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HLA-B∗46:01 was formed by an intergenic mini-conversion, between HLA-B∗15:01 and HLA-C∗01:02, in Southeast Asia during the last 50,000 years, and it has since become the most common HLA-B allele in the region. A functional effect of the mini-conversion was introduction of the C1 epitope into HLA-B∗46:01, making it an exceptional HLA-B allotype that is recognized by the C1-specific natural killer (NK) cell receptor KIR2DL3. High-resolution mass spectrometry showed that HLA-B∗46:01 has a low-diversity peptidome that is distinct from those of its parents. A minority (21%) of HLA-B∗46:01 peptides, with common C-terminal characteristics, form ligands for KIR2DL3. The HLA-B∗46:01 peptidome is predicted to be enriched for peptide antigens derived from Mycobacterium leprae. Overall, the results indicate that the distinctive peptidome and functions of HLA-B∗46:01 provide carriers with resistance to leprosy, which drove its rapid rise in frequency in Southeast Asia. Fil: Hilton, Hugo G.. University of Stanford; Estados Unidos Fil: McMurtrey, Curtis P.. Oklahoma State University; Estados Unidos Fil: Han, Alex S.. University of Stanford; Estados Unidos Fil: Djaoud, Zakia. University of Stanford; Estados Unidos Fil: Guethlein, Lisbeth A.. University of Stanford; Estados Unidos Fil: Blokhuis, Jeroen H.. University of Stanford; Estados Unidos Fil: Pugh, Jason L.. University of Stanford; Estados Unidos Fil: Goyos, Ana. University of Stanford; Estados Unidos Fil: Horowitz, Amir. University of Stanford; Estados Unidos Fil: Buchli, Rico. Pure Protein LLC; Estados Unidos Fil: Jackson, Ken W.. Oklahoma State University; Estados Unidos Fil: Bardet, Wilfred. Oklahoma State University; Estados Unidos Fil: Bushnell, David A.. University of Stanford; Estados Unidos Fil: Robinson, Philip J.. University of Stanford; Estados Unidos Fil: Mendoza, Juan L. University of Stanford; Estados Unidos Fil: Birnbaum, Michael E.. University of Stanford; Estados Unidos Fil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Garcia, K. Christopher. University of Stanford; Estados Unidos Fil: Hildebrand, William H.. Oklahoma State University; Estados Unidos Fil: Parham, Peter. University of Stanford; Estados Unidos

CD4 and CD8 co-receptors play critical roles in T cell development and activation by interacting both with T cell receptors and MHC molecules. Although homologs of these genes have been identified in many jawed vertebrates, there are still unresolved gaps concerning their evolution and specialization in MHC interaction and T cell function. Using experimental and computational procedures we identified CD4, CD8α and CD8β gene homologs both in Xenopus tropicalis, whose full genome has been sequenced, and its sister species Xenopus laevis. Multiple alignments of deduced amino acid sequences reveal a poor conservation of the residues involved in binding of CD4 to MHC class II, and CD8α to class I in non-mammalian species, presumably related to the co-evolutionary pressure of MHC I and II genes. Phylogenetic study suggests that Xenopodinae co-receptor genes are more closely related to their homologs in other tetrapods than those of bony fish. Furthermore, the developmental and cell-specific expression patterns of these genes in X. laevis are very similar to that of mammals. X. laevis CD4 is mainly expressed by peripheral non-CD8 T cells and detected in the thymus as early as four days post-fertilization (dpf) at the onset of thymic organogenesis. CD8β expression is specific to adult surface CD8(+) T cells and thymocytes, and is first detected in the thymus at 5 dpf in parallel with productive TCRγ transrcipts, whereas productive TCRβ and α rearrangements are not detected before 7-9 dpf.

Nonclassical MHC class Ib (class Ib) genes are heterogeneous genes encoding molecules that are structurally similar to classical MHC class Ia molecules but with limited tissue distribution and polymorphism. Mammalian class Ib genes have diverse and often uncharacterized functions, and because of their rapid rate of evolution, class Ib phylogeny is difficult to establish. We have conducted an extensive genomic, molecular, and phylogenetic characterization of class Ib genes in two Xenopodinae amphibian species of different genera that diverged from a common ancestor as long ago as primates and rodents (∼65 million years). In contrast with the unsteadiness of mammalian class Ib genes, our results reveal an unusual degree of conservation of most Xenopodinae class Ib gene lineages, including a novel monogenic lineage represented by the divergent Xenopus laevis XNC10 gene and its unequivocal Silurana (Xenopus) tropicalis orthologue, SNC10. The preferential expression of this gene lineage by thymocytes themselves from the onset of thymic organogenesis is consistent with a specialized role of class Ib in early T cell development and suggests such a function is conserved in all tetrapods.

In jawed vertebrates, the heterogeneous nonclassical MHC class Ib (class Ib) gene family encodes molecules structurally similar to classical MHC class Ia (class Ia) but with more limited tissue distribution and lower polymorphism. In mammals, class Ib gene products are involved in stress responses, malignancy and differentiation of intrathymic CD8 T cells. The frog Xenopus laevis possesses at least 20 class Ib genes (XNCs), and 9 subfamilies have been defined so far. We have characterized two novel subfamilies, XNC10 and XNC11. XNC10 is phylogenetically and structurally distinct from both class Ia and other XNC genes. Besides thymic lymphoid tumors, XNC10 is preferentially expressed by circulating T cells and thymocytes of the CD8 lineage both in adult and in larvae from the onset of thymus organogenesis. XNC11 is expressed only by thymocytes and upregulated by several thymic lymphoid tumors. These data provide the first evidence of the expression of any class Ib genes in Xenopus larvae, and suggests evolutionary relationships between certain class Ib genes, malignancy and CD8 T cell ontogeny.

The heat shock proteins (HSPs) gp96 and hsp70 can elicit potent anti-tumor responses and as such have significant clinical potential. Besides cytotoxic CD8 T cell (CTLs) effectors, evidence suggests that natural killer (NK) cells and other less well-characterized cell types also play a critical role in HSP-mediated anti-tumor responses. Owing to their high degree of phylogenetic conservation, we have proposed that HSPs are ancestral agents of immune surveillance; and postulated that their immunological properties, if important, should have been conserved during evolution. We are investigating this issue using a unique non-mammalian comparative tumor-immunity model in the frog Xenopus, which allows us to focus on the relationship between HSPs, classical MHC class Ia, and non-classical MHC class Ib molecules. In addition to a transplantable lymphoid tumor in genetically defined cloned Xenopus, we are generating transgenic frogs with inducible or knocked-down (RNAi) gene expression.

Modulating natural killer cell functions in human immunity and reproduction are diverse interactions between the killer cell immunoglobulin-like receptors (KIR) of Natural Killer (NK) cells and HLA class I ligands on the surface of tissue cells. Dominant interactions are between KIR2DL1 and the C2 epitope of HLA-C and between KIR2DL2/3 and the C1 epitope of HLA-C. KhoeSan hunter-gatherers of Southern Africa represent the earliest population divergence known and are the most genetically diverse indigenous people, qualities reflected in their KIR and HLA genes. Of the ten KhoeSan KIR2DL1 alleles, KIR2DL1*022 and KIR2DL1*026 likely originated in the KhoeSan, and later were transmitted at low frequency to the neighboring Zulus through gene flow. These alleles arose by point mutation from other KhoeSan KIR2DL1 alleles that are more widespread globally. Mutation of KIR2DL1*001 gave rise to KIR2DL1*022, causing loss of C2 recognition and gain of C1 recognition. This makes KIR2DL1*022 a more avid and specific C1 receptor than any KIR2DL2/3 allotype. Mutation of KIR2DL1*012 gave rise to KIR2DL1*026, causing premature termination of translation at the end of the transmembrane domain. This makes KIR2DL1*026 a membrane-associated receptor that lacks both a cytoplasmic tail and signaling function. At higher frequencies than their parental allotypes, the combined effect of the KhoeSan-specific KIR2DL1*022 and KIR2DL1*026 is to reduce the frequency of strong inhibitory C2 receptors and increase the frequency of strong inhibitory C1 receptors. Because interaction of KIR2DL1 with C2 is associated with risk of pregnancy disorder, these functional changes are potentially advantageous. Whereas all other KhoeSan KIR2DL1 alleles are present on a wide diversity of centromeric KIR haplotypes, KIR2DL1*026 is present on a single KIR haplotype and KIR2DL1*022 is present on two very similar haplotypes. The high linkage disequilibrium across their haplotypes is consistent with a recent emergence for these KIR2DL1 alleles that have distinctive functions., Author Summary The genes that control the response of the human immune system vary enormously between individuals. Understanding the evolution of these genetic differences and how they individualize immune responses is central to understanding how the immune system works in health and disease. In this regard, the KhoeSan of southern Africa are particularly informative because they are genetically diverse, divergent from other modern human populations and have been subject to unique demographic history. In the KhoeSan population, we studied variable genes that control natural killer cell function. We identified two recently evolved, novel gene variants that have unusual function; one completely changed its ligand specificity and the other lost its capacity for signal transduction.

Nonclassical MHC class Ib (class Ib) genes are a family of highly diverse and rapidly evolving genes wherein gene numbers, organization, and expression markedly differ even among closely related species rendering class Ib phylogeny difficult to establish. Whereas among mammals there are few unambiguous class Ib gene orthologs, different amphibian species belonging to the anuran subfamily Xenopodinae exhibit an unusually high degree of conservation among multiple class Ib gene lineages. Comparative genomic analysis of class Ib gene loci of two divergent (~65 million years) Xenopodinae subfamily members Xenopus laevis (allotetraploid) and Xenopus tropicalis (diploid) shows that both species possess a large cluster of class Ib genes denoted as Xenopus/Silurana nonclassical (XNC/SNC). Our study reveals two distinct phylogenetic patterns among these genes: some gene lineages display a high degree of flexibility, as demonstrated by species-specific expansion and contractions, whereas other class Ib gene lineages have been maintained as monogenic subfamilies with very few changes in their nucleotide sequence across divergent species. In this second category, we further investigated the XNC/SNC10 gene lineage that in X. laevis is required for the development of a distinct semi-invariant T cell population. We report compelling evidence of the remarkable high degree of conservation of this gene lineage that is present in all 12 species of the Xenopodinae examined, including species with different degrees of ploidy ranging from 2, 4, 8 to 12 N. This suggests that the critical role of XNC10 during early T cell development is conserved in amphibians.

Aim The click-speaking Khoesan of Southern Africa represent the oldest living lineage and are among the most genetically diverse, of modern human populations. In studying the extent and evolution of human immunogenetic diversity, strong balancing selection has been identified in the Khoesan HLA region. The aim of our study was to investigate the genetic and functional diversity of HLA-C and KIR interactions in the Khoesan and compare them to populations with different demographic and genetic histories. Methods We investigated the allelic diversity of KIR2DL1, 2DL2, 2DL3 (using pyrosequencing) and HLA-C (using Luminex) in 56 unrelated Khoesan hunter-gatherers from Upington, South Africa. To measure ligand-specificity and strength, KIR-Fc fusion proteins were made for each identified allotype and tested against One Lambda Labscreen HLA class I beads. To measure the population-wide combinatorial reactivity of KIR and HLA allotypes we devised a scoring system that accounted for their surface expression, binding and signaling capacity as well as their frequencies. Results We identified two novel alleles of KIR2DL1, which are common in the Khoesan. Analysis of 1000 genomes data showed these alleles are unique to this population. 2DL1 ∗ 022 encodes lysine at position 44 which changes its specificity from HLA-C2 to C1 and 2DL1 ∗ 026 binds HLA-C2 but does not transduce an inhibitory signal because it lacks a cytoplasmic tail. Together these alleles reduce the capacity of 2DL1 to function as a C2 receptor in the Khoesan. When accounting for functional characteristics of receptor/ligand pairs, the population wide reactivity of 2DL1-C2 was much lower in the Khoesan than predicted from allele frequencies alone. In contrast, the modeled reactivity of 2DL3-C1 was unaffected. We observe an inverse correlation between functionally interactive allotypes and ligand frequencies such that combinatorial reactivity is equilibrated across populations. Conclusions The Khoesan have a diverse and unique repertoire of KIR genes that have evolved under strong selective pressure from reproductive and infectious disease. Although allele frequency spectra of both loci differ significantly, the signature of these selective pressures remains in the form of KIR and HLA functional profiles that are equilibrated between human populations.

CD91, the endocytic receptor for alpha2-macroglobulin (alpha2M), mediates the internalization of certain heat shock proteins (hsps) and the cross-presentation of peptides they chaperone by antigen-presenting cells. The phylogenetic conservation of the immunologically active CD91 ligands, alpha2M and hsps, is consistent with the idea of an ancestral system of immune surveillance. We have further explored this hypothesis by taking advantage of the frog Xenopus, and asked how conserved is CD91 and whether the expression of CD91 is differentially modulated during immune responses of class I-positive adult and naturally class I-negative larvae. We have identified a Xenopus CD91 gene homologue that displays high sequence identity (65%) with other CD91 homologues and contains an additional distinctive cytoplasmic NPXY motif. Phylogenetic analysis indicates that CD91 homologues branch as a monophyletic group distinct from other LDLRs; this suggests an origin of CD91 contemporary with that of metazoans. A 14-kb transcript is detected by Northern blotting in most adult and larval tissues, including lymphoid tissues. RT-PCR study reveals that CD91 is expressed in most cell types, including adult macrophages, B and T cells as well as in splenocytes and thymocytes from naturally MHC class I negative larvae. CD91 is markedly up-regulated in vivo by adult peritoneal leukocytes following bacterial and viral stimulation; it is constitutively expressed on class I-negative larval peritoneal leukocytes at high levels and cannot be further upregulated by such stimulation. These data are in agreement with a conserved role of CD91 in immunity.