Tristan I. Evans

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 α4β7, 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.

Simian Immunodeficiency Virus Infection Induces Expansion of α4β7+ and Cytotoxic CD56+ NK Cells

ABSTRACT Herein we demonstrate that chronic simian immunodeficiency virus (SIV) infection induces significant upregulation of the gut-homing marker α4β7 on macaque NK cells, coupled with downregulation of the lymph node-trafficking marker, CCR7. Interestingly, in naïve animals, α4β7 expression was associated with increased NK cell activation and, on CD16+ NK cells, delineated a unique dual-function cytotoxic-CD107a+/gamma interferon (IFN-γ)-secreting population. However, while SIV infection increased CD107a expression on stimulated CD56+ NK cells, α4β7+ and α4β7− 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.

Hypercytotoxicity and rapid loss of NKp44+ innate lymphoid cells during acute SIV infection.

HIV/SIV infections break down the integrity of the gastrointestinal mucosa and lead to chronic immune activation and associated disease progression. Innate lymphoid cells (ILCs), distinguishable by high expression of NKp44 and RORγt, play key roles in mucosal defense and homeostasis, but are depleted from gastrointestinal (GI) tract large bowel during chronic SIV infection. However, less is known about the kinetics of ILC loss, or if it occurs systemically. In acute SIV infection, we found a massive, up to 8-fold, loss of NKp44+ILCs in all mucosae as early as day 6 post-infection, which was sustained through chronic disease. Interestingly, no loss of ILCs was observed in mucosa-draining lymph nodes. In contrast, classical NK cells were not depleted either from gut or draining lymph nodes. Both ILCs and NK cells exhibited significantly increased levels of apoptosis as measured by increased Annexin-V expression, but while classical NK cells also showed increased proliferation, ILCs did not. Interestingly, ILCs, which are normally noncytolytic, dramatically upregulated cytotoxic functions in acute and chronic infection and acquired a polyfunctional phenotype secreting IFN-γ, MIP1-β, and TNF-α, but decreased production of the prototypical cytokine, IL-17. Classical NK cells had less dramatic functional change, but upregulated perforin expression and increased cytotoxic potential. Finally, we show that numerical and functional loss of ILCs was due to increased apoptosis and ROR γt suppression induced by inflammatory cytokines in the gut milieu. Herein we demonstrate the first evidence for acute, systemic, and permanent loss of mucosal ILCs during SIV infection associated with reduction of IL-17. The massive reduction of ILCs involves apoptosis without compensatory de novo development/proliferation, but the full mechanism of depletion and the impact of functional change so early in infection remain unclear.

Accumulation of Cytotoxic CD16+ NK Cells in Simian Immunodeficiency Virus-Infected Lymph Nodes Associated with In Situ Differentiation and Functional Anergy

ABSTRACT Recent evidence suggests that even in treated infections, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication may continue in lymph nodes (LN), serving as a potential virus reservoir. Here we investigated the effects of lentivirus infection on natural killer (NK) cell frequencies, phenotypes, and functions in naive and acutely or chronically SIVmac239-infected rhesus macaques. Compared to that in naive animals, we observed a 3-fold-greater frequency of cytotoxic CD16+ CD56− NK cells in LN of chronically infected macaques. However, NK cells did not appear to be trafficking to LN, as homing markers CD62L and CCR7 did not increase on circulating NK cells during infection. LN NK cells demonstrated enhanced cytotoxicity in acute infection, with 2-fold increases in perforin expression and 3-fold increases in CD107a expression following mitogen stimulation. Lysis of K562 cells by LN NK cells from acutely infected animals was greater than lysis by preinfection samples from the same animals. LN NK cells from chronically infected animals lysed K562 cells more efficiently than LN NK cells from uninfected animals, but importantly, surrogate markers of cytotoxicity in infected macaques were disproportionately greater than ex vivo killing. Furthermore, Tim-3, an indicator of activation and/or exhaustion, was upregulated 3-fold on LN NK cells in chronically infected animals. Collectively, these data suggest that LN NK cells are skewed toward a cytotoxic phenotype during SIV infection but may become dysfunctional and exhausted in chronic disease. IMPORTANCE The accumulation of CD16+ CD56− NK cells in the SIV-infected lymph node without changes in NK homing to the LN could suggest that these cells are differentiating in situ. Surprisingly, this increase in frequency of the cytotoxic subset of NK cells is not accompanied by an increase of similar magnitude in the cytolytic function of LN lymphocytes. This functional modulation, together with the higher Tim-3 expression observed on LN NK cells isolated from chronically infected animals than on those from naive macaques, is indicative of an exhausted phenotype. This exhaustion could contribute to the robust replication of HIV and SIV in the LN during acute and chronic stages of infection, allowing the survival of infected cells and maintenance of a viral reservoir.

SIV-induced Translocation of Bacterial Products in the Liver Mobilizes Myeloid Dendritic and Natural Killer Cells Associated With Liver Damage

Disruption of the mucosal epithelium during lentivirus infections permits translocation of microbial products into circulation, causing immune activation and driving disease. Although the liver directly filters blood from the intestine and is the first line of defense against gut-derived antigens, the effects of microbial products on the liver are unclear. In livers of normal macaques, minute levels of bacterial products were detectable, but increased 20-fold in simian immunodeficiency virus (SIV)-infected animals. Increased microbial products in the liver induced production of the chemoattractant CXCL16 by myeloid dendritic cells (mDCs), causing subsequent recruitment of hypercytotoxic natural killer (NK) cells expressing the CXCL16 receptor, CXCR6. Microbial accumulation, mDC activation, and cytotoxic NK cell frequencies were significantly correlated with markers of liver damage, and SIV-infected animals consistently had evidence of hepatitis and fibrosis. Collectively, these data indicate that SIV-associated accumulation of microbial products in the liver initiates a cascade of innate immune activation, resulting in liver damage.

Bone Marrow–Imprinted Gut-Homing of Plasmacytoid Dendritic Cells (pDCs) in Acute Simian Immunodeficiency Virus Infection Results in Massive Accumulation of Hyperfunctional CD4+ pDCs in the Mucosae

Plasmacytoid dendritic cells (pDCs), a primary source of interferon α (IFN-α), provide a first line of innate immune defense against human immunodeficiency virus infection. However, their kinetics and functions during acute infection are poorly understood. In mucosal tissues of normal rhesus macaques, we found CD4(+) pDCs to be the subset responsible for most IFN-α and tumor necrosis factor α (TNF-α) production in response to Toll-like receptor (TLR) 7/8 stimulation, compared with relatively anergic CD4(-) pDCs. During acute simian immunodeficiency virus (SIV) infection, gut homing was imprinted on pDCs in the bone marrow, resulting in a decline in pDCs from circulation and secondary lymphoid tissues. Although the accumulated pDCs in the gut mucosae had robust cytokine responses to TLR7/8 stimulation in vitro, pDC gut migration occurred after infection and detection of SIV in plasma. Our data suggest that innate pDC responses do not control initial SIV seeding and dissemination but instead may contribute to ongoing immune activation in the gut.

All-trans-Retinoic Acid Imprints Expression of the Gut-Homing Marker α4β7 while Suppressing Lymph Node Homing of Dendritic Cells

ABSTRACT Tissue-directed trafficking of dendritic cells (DCs) as natural adjuvants and/or direct vaccine carriers is highly attractive for the next generation of vaccines and immunotherapeutics. Since these types of studies would undoubtedly be first conducted using nonhuman primate models, we evaluated the ability of all-trans-retinoic acid (ATRA) to induce gut-homing α4β7 expression on rhesus macaque plasmacytoid and myeloid DCs (pDCs and mDCs, respectively). Induction of α4β7 occurred in both a time-dependent and a dose-dependent manner with up to 8-fold increases for mDCs and 2-fold increases for pDCs compared to medium controls. ATRA treatment was also specific in inducing α4β7 expression, but not expression of another mucosal trafficking receptor, CCR9. Unexpectedly, upregulation of α4β7 was associated with a concomitant downregulation of CD62L, a marker of lymph node homing, indicating an overall shift in the trafficking repertoire. These same phenomena occurred with ATRA treatment of human and chimpanzee DCs, suggesting a conserved mechanism among primates. Collectively, these data serve as a first evaluation for ex vivo modification of primate DC homing patterns that could later be used in reinfusion studies for the purposes of immunotherapeutics or mucosa-directed vaccines.

Characterization of Circulating Natural Killer Cells in Neotropical Primates

Despite extensive use of nonhuman primates as models for infectious diseases and reproductive biology, imprecise phenotypic and functional definitions exist for natural killer (NK) cells. This deficit is particularly significant in the burgeoning use of small, less expensive New World primate species. Using polychromatic flow cytometry, we identified peripheral blood NK cells as CD3-negative and expressing a cluster of cell surface molecules characteristic of NK cells (i.e., NKG2A, NKp46, NKp30) in three New World primate species – common marmosets, cotton-top tamarins, and squirrel monkeys. We then assessed subset distribution using the classical NK markers, CD56 and CD16. In all species, similar to Old World primates, only a minor subset of NK cells was CD56+, and the dominant subset was CD56–CD16+. Interestingly, CD56+ NK cells were primarily cytokine-secreting cells, whereas CD56–CD16+ NK cells expressed significantly greater levels of intracellular perforin, suggesting these cells might have greater potential for cytotoxicity. New World primate species, like Old World primates, also had a minor CD56–CD16– NK cell subset that has no obvious counterpart in humans. Herein we present phenotypic profiles of New World primate NK cell subpopulations that are generally analogous to those found in humans. This conservation among species should support the further use of these species for biomedical research.

SIVΔnef vaccination mobilizes systemic and mucosal natural killer cells in Mamu A*01+ macaques

Background Although vaccination with live attenuated SIV is the most effective means of inducing protection against lentiviruses, the immunologic mechanisms responsible remain unclear. Previous studies have yielded conflicting data regarding the role of adaptive immune responses in mediating protection, suggesting that innate immune responses, including natural killer (NK) cells, may play a role.

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.