DC-SIGN on B lymphocytes is required for transmission of HIV-1 to T lymphocytes.

Infection of T cells by HIV-1 can occur through binding of virus to dendritic cell (DC)-specific ICAM-3 grabbing nonintegrin (DC-SIGN) on dendritic cells and transfer of virus to CD4+ T cells. Here we show that a subset of B cells in the blood and tonsils of normal donors expressed DC-SIGN, and that this increased after stimulation in vitro with interleukin 4 and CD40 ligand, with enhanced expression of activation and co-stimulatory molecules CD23, CD58, CD80, and CD86, and CD22. The activated B cells captured and internalized X4 and R5 tropic strains of HIV-1, and mediated trans infection of T cells. Pretreatment of the B cells with anti-DC-SIGN monoclonal antibody blocked trans infection of T cells by both strains of HIV-1. These results indicate that DC-SIGN serves as a portal on B cells for HIV-1 infection of T cells in trans. Transmission of HIV-1 from B cells to T cells through this DC-SIGN pathway could be important in the pathogenesis of HIV-1 infection.

Increased expression of interferon-inducible genes in macaque lung tissues during simian immunodeficiency virus infection.

Pulmonary infections and dysfunction are frequent outcomes during the development of immunodeficiency associated with human immunodeficiency virus type 1 (HIV-1) infection, and obtaining a better understanding of the immunologic changes that occur in lungs following HIV-1 infection will provide a foundation for the development of further intervention strategies. We sought here to identify changes in the pulmonary immune environment that arise during simian immunodeficiency virus (SIV) infection of rhesus macaques, which serves as an excellent model system for HIV-1 infection and disease. To examine the gene expression profiles of macaque lung tissues following infection with the pathogenic SIV/DeltaB670 isolate, we performed cDNA microarray hybridizations with lung total RNAs using two commercially available cDNA arrays and a custom-fabricated, immunologically focused macaque cDNA microarray. In situ hybridization and real-time RT-PCR were performed to provide additional analyses of gene expression. Among the genes exhibiting the highest level of induction in lung tissues were the IFN-gamma-inducible chemokines, CXCL10/IP-10 and CXCL9/Mig. In situ hybridization and real-time RT-PCR strongly supported these findings. Correlation analyses revealed that the levels of expression of IFN-gamma, CXCL9/Mig, and CXCL10/IP-10 mRNAs were all strongly positively correlated, and that CXCL10/IP-10 mRNA and Pneumocystis carinii rRNA were positively correlated. Taken together, these findings demonstrate that inflammatory chemokines are among the most differentially expressed mRNAs in macaque lung tissues during systemic SIV infection of rhesus macaques, and provide insight into the complicated events occurring in the lung tissues during HIV-1 infection in humans.

In situ study of abundant expression of proinflammatory chemokines and cytokines in pulmonary granulomas that develop in cynomolgus macaques experimentally infected with Mycobacterium tuberculosis.

Tuberculosis remains a major public health problem worldwide. Chemokines and cytokines organize and direct infiltrating cells to sites of infection, and these molecules likely play crucial roles in granuloma formation and maintenance. To address this issue, we used in situ hybridization (ISH) to measure chemokine and cytokine mRNA expression levels and patterns directly in lung tissues from cynomolgus macaques (Macaca fascicularis) experimentally infected with a low dose of virulent Mycobacterium tuberculosis. We examined more than 300 granulomas and observed abundant expression of gamma interferon (IFN-gamma)-inducible chemokine mRNAs (CXCL9/monokine induced by IFN-gamma, CXCL10/IFN-gamma-inducible protein, and CXCL11/IFN-gamma-inducible T-cell alpha-chemoattractant) within solid and caseous granulomas, and there was only minimal expression in nongranulomatous regions of tissue. The mRNA expression patterns of IFN-gamma and tumor necrosis factor alpha were examined in parallel, and the results revealed that cytokine mRNA(+) cells were abundant and generally localized to the granulomas. Mycobacterial 16S rRNA expression was also measured by ISH, and the results revealed that there was localization predominantly to the granulomas and that the highest signal intensity was in caseous granulomas. We observed several granulomatous lesions with exceptionally high levels of RNA for mycobacterial 16S rRNA, IFN-gamma, and IFN-gamma-inducible chemokines, suggesting that the local presence of mycobacteria is partially responsible for the upregulation of IFN-gamma-inducible chemokines and recruitment of CXCR3(+) cells, which were also abundant in granulomatous lesions. These results suggest that expression of CXCR3 ligands and the subsequent recruitment of CXCR3(+) cells are involved in granuloma formation and maintenance.

Molecular cloning and sequencing of 25 different rhesus macaque chemokine cDNAs reveals evolutionary conservation among C, CC, CXC, AND CX3C families of chemokines.

Chemokines are small chemoattractant cytokines involved in normal and pathological immune processes. Although extensive nucleotide sequence data are available for human and murine chemokine cDNA sequences, very few data are currently available regarding rhesus macaque sequences. To increase our understanding of immune function in nonhuman primates, we have used reverse-transcription polymerase chain reaction (RT-PCR) to clone and sequence rhesus macaque cDNAs from each of the C, CC, CXC, and CX3C groups of chemokines. Relative to the respective human chemokines, these 25 chemokine cDNA sequences were from 77% to 98% identical. Of the amino acid differences between the rhesus macaque and human chemokines, 51% were species-specific when compared together with the respective murine chemokine sequences. These studies of rhesus macaque chemokine sequences demonstrate that chemokine genes are highly conserved across species, and provide a large foundation for the study of chemokine biology and genetics in nonhuman primates.

Dendritic cell trafficking and antigen presentation in the human immune response to Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is an extraordinarily successful human pathogen, one of the major causes of death by infectious disease worldwide. A key issue for the study of tuberculosis is to understand why individuals infected with Mtb experience different clinical outcomes. To better understand the dynamics of Mtb infection and immunity, we coupled nonhuman primate experiments with a mathematical model we previously developed that qualitatively and quantitatively captures important processes of cellular priming and activation. These processes occur between the lung and the nearest draining lymph node where the key cells mediating this process are the dendritic cells (DC). The nonhuman primate experiments consist of bacteria and cell numbers from tissues of 17 adult cynomolgus macaques (Macaca fascicularis) that were infected with Mtb strain Erdman ( approximately 25 CFU/animal via bronchoscope). The main result of this work is that delays in either DC migration to the draining lymph node or T cell trafficking to the site of infection can alter the outcome of Mtb infection, defining progression to primary disease or latent infection and reactivated tuberculosis. Our results also support the idea that the development of a new generation of treatment against Mtb should optimally elicit a fast DC turnover at the site of infection, as well as strong activation of DCs for maximal Ag presentation and production of key cytokines. This will induce the most protective T cell response.

Restricted SIV replication in rhesus macaque lung tissues during the acute phase of infection.

The extent to which simian immunodeficiency virus (SIV) replication in lung tissues contributes to the pool of viruses replicating during acute infection is incompletely understood. To address this issue, in situ hybridization was used to examine SIV replication in multiple lobes of lung from rhesus macaques infected with pathogenic SIV. Despite widespread viral replication in lymphoid and intestinal tissues, the lungs during acute infection harbored rare productively infected cells. Simultaneous immunohistochemical staining for the monocytic marker, CD68, revealed that SIV RNA(+) cells in lung tissues during acute infection were CD68(-), whereas during AIDS they were predominantly CD68(+) and localized in large foci in caudal lobes. SIV RNA(+) cells in spleen remained CD68(-) throughout disease. Since CD68 is also expressed by subpopulations of dendritic cells (DC), we also examined pulmonary CD68(+) cells for expression of additional DC markers. DC-LAMP mRNA was abundant in lung tissues and expressed predominantly by CD68(-) cells, whereas DC-SIGN mRNA was expressed in only very rare cells, indicating that SIV RNA(+) cells late in disease were most likely macrophages. These studies of SIV/host interactions demonstrate that macaque lung tissues are minimally infected during acute infection, exhibit changes in predominant target cells for infection, and express very little DC-SIGN.

The conserved process of TCR/CD3 complex down-modulation by SIV Nef is mediated by the central core, not endocytic motifs.

The Nef protein of Simian immunodeficiency virus (SIV) associates with multiple T lymphocyte signaling proteins, including the T cell receptor (TCR) zeta chain. We demonstrate here that these interactions are conserved and highly specific. Nefs derived from genetically diverse strains of SIV (SIV(mac)239, SIV(smm)PBj, and SIV(smm)DeltaB670) all interacted with TCR zeta on two separate domains, referred to as SIV Nef interaction domains (SNIDs), as examined in both yeast two-hybrid and glutathione-S-transferase (GST) fusion protein pull-down assays. Multiple HIV-1 Nefs were examined and none interacted with TCR zeta. In contrast, HIV-2(UC1) Nef, similar to SIV Nef, interacted with TCR zeta on two domains, although only the SIV Nefs potently reduced cell-surface expression of the TCR/CD3 complex in T cells. In addition, we examined the abilities of SIV, HIV-2, and HIV-1 Nefs to interact with the cytoplasmic domains of other signaling molecules including CD3epsilon, CD3gamma, and FcepsilonRIgamma, which also contain YxxL motifs, and determined that SIV and HIV-2 Nefs interacted only with TCR zeta, whereas HIV-1 Nef did not interact with any signal-transducing cytoplasmic domain examined. Last, to gain further insight into the mechanism by which Nef down-modulates the TCR/CD3 complex, we mutated or deleted regions on Nef involved in endocytosis, localization of Nef to the plasma membrane, interaction with cellular kinases, or that were conserved among multiple strains of SIV. Mutation of the myristoylation site and a conserved region surrounding a putative PKC phosphorylation site were the only mutations that abrogated Nef-mediated down-modulation of the TCR/CD3 complex. These findings demonstrate there is a spectrum of associations between SIV, HIV-2, and HIV-1 Nefs, and the TCR/CD3 complex, and suggest that down-modulation of the TCR/CD3 complex occurs via association with subsets of cellular proteins that are different from those involved in CD4 and CD28 down-modulation.

Increased expression of the inflammatory chemokine CXC chemokine ligand 9/monokine induced by interferon-gamma in lymphoid tissues of rhesus macaques during simian immunodeficiency virus infection and acquired immunodeficiency syndrome.

Chemokines are important mediators of cell trafficking during immune inductive and effector activities, and dysregulation of their expression might contribute to the pathogenesis of human immunodeficiency virus type 1 and the related simian immunodeficiency virus (SIV). To understand better the effects of SIV infection on lymphoid tissues in rhesus macaques, we examined chemokine messenger RNA (mRNA) expression patterns by using DNA filter array hybridization. Of the 34 chemokines examined, the interferon gamma (IFN-gamma)-inducible chemokine CXC chemokine ligand 9/monokine induced by interferon-gamma (CXCL9/Mig) was one of the most highly up-regulated chemokines in rhesus macaque spleen tissue early after infection with pathogenic SIV. The relative levels of expression of CXCL9/Mig mRNA in spleen and lymph nodes were significantly increased after infection with SIV in both quantitative image capture and analysis and real-time reverse transcriptase-polymerase chain reaction assays. In addition, in situ hybridization for CXCL9/Mig mRNA revealed that the patterns of expression were altered after SIV infection. Associated with the increased expression of CXCL9/Mig were increased numbers of IFN-gamma mRNA-positive cells in tissues and reduced percentages of CXC chemokine receptor (CXCR) 3(+)/CD3(+) and CXCR3(+)/CD8(+) lymphocytes in peripheral blood. We propose that SIV replication in vivo initiates IFN-gamma-driven positive-feedback loops in lymphoid tissues that disrupt the trafficking of effector T lymphocytes and lead to chronic local inflammation, thereby contributing to immunopathogenesis.