Search

Polypeptide vaccines effectively activate human T cells but suffer from poor biological stability, which confines both transport logistics and in vivo therapeutic activity. Synthetic biology has the potential to address these limitations through the generation of highly stable antigenic "mimics" using subunits that do not exist in the natural world. We developed a platform based on D-amino acid combinatorial chemistry and used this platform to reverse engineer a fully artificial CD8+ T cell agonist that mirrored the immunogenicity profile of a native epitope blueprint from influenza virus. This nonnatural peptide was highly stable in human serum and gastric acid, reflecting an intrinsic resistance to physical and enzymatic degradation. In vitro, the synthetic agonist stimulated and expanded an archetypal repertoire of polyfunctional human influenza virus-specific CD8+ T cells. In vivo, specific responses were elicited in naive humanized mice by subcutaneous vaccination, conferring protection from subsequent lethal influenza challenge. Moreover, the synthetic agonist was immunogenic after oral administration. This proof-of-concept study highlights the power of synthetic biology to expand the horizons of vaccine design and therapeutic delivery.

CD8+ T-cells play a role in the pathogenesis of autoimmune diseases such as multiple sclerosis and type 1 diabetes. However, drugs that target the entire CD8+ T-cell population are not desirable because the associated lack of specificity can lead to unwanted consequences, most notably an enhanced susceptibility to infection. Here, we show that autoreactive CD8+ T-cells are highly dependent on CD8 for ligand-induced activation via the T-cell receptor (TCR). In contrast, pathogen-specific CD8+ T-cells are relatively CD8-independent. These generic differences relate to an intrinsic dichotomy that segregates self-derived and exogenous antigen-specific TCRs according to the monomeric interaction affinity with cognate peptide-major histocompatibility complex class I (pMHCI). As a consequence, "blocking" anti-CD8 antibodies can suppress autoreactive CD8+ T-cell activation in a relatively selective manner. These findings provide a rational basis for the development and in vivo assessment of novel therapeutic strategies that preferentially target disease-relevant autoimmune responses within the CD8+ T-cell compartment.

In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein-Barr virus (EBV) latency-II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV(+) cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA-A*02 is protective in EBV(+) cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA-A*02(-) versus HLA-A*02(+) EBV(+) cHL patients, suggesting that LMP2A-specific CD8(+) T cell anti-tumoral immunity may be relatively ineffective in HLA-A*02(-) EBV(+) cHL. To ascertain the impact of HLA class I on EBV latency antigen-specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV(+) cHL, the magnitude of ex-vivo LMP1/2A-specific CD8(+) T cell responses was elevated in HLA-A*02(+) patients. Furthermore, in a controlled in-vitro assay, LMP2A-specific CD8(+) T cells from healthy HLA-A*02 heterozygotes expanded to a greater extent with HLA-A*02-restricted compared to non-HLA-A*02-restricted cell lines. In an extensive analysis of HLA class I-restricted immunity, immunodominant EBNA3A/3B/3C-specific CD8(+) T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A-specific responses were confined largely to HLA-A*02. Our results demonstrate that HLA-A*02 mediates a modest, but none the less stronger, EBV-specific CD8(+) T cell response than non-HLA-A*02 alleles, an effect confined to EBV latency-II antigens. Thus, the protective effect of HLA-A*02 against EBV(+) cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency-II antigen-specific CD8(+) T cell hierarchies.

To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.

Exposure to naturally occurring variants of herpesviruses in clinical settings can have a dramatic impact on anti-viral immunity. Here we have evaluated the molecular imprint of variant peptide-MHC complexes on the T-cell repertoire during human cytomegalovirus (CMV) infection and demonstrate that primary co-infection with genetic variants of CMV was coincident with development of strain-specific T-cell immunity followed by emergence of cross-reactive virus-specific T-cells. Cross-reactive CMV-specific T cells exhibited a highly conserved public T cell repertoire, while T cells directed towards specific genetic variants displayed oligoclonal repertoires, unique to each individual. T cell recognition foot-print and pMHC-I structural analyses revealed that the cross-reactive T cells accommodate alterations in the pMHC complex with a broader foot-print focussing on the core of the peptide epitope. These findings provide novel molecular insight into how infection with naturally occurring genetic variants of persistent human herpesviruses imprints on the evolution of the anti-viral T-cell repertoire.

T cell repertoire diversity and clonotype follow-up in vaccination, cancer, infectious and immune diseases represent a major challenge owing to the enormous complexity of the data generated. Here we describe a next generation methodology, which combines 5'RACE PCR, 454 sequencing and, for analysis, IMGT, the international ImMunoGeneTics information system (IMGT), IMGT/HighV-QUEST web portal and IMGT-ONTOLOGY concepts. The approach is validated in a human case study of T cell receptor beta (TRB) repertoire, by chronologically tracking the effects of influenza vaccination on conventional and regulatory T cell subpopulations. The IMGT/HighV-QUEST paradigm defines standards for genotype/haplotype analysis and characterization of IMGT clonotypes for clonal diversity and expression and achieves a degree of resolution for next generation sequencing verifiable by the user at the sequence level, while providing a normalized reference immunoprofile for human TRB.

In comparison to human leukocyte antigen (HLA) polymorphism, the impact of allelic sequence variation within T cell receptor (TCR) loci is much less understood. Particular TCR loci have been associated with autoimmunity, but the molecular basis for this phenomenon is undefined. We examined the T cell response to an HLA-B*3501-restricted epitope (HPVGEADYFEY) from Epstein-Barr virus (EBV), which is frequently dominated by a TRBV9*01(+) public TCR (TK3). However, the common allelic variant TRBV9*02, which differs by a single amino acid near the CDR2beta loop (Gln55-->His55), was never used in this response. The structure of the TK3 TCR, its allelic variant, and a nonnaturally occurring mutant (Gln55-->Ala55) in complex with HLA-B*3501(HPVGEADYFEY) revealed that the Gln55-->His55 polymorphism affected the charge complementarity at the TCR-peptide-MHC interface, resulting in reduced functional recognition of the cognate and naturally occurring variants of this EBV peptide. Thus, polymorphism in the TCR loci may contribute toward variability in immune responses and the outcome of infection.

Human leukocyte antigen (HLA) gene polymorphism plays a critical role in protective immunity, disease susceptibility, autoimmunity, and drug hypersensitivity, yet the basis of how HLA polymorphism influences T cell receptor (TCR) recognition is unclear. We examined how a natural micropolymorphism in HLA-B44, an important and large HLA allelic family, affected antigen recognition. T cell-mediated immunity to an Epstein-Barr virus determinant (EENLLDFVRF) is enhanced when HLA-B*4405 was the presenting allotype compared with HLA-B*4402 or HLA-B*4403, each of which differ by just one amino acid. The micropolymorphism in these HLA-B44 allotypes altered the mode of binding and dynamics of the bound viral epitope. The structure of the TCR-HLA-B*4405(EENLLDFVRF) complex revealed that peptide flexibility was a critical parameter in enabling preferential engagement with HLA-B*4405 in comparison to HLA-B*4402/03. Accordingly, major histocompatibility complex (MHC) polymorphism can alter the dynamics of the peptide-MHC landscape, resulting in fine-tuning of T cell responses between closely related allotypes.

The energetic bases of T cell recognition are unclear. Here, we studied the 'energetic landscape' of peptide-major histocompatibility complex (pMHC) recognition by an immunodominant alphabeta T cell receptor (TCR). We quantified and evaluated the effect of natural and systematic substitutions in the complementarity-determining region (CDR) loops on ligand binding in the context of the structural detail of each component of the immunodominant TCR-pMHC complex. The CDR1 and CDR2 loops contributed minimal energy through direct recognition of the antigen and instead had a chief function in stabilizing the ligated CDR3 loops. The underlying energetic basis for recognition lay in the CDR3 loops. Therefore the energetic burden of the CDR loops in the TCR-pMHC interaction is variable among TCRs, reflecting the inherent adaptability of the TCR in ligating different ligands.

Thousands of potentially antigenic peptides are encoded by an infecting pathogen; however, only a small proportion induce measurable CD8(+) T cell responses. To investigate the factors that control peptide immunogenicity, we have examined the cytotoxic T lymphocyte (CTL) response to a previously undefined epitope ((77)APQPAPENAY(86)) from the BZLF1 protein of Epstein-Barr virus (EBV). This peptide binds well to two human histocompatibility leukocyte antigen (HLA) allotypes, HLA-B*3501 and HLA-B*3508, which differ by a single amino acid at position 156 ((156)Leucine vs. (156)Arginine, respectively). Surprisingly, only individuals expressing HLA-B*3508 show evidence of a CTL response to the (77)APQPAPENAY(86) epitope even though EBV-infected cells expressing HLA-B*3501 process and present similar amounts of peptide for CTL recognition, suggesting that factors other than peptide presentation levels are influencing immunogenicity. Functional and structural analysis revealed marked conformational differences in the peptide, when bound to each HLA-B35 allotype, that are dictated by the polymorphic HLA residue 156 and that directly affected T cell receptor recognition. These data indicate that the immunogenicity of an antigenic peptide is influenced not only by how well the peptide binds to major histocompatibility complex (MHC) molecules but also by its bound conformation. It also illustrates a novel mechanism through which MHC polymorphism can further diversify the immune response to infecting pathogens.

Unusually long major histocompatibility complex (MHC) class I-restricted epitopes are important in immunity, but their 'bulged' conformation represents a potential obstacle to alphabeta T cell receptor (TCR)-MHC class I docking. To elucidate how such recognition is achieved while still preserving MHC restriction, we have determined here the structure of a TCR in complex with HLA-B(*)3508 presenting a peptide 13 amino acids in length. This complex was atypical of TCR-peptide-MHC class I interactions, being dominated at the interface by peptide-mediated interactions. The TCR assumed two distinct orientations, swiveling on top of the centrally bulged, rigid peptide such that only limited contacts were made with MHC class I. Although the TCR-peptide recognition resembled an antibody-antigen interaction, the TCR-MHC class I contacts defined a minimal 'generic footprint' of MHC-restriction. Thus our findings simultaneously demonstrate the considerable adaptability of the TCR and the 'shape' of MHC restriction.

HLA-B*4402 and B*4403 are naturally occurring MHC class I alleles that are both found at a high frequency in all human populations, and yet they only differ by one residue on the alpha2 helix (B*4402 Asp156-->B*4403 Leu156). CTLs discriminate between HLA-B*4402 and B*4403, and these allotypes stimulate strong mutual allogeneic responses reflecting their known barrier to hemopoeitic stem cell transplantation. Although HLA-B*4402 and B*4403 share >95% of their peptide repertoire, B*4403 presents more unique peptides than B*4402, consistent with the stronger T cell alloreactivity observed toward B*4403 compared with B*4402. Crystal structures of B*4402 and B*4403 show how the polymorphism at position 156 is completely buried and yet alters both the peptide and the heavy chain conformation, relaxing ligand selection by B*4403 compared with B*4402. Thus, the polymorphism between HLA-B*4402 and B*4403 modifies both peptide repertoire and T cell recognition, and is reflected in the paradoxically powerful alloreactivity that occurs across this "minimal" mismatch. The findings suggest that these closely related class I genes are maintained in diverse human populations through their differential impact on the selection of peptide ligands and the T cell repertoire.

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and beta(2)-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

Cytotoxic T cells (CTL) represent the major defense mechanism against the spread of virus infection. It is believed that the pore-forming protein, perforin, facilitates the entry of a series of serine proteases (particularly granzyme B) into the target cell which ultimately leads to DNA fragmentation and apoptosis. We demonstrate here that during CTL-mediated cytolysis the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an enzyme implicated in the repair of double strand breaks in DNA, is specifically cleaved by an interleukin (IL)-1 beta-converting enzyme (ICE)-like protease. A serine protease inhibitor, 3,4-dichloroisocoumarin (DCl), which is known to block granzyme B activity, inhibited CTL-induced apoptosis and prevented the degradation of DNA-PKcs in cells but failed to prevent the degradation of purified DNA-PKcs by CTL extracts. However, Tyr-Val-Ala-Asp-CH2Cl (YVAD-CMK) and other cysteine protease inhibitors prevented the degradation of purified DNA-PKcs by CTL extracts. Furthermore, incubation of DNA-PKcs with granzyme B did not produce the same cleavage pattern observed in cells undergoing apoptosis and when this substrate was incubated with either CTL extracts or the ICE-like protease, CPP32. Sequence analysis revealed that the cleavage site in DNA-PKcs during CTL killing was the same as that when this substrate was exposed to CPP32. This study demonstrates for the first time that the cleavage of DNA-PKcs in this intact cell system is exclusively due to an ICE-like protease.