Gut dendritic cell activation links an altered colonic microbiome to mucosal and systemic T-cell activation in untreated HIV-1 infection

HIV-1-associated disruption of intestinal homeostasis is a major factor contributing to chronic immune activation and inflammation. Dendritic cells (DCs) are crucial in maintaining intestinal homeostasis, but the impact of HIV-1 infection on intestinal DC number and function has not been extensively studied. We compared the frequency and activation/maturation status of colonic myeloid DC (mDC) subsets (CD1c⁺ and CD1c^neg) and plasmacytoid DCs in untreated HIV-1-infected subjects with uninfected controls. Colonic mDCs in HIV-1-infected subjects had increased CD40 but decreased CD83 expression, and CD40 expression on CD1c⁺ mDCs positively correlated with mucosal HIV-1 viral load, with mucosal and systemic cytokine production, and with frequencies of activated colon and blood T cells. Percentage of CD83⁺CD1c⁺ mDCs negatively correlated with frequencies of interferon-γ-producing colon CD4⁺ and CD8⁺ T cells. CD40 expression on CD1c⁺ mDCs positively associated with abundance of high prevalence mucosal Prevotella copri and Prevotella stercorea but negatively associated with a number of low prevalence mucosal species, including Rumminococcus bromii. CD1c⁺ mDC cytokine production was greater in response to in vitro stimulation with Prevotella species relative to R. bromii. These findings suggest that, during HIV infection, colonic mDCs become activated upon exposure to mucosal pathobiont bacteria leading to mucosal and systemic immune activation.     

Role of dendritic cells infected with human herpesvirus 6 in virus transmission to CD4 T cells

Human herpesvirus 6 (HHV-6) is a ubiquitous betaherpesvirus that predominantly infects and replicates in CD4⁺ T lymphocytes. However, the mechanism of HHV-6 transmission to T cells from the peripheral mucosa is unknown. Here we found that dendritic cells (DCs) can transmit HHV-6 to T cells, resulting in productive infection. In immature monocyte-derived DCs (MDDCs) infected with HHV-6, viral early and late antigens were expressed, and nucleocapsids containing a DNA core were observed, although few virions were detected in the cytoplasm by electron microscopy, indicating that the maturation of HHV-6 virions may be incomplete in MDDCs. However, HHV-6 transmission from MDDCs to stimulated CD4⁺ T cells occurred efficiently in coculture of these cells, but not from MDDCs culture supernatants. This transmission was partially inhibited by treating the DCs with a viral DNA synthesis blocker, indicating that viral replication in MDDCs is required for this transmission. Furthermore, myeloid DCs and plasmacytoid DCs infected with HHV-6 could also transmit the virus to stimulated T cells. Thus, DCs may be the first cell population targeted by HHV-6 and could play an important role in the virus' transmission to T cells for their further propagation.     

Crosstalk between human DC subsets promotes antibacterial activity and CD8+ T‐cell stimulation in response to bacille Calmette‐Guérin

To date, little is known about the unique contributions of specialized human DC subsets to protection against tuberculosis (TB). Here, we focus on the role of human plasmacytoid (p)DCs and myeloid (m)DCs in the immune response to the TB vaccine bacille Calmette‐Guérin (BCG). Ex vivo DC subsets from human peripheral blood were purified and infected with BCG expressing GFP to distinguish between infected and noninfected cells. BDCA‐1⁺ myeloid DCs were more susceptible than BDCA‐3⁺ mDCs to BCG infection. Plasmacytoid DCs have poor phagocytic activity but are equipped with endocytic receptors and can be activated by bystander stimulation. Consequently, the mutual interaction of the two DC subsets in response to BCG was analyzed. We found that pDCs were activated by BCG‐infected BDCA‐1⁺ mDCs to upregulate maturation markers and to produce granzyme B, but not IFN‐α. Reciprocally, the presence of activated pDCs enhanced mycobacterial growth control by infected mDCs and increased IL‐1β availability. The synergy between the two DC subsets promoted BCG‐specific CD8⁺ T‐cell stimulation and the role of BCG‐infected BDCA‐1⁺ mDCs could not be efficiently replaced by infected BDCA‐3⁺ mDCs in the crosstalk with pDCs. We conclude that mDC–pDC crosstalk should be exploited for rational design of next‐generation TB vaccines.     

Activation of cord blood myeloid dendritic cells by Trypanosoma cruzi and parasite-specific antibodies,proliferation of CD8+ T cells,and production of IFN-γ

We previously reported that Trypanosoma cruzi, the agent of Chagas disease, induces in congenitally infected fetuses a strong, adult-like parasite-specific CD8⁺ T cell response producing IFN-γ (Hermann et al. in Blood 100:2153–2158, 2002). This suggests that the parasite is able to overcome the immaturity of neonatal antigen presenting cells, an issue which has not been previously addressed. We therefore investigated in vitro the ability of T. cruzi to activate cord blood DCs and compared its effect to that on adult cells. We show that T. cruzi induces phenotypic maturation of cord blood CD11c⁺ myeloid DCs (mDCs), by enhancing surface expression of CD40, CD80, and CD83, and that parasite-specific IgG purified from cord blood of neonates born to T. cruzi-infected mothers amplify such expression. CD83, considered as the best marker of mature DCs, reaches higher level on cord blood than on adult mDCs. Allo-stimulation experiments showed that T. cruzi-activated cord blood mononuclear cells enriched in DCs (eDCs) stimulate proliferation of cord blood and adult CD3⁺ T cells to a similar extent. Of note, T. cruzi-activated eDCs from cord blood trigger more potent proliferation of CD8⁺ than CD8⁻ (mainly CD4⁺) adult T cells, a feature not observed with adult eDCs. T cell proliferation is associated with IFN-γ release and down-regulation of IL-13 production. These data show that T. cruzi potently activates human cord blood mDCs and endows eDCs to trigger CD8⁺ T cell proliferation and favor type 1 immune response. Interestingly, maternal antibodies can strengthen the development of mature DCs that might contribute to overcome the immunological immaturity associated with early life.     

Effect of Bacillus anthracis virulence factors on human dendritic cell activation

Bacillus anthracis possesses three primary virulence factors: capsule, lethal toxin (LT), and edema toxin (ET). Dendritic cells (DCs) are critical to innate and acquired immunity and represent potential targets for these factors. We examined the ability of B. anthracis spores and bacilli to stimulate human monocyte-derived DC (MDDC), primary myeloid DC (mDC), and plasmacytoid DC (pDC) cytokine secretion. Exposure of MDDCs and mDCs to spores or vegetative bacilli of the genetically complete strain UT500 induced significantly increased cytokine secretion. Spores lacking genes required for capsule biosynthesis stimulated significantly higher cytokine secretion than UT500 spores from mDCs, but not MDDCs. In contrast, bacilli lacking capsule stimulated significantly higher cytokine secretion than UT500 bacilli in both MDDCs and mDCs. Spores or bacilli lacking both LT and ET stimulated significantly higher cytokine secretion than UT500 spores or bacilli, respectively, in both mDCs and MDDCs. pDCs exposed to spores or bacilli did not produce significant amounts of cytokines even when virulence factors were absent. In conclusion, B. anthracis employs toxins as well as capsule to inhibit human MDDC and mDC cytokine secretion, whereas human pDCs respond poorly even when capsule or both toxins are absent.     

Pattern recognition receptor expression and maturation profile of dendritic cell subtypes in human tonsils and lymph nodes

Dendritic cells (DCs) with capacity of antigen cross-presentation are of key interest for immunotherapy against cancer as they can induce antigen-specific cytotoxic T lymphocyte (CTL) responses. This study describes frequencies of DC subtypes in human tonsils and lymph nodes, and phenotypic aspects that may be targeted by adjuvant measures.From human tonsils and neck lymph nodes, DCs were identified through flow cytometry, and subsets of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were investigated. Maturity status was assessed and surface receptors with CTL-promoting potentials were studied.CD123⁺ pDCs as well as CD1c⁺, CD141⁺, and CD1c^-CD141^- mDCs were detected in tonsils and lymph nodes. Both sites featured a similar presence of DC subsets, with CD123⁺ pDC being dominant and CD141⁺ mDCs least frequent. Based on CD80/CD86 expression, all DC subtypes featured a low degree of maturation. Expression of pattern recognition receptors (PRRs) CD206, CD207, DC-SIGN, TLR2, and TLR4, as well as the chemokine receptor XCR1, indicated DC subset-specific receptor profiles.We conclude that tonsils and lymph nodes share common features in terms of DC subset frequency and maturation as well as PRR and XCR1 expression pattern. Our work suggests that both sites may be considered for vaccine deposition in DC-mediated immunotherapy.     

The allergenic yeast Malassezia furfur induces maturation of human dendritic cells

BACKGROUND: The yeast Malassezia furfur (M. furfur), present in the normal microflora of human skin, can act as an allergen that incites specific IgE reactivity and T cell proliferation in atopic dermatitis (AD) patients. The role of antigen presenting dendritic cells (DCs) in the onset and maintenance of AD is not well established. OBJECTIVE: The objective of the present study was to assess whether the interaction of M. furfur with human DCs will result in DC maturation, cytokine production and lymphocyte proliferation. METHODS: Monocyte-derived dendritic cells (MDDCs) were generated from human peripheral blood. Immature MDDCs were cultured with or without M. furfur or plastic beads, and with or without CD40L stimulation. Interaction of yeast cells by MDDCs was studied by time-lapse photography and cytokines were detected in culture supernatants with ELISA. The ability of MDDCs pre-incubated with M. furfur to induce proliferation in autologous lymphocytes was measured by [(3)H]-thymidine incorporation. RESULTS: Time-lapse photography showed that the majority of immature MDDCs internalized whole M. furfur yeast cells within 1 h. The presence of M. furfur induced maturation (CD83 expression) of MDDCs, and up-regulation of the costimulatory molecules CD80 and CD86. Production of TNF-alpha, IL-1 beta and IL-18 by MDDCs increased significantly (P < 0.05 for TNF-alpha and IL-1 beta, and P < 0.01 for IL-18) after the addition of M. furfur, while IL-10 and IL-12p70 levels remained unaltered. The CD40L-stimulated IL12p70 production by MDDCs was decreased in the presence of M. furfur (P < 0.05). Finally, immature MDDCs pre-incubated with M. furfur induced a proliferative response in autologous CD14-depleted peripheral blood mononuclear cells, in a dose-dependent manner. CONCLUSION: The data indicate that immature MDDCs can internalize the opportunistic yeast M. furfur. This process was associated with MDDC maturation, production of pro-inflammatory and immunoregulatory cytokines, which might favour induction of a Th2-type immune response, and a capacity to stimulate lymphocyte proliferation. This chain of events most likely contributes to the inflammatory reaction in AD.     

Activated CD56(+) lymphocytes (NK+NKT) mediate immunomodulatory and anti-viral effects during Japanese encephalitis virus infection of dendritic cells in-vitro

Japanese encephalitis virus (JEV) remains one of the major causative agents of pediatric encephalitis. Interaction of dendritic cells (DCs) with innate lymphocytes (NK and NKT) represents a crucial event during anti-viral innate immune response. In the current study, we have tried to understand the interaction between JEV, human monocyte derived DCs (MDDCs), and CD56⁺ cells (NK+NKT) in-vitro. We have used two JEV strains (i) JE057434 (neurovirulent, wild-type) and (ii) SA14-14-2 (non-neurovirulent, live-attenuated vaccine) to investigate the effect of viral virulence on the functional status of primary human MDDCs. Our preliminary results indicate that replicating JEV induces MDDCs maturation via PI3K and p38 pathways. We also show that the presence of IL2-activated CD56⁺ cells impart both immunomodulatory and anti-viral effects on DCs infected with JEV. Mechanistic studies illustrate that, IL2-activated CD56⁺ lymphocytes mediated immunomodulation occurs through direct cell-to-cell contact and TNFα, while the anti-viral effect is dependent on direct cell-to-cell contact.     

siRNA Knockdown of PD-L1 and PD-L2 in Monocyte-Derived Dendritic Cells only Modestly Improves Proliferative Responses to Gag by CD8<Superscript>+</Superscript> T Cells from HIV-1-Infected Individuals

Introduction  Due to their capacity to elicit and regulate immunity, dendritic cells (DCs) are important targets to improve vaccination. Knowing that programmed death-1 (PD-1) high virus-specific T cells become functionally exhausted during chronic exposure to human immunodeficiency virus-1 (HIV-1), the development of a therapeutic DC-based HIV-1 vaccine might include strategies that downregulate PD-L1 and PD-L2 counter-receptors. Methods  After showing that monocyte-derived DCs rapidly upregulated PD-L1 and PD-L2 expression upon maturation with a variety of stimuli, e.g., Toll-like receptor ligands and cytokines, we determined that PD-L1 and PD-L2 expression could be knocked down by electroporation of a single small interfering RNA (siRNA) sequence twice at the monocyte and immature stages of DC development. This knockdown approached completion and was specific and lasting for several days. Results  We then added the PD-L1 and PD-L2 silenced monocyte-derived DCs to peripheral blood mononuclear cells from HIV-1-infected individuals along with pools of 15-mer HIV-1 Gag p24 peptides. However, in cultures from six patients, there was only a modest enhancing effect of PD-L1 and PD-L2 silencing on CD8⁺ T cell proliferative responses to the DCs. Discussion  These findings suggest that, in monocyte-derived DCs, additional strategies than PD-L1 or PD-L2 blockade will be needed to improve the function of PD-1 high T cells.     

Urban particulate matter stimulation of human dendritic cells enhances priming of naive CD8 T lymphocytes

Epidemiological studies have consistently shown associations between elevated concentrations of urban particulate matter (UPM) air pollution and exacerbations of asthma and chronic obstructive pulmonary disease, which are both associated with viral respiratory infections. The effects of UPM on dendritic cell (DC) ‐stimulated CD4 T lymphocytes have been investigated previously, but little work has focused on CD8 T‐lymphocyte responses despite their importance in anti‐viral immunity. To address this, we examined the effects of UPM on DC‐stimulated naive CD8 T‐cell responses. Expression of the maturation/activation markers CD83, CCR7, CD40 and MHC class I on human myeloid DCs (mDCs) was characterized by flow cytometry after stimulation with UPMin vitro in the presence/absence of granulocyte–macrophage colony‐stimulating factor (GM‐CSF). The capacity of these mDCs to stimulate naive CD8 T‐lymphocyte responses in allogeneic co‐culture was then assessed by measuring T‐cell cytokine secretion using cytometric bead array, and proliferation and frequency of interferon‐γ (IFN‐γ)‐producing T lymphocytes by flow cytometry. Treatment of mDCs with UPM increased expression of CD83 and CCR7, but not MHC class I. In allogeneic co‐cultures, UPM treatment of mDCs enhanced CD8 T‐cell proliferation and the frequency of IFN‐γ⁺ cells. The secretion of tumour necrosis factor‐α, interleukin‐13, Granzyme A and Granzyme B were also increased. GM‐CSF alone, and in concert with UPM, enhanced many of these T‐cell functions. The PM‐induced increase in Granzyme A was confirmed in a human experimental diesel exposure study. These data demonstrate that UPM treatment of mDCs enhances priming of naive CD8 T lymphocytes and increases production of pro‐inflammatory cytokines. Such UPM‐induced stimulation of CD8 cells may potentiate T‐lymphocyte cytotoxic responses upon concurrent airway infection, increasing bystander damage to the airways.     

Thyroglobulin-pulsed human monocyte-derived dendritic cells induce CD4+ T cell activation

Although thyroglobulin (Tg) would be expected to act as a tumor-associated antigen that might be exploitable by immunotherapy against thyroid cancers, it remains unclear how to effectively enhance the immune response to Tg in human since it is a self-component glycoprotein. We therefore tested whether and how human peripheral blood (PB) monocyte-derived dendritic cells (DCs) pulsed with human (h)Tg would induce activation of hTg-specific T cells. We found that immature DCs (iDCs) exhibited a higher endocytic capacity for fluorescein isothiocyanate-conjugated hTg than did mature DCs (mDCs). Although freshly isolated T cells responded poorly to mDCs, hTg-primed T cells responded much more strongly to hTg pulsed mDCs, which selectively induced IFN-gamma-secreting T cells. These results suggest that hTg-pulsed mDCs enhance the responses of Tg-specific T cells, raising the possibility that vaccination with hTg-pulsed mDCs may be an effective approach as immunotherapy to potentiate thyroid cancer specific therapy.     

Fimbriated Porphyromonas gingivalis is more efficient than fimbria-deficient P. gingivalis in entering human dendritic cells in vitro and induces an inflammatory Th1 effector response

Porphyromonas gingivalis is a fimbriated mucosal pathogen implicated in chronic periodontitis (CP). The fimbriae are required for invasion of the gingival mucosa and for induction of CP in animal models of periodontitis. CP is associated with infection of immature dendritic cells (DCs) by P. gingivalis in situ and with increased numbers of dermal DCs (DDCs) and mature DCs in the lamina propria. The role of fimbriae in gaining entry into human DCs and how this modulates the inflammatory and effector immune responses, however, have not been explored. To address this, we generated monocyte-derived DCs (MDDCs) in vitro which phenotypically and functionally resemble DDCs. We show here that virulent fimbriated P. gingivalis 381, in contrast to its fimbria-deficient mutant, P. gingivalis DPG3, efficiently gains entry to MDDCs in a manner dependent on active cell metabolism and cytoskeletal rearrangement. In addition, uptake of 381, unlike DPG3, induces DCs to undergo maturation, upregulate costimulatory molecules, and secrete inflammation cytokines interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor alpha, IL-10, and IL-12. Moreover, MDDCs pulsed with 381 also stimulated a higher autologous mixed lymphocyte reaction and induced a Th1-type response, with gamma interferon (IFN-gamma) being the main cytokine. Monocytes used as controls demonstrated fimbria-dependent uptake of 381 as well but produced low levels of inflammatory cytokines compared to MDDCs. When MDDCs were pulsed with recombinant fimbrillin of P. gingivalis (10 micro g/ml), maturation of MDDCs was also induced; moreover, matured MDDCs induced proliferation of autologous CD4(+) T cells and release of IFN-gamma. Thus, these results establish the significance of P. gingivalis fimbriae in the uptake of P. gingivalis by MDDCs and in induction of immunostimulatory Th1 responses.     

Chinese human immunodeficiency virus-1 patients with different routes of transmission exhibit altered expression levels of blood dendritic cell subpopulations

Dendritic cells (DCs) play a pivotal role in the pathogenesis of human immunodeficiency virus-1 (HIV-1). Reduced numbers of blood DCs have been observed in individuals with chronic HIV-1 infection. In the present study, we analyzed the expression levels of monocytes, myeloid dendritic cell (mDC) precursors, mDCs, and plasmacytoid dendritic cells (pDCs), in HIV-1-infected patients in China who were infected via different routes of transmission, including heterosexual and homosexual sexual contact, and blood transmission through importation of blood or blood products, to further elucidate their role in HIV. Compared with HIV-negative individuals (n?=?40), relative levels of CD11c+CD14?mDCs, CD11c++CD123(low) mDCs, and CD11c?CD123+ pDCs in total peripheral blood mononuclear cells (PBMCs) were significantly lower in all HIV patients (n?=?93), and in those with blood transmission (n?=?26) and heterosexual transmission (n?=?43), while relative levels of CD11c+CD14?mDCs were significantly lower in HIV patients infected via homosexual transmission (n?=?24). The results of correlation analysis demonstrated a significant negative correlation between CD4+ T-cell counts and the relative levels of CD11c++CD123(low) mDCs in HIV-I patients infected via blood transmission. There was no significant correlation between CD4+ T-cell counts and the expression level of other DC subpopulations in PBMCs from HIV patients. The results of this study suggest that HIV-1 patients with different routes of transmission exhibit altered expression levels of blood DC subpopulations, which contributes to dysregulated immune responses and pathogenesis of HIV-1.     

Production of a Dendritic Cell-Based Vaccine Containing Inactivated Autologous Virus for Therapy of Patients with Chronic Human Immunodeficiency Virus Type 1 Infection

In preparation for a pilot clinical trial in patients with chronic human immunodeficiency virus type 1 (HIV-1) infection, a novel dendritic cell (DC)-based vaccine is being manufactured. The trial will test the hypothesis that isolated endogenous virus presented by DCs serves as a potent immunogen for activation of CD8⁺ and CD4⁺ T cells specific for a broad range of autologous HIV-1 antigens. Production of the vaccine under good manufacture practice conditions involves (i) autologous virus isolation; (ii) superinfection of CD4⁺ T cells with the virus; (iii) inactivation of the virus in CD4⁺ T cells, T-cell apoptosis, and coincubation of T cells with autologous DCs; and (iv) product testing and release. Endogenous virus was isolated from peripheral blood-derived CD4⁺ T cells of three HIV-1-positive subjects by coincubation with autologous OKT-3-stimulated CD4⁺ T cells. CD4⁺ T-cell supernatants were tested for p24 levels by enzyme-linked immunosorbent assay (>25 ng/ml) and for the 50% tissue culture infective doses (TCID₅₀; which ranged from 4,642 to 46,416/ml on day 19 of culture). Autologous CD4⁺ T cells that were separated on immunobeads (>95% purity) and superinfected with virus-expressed p24 (28 to 54%) had TCID₅₀ of >400/ml on days 5 to 10. Virus inactivation with psoralen (20 μg/ml) and UVB irradiation (312 nm) reduced the TCID₅₀ of the supernatants from 199,986 to 11/ml (>99%). 7-Amino-actinomycin D-positive, annexin V-positive CD4⁺ T cells were fed to autologous DCs generated by using the Elutra cell separation system and the Aastrom system. Flow analysis showed that DC loading was complete in 24 h. On the basis of these translational results and experience with the generation of DCs from HIV-1-infected patients in a previous clinical trial, the Investigational New Drug application for clinical vaccination was submitted and approved by the FDA (application no. BB-IND-13137).Antiretroviral therapy (ART) has been widely used to suppress human immunodeficiency virus type 1 (HIV-1) replication and increase the number of CD4⁺ T cells in patients with HIV-1 infection. However, in most of these patients, the recovery of anti-HIV-1-specific T-cell function is incomplete. As the complete restoration of T-cell immune function is considered to be necessary for effective control of the viral infection, additional measures aimed at the bolstering of the HIV-1-specific adaptive immunity in patients treated with ART are being evaluated.Dendritic cells (DCs) are the most potent antigen-presenting cells that can both prime and sustain memory responses (24, 28). DCs have been used increasingly frequently in vaccines against cancer and viral infections (4, 13, 20). Previous studies from our group showed that DCs derived from the blood of subjects with chronic progressive HIV-1 infection and not receiving ART were able to stimulate anti-HIV-1 reactivity (5). HIV-1-reactive CD8⁺ T cells are detectable in the peripheral circulation of subjects receiving ART following in vitro activation with many types of HIV-1 antigens, including HIV-1 proteins, HIV-1 peptides, and virus-infected apoptotic cell-loaded matured DCs (6, 10, 14, 22, 23, 31). We hypothesized that it may be possible to reconstitute the reactivity of naïve and memory virus-specific T cells by delivering to patients autologous DCs engineered ex vivo to express and present known immunodominant peptides of HIV-1. To this end, we have recently completed a phase I clinical protocol in which autologous monocyte-derived DCs were pulsed with a mix of three HIV-1 peptides (Gag, Pol, and Env) and one influenza A virus (matrix) major histocompatibility complex class I supertype peptide and delivered as vaccines to 18 HIV-1-infected, ART-treated subjects (5). This vaccination strategy was found to be safe and feasible and resulted in a transient but significant increase in the frequency of CD8⁺ T cells specific for HIV-1 peptides present in the vaccine (5). On the basis of the results of this trial, we have been considering a strategy of stimulating HIV-1-specific, naïve CD8⁺ and CD4⁺ T cells by priming them with DCs engineered to express autologous HIV-1 (19). The rationale for this strategy is that autologous virus represents a large repertoire of the host''s diverse HIV-1 antigen pool and offers the potential to elicit the most specific, broadest, and most effective immune responses for each subject''s quasispecies of HIV-1, thus increasing vaccine efficacy.In this report, we provide evidence that the production of an antiviral vaccine containing autologous DCs fed with inactivated HIV-1-infected, autologous, apoptotic CD4⁺ T cells is feasible, can be successfully accomplished in a good manufacture practice facility, and can be scaled up for therapeutic delivery to HIV-positive (HIV-1⁺) patients. The production process consists of several steps: (i) isolation of autologous virus from the peripheral blood of HIV-1-infected subjects; (ii) superinfection of autologous enriched CD4⁺ CD8⁻ T cells with viral supernatants; (iii) virus inactivation by psoralen and UVB irradiation; (iv) testing for p24 levels and the residual HIV-1 load by determining the 50% tissue culture infective doses (TCID₅₀) for apoptotic CD4⁺ T cells; and (v) loading of autologous DCs with apoptotic, HIV-1-infected CD4⁺ T cells. Although this process is complex, it has been successfully scaled up for therapeutic vaccine production.     

CD4+ T-cell anergy induced by lin− CD117(c-kit)+ stem cell-derived immature dendritic cells loaded with nuclear antigen derived from Trypanosoma equiperdum

Dendritic cells (DCs) are professional antigen-presenting cells, which have the extraordinary capacity to initiate naïve T-cell-mediated primary immune responses. To investigate the role of DCs in the induction of antigen-specific tolerance, the immature DCs (imDCs) and mature DCs (mDCs) were generated in vitro from lin^? CD117(c-kit)⁺ stem cells isolated from mice bone marrow. Flow cytometry and confocal microscopy were used to characterize the phenotypes of DCs. These cells were loaded with nuclear antigen derived from Trypanosoma equiperdum and then co-cultured with naïve CD4⁺ T cells. It was found that imDC-treated T cells had lower proliferation level and cytokine expression of interleukin (IL)-2, IL-4, IL-12, and interferon-γ compared with mDC-treated T cells. These results demonstrated that the maturation status of DCs is critical for preventing the production of autoantibodies.     

Plasmacytoid and myeloid dendritic cells with a partial activation phenotype accumulate in lymphoid tissue during asymptomatic chronic HIV-1 infection

Dendritic cells (DCs) from HIV-1-infected individuals display numeric and functional defects, and recent evidence suggests that HIV-1 can directly and indirectly activate DCs in vitro. The in vivo activation state and compartmentalization of DC subsets during HIV-1 infection remain poorly understood, however. We evaluated phenotypic and functional characteristics of myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) directly ex vivo in peripheral blood and lymphoid tissue from HIV-1-infected and HIV-seronegative individuals. Analysis of a wide range of chemokine receptors and activation/maturation markers on circulating DCs from viremic HIV-1-infected donors revealed a phenotype indicative of partial activation. Yet, blood DCs from viremic subjects still achieved full maturation when stimulated in vitro. In addition, blood pDCs from viremic individuals had a reduced capacity to migrate to CXCL12 in vitro. Total numbers of both DC subsets were increased in lymph nodes of asymptomatic untreated HIV-1-infected subjects, consistent with DC accumulation in the lymphoid compartment. Lymph node DCs also expressed high levels of CD40 in the absence of increases of other typical activation/maturation markers. Activation and depletion of DCs in blood with accumulation in lymphoid tissue may contribute to HIV-associated chronic immune activation and T-cell dysfunction.     

Effect of intrauterine HIV-1 exposure on the frequency and function of uninfected newborns' dendritic cells

Immaturity of the neonatal immune system is considered an underlying factor for enhanced severity of infections in newborns. Functional defects of neonatal antigen-presenting cells lead to defective T-cell responses. T cells from uninfected neonates exposed in utero to HIV-1 (EU) exhibit phenotypic and functional alterations; however, the function of their circulating dendritic cells (DCs) has not been characterized. We hypothesized that an HIV-1-infected maternal environment may influence the infants' DC number, phenotype and function. EU exhibited a higher percentage of myeloid DCs (mDCs) than unexposed neonates, although this frequency remained lower than that observed in adults. Plasmacytoid DC (pDC) frequencies were similar in all groups, although both groups of infants tended to have lower frequencies than adults. After LPS stimulation, mDCs from EU up-regulated CD80, CD86 and B7-H1, whereas mDCs from unexposed infants upregulated B7-H1, but not CD80/CD86, and adult mDCs up-regulated mainly CD80 and CD86. IFN-alpha production was similar in all groups, indicating a normal pDC function. Therefore, in utero exposure to HIV-1 induces quantitative and qualitative changes in neonatal DCs, particularly in mDCs, which might be associated with alterations observed in T cells from these EU.     

Macropinocytosis and cytoskeleton contribute to dendritic cell-mediated HIV-1 transmission to CD4+ T cells

Dendritic cells (DCs) are among the first immune cells to encounter HIV-1 at the initial infection. DCs efficiently transfer HIV-1 to CD4⁺ T cells via infectious or virological synapses formed between DCs and T cells. Retroviruses exploit the cytoskeletal network to facilitate viral infection and dissemination; however, the role of the cytoskeleton in DC-mediated HIV-1 transmission is unknown. Here, we report that intact cytoskeleton is essential for DC-mediated HIV-1 transmission to CD4⁺ T cells. We found that macropinocytosis of HIV-1 contributes to DC-mediated HIV-1 endocytosis and transmission. Blocking HIV-1 macropinocytosis and disrupting actin or microtubules in DCs with specific inhibitors significantly prevented DC-mediated HIV-1 trans-infection of CD4⁺ T cells. Altered HIV-1 trafficking and impaired formation of virological synapses primarily accounted for the inhibition of viral transmission by cytoskeletal inhibitors. Our results provide new insights into the mechanisms underlying DC-mediated HIV-1 transmission to CD4⁺ T cells via the cytoskeletal network.