Rapid single-cell identification of Epstein–Barr virus-specific T-cell receptors for cellular therapy

Background and aimsEpstein–Barr virus (EBV) is associated with solid and hematopoietic malignancies. After allogeneic stem cell transplantation, EBV infection or reactivation represents a potentially life-threatening condition with no specific treatment available in clinical routine. In vitro expansion of naturally occurring EBV-specific T cells for adoptive transfer is time-consuming and influenced by the donor's T-cell receptor (TCR) repertoire and requires a specific memory compartment that is non-existent in seronegative individuals.The authors present highly efficient identification of EBV-specific TCRs that can be expressed on human T cells and recognize EBV-infected cells.Methods and ResultsMononuclear cells from six stem cell grafts were expanded in vitro with three HLA-B*35:01- or four HLA-A*02:01-presented peptides derived from six EBV proteins expressed during latent and lytic infection. Epitope-specific T cells expanded on average 42-fold and were single-cell-sorted and TCRαβ-sequenced. To confirm specificity, 11 HLA-B*35:01- and six HLA-A*02:01-restricted dominant TCRs were expressed on reporter cell lines, and 16 of 17 TCRs recognized their presumed target peptides. To confirm recognition of virus-infected cells and assess their value for adoptive therapy, three selected HLA-B*35:01- and four HLA-A*02:01-restricted TCRs were expressed on human peripheral blood lymphocytes. All TCR-transduced cells recognized EBV-infected lymphoblastoid cell lines.ConclusionsThe authors’ approach provides sets of EBV epitope-specific TCRs in two different HLA contexts. Resulting cellular products do not require EBV-seropositive donors, can be adjusted to cell subsets of choice with exactly defined proportions of target-specific T cells, can be tracked in vivo and will help to overcome unmet clinical needs in the treatment and prophylaxis of EBV reactivation and associated malignancies.     

Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions

Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157-165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3alpha and CDR2beta amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1(+)/HLA-A*02(+) tumor cell lines by TCR gene-modified CD4(+) T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3alpha chain was identified that enhanced Ag-specific reactivity in gene-modified CD4(+) and CD8(+) T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27-35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4(+) T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.     

MHC allele-specific molecular features determine peptide/HLA-A2 conformations that are recognized by HLA-A2-restricted T cell receptors

The structures of alphabeta TCRs bound to complexes of class I MHC molecules and peptide show that the TCRs make multiple contacts with the alpha1 and alpha2 helixes of the MHC. Previously we have shown that the A6 TCR in complex with the HLA-A2/Tax peptide has 15 contact sites on HLA-A2. Single amino acid mutagenesis of these contact sites demonstrated that mutation of only three amino acids clustered on the alpha1 helix (R65, K66, A69) disrupted recognition by the A6 TCR. In the present study we have asked whether TCRs that recognize four other peptides presented by HLA-A2 interact with the MHC in identical, similar, or different patterns as the A6 TCR. Mutants K66A and Q155A had the highest frequency of negative effects on lysis. A subset of peptide-specific CTL also selectively recognized mutants K66A or Q155A in the absence of exogenous cognate peptides, indicating that these mutations affected the presentation of endogenous peptide/HLA-A2 complexes. These findings suggest that most HLA-A2-restricted TCRs recognize surfaces on the HLA-A2/peptide complex that are dependent upon the side chains of K66 and Q155 in the central portion of the peptide binding groove. Crystallographic structures of several peptide/HLA-A2 structures have shown that the side chains of these critical amino acids that make contact with the A6 TCR also contact the bound peptide. Collectively, our results indicate that the generalized effects of changes at these critical amino acids are probably due to the fact that they can be directly contacted by TCRs as well as influence the binding and presentation of the bound peptides.     

A TCR targeting the HLA-A*0201-restricted epitope of MAGE-A3 recognizes multiple epitopes of the MAGE-A antigen superfamily in several types of cancer

Adoptive immunotherapy using TCR-engineered PBLs against melanocyte differentiation Ags mediates objective tumor regression but is associated with on-target toxicity. To avoid toxicity to normal tissues, we targeted cancer testis Ag (CTA) MAGE-A3, which is widely expressed in a range of epithelial malignancies but is not expressed in most normal tissues. To generate high-avidity TCRs against MAGE-A3, we employed a transgenic mouse model that expresses the human HLA-A*0201 molecule. Mice were immunized with two HLA-A*0201-restricted peptides of MAGE-A3: 112-120 (KVAELVHFL) or MAGE-A3: 271-279 (FLWGPRALV), and T cell clones were generated. MAGE-A3-specific TCR α- and β-chains were isolated and cloned into a retroviral vector. Expression of both TCRs in human PBLs demonstrated Ag-specific reactivity against a range of melanoma and nonmelanoma tumor cells. The TCR against MAGE-A3: 112-120 was selected for further development based on superior reactivity against tumor target cells. Interestingly, peptide epitopes from MAGE-A3 and MAGE-A12 (and to a lesser extent, peptides from MAGE-A2 and MAGE-A6) were recognized by PBLs engineered to express this TCR. To further improve TCR function, single amino acid variants of the CDR3 α-chain were generated. Substitution of alanine to threonine at position 118 of the α-chain in the CDR3 region of the TCR improved its functional avidity in CD4 and CD8 cells. On the basis of these results, a clinical trial is planned in which patients bearing a variety of tumor histologies will receive autologous PBLs that have been transduced with this optimized anti-MAGE-A3 TCR.     

Antigen-driven patterns of TCR bias are shared across diverse outcomes of human hepatitis C virus infection

Hepatitis C virus (HCV) infection causes significant morbidity and mortality worldwide. T cells play a central role in HCV clearance; however, there is currently little understanding of whether the disease outcome in HCV infection is influenced by the choice of TCR repertoire. TCR repertoires used against two immunodominant HCV determinants--the highly polymorphic, HLA-B*0801 restricted (1395)HSKKKCDEL(1403) (HSK) and the comparatively conserved, HLA-A*0101-restricted, (1435)ATDALMTGY(1443) (ATD)--were analyzed in clearly defined cohorts of HLA-matched, HCV-infected individuals with persistent infection and HCV clearance. In comparison with ATD, TCR repertoire selected against HSK was more narrowly focused, supporting reports of mutational escape in this epitope, in persistent HCV infection. Notwithstanding the Ag-driven divergence, T cell repertoire selection against either Ag was comparable in subjects with diverse disease outcomes. Biased T cell repertoires were observed early in infection and were evident not only in persistently infected individuals but also in subjects with HCV clearance, suggesting that these are not exclusively characteristic of viral persistence. Comprehensive clonal analysis of Ag-specific T cells revealed widespread use of public TCRs displaying a high degree of predictability in TRBV/TRBJ gene usage, CDR3 length, and amino acid composition. These public TCRs were observed against both ATD and HSK and were shared across diverse disease outcomes. Collectively, these observations indicate that repertoire diversity rather than particular Vβ segments are better associated with HCV persistence/clearance in humans. Notably, many of the anti-HCV TCRs switched TRBV and TRBJ genes around a conserved, N nucleotide-encoded CDR3 core, revealing TCR sequence mosaicism as a potential host mechanism to combat this highly variant virus.     

Conserved CDR3 regions in T-cell receptor (TCR) CD8(+) T cells that recognize the Tax11-19/HLA-A*0201 complex in a subject infected with human T-cell leukemia virus type 1: relationship of T-cell fine specificity and major histocompatibility complex/peptide/TCR crystal structure

We investigated the T-cell receptor (TCR) repertoire of CD8(+) T cells that recognize the Tax11-19 immunodominant epitope of Tax protein expressed by human T-cell leukemia virus (HTLV-1) that is implicated in the disease HTLV-1-associated myelopathy (HAM/TSP). A panel of Tax11-19-reactive CD8(+) T-cell clones was generated by single-cell cloning of Tax11-19/HLA-A*0201 tetramer-positive peripheral blood lymphocytes from an HTLV-1-infected individual. The analyses of TCR usage revealed that the combination of diverse TCR alpha and beta chains could be used for the recognition of Tax11-19 but the major population of T-cell clones (15 of 24 clones) expressed the TCR V beta 13S1 and V alpha 17 chain. We found striking similarities in CDR3 regions of TCR alpha and beta chains between our major group of CD8(+) T-cell clones and those originating from different subjects as previously reported, including TCRs with resolved crystal structures. A 3-amino-acid sequence (PG-G) in the CDR3 region of the V beta chain was conserved among all the Tax11-19-reactive T-cell clones expressing V beta 13S1 and V alpha 17 chains. Conserved amino acids in the CDR3 region do not directly contact the Tax11-19 peptide, as corroborated by the crystal structure of B7-TCR, a TCR that is almost identical to VB13S1 clones isolated in this study. Analysis of fine peptide specificity using altered peptide ligands (APL) of Tax11-19 revealed a similar recognition pattern among this panel of T-cell clones. These data suggest that the PG-G amino acids in the CDR3 beta loop provide a structural framework necessary for the maintenance of the tertiary TCR structure.     

TCRs used in cancer gene therapy cross-react with MART-1/Melan-A tumor antigens via distinct mechanisms

T cells engineered to express TCRs specific for tumor Ags can drive cancer regression. The first TCRs used in cancer gene therapy, DMF4 and DMF5, recognize two structurally distinct peptide epitopes of the melanoma-associated MART-1/Melan-A protein, both presented by the class I MHC protein HLA-A*0201. To help understand the mechanisms of TCR cross-reactivity and provide a foundation for the further development of immunotherapy, we determined the crystallographic structures of DMF4 and DMF5 in complex with both of the MART-1/Melan-A epitopes. The two TCRs use different mechanisms to accommodate the two ligands. Although DMF4 binds the two with a different orientation, altering its position over the peptide/MHC, DMF5 binds them both identically. The simpler mode of cross-reactivity by DMF5 is associated with higher affinity toward both ligands, consistent with the superior functional avidity of DMF5. More generally, the observation of two diverging mechanisms of cross-reactivity with the same Ags and the finding that TCR-binding orientation can be determined by peptide alone extend our understanding of the mechanisms underlying TCR cross-reactivity.     

Highly Divergent T-cell Receptor Binding Modes Underlie Specific Recognition of a Bulged Viral Peptide bound to a Human Leukocyte Antigen Class I Molecule

Human leukocyte antigen (HLA)-I molecules can present long peptides, yet the mechanisms by which T-cell receptors (TCRs) recognize featured pHLA-I landscapes are unclear. We compared the binding modes of three distinct human TCRs, CA5, SB27, and SB47, complexed with a “super-bulged” viral peptide (LPEPLPQGQLTAY) restricted by HLA-B*35:08. The CA5 and SB27 TCRs engaged HLA-B*35:08^LPEP similarly, straddling the central region of the peptide but making limited contacts with HLA-B*35:08. Remarkably, the CA5 TCR did not contact the α1-helix of HLA-B*35:08. Differences in the CDR3β loop between the CA5 and SB27 TCRs caused altered fine specificities. Surprisingly, the SB47 TCR engaged HLA-B*35:08^LPEP using a completely distinct binding mechanism, namely “bypassing” the bulged peptide and making extensive contacts with the extreme N-terminal end of HLA-B*35:08. This docking footprint included HLA-I residues not observed previously as TCR contact sites. The three TCRs exhibited differing patterns of alloreactivity toward closely related or distinct HLA-I allotypes. Thus, the human T-cell repertoire comprises a range of TCRs that can interact with “bulged” pHLA-I epitopes using unpredictable strategies, including the adoption of atypical footprints on the MHC-I.     

Conformational melding permits a conserved binding geometry in TCR recognition of foreign and self molecular mimics

Molecular mimicry between foreign and self Ags is a mechanism of TCR cross-reactivity and is thought to contribute to the development of autoimmunity. The αβ TCR A6 recognizes the foreign Ag Tax from the human T cell leukemia virus-1 when presented by the class I MHC HLA-A2. In a possible link with the autoimmune disease human T cell leukemia virus-1-associated myelopathy/tropical spastic paraparesis, A6 also recognizes a self peptide from the neuronal protein HuD in the context of HLA-A2. We found in our study that the complexes of the HuD and Tax epitopes with HLA-A2 are close but imperfect structural mimics and that in contrast with other recent structures of TCRs with self Ags, A6 engages the HuD Ag with the same traditional binding mode used to engage Tax. Although peptide and MHC conformational changes are needed for recognition of HuD but not Tax and the difference of a single hydroxyl triggers an altered TCR loop conformation, TCR affinity toward HuD is still within the range believed to result in negative selection. Probing further, we found that the HuD-HLA-A2 complex is only weakly stable. Overall, these findings help clarify how molecular mimicry can drive self/nonself cross-reactivity and illustrate how low peptide-MHC stability can permit the survival of T cells expressing self-reactive TCRs that nonetheless bind with a traditional binding mode.     

Peptide recognition by two HLA-A2/Tax11-19-specific T cell clones in relationship to their MHC/peptide/TCR crystal structures

The crystal structures of two human TCRs specific for a HTLV-I Tax peptide bound to HLA-A2 were recently determined, for the first time allowing a functional comparison of TCRs for which the MHC/peptide/TCR structures are known. Extensive amino acid substitutions show that the native Tax residues are optimal at each peptide position. A prominent feature of the TCR contact surface is a deep pocket that accommodates a tyrosine at position 5 of the peptide. For one of these TCRs, this pocket is highly specific for aromatic residues. In the other TCR structure, this pocket is larger, allowing many different residues to be accommodated. The CTL clones also show major differences in the specificity for several other peptide residues, including side chains that are not directly contacted by the TCR. Despite the specificity of these clones, peptides that are distinct at five or six positions from Tax11-19 induce CTL activity, indicating that substantial changes of the peptide surface are tolerated. Human peptides with limited sequence homology to Tax11-19 represent partial TCR agonists for these CTL clones. The distinct functional properties of these CTL clones highlight structural features that determine TCR specificity and cross-reactivity for MHC-bound peptides.     

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition

T cells use the αβ T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the “altered self” mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity.     

Distinct molecular mechanisms account for the specificity of two different T-cell receptors

Analysis of the thermodynamics of the interactions between the D3 T-cell receptor (TCR) and its natural ligand, an HIV peptide bound to a HLA-A0201 (HLA-A2) major histocompatibility complex (MHC) protein, shows both similarities and striking differences when compared with the 2B4 TCR binding to its peptide-MHC ligand. The equilibrium thermodynamic parameters of both reactions are consistent with a conformational adjustment at the binding interface during the formation of specific TCR-peptide-MHC complexes. However, osmolytic reagents that dehydrate protein surfaces have profoundly different effects on the strength of the two reactions, indicating that water molecules make very different contributions-enhancing the binding of D3 TCR but weakening the binding of 2B4 TCR. The use of these different mechanisms by TCRs to recognize ligands might be an important means augmenting their inherent cross-reactivity.     

Unraveling a hotspot for TCR recognition on HLA-A2: evidence against the existence of peptide-independent TCR binding determinants

T cell receptor (TCR) recognition of peptide takes place in the context of the major histocompatibility complex (MHC) molecule, which accounts for approximately two-thirds of the peptide/MHC buried surface. Using the class I MHC HLA-A2 and a large panel of mutants, we have previously shown that surface mutations that disrupt TCR recognition vary with the identity of the peptide. The single exception is Lys66 on the HLA-A2 alpha1 helix, which when mutated to alanine disrupts recognition for 93% of over 250 different T cell clones or lines, independent of which peptide is bound. Thus, Lys66 could serve as a peptide-independent TCR binding determinant. Here, we have examined the role of Lys66 in TCR recognition of HLA-A2 in detail. The structure of a peptide/HLA-A2 molecule with the K66A mutation indicates that although the mutation induces no major structural changes, it results in the exposure of a negatively charged glutamate (Glu63) underneath Lys66. Concurrent replacement of Glu63 with glutamine restores TCR binding and function for T cells specific for five different peptides presented by HLA-A2. Thus, the positive charge on Lys66 does not serve to guide all TCRs onto the HLA-A2 molecule in a manner required for productive signaling. Furthermore, electrostatic calculations indicate that Lys66 does not contribute to the stability of two TCR-peptide/HLA-A2 complexes. Our findings are consistent with the notion that each TCR arrives at a unique solution of how to bind a peptide/MHC, most strongly influenced by the chemical and structural features of the bound peptide. This would not rule out an intrinsic affinity of TCRs for MHC molecules achieved through multiple weak interactions, but for HLA-A2 the collective mutational data place limits on the role of any single MHC amino acid side-chain in driving TCR binding in a peptide-independent fashion.     

TCR alpha genes direct MHC restriction in the potent human T cell response to a class I-bound viral epitope

The underlying generic properties of alphabeta TCRs that control MHC restriction remain largely unresolved. To investigate MHC restriction, we have examined the CTL response to a viral epitope that binds promiscuously to two human leukocyte Ags (HLAs) that differ by a single amino acid at position 156. Individuals expressing either HLA-B*3501 (156Leucine) or HLA-B*3508 (156Arginine) showed a potent CTL response to the 407HPVGEADYFEY417 epitope from EBV. Interestingly, the response was characterized by highly restricted TCR beta-chain usage in both HLA-B*3501+ and HLA-B*3508+ individuals; however, this conserved TRBV9+ beta-chain was associated with distinct TCR alpha-chains depending upon the HLA-B*35 allele expressed by the virus-exposed host. Functional assays confirmed that TCR alpha-chain usage determined the HLA restriction of the CTLs. Structural studies revealed significant differences in the mobility of the peptide when bound to HLA-B*3501 or HLA-B*3508. In HLA-B*3501, the bulged section of the peptide was disordered, whereas in HLA-B*3508 the bulged epitope adopted an ordered conformation. Collectively, these data demonstrate not only that mobile MHC-bound peptides can be highly immunogenic but can also stimulate an extremely biased TCR repertoire. In addition, TCR alpha-chain usage is shown to play a critical role in controlling MHC restriction between closely related allomorphs.     

Molecular properties of gp100‐reactive T‐cell receptors drive the cytokine profile and antitumor efficacy of transgenic host T cells

To study the contribution of T‐cell receptors (TCR) to resulting T‐cell responses, we studied three different human αβ TCRs, reactive to the same gp100‐derived peptide presented in the context of HLA‐A*0201. When expressed in primary CD8 T cells, all receptors elicited classic antigen‐induced IFN‐γ responses, which correlated with TCR affinity for peptide–MHC in the order T4H2 > R6C12 > SILv44. However, SILv44 elicited superior IL‐17A release. Importantly, in vivo, SILv44‐transgenic T cells mediated superior antitumor responses to 888‐A2 + human melanoma tumor cells upon adoptive transfer into tumor‐challenged mice while maintaining IL‐17 expression. Modeling of the TCR ternary complexes suggested architectural differences between SILv44 and the other complexes, providing a potential structural basis for the observed differences. Overall, the data reveal a more prominent role for the T‐cell receptor in defining host T‐cell physiology than traditionally assumed, while parameters beyond IFN‐γ secretion and TCR affinity ultimately determine the reactivity of tumor‐reactive T cells.     

MHC recognition by hapten-specific HLA-A2-restricted CD8+ CTL

T cell recognition by peptide-specific alphabeta TCRs involves not only recognition of the peptide, but also recognition of multiple molecular features on the surface of the MHC molecule to which the peptide has been bound. We have previously shown that TCRs that are specific for five different peptides presented by HLA-A2 recognize similar molecular features on the surface of the alpha1 and alpha2 helices of the HLA-A2 molecule. We next asked whether these same molecular features of the HLA-A2 molecule would be recognized by hapten-specific HLA-A2-restricted TCRs, given that hapten-specific T cells frequently show reduced MHC dependence/restriction. The results show that a panel of CD8+ CTL that are specific for the hapten DNP bound to two different peptides presented by HLA-A2 do the following: 1) show stringent MHC restriction, and 2) are largely affected by the same mutations on the HLA-A2 molecule that affected recognition by peptide-specific CTL. A small subset of this panel of CD8+ CTL can recognize a mutant HLA-A2 molecule in the absence of hapten. These data suggest that TCR recognition of a divergent repertoire of ligands presented by HLA-A2 is largely dependent upon common structural elements in the central portion of the peptide-binding site.     

Peptide fine specificity of anti-glycoprotein 100 CTL is preserved following transfer of engineered TCR alpha beta genes into primary human T lymphocytes

TCR with known antitumor reactivity can be genetically introduced into primary human T lymphocytes and provide promising tools for immunogene therapy of tumors. We molecularly characterized two distinct TCRs specific for the same HLA-A2-restricted peptide derived from the melanocyte differentiation Ag gp100, yet exhibiting different stringencies in peptide requirements. The existence of these two distinct gp100-specific TCRs allowed us to study the preservation of peptide fine specificity of native TCRalphabeta when engineered for TCR gene transfer into human T lymphocytes. Retroviral transduction of primary human T lymphocytes with either one of the two sets of TCRalphabeta constructs enabled T lymphocytes to specifically kill and produce TNF-alpha when triggered by native gp100(pos)/HLA-A2(pos) tumor target cells as well as gp100 peptide-loaded HLA-A2(pos) tumor cells. Peptide titration studies revealed that the cytolytic efficiencies of the T lymphocyte transductants were in the same range as those of the parental CTL clones. Moreover, primary human T lymphocytes expressing either one of the two engineered gp100-specific TCRs show cytolytic activities in response to a large panel of peptide mutants that are identical with those of the parental CTL. The finding that two gp100-specific TCR, derived from two different CTL, can be functionally introduced into primary human T lymphocytes without loss of the Ag reactivity and peptide fine specificity, holds great promise for the application of TCR gene transfer in cancer immunotherapy.     

Determinants of public T cell responses

Historically, sharing T cell receptors (TCRs) between individuals has been speculated to be impossible, considering the dramatic discrepancy between the potential enormity of the TCR repertoire and the limited number of T cells generated in each individual. However, public T cell response, in which multiple individuals share identical TCRs in responding to a same antigenic epitope, has been extensively observed in a variety of immune responses across many species. Public T cell responses enable individuals within a population to generate similar antigen-specific TCRs against certain ubiquitous pathogens, leading to favorable biological outcomes. However, the relatively concentrated feature of TCR repertoire may limit T cell response in a population to some other pathogens. It could be a great benefit for human health if public T cell responses can be manipulated. Therefore, the mechanistic insight of public TCR generation is important to know. Recently, high-throughput DNA sequencing has revolutionized the study of immune receptor repertoires, which allows a much better understanding of the factors that determine the overlap of TCR repertoire among individuals. Here, we summarize the current knowledge on public T-cell response and discuss future challenges in this field.     

Knowledge-based virtual screening of HLA-A*0201-restricted CD8+ T-cell epitope peptides from herpes simplex virus genome

A novel knowledge-based method is developed to virtually screen potential HLA-A?0201 binders from large-scale peptide candidates. This method utilizes the information from both the crystal structures and experimental affinities of various peptides bound with HLA-A*0201 to construct a single-position mutation free energy profile for accurately characterizing HLA-A*0201-peptide interaction and for effectively predicting the binding affinities of peptides to HLA-A*0201. We employ this method to analyze physicochemical properties and structural implication underlying the specific recognition and association between the HLA-A*0201 and a large panel of peptide segments generated from the herpes simplex virus type 1 (HSV-1) genome, and to evaluate the binding potencies of these peptide candidates to HLA-A*0201. As a result, 288 out of 38,020 candidates are predicted as the potential high-affinity binders of HLA-A*0201, from which three most promising peptides are picked out for further development of potent vaccines against HSV-1. In addition, we also demonstrate that this newly proposed method can successfully identify 8 known binders and 3 known nonbinders from the glycoproteins D and K of HSV-1.     

Recognition of variant HIV-1 epitopes from diverse viral subtypes by vaccine-induced CTL

Recognition by CD8(+) T lymphocytes (CTL) of epitopes that are derived from conserved gene products, such as Gag and Pol, is well documented and conceptually supports the development of epitope-based vaccines for use against diverse HIV-1 subtypes. However, many CTL epitopes from highly conserved regions within the HIV-1 genome are highly variable, when assessed by comparison of amino acid sequences. The TCR is somewhat promiscuous with respect to peptide binding, and, as such, CTL can often recognize related epitopes. In these studies, we evaluated CTL recognition of five sets of variant HIV-1 epitopes restricted to HLA-A*0201 and HLA-A*1101 using HLA transgenic mice. We found that numerous different amino acid substitutions can be introduced into epitopes without abrogating their recognition by CTL. Based on our findings, we constructed an algorithm to predict those CTL epitopes capable of inducing responses in the HLA transgenic mice to the greatest numbers of variant epitopes. Similarity of CTL specificity for variant epitopes was demonstrated for humans using PBMC from HIV-1-infected individuals and CTL lines produced in vitro using PBMC from HIV-1-uninfected donors. We believe the ability to predict CTL epitope immunogenicity and recognition patterns of variant epitopes can be useful for designing vaccines against multiple subtypes and circulating recombinant forms of HIV-1.