Masters of Immunology

ab TCR-Mediated Recognition: Relevance toTumor-Antigen Discovery and CancerImmunotherapyEllis L. Reinherz1,2

Abstract

ab T lymphocytes sense perturbations in host cellular bodycomponents induced by infectious pathogens, oncogenic trans-formation, or chemical or physical damage.Millions to billions ofthese lymphocytes are generated through T-lineage developmentin the thymus, each endowed with a clonally restricted surfaceT-cell receptor (TCR). An individual TCR has the capacity torecognize a distinct "foreign" peptide among the myriad ofantigens that the mammalian host must be capable of detecting.TCRs explicitly distinguish foreign from self-peptides bound tomajor histocompatibility complex (MHC) molecules. This is adaunting challenge, given that the MHC-linked peptidome con-sists of thousands of distinct peptides with a relevant nonselftarget antigen often embedded at low number, among orders of

magnitude higher frequency self-peptides. In this Masters ofImmunology article, I review how TCR structure and attendantmechanobiology involving nonlinear responses affect sensitivityas well as specificity to meet this requirement. Assessment ofhuman tumor-cell display using state-of-the-artmass spectrometryphysical detection methods that quantify epitope copy numbercan help to provide information about requisite T-cell functionalavidity affording protection and/or therapeutic immunity. Futurerational CD8 cytotoxic T-cell–based vaccinesmay follow, targetingvirally induced cancers, other nonviral immunogenic tumors, andpotentially even nonimmunogenic tumors whose peptide displaycan be purposely altered by MHC-binding drugs to stimulateimmune attack. Cancer Immunol Res; 3(4); 305–12. �2015 AACR.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Editor's DisclosuresThe following editor(s) reported relevant financial relationships. G. Dranoff—None.

CME Staff Planners' DisclosuresThe members of the planning committee have no real or apparent conflicts of interest to disclose.

Learning ObjectivesResearch on the fundamental aspects of ab T-cell receptor (TCR) structure and function has informed infectious disease and oncology

disciplines about the nature of cognate antigen recognition by the T-cell adaptive immune system. This information, in turn, will lead to

effective development of CD8 T cell–based vaccines for preventive and immunotherapeutic purposes. Upon completion of this activity, the

participant should gain abasic knowledgeof themolecular structure of theabTCRand themechanobiology that allows a TCR to recognize a

distinct foreign peptide among myriad of antigens bound to the major histocompatibility complex with the required sensitivity and

specificity for host protection.

Acknowledgment of Financial or Other SupportThis activity does not receive commercial support.

IntroductionAdaptive immunity endows mammals and other jawed verte-

brates with precursors of T (thymus-derived) and B (bonemarrow–derived) lymphocytes able to generate a repertoire of clonotypicantigen receptors [T-cell receptor (TCR) and B-cell receptor (BCR)]of immense diversity from somatic rearrangements of variable genesegments (VDJ andVJ recombination). Spatiotemporally controlleddifferentiation and selection processes of those cells shape twocomplementary lineages of the immune system, offering protectionwith exquisite specificity, sensitivity, and long-term memory.

1Laboratory of Immunobiology andDepartment of Medical Oncology,Dana-FarberCancer Institute, Boston,Massachusetts. 2DepartmentofMedicine, Harvard Medical School, Boston, Massachusetts.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Ellis L. Reinherz, Dana-Farber Cancer Institute, 44Binney Street, Boston, MA 02115. Phone: 617-632-3412; Fax: 617-632-3351;E-mail: ellis_reinherz@dfci.harvard.edu

doi: 10.1158/2326-6066.CIR-15-0042

�2015 American Association for Cancer Research.

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Key discoveries during the last 50 years have unraveled thecellular andmolecular nature of adaptive immunity. In the 1960s,T and B lymphocytes were identified, and their interactions wereshown to be essential for antibody production (1, 2). The basicparadigm of immunoglobulin (Ig) gene rearrangements thatgenerate antibody diversity was revealed in 1976 (3). The "dual"specificity of T cells for a foreign peptide and a self-MHCmoleculeby functional studies was discovered and clearly noted to bedistinct from the "single" specificity of antibody recognition offoreign proteins (4, 5). This realization then led to an intenseeffort to understand the molecular puzzle represented by selfversus nonself discrimination and the receptor and ancillarymolecules on T cells responsible for this unusual recognition.

The discovery of how to expand T cells in vitro, via IL2-depen-dent T-cell cloning (6), in conjunctionwithmonoclonal antibody(7) and flow cytometry screening (8) technologies plus in vitrofunctional analyses, was decisive in molecular identification forthe long sought after TCR. A key set of advances came in the early1980s with the identification in humans of a clonotypic disulfide-linked heterodimer, the Ti ab TCR heterodimer, which, togetherwith CD3molecules, was essential for the peptide-MHC (pMHC)recognition and cellular activation (9–14). Biochemical evidenceshowed that, similar to Ig molecules, both Ti a and b chainspossessed variable and constant regions (9, 10). A comparable abTi was also identified by Kappler and Marrack in the mouse withsimilar cognate immune recognition features (15, 16). Thosemurine studies supported the earlier conjecture by Allison andcolleagues of a potential TCR-related molecule detected on amurine T-cell lymphoma (17). cDNAs for the TCRab genes wereobtained from the cloning efforts led by Davis and Mak (18–20)in mice and humans, respectively, identifying the b chain asshown by protein sequence (21). These studies showed that TCRcombinatorial diversity was generated by the same type of site-specific gene recombination mechanisms as with Ig genes, butwithout somatic hypermutation and led to identification of asecond type of TCR, the gd TCR (reviewed in ref. 3).

CD4 and CD8, surface molecules identified during the sameperiod, were recognized as coreceptors that optimize TCR recog-nition and T-cell activation via interaction with monomorphicsegments of MHC class II and class I molecules, respectively(22, 23). The "dual recognition" puzzle was solved when it wasshown that MHC class I and class II proteins bound foreign andself-peptides derived from degradation of intracellular or exoge-nous proteins and that such complexes could be recognized by theTCR (24). Three-dimensional structures of peptides complexedwith MHC molecules (pMHC) were defined (25, 26), as werestructures ofabTCRheterodimers in complexwithpMHC ligands(27–30).

Self versus nonself discrimination is at the core of T-lym-phocyte recognition. ab TCRs ligate foreign peptides bound toself-MHC, resulting in specific T-cell activation, proliferation,and effector function. In contrast, aside from homeostaticproliferation, self-peptide/self-MHC complexes are ignored andthus, activation inert. This preoccupation with foreign epitopesby ab T cells is generated in the thymus, where self-reactivethymocytes are deleted by an apoptotic negative selection mech-anism (ref. 31, and reviewed in ref. 32). Any residual self-reactive Tcells that escape deletion are controlled by regulatory T cells inthe peripheral lymphoid and tissue compartments (33).

If advances in molecular T-cell immunology can be rationallyapplied to tumor prevention and immunotherapy through vac-

cination, then precise identification and targeting of tumor anti-gens leading to destruction of cancer should be possible. Therecent success of chimeric antigen receptors using Ig-relatedectodomains and TCR signaling cytoplasmic components toinduce dramatic tumor remission in otherwise incurable patientssupports the validity of T-cell–based strategies (34). Eliminatingthe toxicities and cost of personalized gene therapy is a significantadvantage of an effective cytotoxic T cell (CTL)–based vaccineimmunotherapy that can inducehigh-avidity, tumor-specificCD8T cells. Fulfilling this promise requires a deep understanding ofadaptive T-cell recognition and the nuances of T-cell biology andmolecular structure.

The HLA Peptidome ChallengeIn humans, the MHC molecules are called HLA proteins.

Different people express versions encoded by different genevariants (alleles). In general, peptides associate with HLA mole-cules by inserting parts of their amino acid residues into a set of sixbinding pockets (termed A–F) in HLA. The structure of thesepockets is highly allele specific, thereby dictating peptide-bindingpreferences for each HLA molecule.

As shown in Fig. 1A, in the cytoplasmofmost human cells, self-proteins as well as foreign proteins, such as those from a virus thathas infected a cell, are cleaved intomultiple peptides by a complexcalled the proteasome. A subset of these peptides is then carried bytransporters associated with antigen processing (TAP) proteins tothe endoplasmic reticulum. Here, an even more limited set ofpeptides is loaded onto HLAmolecules, which are transported tothe cell surface. These surface-displayed HLA-peptide complexesare recognized by TCRs on the surface of T cells. Specific CD8þ Tcells can recognize the infected cells and induce a lytic programthat kills these virally infected targets.

The MHC-bound peptides recognized by T cells are typically 8to 11 amino acids in length with single amino acid substitutionsreadily sensed and discriminated by a TCR. The entire array ofMHC-linked peptides is referred to as the HLA peptidome inhumans. During immune surveillance, an individual high-avidityT cell has the capacity to detect one to several copies of a specificpMHC on the antigen-presenting cell (APC) that expresses100,000 chemically similar pMHC molecules (35). Discrimina-tion of foreign versus myriad self-peptides is manifested withprecision; if this were not the case, then either autoimmunity orimmunodeficiency would result. Figure 1B shows a tumor-cellsurface display with only one tumor antigen surface expressedamong a huge number of self-pMHC molecules. How TCRs arecapable of mediating such exquisite specificity and sensitivitywas previously a mystery, especially in view of the fact thatunlike with B-cell receptors, there is no somatic hypermutationof TCR genes. Monomeric TCR-pMHC affinities are orders ofmagnitude weaker than high-affinity antibody–antigen inter-actions (36). As described below, however, the elucidation ofthe TCR as a mechanotransducer with dynamic, nonlinearresponses explains this behavior.

TCR StructureFigure 2 offers a model of the TCR complex of the ab hetero-

dimer consisting of V (variable) and C (constant) modules, theCD3eg and CD3ed ectodomains (37), defines a plausible subunittopology, and emphasizes its glycan richness. The multiple

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N-linked glycan adducts of the TCR complex (Fig. 2B) help guidepMHC ligands to the TCR recognition surface, reducing entropicpenalties by directing binding to the exposed, glycan-free com-plementarity-determining region loops at the top of the structure.Given that CD3z has only a 9-amino acid–long ectosegment, it isomitted from Fig. 2 as are the connecting peptides (CP). Thisrendering incorporates the consequences of several known TCRcharacteristics: (i) putative transmembrane charge pairs involvingTCR subunit chain association (Fig. 2A) with CD3e-CD3d-TCRa-CD3z-CD3z as one cluster and CD3e-CD3g-TCRb as a secondcluster (38, 39); (ii) extracellular domain associations involvingother in vitro chain association data (40–42); and (iii) proximityof one CD3e subunit to the TCR Cb FG loop (designated by anasterisk (�) in Fig. 2; ref. 43). Evident in Fig 2A and B is the centralposition of the TCRab heterodimer with a vertical dimension of80Å projecting from the cell membrane, flanked on either side bythe shorter (40Å) CD3 heterodimers, CD3ed on the "left" TCRaside and CD3eg on the "right" TCRb side. The width of the CD3edand CD3eg components, 50 Å and 55 Å, respectively, is compa-rable in size with that of the TCRab heterodimer (58 Å), andtogether (excluding glycans) span �160 Å. These flanking CD3ectodomain components will likely impede lateral movement ofthe TCRab heterodimer upon pMHC binding. The 5– to 10–amino acid squat and rigid CD3 CP segments (44) contrastsharply with the long (19–26aa) and flexible TCR a and b CPlinking their respective constant domains to the transmembranesegments (Fig. 2A).

This contrasting array suggested that the ab TCR heterodimermay bend and extend relative to the squat and rigid CD3 hetero-dimers. For example, if as shown in Fig. 2C (from right to left), the

pMHC on the APC is first ligated by a specific TCR, then as the Tcell continues to move prior to a stopmovement signal mediatedthrough inside-out integrin affinity upregulation, pMHC func-tions as a force transducing handle to pull on the TCRab hetero-dimer. This force is amplified and exerted on CD3eg by the leverarm aided by the Cb FG loop (depicted in magenta) where theTCRb transmembrane acts as a fulcrum. Supplementary MoviesS1 and S2 reveal the large forces that result from T-cell immunesurveillance of epithelial surfaces and the likely impact of thismovement on the TCR complex quaternary conformationalchanges. Signal transduction involving common structural sub-unit conformational rearrangement rather than alterations withinclonotypic ab heterodimers per se offers a basic activation mech-anism common to all TCRs.

MechanotransductionThe simple notion that the TCR is a mechanosensor has been

codified by several independent studies. Kim and colleagues (44)provided the first direct evidence of the influence of mechanicalforce in TCR activation. Using an optically trapped bead coatedwith pMHC or anti-CD3 monoclonal antibody for engaging theTCR, T cellsweremechanically triggered by applying anoscillatingtangential force to the cell surface while monitoring their activa-tion via intracellular calcium flux. Importantly, piconewton (pN)force application with cognate pMHC but not irrelevant pMHCtriggered activation. Additional mechanosensor evidence wasprovided in studies by Li and colleagues using a micropipette todemonstrate shear force associated with activation (45) andHusson and colleagues employing a biomembrane force probe

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Figure 1.Processing and presentation ofHLA-bound self- and foreign peptides.A, peptides are generated throughproteolysis in the proteasome,transported to the endoplasmicreticulum by TAP, associated withHLA molecules therein, and thenexported and cell surface displayed.B, artistic rendition of 50,000 to100,000 pMHC molecules on a cellsurface (blue/purple) with a singletumor antigen (yellow) among theMHC-bound peptidome, emphasizingthe daunting challenge of TCR-basedrecognition. This figure was renderedby Steve Moskowitz of AdvancedMedical Graphics, Boston, MA.

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(BFP) to reveal pushing and pulling associated with T-cell trig-gering (46). Triggering was also shown by Judokusumo andcolleagues to depend on substrate stiffness (47). Prediction thata nonlinear mechanical response such as catch-bond formationmight facilitate TCR-based recognition (44) was elegantly con-firmed by Liu and colleagues using a BFP (48).

Physical forces are generated through T-lymphocyte move-ment during immune surveillance as well as by cytoskeletalrearrangements at the immunological synapse following cessa-tion of cell migration (ref. 49 and references therein). Neverthe-less, the mechanistic explanation for how TCRs distinguishbetween foreign and self-peptides bound to a given MHC mol-ecule has been unclear: peptide residues themselves comprisefew of the TCR contacts on the pMHC, and pathogen-derivedpeptides are scant among myriad self-peptides bound to thesame MHC class arrayed on infected cells, as noted above. Usingoptical tweezers and DNA tether spacer technology that permitpN force application and nm scale precision, Das and colleagues(49) have determined how bioforces relate to self- versus nonselfdiscrimination. Single-molecule analyses involving isolated abheterodimers as well as complete TCR complexes on T lympho-cytes reveal that the FG loop in the b subunit constant domain(Fig. 2; depicted in magenta) allosterically controls both thevariable domain module's catch-bond lifetime and peptidediscrimination via force-driven conformational transition. Incontrast with slip bonds that release under physical load, catchbonds become stronger so that TCR-pMHC single-bond lifetimesextend. For a representative CD8-derived ab TCR heterodimerlike N15 that binds a rabies family vesicular stomatitis viruspeptide, the bond lifetime goes from 0.3 seconds at zero force to

>3 seconds at 15 pN. Such low forces are readily achievable inbiologic systems.

Ligation of the relevant TCRab heterodimer initiates a cascadeof T-cell signaling events following exposure of the immunore-ceptor tyrosine-based activation motif (ITAM) elements in thecytoplasmic tail of thenoncovalently associated subunits (CD3eg ,CD3ed, and CD3zz) comprising the TCR complex in 1:1:1:1dimer stoichiometry. This accessibility allows the active kinase,Lck, to bind and phosphorylate ITAMs followed by recruitmentand activation of a second tyrosine kinase, ZAP-70 (50–53). Inturn, multiple downstream pathways are engaged including tran-scriptional regulators controlling activation and differentiation ofT cells (54, 55). Thymocyte development is also regulated by theTCR–pMHC interaction as it relates to repertoire selection(reviewed in ref. 32). The extended bond lifetime under forcewill foster greater subunit conformational alterations and mem-brane-lipid perturbation and facilitate exposure of sequesteredCD3 cytoplasmic tail ITAMs.

Kinetic proofreading has been described as a mechanism per-mitting biologic systems to finely discriminate between ligandswhich show small differences in their affinity (56, 57). Clearly, thenonequilibrium force-driven TCR–pMHC bond lifetime charac-teristics noted above allow considerable discrimination betweenself-pMHC versus foreign-pMHC complexes. The differentialligation kinetics and attendant conformational changes are suf-ficient to afford major signaling differences between stimulatoryand nonstimulatory pMHC ligands. This discrimination is madeeven more robust in the formation of the signalosome at theimmunological synapse. There in the immunological synapse,signaling is influenced by TCR ligation in both time and space,

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A

C

BFigure 2.ab TCR complex interaction withpMHC. A, TCR components[ectodomains, stalk CPs (depicted asred, blue, chartreuse, and turquoiselines), transmembrane (TM)segments, and cytoplasmic tails) arelabeled and shown in distinct colors.An enlarged view is shown in the box.Note that the single acid residue ineach CD3 subunit is omitted here forclarity. The pMHC on the APC and theinteracting CD8 coreceptor are notcolored. In each panel, the Cb FG loopis depicted by an asterisk (�). B, lateralview of TCR receptor components inribbon form (PDB:IFND, 1XM and1JBJ) oriented above the T-cellmembrane (gray). Adducted sugarsare depicted in beige in space-filling(CPK) representation. CD3zz isabsent since it lacks ectodomains.C, force on TCR–pMHC interactioninitiates signaling (pMHC, orange;Cb FG loop, magenta; and TCRcomplex, other colors). This figurewas rendered by Steve Moskowitzof Advanced Medical Graphics,Boston, MA. See accompanyingSupplementary Movies S1 and S2.

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further enhancing discrimination between foreign-pMHC versusself-pMHC interaction. Explicitly, T-cell triggering might occur asa result of one foreign-pMHC ligand interacting with several TCRsthrough serial engagement, or alternatively, more than one copyof the same foreign-pMHC interacting with several TCRs inproximity. Given the specificity of the TCR mechanosensingmechanism under force, it is highly improbable that repeated"mistakes" would follow and result in false activation. In con-clusion to this section, it is clear that the TCR signaling behaviorcannot be readily rationalized in the absence of force. Solutionbinding may be strong between a TCR ab heterodimer andpMHC, but cellular activation may be nevertheless absent (58).The way in which a TCR binds to pMHC and the latter is coligatedby the coreceptor (CD8 in the example shown in Fig. 2A) placesrestrictions on the permissible TCR-binding orientations topMHC. TCR and coreceptor bind to the same pMHC, a so-calledbidentate interaction, positioning lck associated with the core-ceptor to phosphorylate ITAMs of the CD3 cytoplasmic tails.

Functional AvidityGiven the TCR mechanosensor properties noted above, CTL

targeting need not be dependent on display of a large number ofepitopes by single tumor cell or infected cell. The key for protectiveT-cell effector function is the requisite match between the T cell'sfunctional avidity and the relevant epitope copy number pertarget cell. Functional avidity refers to the sensitivity of a particularT cell to be triggered by pMHC on an APC (or target) and tomediate its function. High-avidity T cells are capable of recogniz-ing a very small number of pMHC/target cells, whereas low-avidity T cells may require hundreds or even thousands ofsuch recognition effects (59–61). Without such relevant avidity,effective cytolysis will not follow. Avidity is dependent on theTCR–pMHC interaction, CD8 coreceptor expression, intracellularsignaling molecules, and other factors (62). With respect to theTCR, the Vb and Va gene repertoires as well as the nature ofthe antigen itself contribute. Unfeatured pMHCI molecule sur-faces, like influenza A M158–66 bound to HLA-A�0201, stronglyelicit T cells which, although plentiful, comprise a low-avidityimmunodominant response (63, 64). As such, display require-ments on infected epithelium for activation of those CTLsmay beat toohigh a copynumber to be achievedduring natural infection.Consequently, this CTL response would be nonprotective. Imag-ine that a high-avidity T cell recognizes its target with a 20/20vision while the low-avidity T cell has 20/200, i.e., is legally blind.Identifying correct targets and deploying useful CTLs at a tumorsite or nidas of infection are critical. In contrast, if ineffective T cellsmove into the site, it is counterproductive as T-cell infiltration is offinite magnitude within tissues and impedes deployment ofprotective CTLs. Moreover, high functional avidity T-cell interac-tions confer a cellular state refractory to inhibitors like TGFb (65).

Physical Detection and Quantification ofHLA-Bound Peptides

Until very recently, the primary approach for peptide identifi-cation has been to isolate antigen-experienced T cells from sometissue compartment and demonstrate that these cells functionally"recognize" specific pMHC using an activation-readout such ascytokine production. Beyond technical issues delineated else-where (66), this "reverse immunology" identifies only antigenic

peptides and not those nonantigenic foreign peptides that aredisplayed as surface pMHC.However, nonantigenic peptidesmaybe stealth as a consequence of immunodominance of othersegments (i.e., M158–66 noted above), but such peptides may bereadily capable of generating protective immune responses if theyare identified and then used in vaccine formulations. A wayaround this limitation is the use of physical identification meth-ods, such as mass spectrometry (MS), that have overcome ana-lytical challenges posed by the complex set of peptides bound toMHC as derived from protein metabolism and displayed at thecell surface (refs. 67–69 and reviewed in ref. 66). The dynamicrange of detection, defined as the target's fraction of the total ionflux, has been directly estimated and is on the order of 105.The sensitivity of detection compares well with the most sensitiveT-cell clonewe have generated. A high-avidity T cell cannot exceeda limit lower than one target pMHC among 100,000 irrelevantpMHCs per target cell. The theory of the method and its use inidentifying HPV-16 antigens have been described (67–69). Rapidelectronic data capture, high-resolution mass spectrometers, andinformation-rich precursor and ion fragment beams allow deep-targeted interrogation and reinterrogation of precious samplesafter the sample is acquired and data archived (70).

Anobjective for a therapeutic antitumor vaccine is to focusCTLson HLA-bound peptides restricted to the cancer cell and deployhigh-avidity CTLs at the tumor site to foster elimination of thecancer. The cellular scale and sensitivity of the aforementionedMSdetection methods permits such identification. An example is theset of cancers caused by human papillomavirus 16 (HPV-16). Ofnote, in cases in which HPV-16 infection has induced epithelialtransformation and cervical cancer in HLA-A�0201 hosts, only asingle epitope from the E7 oncogene product, E711–19, is naturallyprocessed and presented by this allele on those tumor cells.Pointedly, although E711–20 is capable of binding equivalentlyto this same HLA allele, the 10-mer peptide, unlike the 9-mer, isnot displayed on the primary epithelial cells. In contrast, when alarge fragment of E7 as a synthetic peptide is exogenously added toHLA-A�0201 professional APCs, the E711–20 is displayed to a verylarge extent (67). Because T cells are not strongly cross-reactive andare particularly specified to a single peptide length (71, 72), thisdiscordance misguides the immune response. It also explains, insignificant part, why the 10-mer vaccinewaswithout clinical effectin a therapeutic HPV-16 cancer trial (73). These data also cautionthat what is directly presented by tumor cells versus cross-pre-sented by dendritic cells is not necessarily the same. Quantifyingcopy number of pMHC complexes per cell has also been imple-mented with this approach (68, 70), providing insight into CTLfunctional avidity requirements.

A Path for Creating CTL-TargetingVaccines

More than 20% of tumors worldwide are caused by viruses, withhigh-risk humanpapillomaviruses alone responsible for >5%of allcancers (74). A CTL-based approach should prove useful for com-bating infectious diseases in general and those 20% of cancerscaused by viruses in particular. In this regard, there are fourcomponents required tocreate aneffectiveCD8-basedT-cell vaccinepipeline: (i) facile bioinformatic prediction of conserved proteinsegments of pathogens that include potential T-cell epitopes fromvariable viral strains; (ii) physical detection MS methodologies todetermine which of the predicted epitopes are actually arrayed on

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infected cells and APCs for T-cell recognition by TCRs; (iii) nano-vaccine technology to deliver conserved T-cell epitope payloadsincluding adjuvants to APCs for stimulating epitope display inappropriate lymphoid tissue at optimal density and duration; and(iv) insightful memory T-cell biology arising from transcriptomics,proteomics, and other molecular analyses of CD8 subsets definingtheirdevelopmentandelucidating the rulesofphysical deploymentinto tissue compartments. Collectively, these technologies andknowledge will create vaccines that elicit potent CD8 memory Tcells with effector function that reside at sites of potential viralattack. Such resident-memory T cells (TRM) are positioned forimmediate action and are in turn reinforced by subsequent recruit-ment of T effector (TEM) and T central memory (TCM) cells fromblood and secondary lymphoid tissues (75, 76). In this manner, aprompt immune response is engendered that minimizes viralreplication with attendant pathologic consequences.

Of note, in addition to the above MS technologies, computa-tional methods are already available. These offer bioinformatictools and databases to focus on peptide epitopes conservedamong diverse strains of a given virus, predict peptide bindingto multiple HLA alleles, and estimate population coverage basedon HLA frequencies (77, 78, and references therein). Vaccinetechnology using synthetic materials to target organs, tissues, orcells and deliver concurrently epitopes and immunomodulatorypayloads also exists (79). The above principles can be applied toimmunogenic tumors as well as nonimmunogenic tumors asdescribed in the section below.

Regulation of Immune ResponsesThe effectiveness of adaptive immunity is predicated not only

on the nature of cognate antigen recognition via TCR–pMHCinteraction but, in addition, on the various pathways that sup-press T-cell activation. These anti-inflammatory mechanismsinvolve inhibitory small molecules like adenosine and carbonmonoxide, proteins such as CTLA-4 and PD-1, and cells includingregulatory T cells (reviewed in refs. 80–82). It seems most prob-able, therefore, that a combination of focused CD8-based vaccineadministration in conjunction with immunotherapy to blockinhibitory pathways will elicit the greatest antitumor response.Because CD4 T-cell help is important for optimal T-cell function(see an overview in ref. 83), an epitope activating CD4 T cellsshould also be provided in the vaccination protocol.

Conclusions and Future ApplicationsThe above MS technology will permit identification of rel-

evant viral epitopes derived from acute infectious pathogens as

well as virally induced cancers. In turn, molecular TCR cloningmethods can be used to identify those TCRs on naturallyderived and vaccine-elicited T cells to determine if necessaryavidity thresholds have been achieved, either by assessingfunctional avidity of TCR transfectants and cytokine productionreadouts or alternatively, single-molecule quantification ofTCR–pMHC interactions (49).

For nonviral tumors that are immunogenic, whole exome and/or transcriptome sequencing of individual tumors in conjunctionwith MS can identify mutant peptides for vaccine formulation inan individualized patient approach. The feasibility of such astrategy has been recently shown in a mouse model (84).

Lastly, for nonimmunogenic tumors, induction of the expres-sion of multiple neopeptide epitopes could target a polyclonalCTL attack against a cancer. In this regard, the antiviral HIV drugabacavir binds tooneHLAmolecule andalters its peptide-bindingcharacteristics, causing the HLA molecule to load and display anew range of peptides on the cell surface. Because this repertoireincludes self-peptides that were not displayed during T-cell devel-opment, the immune system contains T cells that can recognizethese antigens and launch an immune attack against the cell (85).The abacavir interaction is specific to the HLA-B�57:01 allele's Fpeptide binding pocket, but other drugs targeting products ofother HLA alleles exist. In principle, these chemicals and medic-inal compounds specific for various alleles could be directed in arestricted fashion to tumor cells bymonoclonal antibody or othermeans to alter the tumor peptidome and to target the cancer forCTL destruction. Such specificity would mitigate systemic allergicreactions, focusing only on the tumor. The use of the power andspecificity of the immune system is being exploited for tumorimmunotherapywith some exciting results. Asmore details aboutthe adaptive T-cell immune system and its regulation are uncov-ered, this strategy is likely to rise exponentially.

AcknowledgmentsThe author thanks allmembers of the Laboratory of Immunobiology for their

efforts and thoughtful insights.

Grant SupportE.L. Reinherz was supported by an SU2C-Farrah Fawcett FoundationHuman

Papillomavirus (HPV) Translational Cancer Research Team Grant (grant num-ber SU2C-AACR-DT13-14) and by the NIH (grant no. NIH UO1 AI90043).Stand Up To Cancer is a program of the Entertainment Industry Foundationadministered by the American Association for Cancer Research. The FarrahFawcett Foundation is a 501(c)(3) nonprofit dedicated to cancer research,prevention, and patient support.

Received February 5, 2015; accepted February 16, 2015; published onlineApril 6, 2015.

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TCR-Mediated Recognition: Relevance to Tumor-Antigenβα

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