We compared TLR responsiveness in PBMC from HIV-1-infected and uninfected individuals using the TLR agonists: TLR7 (3M-001), TLR8 (3M-002) and TLR7/8 (3M-011). Activation and maturation of plasmacytoid dendritic cells (pDC)were measured by evaluating CD86, CD40 and CD83 expression and myeloid dendritic cell (mDC) activation was measured by evaluating CD40 expression. All agonists tested induced activation and maturation of pDC in PBMC cultures of cells from HIV+ and HIV− individuals. The TLR7 agonist induced significantly less pDC maturation in cells from HIV+ individuals. Quantitative assessment of secreted IFN-α and pro-inflammatory cytokines at the single cell level showed that pDC from HIV+ individuals stimulated with TLR7 and TLR7/8 induced IFN-α. TLR8 and TLR7/8 agonists induced IL-12 and COX-2 expression in mDC from HIV+ and HIV− individuals. Understanding pDC and mDC activation and maturation in HIV-1 infection could lead to more rational development of immunotherapeutic strategies to stimulate the adaptive immune response to HIV-1.

Antibodies that mediate human immunodeficiency virus (HIV)-specific antibody-dependent cell-mediated cytotoxicity (ADCC) are present in the cervical fluid of many HIV-positive women; however, the role that these antibodies play in host defense against HIV is not known. To understand the contribution of ADCC in cervical secretions as a protective mechanism against HIV, we evaluated ADCC titers in paired serum and cervical-lavage (CVL) samples from >300 HIV-1-positive women who participated in the multicenter Division of AIDS Treatment Research Initiative Study 009. The present study demonstrates that women with CVL ADCC activity had lower genital viral loads than did women with serum ADCC activity only. Women with CVL ADCC activity were likely to have HIV-1 gp120-specific immunoglobulin (Ig) G, but not IgA, in their cervical fluid. This finding suggests that specific IgG in cervical fluid can mediate ADCC activity that inversely correlates with genital viral load.

We previously reported elevation of natural killer (NK) cells in women with recurrent spontaneous abortion (RSA) of immune etiology. In this study, we investigated the effect of intravenous immunoglobulin G (IVIg) on peripheral blood NK activity in vivo in women with RSA. Blood was drawn prior to and 7-11 days after IVIg therapy in eight women with RSA. NK activity was measured using K562 as target cells for 51Cr-release assays. Serum IgG concentrations were also measured. All received 400 mg/kg/day of IVIg for 3 consecutive days. 1) Seven of eight women became pregnant. Five delivered a live born infant. Three out of five women (60%) who delivered a live born infant showed a significant inhibition of NK cytotoxicity post IVIg and the rest did not show any changes; 2) NK cytotoxicity was significantly increased in a woman who miscarried again; 3) A woman who miscarried a chromosomally abnormal fetus showed a significant inhibition of NK cytotoxicity after IVIg; and 4) Serum IgG concentration increased significantly from 9.3 +/- 3.0 mg/ml to 23.5 +/- 5.1 mg/ml post IVIg therapy. IVIg effectively inhibits peripheral blood NK activity in vivo. These results are consistent with our previous finding showing that IVIg inhibits NK cell activity in vitro. Women with RSA and elevated NK cells may benefit from IVIg treatment.

C-reactive protein (CRP) is a major human acute phase protein composed of five identical, 21,500 mol wt subunits (1, 2). CRP is detectable on the surface of ~4% of normal PBL. CRP binds its physiological ligands in a Ca++-dependent manner; removal of Ca ++ does not alter the presence of CRP on the lymphocyte surface (3). Recently, investigators in this laboratory have reported (4) substantial inhibition of NK activity when effector cells were treated with anti-CRP. Although the liver is the site of CRP production contributing to the acute phase response (5), data presented here suggests that some iymphocytes produce small amounts of CRP and express it on their surface. Surface CRP (S-CRP) is not acquired exogenously from trace levels of CRP in normal serum. Materials and MethodsIsolation of PBMC. PBMC were separated from whole blood on Ficoll-Hypaque. They were then washed and resuspended in balanced salt solution (BSS) containing 0.05% gelatin. Enrichment of PBL and Large Granular Lymphocytes (LGL). PBL were incubated inRPMI containing 90% FCS and 10 mg/ml carbonyl iron with rotation at 37°C for 45 rain. Phagocytic cells were removed with a magnet. Mononuclear phagocyte-depleted PBL were enriched for LGL by layering onto discontinuous Percoll gradients (6). Labeling PBL with lZ~l-conjugated Antibodies. Goat anti-CRP was iodinated using the lactoperoxidase method (7). Monocyte-depleted PBL were incubated with ~25I-anti-CRP for 30 min at 37°C. Antibody-labeled cells were centrifuged on Percoll gradients; lymphocytes were harvested and counted on a gamma counter. Anti-CRP Pretreatment of PBL. PBL, LGL or target cells were resuspended in RPMI containing 0.05% gelatin and incubated for 30 min at 37°C in the presence or absence of various dilutions of anti-CRP. K562 Target Cells. K562 cells used as target cells were labeled with 5LCr by incubating 5 )< 10 6 cells/ml in RPMI containing 100 #Ci of 5~Cr at 37°C for 1 h. 51Cr-release Assay. A 4-h ~Cr-release assay using 5 × 105 effector cells and 104 5tCr-labeled K562 were incubated in 0.2 ml RPMI containing 0.05% gelatin at 37°C. After 4This work was supported by grants CA34670 and RRO5366 from the National Institutes of Health, Bethesda, MD.J. ExP. MEIg.

No abstract

SummaryOligodeoxynucleotides (ODN) with unmethylated deoxycytidyl-deoxyguanosine dinucleotides (CpG-ODNs) stimulate Toll-like receptor 9 (TLR9) in plasmacytoid dendritic cells (pDC) and B cells and activate innate and adaptive immunity. Three classes of synthetic CpG-ODNs, class A, B and C, activate cells through TLR9; our goal was to evaluate their effect on cells from human immunodeficiency virus (HIV)-1 + individuals. We compared the frequencies and the unstimulated activation status of immune effector cells in HIV-1 + and HIV-1 -individuals. Fewer pDC, myeloid dendritic cells (mDC), B cells, natural killer (NK) cells and invariant natural killer T cells (iNKT) were present in HIV-1 + peripheral blood mononuclear cells (PBMC) and their baseline activation status was higher than HIV-1 -PBMC. Exposure of HIV-1 + PBMC to all classes of CpG-ODNs led to activation and maturation of pDC based on CD86, CD80, and CD83 expression similar to that of cells from HIV-1 -individuals. The percentage of CpG-ODN stimulated pDC that express CD40 was dramatically higher when cells were obtained from HIV-1 + than from HIV-1 -individuals. B-lymphocytes were activated similarly in HIV-1 + and HIV-1 -individuals. mDC, NK and iNKT cell, which lack TLR9, were indirectly activated. Interferon-a (IFN-a) and interferon inducible protein 10 (IP-10) secretion was induced by class A or C but not class B CpG-ODN, but the concentrations were less than those produced by HIV-1 -PBMC. HIV-1 infected individuals have fewer innate effector cells that are chronically activated, but these cells can be further activated by CpG-ODN, which suggests that synthetic CpG-ODNs could be used to enhance the immune system in HIV-1 infected individuals.

Freshly isolated PBMC are broadly used as effector cells in functional assays that evaluate antibody-dependent cell mediated cytotoxicity (ADCC) and NK activity; however, they introduce natural-individual donor-to-donor variability. Cryopreserved PBMC provide a more consistent source of effectors than fresh cells in cytotoxicity assays. Our objective was to determine the effects of cryopreservation of effector PBMC on cell frequency, and on the magnitude and specificity of ADCC and NK activity. Fresh, frozen/overnight rested and frozen/not rested PBMC were used as effector cells in 51Cr-release and CD107a degranulation assays. Frozen/overnight rested PBMC had higher ADCC and NK activity in both assays when compared to fresh PBMC; however, when using frozen/not rested PBMC, ADCC and NK activities were significantly lower than fresh PBMC. Background CD107a degranulation in the absence of target cell stimulation was greater in PBMC that were frozen/not rested when compared to fresh PBMC or PBMC that were frozen overnight and rested. The percentages of CD16+CD56dim NK cells and CD14+ monocytes were lower in PBMC that were frozen and rested overnight than in fresh PBMC. CD16 expression on CD56dim NK cells was similar for all PBMC treatments. PBMC that were frozen and rested overnight were comparable to fresh PBMC effectors. PBMC that were frozen and used immediately when evaluating ADCC or NK activity using either a 51Cr-release assay or a CD107a degranulation assay had the lowest activity. Clinical studies of antibodies that mediate ADCC would benefit from using effector cells that have been frozen, thawed and rested overnight prior to assay.

Studies were designed to determine whether cervical antibodies in human immunodeficiency virus type 1 (HIV-1)-infected women participate in antibody-dependent cell-mediated cytotoxicity (ADCC). Serum and cervical lavage fluid (CVL) ADCC titers were compared with plasma virus load and CD4 cell number in 45 infected and 10 uninfected women from the Women's Interagency HIV Study. Serum and CVL were incubated with normal peripheral blood lymphocytes and HIV-1 gp120-bearing target cells in a standard (51)Cr-release assay. When stringent criteria were used to define ADCC activity, 63% had activity in > or = 1 fluid sample, 56% had serum titers, and 16% had CVL titers. Serum titers did not predict CVL titers. Three women with CVL ADCC had no serum ADCC, which suggests that ADCC antibodies may be produced locally. ADCC antibodies are present in the cervicovaginal fluids, which indicates that this form of innate immunity can contribute to mucosal defense against HIV-1.