Linda L. Baum

Intravenous Immunoglobulin Inhibits Natural Killer Cell Activity In Vivo in Women With Recurrent Spontaneous Abortion

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.

Dendritic cells from HIV-1 infected individuals are less responsive to toll-like receptor (TLR) ligands

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-alpha and pro-inflammatory cytokines at the single cell level showed that pDC from HIV+ individuals stimulated with TLR7 and TLR7/8 induced IFN-alpha. 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.

Women with cervicovaginal antibody-dependent cell-mediated cytotoxicity have lower genital HIV-1 RNA loads.

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.

Impact of class A, B and C CpG‐oligodeoxynucleotides on in vitro activation of innate immune cells in human immunodeficiency virus‐1 infected individuals

Oligodeoxynucleotides (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‐α (IFN‐α) 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.

Antibody-Dependent Cell-Mediated Cytotoxicity in Cervical Lavage Fluids of Human Immunodeficiency Virus Type 1-Infected Women

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 Womens 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.

The relationship of T-regulatory cell subsets to disease stage, immune activation, and pathogen-specific immunity in HIV infection.

Background:Human immunodeficiency virus (HIV) infection is associated with abnormalities in T-regulatory (T-reg) cells, but the effect of HIV on the naive (CD45RO−) and memory (CD45RO+) CD25+CD127loCD4+ T-reg cell subsets has not been defined. Methods:We measured the absolute number and relative percentage of total, naive, and memory T-reg cells in HIV-infected subjects and compared these parameters with their CD4+ T cells, viral load, levels of immune activation, and pathogen-specific immunity. Results:HIV infection was associated with an increased percentage of memory CD25+CD127loCD4+ T-reg cells and a decreased percentage of naive CD25+CD127loCD4+ T-reg cells as CD4+ T cells declined. The level of HIV viremia inversely correlated with total, memory, and naive CD25+CD127loCD4+ T-reg cell numbers and percentage of naive CD25+CD127loCD4+ T-reg cells. Lower total, memory, and naive CD25+CD127loCD4+ T-cell numbers were associated with higher levels of immune activation, whereas a higher percentage of CD25+CD127loCD4+ T-reg cells was associated with lower Candida- and HIV-specific immune responses. Conclusions:These observations suggest that CD25+CD127loCD4+ T-reg cells contribute to the immunodeficiency seen in HIV disease.

Proinflammatory and type 1 cytokine expression in cervical mucosa during HIV-1 and human papillomavirus infection.

Suppression of immune activation and increased inflammation are prevalent during viral infection. To investigate the role of inflammation in HIV transmission, we studied the infectious and inflammatory milieu in cervical mucosa from HIV-1- and human papillomavirus (HPV)-coinfected and HPV-monoinfected women. The numbers of cytokine-, chemokine-, and p24-expressing cells were determined using in situ imaging analysis and intracellular staining of p24 antigen. Significantly higher expression of the proinflammatory cytokines, interleukin (IL)-1α/β, was seen in cervical tissue from HIV/HPV-coinfected as compared with HPV-monoinfected tissues, whereas IL-2- and interferon (IFN)-γ-expressing cells were higher in HPV-monoinfected tissues. IL-10 was low in both groups, whereas IL-4 was significantly higher in HPV-monoinfected and HIV/HPV-coinfected tissues than in HIV/HPV-negative controls. RANTES and macrophage inflammatory protein (MIP)-1β but not MIP-1α were significantly higher in the genital tract of HIV/HPV-coinfected as compared with HPV-monoinfected individuals and controls. HIV/HPV-coinfected tissues had a higher level of human leukocyte antigen D-related (HLA-DR)-expressing dendritic cells (DCs). There was a positive correlation between the number of CD4+ and CD8+ T cells as well as CD1a, IL-1α, and RANTES expression and p24 antigen-expressing cells in the HIV/HPV-coinfected tissues. These findings suggest the persistence of immune activation and inflammation in the genital tract of women with HPV monoinfection and in HIV-infected women coinfected with HPV.

Role of Humoral Immunity in Host Defense Against HIV

Individuals infected with HIV-1 and nearly everyone vaccinated with HIV-1 vaccines will, in time, generate antibodies against viral proteins. These antibodies do not resolve natural infection, and vaccine candidates that successfully stimulate the production of high titers of neutralizing antibodies have failed to protect against infection. In spite of this, antibodies continue to be a focus of vaccine research. One reason for the continued interest in antibodies is the failure of a vaccine engineered to generate cell-mediated immunity against HIV. Successful protective immunity against most intracellular pathogens involves several arms of the immune response. A successful vaccine should also stimulate both protective cell-mediated immunity and specific antibody. Efforts should be directed toward making a vaccine that will stimulate the production of 1) more antibody, 2) more broadly cross-reactive neutralizing antibody (broadly neutralizing antibodies), and 3) antibody with a particular functional activity (antibody-dependent cell-mediated cytotoxicity; catalytic antibodies).

Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in 51Cr-release and CD107a assays

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 (51)Cr-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(+)CD56(dim) NK cells and CD14(+) monocytes were lower in PBMC that were frozen and rested overnight than in fresh PBMC. CD16 expression on CD56(dim) 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 (51)Cr-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.

Advances in CD4 cell enumeration in resource-poor countries

Purpose of reviewTo review recent progress in the development and evaluation of rapid CD4 cell assays needed to treat HIV-infected individuals in resource-poor countries. Recent findingsField tests indicate that many simplified flow-based assays provide CD4 cell numbers comparable to the current standard, FACSCount, at a lower cost. Most are intended for use at centralized clinics, but some new assays are portable. The manual assays, Dynal T4 Quant and cyto-spheres, are useful when only a few tests are performed daily. Several new formats include a manual rosetting method that uses a unique anti-CD4 cell monoclonal antibody that does not bind to monocytes. Microchip digital imaging and immunochromatography assays are being developed with the goal of being less expensive, more portable and easier than current methods. Reagents that increase the stability of blood samples and clinical reagents in tropical climates are being field tested. SummaryThe availability of antiretroviral medications increases the need for CD4 cell assays to monitor disease progression and the efficacy of therapy in resource-poor countries. Simplified flow cytometry is needed in centralized clinics, but there is a critical need for even less expensive easier methodology that can be used in smaller laboratories and in rural villages.