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Celiac disease is a chronic inflammatory condition of the small intestine caused by aberrant adaptive immune response to gluten protein from wheat and related cereal plants. Over 90% of celiac disease patients carry the HLA-DQ2.5 allotype and HLA-DQ2.5 presents gluten peptides to gluten-reactive CD4+ T cells in celiac disease patients. A large number of HLA-DQ2.5-restricted gluten T cell epitopes have been identified over the years. These epitopes are in general proline-rich and contain at least one glutamic acid residue that is generated from glutamine in the native gluten protein by deamidation. The deamidation is mediated by the enzyme transglutaminase 2 (TG2). It has been shown that the same T cell could recognize several different HLA-DQ2.5-restricted gluten T cell epitopes due to sequence similarities. In this paper, we demonstrate that three T cell clones derived from duodenal biopsies of different celiac disease patients are able to respond to at least five different gluten T cell epitopes within the DQ2.5-glia-γ4 epitope family, including two novel epitopes. [ABSTRACT FROM AUTHOR]

The chicken major histocompatibility complex (MHC, also known as the BF-BL region of the B locus) is notably small and simple with few genes, most of which are involved in antigen processing and presentation. There are two classical class I genes, of which only BF2 is well and systemically expressed as the major ligand for cytotoxic T lymphocytes (CTLs). The other class I gene, BF1, is believed to be primarily a natural killer (NK) cell ligand. Among most standard chicken MHC haplotypes examined in detail, BF1 is expressed tenfold less than BF2 at the RNA level due to defects in the promoter or in a splice site. However, in the B14 and typical B15 haplotypes, BF1 RNA was not detected, and here, we show that a deletion between imperfect 32 nucleotide direct repeats has removed the BF1 gene entirely. The phenotypic effects of not having a BF1 gene (particularly on resistance to infectious pathogens) have not been systematically explored, but such deletions between short direct repeats are also found in some BF1 promoters and in the 5′ untranslated region (5′UTR) of some BG genes found in the BG region of the B locus. Despite the opposite transcriptional orientation of homologous genes in the chicken MHC, which might prevent the loss of key genes from a minimal essential MHC, it appears that small direct repeats can still lead to deletion. [ABSTRACT FROM AUTHOR]

Upon recognition of peptide-MHC complexes by T cell receptors (TCR), the cognate T cells expand and differentiate into effector T cells to generate protective immunity. Despite the fact that any immune response generates a diverse set of TCR clones against a particular epitope, only a few clones are highly expanded in any immune response. Previous studies observed that the highest frequency clones usually control viral infections better than subdominant clones, but the reasons for this dominance among T cell clones are still unclear. Here, we used publicly available TCR amino acid sequences to study which factors determine whether a response becomes immunodominance (ID) per donor; we classified the largest T cell clone as the epitope-specific dominant clone and all the other clones as subdominant responses (SD). We observed a distinctively hydrophobic CDR3 in ID responses against a dominant epitope from influenza A virus, compared to the SD responses. The common V-J combinations were shared between ID and SD responses, suggesting that the biased V-J recombination events are restricted by epitope specificity; thus, the immunodominance is not directly determined by a bias combination of V and J genetic segments. Our findings reveal a close similarity of global sequence properties between dominant and subdominant clones of epitope-specific responses but detectable distinctive amino acid enrichments in ID. Taken together, we believe this first comparative study of immunodominant and subdominant TCR sequences can guide further studies to resolve factors determining the immunodominance of antiviral as well as tumor-specific T cell responses. [ABSTRACT FROM AUTHOR]

Affinity and stability of peptides bound by major histocompatibility complex (MHC) class I molecules are important factors in presentation of peptides to cytotoxic T lymphocytes (CTLs). In silico prediction methods of peptide-MHC binding followed by experimental analysis of peptide-MHC interactions constitute an attractive protocol to select target peptides from the vast pool of viral proteome peptides. We have earlier reported the peptide binding motif of the porcine MHC-I molecules SLA-1*04:01 and SLA-2*04:01, identified by an ELISA affinity-based positional scanning combinatorial peptide library (PSCPL) approach. Here, we report the peptide binding motif of SLA-3*04:01 and combine two prediction methods and analysis of both peptide binding affinity and stability of peptide-MHC complexes to improve rational peptide selection. Using a peptide prediction strategy combining PSCPL binding matrices and in silico prediction algorithms (NetMHCpan), peptide ligands from a repository of 8900 peptides were predicted for binding to SLA-1*04:01, SLA-2*04:01, and SLA-3*04:01 and validated by affinity and stability assays. From the pool of predicted peptides for SLA-1*04:01, SLA-2*04:01, and SLA-3*04:01, a total of 71, 28, and 38 % were binders with affinities below 500 nM, respectively. Comparison of peptide-SLA binding affinity and complex stability showed that peptides of high affinity generally, but not always, produce complexes of high stability. In conclusion, we demonstrate how state-of-the-art prediction and in vitro immunology tools in combination can be used for accurate selection of peptides for MHC class I binding, hence providing an expansion of the field of peptide-MHC analysis also to include pigs as a livestock experimental model. [ABSTRACT FROM AUTHOR]

Memory programming of cytotoxic T cells (CTLs) by inflammatory cytokines can be regulated by mammalian target of rapamycin (mTOR). We have shown that inhibition of mTOR during CTL activation leads to the enhancement of memory, but the molecular mechanisms remain largely unknown. Using high-throughput RNA-Seq, we identified genes and functions in mouse CTLs affected by mTOR inhibition through rapamycin. Of the 43,221 identified transcripts, 184 transcripts were differentially expressed after rapamycin treatment, corresponding to 128 annotated genes. Of these genes, 114 were downregulated and only 14 were upregulated. Most importantly, 50 of them are directly related to cell death and survival. In addition, several genes such as CD62L are related to migration. Furthermore, we predicted downregulation of transcriptional regulators based on the total differentially expressed genes, as well as the subset of apoptosis-related genes. Quantitative PCR confirmed the differential expressions detected in RNA-Seq. We conclude that the regulatory function of rapamycin may work through inhibition of multiple genes related to apoptosis and migration, which enhance CTL survival into memory. [ABSTRACT FROM AUTHOR]

Multiple factors determine the ability of a peptide to elicit a cytotoxic T cell lymphocyte response. Binding to a major histocompatibility complex class I (MHC-I) molecule is one of the most essential factors, as no peptide can become a T cell epitope unless presented on the cell surface in complex with an MHC-I molecule. As such, peptide-MHC (pMHC) binding affinity predictors are currently the premier methods for T cell epitope prediction, and these prediction methods have been shown to have high predictive performances in multiple studies. However, not all MHC-I binders are T cell epitopes, and multiple studies have investigated what additional factors are important for determining the immunogenicity of a peptide. A recent study suggested that pMHC stability plays an important role in determining if a peptide can become a T cell epitope. Likewise, a T cell propensity model has been proposed for identifying MHC binding peptides with amino acid compositions favoring T cell receptor interactions. In this study, we investigate if improved accuracy for T cell epitope discovery can be achieved by integrating predictions for pMHC binding affinity, pMHC stability, and T cell propensity. We show that a weighted sum approach allows pMHC stability and T cell propensity predictions to enrich pMHC binding affinity predictions. The integrated model leads to a consistent and significant increase in predictive performance and we demonstrate how this can be utilized to decrease the experimental workload of epitope screens. The final method, NetTepi, is publically available at . [ABSTRACT FROM AUTHOR]

The CD1 family is a group of non-polymorphic MHC class I-like molecules that present lipid-based antigens to T cells. Previous work in our laboratory demonstrated that cytotoxic T lymphocytes from immune adult horses recognize lipids from the cell wall of an important equine pathogen, Rhodococcus equi. These findings suggest an important role for the equine CD1 antigen presentation system in protective immune responses to microbial pathogens in the horse. In this study, we characterized and mapped the equine CD1 gene cluster. The equine genome was found to contain 13 complete CD1 genes; seven genes were classified as homologues of human CD1a, two CD1b, one CD1c, one CD1d, and two CD1e, making it the largest CD1 family to date. All but one of the eqCD1 molecules were expressed in all antigen-presenting cells investigated. The major amino acid differences between equine CD1 isoforms are located in the predicted antigen binding site, suggesting that a variety of lipid antigens can be presented. R. equi survives and replicates within professional phagocytes by arresting phagosome maturation between the early endosome and late phagosome. Based on the absence of a tyrosine sorting motif in all eqCD1a, CD1a molecules are predicted to co-localize with R. equi in the early endosome. Here, they could acquire lipid antigen and present it to T lymphocytes. The extraordinarily large number of CD1 molecules in the horse may reflect their crucial role in immunity to R. equi. [ABSTRACT FROM AUTHOR]

A novel gene involved in cytotoxicity, NKLAM [for "natural killer (NK) lytic-associated molecule"], has recently been identified in human NK cells. Its expression correlates with the cytolytic activity of both NK cells and cytotoxic T cells (CTLs); treatment of these cells with NKLAM-specific antisense oligonucleotides inhibits their cytotoxic function. NKLAM encodes a zinc finger protein that resides in NK cytolytic granules. Here, we identified a second human NKLAM transcript that differs from the original only at the 3′ end, extending the open reading frame, and therefore encoding a significantly longer protein (731 residues compared with 587). The genomic structure of human NKLAM indicates that these two isoforms are products of alternative splicing. Both forms of NKLAM protein are expressed in NK cells, with the larger protein predominant. Human NKLAM cDNA was used to isolate mouse NKLAM from a C57BL/6 spleen cDNA library. There is a single NKLAM isoform in the mouse which shares over 89% nucleotide and 94% amino acid homology with the larger human form of NKLAM. The genomic organization of NKLAM is similar in both mouse and human. As with human NKLAM, mouse NKLAM mRNA is selectively expressed in CD8+ T cells NK cells, and activated peritoneal macrophages and further induced by cytokines that enhance the cytotoxic activity of these cells. These results indicate that human and mouse NKLAM are highly conserved both structurally and functionally, reinforcing the premise that this gene plays an important role in cellular cytotoxicity. [ABSTRACT FROM AUTHOR]