Polymorphic sites preferentially avoid co-evolving residues in MHC class I proteins.

Major histocompatibility complex class I (MHC-I) molecules are critical to adaptive immune defence mechanisms in vertebrate species and are encoded by highly polymorphic genes. Polymorphic sites are located close to the ligand-binding groove and entail MHC-I alleles with distinct binding specificities. Some efforts have been made to investigate the relationship between polymorphism and protein stability. However, less is known about the relationship between polymorphism and MHC-I co-evolutionary constraints. Using Direct Coupling Analysis (DCA) we found that co-evolution analysis accurately pinpoints structural contacts, although the protein family is restricted to vertebrates and comprises less than five hundred species, and that the co-evolutionary signal is mainly driven by inter-species changes, and not intra-species polymorphism. Moreover, we show that polymorphic sites in human preferentially avoid co-evolving residues, as well as residues involved in protein stability. These results suggest that sites displaying high polymorphism may have been selected during vertebrates' evolution to avoid co-evolutionary constraints and thereby maximize their mutability

Investigating celiac disease using recombinant soluble MHC class II molecules

Celiac disease (CD) is a chronic intestinal inflammation showing a strong association to HLA-DQ2.5. This thesis focused on the use of soluble MHC class II tetramers to examine the underlying mechanism for this selective CD association and also to develop a potential diagnostic tool based on the detection of gluten specific T cells. DQ2.5 is associated with atypically high amounts of Ii-chain derived CLIP peptides. This phenotype was assigned to an abnormally low interaction with the peptide editing molecule HLA-DM, and high kinetic stability of CLIP peptides to DQ2.5. HLA-DQ2.2 is a highly similar molecule, but does not show strong CD association. Interestingly, this molecule displayed a CLIP low phenotype due to a drastically reduced kinetic stability for CLIP. This was assigned to a polymorphic residue causing the gain/loss of a hydrogen bond involved in peptide binding. In addition, the application of a short bread challenge allowed for the detection of gluten reactive T cells in peripheral blood of CD patients using flow cytometry and MHC tetramers. This opens for the use of MHC tetramers as a diagnostic tool for diseases where the antigen and MHC restriction elements are known

Implications of the polymorphism of HLA-G on its function, regulation, evolution and disease association

The HLA-G gene displays several peculiarities that are distinct from those of classical HLA class I genes. The unique structure of the HLA-G molecule permits a restricted peptide presentation and allows the modulation of the cells of the immune system. Although polymorphic sites may potentially influence all biological functions of HLA-G, those present at the promoter and 3′ untranslated regions have been particularly studied in experimental and pathological conditions. The relatively low polymorphism observed in the MHC-G coding region both in humans and apes may represent a strong selective pressure for invariance, whereas, in regulatory regions several lines of evidence support the role of balancing selection. Since HLA-G has immunomodulatory properties, the understanding of gene regulation and the role of polymorphic sites on gene function may permit an individualized approach for the future use of HLA-G for therapeutic purposes

Expanding the immune self : impact of non-canonical translation on the repertoire of MHC I-associated peptides

Les molécules du complexe majeur d’histocompatibilité de classe I (MHC I) sont des glycoprotéines de surface exprimées par la majorité des cellules nucléés de notre organisme. Ces molécules servent à exposer une vue intégrative de l’état interne de nos cellules (soi immunitaire) via la présentation de courts peptides (MAPs) générés lors de la dégradation des protéines cytosoliques par le protéasome. Le répertoire des MAPs de chaque cellule, véritable carte d’identité peptidique, est constamment passé en revue par nos lymphocytes T CD8+, cellules centrales du système immunitaire, afin de débusquer et d’éliminer toute cellule anormale, e.g., celles présentant des MAPs d’origine virale ou tumorale (TSAs). Au vu du nombre grandissant d’articles démontrant que des ARNs autres que les ARNs messagers peuvent être traduits, nous avons décidé d’évaluer l’impact de ces mécanismes de traduction non-canonique sur le répertoire des MAPs présentés par des cellules B. En développant une approche protéogénomique, i.e., combinant spectrométrie de masse et séquençage d’ARN à haut-débit, nous avons pu démontrer qu’environ 10 % des MAPs présentés par nos cellules B dérivent d’évènements de traduction non-canonique incluant (i) la traduction d’ARNs messagers dans un cadre de lecture alternatif ou (ii) la traduction de régions ou ARNs supposés non-codants. L’analyse subséquente des caractéristiques de ces MAPs dits « cryptiques » suggère que leur biogenèse diffère de celle des MAPs conventionnels, les MAPs cryptiques étant principalement encodés par des ARNs instables produisant de courtes protéines dont la dégradation ne semble pas exiger l’intervention du protéasome. Sachant que la déméthylation globale du génome des cellules cancéreuses permet l’expression d’un plus grand bassin d’ARNs non-codants, nous avons supposé que ces cellules pourraient présenter de nombreux MAPs (et TSAs) cryptiques. En adaptant notre approche protéogénomique, nous avons pu analyser le répertoire des MAPs de cellules cancéreuses, incluant celui de deux lignées tumorales de souris (EL4 et CT26) et sept échantillons primaires humains (quatre leucémies aigues lymphoblastiques B et trois biopsies de cancer du poumon). Cette analyse nous a permis de découvrir qu’environ 90% des TSAs sont des TSAs cryptiques. Ayant observé que la plupart de ces TSAs dérivent de séquences normales dont l’expression est restreinte aux cellules tumorales, comme les retroéléments endogènes, il est plausible que ces TSAs soient partagés par plusieurs patients. Enfin, nos études chez la souris nous ont permis de démontrer qu’au moins deux facteurs influencent positivement le potentiel protectif d’un TSA in vivo : l’expression de cet antigène par les cellules cancéreuses et la fréquence des lymphocytes T capables de le reconnaître. En conclusion, le recours à la protéogénomique pour analyser les MAPs présentés par les cellules normales et cancéreuses nous a permis de démontrer que les MAPs cryptiques contribuent significativement au bassin de peptides constituant le soi immunitaire et qu’ils permettent aux lymphocytes T CD8+ d’effectuer une surveillance immunitaire plus efficace.On their surface, nucleated cells present major histocompatibility complex class I (MHC I) molecules in complex with short peptides, that we will refer to as MHC I-associated peptides (MAPs). These MAPs derive from the degradation of cytosolic proteins by the proteasome and provide an integrative view of the inner state of cells to CD8+ T cells, which can, in turn, eliminate abnormal cells, e.g., those presenting viral MAPs or tumor-specific antigens (TSAs). With the growing body of evidence suggesting that translation does occur outside of protein-coding transcripts, we tried to evaluate the impact of non-canonical translation on the repertoire of MAPs. Combining RNA-sequencing and mass spectrometry to analyze the MAP repertoire of B-lymphoblastoid cell lines, we uncovered that ~ 10 % of the MAP repertoire derives from such non-canonical translation events, including (i) the out-of-frame translation of protein-coding transcripts or (ii) the translation of non-coding regions (UTRs, introns, etc.) or transcripts (antisense, pseudogene, etc.). Interestingly, our data suggest that the biogenesis of cryptic and conventional MAPs differs, as cryptic MAPs derive from unstable transcripts generating short proteins that might be degraded in a proteasome-independent fashion. Because the global DNA hypomethylation observed in cancer cells tend to de-repress non-coding transcripts, we developed another proteogenomic approach to probe the cryptic MAP repertoire of two murine cancer cell lines (EL4 and CT26) and seven humor primary tumor samples (four B-lineage acute lymphoblastic leukemias and three lung tumor biopsies). This second analysis revealed that ~ 90% of TSAs are cryptic TSAs. Interestingly, most of those TSAs derived from cancer-restricted yet non-mutated sequences, such as endogenous retroelements, thereby suggesting that such TSAs could be shared between patients. Lastly, our validation study in mice demonstrated that at least two parameters can influence the in vivo protective effect of TSAs, namely TSA expression in cancer cells and the frequency of TSA-specific T cells. Altogether, our proteogenomic studies on the MAP repertoire of normal and cancer cells demonstrate that cryptic MAPs significantly expand the immune self and, consequently, the scope of CD8+ T cell immunosurveillance

Unveiling the murine t-haplotype’s extent and emergence of diversity in MHC class II genes

Genes of the major histocompatibility complex locus that present pathogen peptides to T-Lymphocytes are among the most polymorphic genes in mammals. Within the major histocompatibility complex diversity is under positive selection and new alleles are generated by point mutations and recombination events. In some house mice (Mus musculus) however the major histocompatibility complex (called H-2) is part of the t-haplotype, a meiotic driver located on Chromosome 17 carried by 10 to 40 percent of the natural population. Meiotic drivers are selfish chromosomal arrangements defined by the deviation of Mendelian ratio of inheritance, meaning that they are over-proportionally transmitted to offspring. As such purifying selection on meiotic drivers is reduced and deleterious mutations can accumulate although outside of lethal alleles few non-synonymous mutations are observed. Additionally meiotic drivers often show strong linkage disequilibrium which is the result of reduced recombination between the wildtype chromo ome and the chromosome carrying the genetic driver often caused by structural variations between chromosomes such as inversions. In this thesis, the physical extent of the t-haplotype inversions is resolved using optical mapping at higher resolution than ever done before. Evidence for a fifth inversion in the t-haplotype of Mus musculus domesticus was found. Also the allelic diversity of the MHC class II gene H2Aa in t-haplotype individuals of the subspecies Mus musculus musculus and Mus musculus domesticus was described based on sanger sequenced exons. The degree of diversity found here indicates that recombination between the t-haplotype and the wildtype might play an important role in diversifying this H2Aa exon. Lastly, to uncover de novo meiotic recombination events within the H2Aa gene and the Prdm9 ZnF Array, which determines the placement of meiotic hotspots, Nanopore Sequencing was implemented. To identify the original template of sequenced amplicons the Primer ID as presented by Jabara et al., 2011 as included in the Primers for amplifying gene regions to be sequenced. However due to low coverage with Primer IDs no definite de novo recombination events could be defined, leaving the use of Primer IDs in Nanopore Sequencing up to discussion

Histocompatibility

This book presents some recent researches related to histocompatibility for scientists interested in this field. It includes 10 chapters, in different topics, prepared by Sundararajulu Panneerchelvam and Mohd Nor Norazmi; Giada Amodio and Silvia Gregori; Adema Ribic; Bahaa K. A. Abdel-Salam; Kai-Fu Tang; Roberto Biassoni, Irene Vanni and Elisabetta Ugolotti; Wei-Cheng Yang, Lien-Siang Chou and Jer-Ming Hu; Shatrah Othman and Rohana Yusof; Masahiro Hirayama, Eiichi Azuma and Yoshihiro Komada; Gustav Roder, Linda Geironson, Elna Follin, Camilla Thuring and Kajsa Paulsson

Organization of the class I region of the bovine major histocompatibility complex (BoLA) and the characterization of a class I frameshift deletion (BoLA-Adel) prevalent in feral bovids

The major histocompatibility complex (MHC) is a genomic region containing genes of immunomodulatory importance. MHC class I genes encode cell-surface glycoproteins that present peptides to circulating T cells, playing a key role in recognition of self and non-self. Studies of MHC loci in vertebrates have examined levels of polymorphism and molecular evolutionary processes generating diversity. The bovine MHC (BoLA) has been associated with disease susceptibility, resistance and progression. To delineate mechanisms by which MHC class I genes evolved to function optimally in a species like cattle, it is necessary to study genomic organization of BoLA to define gene content, and investigate characteristics of expressed class I molecules. This study describes development of a physical map of BoLA class I region derived from screening two BAC libraries, isolating positive clones and confirming gene content, order and chromosomal location through PCR, novel BAC end sequencing techniques, and selected BAC shotgun cloning and/or sequencing and FISH analysis. To date, this is the most complete ordered BAC array encompassing the BoLA class I region from the class III boundary to the extended class I region. Characterization of a frameshift allele exhibiting trans-species polymorphism in Bos and Bison by flow cytometry, real-time RT-PCR, 1D and 2D gel analysis is also described. This frameshift allele encodes an early termination signal within the antigen recognition site (ARS) of exon 3 of the BoLA BSA-Adel class I gene predicting a truncated class I protein that is soluble. An ability to assess MHC diversity in populations and provision of animals with defined MHC haplotypes and genetic content for experimental research is necessary in developing a basis upon which to build functional studies to elucidate associations between haplotype and disease in bovids. The BoLA class I region is immunologically important for disease association studies in an economically important species. This study provides knowledge of gene content and organization within the class I MHC region in cattle, providing a template for more detailed analysis and elucidation of complex disease associations through functional genomics and comparative analysis, as well as evolution of the MHC in bovids to optimize a populationÂs immune response

An Examination of MHC, Peptide, and TCR Interactions

T cell receptors (TCR) bind to peptides from various sources on MHC (Major Histocompatibility Complex) molecules. A long-standing goal in the field is to understand the mechanisms of MHC-peptide exchange and MHC-TCR interactions. Here, I present work from three uniquely different systems that address the following: HLA-DR1 conformational stability, self-tolerant mechanisms of TCRs isolated from self-reactive TCR transgenic mice, and TCR cross-reactivity mechanisms between LCMV and VV. First, I present a crystal structure of HLA-DR1 in complex with A1L9 peptide, a peptide with two amino acid substitutions from the parental peptide. The singly substituted A1 peptide, which has a pocket 1 alanine substitution, decreases intrinsic half-life between MHC-peptide and increases susceptibility to HLA-DM mediated peptide exchange. This data agrees with previous models of HLA-DM-mediated peptide exchange in which the major determinant is located at the HLA-DR1 pocket 1. However, the L9 substituted peptide, which has a pocket 9 leucine substitution, displays the opposite phenotype: increased intrinsic half-life and decreased HLA-DM susceptibility. The crystal structure presented here shows that HLA-DR1 in complex with a doubly substituted peptide, A1L9, is in the same conformation as HLA-DR1 with the wild-type peptide, demonstrating that pocket 9 residues can rescue pocket 1 residue binding deficiencies and that HLA-DR1 stability is determined by amino acids along the peptide, not only at pocket 1. Next, I present crystal structures of two self-tolerant TCRs in complex with IAb-3K pMHC. To elucidate molecular mechanism for self-reactivity and self-tolerance, the TCRs J809.B5 and 14.C6 are compared to each other and its parental self-reactive TCR, YAe-62.8. In comparison to YAe-62.8, J809.B5 interacts with the same pMHC, but utilizes more peptide specific interactions, a mechanism that may distinguish self-reactive receptors from self-tolerant receptors. Additionally, the crystal structure of 14.C6 TCR, which bears a different CDR3α sequence from J809.B5, demonstrates that CDR3 sequences can modulate interactions of germline encoded CDR1 and CDR2 loops. Together, these results highlight that in addition to CDR3 VDJ recombination, diversity is generated in the mature TCR repertoire by differential chain pairing, either of which can affect the interactions of germline encoded CDR loops. Next, I present a detailed analysis of cross-reactive TCRs between Kb-GP34 and Kb-A11R. The mature LCMV-immune repertoire was analyzed by DNA deep sequencing of TCRβ CDR3 sequences, which led to the identification of new cross-reactive sequence motifs. Cross-reactive sequence motifs varied by each Vβ gene, suggesting a role of CDR1, CDR2, and CDR3 loop interplay in cross-reactivity. Lastly, I present the crystal structures of a GP34/A11R cross-reactive TCR in complex with both Kb-GP34 and Kb-A11R. Analysis of the crystal structures revealed that the two complexes are largely the same, despite differences in peptide sequences. Surprisingly, the TCR to peptide interactions were dominated by three out of eight peptide side-chains. Cross-reactivity between these two complexes is likely due to a large amount of interactions from TCR to MHC compared to interactions of TCR to peptide. We note two unique MHC-peptide interactions that may allow Kb to be an allele prone to cross-reactivity. The first is an interaction at the C-terminus of the A11R peptide which pulls A11R P7 asparagine away from TCR interactions. The second interaction is from an arginine at position 155, which sits at the interface between TCRα and TCRβ , and contributes the most buried surface area in the interaction interface. Because Kb’s arginine 155 is a long side chain that hydrogen bonds with the peptide backbone, and is also at the center of the TCR-peptide interface, GP34 and A11R peptide sequence differences may be occluded from TCR discrimination by Kb presentation. The data presented in this dissertation demonstrate that interactions between MHC-peptide and MHC-TCR act harmoniously and coopertively, whereby proximal interactions are affected by interactions elsewhere. While previous models of HLA-DR/HLA-DM interactions demonstrate the importance of interactions at HLA-DR1 pocket 1, I showed that pocket 9 also contributes to HLA-DR stability and therefore, HLA-DM susceptibility. I also showed that TCR CDR3 loop sequences affect germline CDR1/CDR2 loop interactions and vice versa. Lastly, I showed that allele specific MHC side chain interactions with the bound peptide influence TCR ligand binding and hence, TCR cross-reactivity