Identification of the major histocompatibility complex class-II DM and DO alleles in a cohort of northern pig-tailed macaques (Macaca leonina).

The northern pig-tailed macaque (Macaca leonina) has been considered as an independent species from the pig-tailed macaque group. We have previously reported that this species macaque has the potential to be a useful animal model in HIV/AIDS pathogenesis and vaccine studies due to its susceptibility to HIV-1. To develop this animal into a potential HIV/AIDS model, we have studied the classical MHC genes of this animal. In this study, the non-classical MHC genes Malo-DM and Malo-DO alleles were first characterized by sequencing and cloning in 12 unrelated northern pig-tailed macaques. A total of 20 full-length sequences identified include 4 Malo-DMA, 5 Malo-DMB, 7 Malo-DOA, and 4 Malo-DOB alleles. Most of these allele sequences were shared between northern pig-tailed macaque and other macaque species in exon 2. The full-length MHC-DM and MHC-DO sequences provide more comprehensive analysis of immunogenetics of northern pig-tailed macaques and increase the value of the macaques in further biomedical studies.

Characterization of classical major histocompatibility complex (MHC) class II genes in northern pig-tailed macaques (Macaca leonina).

The northern pig-tailed macaque (Macaca leonina) has been identified as an independent species from the pig-tailed macaque group. The species is a promising animal model for HIV/AIDS pathogenesis and vaccine studies due to susceptibility to HIV-1. However, the major histocompatibility complex (MHC) genetics in northern pig-tailed macaques remains poorly understood. We have previously studied the MHC class I genes in northern pig-tailed macaques and identified 39 novel alleles. Here, we describe the MHC class II alleles in all six classical loci (DPA, DPB, DQA, DQB, DRA, and DRB) from northern pig-tailed macaques using a sequence-based typing method for the first time. A total of 60 MHC-II alleles were identified of which 27 were shared by other macaque species. Additionally, northern pig-tailed macaques expressed a single DRA and multiple DRB genes similar to the expression in humans and other macaque species. Polymorphism and positive selection were detected, and phylogenetic analysis suggested the presence of a common ancestor in human and northern pig-tailed macaque MHC class II allelic lineages at the DQA, DQB, and DRB loci. The characterization of full-length MHC class II alleles in this study significantly improves understanding of the immunogenetics of northern pig-tailed macaques and provides the groundwork for future animal model studies.

α3-Deletion Isoform of HLA-A11 Modulates Cytotoxicity of NK Cells: Correlations with HIV-1 Infection of Cells.

Alternative splicing occurs frequently in many genes, especially those involved in immunity. Unfortunately, the functions of many alternatively spliced molecules from immunologically relevant genes remain unknown. Classical HLA-I molecules are expressed on almost all nucleated cells and play a pivotal role in both innate and adaptive immunity. Although splice variants of HLA-I genes have been reported, the details of their functions have not been reported. In the current study, we determined the characteristics, expression, and function of a novel splice variant of HLA-A11 named HLA-A11svE4 HLA-A11svE4 is located on the cell surface without ß2-microglobulin (ß2m). Additionally, HLA-A11svE4 forms homodimers as well as heterodimers with HLA-A open conformers, instead of combining with ß2m. Moreover, HLA-A11svE4 inhibits the activation of NK cells to protect target cells. Compared with ß2m and HLA-A11, the heterodimer of HLA-A11svE4 and HLA-A11 protected target cells from lysis by NK cells more effectively. Furthermore, HLA-AsvE4 expression was upregulated by HIV-1 in vivo and by HSV, CMV, and hepatitis B virus in vitro. In addition, our findings indicated that HLA-A11svE4 molecules were functional in activating CD8+ T cells through Ag presentation. Taken together, these results suggested that HLA-A11svE4 can homodimerize and form a novel heterodimeric complex with HLA-A11 open conformers. Furthermore, the data are consistent with HLA-A11svE4 playing a role in the immune escape of HIV-1.

Cloning, sequencing, and polymorphism analysis of novel classical MHC class I alleles in northern pig-tailed macaques (Macaca leonina).

The northern pig-tailed macaque (Macaca leonina) has been confirmed to be an independent species from the pig-tailed macaque group of Old World monkey. We have previously reported that the northern pig-tailed macaques were also susceptible to HIV-1. Here, to make this animal a potential HIV/AIDS model and to discover the mechanism of virus control, we attempted to assess the role of major histocompatibility complex (MHC) class I-restricted immune responses to HIV-1 infection, which was associated with viral replication and disease progression. As an initial step, we first cloned and characterized the classical MHC class I gene of northern pig-tailed macaques. In this study, we identified 39 MHC class I alleles including 17 MHC-A and 22 MHC-B alleles. Out of these identified alleles, 30 were novel and 9 were identical to alleles previously reported from other macaque species. The MHC-A and MHC-B loci were both duplicates as rhesus macaques and southern pig-tailed macaques. In addition, we also detected the patterns of positive selection in northern pig-tailed macaques and revealed the existence of balance selection with 20 positive selection sites in the peptide binding region. The analysis of B and F peptide binding pockets in northern and southern pig-tailed macaques and rhesus macaques suggested that they were likely to share a few common peptides to present. Thus, this study provides important MHC immunogenetics information and adds values to northern pig-tailed macaques as a promising HIV/AIDS model.

A splice variant of HLA-A with a deletion of exon 3 expressed as nonmature cell-surface glycoproteins forms a heterodimeric structure with full-length HLA-A.

Alternatively spliced isoforms of the major histocompatibility complex (MHC) class I genes have been reported in many different species and therefore alternative splicing has been observed to be an additional layer of diversity in the MHC class I region. Here we show the characterization of a HLA-A splice variant in the human peripheral blood mononuclear cells (named "HLA-AΔE3"). This transcript is characterized by the deletion of exon 3 that encodes the α2 domain of the full-length HLA-A protein. Cell surface biotinylation experiments indicated that HLA-AΔE3 is able to be transported to the cell surface, as a 34-KDa glycoprotein that is totally sensitive to endoglycosidase-H treatment. Under nonreducing conditions, HLA-AΔE3 can form disulfide-linked homodimers on the cell surface. Furthermore, co-immunoprecipitation studies revealed that HLA-AΔE3 could interact with full-length HLA-A, forming a heterodimeric complex. These findings suggest that the splice variants of HLA-A under steady-state conditions may have an important function in regulating immune homeostasis.

Molecular characterization, balancing selection, and genomic organization of the tree shrew (Tupaia belangeri) MHC class I gene.

The major histocompatibility complex (MHC) class I genes play a pivotal role in the adaptive immune response among vertebrates. Accordingly, in numerous mammals the genomic structure and molecular characterization of MHC class I genes have been thoroughly investigated. To date, however, little is known about these genes in tree shrews, despite the increasingly popularity of its usage as an animal model. To address this deficiency, we analyzed the structure and characteristic of the tree shrew MHC class I genes (Tube-MHC I) and performed a comparative gene analysis of the tree shrew and other mammal species. We found that the full-length cDNA sequence of the tree shrew MHC class I is 1074bp in length. The deduced peptide is composed of 357 amino acids containing a leader peptide, an α1 and α2 domain, an α3 domain, a transmembrane domain and a cytoplasmic domain. Among these peptides, the cysteines, CD8(+) interaction and N-glycosylation sites are all well conserved. Furthermore, the genomic sequence of the tree shrew MHC class I gene was identified to be 3180bp in length, containing 8 exons and 7 introns. In 21 MHC class I sequences, we conducted an extensive study of nucleotide substitutions. The results indicated that in the peptide binding region (PBR) the rate of non-synonymous substitutions (dN) to synonymous substitutions (dS) was greater than 1, suggesting balancing selection at the PBR. These findings provide valuable contributions in furthering our understanding of the structure, molecular polymorphism, and function of the MHC class I genes in tree shrews, further improving their utility as an animal model in biomedical research.

Identification and characterization of a novel splice variant of rhesus macaque MHC IA.

Major histocompatibility complex class I (MHC I) molecules play a pivotal role in the immune recognition to intracellular pathogens. A number of important splice variants have already been characterized for these molecules in different species, suggesting their important roles in modulation of immune responses. In this study, we have identified and characterized a novel alternatively spliced form of rhesus macaque MHC IA (designated MHC IA-sv2) that lacks exons coding for the α2 and α3 domains. Despite lacking the α2 and α3 domains, MHC IA-sv2 is targeted to the cell surface, as a 23-kDa glycoprotein that is totally susceptible to endoglycosidase-H digestion and is reduced to 18kDa after deglycosylation with PNGase F. In contrast, the full-length MHC IA reaches the cell surface as a 43-kDa protein of form with complex-type N-glycosylation (endoglycosidase-H resistant). Moreover, we provide evidence here that MHC IA-sv2 can self-associate, forming homodimers, or associate with the fully mature MHC IA molecule, forming a heterodimeric structure in mammalian cells. These data demonstrate that the formation of heterodimers may have some functional implications in the fine tuning of MHC IA-mediated innate and adaptive immune responses.

The ß2-microglobulin-free heterodimerization of rhesus monkey MHC class I A with its normally spliced variant reduces the ubiquitin-dependent degradation of MHC class I A.

The MHC class I (MHC I) molecules play a pivotal role in the regulation of immune responses by presenting antigenic peptides to CTLs and by regulating cytolytic activities of NK cells. In this article, we show that MHC I A in rhesus macaques can be alternatively spliced, generating a novel MHC I A isoform (termed "MHC I A-sv1") devoid of α(3) domain. Despite the absence of ß2-microglobulin (ß2m), the MHC I A-sv1 proteins reached the cell surface of K562-transfected cells as endoglycosidase H-sensitive glycoproteins that could form disulfide-bonded homodimers. Cycloheximide-based protein chase experiments showed that the MHC I A-sv1 proteins were more stable than the full-length MHC I A in transiently or stably transfected cell lines. Of particular interest, our studies demonstrated that MHC I A-sv1 could form ß2m-free heterodimers with its full-length protein in mammalian cells. The formation of heterodimers was accompanied by a reduction in full-length MHC I A ubiquitination and consequent stabilization of the protein. Taken together, these results demonstrated that MHC I A-sv1 and MHC I A can form a novel heterodimeric complex as a result of the displacement of ß2m and illustrated the relevance of regulated MHC I A protein degradation in the ß2m-free heterodimerization-dependent control, which may have some implications for the MHC I A splice variant in the fine tuning of classical MHC I A/TCR and MHC I A/killer cell Ig-like receptor interactions.

Dynamics and functions of CD4⁺CD25 (high) regulatory T lymphocytes in Chinese rhesus macaques during the early stage of infection with SIVmac239.

CD4(+)CD25(high) regulatory T cells (Treg), which are a specialized subset of T cells, play an important role in the prevention of autoimmune diseases, maintenance of immune system homeostasis and tolerance to self-antigens. Chinese rhesus macaques (CRMs) are widely used in preclinical research on potential therapeutic drugs, vaccines and mechanisms of human diseases. However, the basic immunological characterization of Treg cells of CRMs has not been well established. To characterize Treg cells, peripheral blood of 43 adult CRMs was analyzed for CD4+ T lymphocytes by flow cytometry. It was found that Treg cells ranged from 1.52% to 11.1% of CD4+ T cells, and the average value was 5.7%. With our SIV-infected CRM model, through further studies, it was found that Treg cells in peripheral blood increased both in relative and absolute quantities. Moreover, Treg cells maintained their functions by suppressing Th1 cytokine secretion of their target cells. The results show that Treg cells might render cellular immunity against SIV viruses dysfunctional during the early stage after infection.

Dendritic cell subsets dynamics and cytokine production in SIVmac239-infected Chinese rhesus macaques.

BACKGROUND: Several studies have demonstrated that SIV infection progresses more slowly to experimental AIDS in Chinese rhesus macaques (Ch Rhs) than in Indian rhesus macaques (Ind Rhs). Here we investigated the dynamic and functional changes in dendritic cell (DC) subsets in SIVmac239-infected Ch Rhs. RESULTS: The numbers of both mDC and pDC strongly fluctuated but were not significantly changed during the acute and chronic phases of infection. However, the concentration of both poly (I:C)-induced IL-12 and HSV-1-induced IFN-α significantly increased in the acute phase of infection but returned to normal levels at the chronic phase of infection. The peak of IFN-α emerged earlier than that of IL-12, and it had a significantly positive correlation with IL-12, which indicated that IFN-α may initiate the immune activation. We also found that only the concentration of IFN-α was positively correlated with CD4+ T-cell counts, but it was negatively correlated with viral load. CONCLUSION: High levels of IFN-α in the early stage of infection may contribute to effective control of virus replication, and normal levels of IFN-α during chronic infection may help Ch Rhs resist the disease progression. The change in DC subsets dynamics and cytokine production may help further our understanding of why Ch Rhs are able to live longer without progressing to an AIDS-like illness.