The shaping of T cell receptor recognition by self-tolerance.

During selection of the T cell repertoire, the immune system navigates the subtle distinction between self-restriction and self-tolerance, yet how this is achieved is unclear. Here we describe how self-tolerance toward a trans-HLA (human leukocyte antigen) allotype shapes T cell receptor (TCR) recognition of an Epstein-Barr virus (EBV) determinant (FLRGRAYGL). The recognition of HLA-B8-FLRGRAYGL by two archetypal TCRs was compared. One was a publicly selected TCR, LC13, that is alloreactive with HLA-B44; the other, CF34, lacks HLA-B44 reactivity because it arises when HLA-B44 is coinherited in trans with HLA-B8. Whereas the alloreactive LC13 TCR docked at the C terminus of HLA-B8-FLRGRAYGL, the CF34 TCR docked at the N terminus of HLA-B8-FLRGRAYGL, which coincided with a polymorphic region between HLA-B8 and HLA-B44. The markedly contrasting footprints of the LC13 and CF34 TCRs provided a portrait of how self-tolerance shapes the specificity of TCRs selected into the immune repertoire.

Epstein-Barr virus isolates retain their capacity to evade T cell immunity through BNLF2a despite extensive sequence variation.

The Epstein-Barr virus (EBV)-encoded immune evasion protein BNLF2a inhibits the transporter associated with antigen processing (TAP), thereby downregulating HLA class I expression at the cell surface. As a consequence, recognition of EBV-infected cells by cytotoxic T cells is impaired. Here, we show that sequence polymorphism of the BNLF2a protein is observed with natural EBV isolates, with evidence for positive selection. Despite these mutations, the BNLF2a variants efficiently reduce cell surface HLA class I levels. This conservation of BNLF2a function during evolution of EBV implies an important role for the viral TAP inhibitor in preventing T cell recognition during viral infection.

Cross-recognition of HLA DR4 alloantigen by virus-specific CD8+ T cells: a new paradigm for self-/nonself-recognition.

The ability of CD8(+) T cells to engage a diverse range of peptide-major histocompatibility complex (MHC) complexes can also lead to cross-recognition of self and nonself peptide-MHC complexes and thus directly contribute toward allograft rejection or autoimmunity. Here we present a novel form of cross-recognition by herpes virus-specific CD8(+) cytotoxic T cells that challenges the current paradigm of self/non-self recognition. Functional characterization of a human leukocyte antigen (HLA) Cw*0602-restricted cytomegalovirus-specific CD8(+) T-cell response revealed an unusual dual specificity toward a pp65 epitope and the alloantigen HLA DR4. This cross-recognition of HLA DR4 alloantigen was critically dependent on the coexpression of HLA DM and was preferentially directed toward the B-cell lineage. Furthermore, allostimulation of peripheral blood lymphocytes with HLA DRB*0401-expressing cells rapidly expanded CD8(+) T cells, which recognized the pp65 epitope in the context of HLA Cw*0602. T-cell repertoire analysis revealed 2 dominant populations expressing T-cell receptor beta variable (TRBV)4-3 or TRBV13, with cross-reactivity exclusively mediated by the TRBV13(+) clonotypes. More importantly, cross-reactive TRBV13(+) clonotypes displayed markedly lower T-cell receptor binding affinity and a distinct pattern of peptide recognition, presumably mimicking a structure presented on the HLA DR4 allotype. These results illustrate a novel mechanism whereby virus-specific CD8(+) T cells can cross-recognize HLA class II molecules and may contribute toward allograft rejection and/or autoimmunity.

An HLA-A2-restricted T-cell epitope mapped to the BNLF2a immune evasion protein of Epstein-Barr virus that inhibits TAP.

The early lytic cycle protein of Epstein-Barr virus (EBV), BNLF2a, has recently been shown to play a critical role in immune evasion by inhibiting the peptide transporter associated with antigen processing (TAP), thereby blocking antigen-specific CD8(+) T-cell recognition of many lytic cycle antigens. Surprisingly, we now show that a peptide ((50)VLFGLLCLL(58)) from the hydrophobic C-terminal region of this small (60-amino-acid) EBV protein is efficiently presented by the common class I allele HLA-A2 for recognition by cytotoxic T lymphocytes. The mechanism for this unexpected finding was revealed by experiments showing that this epitope is processed and presented independently of TAP.

Preferential binding of unusually long peptides to MHC class I and its influence on the selection of target peptides for T cell recognition.

A classic feature of antigen presentation for CD8+ T cell recognition is that MHC class I molecules generally present peptides of 8-10 amino acids in length. However, recent studies have demonstrated that peptides of >10 residues play a significant role in immune surveillance by T cells restricted by some HLA class I alleles. In the present study, we describe several examples of unusually long viral peptides of 11 or 12 residues, recognized by CTLs in the context of HLA-B35. Interestingly, all these immunogenic peptides completely encompass shorter canonical length sequences that conform to the HLA-B35 binding motif, but which fail to stimulate detectable T cell responses. The mechanism for this phenomenon appears to involve the preferential binding to HLA-B35 of the atypically long CD8+ T cell target peptides over the overlapping canonical length sequences. These data suggest that the peptide length specificity of some HLA class I alleles is broad, allowing peptides of >10 residues to sometimes dominate over canonical length class I ligands as targets for T cell recognition.

Susceptibility of wild-caught Lutzomyia longipalpis (Diptera: Psychodidae) sand flies to insecticide after an extended period of exposure in western São Paulo, Brazil.

BACKGROUND: In Brazil, members of the sand fly species complex Lutzomyia longipalpis transmit Leishmania infantum, a protist parasite that causes visceral leishmaniasis. Male Lu. longipalpis produce a sex pheromone that is attractive to both females and males. During a cluster randomised trial, to determine the combined effect of synthetic sex-aggregation pheromone and insecticide on Le. infantum transmission Lu. longipalpis had been continuously exposed to insecticide for 30 months. The objective of this study was to determine the effect of continuous exposure to the insecticides used in the trial on the susceptibility of Lu. longipalpis population. METHODS: During the trial the sand flies had been exposed to either lambda-cyhalothrin [pheromone + residual insecticide spray (PI)], deltamethrin [dog collars (DC)] or no insecticide [control (C)], for 30 months (November 2012 to April 2015). The insecticide treatment regime was kept in place for an additional 12 months (May 2015-April 2016) during this susceptibility study. Sand flies collected from the field were exposed to WHO insecticide-impregnated papers cyhalothrin (0.05%), deltamethrin (0.5%) and control (silicone oil) in a modified WHO insecticide exposure trial to determine their susceptibility. RESULTS: We collected 788 Lu. longipalpis using CDC-light traps in 31 municipalities across the three trial arms. Probit analysis showed that the knockdown times (KDTs) of Lu. longipalpis collected from the lambda-cyhalothrin exposed PI-arm [KDT50: 31.1 min, confidence interval (CI): 29.6-32.6 and KDT90: 44.2 min, CI: 42.1-46.7] were longer than the KDTs from the non-insecticide-treated C-arm (KDT50: 26.3 min, CI: 25.1-27.6 and KDT90: 38.2, CI: 36.5-40.2) (no-overlapping 95% CIs). KDTs of Lu. longipalpis collected from the deltamethrin exposed DC-arm had similar values (KDT50: 13.7 min, CI: 10.1-16.2 and KDT90: 26.7 min, CI: 21.8-30.6) to those for the C-arm (KDT50: 13.5 min; CI: 12.2-14.8 and KDT90: 23.2 min, CI: 21.4-25.4) (overlapping CIs). The wild-caught unexposed Lu. longipalpis (C-arm), took approximately twice as long to knock down as laboratory-colonised specimens for both insecticides. CONCLUSIONS: Our study reveals slight changes in KDT, in sand flies after prolonged exposure to lambda-cyhalothrin in the presence of pheromone. These changes are not considered to have reached the reference levels indicative of resistance in sand flies suggesting that pheromone and insecticide treatment at the level indicated in this study do not constitute a significant risk of increased insecticide resistance. Prolonged exposure to deltamethrin in dog collars did not result in changes to KDT.

Sand fly synthetic sex-aggregation pheromone co-located with insecticide reduces the incidence of infection in the canine reservoir of visceral leishmaniasis: A stratified cluster randomised trial.

OBJECTIVE: To evaluate the efficacy of a synthetic sex-aggregation pheromone of the sand fly vector Lu. longipalpis, co-located with residual insecticide, to reduce the infection incidence of Leishmania infantum in the canine reservoir. METHODS: A stratified cluster randomised trial was designed to detect a 50% reduction in canine incident infection after 24 months in 42 recruited clusters, randomly assigned to one of three intervention arms (14 cluster each): synthetic pheromone + insecticide, insecticide-impregnated dog collars, or placebo control. Infection incidence was measured by seroconversion to anti-Leishmania serum antibody, Leishmania parasite detection and canine tissue parasite loads. Changes in relative Lu. longipalpis abundance within households were measured by setting three CDC light traps per household. RESULTS: A total 1,454 seronegative dogs were followed-up for a median 15.2 (95% C.I.s: 14.6, 16.2) months per cluster. The pheromone + insecticide intervention provided 13% (95% C.I. 0%, 44.0%) protection against anti-Leishmania antibody seroconversion, 52% (95% C.I. 6.2%, 74·9%) against parasite infection, reduced tissue parasite loads by 53% (95% C.I. 5.4%, 76.7%), and reduced household female sand fly abundance by 49% (95% C.I. 8.2%, 71.3%). Variation in the efficacy against seroconversion varied between trial strata. Equivalent protection attributed to the impregnated-collars were 36% (95% C.I. 14.4%, 51.8%), 23% (95% C.I. 0%, 57·5%), 48% (95% C.I. 0%, 73.4%) and 43% (95% C.I. 0%, 67.9%), respectively. Comparison of the two interventions showed no statistically consistent differences in their efficacies; however, the errors were broad for all outcomes. Reductions in sand fly numbers were predominant where insecticide was located (chicken and dog sleeping sites), with no evidence of insecticide-induced repellence onto humans or dogs. CONCLUSION: The synthetic pheromone co-located with insecticide provides protection particularly against canine L. infantum parasite transmission and sand fly vector abundance. The effect estimates are not dissimilar to those of the insecticide-impregnated collars, which are documented to reduce canine infection incidence, human infection and clinical VL disease incidence, in different global regions. The trialled novel lure-and-kill approach is a low-cost potential vector control tool against ZVL in the Americas.

Attraction of Lutzomyia longipalpis to synthetic sex-aggregation pheromone: Effect of release rate and proximity of adjacent pheromone sources.

In South America, the Protist parasite that causes visceral leishmaniasis, a potentially fatal human disease, is transmitted by blood-feeding female Lutzomyia longipalpis sand flies. A synthetic copy of the male produced sex-aggregation pheromone offers new opportunities for vector control applications. We have previously shown that the pheromone placed in plastic sachets (lures) can attract both females and males to insecticide treated sites for up to 3 months. To use the pheromone lure in a control program we need to understand how the application of lures in the field can be optimised. In this study we investigated the effect of increasing the number of lures and their proximity to each other on their ability to attract Lu. longipalpis. Also for the first time we applied a Bayesian log-linear model rather than a classic simple (deterministic) log-linear model to fully exploit the field-collected data. We found that sand fly response to pheromone is significantly related to the quantity of pheromone and is not influenced by the proximity of other pheromone sources. Thus sand flies are attracted to the pheromone source at a non-linear rate determined by the amount of pheromone being released. This rate is independent of the proximity of other pheromone releasing traps and indicates the role of the pheromone in aggregation formation. These results have important implications for optimisation of the pheromone as a vector control tool and indicate that multiple lures placed in relatively close proximity to each other (5 m apart) are unlikely to interfere with one another.

A temporal comparison of sex-aggregation pheromone gland content and dynamics of release in three members of the Lutzomyia longipalpis (Diptera: Psychodidae) species complex.

BACKGROUND: Lutzomyia longipalpis is the South American vector of Leishmania infantum, the etiologic agent of visceral leishmaniasis (VL). Male L. longipalpis produce a sex-aggregation pheromone that is critical in mating, yet very little is known about its accumulation over time or factors involved in release. This laboratory study aimed to compare accumulation of pheromone over time and determine factors that might influence release in three members of the L. longipalpis species complex. METHODOLOGY/PRINCIPAL FINDINGS: We investigated male sex-aggregation pheromone gland content at different ages and the release rate of pheromone in the presence or absence of females under different light conditions by gas chromatography-mass spectrometry (GC-MS). Pheromone gland content was determined by extraction of whole males and pheromone release rate was determined by collection of headspace volatiles. Pheromone gland content appeared age-related and pheromone began to accumulate between 6 to 12 h post eclosion and gradually increased until males were 7-9 days old. The greatest amount was detected in 9-day old Campo Grande males ((S)-9-methylgermacrene-B; X ± SE: 203.5 ± 57.4 ng/male) followed by Sobral 2S males (diterpene; 199.9 ± 34.3) and Jacobina males ((1S,3S,7R)-3-methyl-α-himachalene; 128.8 ± 30.3) at 7 days old. Pheromone release was not continuous over time. During a 4-hour period, the greatest quantities of pheromone were released during the first hour, when wing beating activity was most intense. It was then substantially diminished for the remainder of the time. During a 24 h period, 4-5 day old male sand flies released approximately 63 ± 11% of the pheromone content of their glands, depending on the chemotype. The presence of females significantly increased pheromone release rate. The light regime under which the sand flies were held had little influence on pheromone release except on Sobral 2S chemotype. CONCLUSIONS/SIGNIFICANCE: Accumulation of pheromone appears to occur at different rates in the different chemotypes examined and results in differing amounts being present in glands over time. Release of accumulated pheromone is not passive, but depends on biotic (presence of females) and abiotic (light) circumstances. There are marked differences in content and release between the members of the complex suggesting important behavioural, biosynthetic and ecological differences between them.

Allelic polymorphism in the T cell receptor and its impact on immune responses.

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.

The peptide length specificity of some HLA class I alleles is very broad and includes peptides of up to 25 amino acids in length.

The major ligands presented by MHC class I molecules after natural antigen processing are peptides of eight to ten residues in length, and it is widely accepted that the binding preferences of MHC class I molecules play a dominant role in dictating this classic feature of antigen presentation. In this report, we have reassessed the peptide size specificity of class I human leukocyte antigens (HLAs). By lengthening previously defined T cell epitopes by central amino acid insertion, we demonstrate that the peptide length specificity of some common HLA class I alleles (HLA-B*3501, B*0702 and A*2402) is very broad, and includes peptides of up to 25 residues. These data suggest that the length limitation of naturally processed MHC class I-associated peptides is primarily controlled by peptide availability after antigen processing rather than the binding specificity of MHC class I molecules. Furthermore, the findings provide an explanation for recent reports highlighting that epitopes of >10 amino acids play a minor but significant role in virus-specific immune surveillance by CD8(+) T cells.

Natural micropolymorphism in human leukocyte antigens provides a basis for genetic control of antigen recognition.

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.

Widespread sequence variation in Epstein-Barr virus nuclear antigen 1 influences the antiviral T cell response.

Epstein-Barr virus (EBV) nuclear antigen (EBNA) 1 is perhaps the most widely studied EBV protein, because of its critical role in maintaining the EBV episome and its expression in all EBV-associated malignancies. Much of this research has focused exclusively on the EBV wild-type (wt) strain (B95-8). Sequence analysis of the gene encoding for EBNA1 in EBV isolates from 43 Caucasians has now revealed considerable EBNA1 sequence divergence from the EBV wt strain in the majority of isolates from this population group. Importantly, T cell recognition of an endogenously processed HLA-B8 - binding EBNA1 epitope was greatly influenced by this sequence polymorphism.

Predictable alphabeta T-cell receptor selection toward an HLA-B*3501-restricted human cytomegalovirus epitope.

Human cytomegalovirus (HCMV) elicits a very large burden on the immune system, with approximately one in ten T cells being reserved solely to manage this infection. However, information on the clonotypic composition of these vast T-cell populations is limited. In this study, we sequenced 116 T-cell receptor (TcR) alpha/beta-chains specific for the highly immunogenic HLA-B*3501-resticted epitope IPSINVHHY from the pp65 antigen. Interestingly, T cells recovered from all donors bore an identical or near-identical TRBV28/TRBJ1-4/TRAV17/TRAJ33 TcR. The ability to predict the responding alphabeta TcR repertoire before viral infection should prove a powerful tool for basic and clinical immunology.

The impact of HLA-B micropolymorphism outside primary peptide anchor pockets on the CTL response to CMV.

The factors controlling epitope selection in the T cell response to persistent viruses are not fully understood, and we have examined this issue in the context of four HLA-B*35-binding peptides from the pp65 antigen of human cytomegalovirus, two of which are previously undescribed. Striking differences in the hierarchy of immunodominance between these four epitopes were observed in healthy virus carriers expressing HLA-B*3501 versus B*3508, two HLA-B allotypes that differ by a single amino acid at position 156 (HLA-B*3501, (156)Leucine; HLA-B*3508, (156)Arginine) that projects from the alpha2 helix into the centre of the peptide-binding groove. While HLA-B*3501(+) individuals responded most strongly to the (123)IPSINVHHY(131) and (366)HPTFTSQY(373) epitopes, HLA-B*3508(+) individuals responded preferentially to (103)CPSQEPMSIYVY(114) and (188)FPTKDVAL(195). By comparing peptide-MHC association and disassociation rates with peptide immunogenicity, it was clear that dissociation rates correlate more closely with the hierarchy of immunodominance among the four pp65 peptides. These findings demonstrate that MHC micropolymorphism at positions outside the primary anchor residue binding pockets can have a major impact on determinant selection in antiviral T cell responses. Such influences may provide the evolutionary pressure that maintains closely related MHC molecules in diverse human populations.

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.