Restoration of TLR3-activated myeloid dendritic cell activity leads to improved natural killer cell function in chronic hepatitis B virus infection

There is increasing evidence that the function of NK cells in patients with chronic hepatitis B (CHB) infection is impaired. The underlying mechanism for the impaired NK cell function is still unknown. Since myeloid dendritic cells (mDC) are potent inducers of NK cells, we investigated the functional interaction of mDC and NK cells in CHB and the influence of antiviral therapy. Blood BDCA1(+) mDC and NK cells were isolated from 16 healthy controls or 39 CHB patients at baseline and during 6 months of antiviral therapy. After activation of mDC with poly(I · C) and gamma interferon (IFN-γ), mDC were cocultured with NK cells. Phenotype and function were analyzed in detail by flow cytometry and enzyme-linked immunosorbent assay. Our findings demonstrate that on poly(I · C)/IFN-γ-stimulated mDC from CHB patients, the expression of costimulatory molecules was enhanced, while cytokine production was reduced. In cocultures of poly(I · C)/IFN-γ-stimulated mDC and NK cells obtained from CHB patients, reduced mDC-induced NK cell activation (i.e., CD69 expression) and IFN-γ production compared to those in healthy individuals was observed. Antiviral therapy normalized mDC activity, since decreased expression of CD80 and CD86 on DC and of HLA-E on NK cells was observed, while poly(I · C)/IFN-γ-induced cytokine production by mDC was enhanced. In parallel, successful antiviral therapy resulted in improved mDC-induced NK cell activation and IFN-γ production. These data demonstrate that CHB patients display a diminished functional interaction between poly(I · C)/IFN-γ activated mDC and NK cells due to impaired mDC function, which can be partially restored by antiviral therapy. Enhancing this reciprocal interaction could reinforce the innate and thus the adaptive T cell response, and this may be an important step in achieving effective antiviral immunity.     

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.     

Increased natural killer cell activity in viremic HIV-1 infection

NK cells are a subset of granular lymphocytes that are critical in the innate immune response to infection. These cells are capable of killing infected cells and secreting integral cytokines and chemokines. The role that this subset of cytolytic cells plays in HIV infection is not well understood. In this study, we dissected the function of NK cells in viremic and aviremic HIV-1-infected subjects, as well as HIV-1-negative control individuals. Despite reduced NK cell numbers in subjects with ongoing viral replication, these cells were significantly more active in secreting both IFN-gamma and TNF-alpha than NK cells from aviremic subjects or HIV-1-negative controls. In addition, NK cells in subjects with detectable viral loads expressed significantly higher levels of CD107a, a marker of lysosomal granule exocytosis. The expression of CD107a correlated with NK cell-mediated cytokine secretion and cytolytic activity as well as with the level of viral replication, suggesting that CD107a represents a good marker for the functional activity of NK cells. Finally, killer Ig-related receptor+ NK cells were stable or elevated in viremic subjects, while the numbers of CD3-/CD56+/CD94+ and CD3-/CD56+/CD161+ NK cells were reduced. Taken together, these data demonstrate that viremic HIV-1 infection is associated with a reduction in NK cell numbers and a perturbation of NK cell subsets, but increased overall NK cell activity.     

Compartmental imbalance and aberrant immune function of blood CD123+ (plasmacytoid) and CD11c+ (myeloid) dendritic cells in atopic dermatitis

Atopic dermatitis (AD) is a pruritic, chronically relapsing skin disease in which Th2 cells play a crucial role in cutaneous and extracutaneous immune reactions. In humans, CD11c+CD123- myeloid dendritic cells (mDC) and CD11c-CD123+ plasmacytoid DC (pDC) orchestrate the decision-making process in innate and acquired immunity. Since the number and function of these blood dendritic cell (DC) subsets reportedly reflect the host immune status, we studied the involvement of the DC subsets in the pathogenesis of AD. Patients with AD had an increased DC number and a low mDC:pDC ratio with pDC outnumbering mDC in the peripheral blood compared with normal subjects and psoriasis patients (a Th1 disease model group). The mDC:pDC ratio was correlated with the total serum IgE level, the ratio of IFN-gamma-producing blood cells:IL-4-producing blood cells, and the disease severity. In vitro allogeneic stimulation of naive CD4+ cells with atopic DC showed that the ability of pDC for Th1 induction was superior or comparable to that of mDC. In skin lesions, pDC infiltration was in close association with blood vessels expressing peripheral neural addressins. Therefore, compartmental imbalance and aberrant immune function of the blood DC subsets may deviate the Th1/Th2 differentiation and thus induce protracted allergic responses in AD.     

Differential restoration of myeloid and plasmacytoid dendritic cells in HIV-1-infected children after treatment with highly active antiretroviral therapy

Numerical and functional deficits in myeloid (mDC) and plasmacytoid dendritic cell (pDC) subsets have been found in both adult and pediatric HIV-1 carriers. Whether these impaired DC subsets can be restored after treatment with highly active antiretroviral therapy (HAART) is currently unknown, especially in HIV-1-infected children. In this report, we characterized mDC and pDC subsets in 18 HIV-1-infected children who received HAART treatment and compared them with those in 6 untreated HIV-1-infected children and 27 HIV-1-uninfected healthy children. Among children treated with HAART, 11 were found to suppress HIV-1 replication successfully below the detection limit (HAART-suppressed group) while the remaining 7 failed (HAART-failure group). In HAART-suppressed children, a gradual and complete restoration of the frequency and function of mDCs was observed while the recovery of pDCs was only partial. However, mDC and pDC subsets in HARRT failure children were indistinguishable from the HAART-naive infected children. We also found that mDC frequency and IFN-alpha-releasing capacity of pDC positively correlated with CD4 T cell percentages in all HIV-1-infected children. In HAART-naive children, the mDC frequency correlated the HIV-1-specific CTL frequency. Our findings suggest that HAART has a differential impact on the restoration of mDC and pDC subsets. These findings may help guide the development of HIV-1-specific immune therapy aimed at fully restoring host immune function in chronically HIV-1-infected children.     

Parallel loss of myeloid and plasmacytoid dendritic cells from blood and lymphoid tissue in simian AIDS

The loss of myeloid (mDC) and plasmacytoid dendritic cells (pDC) from the blood of HIV-infected individuals is associated with progressive disease. It has been proposed that DC loss is due to increased recruitment to lymph nodes, although this has not been directly tested. Similarly as in HIV-infected humans, we found that lineage-negative (Lin(-)) HLA-DR(+)CD11c(+)CD123(-) mDC and Lin(-)HLA-DR(+)CD11c(-)CD123(+) pDC were lost from the blood of SIV-infected rhesus macaques with AIDS. In the peripheral lymph nodes of SIV-naive monkeys the majority of mDC were mature cells derived from skin that expressed high levels of HLA-DR, CD83, costimulatory molecules, and the Langerhans cell marker CD1a, whereas pDC expressed low levels of HLA-DR and CD40 and lacked costimulatory molecules, similar to pDC in blood. Surprisingly, both DC subsets were depleted from peripheral and mesenteric lymph nodes and spleens in monkeys with AIDS, although the activation status of the remaining DC subsets was similar to that of DC in health. In peripheral and mesenteric lymph nodes from animals with AIDS there was an accumulation of Lin(-)HLA-DR(moderate)CD11c(-)CD123(-) cells that resembled monocytoid cells but failed to acquire a DC phenotype upon culture, suggesting they were not DC precursors. mDC and pDC from the lymphoid tissues of monkeys with AIDS were prone to spontaneous death in culture, indicating that apoptosis may be a mechanism for their loss in disease. These findings demonstrate that DC are lost from rather than recruited to lymphoid tissue in advanced SIV infection, suggesting that systemic DC depletion plays a direct role in the pathophysiology of AIDS.     

Plasmacytoid Dendritic Cells Suppress HIV-1 Replication but Contribute to HIV-1 Induced Immunopathogenesis in Humanized Mice

The role of plasmacytoid dendritic cells (pDC) in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis remains unclear. HIV-1 infection in the humanized mouse model leads to persistent HIV-1 infection and immunopathogenesis, including type I interferons (IFN-I) induction, immune-activation and depletion of human leukocytes, including CD4 T cells. We developed a monoclonal antibody that specifically depletes human pDC in all lymphoid organs in humanized mice. When pDC were depleted prior to HIV-1 infection, the induction of IFN-I and interferon-stimulated genes (ISGs) were abolished during acute HIV-1 infection with either a highly pathogenic CCR5/CXCR4-dual tropic HIV-1 or a standard CCR5-tropic HIV-1 isolate. Consistent with the anti-viral role of IFN-I, HIV-1 replication was significantly up-regulated in pDC-depleted mice. Interestingly, the cell death induced by the highly pathogenic HIV-1 isolate was severely reduced in pDC-depleted mice. During chronic HIV-1 infection, depletion of pDC also severely reduced the induction of IFN-I and ISGs, associated with elevated HIV-1 replication. Surprisingly, HIV-1 induced depletion of human immune cells including T cells in lymphoid organs, but not the blood, was reduced in spite of the increased viral replication. The increased cell number in lymphoid organs was associated with a reduced level of HIV-induced cell death in human leukocytes including CD4 T cells. We conclude that pDC play opposing roles in suppressing HIV-1 replication and in promoting HIV-1 induced immunopathogenesis. These findings suggest that pDC-depletion and IFN-I blockade will provide novel strategies for treating those HIV-1 immune non-responsive patients with persistent immune activation despite effective anti-retrovirus treatment.     

Identification of novel dendritic cell subset markers in human blood

Human dendritic cells (DC) are key regulators of innate and adaptive immunity that can be divided in at least three major subpopulations: plasmacytoid DC (pDC), myeloid type 1 DC (mDC1) and myeloid type 2 DC (mDC2) exhibiting different functions. However, research, diagnostic and cell therapeutic studies on human DC subsets are limited because only few DC subset markers have been identified so far. Especially mDC2 representing the rarest blood DC subset are difficult to be separated from mDC1 and pDC due to a paucity of mDC2 markers. We have combined multiparameter flow cytometry analysis of human blood DC subsets with systematic expression analysis of 332 surface antigens in magnetic bead-enriched blood DC samples. The initial analysis revealed eight novel putative DC subset markers CD26, CD85a, CD109, CD172a, CD200, CD200R, CD275 and CD301 that were subsequently tested in bulk peripheral blood mononuclear cell (PBMC) samples from healthy blood donors. Secondary analysis of PBMC samples confirmed three novel DC subset markers CD26 (dipeptidyl peptidase IV), CD85a (Leukocyte immunoglobulin-like receptor B3) and CD275 (inducible costimulator ligand). CD85a is specifically expressed in mDC1 and CD26 and CD275 represent novel mDC2 markers. These markers will facilitate human DC subset discrimination and additionally provide insight into potentially novel DC subset-specific functions.     

Dendritic cells infiltrating human non-small cell lung cancer are blocked at immature stage

The efficacy of immune response to control human cancer remains controversial. It is particularly debated whether and to what extent the capacity of tumor-infiltrating dendritic cells (DC) to drive immunization can be turned off by transformed cells, leading to tumor-specific tolerance rather than immunization. To address this issue, we have characterized the DC isolated from human non-small cell lung cancer (NSCLC). These biopsy specimens contained CD11c(high) myeloid DC (mDC), but also CD11c(-) plasmacytoid DC (pDC) and a third DC subset expressing intermediate level of CD11c. Compared with peripheral blood, CD11c(high) tumor-infiltrating DC (TIDC) displayed a "semi-mature" phenotype, and TLR4 or TLR8 stimulation drove them to mature partially and to secrete limited amounts of cytokines. In contrast, most tumor-infiltrating pDC were immature but underwent partial maturation after TLR7 activation, whereas TLR9 ligation triggered low secretion of IFN-alpha. CD11c(int) mDC represented approximately 25% of total DC in tumoral and peritumoral tissues and expressed low levels of costimulatory molecules contrasting with high levels of the immunoinhibitory molecule B7-H1. Finally, the poor APC function of total TIDC even after TLR stimulation and the migratory response of both tumor-infiltrating mDC and pDC toward CCL21 and SDF-1 in vitro suggested their ability to compromise the tumor-specific immune response in draining lymph nodes in vivo. Further studies will be required to establish the specific role of the three TIDC subsets in tumor immunity and to draw conclusions for the design of therapeutic strategies.     

NK cell activation by dendritic cells is dependent on LFA-1-mediated induction of calcium-calmodulin kinase II: inhibition by HIV-1 Tat C-terminal domain.

In this study, we show that binding to autologous dendritic cells (DC) induces a calcium influx in NK cells, followed by activation of the calcium-calmodulin kinase II (CAMKII), release of perforin and granzymes, and IFN-gamma secretion. CAMKII is induced via LFA-1: indeed, oligomerization of LFA-1 leads to CAMKII induction in NK cells. Moreover, release of lytic enzymes and cytotoxic activity is strongly reduced by masking LFA-1 or by adding CAMKII inhibitors such as KN62 and KN93, at variance with the inactive compound KN92. NK cell-mediated lysis of DC and IFN-gamma release by NK cells upon NK/DC contact are inhibited by exogenous HIV-1 Tat: the protein blocks calcium influx and impairs CAMKII activation elicited via LFA-1 in NK cells, eventually inhibiting degranulation. Experiments performed with synthetic, overlapping Tat-derived peptides showed that the C-terminal domain of the protein is responsible for inhibition. Finally, both KN62 and Tat reduced the extension of NK/DC contacts, possibly affecting NK cell granule polarization toward the target. These data provide evidence that exogenous Tat inhibits NK cell activation occurring upon contact with DC: this mechanism might contribute to the impairment of natural immunity in HIV-1 infection.     

Surface phenotype and rapid quantification of blood dendritic cell subsets in the rhesus macaque

Background  The study of dendritic cell (DC) biology in the rhesus macaque is becoming increasingly important but is limited by incomplete characterization and the lack of a rapid assay to quantify cells.
Methods  We characterized the surface phenotype of myeloid (mDC) and plasmacytoid DC (pDC) subsets in healthy rhesus macaque blood and developed a flow cytometry-based assay for absolute DC determinations.
Results  Rhesus CD11c⁺ mDC were CD16⁺ CD11b⁺ CD56^lo CD8⁻ CD1c⁻ whereas CD123⁺ pDC lacked expression of these markers. Precise DC determinations were performed using a rapid two-step assay combining the analysis of whole blood and peripheral blood leukocytes (PBL).
Conclusions  Antibodies to CD11b, CD56 and CD16 must be omitted from the lineage antibody cocktail to prevent inadvertent gating-out of DC when analyzing rhesus blood. The combined whole-blood/PBL quantification assay will be invaluable for the rapid and repeated monitoring of blood DC counts in this species.     

The reciprocal interaction of NK cells with plasmacytoid or myeloid dendritic cells profoundly affects innate resistance functions

A reciprocal activating interaction between NK cells and dendritic cells (DC) has been suggested to play a role in the functional regulation of these cells in immunity, but it has been studied only using in vitro generated bone marrow- or monocyte-derived DC. We report that human peripheral blood plasmacytoid DC (pDC) and myeloid DC are necessary to induce NK cell function depending on the type of microbial stimulus. pDC and myeloid DC are required for strongly increased NK cytolytic activity and CD69 expression, in response to inactivated influenza virus or CpG-containing oligonucleotides and poly(I:C), respectively. Secreted type I IFN is required and sufficient for the augmentation of NK cell cytolytic activity in the coculture with pDC or myeloid DC, whereas CD69 expression is dependent on both type I IFN and TNF. In addition, in response to poly(I:C), myeloid DC induce NK cells to produce IFN-gamma through a mechanism dependent on both IL-12 secretion and cell contact between NK cells and myeloid DC, but independent of type I IFN. IL-2-activated NK cells have little to no cytolytic activity for immature myeloid DC and pDC, but are able to induce maturation of these cells. Moreover, IL-2-activated NK cells induce, in the presence of a suboptimal concentration of CpG-containing oligonucleotides, a strong IFN-alpha and TNF production. These data suggest that the reciprocal functional interaction between NK cells and either pDC or myeloid DC may play an important physiological role in the regulation of both innate resistance and adaptive immunity to infections.     

Severe and persistent depletion of circulating plasmacytoid dendritic cells in patients with 2009 pandemic H1N1 infection

Background

Dysregulation of host immune responses plays a critical role in the pathogenesis of severe 2009 pandemic H1N1 infection. Whether H1N1 virus could escape innate immune defense in vivo remains to be investigated. The aim of this study was to evaluate the pattern of innate immune response during human 2009 H1N1 infection. We performed the enumeration of circulating myeloid dendritic cells (mDC) and plasmacytoid DC (pDC) in blood from patients with H1N1 pneumonia shortly after the onset of symptoms and during follow-up at different intervals of time. The analysis of CD4 and CD8 count, CD38 T-cell activation marker and serum cytokine/chemokine plasma levels was also done.

Methodology/Principal Findings

Blood samples were collected from 13 hospitalized patients with confirmed H1N1-related pneumonia at time of admission and at weeks 1, 4, and 16 of follow-up. 13 healthy donors were enrolled as controls. In the acute phase of the disease, H1N1-infected patients exhibited a significant depletion in both circulating pDC and mDC in conjunction with a decrease of CD4 and CD8 T cell count. In addition, we found plasmatic hyperproduction of IP-10 and RANTES, whereas increase in T-cell immune activation was found at all time points. When we assessed the changes in DC count over time, we observed a progressive normalization of mDC number. On the contrary, H1N1-infected patients did not achieve a complete recovery of pDC count as values remained lower than healthy controls even after 16 weeks of follow-up.

Conclusions

H1N1 disease is associated with a profound depletion of DC subsets. The persistence of pDC deficit for several weeks after disease recovery could be due to H1N1 virus itself or to a preexisting impairment of innate immunity.     

Frequent and strong antibody-mediated natural killer cell activation in response to HIV-1 Env in individuals with chronic HIV-1 infection

Natural killer (NK) cells play a critical role in the control of HIV-1 infection, and NK cells that respond to HIV-1 peptides have been recently described. However, the mechanisms by which NK cells recognize HIV-1 antigens are not fully understood. We investigated NK cell activation in response to HIV-1 peptides during early and chronic HIV-1 clade B infection using a whole-blood assay and multiparameter flow cytometry. Antibody-mediated NK cell activation in response to HIV-1 peptides was not detected in HIV-1-uninfected individuals. In contrast, 79% of individuals with chronic infection and 22% of individuals with early infection had detectable gamma interferon (IFN-γ) NK cell responses to HIV-1 antigens (P < 0.00001). IFN-γ- and tumor necrosis factor alpha (TNF-α)-producing NK cells most frequently targeted Env gp120 (median of 4% and range of 0 to 31% of all NK cells). NK cells rarely targeted other HIV-1 proteins such as Gag, Pol, and Nef. Antibody-mediated NK cell responses to peptides mapped predominantly to Env protein, required the presence of plasma or plasma IgG, and resulted in lower CD16 expression on NK cells, suggesting an antibody-mediated activation of NK cells. Further studies are needed to assess the consequences of these antibody-mediated NK cell responses for HIV-1 disease progression and vaccine-induced protection from infection.     

Dendritic cell type determines the mechanism of bystander suppression by adaptive T regulatory cells specific for the minor antigen HA-1

One hallmark of acquired tolerance is bystander suppression, a process whereby Ag-specific (adaptive) T regulatory cells (TR) inhibit the T effector cell response both to specific Ag and to a colocalized third-party Ag. Using peripheral blood T cells from recipients of HLA-identical kidney transplants as responders in the trans vivo-delayed type hypersensitivity assay, we found that dendritic cells (DC), but not monocyte APCs, could mediate bystander suppression of EBV-specific recall response. When HA-1(H) peptide was added to mixtures of plasmacytoid DC (pDC) and T cells, bystander suppression of the response to a colocalized recall Ag occurred primarily via indolamine-2,3-dioxygenase (IDO) production. Similarly, addition of HA-1(H) peptide to cocultures of T cells and pDC, but not myeloid DC (mDC), induced IDO activity in vitro. When mDC presented HA-1(H) peptide to Ag-specific CD8+ TR, cytokine release (TGF-beta, IL-10, or both) was the primary mode of bystander suppression. Bystander suppression via mDC was reversed not only by Ab to TGF-beta and its receptor on T cells, but also by Ab to thrombospondin-1. EBV addition did not induce IDO or thrombospondin-1 in T-DC cocultures, suggesting that these DC products are not induced by T effector cells, but only by TR cells. These results shed light upon the mechanism of bystander suppression by donor Ag-specific TR in patients with organ transplant tolerance and underscores the distinct and critical roles of mDC and pDCs in this phenomenon.     

The CD85j+ NK cell subset potently controls HIV-1 replication in autologous dendritic cells

Natural killer (NK) cells and dendritic cells (DC) are thought to play critical roles in the first phases of HIV infection. In this study, we examined changes in the NK cell repertoire and functions occurring in response to early interaction with HIV-infected DC, using an autologous in vitro NK/DC coculture system. We show that NK cell interaction with HIV-1-infected autologous monocyte-derived DC (MDDC) modulates NK receptor expression. In particular, expression of the CD85j receptor on NK cells was strongly down-regulated upon coculture with HIV-1-infected MDDC. We demonstrate that CD85j(+) NK cells exert potent control of HIV-1 replication in single-round and productively HIV-1-infected MDDC, whereas CD85j(-) NK cells induce a modest and transient decrease of HIV-1 replication. HIV-1 suppression in MDCC by CD85j(+) NK cells required cell-to-cell contact and did not appear mediated by cytotoxicity or by soluble factors. HIV-1 inhibition was abolished when NK-MDDC interaction through the CD85j receptor was blocked with a recombinant CD85j molecule, whereas inhibition was only slightly counteracted by blocking HLA class I molecules, which are known CD85j ligands. After masking HLA class I molecules with specific antibodies, a fraction of HIV-1 infected MDDC was still strongly stained by a recombinant CD85j protein. These results suggest that CD85j(+) NK cell inhibition of HIV-1 replication in MDDC is mainly mediated by CD85j interaction with an unknown ligand (distinct from HLA class I molecules) preferentially expressed on HIV-1-infected MDDC.     

Distinct responses of splenic dendritic cell subsets to infection with Listeria monocytogenes: maturation phenotype, level of infection, and T cell priming capacity ex vivo

To determine the relative contributions of DC subsets in the development of protective immunity to Listeria monocytogenes we examined the relationship between maturation, bacterial burden, and T cell priming capacity of four well characterized subsets of splenic DC following infection with Lm. CD8α⁺, CD4⁺, and CD8α⁻CD4⁻ DC and the B220⁺ plasmacytoid DC (pDC) were compared for abundance and costimulatory molecule expression at 24, 48, and 72 h post i.v. infection. We further determined the bacterial burden associated with each DC subset and their relative capacities to prime CD8⁺ T cells at 24 hpi. The CD8α⁺ DC displayed the highest level of maturation, association with live bacteria, and T cell activation potential. Second, the CD4⁺ DC were also mature, yet were associated with fewer bacteria, and stimulated T cell proliferation, but not IFN-γ production. The CD8α⁻CD4⁻ DC showed a modest maturation response and were associated with a high number of bacteria, but failed to induce T cell proliferation ex vivo. pDC displayed a strong maturation response, but were not associated with detectable bacteria and also failed to stimulate T cell activation. Finally, we measured the cytokine responses in these subsets and determined that IL-12 was produced predominantly by the CD8⁺ DC, correlating with the ability of this subset DC to induce IFN-γ production in T cells. We conclude that Listeria-specific CD8⁺ T cell activation in the spleen is most effectively achieved by infection-induced maturation of the CD8α⁺ DC subset.     

Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules, HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells.     

NK cells promote type 1 T cell immunity through modulating the function of dendritic cells during intracellular bacterial infection

Dendritic cells (DC) play a key role in establishing protective adaptive immunity in intracellular bacterial infections, but the cells influencing DC function in vivo remain unclear. In this study, we investigated the role of NK cells in modulating the function of DC using a murine Chlamydia infection model. We found that the NK cell-depleted mice showed exacerbated disease after respiratory tract Chlamydia muridarum infection, which was correlated with altered T cell cytokine profile. Furthermore, DC from C. muridarum-infected NK-depleted mice (NK(-)DC) exhibited a less mature phenotype compared with that of DC from the infected mice without NK depletion (NK(+)DC). NK(-)DC produced significantly lower levels of both IL-12 and IL-10 than those of NK(+)DC. Moreover, NK(-)DC showed reduced ability to direct primary and established Ag-specific Th1 CD4(+) T cell responses in DC-T coculture systems. More importantly, adoptive transfer of NK(-)DC, in contrast to NK(+)DC, failed to induce type 1 protective immunity in recipients after challenge infection. Finally, NK cells showed strong direct enhancing effect on IL-12 production by DC in an NK-DC coculture system, which was partially reduced by blocking NKG2D receptors signaling and virtually abolished by neutralizing IFN-γ activity. The data demonstrate a critical role of NK cells in modulating DC function in an intracellular bacterial infection.