Objective: CD14+CD16+ monocytes are an important cellular target for HIV-1 entry and expand in the peripheral blood of HIV-infected individuals. Because CD14+CD16+ monocytes are a heterogeneous population and consist of CD14highCD16+ and CD14lowCD16+ subsets, we evaluated the effects of HIV infection on distinct subsets of CD16+ monocytes.
Methods: Untreated HIV-infected patients were recruited to investigate the relationship between the proportions of monocyte subsets with plasma viral loads and CD4+ T-cell counts. Patients receiving highly active antiretroviral therapy (HAART) were followed up in a cross-sectional and a longitudinal study.
Results: Compared with CD14lowCD16+, CD14highCD16+ monocytes showed higher levels of CD64 and HLA-DR antigens, which imply that these 2 distinct subsets have different immunoregulatory phenotypes. In HAART-naive patients, elevated proportions of CD14highCD16+ monocytes were correlated with increased viral loads and decreased CD4+ T-cell counts, whereas CD14lowCD16+ monocytes did not show such correlation with disease progression. Of importance, HAART recovered the proportion of CD14highCD16+ monocytes, whereas CD14lowCD16+ monocytes did not decrease during 1 year of antiviral therapy.
Conclusions: Taken together, our observations elucidate distinct immune responses of monocyte subsets during HIV infection and antiviral therapy and provide new insight into the roles of innate immunity in HIV-related pathogenesis.
From the *Institute of Infectious Diseases, Capital Medical University, Beijing, China; †Division of Infectious Diseases, Beijing Ditan Hospital, Beijing, China; and ‡Division of Treatment and Care, National Center for AIDS, Chinese Center for Disease Control and Prevention, Beijing, China.
Received for publication June 18, 2009; accepted September 18, 2009.
Supported by the Chinese National Basic Research Program (973 Program, 2006CB504201).
Presented at the Third Ditan International Conference on Infectious Diseases, July 30 to August 2, 2009, Beijing, China.
J. Han and B. Wang contributed equally to this study.
Conflicts of interest: No conflict.
Correspondence to: Hui Zeng, MD, PhD and Hongxin Zhao, MD, MPH, Institute of Infectious Diseases, Beijing Ditan Hospital, Jingshundongjie 8, Beijing 100015, China (e-mail: email@example.com and firstname.lastname@example.org).
Mononuclear phagocytic cells comprise monocytes, macrophages, and myeloid dendritic cells (mDCs). Circulating monocytes represent 5%-10% of peripheral blood leukocytes in humans and may act as precursor cells that give rise to mature macrophages and mDCs.1 Unlike CD4+ T cells, which undergo apoptotic death after HIV infection, mononuclear phagocytic cells are resistant to the cytopathic effect of HIV.2-5 Therefore, these cells can accumulate a large number of virions for a long period and remain as important reservoirs of actively replicating virus.
However, different types of mononuclear phagocytic cells show distinct susceptibility to HIV infection. The proportion of tissue macrophages that harbor HIV-1 has been reported to be as high as 50% in HIV-infected patients, although HIV-1 can be isolated from only a small (<1%) subpopulation of monocytes, which implies that circulating monocytes are much less susceptible to HIV infection.6,7 Human monocytes circulate in peripheral blood for less than 3 days before differentiating into macrophages in tissues.8,9 Therefore, the persistence of HIV-1 in blood monocytes implies ongoing renewal of infected monocytes by virus replication and/or recent infection in monocytes or their precursor cells.10 Recently, the insusceptibility of monocytes to HIV infection has been explained by their heterogeneity.11 According to distinct phenotypic characteristics and immune functions, circulating monocytes are subdivided into CD14+CD16− classic monocytes with a phenotype that resembles the original description of monocytes and CD14+CD16+ resident monocytes with a phenotype that resembles mature tissue-resident macrophages. The majority of peripheral blood monocytes (about 90%) are classic monocytes.1,11-14 Despite representing only 5%-10% of circulating monocytes, CD14+CD16+ monocytes are an important cellular target for HIV-1 entry. This is because these cells express higher levels of chemokine receptor CCR5 and they are more permissive for productive HIV-1 infection in vivo and in vitro than classic monocytes are.11,15,16 More importantly, several studies have shown a dramatic expansion of the proportion of CD16+ monocytes in the peripheral blood of HIV-1-infected patients.15,17,18 Circulating CD16+ monocytes can be recruited into a variety of tissues and differentiate into mature macrophages or mDCs; therefore, the increased number of CD16+ monocytes are proposed to act as a continuing source of HIV persistence.10,19
Given that CD16+ monocytes play important roles in HIV infection, these cells have gained considerable attention. The heterogeneity of CD16+ monocytes has been demonstrated by a number of studies on their surface markers, immunoregulatory ability, and differentiation potential.20-23 Despite well-characterized immune function under normal conditions, the effects of HIV infection on distinct subsets of CD16+ monocytes remain incompletely understood. In the present study, we demonstrated that CD14highCD16+ and CD14lowCD16+ monocytes differed in phenotypic characteristics and HIV responsiveness. Only the proportion of CD14highCD16+ monocytes was correlated positively with plasma HIV viral load and negatively with CD4+ T-cell count in HIV-infected patients. More importantly, the proportion of CD14highCD16+ but not CD14lowCD16+ monocytes recovered gradually in patients with AIDS who received highly active antiretroviral therapy (HAART). Our data indicate that CD14highCD16+ cells are more responsive to HIV infection and perform immunoregulatory functions during HAART.
Study Subjects, Follow-up, and HAART
One hundred and ten HIV-infected patients and 68 healthy controls in the present study were enrolled at the Division of Infectious Diseases, Beijing Ditan Hospital, Beijing, China from January 2006 to December 2008. The study was approved by the Committee of Ethics at Beijing Ditan Hospital. All human blood samples were collected with informed consent. HIV-1 infection was defined by the presence of HIV antibodies using enzyme-linked immunosorbent assay and confirmed by Western blotting. At screening, subjects had a clinical assessment, and routine laboratory tests were performed. These patients were followed up every 12 weeks in Beijing Ditan Hospital, and blood samples were collected for the measurement of plasma viral load, CD4+ T-cell count, and monocyte subsets at each visit. No patients in the cohort had contraindications to HAART (ie, active additional infections, severe anemia, or elevated liver enzymes).
Among the patients, 97 HAART-naive chronic HIV-infected individuals were enrolled for the study of the relationship between monocyte subsets and immunological parameters during disease progression. To monitor the dynamics of monocyte subsets during HAART, 110 patients with CD4+ T-cell counts <350 cells per microliter started HAART that consisted of 2 nucleotide reverse transcriptase inhibitors and a nonnucleoside reverse transcriptase inhibitor, in a cross-sectional study. To confirm our results, 48 patients who received HAART with baselines of monocyte subsets were included for longitudinal study.
Plasma HIV-1 Viral Load and CD4+ T-Cell Count
HIV-1 RNA levels and CD4+ T-cell counts were determined in a single laboratory using standard methodology, and both were included in the national quality assurance programs twice a year. Peripheral blood was collected in Na2-EDTA tubes from HIV-infected patients and processed within 2 hours. Plasma HIV-1 RNA copy number was measured using the Standard Amplicor HIV Monitor assay, version 1.5 (Roche Diagnostics, Indianapolis, IN). The assay employed a competitive reverse transcriptase-polymerase chain reaction methodology and was performed according to the manufacturer's instructions.
CD4+ T-cell count was determined in EDTA-treated whole blood and was performed using a standard flow cytometry technique with a TruCOUNT tube in routine laboratories (BD Biosciences, San Jose, CA). The absolute numbers of lymphocytes stained for CD45/CD3/CD4, and CD45/CD3/CD8 were analyzed with MultiSET software (BD Biosciences).
Immunophenotypic Characterization of Monocytes
Whole blood was collected for the surface staining. After red blood cell lysis, the following monoclonal antibodies were used: anti-human CD14, CD16, CD64, CD80, CD86, and HLA-DR (BD Biosciences). Matched isotype antibodies were used as negative controls. Monocytes were gated on the basis of forward scatter/side scatter characteristics and CD14/CD16 expression pattern. CD64, CD80, HLA-DR, and CD86 expression was then measured in these gated populations of 20,000 total events. Samples were analyzed on FACSCalibur (BD Biosciences) using CellQuest software. The mean fluorescence intensity was used for comparison of analyzed samples. For HIV-infected subjects, the flow cytometry results were analyzed in a manner blinded to the CD4+ T-cell counts and plasma HIV-1 viral load results.
All statistical analysis was performed using SPSS 14.0 software. Data are expressed as mean ± SD. Flow cytometric data of monocyte subsets from different stages of infection were analyzed using the 1-way analysis of variance test. P values were derived from the 1-way analysis of variance test to determine differences among healthy individuals and HIV-infected patients. The correlation among CD4+ T-cell counts, plasma HIV-1 viral load, and the proportions of monocyte subsets was analyzed with the Pearson correlation. For all comparisons, P < 0.05 was considered to be statistically significant.
Phenotypic Characterization of CD14lowCD16+ and CD14highCD16+ Monocytes
Previous studies have shown that CD16+ monocyte subsets are more permissive for HIV infection.15 In the present study, 3 subsets of peripheral blood monocytes were delineated according to CD14/CD16 expression patterns: CD14highCD16−, CD14highCD16+, and CD14lowCD16+ (Fig. 1A). To characterize the phenotypes of circulating CD14highCD16+ and CD14lowCD16+ monocyte subsets in healthy individuals, we analyzed the expression pattern of additional surface molecules that have been reported to relate to immune functions in monocyte subsets, including Fcγ receptor I (CD64), major histocompatibility complex molecules (HLA-DR), and B7 molecules (CD80 and CD86). Compared with CD14lowCD16+subsets, CD14highCD16+ expressed similar levels of CD86 but higher levels of CD64, HLA-DR, and CD80 (Fig. 1B). Of note, CD64 and HLA-DR, which are closely related to the differentiation and immune function of monocytes,21,22,24 were the 2 molecules that showed the most dramatic difference between CD14highCD16+ and CD14lowCD16+ subsets (2.98-fold higher for CD64 and 2.64-fold higher for HLA-DR). Therefore, CD14highCD16+ cells resembled the CD14+CD16+CD64+ monocytes with an immunoregulatory monocyte phenotype and an intermediate phenotype between that of monocytes and dendritic cells (DCs), as described previously.1,21 Because CD14highCD16+ and CD14lowCD16+ subsets showed different phenotypic characteristics, they might possess distinct immunoregulatory functions during HIV infection and AIDS progression.
CD14highCD16+ Rather Than CD14lowCD16+ Monocytes Were Correlated With Disease Progression in HAART-Naive HIV-Infected Patients
HIV-1 may lead to an increase in CD16+ monocytes; therefore, we investigated whether there was an increase in CD14high and CD14low subsets of CD16+ monocytes. To minimize the effects of other pathogens, we recruited HAART-naive HIV-infected patients without active opportunistic infections (n = 97) and with a range of CD4+ T-cell counts (4-705 cells/μL) and plasma viral loads (575-1,445,440 copies/mL). Similar to previous studies, the proportion of CD16+ monocytes significantly increased in HIV-infected patients in comparison with healthy controls, with a concomitant decline in the CD14highCD16− subset (P < 0.0001; Fig. 2A). Consistent with the observed increase in CD16+ monocytes (Fig. 2B), we observed a significant increase in the relative proportion of CD14highCD16+ and CD14lowCD16+ subsets (Figs. 2C, D).
Next, correlation analysis was performed to investigate the relationship between the proportions of different monocyte subsets and activated replication of HIV. The proportion of CD14highCD16+ monocytes correlated positively with plasma HIV viral load (P = 0.001, r = 0.346) (Fig. 3; left panel). In contrast, no correlation was found between CD14lowCD16+ monocytes and HIV viral load (P = 0.447; r = −0.083) (Fig. 3; left panel). These results indicated that only expansion of the CD14highCD16+ monocyte subset was related to the level of HIV replication.
Given that HIV infection results in a decrease in CD4+ T-cell number but an increase in CD16+ monocytes, an inverse correlation would be expected between monocyte subsets and CD4+ T-cell count. However, we observed that the proportion of CD14highCD16+ monocytes correlated negatively with CD4+ T-cell counts, whereas no association between percentage of CD14lowCD16+ monocytes and CD4+ T-cell counts was detected (Fig. 3; right panels). These findings demonstrated that the CD14highCD16+ subset was correlated with disease progression in HAART-naive HIV-infected patients.
HAART Recovered the Proportion of CD14highCD16+ but Not CD14lowCD16+ Monocytes in Patients With AIDS
Antiretroviral therapy not only reduces plasma HIV viral load but also partially recovers immune parameters, including CD4+ T-cell counts, CD4+/CD8+ ratios, production of IL-2, and IL-2 reactivity of lymphocytes.25 To determine whether antiretroviral therapy recovered monocyte subsets, we analyzed the dynamic changes in monocyte subsets in patients receiving HAART (n = 110) in a cross-sectional study and elucidated the relationship between the monocyte subsets and plasma HIV RNA levels and/or CD4+ T-cell counts. Complete viral suppression was achieved and maintained, as shown by plasma viral load of <50 copies per milliliter within 24 weeks (Fig. 4, left panel). Along with the decrease in plasma HIV viral load, the CD4+ T-cell count increased gradually until reaching a plateau after 24 weeks of treatment. During regular follow-up at 12, 24, 36, and 48 weeks after initiating HAART, we observed that the proportion of circulating CD14high CD16+ monocytes declined gradually (from 7.32% ± 4.71% to 5.16% ± 2.55%; Fig. 4, left panel). However, it remained above the level seen in healthy controls until 48 weeks (Fig. 4, left panel), which was paralleled with a slight increase in the proportion of CD14highCD16− monocytes (from 86.31% ± 6.21% to 87.07% ± 5.91%; Fig. 4, left panel). Strikingly, despite the recovery in CD14highCD16− and CD14highCD16+ monocytes, the proportion of CD14lowCD16+ monocytes did not decrease during 1 year of antiviral therapy. In fact, the proportion of CD14lowCD16+ monocytes even slightly increased from 6.38% ± 3.71% to 7.77% ± 5.01% (Fig. 4, left panel), which suggested that this population responded differently to antiviral therapy.
To confirm that HAART induced changes in monocyte subsets, we recruited 48 HAART-naive patients with a CD4+ T-cell count of <350 cells per microliter for a longitudinal study. Pre-HAART and post-HAART blood samples were monitored. In line with the data from the cross-sectional study, a trend toward a decline in CD14highCD16+ percentage, along with an increased proportion of CD14highCD16− monocytes, was observed in the longitudinal study (Fig. 4, right panel). By contrast, an increased proportion of CD14lowCD16+ subset was observed at the time point of 36 weeks after HAART. Collectively, these data indicated that CD14highCD16+ and CD14lowCD16+ monocytes might respond differently to antiviral therapy. The CD14highCD16+ subset was in association with HIV disease progression and normalized after HAART.
Chronic HIV infection is associated with HIV replication and a number of immune dysfunctions, including loss of CD4+ T cells, impaired CD8+ T-cell activity, and persistent immune activation.26 In contrast to HIV-induced CD4 lymphopenia, CD14+CD16+ monocytes, the minority of those that are more susceptible to HIV infection, increase in HIV-infected individuals.15 Because CD14+CD16+ monocytes are a heterogeneous population and consist of CD14low and CD14high subsets, the present study was designed to evaluate the relationship between monocyte subsets and key markers of disease progression, that is, plasma viral load and CD4+ T-cell count.
We reported that CD14lowCD16+ and CD14highCD16+ monocyte subsets were both elevated in HIV-infected patients. However, the CD14highCD16+ subset was uniquely and closely correlated with plasma viral load and CD4+ T-cell count in HAART-naive patients. More importantly, increased numbers of CD14highCD16+ monocytes were reduced by HAART, along with HIV viral load and CD4+ T-cell count. In addition to the previously described higher susceptibility of CD16+ monocytes to HIV infection,15 our study provides new evidence of the linkage between CD14highCD16+ monocytes and pathogenesis of AIDS.
Previous studies have demonstrated that HIV-induced immune activation is the major driving force of CD4+ T-cell depletion.27,28 The large increase in CD14highCD16+ monocytes observed in HIV-infected patients might be caused by many factors, including viral replication and the cellular immune response to HIV. Because monocytes are the major cellular component of the innate immune response, expansion of CD16+ monocytes is very likely driven by viral replication. This notion is supported by former findings of higher susceptibility of CD16+ monocytes to HIV15,29 and our positive correlation between the CD14highCD16+ subset and viral load. The hypothesis is further strengthened by the observation that an increased number of CD14highCD16+ monocytes in HIV-infected patients were recovered by HAART.
However, we observed clear differences between CD14highCD16+ and CD14lowCD16+ monocyte subsets with regard to their correlation with viral load and responsiveness to antiviral therapy, which suggests that HIV replication leads to different immune responses in these 2 subsets. Although the potential effects of HIV-1 on different monocyte subsets are a subject of interest, the limited number of CD16+ monocytes precludes detailed functional analysis of the immunoregulatory discrepancies between CD14highCD16+ and CD14lowCD16+ subsets in HIV-infected patients. Fortunately, phenotypic and functional characterization of distinct monocyte subsets has been studied extensively, which has revealed surface markers that are linked with immune functions and differentiation deviation.1,13-15,21,23,29-34 In line with close correlation with disease progression, CD14highCD16+ monocytes showed higher levels of CD64 and HLA-DR, which provided strong evidence that they resembled a minor subset with an immunoregulatory phenotype. Several groups have demonstrated that compared with CD14+CD16+CD64− cells, CD14+CD16+CD64+ cells and CD14highCD16−CD64+ subsets have a similarly higher phagocytic activity and production of proinflammatory cytokines, including tumor necrosis factor and IL-6. Although the expression of HLA-DR and CD86 is higher in CD14+CD16+CD64+ than in CD14+CD16+CD64− subsets, their antigen-presenting activity is comparable. In contrast, CD14+CD16+CD64+ cells exhibit greater stimulatory activity in mixed lymphocyte reactions than CD14lowCD16+CD64+ monocytes. Moreover, the unique combination of typical monocyte and DC features in CD14+CD16+CD64+ cells suggests a more mature mDC precursor-like nature or an intermediate phenotype between that of monocytes and DCs.21,22,24,35 Therefore, the differences between the CD14+CD16+CD64+ and CD14+CD16+CD64− subsets in HIV-infected patients can be interpreted as a consequence of their distinct immune response during chronic HIV infection. Alternatively, dysregulation of monocyte subsets in HIV-infected individuals may contribute to persistent immune activation and/or negative immunoregulation. Whether CD14highCD16+ monocyte accumulation in the peripheral blood of HIV-infected patients results in immune hyperactivation, or a feedback mechanism to limit immune-mediated damage to the host, is an important question to be addressed by future functional studies.
Unlike the cytopathic effects of HIV-1 on T cells, circulating monocytes can accumulate large numbers of virions without cell death. For this reason, monocytes may be involved in HIV-1 persistence and increase the susceptibility of resting T cells to HIV-1 infection,19 especially in patients with late-stage disease and opportunistic infections.7 Expansion of the CD16+ subset increases the number of HIV-susceptible cells in the peripheral blood. Given the fact that peripheral blood monocytes migrate into tissues and differentiate locally into functionally distinct macrophages or mDCs, HIV-1 infection of monocytes is important for virus dissemination upon recruitment into peripheral tissues.36 CD16+ monocytes resemble a more advanced stage of tissue macrophage and DC differentiation than CD16− monocytes.21,22 Therefore, an increased number of CD16+ monocytes is thought to increase the number of HIV-infected macrophages and mDCs in lymphoid organs. However, it is an important question whether CD14highCD16+ or CD14lowCD16+ monocytes are correlated with alterations in their descendants in lymphoid organs and tissues, especially whether the residual CD14highCD16+ monocytes after HAART contribute to HIV-susceptible tissue macrophages and mDCs. Addressing such questions will necessitate assessing the number and function of mature macrophages and DCs in lymphoid organs and tissues within infected hosts at different stages of infection.
Collectively, our observations elucidated the distinct immune responses of monocyte subsets during HIV infection and antiviral therapy. CD14highCD16+ is the most responsive monocyte subset to HIV infection; therefore, it might be a biomarker for disease progression. Meanwhile, new therapeutic strategies with greater potency against viral production within different blood monocyte subsets, especially the CD14lowCD16+ monocytes, might enhance the efficacy of antiretroviral therapy.
We thank Dr. Linqi Zhang for the preparation of this article.
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