlie it are yet to PAgs, such as isopentenyl pyrophosphate (IPP), is a result of be elucidated. The role of cd T cells in infection is well estab- metabolic dysregulation that commonly occurs in tumour lished and has been amply reviewed elsewhere.16 More recent cells18 and therefore the enzymes of the mevalonate pathway discoveries suggest that cd T cells play a role in anticancer are a valid target for anticancer drugs (reviewed in Clendening immunity, and it has been known for over a decade that mice and Penn19). Importantly, PAgs have long been known to be lacking the cd TCR are more susceptible to some cancers recognised by cd T cells expressing Vc9Vd2 TCR. The presence through unknown mechanisms.17 Here, we review recent ad- of this peripheral blood subset of cd T cells is restricted to vances in discovering cancer-associated antigens recognised by higher primates, with a few exceptions.20 Importantly, mice human cd T cells and the evidence that there is a broad spec- and other rodents do not possess any corresponding T cell trum of cd T cell ligands waiting to be discovered. We also subsets that respond to PAgs. provide a brief overview of therapeutic applications of cd T The mode of PAg recognition has only recently been cells in cancer immunotherapy. resolved. Early studies indicated that recognition of PAgs by human Vc9Vd2 T cells required cell surface presentation by a cd TCR RECOGNITION OF CANCER-ASSOCIATED species-specific molecule.21 One of the proposed molecules ANTIGENS presenting the antigens was F1-ATPase which is expressed on Known ligands of human cd TCR are scarce when compared with the surface of a wide range of tumour cells.22 Further research the vast spectrum of antigens recognised by ab T cells. However, suggested that PAgs could bind F1-ATPase in the form of a significant progress in this area has been made in the recent years, nucleotide derivative, increasing the efficiency of T cell activa- linking the observed recognition of tumour cells to specific tion. However, as antibody blocking of F1-ATPase did not ligands confirmed by biochemical and biophysical data. The main abrogate T cell recognition, other molecules were thought to targets encompass phosphorylated prenyl antigens, endo- and be involved in PAg presentation.23 The cell surface-expressed exogenous lipids presented by CD1-family proteins, and cell stress Butyrophilin molecules are encoded within the MHC class I molecules that can indicate DNA damage, viral infection or locus and offer attractive potential candidates as PAg-present- malignant transformation. ing molecules. Butyrophilin-3A (BTN3A/CD277) is present in humans in three isoforms (BTN3A1, BTN3A2 and BTN3A3).24 Recognition of phosphoantigens Recent studies have shown that antibodies specific for BTN3A1 Phosphorylated isoprenoid metabolites, commonly referred to could either mimic PAg-mediated activation of the TCR (anti- as phosphoantigens (PAgs), can be produced by both bacterial body 20.1) or abrogate this stimulatory effect (antibody

Cellular & Molecular Immunology cd T cells for cancer immunotherapy M Legut et al. 659

103.2).25 Biophysical analysis of the underlying interactions sug- mechanisms need not be mutually exclusive and it remains gested that 20.1 antibody induced/stabilised a TCR-activating possible that PAgs bind to both the intracellular and extra- conformation of BTN3A1.26 Additionally, PAgs and 20.1 anti- cellular domains of BTN3A1 to provide a failsafe against body activated the same intracellular signalling pathways in the aberrant TCR triggering and autoimmunity. The binding responding cd T cells, suggesting a common recognition pro- affinities of IPP to BTN3A1 and the Vc9Vd2 TCR interaction cess.27 Interestingly, transduction studies revealed that while with BTN3A1-IPP are summarised in Table 1. Further invest- the expression of BTN3A1 alone is sufficient for the activation igation will be required to determine the exact mechanism of mediated by 20.1, additional genes located on chromosome antigen presentation by BTN3A1. 6 are required for PAg-mediated recognition.28 These studies suggest the possible existence of molecules responsible for effi- cd T cells recognise specific ligands in the context of antigen- cient loading of PAgs into butyrophilin-3A1 in a conceptually presenting molecules from the CD1 family similar manner to loading peptides into MHC, or that there is a Four members of the human CD1 family of proteins (CD1a– requirement for a co-stimulatory ligand. Two main models CD1d) present both endogenous and exogenous lipids that can explaining the role of BTN3A1 have been proposed, involving be recognised by both ab and cd T cells.32 Early reports demon- intracellular or extracellular binding of pyrophosphate antigens strated that mucosal cd T cell clones showed broad reactivity (Figure 3a). Sandstrom et al. demonstrated that BTN3A1 acts towards these four CD1 isoforms loaded with exogenous phos- as a sensor of intracellular PAgs by binding them in a surface pholipids.33 Further studies showed that T cells expressing the pocket located in an intracellular domain termed B30.2.29 Vd3 chain were capable of killing tumour cells expressing This result was confirmed by the crystal structure of the CD1d (but not other CD1 isoforms) without addition of exo- B30.2-PAg complex (Figure 3c) and by mutational analysis. genous lipids.34 Additionally, the cd T cell compartment of Introduction of the B30.2 domain of BTN3A1 into the non- peripheral blood was shown to recognise CD1d in complex activating isoform BTN3A3 was shown to confer PAg binding with self-lipids termed sulphatides (sulphated galactosylcera- and T cell stimulation. This result falls in line with observa- mides).35 CD1d-reactive cd T cells expressed similar Vd1-Jd1 tions by Wang et al. who demonstrated a lack of high-affinity chains paired with different Vc chains. Notably, some of the binding of PAgs to the extracellular regions of BTN3A1.30 In generated clones could bind CD1d without any loaded lipid. direct contrast, De Libero and colleagues resolved the crystal Recent studies provided the structural basis for CD1d-sulpha- structure of two PAgs (IPP and HMBPP) in complex with the tide recognition, demonstrating that the binding occurred extracellular domain of BTN3A1 where the antigen was solely via the d chain of the TCR – the germline-encoded resi- bound in a shallow surface pocket (Figure 3b).31 The binding dues made contact with the CD1d molecule whereas the 36 affinity of Vc9Vd2 TCR to BTN3A1 was relatively weak (KD CDR3d loop interacted with sulphatide (Figure 4a). As the , 0.1 mM) but increased in the presence of IPP. These two CDR3 region is the most variable part of the cd TCR, it is

IPP

BTN3A1

B30.2 domain

Loading (additional proteins involved? c ) Mevalonate pathway enzymes Downstream IPP products

Aminobisphosphonates Metabolic deregulation

Figure 3 (a) Schematic representation of the phosphoantigen presentation pathways by human cells showing phosphoantigen (IPP) binding in the extracellular pocket of BTN3A1 (b) and phosphoantigen (cHDMAPP) binding in the intracellular domain (B30.2) of BTN3A1 (C). PDB IDs: 4JKW (B)31 and 4N7U (C).29

Cellular & Molecular Immunology cd T cells for cancer immunotherapy M Legut et al. 660

Table 1 The interaction affinities of IPP binding to the intra- shown to present a novel class of immunogenic self-lipids, cellular or extracellular domains of BTN3A1, and of TCR bind- called methyl-lysophosphatidic acids (mLPAs). Recognition ing to BNT3A1 in presence of IPP. of CD1c-mLPA complex by cd T cells has yet to be demonstrated,42 but recent studies showing that CD1 family members Interaction KD (mM) Ref. can present self-lipids make these molecules attractive targets IPP: extracellular BTN3A1 69.9 31 for future exploration. IPP:B30.20 <500 29 Vc9Vd2 TCR:BTN3A1(IPP) 340 31 cd TCR recognition of general stress ligands cd T cells are known to expand in response to some viral infec- possible that a subset of cd T cells can recognise diverse lipid tions, with cytomegalovirus (CMV) providing the best-studied cargoes presented by CD1 molecules, in a similar way to ab example to date. CMV infection results in preferential expan- TCR recognition of peptides bound to MHC. A similar mode sion of cd T cells expressing Vd1, Vd3 and Vd5 (commonly of interaction was reported for CD1d-a-galactosylceramide termed ‘non-Vd2 T cells’).43,44 These cells can also recognise (aGalCer) and a Vd1 TCR (9C2) where CD1d was bound by and lyse intestinal tumour cells, in line with the fact that they germline-encoded residues in the d chain while the lipid was are enriched in epithelial tissues where they perform stress 37 recognised by the hypervariable CDR3c loop (Figure 4b). surveillance.44 Interestingly, infection with CMV, regarded as Interestingly, Pellicci et al. have recently discovered a novel T a treatment-related complication in kidney transplant recipi- cell subgroup recognising CD1d-aGalCer complex, expressing ents, serves a protective role against cancer in those patients.45 aVd1 domain linked to permissive Ja-Ca segments, paired Taken together with the fact that Vd2neg compartment is selec- with diverse TCR-b chains (termed d/ab T cells).38 Again, tively expanded in CMV carriers and does not diminish with the binding of the TCR to CD1d was mainly driven by germ- age, it is possible that CMV-mediated activation of cd T cells line-encoded residues in the Vd1 domain while the specific confers an adaptive-like, lifelong reduction of cancer risk.46 recognition of the bound lipid was exclusively mediated by The ligands of dual reactive cd T cell clones recognising the b chain of the TCR (Figure 4c). The affinity of the inter- CMV-infected and transformed cells remain unknown, with action between TCRd/ab and CD1d-aGalCer was, however, one notable exception. Recently, De´chanet-Merville and collea- much higher than described for 9C2 cd TCR (the binding gues identified the ligand of one of such clone, named LES. The affinities of TCRs to CD1d complexed with lipid ligands are LES cd T cell clone expressed Vc4andVd5TCRchainsandwas summarised in Table 2). Importantly, functional d/ab T cells substantially enriched (25% of peripheral blood T cells) in a comprise a high proportion of human CD1d-reactive T cells. CMV-infected transplant recipient. LES was shown to recognise aGalCer has been so far investigated only in context of boosting a range of solid tumour lines and CMV-infected cells via its TCR NK T cell anticancer activity,39 but it could potentially be used rather than natural killer (NK)-type receptors. The LES TCR was to activate aGalCer-reactive cd T cells, similarly to using PAgs. demonstrated to bind endothelial protein C receptor (EPCR).47 Notably, a recent study showed that small quantities of EPCR exhibits sequence and structural homology with the aGalCer and other a-glycosylceramides can be endogenously MHC-like protein family CD1, and can present phospholipids produced in mammalian species to modulate the immune res- bound in the antigen-presenting groove.48 EPCR plays a dual ponse.40 Additionally, CD1d may be an important target for role in cancer, as it can both promote and inhibit metastases, cancer immunotherapy as its high expression was reported on presumably depending on whether it is expressed on tumour chronic lymphocytic leukaemia cells, correlating with disease cells or endothelium (reviewed in Mohan Rao et al.49). progression.41 Similarly, CD1c on acute leukaemia cells was Additionally, EPCR signalling can activate antiapoptotic or

abVγ4Vδ1 TCR Vγ5Vδ1 TCR c Vδ1/CαVβ2 TCR

CD1d-sulphatideCD1d-αGalCer CD1d-αGalCer

Figure 4 Complex formation between TCR and CD1 ligands. CDR loops are colour coded as in Figure 2: CDR1d-red, CDR2d-green, CDR3d-blue, CDR1c-yellow, CDR2c-cyan and CDR3c-orange. The blue loop in panel C is CDR3d/a. PDB IDs: 4MNG (A),36 4LHU (B),37 4WO4 (C).38

Cellular & Molecular Immunology cd T cells for cancer immunotherapy M Legut et al. 661

Table 2 The interaction affinities of TCR binding the CD1d- strated that hMSH2 can be a ligand for some Vd2 cd T cells lipid complex. when it is ectopically expressed, inducing cytotoxicity and IFN-c production.54 Surface expression of hMSH2 can be TCR Ligand KD (mM) PDB code Ref. induced by Epstein-Barr virus (EBV) infection,54 oxidative Vc4Vd1 CD1d- 5.6 4MNG 36 stress and proinflammatory cytokines such as IL-18 corrob- sulphatide orating its role in generalised stress. Vc5Vd1 CD1d- 16 4LHU 37 aGalCer Interestingly, oxidative stress promoted the expression of Vd1/Ca Vb2 CD1d- 0.066 4WO4 38 MIC A in a similar manner to hMSH2, involving p38/JNK 55 aGalCer signalling pathways. Another group reported MIC A/B upregulation resulting from ataxia telangiectasia mutated (ATM) protein kinase signalling in response to DNA damage, contrib- 56 proapoptotic pathways through unresolved mechanisms. uting to cd T cell-mediated lysis of ovarian cancer cell lines. Importantly, direct binding of the LES TCR to EPCR was con- Additionally, the level of MIC A expression on breast cancer cell lines correlated with their susceptibility to cd Tcell-mediated firmed by surface plasmon resonance, showing relatively low 57 < m cytotoxicity. Induction of surface expression of MIC A/B was binding affinity (KD 90 M) (Table 3). EPCR, however, does 58 not appear to be a common, innate-like target for dual reactive also reported in primary glioblastoma after chemotherapy. cd T cell clones, as the recognition of the tumour lines by such EBV infection induces MIC A expression as well, showing fur- clones, isolated from other patients, was not abrogated by anti- ther similarities between expression of MIC and hMSH2. body blocking of EPCR. Moreover, the LES TCR recognition cd TCRS RECOGNISE TUMOURS IN A CO-STIMULATORY was mediated by the hypervariable CDR3 loop of the Vc4chain, STRESS CONTEXT supporting the hypothesis that EPCR binding was part of an As mentioned above, the recognition of cell stress markers by adaptive response. Interestingly, the expression of EPCR was not cd T cells appears not to be driven solely by the interaction sufficient to induce T cell activation, suggesting that functional between the TCR and its cognate ligand but rather requires a recognition required a cell stress context and additional co-sti- wider stress context provided by co-stimulatory receptors on mulatory molecules. Indeed, CMV infection conferred the the T cell and additional self-antigens. In the case of the LES recognition of fibroblasts and endothelial cells without altering clone recognising EPCR, part of the co-stimulatory effect was EPCR expression suggesting that recognition of this receptor provided by LFA-1 on the T cells binding to ICAM-1 (over- occurs in a stress-related co-stimulatory context. The identity expressed as a result of CMV infection).47 CD2 interaction with of these co-stimulatory ligands remains unknown. CD58 (LFA-3) was also implicated. Blocking of TCR binding to EPCR is not the only cell stress marker that requires an EPCR completely abrogated the recognition while disrupting additional co-stimulatory context for efficient recognition by the ICAM-1-LFA-1 and CD2-CD58 axes merely decreased T cd T cells. Other examples described so far encompass the 50 cell activation, demonstrating the dominance of TCR signalling human homologue of the bacterial MutS (hMSH2) protein, and the possible co-existence of complementary co-stimula- being a part of the DNA mismatch repair system (MMR), and 51 tory pathways. Furthermore, another group showed that the MHC class I polypeptide-related sequence A (MIC A), a CD8aa homodimer could act as a coreceptor for recognition of broadly recognised stress marker. Both molecules are recog- CMV-infected cells by Vd1 TCR chains (Figure 5a).59 This nised by both highly variable cd TCR and the invariant observation is in line with the findings that T cells expressing NKG2D receptor (discussed in the next section). hMSH2 is a CD8aa play an important role in immunosurveillance of the key protein involved in repairing mutations resulting from epithelial tissues against viral60 and bacterial61 infections. DNA recombination or replication. Unsurprisingly, defects The requirement for co-stimulation is well established for ab in hMSH2 have been reported in various tumour histologies T cells and it has been known for some time that CD8ab het- 52 including lung, colon, breast and prostate cancers. Although erodimer acts to co-receive peptide-MHC class I, stabilising the hMSH2 is normally restricted to the nucleus, it can be trans- TCR interaction62 and ensuring full phosphorylation of the 53 located during cellular stress. Recent studies have demon- CD3f chain63 to increase sensitivity to antigen by up to a mil- lion-fold64 (reviewed in Cole et al.65). Thus the ab T cell co- receptors, CD4 and CD8, ensure that this T cell subset is MHC- Table 3 cd TCR and NKG2D binding affinities to stress- restricted66,67 and determine the fate of the developing T cell.68 related ligands EPCR, hMSH2 and MIC A. In an analogous fashion, co-stimulation via stress-induced Receptor Ligand KD (mM) Ref. ligands might maintain the correct focus and function of the Vc4Vd5 TCR EPCR 96 47 cd T cell compartment. In keeping with this concept, both (LES) hMSH2 and MIC A/B are dually recognised by cognate TCRs Vd2 TCRs hMSH2 N/A 54 and the NKG2D receptor (Figure 5b). NKG2D is commonly NKG2D hMSH2 0.132 54 expressed on the surface of NK cells, ab T cells and cd T cells, Vc4Vd1 TCR MIC A 110–900 70 and binds to cell stress molecules from the UL16-binding NKG2D MIC A 0.3–21 70 protein (ULBP1–6) family (reviewed in Ullrich et al.69). The

Cellular & Molecular Immunology cd T cells for cancer immunotherapy M Legut et al. 662

a b γδ TCR NKG2D CD8αα γδ TCR LFA-1

CD2 1 2

ICAM-1 LFA-3 hMSH2 ? MIC A EPCR

EBV infection CMV infection Oxidative stress Malignant transformation lnflammation DNA damage

Figure 5 Recognition of cd TCR ligands requires a cell stress context. (a) Recognition of EPCR requires co-stimulatory ligands on the surface of the target cells, and accessory molecules on the surface of the T cell. (b) Both hMSH2 and MIC A become upregulated and ectopically expressed in response to cell stress stimuli and are dually recognised by NKG2D and TCR. The sequential model of recognition implies that the initial contact provided by a transient high-affinity NKG2D–ligand interaction (1) is followed by formation of a stable low affinity TCR-ligand complex (2).

interactions of germline-encoded NKG2D with such diverse HARNESSING cd T CELLS FOR THERAPY ligands are possible because of conserved fragments within Stimulating cd T cells in vivo with phosphoantigens the a1a2 superdomain in the ligands. The involvement of As mentioned above, the Vc9Vd2 T cell subset that predomi- the cd TCR and NKG2D in recognition of the MIC A molecule nates in human peripheral blood is capable of responding to has recently been studied. Xu et al. showed that both TCR and prenyl pyrophosphates. This feature has been employed to redir- NKG2D bound overlapping fragments of MIC A, with differ- ect Vc9Vd2 T cells to tumours by manipulating isoprenoid ent affinity and kinetics – for NKG2D, the affinity was much metabolism in the cancer cells. Such manipulation can be 70 higher than for the TCR (Table 3). Nevertheless, the complex achieved using aminobisphosphonates (e.g. zoledronate, pami- between TCR and MIC A was particularly stable, suggesting a dronate, risedronate) which are structural analogues of prenyl sequential model of binding where the initial contact by pyrophosphates that contain an amino moiety. As a result, ami- NKG2D is followed by the more stable TCR:MIC A complex. nobisphosphonates act indirectly by inhibiting farnesyl pyro- The role of TCR chains in complex formation remains to be phosphate synthase (FPPS) in the mevalonate pathway leading fully resolved as the studies implicated partial contributions of to the accumulation of prenyl pyrophosphate substrates.75,76 70 the germline-encoded CDR loops from Vd1 TCR while other Recently, Idrees et al. screened over 50 tumour cell lines, show- studies showed that MIC A-associated recognition ing a direct correlation between zoledronate-induced FPPS of tumour cells could be mediated by both Vd171 and inhibition and tumour recognition by Vc9Vd2Tcells.77 The Vd256,57,72 TCRs. Notably, TCR engagement has been shown FPPS-inhibiting concentration of zoledronate was on average to be indispensable for cd T cell-mediated cytotoxicity whereas two orders of magnitude lower than the concentration required NKG2D played only a co-stimulatory role; thus indicating the for inhibition of tumour proliferation, thus indicating that T cell importance of TCR:MIC A complex formation.73 ULBP mole- activation was not the result of cell death. Another group cules may be recognised in a similar manner as it has been showed that risedronate caused PAg accumulation in the shown that ULBP-4 engages both NKG2D and Vc9Vd2 tumour cells which were then recognised by Vc9Vd2 T cells.78 TCRs.74 Upon recognition, T cells produced IFNc which caused upre- The sequential recognition of different targets by cd T cells gulation of ICAM-1 on tumour cells, contributing to a posi- could therefore play an important role in immunosurveillance tive feedback loop and subsequent cytotoxicity. Additionally, as it allows the T cell to rapidly scan the target cells for the signs aminobisphosphonates,79 as well as conventional chemotherapy, of stress, indicating a possible infection or transformation. The can sensitise the neoplastic cells to cytototoxic activity of requirement for a multicomponent stress context for full T Vc9Vd2 T cells. This may be an attractive therapeutic option cell activation could then provide fail-safe protection against for eradication of cancer-initiating cells, which are often resist- autoimmunity. The apparent co-existence of diverse co-stimu- ant to conventional therapy and can contribute to the relapse latory axes decreases the chances of immune evasion. of the disease. Finally, cd T cells are known to acquire the

Cellular & Molecular Immunology cd T cells for cancer immunotherapy M Legut et al. 663 phenotype of professional antigen-presenting cells upon activa- (Figure 6c).92,93 Additionally, cd T cells transduced with ab tion.80 Therefore, they may be used as a potent anticancer vac- TCRs retain the functionality of their original TCR and cine in addition to their broad antineoplastic cytotoxicity. respond to stimuli transferred via either TCR with rapid, cd- like kinetics.94 The main obstacle associated with ab TCR trans- Redirecting T cells to tumours fer, however, is that cd T cells usually do not express CD4 or In the absence of known ligands, cd T cells can be redirected to CD8 co-receptors required for the efficient recognition of pep- tumours using antibodies. The efficacy of bispecific antibodies, tide-MHC. Thus efficient function might require co-transduc- where one part recognised a tumour surface marker (for tion with a coreceptor95 or use of enhanced affinity TCRs.96 It is example, EpCAM on liver tumours or HER2/neu on pancreatic also possible to transduce peripheral lymphocytes (both of cd cancer) while the other binding site targets CD3 or the Vc9 and ab origin) with a specific cd TCR. Zhao et al. demonstrated chain of the TCR, has been demonstrated in pre-clinical mod- that T cells transduced with a Vc9Vd2 TCR, modified with a els.81,82 An interesting approach was described by Zheng et al. CDR3d loop specific for unidentified antigens on ovarian car- where they cloned out the extracellular domains of a Vc9Vd2 cinoma, exerted an anticancer activity in vivo.97 TCR from ovarian cancer tumour-infiltrating lymphocytes Similarly, the transduction of a PAg-reactive Vc9Vd2TCR (TILs) and conjugated them with Fc domain of human into ab T cells successfully redirected them towards cancer cells, IgG1.83 This bispecific construct bound to a range of ovarian as well as led to downregulation of their endogenous ab TCRs, cancer cells, recognising an unknown ubiquitous antigen, and thus abrogating alloreactive responses.98 It is therefore likely that mediated the killing of the cells via antibody-dependent cellular the detailed characterisation of cancer-specific cd TCRs will cytotoxicity (ADCC). ADCC can be mediated by binding of bring about new studies examining the efficacy of such TCRs CD16 (FccRIII) to the Fc region of IgGs, constituting yet in the TCR gene transduction setting. another means of target recognition by cd T cells, in addition to TCR and NKG2D. CD16 can be upregulated on both Vd2pos Generating clinically relevant numbers of tumour-reactive and Vd2neg T cells, depending on circumstances, while binding cd T cells of its target may trigger either cytotoxicity or other effector The main hurdle that needs to be overcome in order to use cd T functions (e.g. IFNc secretion).84,85 A similar approach cells as a therapeutic is obtaining these cell in clinically mean- involves transducing T cells with chimeric antigen receptors ingful numbers. For ab T cell-based therapies, this is a relatively (CARs; Figure 6). CARs are usually derived from single chain straightforward task as the requirements for effective ab T cell Fv fragments of antibodies, thus enabling the CAR-transduced expansion have been widely studied, and the cognate ligands T cells to recognise conformational epitopes independently of can be readily used to stimulate the expanding cells.99 In the their TCR (recently reviewed in Maus et al.86). To date, most case of cd T cells, the only known common ligands are PAgs CAR transduction experiments have been conducted on ab T which are recognised by the Vc9Vd2 subset. As aminobispho- cells – nevertheless, cd T cells are also an appealing target, due sphonates such as zoledronate cause accumulation of PAgs in to their broad antitumour cytotoxicity and numerous effector the cells, they can be used to preferentially expand that T cell functions. Recently, Deniger et al. have transduced polyclonal subset both ex vivo, prior to re-infusion, and directly in cd T cells with a CD19-specific CAR, demonstrating their effi- patients. However, PAgs and aminobisphosphonates expand cacy in killing CD191 leukaemia lines.87 CAR-mediated signal- only the Vc9Vd2 subset of cd T cells, and this bias may have ling resulted in a similar cytokine secretion profile as TCR a negative impact on clinical efficacy. In an alternative approach, activation, and induced unbiased expansion of cd T cell sub- immobilised anti-cd TCR antibody has been demonstrated to sets. Notably, CAR technology has been combined with the promote the expansion of all cd T cell subsets, without modulat- generation of induced pluripotent stem cells (iPSC) from ing their antitumour function.100,101 This method, however, may human peripheral blood T cells.88 Such cells exhibited a pheno- lead to mitogen-induced T cell death and has not yet been used in type closest to cd T cells of all lymphocyte lineages and exerted a published clinical trial. Deniger et al. have recently proved that T similar in vivo antitumour activity to CAR-transduced cd T cells with polyclonal cd TCR repertoires can be expanded to large cells. numbers (.109) using IL-2, IL-21 and clinical grade artificial Finally, T cells can be redirected to tumours by introducing antigen-presenting cells, engineered to express co-stimulatory an exogenous TCR of known anticancer specificity into molecules CD19, CD64, CD86, CD137L and membrane-bound patient-derived peripheral lymphocytes prior to adoptive IL-15.102 Notably, the antigen-presenting cells were derived from transfer of these cells. Most TCR gene studies have involved a tumour line K562, broadly recognised by cd Tcells,andc- transduction of ab TCRs into ab T cells.89 However, this strat- irradiated prior to T cell exposure. The irradiation step could egy comes with the inherent risk of ab TCR mispairing between potentially enhance the recognition even further, as it leads to the endo- and exogenous TCR chains, resulting in receptors of expression of cell stress molecules. Polyclonal cd T cell lines unknown and potentially autoreactive specificities (Figure 6b). showed superior toxicity compared to those expressing only Such autoreactivity has been observed both in ex vivo human Vd2 chains, thus highlighting the possibility of enhancing cd T samples90 and in mouse models.91 cd T cells offer an attractive cell-focused immunotherapy. Similarly, Anderson and colleagues way to circumvent this problem as tumour-reactive ab TCRs used the same antigen-presenting cell line to generate unbiased cd can be introduced into these cells without the risk of mispairing T cell products from peripheral blood of cancer patients.84

Cellular & Molecular Immunology cd T cells for cancer immunotherapy M Legut et al. 664

a γδ T cells can be retargeted via CAR. γδ TCR

γδ T cancer cells target cell CAR

b αβ TCR gene transfer to αβ T cells may result in mispaired TCRs of unknown specificities.

transduced αβ TCR off-target αβ T cell ? cancer cells target cell

mispaired native αβ TCRs αβ TCR

c αβ TCR gene transfer to γδ T cells does not lead to TCR mispairing. transduced αβ TCR γδ T off-target cancer ? cells target cell cell

native γδ TCR

Figure 6 Genetic modification of cd T cells for adoptive therapy approaches to cancer. (a) cd T cells can be redirected to kill cancer cells using a chimeric antigen receptor (CAR) made from an antibody that targets a tumour-specific molecule at the cancer cell surface. (b) ab T cells can be redirected to kill cancer cells by transducing them with a cancer-specific ab TCR. Such ab TCR gene transfer could result in the expression of up to four different ab TCRs at the T cell surface: (i) the endogenous TCR; (ii) the transduced TCR; (iii) a hybrid TCR consisting of the endogenous TCRa chain paired with the transduced TCRb chain; and (iv) endogenous TCRb chain paired with the transduced TCRa chain. Neither hybrid will have undergone the rigours of thymic selection and therefore these TCRs have the potential of being autoreactive. (c) Transduction of cd T cells with an ab TCR provides a means of circumventing the potential mispairing problem seen in b.

Finally, manipulations of the CD3 co-receptor can be applied A similar study by Kunzmann and colleagues showed no objective to increase therapeutic efficacy of cd T cells. Dopfer et al. response in patients with solid tumours (melanoma and renal cell recently showed that two anti-CD3 antibodies affect the func- carcinoma), and a partial response in acute myeloid leukaemia tional activity of cd T cells differentially.103 The reported results patients.105 Surprisingly, the treatment resulted in increased vas- indicated that an anti-CD3 antibody, OKT3, induced strong cular endothelial growth factor levels, an indication of augmented proliferation and cytokine secretion with decreased cytotoxic angiogenesis, having a negative impact on the therapeutic out- potential, while another anti-CD3 antibody, UCHT1, enhanced come. No severe side effects (apart from grade 4 fever) were tumour killing via Ras/Erk and PI3K/Akt pathways, without an observed. A recent phase IV clinical trial of zoledronate in can- increase in cytokine secretion or proliferation. These results cer-free patients demonstrated that the severity of transient might pave the way for new therapeutic approaches focused inflammation-related side effects, caused by the treatment, could on manipulation for CD3-mediated signal transduction to easily be predicted by ex vivo measurements of IFN-c production favour expansion of large numbers of cd T cells, with desired by zoledronate-activated peripheral blood cells.106 Notably, effector functions such as cytotoxicity. repeated zoledronate treatment led to the depletion of the central memory subset of blood Vc9Vd2 T cells, as well as a long-lasting Lessons learned from clinical trials decrease of their absolute count.106,107 This phenomenon may be Numerous attempts to use cd T cells in cancer immunotherapy caused by peripheral blood neutrophils which have been shown to have been made over the past decade, with variable efficacy and ingest zoledronate and subsequently inhibit the proliferation of a good overall safety profile. the T cells by production of hydrogen peroxide.108 Repeated Two recent trials investigated the anticancer effect of zoledro- stimulation with PAgs, either accumulated in response to amino- nate combined with low dose IL-2. In renal cell carcinoma, the bisphosphonates or as a result of an accelerated mevalonate path- combined treatment resulted only in a minor expansion of Vd2pos way, can also lead to a differentiation shift of Vc9Vd2 T cells and cells which diminished after repeated treatment cycles without functional exhaustion.109 Zoledronate therefore appears to be achieving the objective response in any of the enrolled patients.104 insufficient to generate an effective antitumour response per

Cellular & Molecular Immunology cd T cells for cancer immunotherapy M Legut et al. 665 se – nevertheless, two multicentre studies demonstrated that in the maintenance of immune integrity. cd T cells are known treatment with zoledronate combined with chemotherapy to play an important role in infection16; further recent discov- improved the survival of breast cancer and multiple myeloma eries have shown that this T cell lineage can respond to stress patients.110,111 signals and thereby play an important role in cancer immuno- The adoptive cell transfer (ACT) of ex vivo expanded cd T surveillance. Indeed, mice lacking the cd TCR have increased cells appears to be a more effective treatment option than using cancer risk.17 Despite the importance of cd T cells, very few zoledronate as a single agent. In metastatic renal cell carcin- ligands for the human cd TCR have been confirmed by bio- oma, two groups reported re-infusing autologous PAg- chemical and biophysical data and this field is still in its infancy. pos 112,113 expanded Vd2 T cells. When the T cells were infused Interestingly, certain similarities exist between the few known with IL-2 only, 6 out of 10 patients experienced stabilisation of human cd TCR ligands that might point to some generalised the disease whereas the infusion of the cells with both IL-2 and mechanisms by which these cells patrol the periphery. zoledronate resulted in 5 out of 10 disease stabilisations and 1 BTN3A1, MIC A/B and CD1-family proteins belong to the complete response. However, as both studies included only a immunoglobulin superfamily (while EPCR is highly homolog- small number of patients, it is possible that the observed super- ous to CD1d), and both infections and cellular stress seem to ior efficacy of using both IL-2 and zoledronate was purely play a role in activating cd T cells via these immunoglobulin coincidental. When cells were expanded using IL-2 and zole- structures. Microbial infections lead to a stimulatory confor- dronate but infused without further exogenous stimulation, mational change and/or direct metabolite presentation by the partial response was observed in 3 out of 10 patients with 114 BTN3A1 while herpesvirus (CMV, EBV) infections cause upre- various solid tumours. As the patients were treated with gulation of EPCR, MIC A and hMSH2, respectively. Similarly, chemotherapy together with ACT, the response could be con- cell stress, including neoplasms, can involve deregulation of the tributed to the additive/synergistic effect of the combination mevalonate pathway122 and subsequent PAg activation of therapy. A similar effect was observed by Nicol and colleagues BNT3A1 in addition to ectopic expression of cell stress markers as the complete response (1/17) and partial response (2/17) such as EPCR, MIC and hMSH2, while CD1-family proteins patients received both cell infusion and chemo/hormone ther- 115 can present endogenous lipids acting as a marker of malig- apy. However, the patient cohorts in these studies were small nant transformation (CD1c)42 or viral infection (CD1d).123 and highly diverse, in terms of cancer type, additional therapy Interestingly, both CD1d and EPCR are capable of lipid pre- and composition of the T cell infusion. These variables make it sentation, and both can be recognised by cd T cell clones difficult to assign the exact clinical contribution of cd T cells. regardless of the lipid cargo – thus indicating that both mole- ACT of cd T cells and other cytotoxic cell types can also increase cules can act as an immunogenic marker per se.35,47 the efficacy of radiofrequency ablation.116 The aforementioned Requirement for a wider cell stress context for cd T cell recog- trials utilised patients’ autologous peripheral blood T cells for nition potentially restrains these immune cells and provides a ACT – however, in some instances the effector functions of failsafe against autoimmune reactions. Cancer-associated cd autologous T cells are severely impaired.109 A recent study by TCR targets are essentially self-antigens. Accordingly, cd Wilhelm et al. demonstrated that it is possible to infuse patients TCR interactions with the known targets are of low affinity with a cd T cell product obtained from haploidentical donors, –4 –6 (KD 5 10 –10 M; Tables 1–3), similar to ab TCR affinities with no evidence of graft versus host disease, showing substan- 124,125 tial clinical efficacy (3 out of 4 patients with advanced haema- for self-derived peptides. To date, only two structures of 117 cd tological malignancies experienced complete regression). human TCR in complex with a ligand have been resolved The donor cd T cells persisted for 28 days and expanded in (Figure 4). In both cases (CD1d-sulphatide and CD1d- vivo, in response to exogenous IL-2 and zoledronate. Another aGalCer), the germline-encoded CDR loops engaged the anti- group also showed that prolonged persistence of infused cd T gen-presenting CD1d molecule while hypervariable CDR3 cells can occur without IL-2 supplementation, probably relying loops made contacts with the lipid ligand, suggesting that dis- on endogenous IL-15.118 Moreover, exogenous IL-18 can be a crimination between subtly different lipid cargoes is possible. potent co-stimulator of cd T cell proliferation.119,120 In contrast, PAg recognition may be mediated mostly by germ- Overall, while cd T cell therapy has a good safety profile, line-encoded CDR loops from Vd2 and/or Vc9 chains as PAg- clinical performance to date has been underwhelming.121 BTN3A1 can be recognised by a wide range of different Vd2 Anticipated discoveries of the various ligands recognised on Vc9 T cell clones. However, the exact mode of PAg recognition tumour cells by the Vd2neg T cell subset will lead to a better awaits a BTN3A1: cd TCR complex structure. understanding of how these cells operate and an increased In summary, cd T cell therapy for cancer has demonstrated a capacity to beneficially harness these mechanisms for immu- good overall safety profile although results to date have not lived notherapy. up to their early promise. Nevertheless, we anticipate that many more ligands will be found for these important immune cells in CONCLUSIONS AND FURTHER DIRECTIONS the next 5 years thereby greatly expanding the therapeutic hori- The evolutionary conservation of cd T cells in all jawed verte- zon. Therapeutic approaches to cancer treatment based around brates and of an analogous third lymphocyte lineage in their the ab TCR in gene transfer99 or as soluble bispecifics126,127 have jawless ancestors2 attests to the critical importance of these cells burgeoned in the last two years. Importantly, cd TCRs do not

Cellular & Molecular Immunology cd T cells for cancer immunotherapy M Legut et al. 666

appear to suffer the limitation of being HLA-restricted so might in selected species like alpaca (Vicugna pacos). Immunogenetics 2014; 66: 243–254. offer exciting possibilities for new off-the-shelf, pan-population 21 Green AE et al. Recognition of nonpeptide antigens by human V cancer immunotherapies in the very near future. gamma 9V delta 2 T cells requires contact with cells of human origin. Clin Exp Immunol 2004; 136: 472–482. ACKNOWLEDGEMENT 22 Scotet E et al. Tumor recognition following Vc9Vd2 T cell receptor Mateusz Legut is a Cancer Research UK funded PhD student, David interactions with a surface F1-ATPase-related structure and apolipoprotein A-I. Immunity 2005; 22: 71–80. Cole is a Wellcome Trust Career Development Fellow and Andrew 23 Mookerjee-Basu J et al. F1-adenosine triphosphatase displays Sewell is a Wellcome Trust Senior Investigator. properties characteristic of an antigen presentation molecule for V 9V 2 T Cells. J Immunol 2010; 184: 6920–6928. 24 Afrache H, Gouret P, Ainouche S, Pontarotti P, Olive D. The butyrophilin (BTN) gene family: from milk fat to the regulation of the immune response. Immunogenetics 2012; 64: 781–794. 1 Davis MM, Bjorkman PJ. T-cell antigen receptor genes and T-cell 25 Harly C et al. Key implication of CD277/butyrophilin-3 (BTN3A) in recognition. Nature 1988; 334: 395–402. cellular stress sensing by a major human T-cell subset. Blood 2012; 2HiranoMet al. Evolutionary implications of a third lymphocyte 120: 2269–2279. lineage in lampreys. Nature 2013; 501: 435–438. 26 Palakodeti A et al. The molecular basis for modulation of human V 9V 3LefrancMPet al.IMGT , the international ImMunoGeneTics H 2 T cell responses by CD277/butyrophilin-3 (BTN3A)-specific information system 25 years on. Nucleic Acids Res 2014. H antibodies. J Biol Chem 2012; 287: 32780–32790. doi:10.1093/nar/gku1056 27 Decaup E et al. Phosphoantigens and butyrophilin 3A1 induce 4 Lefranc, M.-P. APPENDIX 1O nomenclature of the human T cell similar intracellular activation signaling in human TCRVc91 cd T receptor genes. In: Coligan JE, Bierer BE, Margulies DH, Shevach lymphocytes. Immunol Lett 2014; 161: 133–137. EM, Strober WW (eds.) Current Protocols in Immunology. John Wiley 28 Rian˜o F et al.Vc9Vd2 TCR-activation by phosphorylated antigens & Sons, 2001. Available from: http://doi.wiley.com/10.1002/ requires butyrophilin 3 A1 (BTN3A1) and additional genes on 0471142735.ima01os40 human chromosome 6: antigen processing. Eur J Immunol 2014; 5 Clark SP, Arden B, Kabelitz D, Mak TW. Comparison of human and 44: 2571–2576. mouse T-cell receptor variable gene segment subfamilies. 29 Sandstrom A et al. The intracellular B30.2 domain of butyrophilin Immunogenetics 1995; 42: 531–540. 3A1 binds phosphoantigens to mediate activation of human Vc9Vd2 6 Hayday AC. cd cells: a right time and a right place for a conserved T Cells. Immunity 2014; 40: 490–500. third way of protection. Annu Rev Immunol 2000; 18: 975–1026. 30 Wang H et al. Butyrophilin 3A1 plays an essential role in Prenyl 7 Davis MM. The evolutionary and structural ‘logic’ of antigen receptor pyrophosphatestimulationofhumanV2V2Tcells.J Immunol diversity. Semin Immunol 2004; 16: 239–243. 2013; 191: 1029–1042. 8 Rock EP, Sibbald PR, Davis MM, Chien YH. CDR3 length in antigen- 31 Vavassori S et al. Butyrophilin 3A1 binds phosphorylated antigens specific immune receptors. J Exp Med 1994; 179: 323–328. and stimulates human cd T cells. Nat Immunol 2013; 14: 908–916. 9 Tuovinen H et al. gammadelta T cells develop independently of Aire. 32 Porcelli SA, Modlin RL. The CD1 system: antigen-presenting Cell Immunol 2009; 257: 5–12. molecules for T cell recognition of lipids and glycolipids. Annu Rev 10 Burtrum DB, Kim S, Dudley EC, Hayday AC, Petrie HT. TCR gene Immunol 1999; 17: 297–329. recombination and alpha beta-gamma delta lineage divergence: 33 Russano AM et al. CD1-restricted recognition of exogenous and self- productive TCR-beta rearrangement is neither exclusive nor lipid antigens by duodenal gammadelta1 T lymphocytes. J Immunol preclusive of gamma delta cell development. J Immunol 1996; 2007; 178: 3620–3626. 157: 4293–4296. 34 Mangan BA et al. Cutting edge: CD1d restriction and Th1/Th2/Th17 11 Zarin P, Wong GW, Mohtashami M, Wiest DL, Zuniga-Pflucker JC. cytokine secretion by human V 3 T cells. J Immunol 2013; 191: 30– Enforcement of -lineage commitment by the pre-T-cell receptor in 34. precursors with weak -TCR signals. Proc Natl Acad Sci USA 2014; 35 Bai L et al. The majority of CD1d-sulfatide-specific T cells in human 111: 5658–5663. blood use a semiinvariant Vd1 TCR: clinical immunology. Eur J 12 Hayes SM, Love PE. Strength of signal: a fundamental mechanism Immunol 2012; 42: 2505–2510. for cell fate specification. Immunol Rev 2006; 209: 170–175. 36 Luoma AM et al. Crystal structure of Vd1 T cell receptor in complex 13 Barbee SD et al. Skint-1 is a highly specific, unique selecting with CD1d-sulfatide shows MHC-like recognition of a self-lipid by component for epidermal T cells. Proc Natl Acad Sci USA 2011; human cd T cells. Immunity 2013; 39: 1032–1042. 108: 3330–3335. 37 Uldrich AP et al. CD1d-lipid antigen recognition by the cd TCR. Nat 14 Coffey F et al. The TCR ligand-inducible expression of CD73 marks Immunol 2013; 14: 1137–1145. lineage commitment and a metastable intermediate in effector 38 Pellicci DG et al. The molecular bases of d/ab T cell-mediated specification. J Exp Med 2014; 211: 329–343. antigen recognition. J Exp Med 2014; 211: 2599–2615. 15 Narayan K et al. Intrathymic programming of effector fates in three 39 Cerundolo V, Silk JD, Masri SH, Salio M. Harnessing invariant NKT molecularly distinct cd T cell subtypes. Nat Immunol 2012; 13: cells in vaccination strategies. Nat Rev Immunol 2009; 9: 28–38. 511–518. 40 Kain L et al. The identification of the endogenous ligands of natural 16 Zheng J, Liu Y, Lau YL, Tu W. cd-T cells: an unpolished sword in killer T cells reveals the presence of mammalian a-linked human anti-infection immunity. Cell Mol Immunol 2013; 10: 50– glycosylceramides. Immunity 2014; 41: 543–554. 57. 41 Bojarska-Junak A et al.CD1dexpressionishigherinchronic 17 Girardi M et al. Regulation of cutaneous malignancy by gammadelta lymphocytic leukemia patients with unfavorable prognosis. Leuk T cells. Science 2001; 294: 605–609. Res 2014; 38: 435–442. 18 Gober HJ et al. Human T cell receptor cells recognize endogenous 42 Lepore M et al. A novel self-lipid antigen targets human T cells mevalonate metabolites in tumor cells. J Exp Med 2003; 197: 163– against CD1c1 leukemias. J Exp Med 2014; 211: 1363–1377. 168. 43 De´chanet J et al. Implication of gammadelta T cells in the human 19 Clendening JW, Penn LZ. Targeting tumor cell metabolism with immune response to cytomegalovirus. J Clin Invest 1999; 103: statins. Oncogene 2012; 31: 4967–4978. 1437–1449. 20 Karunakaran MM, Go¨bel TW, Starick L, Walter L, Herrmann T. Vc9 44 Halary F. Shared reactivity of V 2neg T cells against cytomegalovirus- and Vd2 T cell antigen receptor genes and butyrophilin 3 (BTN3) infected cells and tumor intestinal epithelial cells. J Exp Med 2005; emerged with placental mammals and are concomitantly preserved 201: 1567–1578.

Cellular & Molecular Immunology cd T cells for cancer immunotherapy M Legut et al. 667

45 Couzi L et al. Cytomegalovirus-induced T cells associate with 66 Van Laethem F, Tikhonova AN, Singer A. MHC restriction is imposed reduced cancer risk after kidney transplantation. JAmSoc on a diverse T cell receptor repertoire by CD4 and CD8 co-receptors Nephrol 2010; 21: 181–188. during thymic selection. Trends Immunol 2012; 33: 437–441. 46 Alejenef A et al. Cytomegalovirus drives Vd2neg cd T cell inflation in 67 Van Laethem F et al. Deletion of CD4 and CD8 coreceptors permits many healthy virus carriers with increasing age: CMV distorts cd T generation of alphabetaT cells that recognize antigens indepen- cells over time. Clin Exp Immunol 2014; 176: 418–428. dently of the MHC. Immunity 2007; 27: 735–750. 47 Willcox CR et al. Cytomegalovirus and tumor stress surveillance by 68 Adoro S et al. Coreceptor gene imprinting governs thymocyte lineage binding of a human cd T cell antigen receptor to endothelial protein C fate. EMBO J 2012; 31: 366–377. receptor. Nat Immunol 2012; 13: 872–879. 69 Ullrich E, Koch J, Cerwenka A, Steinle A. New prospects on the 48 Oganesyan V. The crystal structure of the endothelial protein C NKG2D/NKG2DL system for oncology. Oncoimmunology 2013; 2: receptor and a bound phospholipid. JBiolChem2002; 277: e26097. 24851–24854. 70 Xu B et al. Crystal structure of a T-cell receptor specific for the 49 Mohan Rao LV, Esmon CT, Pendurthi UR. Endothelial cell protein C human MHC class I homolog MICA. Proc Natl Acad Sci USA receptor: a multiliganded and multifunctional receptor. Blood 2011; 108: 2414–2419. 2014; 124: 1553–1562. 71 Knight A et al. CMV-independent lysis of glioblastoma by ex vivo 50 Chen H et al. Identification of human T cell receptor gammadelta- expanded/activated Vd11 cd T cells. PLoS One 2013; 8: e68729. recognized epitopes/proteins via CDR3delta peptide-based 72 Xiang Z et al. Targeted activation of human Vc9Vd2-T cells controls immunobiochemical strategy. JBiolChem2008; 283: 12528– Epstein-Barr virus-induced B cell lymphoproliferative disease. 12537. Cancer Cell 2014; 26: 565–576. doi:10.1016/j.ccr.2014.07.026. 51 Groh V, Steinle A, Bauer S, Spies T. Recognition of stress-induced 73 Yin S et al. Vav1-phospholipase C- 1 (Vav1-PLC- 1) pathway initiated MHC molecules by intestinal epithelial gammadelta T cells. Science by T cell antigen receptor (TCR) activation is required to overcome 1998; 279: 1737–1740. inhibition by ubiquitin ligase Cbl-b during T cell cytotoxicity. J Biol 52 Martin SA, Lord CJ, Ashworth A. Therapeutic targeting of the DNA Chem 2013; 288: 26448–26462. mismatch repair pathway. Clin Cancer Res 2010; 16: 5107–5113. 74 Kong Y et al. The NKG2D ligand ULBP4 binds to TCR 9/2 and 53 Nadin SB, Cuello-Carrio´n FD, Sottile ML, Ciocca DR, Vargas-Roig induces cytotoxicity to tumor cells through both TCR and NKG2D. LM. Effects of hyperthermia on Hsp27 (HSPB1), Hsp72 (HSPA1A) Blood 2009; 114: 310–317. and DNA repair proteins hMLH1 and hMSH2 in human colorectal 75 Hewitt RE et al. The bisphosphonate acute phase response: rapid cancer hMLH1-deficient and hMLH1-proficient cell lines. Int J and copious production of proinflammatory cytokines by peripheral Hyperth Off J Eur Soc Hyperthermic Oncol North Am Hyperth blood gd T cells in response to aminobisphosphonates is inhibited by Group 2012; 28: 191–201. statins. Clin Exp Immunol 2005; 139: 101–111. 54 Dai Y, Chen H, Mo C, Cui L, He W. Ectopically expressed human 76 Wang H et al. Indirect stimulation of human V 2V 2 T cells through tumor biomarker MutS homologue 2 is a novel endogenous ligand alterations in isoprenoid metabolism. J Immunol 2011; 187: 5099– that is recognized by human T cells to induce innate anti-tumor/virus 5113. immunity. J Biol Chem 2012; 287: 16812–16819. 77 Idrees ASM et al. Comparison of cd T cell responses and farnesyl 55 Mo C, Dai Y, Kang N, Cui L, He W. Ectopic expression of human MutS diphosphate synthase inhibition in tumor cells pretreated with homologue 2 on renal carcinoma cells is induced by oxidative stress zoledronic acid. Cancer Sci 2013: 104: 536–542. with interleukin-18 promotion via p38 mitogen-activated protein 78 Benzaid I, Monkkonen H, Bonnelye E, Monkkonen J, Clezardin P. In kinase (MAPK) and c-Jun N-terminal kinase (JNK) signaling vivo phosphoantigen levels in bisphosphonate-treated human breast pathways. J Biol Chem 2012; 287: 19242–19254. tumors trigger V 9V 2 T-cell antitumor cytotoxicity through ICAM-1 56 Lu J et al. Induction of ATM/ATR pathway combined with Vc2Vd2T engagement. Clin Cancer Res 2012; 18: 6249–6259 cells enhances cytotoxicity of ovarian cancer cells. Biochim Biophys Acta BBA – Mol Basis Dis 2014; 1842: 1071–1079. 79 Todaro M et al. Chemotherapy sensitizes colon cancer initiating cells 57 Aggarwal R et al. Human Vc2Vd2 T cells limit breast cancer growth to Vc9Vd2 T cell-mediated cytotoxicity. PLoS One 2013; 8: by modulating cell survival-, apoptosis-related molecules and e65145. microenvironment in tumors: human Vc2Vd2 T cells limit breast 80 Brandes M. Professional antigen-presentation function by human gd cancer growth. Int J Cancer 2013; 133: 2133–2144. T cells. Science 2005; 309: 264–268. 58 Lamb LS et al. Engineered drug resistant cd T cells kill glioblastoma 81 Hoh A et al. The activity of cd T cells against paediatric liver tumour cell lines during a chemotherapy challenge: a strategy for combining cells and spheroids in cell culture. Liver Int 2013; 33: 127–136. chemo- and immunotherapy. PLoS One 2013; 8: e51805. 82 Oberg HH et al. Novel bispecific antibodies increase T-cell 59 Scheper W et al. cdT cells elicited by CMV reactivation after allo-SCT cytotoxicity against pancreatic cancer cells. Cancer Res 2014; 74: cross-recognize CMV and leukemia. Leukemia 2013; 27: 1328– 1349–1360. 1338. 83 Zheng J, Guo Y, Ji X, Cui L, He W. A novel antibody-like TCRcd-Ig 60 Zhu J et al. Immune surveillance by CD8aa1 skin-resident T cells in fusion protein exhibits antitumor activity against human ovarian human herpes virus infection. Nature 2013; 497: 494–497. carcinoma. Cancer Lett 2013; 341: 150–158. 61 Van Kaer L et al. CD8aa1 innate-type lymphocytes in the intestinal 84 Fisher J et al. Neuroblastoma killing properties of V-delta 2 and V- epithelium mediate mucosal immunity. Immunity 2014; 41: 451– delta2 negative gamma delta T cells following expansion by artificial 464. antigen presenting cells. Clin Cancer Res 2014; 20: 5720–5732. 62 Wooldridge L et al. Interaction between the CD8 coreceptor and doi:10.1158/1078-0432.CCR-13-3464. major histocompatibility complex class I stabilizes T cell receptor- 85 Couzi L et al. Antibody-dependent anti-cytomegalovirus activity of antigen complexes at the cell surface. JBiolChem2005; 280: human T cells expressing CD16 (Fc RIIIa). Blood 2012; 119: 1418– 27491–27501. 1427. 63 Purbhoo MA et al. The human CD8 coreceptor effects cytotoxic T cell 86 Maus MV, Grupp SA, Porter DL, June CH. Antibody-modified T cells: activation and antigen sensitivity primarily by mediating complete CARs take the front seat for hematologic malignancies. Blood 2014; phosphorylation of the T cell receptor zeta chain. J Biol Chem 2001; 123: 2625–2635. 276: 32786–32792. 87 Deniger DC et al. Bispecific T-cells expressing polyclonal repertoire 64 Holler PD, Kranz DM. Quantitative analysis of the contribution of of endogenous cd T-cell receptors and introduced CD19-specific TCR/pepMHC affinity and CD8 to T cell activation. Immunity chimeric antigen receptor. Mol Ther 2013; 21: 638–647. 2003; 18: 255–264. 88 Themeli M et al. Generation of tumor-targeted human T lymphocytes 65 Cole DK et al. The molecular determinants of CD8 co-receptor from induced pluripotent stem cells for cancer therapy. Nat function. Immunology 2012; 137: 139–148. Biotechnol 2013; 31: 928–933.

Cellular & Molecular Immunology cd T cells for cancer immunotherapy M Legut et al. 668

89 Morgan RA et al. Cancer regression in patients after transfer of follow-up from the ABCSG-12 randomised trial. Lancet Oncol genetically engineered lymphocytes. Science 2006; 314: 126–129. 2011; 12: 631–641. 90 Van Loenen MM et al. Mixed T cell receptor dimers harbor potentially 111 Morgan GJ et al. First-line treatment with zoledronic acid as harmful neoreactivity. Proc Natl Acad Sci USA 2010; 107: 10972– compared with clodronic acid in multiple myeloma (MRC 10977. Myeloma IX): a randomised controlled trial. The Lancet 2010; 91 Bendle GM et al. Lethal graft-versus-host disease in mouse models 376: 1989–1999. of T cell receptor gene therapy. Nat Med 2010; 16: 565–570, 1p 112 Bennouna J et al. Phase-I study of Innacell cdTM, an autologous cell- following 570. therapy product highly enriched in c9d2 T lymphocytes, in 92 Van der Veken LT et al. Alphabeta T-cell receptor engineered combination with IL-2, in patients with metastatic renal cell gammadelta T cells mediate effective antileukemic reactivity. carcinoma. Cancer Immunol Immunother 2008; 57: 1599–1609. Cancer Res 2006; 66: 3331–3337. 113 Kobayashi H, Tanaka Y, Yagi J, Minato N, Tanabe K. Phase I/II study 93 Van der Veken LT et al. Alpha beta T cell receptor transfer to gamma of adoptive transfer of cd T cells in combination with zoledronic acid delta T cells generates functional effector cells without mixed TCR and IL-2 to patients with advanced renal cell carcinoma. Cancer dimers in vivo. J Immunol 2009; 182: 164–170. Immunol Immunother 2011; 60: 1075–1084. 94 Hiasa A et al. Rapid alphabeta TCR-mediated responses in 114 Noguchi A et al. Zoledronate-activated Vc9cd Tcell-based gammadelta T cells transduced with cancer-specific TCR genes. immunotherapy is feasible and restores the impairment of cd T Gene Ther 2009; 16: 620–628. cells in patients with solid tumors. Cytotherapy 2011; 13: 92–97. 95 Xue SA et al. Human MHC class I-restricted high avidity CD4(1)T 115 Nicol AJ et al. Clinical evaluation of autologous gamma delta T cell- cells generated by co-transfer of TCR and CD8 mediate efficient based immunotherapy for metastatic solid tumours. Br J Cancer tumor rejection in vivo. Oncoimmunology 2013; 2: e22590. 2011; 105: 778–786. 96 Tan MP et al. TCR binding affinity governs the functional profile of 116 Cui J et al. Combination of radiofrequency ablation and sequential cancer-specific CD8(1) T cells. Clin Exp Immunol 2014. doi:10. cellular immunotherapy improves progression-free survival for 1111/cei.12570. patients with hepatocellular carcinoma: radiofrequency ablation 97 Zhao H, Xi X, Cui L, He W. CDR3d -grafted c9d2T cells mediate with cellular immunotherapy for HCC. Int J Cancer 2014; 134: effective antitumor reactivity. Cell Mol Immunol 2012; 9: 147–154. 342–351. 98 Marcu-Malina V et al. Redirecting T cells against cancer cells by 117 Wilhelm M et al. Successful adoptive transfer and in vivo expansion transfer of a broadly tumor-reactive T-cell receptor. Blood 2011; of haploidentical cd T cells. J Transl Med 2014; 12: 45. 118: 50–59. 118 Izumi T et al. Ex vivo characterization of cd T-cell repertoire in 99 Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy for patients after adoptive transfer of Vc9Vd2 T cells expressing the cancer: harnessing the T cell response. Nat Rev Immunol 2012; 12: interleukin-2 receptor b-chain and the common c-chain. 269–281. Cytotherapy 2013; 15: 481–491. 100 Kang N et al. Adoptive immunotherapy of lung cancer with 119 Nussbaumer O et al. Essential requirements of zoledronate-induced immobilized anti-TCRgammadelta antibody-expanded human cytokine and T cell proliferative responses. J Immunol 2013; 191: gammadelta T-cells in peripheral blood. Cancer Biol Ther 2009; 1346–1355. 8: 1540–1549. 120 Sugie T et al. Zoledronic acid-induced expansion of cd T cells from 101 Zhou J, Kang N, Cui L, Ba D, He W. Anti-cd TCR antibody-expanded early-stage breast cancer patients: effect of IL-18 on helper NK cd T cells: a better choice for the adoptive immunotherapy of cells. Cancer Immunol Immunother 2013; 62: 677–687. lymphoid malignancies. Cell Mol Immunol 2012; 9: 34–44. 121 Wada I et al. Intraperitoneal injection of in vitro expanded V c 9V d 2 102 Deniger DC et al. Activating and propagating polyclonal gamma delta T T cells together with zoledronate for the treatment of malignant cells with broad specificity for malignancies. Clin Cancer Res 2014; ascites due to gastric cancer. Cancer Med 2014; 3: 362–375. 20: 5708–5719. doi:10.1158/1078-0432.CCR-13-3451. 122 Clendening JW et al. Dysregulation of the mevalonate pathway 103 Dopfer EP et al. The CD3 conformational change in the cd T cell promotes transformation. Proc Natl Acad Sci USA 2010; 107: receptor is not triggered by antigens but can be enforced to enhance 15051–15056. tumor killing. Cell Rep 2014; 7: 1704–1715. 123 Priatel JJ, Chung BK, Tsai K, Tan R. Natural killer T cell strategies to 104 Lang JM et al. Pilot trial of interleukin-2 and zoledronic acid to combat Epstein–Barr virus infection. Oncoimmunology 2014; 3: augment cd T cells as treatment for patients with refractory renal cell e28329. carcinoma. Cancer Immunol Immunother 2011; 60: 1447–1460. 124 Cole DK et al. Human TCR-binding affinity is governed by MHC class 105 Kunzmann V et al. Tumor-promoting versus tumor-antagonizing restriction. J Immunol 2007; 178: 5727–5734. roles of cd T cells in cancer immunotherapy: results from a 125 Aleksic M et al. Different affinity windows for virus and cancer- prospective phase I/II trial. J Immunother 2012; 35: 205–213. specific T-cell receptors: implications for therapeutic strategies. 106 Welton JL et al. Monocytes and cd T cells control the acute-phase Eur J Immunol 2012; 42: 3174–3179. response to intravenous zoledronate: insights from a phase IV safety 126 Liddy N et al. Monoclonal TCR-redirected tumor cell killing. Nat Med trial. J Bone Miner Res 2013; 28: 464–471. 2012; 18: 980–987. 107 Rossini M et al. Long-term effects of amino-bisphosphonates on 127 Sheridan C. Monoclonal T-cell receptor drugs pique pharma’s circulating cd T cells. Calcif Tissue Int 2012; 91: 395–399. interest. Nat Biotechnol 2013; 31: 950–951. 108 Kalyan S, Chandrasekaran V, Quabius ES, Lindhorst TK, Kabelitz D. Neutrophil uptake of nitrogen-bisphosphonates leads to the This work is licensed under a Creative Commons suppression of human peripheral blood cd T cells. Cell Mol Life Attribution 3.0 Unported License. The images or other Sci 2014; 71: 2335–2346. third party material in this article are included in the article’s Creative 109 Coscia M et al. Dysfunctional V 9V 2 T cells are negative Commons license, unless indicated otherwise in the credit line; if the prognosticators and markers of dysregulated mevalonate pathway activity in chronic lymphocytic leukemia cells. Blood 2012; 120: material is not included under the Creative Commons license, users 3271–3279. will need to obtain permission from the license holder to reproduce 110 Gnant M et al. Adjuvant endocrine therapy plus zoledronic acid in the material. To view a copy of this license, visit http:// premenopausal women with early-stage breast cancer: 62-month creativecommons.org/licenses/by/3.0/

Cellular & Molecular Immunology