Impact of HLA class I functional divergence on HIV control.

Heterozygosity of Human leukocyte antigen (HLA) class I genes is linked to beneficial outcomes after HIV infection, presumably through greater breadth of HIV epitope presentation and cytotoxic T cell response. Distinct allotype pairs, however, differ in the extent to which they bind shared sets of peptides. We developed a functional divergence metric that measures pairwise complementarity of allotype-associated peptide binding profiles. Greater functional divergence for pairs of HLA-A and/or HLA-B allotypes was associated with slower AIDS progression and independently with enhanced viral load control. The metric predicts immune breadth at the peptide level rather than gene level and redefines HLA heterozygosity as a continuum differentially affecting disease outcome. Functional divergence may affect response to additional infections, vaccination, immunotherapy, and other diseases where HLA heterozygote advantage occurs.

HLA class I signal peptide polymorphism determines the level of CD94/NKG2-HLA-E-mediated regulation of effector cell responses.

Human leukocyte antigen (HLA)-E binds epitopes derived from HLA-A, HLA-B, HLA-C and HLA-G signal peptides (SPs) and serves as a ligand for CD94/NKG2A and CD94/NKG2C receptors expressed on natural killer and T cell subsets. We show that among 16 common classical HLA class I SP variants, only 6 can be efficiently processed to generate epitopes that enable CD94/NKG2 engagement, which we term 'functional SPs'. The single functional HLA-B SP, known as HLA-B/-21M, induced high HLA-E expression, but conferred the lowest receptor recognition. Consequently, HLA-B/-21M SP competes with other SPs for providing epitope to HLA-E and reduces overall recognition of target cells by CD94/NKG2A, calling for reassessment of previous disease models involving HLA-B/-21M. Genetic population data indicate a positive correlation between frequencies of functional SPs in humans and corresponding cytomegalovirus mimics, suggesting a means for viral escape from host responses. The systematic, quantitative approach described herein will facilitate development of prediction algorithms for accurately measuring the impact of CD94/NKG2-HLA-E interactions in disease resistance/susceptibility.

Distinct frequency patterns of LILRB3 and LILRA6 allelic variants in Europeans.

The leukocyte immunoglobulin-like receptor (LILR)B3 and LILRA6 genes encode homologous myeloid inhibitory and activating orphan receptors, respectively. Both genes exhibit a strikingly high level of polymorphism at the amino acid level and LILRA6 (but not LILRB3) displays copy number variation (CNV). Although multiple alleles have been reported for both genes, limited data is available on frequencies of these alleles among humans. We have sequenced LILRB3/A6 exons encoding signal peptides and ectodomains in 91 healthy blood donors of European descent who carry one or two copies of LILRA6 per diploid genome. Analysis of haplotypes among individuals with two LILRA6 copies, representing the majority in this cohort (N = 86), shows that common LILRB3 and LILRA6 alleles encode some distinct amino acid sequences in homologous regions of the receptors, which could potentially impact their respective functions differentially. Comparison of sequences in individuals with one vs. two copies of LILRA6 supports non-allelic homologous recombination between LILRB3 and LILRA6 as a mechanism for generating LILRA6 CNV and LILRB3 diversity. These data characterize LILRB3/LILRA6 genetic variation in more detail than previously described and underscore the need to determine their ligands.

Population-specific diversity of the immunoglobulin constant heavy G chain (IGHG) genes.

Human immunoglobulin G (IgG) molecules, IgG1, IgG2 and IgG3, exhibit substantial inter-individual variation in their constant heavy chain regions, as discovered by serological methods. This polymorphism is encoded by the IGHG1, IGHG2, and IGHG3 genes and may influence antibody function. We sequenced the coding fragments of these genes in 95 European Americans, 94 African Americans, and 94 Black South Africans. Striking differences were observed between the population groups, including extremely low amino acid sequence variation in IGHG1 among South Africans, and higher IGHG2 and IGHG3 diversity in individuals of African descent compared to individuals of European descent. Molecular definition of the loci illustrates a greater level of allelic polymorphism than previously described, including the presence of common IGHG2 and IGHG3 variants that were indistinguishable serologically. Comparison of our data with the 1000 Genome Project sequences indicates overall agreement between the datasets, although some inaccuracies in the 1000 Genomes Project are likely. These data represent the most comprehensive analysis of IGHG polymorphisms across major populations, which can now be applied to deciphering their functional impact.

Identification and Characterization of Antigen-Specific CD8+ T Cells Using Surface-Trapped TNF-α and Single-Cell Sequencing.

CD8+ T cells are key mediators of antiviral and antitumor immunity. The isolation and study of Ag-specific CD8+ T cells, as well as mapping of their MHC restriction, has practical importance to the study of disease and the development of therapeutics. Unfortunately, most experimental approaches are cumbersome, owing to the highly variable and donor-specific nature of MHC-bound peptide/TCR interactions. Here we present a novel system for rapid identification and characterization of Ag-specific CD8+ T cells, particularly well suited for samples with limited primary cells. Cells are stimulated ex vivo with Ag of interest, followed by live cell sorting based on surface-trapped TNF-α. We take advantage of major advances in single-cell sequencing to generate full-length sequence data from the paired TCR α- and ß-chains from these Ag-specific cells. The paired TCR chains are cloned into retroviral vectors and used to transduce donor CD8+ T cells. These TCR transductants provide a virtually unlimited experimental reagent, which can be used for further characterization, such as minimal epitope mapping or identification of MHC restriction, without depleting primary cells. We validated this system using CMV-specific CD8+ T cells from rhesus macaques, characterizing an immunodominant Mamu-A1*002:01-restricted epitope. We further demonstrated the utility of this system by mapping a novel HLA-A*68:02-restricted HIV Gag epitope from an HIV-infected donor. Collectively, these data validate a new strategy to rapidly identify novel Ags and characterize Ag-specific CD8+ T cells, with applications ranging from the study of infectious disease to immunotherapeutics and precision medicine.

HLA tapasin independence: broader peptide repertoire and HIV control.

Human leukocyte antigen (HLA) class I allotypes vary in their ability to present peptides in the absence of tapasin, an essential component of the peptide loading complex. We quantified tapasin dependence of all allotypes that are common in European and African Americans (n = 97), which revealed a broad continuum of values. Ex vivo examination of cytotoxic T cell responses to the entire HIV-1 proteome from infected subjects indicates that tapasin-dependent allotypes present a more limited set of distinct peptides than do tapasin-independent allotypes, data supported by computational predictions. This suggests that variation in tapasin dependence may impact the strength of the immune responses by altering peptide repertoire size. In support of this model, we observed that individuals carrying HLA class I genotypes characterized by greater tapasin independence progress more slowly to AIDS and maintain lower viral loads, presumably due to increased breadth of peptide presentation. Thus, tapasin dependence level, like HLA zygosity, may serve as a means to restrict or expand breadth of the HLA-I peptide repertoire across humans, ultimately influencing immune responses to pathogens and vaccines.

HLA class I and KIR genes do not protect against HIV type 1 infection in highly exposed uninfected individuals with hemophilia A.

A recent genome-wide association study (GWAS) involving patients with hemophilia A who were exposed to but uninfected with human immunodeficiency virus type 1 (HIV-1) did not reveal genetic variants associated with resistance to HIV-1 infection, beyond homozygosity for CCR5-Δ32. Since variation in HLA class I and KIR genes is not well interrogated by standard GWAS techniques, we tested whether these 2 loci were involved in protection from HIV-1 infection in the same hemophilia cohort, using controls from the general population. Our data indicate that HLA class I alleles, presence or absence of KIR genes, and functionally relevant combinations of the HLA/KIR genotypes are not involved in resistance to parenterally transmitted HIV-1 infection.

LILRB2 interaction with HLA class I correlates with control of HIV-1 infection.

Natural progression of HIV-1 infection depends on genetic variation in the human major histocompatibility complex (MHC) class I locus, and the CD8+ T cell response is thought to be a primary mechanism of this effect. However, polymorphism within the MHC may also alter innate immune activity against human immunodeficiency virus type 1 (HIV-1) by changing interactions of human leukocyte antigen (HLA) class I molecules with leukocyte immunoglobulin-like receptors (LILR), a group of immunoregulatory receptors mainly expressed on myelomonocytic cells including dendritic cells (DCs). We used previously characterized HLA allotype-specific binding capacities of LILRB1 and LILRB2 as well as data from a large cohort of HIV-1-infected individuals (N = 5126) to test whether LILR-HLA class I interactions influence viral load in HIV-1 infection. Our analyses in persons of European descent, the largest ethnic group examined, show that the effect of HLA-B alleles on HIV-1 control correlates with the binding strength between corresponding HLA-B allotypes and LILRB2 (p = 10(-2)). Moreover, overall binding strength of LILRB2 to classical HLA class I allotypes, defined by the HLA-A/B/C genotypes in each patient, positively associates with viral replication in the absence of therapy in patients of both European (p = 10(-11)-10(-9)) and African (p = 10(-5)-10(-3)) descent. This effect appears to be driven by variations in LILRB2 binding affinities to HLA-B and is independent of individual class I allelic effects that are not related to the LILRB2 function. Correspondingly, in vitro experiments suggest that strong LILRB2-HLA binding negatively affects antigen-presenting properties of DCs. Thus, we propose an impact of LILRB2 on HIV-1 disease outcomes through altered regulation of DCs by LILRB2-HLA engagement.

Diversity of the human LILRB3/A6 locus encoding a myeloid inhibitory and activating receptor pair.

Leukocyte immunoglobulin-like receptor (LILR)B3 and LILRA6 represent a pair of inhibitory/activating receptors with identical extracellular domains and unknown ligands. LILRB3 can mediate inhibitory signaling via immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic tail whereas LILRA6 can signal through association with an activating adaptor molecule, FcRγ, which bears a cytoplasmic tail with an immunoreceptor tyrosine-based activation motif. The receptors are encoded by two highly polymorphic neighboring genes within the leukocyte receptor complex on human chromosome 19. Here, we report that the two genes display similar levels of single nucleotide polymorphisms with the majority of polymorphic sites being identical. In addition, the LILRA6 gene exhibits copy number variation (CNV) whereas LILRB3 does not. A screen of healthy Caucasians indicated that 32 % of the subjects possessed more than two copies of LILRA6, whereas 4 % have only one copy of the gene per diploid genome. Analysis of mRNA expression in the major fractions of PBMCs showed that LILRA6 is primarily expressed in monocytes, similarly to LILRB3, and its expression level correlates with copy number of the gene. We suggest that the LILRA6 CNV may influence the level of the activating receptor on the cell surface, potentially affecting signaling upon LILRB3/A6 ligation.

On stand by: host genetics of HIV control.

The impact of host genetic variation on determining the differential outcomes after HIV infection has been studied by two approaches: targeting of candidate genes and genome-wide association studies (GWASs). The overlap in genetic variants that has been identified by these two means has essentially been restricted to variants near to the human leukocyte antigen (HLA) class I genes, although variation in the CCR5 locus, which was first shown to have an effect on HIV outcomes using the candidate gene approach, does reach significance genome-wide when very large samples sizes (i.e. thousands) are used in GWAS. Overall, many of the variants identified by the candidate gene approach are likely to be spurious, as no additional variants apart from a novel variant near the HLA-C gene have been consistently identified by GWAS. Variants with low frequency and/or low impact on HIV outcomes are likely to exist in the genome and there could be many of them, but these are not identifiable, given current GWAS sample sizes. Several loci centrally involved in the immune response, including the immunoglobulin genes, T-cell receptor loci, or leukocyte receptor complex, are either poorly covered on the GWAS chips or difficult to interpret due to their repetitive nature and/or the presence of insertion/deletion polymorphisms in the region. These loci warrant further interrogation, but genetic characterization of these regions across a range of individuals will first be required. Finally, synergistic interactions between loci may affect outcome after infection, as suggested by associations of specific, functionally relevant HLA and killer cell immunoglobulin-like receptor variants with HIV disease outcomes, and these require further consideration as well.

HLA/KIR restraint of HIV: surviving the fittest.

Multiple epidemiological studies have demonstrated associations between the human leukocyte antigen (HLA) loci and human immunodeficiency virus (HIV) disease, and more recently the killer cell immunoglobulin-like (KIR) locus has been implicated in differential responses to the virus. Genome-wide association studies have convincingly shown that the HLA class I locus is the most significant host genetic contributor to the variation in HIV control, underscoring a central role for CD8 T cells in resistance to the virus. However, both genetic and functional data indicate that part of the HLA effect on HIV is due to interactions between KIR and HLA genes, also implicating natural killer cells in defense against viral infection and viral expansion prior to initiation of an adaptive response. We review the HLA and KIR associations with HIV disease and the progress that has been made in understanding the mechanisms that explain these associations.

Novel KIR3DL1 alleles and their expression levels on NK cells: convergent evolution of KIR3DL1 phenotype variation?

KIR3DL1 shows extensive polymorphism, and its variation has functional significance in terms of cell-surface expression levels and inhibitory capacity. We characterized nine KIR3DL1 alleles (*022, *028, *029, *033, *035, *051, *052, *053, and *054), four of which were identified for the first time in this study, and compared them to known alleles in phylogenetic analysis. Blood was available from eight individuals with these alleles, and cell-surface expression on NK cells could be determined for six of them using the KIR3DL1-specific Ab DX9. Four of the alleles were expressed at clearly detectable levels, and two others showed exceptionally low levels of expression. Site-directed mutagenesis demonstrated that single amino acid changes can result in either diminished or enhanced DX9 staining compared with the respective related KIR3DL1 allotypes. These results raise the possibility that KIR3DL1 evolution maintains variation in KIR3DL1 cell-surface expression levels, potentially due to the effect of such variation on functional capacity.

The killer immunoglobulin-like receptor gene cluster: tuning the genome for defense.

Killer immunoglobulin-like receptors (KIRs) are molecules expressed on the surface of natural killer (NK) cells, which play an important role in innate immunity. KIR recognition of major histocompatability complex (MHC) class I allotypes represents one component of the complex interactions between NK cells and their targets in determining NK cell reactivity. KIRs are encoded by a gene cluster at human chromosome 19q13.4. Despite their high degree of sequence identity, KIR genes encode proteins that have diverse recognition patterns (specific HLA class I allotypes) and confer opposing signals (activating or inhibitory) to the NK cell. The KIR gene cluster is highly polymorphic, with individual genes exhibiting allelic variability and individual haplotypes differing in gene content. The polymorphism of the KIR locus parallels that of the MHC, facilitating the adaptation of the immune system to a dynamic, challenging environment. This variation is associated with a growing number of human diseases, which is likely to extend to levels observed for the HLA loci. Here we review current progress in understanding KIR biology and genetics.

Consistent effects of TSG101 genetic variability on multiple outcomes of exposure to human immunodeficiency virus type 1.

Tumor susceptibility gene 101 (TSG101) encodes a host cellular protein that is appropriated by human immunodeficiency virus type 1 (HIV-1) in the budding process of viral particles from infected cells. Variation in the coding or noncoding regions of the gene could potentially affect the degree of TSG101-mediated release of viral particles. While the coding regions of the gene were found to lack nonsynonymous variants, two polymorphic sites in the TSG101 5' area were identified that were associated with the rate of AIDS progression among Caucasians. These single-nucleotide polymorphisms (SNPs), located at positions -183 and +181 relative to the translation start, specify three haplotypes termed A, B, and C, which occur at frequencies of 67%, 21%, and 12%, respectively. Haplotype C is associated with relatively rapid AIDS progression, while haplotype B is associated with slower disease progression. Both effects were dominant over the intermediate haplotype A. The haplotypes also demonstrated parallel effects on the rate of CD4 T-cell depletion and viral load increase over time, as well as a possible influence on HIV-1 infection. The data raise the hypothesis that noncoding variation in TSG101 affects the efficiency of TSG101-mediated release of viral particles from infected cells, thereby altering levels of plasma viral load and subsequent disease progression.

Novel member of the CD209 (DC-SIGN) gene family in primates.

Two CD209 family genes identified in humans, CD209 (DC-SIGN) and CD209L (DC-SIGNR/L-SIGN), encode C-type lectins that serve as adhesion receptors for ICAM-2 and ICAM-3 and participate in the transmission of human and simian immunodeficiency viruses (HIV and SIV, respectively) to target cells in vitro. Here we characterize the CD209 gene family in nonhuman primates and show that recent evolutionary alterations have occurred in this family across primate species. All of the primate species tested, specifically, Old World monkeys (OWM) and apes, have orthologues of human CD209. In contrast, CD209L is missing in OWM but present in apes. A third family member, that we have named CD209L2, was cloned from rhesus monkey cDNA and subsequently identified in OWM and apes but not in humans. Rhesus CD209L2 mRNA was prominently expressed in the liver and axillary lymph nodes, although preliminary data suggest that levels of expression may vary among individuals. Despite a high level of sequence similarity to both human and rhesus CD209, rhesus CD209L2 was substantially less effective at binding ICAM-3 and poorly transmitted HIV type 1 and SIV to target cells relative to CD209. Our data suggest that the CD209 gene family has undergone recent evolutionary processes involving duplications and deletions, the latter of which may be tolerated because of potentially redundant functional activities of the molecules encoded by these genes.

Rhesus macaque dendritic cells efficiently transmit primate lentiviruses independently of DC-SIGN.

Here, we describe the isolation and characterization of the rhesus macaque homolog for human DC-SIGN, a dendritic cell-specific C-type lectin. mac-DC-SIGN is 92% identical to hu-DC-SIGN. mac-DC-SIGN preserves the virus transmission function of hu-DC-SIGN, capturing and efficiently transducing simian and human immunodeficiency virus to target CD4(+) T cells. Surprisingly, however, mac-DC-SIGN plays no discernable role in the ability of rhesus macaque dendritic cells to capture and transmit primate lentiviruses. Expression and neutralization analyses suggest that this process is DC-SIGN independent in macaque, although the participation of other lectin molecules cannot be ruled out. The ability of primate lentiviruses to effectively use human and rhesus dendritic cells in virus transmission without the cells becoming directly infected suggests that these viruses have taken advantage of a conserved dendritic cell mechanism in which DC-SIGN family molecules are significant contributors but not the only participants.