SIV infection and ARV treatment reshape the transcriptional and epigenetic profile of naïve and memory T cells in vivo.

Human and simian immunodeficiency viruses (HIV and SIV) are lentiviruses that reverse transcribe their RNA genome with subsequent integration into the genome of the target cell. How progressive infection and administration of antiretrovirals (ARVs) longitudinally influence the transcriptomic and epigenetic landscape of particular T cell subsets, and how these may influence the genetic location of integration are unclear. Here, we use RNAseq and ATACseq to study the transcriptomics and epigenetic landscape of longitudinally sampled naïve and memory CD4+ and CD8+ T cells in two species of non-human primates prior to SIV infection, during chronic SIV infection, and after administration of ARVs. We find that SIV infection leads to significant alteration to the transcriptomic profile of all T cell subsets that are only partially reversed by administration of ARVs. Epigenetic changes were more apparent in animals with longer periods of untreated SIV infection and correlated well with changes in corresponding gene expression. Known SIV integration sites did not vary due to SIV status but did contain more open chromatin in rhesus macaque memory T cells, and the expression of proteasome-related genes at the pre-SIV timepoint correlated with subsequent viremia.IMPORTANCEChronic inflammation during progressive human and simian immunodeficiency virus (HIV and SIV) infections leads to significant co-morbidities in infected individuals with significant consequences. Antiretroviral (ARV)-treated individuals also manifest increased levels of inflammation which are associated with increased mortalities. These data will help guide rational development of modalities to reduce inflammation observed in people living with HIV and suggest mechanisms underlying lentiviral integration site preferences.

Chimpanzee and pig-tailed macaque iPSCs: Improved culture and generation of primate cross-species embryos.

As our closest living relatives, non-human primates uniquely enable explorations of human health, disease, development, and evolution. Considerable effort has thus been devoted to generating induced pluripotent stem cells (iPSCs) from multiple non-human primate species. Here, we establish improved culture methods for chimpanzee (Pan troglodytes) and pig-tailed macaque (Macaca nemestrina) iPSCs. Such iPSCs spontaneously differentiate in conventional culture conditions, but can be readily propagated by inhibiting endogenous WNT signaling. As a unique functional test of these iPSCs, we injected them into the pre-implantation embryos of another non-human species, rhesus macaques (Macaca mulatta). Ectopic expression of gene BCL2 enhances the survival and proliferation of chimpanzee and pig-tailed macaque iPSCs within the pre-implantation embryo, although the identity and long-term contribution of the transplanted cells warrants further investigation. In summary, we disclose transcriptomic and proteomic data, cell lines, and cell culture resources that may be broadly enabling for non-human primate iPSCs research.

Chromosome-level de novo assembly of the pig-tailed macaque genome using linked-read sequencing and HiC proximity scaffolding.

BACKGROUND: Macaque species share >93% genome homology with humans and develop many disease phenotypes similar to those of humans, making them valuable animal models for the study of human diseases (e.g., HIV and neurodegenerative diseases). However, the quality of genome assembly and annotation for several macaque species lags behind the human genome effort. RESULTS: To close this gap and enhance functional genomics approaches, we used a combination of de novo linked-read assembly and scaffolding using proximity ligation assay (HiC) to assemble the pig-tailed macaque (Macaca nemestrina) genome. This combinatorial method yielded large scaffolds at chromosome level with a scaffold N50 of 127.5 Mb; the 23 largest scaffolds covered 90% of the entire genome. This assembly revealed large-scale rearrangements between pig-tailed macaque chromosomes 7, 12, and 13 and human chromosomes 2, 14, and 15. We subsequently annotated the genome using transcriptome and proteomics data from personalized induced pluripotent stem cells derived from the same animal. Reconstruction of the evolutionary tree using whole-genome annotation and orthologous comparisons among 3 macaque species, human, and mouse genomes revealed extensive homology between human and pig-tailed macaques with regards to both pluripotent stem cell genes and innate immune gene pathways. Our results confirm that rhesus and cynomolgus macaques exhibit a closer evolutionary distance to each other than either species exhibits to humans or pig-tailed macaques. CONCLUSIONS: These findings demonstrate that pig-tailed macaques can serve as an excellent animal model for the study of many human diseases particularly with regards to pluripotency and innate immune pathways.

Differential regulation of TREM2 and CSF1R in CNS macrophages in an SIV/macaque model of HIV CNS disease.

HIV-associated neuroinflammation is primarily driven by CNS macrophages including microglia. Regulation of these immune responses, however, remains to be characterized in detail. Using the SIV/macaque model of HIV, we evaluated CNS expression of triggering receptor expressed on myeloid cells 2 (TREM2) which is constitutively expressed by microglia and contributes to cell survival, proliferation, and differentiation. Loss-of-function mutations in TREM2 are recognized risk factors for neurodegenerative diseases including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Nasu-Hakola disease (NHD); recent reports have also indicated a role for TREM2 in HIV-associated neuroinflammation. Using in situ hybridization (ISH) and qRT-PCR, TREM2 mRNA levels were found to be significantly elevated in frontal cortex of macaques with SIV encephalitis compared with uninfected controls (P = 0.02). TREM2 protein levels were also elevated as measured by ELISA of frontal cortex tissue homogenates in these animals. Previously, we characterized the expression of CSF1R (colony-stimulating factor 1 receptor) in this model; the TREM2 and CSF1R promoters both contain a PU.1 binding site. While TREM2 and CSF1R mRNA levels in the frontal cortex were highly correlated (Spearman R = 0.79, P < 0.001), protein levels were not well correlated. In SIV-infected macaques released from ART to study viral rebound, neither TREM2 nor CSF1R mRNA increased with rebound viremia. However, CSF1R protein levels remained significantly elevated unlike TREM2 (P = 0.02). This differential expression suggests that TREM2 and CSF1R play unique, distinct roles in the pathogenesis of HIV CNS disease.

Macaque interferon-induced transmembrane proteins limit replication of SHIV strains in an Envelope-dependent manner.

HIV-1 does not persistently infect macaques due in part to restriction by several macaque host factors. This has been partially circumvented by generating chimeric SIV/HIV-1 viruses (SHIVs) that encode SIV antagonist of known restriction factors. However, most SHIVs replicate poorly in macaques unless they are further adapted in culture and/or macaques (adapted SHIVs). Therefore, development of SHIVs encoding HIV-1 sequences derived directly from infected humans without adaptation (unadapted SHIVs) has been challenging. In contrast to the adapted SHIVs, the unadapted SHIVs have lower replication kinetics in macaque lymphocytes and are sensitive to type-1 interferon (IFN). The HIV-1 Envelope (Env) in the chimeric virus determines both the reduced replication and the IFN-sensitivity differences. There is limited information on macaque restriction factors that specifically limit replication of the more biologically relevant, unadapted SHIV variants. In order to identify the IFN-induced host factor(s) that could contribute to the inhibition of SHIVs in macaque lymphocytes, we measured IFN-induced gene expression in immortalized pig-tailed macaque (Ptm) lymphocytes using RNA-Seq. We found 147 genes that were significantly upregulated upon IFN treatment in Ptm lymphocytes and 31/147 were identified as genes that encode transmembrane helices and thus are likely present in membranes where interaction with viral Env is plausible. Within this group of upregulated genes with putative membrane-localized proteins, we identified several interferon-induced transmembrane protein (IFITM) genes, including several previously uncharacterized Ptm IFITM3-related genes. An evolutionary genomic analysis of these genes suggests the genes are IFITM3 duplications not found in humans that are both within the IFITM locus and also dispersed elsewhere in the Ptm genome. We observed that Ptm IFITMs are generally packaged at higher levels in unadapted SHIVs when compared to adapted SHIVs. CRISPR/Cas9-mediated knockout of Ptm IFITMs showed that depletion of IFITMs partially rescues the IFN sensitivity of unadapted SHIV. Moreover, we found that the depletion of IFITMs also increased replication of unadapted SHIV in the absence of IFN treatment, suggesting that Ptm IFITMs are likely important host factors that limit replication of unadapted SHIVs. In conclusion, this study shows that Ptm IFITMs selectively restrict replication of unadapted SHIVs. These findings suggest that restriction factors including IFITMs vary in their potency against different SHIV variants and may play a role in selecting for viruses that adapt to species-specific restriction factors.

Structural basis of chimpanzee APOBEC3H dimerization stabilized by double-stranded RNA.

APOBEC3H (A3H) is a mammal-specific cytidine deaminase that potently restricts the replication of retroviruses. Primate A3Hs are known to exert key selective pressures against the cross-species transmission of primate immunodeficiency viruses from chimpanzees to humans. Despite recent advances, the molecular structures underlying the functional mechanisms of primate A3Hs have not been fully understood. Here, we reveal the 2.20-Å crystal structure of the chimpanzee A3H (cpzA3H) dimer bound to a short double-stranded RNA (dsRNA), which appears to be similar to two recently reported structures of pig-tailed macaque A3H and human A3H. In the structure, the dsRNA-binding interface forms a specialized architecture with unique features. The analysis of the dsRNA nucleotides in the cpzA3H complex revealed the GC-rich palindrome-like sequence preference for dsRNA interaction, which is largely determined by arginine residues in loop 1. In cells, alterations of the cpzA3H residues critical for the dsRNA interaction severely reduce intracellular protein stability due to proteasomal degradation. This suggests that cpzA3H stability is regulated by the dsRNA-mediated dimerization as well as by unknown cellular machinery through proteasomal degradation in cells. Taken together, these findings highlight unique structural features of primate A3Hs that are important to further understand their cellular functions and regulation.

Novel full-length major histocompatibility complex class I allele discovery and haplotype definition in pig-tailed macaques.

Pig-tailed macaques (Macaca nemestrina, Mane) are important models for human immunodeficiency virus (HIV) studies. Their infectability with minimally modified HIV makes them a uniquely valuable animal model to mimic human infection with HIV and progression to acquired immunodeficiency syndrome (AIDS). However, variation in the pig-tailed macaque major histocompatibility complex (MHC) and the impact of individual transcripts on the pathogenesis of HIV and other infectious diseases is understudied compared to that of rhesus and cynomolgus macaques. In this study, we used Pacific Biosciences single-molecule real-time circular consensus sequencing to describe full-length MHC class I (MHC-I) transcripts for 194 pig-tailed macaques from three breeding centers. We then used the full-length sequences to infer Mane-A and Mane-B haplotypes containing groups of MHC-I transcripts that co-segregate due to physical linkage. In total, we characterized full-length open reading frames (ORFs) for 313 Mane-A, Mane-B, and Mane-I sequences that defined 86 Mane-A and 106 Mane-B MHC-I haplotypes. Pacific Biosciences technology allows us to resolve these Mane-A and Mane-B haplotypes to the level of synonymous allelic variants. The newly defined haplotypes and transcript sequences containing full-length ORFs provide an important resource for infectious disease researchers as certain MHC haplotypes have been shown to provide exceptional control of simian immunodeficiency virus (SIV) replication and prevention of AIDS-like disease in nonhuman primates. The increased allelic resolution provided by Pacific Biosciences sequencing also benefits transplant research by allowing researchers to more specifically match haplotypes between donors and recipients to the level of nonsynonymous allelic variation, thus reducing the risk of graft-versus-host disease.

Analysis of nuclear and organellar genomes of Plasmodium knowlesi in humans reveals ancient population structure and recent recombination among host-specific subpopulations.

The macaque parasite Plasmodium knowlesi is a significant concern in Malaysia where cases of human infection are increasing. Parasites infecting humans originate from genetically distinct subpopulations associated with the long-tailed (Macaca fascicularis (Mf)) or pig-tailed macaques (Macaca nemestrina (Mn)). We used a new high-quality reference genome to re-evaluate previously described subpopulations among human and macaque isolates from Malaysian-Borneo and Peninsular-Malaysia. Nuclear genomes were dimorphic, as expected, but new evidence of chromosomal-segment exchanges between subpopulations was found. A large segment on chromosome 8 originating from the Mn subpopulation and containing genes encoding proteins expressed in mosquito-borne parasite stages, was found in Mf genotypes. By contrast, non-recombining organelle genomes partitioned into 3 deeply branched lineages, unlinked with nuclear genomic dimorphism. Subpopulations which diverged in isolation have re-connected, possibly due to deforestation and disruption of wild macaque habitats. The resulting genomic mosaics reveal traits selected by host-vector-parasite interactions in a setting of ecological transition.

Genetic Characterization of a Captive Colony of Pigtailed Macaques (Macaca nemestrina).

Effective colony management is critical to guarantee the availability of captive NHP as subjects for biomedical research. Pigtailed macaques (Macaca nemestrina) are an important model for the study of human and nonhuman primate diseases and behavior. Johns Hopkins University hosts one of the largest captive colonies of pigtailed macaques in the United States. In this study, we used 56 single-nucleotide polymorphisms (SNP) to characterize this population of pigtailed macaques, understand their population structure, and assess the effectiveness of their colony management. The results demonstrate that the colony has maintained a high level of genetic diversity, with no loss of heterozygosity since its origin, and low levels of inbreeding and genetic subdivision.

Genome-wide mining and comparative analysis of microsatellites in three macaque species.

Microsatellites are found in taxonomically different organisms, and such repeats are related with genomic structure, function and certain diseases. To characterize microsatellites for macaques, we searched and compared SSRs with 1-6 bp nucleotide motifs in rhesus, cynomolgus and pigtailed macaque. A total of 1395671, 1284929 and 1266348 perfect SSRs were mined, respectively. The most frequent perfect SSRs were mononucleotide SSRs. The most GC-content was in dinucleotide SSRs and the least was in the mononucleotide SSRs. Chromosome size was positively correlated with SSR number and negatively correlated with the relative frequency and density of SSRs. The GC content of chromosome SSRs were negatively correlated with relative frequency of SSRs and GC content of chromosome sequences. The features of microsatellite distribution in assembled genomes of the three species were greatly similar, which revealed that the distributional pattern of microsatellites is probably conservative in genus Macaca. The degenerated number of repeat motifs was found to be different in pentanucleotide and hexanucleotide repeats. Species-specific motifs for each macaque were significantly underrepresented. Overall, SSR frequencies of each chromosome in rhesus macaque were higher than in cynomolgus macaque. The maximum repeat times of mono- to pentanucleotide repeats in cynomolgus macaque was more than other two macaques. These results emphasize the genetic diversity and phylogenetic relationship of genus Macaca species. Our data will be beneficial for comparative genome mapping, understanding the distribution of SSRs and genome structure between these animal models, and provide a foundation for further development and identification of more macaque-specific SSRs.

Long-term multilineage engraftment of autologous genome-edited hematopoietic stem cells in nonhuman primates.

Genome editing in hematopoietic stem and progenitor cells (HSPCs) is a promising novel technology for the treatment of many human diseases. Here, we evaluated whether the disruption of the C-C chemokine receptor 5 (CCR5) locus in pigtailed macaque HSPCs by zinc finger nucleases (ZFNs) was feasible. We show that macaque-specific CCR5 ZFNs efficiently induce CCR5 disruption at levels of up to 64% ex vivo, 40% in vivo early posttransplant, and 3% to 5% in long-term repopulating cells over 6 months following HSPC transplant. These genome-edited HSPCs support multilineage engraftment and generate progeny capable of trafficking to secondary tissues including the gut. Using deep sequencing technology, we show that these ZFNs are highly specific for the CCR5 locus in primary cells. Further, we have adapted our clonal tracking methodology to follow individual CCR5 mutant cells over time in vivo, reinforcing that CCR5 gene-edited HSPCs are capable of long-term engraftment. Together, these data demonstrate that genome-edited HSPCs engraft, and contribute to multilineage repopulation after autologous transplantation in a clinically relevant large animal model, an important step toward the development of stem cell-based genome-editing therapies for HIV and potentially other diseases as well.

The complete mitochondrial genome of Southern pig-tailed macaque, Macaca nemestrina, and comparative mitochondrial genomics of Macaca species.

In this study, we report the complete mitochondrial genome sequence of Southern pig-tailed, Macaca nemestrina for the first time. The genome is found to be 16,560 bp in length and has a base composition of A (32.25%), G (12.31%), C (30.51%), and T (24.93%), indicating that the percentage of A + T (57.18%) was higher than G + C (42.82%). Similar to other monkeys, it contains a typically conserved structure including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region (D-loop). Most of the genes were located on the H-strand except for the ND6 gene and 8 tRNA genes. To obtain a more complete understanding of the evolutionary history of Macaca genus, 11 mitochondrial genomes were used for phylogenetic analysis. This mitochondrial sequence reported here would be useful to uncover the monkey's evolution and add a new genetic resource for the genus Macaca.

The effect of exon 7 deletion during the evolution of TRIMCyp fusion proteins on viral restriction, cytoplasmic body formation and multimerization.

TRIMCyp is a fusion protein consisting of the TRIM5 gene product and retrotransposed Cyclophilin A (CypA). Two primate TRIMCyp fusion proteins with varying anti-HIV-1 activities independently evolved in owl monkeys and Old World monkeys. In addition, Old World monkey TRIMCyps lack exon7, which encodes amino acids in the Linker2 region. Previous studies on TRIM5α indicated that this region affects anti-retroviral activity, cytoplasmic body formation, and multimerization. The effects of exon7 deletion on the functions of the TRIMCyp are unclear. In this study, we found that the cytoplasmic bodies and multimers of owl monkey TRIMCyp (omTRIMCyp) are different from those of northern pig-tailed macaque TRIMCyp (npmTRIMCyp). In addition, we demonstrated that exon7 deletion affected cytoplasmic body formation and multimerization. Moreover, we unexpectedly found two chimeric proteins of omTRIMCyp and npmTRIMCyp that failed to block HIV-1 replication, despite the presence of CypA in omTRIMCyp. Further studies indicated that the cytoplasmic bodies and spontaneous multimerization were not responsible for TRIMCyp anti-HIV-1 activity. Moreover, potent viral restriction is associated with higher amounts of monomeric TRIMCyp when the CypA domain is able to recognize and bind to the HIV-1 capsid. Our results suggested that the deletion of exon7 during the evolution of TRIMCyp affected its function.

Survey of major histocompatibility complex class II diversity in pig-tailed macaques.

Pig-tailed macaques (Macaca nemestrina) serve as important models for human infectious disease research. Major histocompatibility complex (MHC) class II molecules are important to this research since they present peptides to CD4+ T cells. Despite the importance of characterizing the MHC-II alleles expressed in model species like pig-tailed macaques, to date, less than 150 MHC-II alleles have been named for the six most common classical class II loci (DRA, DRB, DQA, DQB, DPA, and DPB) in this population. Additionally, only a small percentage of these alleles are full-length, making it impossible to use the known sequence for reagent development. To address this, we developed a fast, high-throughput method to discover full-length MHC-II alleles and used it to characterize alleles in 32 pig-tailed macaques. By this method, we identified 128 total alleles across all six loci. We also performed an exon 2-based genotyping assay to validate the full-length sequencing results; this genotyping assay could be optimized for use in determining MHC-II allele frequencies in large cohorts of pig-tailed macaques.

Cynomolgus and pigtail macaque IgG subclasses: characterization of IGHG genes and computational analysis of IgG/Fc receptor binding affinity.

Macaques are the most widely used experimental nonhuman primate (NHP) species. Rhesus (Macaca mulatta, Macmul), cynomolgus (Macaca fascicularis, Macfas), and pigtail (Macaca nemestrina, Macnem) macaques continue to be popular models for vaccine and infectious diseases research, especially HIV infection and AIDS, and for the development of antibody-based therapeutic strategies. Increased understanding of the immune system of these species is necessary for their optimal use as models of human infections and intervention. In the past few years, the antibody/Fc receptor system has been characterized in a stepwise manner in these species. We have continued this characterization by identifying the four IG heavy gamma (IGHG) genes of Macfas and Macnem in this study. Our results show that these genes share a high degree of similarity with those from other NHP species, while presenting consistent differences when compared to human IGHG genes. Furthermore, comparison of Macfas IGHG genes with those described in other studies suggests the existence of polymorphism. Using sequence- and structure-based computational tools, we performed in silico analysis on multiple polymorphic Macfas IgG and their interactions with human IgG Fc receptors (FcγR), thus predicting that Macfas IGHG polymorphisms influence IgG protein stability and/or binding affinity towards FcγR. The presence of macaque IGHG polymorphisms and macaque/human amino acid changes at locations potentially involved in antibody functional properties indicate the need for cautious design and data interpretation of studies in these models, possibly requiring the characterization of antibody/Fc receptor interactions at the individual level.

Polymorphisms and interspecies differences of the activating and inhibitory FcγRII of Macaca nemestrina influence the binding of human IgG subclasses.

Little is known of the impact of Fc receptor (FcR) polymorphism in macaques on the binding of human (hu)IgG, and nothing is known of this interaction in the pig-tailed macaque (Macaca nemestrina), which is used in preclinical evaluation of vaccines and therapeutic Abs. We defined the sequence and huIgG binding characteristics of the M. nemestrina activating FcγRIIa (mnFcγRIIa) and inhibitory FcγRIIb (mnFcγRIIb) and predicted their structures using the huIgGFc/huFcγRIIa crystal structure. Large differences were observed in the binding of huIgG by mnFcγRIIa and mnFcγRIIb compared with their human FcR counterparts. MnFcγRIIa has markedly impaired binding of huIgG1 and huIgG2 immune complexes compared with huFcγRIIa (His(131)). In contrast, mnFcγRIIb has enhanced binding of huIgG1 and broader specificity, as, unlike huFcγRIIb, it avidly binds IgG2. Mutagenesis and molecular modeling of mnFcγRIIa showed that Pro(159) and Tyr(160) impair the critical FG loop interaction with huIgG. The enhanced binding of huIgG1 and huIgG2 by mnFcγRIIb was shown to be dependent on His(131) and Met(132). Significantly, both His(131) and Met(132) are conserved across FcγRIIb of rhesus and cynomolgus macaques. We identified functionally significant polymorphism of mnFcγRIIa wherein proline at position 131, also an important polymorphic site in huFcγRIIa, almost abolished binding of huIgG2 and huIgG1 and reduced binding of huIgG3 compared with mnFcγRIIa His(131). These marked interspecies differences in IgG binding between human and macaque FcRs and polymorphisms within species have implications for preclinical evaluation of Abs and vaccines in macaques.

The interplay between genome organization and nuclear architecture of primate evolutionary neo-centromeres.

An Evolutionary Neo-Centromere (ENC) is a centromere that emerged in an ectopic region of a chromosome during evolution. It is thought that the old centromere must be inactivated because dicentric chromosomes are not viable. The aim of the present study was to investigate whether 3D arrangement in the interphase nucleus of the novel and old centromeric domains was affected by the repositioning event. The data we present here strongly indicate that the ENC phenomenon does not affect the 3D location of either novel or old centromeres. Very likely, other features, such as gene density, rather than the newly acquired or lost functions, define positioning in the nucleus.

The convergent evolution of blue iris pigmentation in primates took distinct molecular paths.

How many distinct molecular paths lead to the same phenotype? One approach to this question has been to examine the genetic basis of convergent traits, which likely evolved repeatedly under a shared selective pressure. We investigated the convergent phenotype of blue iris pigmentation, which has arisen independently in four primate lineages: humans, blue-eyed black lemurs, Japanese macaques, and spider monkeys. Characterizing the phenotype across these species, we found that the variation within the blue-eyed subsets of each species occupies strongly overlapping regions of CIE L*a*b* color space. Yet whereas Japanese macaques and humans display continuous variation, the phenotypes of blue-eyed black lemurs and their sister species (whose irises are brown) occupy more clustered subspaces. Variation in an enhancer of OCA2 is primarily responsible for the phenotypic difference between humans with blue and brown irises. In the orthologous region, we found no variant that distinguishes the two lemur species or associates with quantitative phenotypic variation in Japanese macaques. Given the high similarity between the blue iris phenotypes in these species and that in humans, this finding implies that evolution has used different molecular paths to reach the same end.

CD34(+) expansion with Delta-1 and HOXB4 promotes rapid engraftment and transfusion independence in a Macaca nemestrina cord blood transplant model.

Umbilical cord blood (CB) transplantation is a promising therapeutic approach but continues to be associated with delayed engraftment and infections. Here, we explored in our macaque CB transplant model expansion and engraftment kinetics of cells expanded with the combination of HOXB4 and Delta-1. CB cells were divided into two equal fractions; one fraction was transduced with HOXB4 yellow fluorescent protein (YFP) and expanded on control OP9 cells, and the other was transduced with HOXB4 green fluorescent protein (GFP) and expanded on Delta-expressing OP9 cells (OP9-DL1). Both fractions were transplanted into myeloablated subjects. Neutrophil and platelet recovery occurred within 7 and 19 days respectively, which was significantly earlier than in our previous study using cells expanded with HOXB4 alone, which resulted in neutrophil recovery within 12 days (P = 0.05) and platelet recovery within 37 days (P = 0.02). Furthermore, two of three animals in the current study remained fully transfusion-independent after transplantation. By day 30, reconstitution of lymphocytes was significantly greater with the HOXB4/OP9-DL1 expanded cells in all animals (P = 0.05). In conclusion, our data show that the combination of OP9-DL1 and HOXB4 can result in increased numbers of repopulating cells, thus leading to rapid engraftment and transfusion independence in macaques transplanted with autologous, expanded CB cells.

The TRIMCyp genotype in four species of macaques in China.

The tripartite motif protein (TRIM)5α/CypA fusion protein TRIMCyp in Old World monkeys is generally considered unable to restrict HIV-1 replication. Monkeys with TRIMCyp can serve as a unique animal model for studies of HIV-1 infection. The present study investigated the distribution and expression status of TRIMCyp in four species of macaques originating from China and its borderlands: pigtail macaques (Macaca nemestrina), rhesus macaques (Macaca mulatta), long-tailed macaques (Macaca fascicularis), and Tibetan macaques (Macaca thibetana). The results revealed that the frequencies of the TRIMCyp genotype were significantly different among different species and even within different populations of the same species. Interestingly, the TRIMCyp genotype was more prevalent among macaques originating from Yunnan and surrounding regions than those from other regions of China. Importantly, TRIMCyp individuals were first identified in Chinese M. mulatta originating from Yunnan, although multiple earlier studies failed to find CypA retrotransposition in this subspecies. Furthermore, TRIMe7-CypA, one of the splicing isoforms of the TRIMCyp transcript was expressed in M. nemestrina and M. mulatta but not M. fascicularis. The intra- and interspecies polymorphisms in the deduced TRIMCyp amino acid sequences of these macaques were also analyzed. Taken together, the data in this study provide important information about the genomic background of TRIMCyp among major species of Chinese macaques.