Lymph node CXCR5+ NK cells associate with control of chronic SHIV infection.

The persistence of virally infected cells as reservoirs despite effective antiretroviral therapy is a major barrier to an HIV/SIV cure. These reservoirs are predominately contained within cells present in the B cell follicles (BCFs) of secondary lymphoid tissues, a site that is characteristically difficult for most cytolytic antiviral effector cells to penetrate. Here, we identified a population of NK cells in macaque lymph nodes that expressed BCF-homing receptor CXCR5 and accumulated within BCFs during chronic SHIV infection. These CXCR5+ follicular NK cells exhibited an activated phenotype coupled with heightened effector functions and a unique transcriptome characterized by elevated expression of cytolytic mediators (e.g., perforin and granzymes, LAMP-1). CXCR5+ NK cells exhibited high expression of FcγRIIa and FcγRIIIa, suggesting a potential for elevated antibody-dependent effector functionality. Consistently, accumulation of CXCR5+ NK cells showed a strong inverse association with plasma viral load and the frequency of germinal center follicular Th cells that comprise a significant fraction of the viral reservoir. Moreover, CXCR5+ NK cells showed increased expression of transcripts associated with IL-12 and IL-15 signaling compared with the CXCR5- subset. Indeed, in vitro treatment with IL-12 and IL-15 enhanced the proliferation of CXCR5+ granzyme B+ NK cells. Our findings suggest that follicular homing NK cells might be important in immune control of chronic SHIV infection, and this may have important implications for HIV cure strategies.

Altered differentiation is central to HIV-specific CD4+ T cell dysfunction in progressive disease.

Dysfunction of virus-specific CD4+ T cells in chronic human infections is poorly understood. We performed genome-wide transcriptional analyses and functional assays of CD4+ T cells specific for human immunodeficiency virus (HIV) from HIV-infected people before and after initiation of antiretroviral therapy (ART). A follicular helper T cell (TFH cell)-like profile characterized HIV-specific CD4+ T cells in viremic infection. HIV-specific CD4+ T cells from people spontaneously controlling the virus (elite controllers) robustly expressed genes associated with the TH1, TH17 and TH22 subsets of helper T cells. Viral suppression by ART resulted in a distinct transcriptional landscape, with a reduction in the expression of genes associated with TFH cells, but persistently low expression of genes associated with TH1, TH17 and TH22 cells compared to the elite controller profile. Thus, altered differentiation is central to the impairment of HIV-specific CD4+ T cells and involves both gain of function and loss of function.

Gene Regulatory Programs Conferring Phenotypic Identities to Human NK Cells.

Natural killer (NK) cells develop from common progenitors but diverge into distinct subsets, which differ in cytokine production, cytotoxicity, homing, and memory traits. Given their promise in adoptive cell therapies for cancer, a deeper understanding of regulatory modules controlling clinically beneficial NK phenotypes is of high priority. We report integrated "-omics" analysis of human NK subsets, which revealed super-enhancers associated with gene cohorts that may coordinate NK functions and localization. A transcription factor-based regulatory scheme also emerged, which is evolutionarily conserved and shared by innate and adaptive lymphocytes. For both NK and T lineages, a TCF1-LEF1-MYC axis dominated the regulatory landscape of long-lived, proliferative subsets that traffic to lymph nodes. In contrast, effector populations circulating between blood and peripheral tissues shared a PRDM1-dominant landscape. This resource defines transcriptional modules, regulated by feedback loops, which may be leveraged to enhance phenotypes for NK cell-based therapies.

Polymorphisms in Rhesus Macaque Tetherin Are Associated with Differences in Acute Viremia in Simian Immunodeficiency Virus Δnef-Infected Animals.

Tetherin (BST-2 or CD317) is an interferon-inducible transmembrane protein that inhibits virus release from infected cells. To determine the extent of sequence variation and the impact of polymorphisms in rhesus macaque tetherin on simian immunodeficiency virus (SIV) infection, tetherin alleles were sequenced from 146 rhesus macaques, including 68 animals infected with wild-type SIVmac239 and 47 animals infected with SIVmac239Δnef Since Nef is the viral gene product of SIV that counteracts restriction by tetherin, these groups afford a comparison of the effects of tetherin polymorphisms on SIV strains that are, and are not, resistant to tetherin. We identified 15 alleles of rhesus macaque tetherin with dimorphic residues at 9 positions. The relationship between these alleles and plasma viral loads was compared during acute infection, prior to the onset of adaptive immunity. Acute viremia did not differ significantly among the wild-type SIV-infected animals; however, differences in acute viral loads were associated with polymorphisms in tetherin among the animals infected with SIVΔnef In particular, polymorphisms at positions 43 and 111 (P43 and H111) were associated with lower acute-phase viral loads for SIVΔnef infection. These observations reveal extensive polymorphism in rhesus macaque tetherin, maintained perhaps as a consequence of variability in the selective pressure of diverse viral pathogens, and identify tetherin alleles that may have an inherently greater capacity to restrict SIV replication in the absence of Nef.IMPORTANCE As a consequence of ongoing evolutionary conflict with viral pathogens, tetherin has accumulated numerous species-specific differences that represent important barriers to the transmission of viruses between species. This study reveals extensive polymorphism in rhesus macaque tetherin and identifies specific alleles that are associated with lower viral loads during the first few weeks after infection with nef-deleted SIV. These observations suggest that the variable selective pressure of viral pathogens, in addition to driving the diversification of tetherin among species, also operates within certain species to maintain sequence variation in tetherin.

Distinct transcriptome profiles of Gag-specific CD8+ T cells temporally correlated with the protection elicited by SIVΔnef live attenuated vaccine.

The live attenuated vaccine (LAV) SIVmac239Δnef (SIVΔnef) confers the best protection among all the vaccine modalities tested in rhesus macaque model of HIV-1 infection. This vaccine has a unique feature of time-dependent protection: macaques are not protected at 3-5 weeks post vaccination (WPV), whereas immune protection emerges between 15 and 20 WPV. Although the exact mechanisms of the time-dependent protection remain incompletely understood, studies suggested that both cellular and humoral immunities contribute to this time-dependent protection. To further elucidate the mechanisms of protection induced by SIVΔnef, we longitudinally compared the global gene expression profiles of SIV Gag-CM9+ CD8+ (Gag-specific CD8+) T cells from peripheral blood of Mamu-A*01+ rhesus macaques at 3 and 20 WPV using rhesus microarray. We found that gene expression profiles of Gag-specific CD8+ T cells at 20 WPV are qualitatively different from those at 3 WPV. At 20 WPV, the most significant transcriptional changes of Gag-specific CD8+ T cells were genes involved in TCR signaling, differentiation and maturation toward central memory cells, with increased expression of CCR7, TCRα, TCRß, CD28 and decreased expression of CTLA-4, IFN-γ, RANTES, granzyme A and B. Our study suggests that a higher quality of SIV-specific CD8+ T cells elicited by SIVΔnef over time contributes to the maturation of time-dependent protection.

Magnitude and Quality of Cytokine and Chemokine Storm during Acute Infection Distinguish Nonprogressive and Progressive Simian Immunodeficiency Virus Infections of Nonhuman Primates.

Acute human immunodeficiency virus (HIV) infection represents a period of intense immune perturbation and activation of the host immune system. Study of the eclipse and viral expansion phases of infection is difficult in humans, but studies in nonprogressive and progressive nonhuman primate (NHP) infection models can provide significant insight into critical events occurring during this time. Cytokines, chemokines, and other soluble immune factors were measured in longitudinal samples from rhesus macaques infected with either SIVmac251 (progressive infection) or SIVmac239Δnef (attenuated/nonprogressive infection) and from African green monkeys infected with SIVsab9315BR (nonpathogenic infection). Levels of acute-phase peak viral replication were highest in SIVmac251 infection but correlated positively with viremia at 3 months postinfection in all three infection models. SIVmac251 infection was associated with stronger corresponding acute-phase cytokine/chemokine responses than the nonprogressive infections. The production of interleukin 15 (IL-15), IL-18, gamma interferon (IFN-γ), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1ß (MIP-1ß), and serum amyloid A protein (SAA) during acute SIVmac251 infection, but not during SIVmac239Δnef or SIVsab9315BR infection, correlated positively with chronic viremia at 3 months postinfection. Acute-phase production of MCP-1 correlated with viremia at 3 months postinfection in both nonprogressive infections. Finally, a positive correlation between the acute-phase area under the curve (AUC) for IL-6 and soluble CD40 ligand (sCD40L) and chronic viremia was observed only for the nonprogressive infection models. While we observed dynamic acute inflammatory immune responses in both progressive and nonprogressive SIV infections, the responses in the nonprogressive infections were not only lower in magnitude but also qualitatively different biomarkers of disease progression. IMPORTANCE: NHP models of HIV infection constitute a powerful tool with which to study viral pathogenesis in order to gain critical information for a better understanding of HIV infection in humans. Here we studied progressive and nonprogressive simian immunodeficiency virus (SIV) infection models in both natural and nonnatural host NHP species. Regardless of the pathogenicity of the virus infection and regardless of the NHP species studied, the magnitude of viremia, as measured by area under the curve, during the first 4 weeks of infection correlated positively with viremia in chronic infection. The magnitude of cytokine and chemokine responses during primary infection also correlated positively with both acute-phase and chronic viremia. However, the pattern and levels of specific cytokines and chemokines produced differed between nonprogressive and progressive SIV infection models. The qualitative differences in the early immune response in pathogenic and nonpathogenic infections identified here may be important determinants of the subsequent disease course.

Reproducing SIVΔnef vaccine correlates of protection: trimeric gp41 antibody concentrated at mucosal front lines.

Vaccination with SIVmac239Δnef provides robust protection against subsequent challenge with wild-type simian immunodeficiency virus (SIV), but safety issues have precluded designing an HIV-1 vaccine based on a live-attenuated virus concept. Safe immunogens and adjuvants that could reproduce identified immune correlates of SIVmac239Δnef protection therefore offer an alternative path for development of an HIV vaccine. Here we describe SIV envelope trimeric gp41 (gp41t) immunogens based on a protective correlate of antibodies to gp41t concentrated on the path of virus entry by the neonatal Fc receptor (FcRn) in cervical vaginal epithelium. We developed a gp41t immunogen-monophosphoryl lipid A adjuvant liposomal nanoparticle for intramuscular (i.m.) immunization and a gp41t-Fc immunogen for intranasal immunization for pilot studies in mice, rabbits, and rhesus macaques. Repeated immunizations to mimic persistent antigen exposure in infection elicited gp41t antibodies in rhesus macaques that were detectable in FcRn+ cervical vaginal epithelium, thus recapitulating one key feature of SIVmac239Δnef vaccinated and protected animals. Although this strategy did not reproduce the system of local production of antibody in SIVmac239Δnef-vaccinated animals, passive immunization experiments supported the concept that sufficiently high levels of antibody can be concentrated by the FcRn at mucosal frontlines, thus setting the stage for assessing protection against vaginal challenge by gp41t immunization.

Multi-functional plasmacytoid dendritic cells redistribute to gut tissues during simian immunodeficiency virus infection.

The objective of this study was to determine the systemic effects of chronic simian immunodeficiency virus (SIV) infection on plasmacytoid dendritic cells (pDCs). pDCs play a critical role in antiviral immunity, but current data are conflicting on whether pDCs inhibit HIV/SIV replication, or, alternatively, contribute to chronic immune activation and disease. Furthermore, previous pDC studies have been complicated by incomplete descriptions of generalized depletion during HIV/SIV infection, and the effects of infection on pDCs outside peripheral blood remain unclear. In scheduled-sacrifice studies of naive and chronically SIV-infected rhesus macaques we evaluated the distribution and functionality of pDCs in multiple tissues using surface and intracellular polychromatic flow cytometry. As previously observed, pDCs were reduced in peripheral blood and spleens, but were also depleted in non-lymphoid organs such as the liver. Interestingly, pDCs accumulated up to fourfold in jejunum, colon and gut-draining lymph nodes, but not in peripheral lymph nodes. Most unexpectedly, SIV infection induced a multi-functional interferon-α, tumour necrosis factor-α, and macrophage inflammatory protein-1ß cytokine secretion phenotype, whereas in normal animals these were generally distinct and separate functions. Herein we show a systemic redistribution of pDCs to gut tissues and gut-draining lymph nodes during chronic SIV infection, coupled to a novel multi-functional cytokine-producing phenotype. While pDC accumulation in the mucosa could aid in virus control, over-production of cytokines from these cells could also contribute to the increased immune activation in the gut mucosa commonly associated with progressive lentivirus infections.

No monkey business: why studying NK cells in non-human primates pays off.

Human NK (hNK) cells play a key role in mediating host immune responses against various infectious diseases. For practical reasons, the majority of the data on hNK cells has been generated using peripheral blood lymphocytes. In contrast, our knowledge of NK cells in human tissues is limited, and not much is known about developmental pathways of hNK cell subpopulations in vivo. Although research in mice has elucidated a number of fundamental features of NK cell biology, mouse, and hNK cells significantly differ in their subpopulations, functions, and receptor repertoires. Thus, there is a need for a model that is more closely related to humans and yet allows experimental manipulations. Non-human primate models offer numerous opportunities for the study of NK cells, including the study of the role of NK cells after solid organ and stem cell transplantation, as well as in acute viral infection. Macaque NK cells can be depleted in vivo or adoptively transferred in an autologous system. All of these studies are either difficult or unethical to carry out in humans. Here we highlight recent advances in rhesus NK cell research and their parallels in humans. Using high-throughput transcriptional profiling, we demonstrate that the human CD56(bright) and CD56(dim) NK cell subsets have phenotypically and functionally analogous counterparts in rhesus macaques. Thus, the use of non-human primate models offers the potential to substantially advance hNK cell research.

SIV infection induces accumulation of plasmacytoid dendritic cells in the gut mucosa.

Multiple studies suggest that plasmacytoid dendritic cells (pDCs) are depleted and dysfunctional during human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infection, but little is known about pDCs in the gut-the primary site of virus replication. Here, we show that during SIV infection, pDCs were reduced 3--fold in the circulation and significantly upregulated the gut-homing marker α4ß7, but were increased 4-fold in rectal biopsies of infected compared to naive macaques. These data revise the understanding of pDC immunobiology during SIV infection, indicating that pDCs are not necessarily depleted, but instead may traffic to and accumulate in the gut mucosa.

In vivo selection of CD4(+) T cells transduced with a gamma-retroviral vector expressing a single-chain intrabody targeting HIV-1 tat.

We evaluated the potential of an anti-human immunodeficiency virus (HIV) Tat intrabody (intracellular antibody) to promote the survival of CD4(+) cells after chimeric simian immunodeficiency virus (SIV)/HIV (SHIV) infection in rhesus macaques. Following optimization of stimulation and transduction conditions, purified CD4(+) T cells were transduced with GaLV-pseudotyped retroviral vectors expressing either an anti-HIV-1 Tat or a control single-chain intrabody. Ex vivo intrabody-gene marking was highly efficient, averaging four copies per CD4(+) cell. Upon reinfusion of engineered autologous CD4(+) cells into two macaques, high levels of gene marking (peak of 0.6% and 6.8% of peripheral blood mononuclear cells (PBMCs) and 0.3% or 2.2% of the lymph node cells) were detected in vivo. One week post cell infusion, animals were challenged with SHIV 89.6p and the ability of the anti-HIV Tat intrabody to promote cell survival was evaluated. The frequency of genetically modified CD4(+) T cells progressively decreased, concurrent with loss of CD4(+) cells and elevated viral loads in both animals. However, CD4(+) T cells expressing the therapeutic anti-Tat intrabody exhibited a relative survival advantage over an 8- and 21-week period compared with CD4(+) cells expressing a control intrabody. In one animal, this survival benefit of anti-Tat transduced cells was associated with a reduction in viral load. Overall, these results indicate that a retrovirus-mediated anti-Tat intrabody provided significant levels of gene marking in PBMCs and peripheral tissues and increased relative survival of transduced cells in vivo.

Vaccine protection against simian immunodeficiency virus in monkeys using recombinant gamma-2 herpesvirus.

Recombinant strains of replication-competent rhesus monkey rhadinovirus (RRV) were constructed in which strong promoter/enhancer elements were used to drive expression of simian immunodeficiency virus (SIV) Env or Gag or a Rev-Tat-Nef fusion protein. Cultured rhesus monkey fibroblasts infected with each recombinant strain were shown to express the expected protein. Three RRV-negative and two RRV-positive rhesus monkeys were inoculated intravenously with a mixture of these three recombinant RRVs. Expression of SIV Gag was readily detected in lymph node biopsy specimens taken at 3 weeks postimmunization. Impressive anti-SIV cellular immune responses were elicited on the basis of major histocompatibility complex (MHC) tetramer staining and gamma interferon enzyme-linked immunospot (ELISPOT) assays. Responses were much greater in magnitude in the monkeys that were initially RRV negative but were still readily detected in the two monkeys that were naturally infected with RRV at the time of immunization. By 3 weeks postimmunization, responses measured by MHC tetramer staining in the two Mamu-A*01(+) RRV-negative monkeys reached 9.3% and 13.1% of all CD8(+) T cells in peripheral blood to the Gag CM9 epitope and 2.3% and 7.3% of all CD8(+) T cells in peripheral blood to the Tat SL8 epitope. Virus-specific CD8(+) T cell responses persisted at high levels up to the time of challenge at 18 weeks postimmunization, and responding cells maintained an effector memory phenotype. Despite the ability of the RRVenv recombinant to express high levels of Env in cultured cells, and despite the appearance of strong anti-RRV antibody responses in immunized monkeys, anti-Env antibody responses were below our ability to detect them. Immunized monkeys, together with three unimmunized controls, were challenged intravenously with 10 monkey infectious doses of SIVmac239. All five immunized monkeys and all three controls became infected with SIV, but peak viral loads were 1.2 to 3.0 log(10) units lower and chronic-phase viral loads were 1.0 to 3.0 log(10) units lower in immunized animals than the geometric mean of unimmunized controls. These differences were statistically significant. Anti-Env antibody responses following challenge indicated an anamnestic response in the vaccinated monkeys. These findings further demonstrate the potential of recombinant herpesviruses as preventive vaccines for AIDS. We hypothesize that this live, replication-competent, persistent herpesvirus vector could match, or come close to matching, live attenuated strains of SIV in the degree of protection if the difficulty with elicitation of anti-Env antibody responses can be overcome.

Gut inflammation and indoleamine deoxygenase inhibit IL-17 production and promote cytotoxic potential in NKp44+ mucosal NK cells during SIV infection.

Natural killer (NK) cells are classically viewed as effector cells that kill virus-infected and neoplastic cells, but recent studies have identified a rare mucosal NK- cell subpopulation secreting the TH17 cytokine IL-22. Here, we report identification of 2 distinct lineages of mucosal NK cells characterized as NKG2A(+)NFIL3(+)RORC(-) and NKp44(+)NFIL3(+)RORC(+). NKG2A(+) NK cells were systemically distributed, cytotoxic, and secreted IFN-γ, whereas NKp44(+) NK cells were mucosae-restricted, noncytotoxic, and produced IL-22 and IL-17. During SIV infection, NKp44(+) NK cells became apoptotic, were depleted, and had an altered functional profile characterized by decreased IL-17 secretion; increased IFN-γ secretion; and, surprisingly, increased potential for cytotoxicity. NKp44(+) NK cells showed no evidence of direct SIV infection; rather, depletion and altered function were associated with SIV-induced up-regulation of inflammatory mediators in the gut, including indoleamine 2,3-dioxygenase 1. Furthermore, treatment of NKp44(+) NK cells with indoleamine 2,3-dioxygenase 1 catabolites in vitro ablated IL-17 production in a dose-dependent manner, whereas other NK-cell functions were unaffected. Thus lentiviral infection both depletes and modifies the functional repertoire of mucosal NK cells involved in the maintenance of gut integrity, a finding that highlights the plasticity of this rare mucosal NK-cell population.

Flow cytometry based identification of simian immunodeficiency virus Env-specific B lymphocytes.

SIV infection of macaques is the most widely employed model for preclinical AIDS vaccine and pathogenesis research. In macaques, high-titer virus-specific antibodies are induced by infection, and antibody responses can drive evolution of viral escape variants. However, neutralizing antibodies (Nabs) induced in response to SIVmac239 and SIVmac251 infection or immunization are generally undetectable or of low titer, and the identification and cloning of potent Nabs from SIVmac-infected macaques remains elusive. Based on recent advances in labeling HIV-specific B lymphocytes [1-3], we have generated recombinant, secreted, soluble SIVmac envelope (Env) proteins (gp120 and gp140) for detection and quantification of SIVmac Env-specific B lymphocytes. In contrast to HIV-1, we found that soluble SIVmac239 gp140 retains the ability to form stable oligomers without the necessity for introducing additional, stabilizing modifications. Soluble oligomeric gp140 reacted with rhesus anti-SIV Env-specific monoclonal antibodies (MAbs), and was used to deplete Env-specific antibodies with SIV neutralization capability from plasma taken from a rhesus macaque immunized with live attenuated SIVmac239∆nef. Soluble gp120 and gp140 bound to SIV-specific immortalized B cells, and to SIV Env-specific B lymphocytes in peripheral blood of immunized animals. These reagents will be useful for analyzing development of Env-specific B cell responses in preclinical studies using SIV-infected or vaccinated rhesus macaques.

Characterization of killer immunoglobulin-like receptor genetics and comprehensive genotyping by pyrosequencing in rhesus macaques.

BACKGROUND: Human killer immunoglobulin-like receptors (KIRs) play a critical role in governing the immune response to neoplastic and infectious disease. Rhesus macaques serve as important animal models for many human diseases in which KIRs are implicated; however, the study of KIR activity in this model is hindered by incomplete characterization of KIR genetics. RESULTS: Here we present a characterization of KIR genetics in rhesus macaques (Macaca mulatta). We conducted a survey of KIRs in this species, identifying 47 novel full-length KIR sequences. Using this expanded sequence library to build upon previous work, we present evidence supporting the existence of 22 Mamu-KIR genes, providing a framework within which to describe macaque KIRs. We also developed a novel pyrosequencing-based technique for KIR genotyping. This method provides both comprehensive KIR genotype and frequency estimates of transcript level, with implications for the study of KIRs in all species. CONCLUSIONS: The results of this study significantly improve our understanding of macaque KIR genetic organization and diversity, with implications for the study of many human diseases that use macaques as a model. The ability to obtain comprehensive KIR genotypes is of basic importance for the study of KIRs, and can easily be adapted to other species. Together these findings both advance the field of macaque KIRs and facilitate future research into the role of KIRs in human disease.

KIR polymorphisms modulate peptide-dependent binding to an MHC class I ligand with a Bw6 motif.

Molecular interactions between killer immunoglobulin-like receptors (KIRs) and their MHC class I ligands play a central role in the regulation of natural killer (NK) cell responses to viral pathogens and tumors. Here we identify Mamu-A1*00201 (Mamu-A*02), a common MHC class I molecule in the rhesus macaque with a canonical Bw6 motif, as a ligand for Mamu-KIR3DL05. Mamu-A1*00201 tetramers folded with certain SIV peptides, but not others, directly stained primary NK cells and Jurkat cells expressing multiple allotypes of Mamu-KIR3DL05. Differences in binding avidity were associated with polymorphisms in the D0 and D1 domains of Mamu-KIR3DL05, whereas differences in peptide-selectivity mapped to the D1 domain. The reciprocal exchange of the third predicted MHC class I-contact loop of the D1 domain switched the specificity of two Mamu-KIR3DL05 allotypes for different Mamu-A1*00201-peptide complexes. Consistent with the function of an inhibitory KIR, incubation of lymphocytes from Mamu-KIR3DL05(+) macaques with target cells expressing Mamu-A1*00201 suppressed the degranulation of tetramer-positive NK cells. These observations reveal a previously unappreciated role for D1 polymorphisms in determining the selectivity of KIRs for MHC class I-bound peptides, and identify the first functional KIR-MHC class I interaction in the rhesus macaque. The modulation of KIR-MHC class I interactions by viral peptides has important implications to pathogenesis, since it suggests that the immunodeficiency viruses, and potentially other types of viruses and tumors, may acquire changes in epitopes that increase the affinity of certain MHC class I ligands for inhibitory KIRs to prevent the activation of specific NK cell subsets.

Quantification of mucosal mononuclear cells in tissues with a fluorescent bead-based polychromatic flow cytometry assay.

Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R=0.9476, P<0.0001). Using this assay, we report enumeration of total CD45(+) leukocytes, CD4(+) and CD8(+) T cells, B cells, NK cells, CD14(+) monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.

SIV-specific CD8+ T cells are enriched in female genital mucosa of rhesus macaques and express receptors for inflammatory chemokines.

PROBLEM: Mucosal T lymphocyte responses in the female reproductive tract, the primary site of HIV transmission in women, may be critical for initial control of virus infection. In addition, characterization of genital immune responses to HIV will be important for the development of a vaccine capable of preventing infection by this route. METHOD OF STUDY: We analyzed lymphocytes isolated from vagina and cervix of chronically SIV-infected macaques for the frequency of SIV Gag tetramer-binding cells and expression of chemokine receptors. RESULTS: We found that the frequency of SIV-specific CD8+ T cell responses was 3- to 30-fold higher in genital tissues than in peripheral blood. SIV-specific CD8+ T cells in genital tissues expressed high levels of CXCR3 and CCR5, chemokine receptors normally expressed on memory T cells that home to inflamed tissues. Cells expressing CXCR3 colocalized with its chemokine ligand CXCL9 [monokine induced by interferon gamma, MIG] in the vaginal lamina propria. CONCLUSION: These results indicate that the frequency of SIV-specific CD8+ T cells in the female genital mucosa is enriched compared with peripheral blood and provide initial information regarding the signals that direct recruitment of T cells to the female reproductive tract.

Simian immunodeficiency virus infection induces expansion of alpha4beta7+ and cytotoxic CD56+ NK cells.

Herein we demonstrate that chronic simian immunodeficiency virus (SIV) infection induces significant upregulation of the gut-homing marker alpha4beta7 on macaque NK cells, coupled with downregulation of the lymph node-trafficking marker, CCR7. Interestingly, in naïve animals, alpha4beta7 expression was associated with increased NK cell activation and, on CD16(+) NK cells, delineated a unique dual-function cytotoxic-CD107a(+)/gamma interferon (IFN-gamma)-secreting population. However, while SIV infection increased CD107a expression on stimulated CD56(+) NK cells, alpha4beta7(+) and alpha4beta7(-) NK cells were affected similarly. These findings suggest that SIV infection redirects NK cells away from the lymph nodes to the gut mucosae but alters NK cell function independent of trafficking repertoires.

CD16- natural killer cells: enrichment in mucosal and secondary lymphoid tissues and altered function during chronic SIV infection.

Natural killer (NK) cells contribute to control of HIV/SIV infection. We defined macaque NK-cell subsets based on expression of CD56 and CD16 and found their distribution to be highly disparate. CD16(+) NK cells predominated in peripheral blood, whereas most mucosal NK cells were CD56(+), and lymph nodes contained both CD56(+) and CD16(-)CD56(-) (double-negative [DN]) subsets. Functional profiles were also distinct among subsets--CD16(+) NK cells expressed high levels of cytolytic molecules, and CD56(+) NK cells were predominantly cytokine-secreting cells, whereas DN NK possessed both functions. In macaques chronically infected with SIV, circulating CD16(+) and DN NK cells were expanded in number and, although markers of cytoxicity increased, cytokine secretion decreased. Notably, CD56(+) NK cells in SIV-infected animals up-regulated perforin, granzyme B, and CD107a. In contrast, the lymph node-homing molecules CD62 ligand (CD62L) and C-C chemokine receptor type 7 (CCR7), which are expressed primarily on CD56(+) and DN NK cells, were significantly down-regulated on NK cells from infected animals. These data demonstrate that SIV infection drives a shift in NK-cell function characterized by decreased cytokine production, expanded cytotoxicity, and trafficking away from secondary lymphoid organs, suggesting that the NK-cell repertoire is not only heterogeneous but also plastic.