The number and distribution of blood dendritic cells in the epidermis and dermis of healthy human subjects

Human blood dendritic cells (BDC) can be divided into three subsets: plasmacytoid DC (PDC) and two myeloid subsets--MDC1 and MDC2. Several studies revealed the presence of both MDC and PDC in blood of healthy subjects, however no precise literature data exist on the number and distribution of BDC in the skin. The aim of our study was to assess the number and distribution of BDC and their subtypes in the healthy skin. The-study included 30 healthy volunteers (age 18-51). Punch biopsies were taken from the buttock skin from each subject, and immunofluorescent staining was performed using monoclonal mouse IgG1 antibodies directed against BDCA-1, BDCA-2, BDCA-3 and BDC-4. The BDC were present both in the epidermis and dermis. PDC were detected mainly in the dermis (mean 1.2 cells per field). Myeloid subtypes were observed mainly in the middle layers of the epidermis and in the upper part of the dermis (mean 1.8 cells per field). The detection of blood dendritic cells in the skin proves their role in immune cutaneous surveillance.     

Persistent decreases in blood plasmacytoid dendritic cell number and function despite effective highly active antiretroviral therapy and increased blood myeloid dendritic cells in HIV-infected individuals

Dendritic cells (DC) have an instrumental role in the activation and function of both innate and adaptive immune responses. In humans, at least two distinct DC subsets have been characterized based on phenotypic markers: the myeloid DC (MDC) and the plasmacytoid DC (PDC). Both subsets are critical producers of cytokines (IL-12 for MDC and type I/II IFNs for PDC) and are functionally different. We show in this study that HIV(+) individuals have a significant decrease in the number of the Lin(-)HLA-DR(+)CD123(+) and BDCA-2(+) PDC compared with uninfected donors (p = 0.0001). HIV(+) individuals also have a sustained impairment in viral-induced IFN-alpha production (p < 0.0001). The decrease of the PDC subsets did not correlate with CD4 count or viral load and was not reversed in subjects under virally suppressive treatment, suggesting an irreversible change after infection. By contrast, the absolute number and median frequency of MDC in HIV-infected individuals were similar to those observed in uninfected controls, while a significant decrease was present in subjects with >5000 HIV-1 copies/ml. The inverse association with viral load of the MDC number, but not of IFN-alpha secretion or the number of PDC, suggests a role for MDC in viral control. Our data suggest that DC subsets are differentially reconstituted during the immune recovery associated with antiviral therapy. The persistent impairment of certain DC subsets may result in a sustained defect in DC-mediated innate immune functions despite an effective treatment regimen.     

Receptor cross-linking on human plasmacytoid dendritic cells leads to the regulation of IFN-alpha production

Plasmacytoid dendritic cells (PDC) are the natural type I IFN-producing cells that produce large amounts of IFN-alpha in response to viral stimulation. During attempts to isolate PDC from human PBMC, we observed that cross-linking a variety of cell surface receptors, including blood DC Ag (BDCA)-2, BDCA-4, CD4, or CD123 with Abs and immunobeads on PDC leads to inhibition of IFN-alpha production in response to HSV. To understand the mechanisms involved, a number of parameters were investigated. Cross-linking did not inhibit endocytosis of soluble Ag by PDC. Flow cytometry for annexin V and activated caspase-3 indicated that PDC are not undergoing apoptosis after receptor cross-linking. Cross-linking of CD123, but not the other receptors, caused the up-regulation of costimulatory molecules CD80 and CD86, as well as the down-regulation of CD62L, indicating PDC maturation. Thus, anti-CD123 Ab may be acting similar to the natural ligand, IL-3. Anti-phosphotyrosine Ab, as well as Ab to the IFN regulatory factor, IRF-7, was used in intracellular flow cytometry to elucidate the signaling pathways involved. Tyrosine phosphorylation occurred after cross-linking BDCA-2 and BDCA-4, but not CD4. Cross-linking did not affect IRF-7 levels in PDC, however, cross-linking BDCA-2, BDCA-4, and CD4, but not CD123, inhibited the ability of IRF-7 to translocate to the nucleus. Taken together, these results suggest that cross-linking BDCA-2, BDCA-4, and CD4 on PDC regulates IFN-alpha production at the level of IRF-7, while the decrease in IFN-alpha production after CD123 cross-linking is due to stimulation of the IL-3R and induction of PDC maturation.     

Relationship between peripheral blood dendritic cells and cytokines involved in the pathogenesis of systemic lupus erythematosus

Recent studies indicate that dendritic cells (DC) and several cytokines are implicated in the induction of autoimmune diseases. In this study we investigated the relationship between the total number of DC (tDC), and their plasmacytoid (pDC) and myeloid (mDC) subpopulations, with serum concentrations of interferons (IFN-alpha and IFN-gamma) and selected cytokines (TNF-alpha, IL-4, IL-6), in patients with systemic lupus erythematosus (SLE) and healthy persons. Subpopulations of DC were determined by the following antigen expression profiles: BDCA-1+\CD11c+\HLA-DR+ (for mDC) and BDCA-2 +\CD123+\HLA-DR+ (for pDC), using flow cytometry. Serum levels of interferons and cytokines were assessed by an enzyme-linked immunosorbent assay (ELISA). The study was performed in 36 SLE patients and 19 healthy volunteers. The mean number of tDC was lower in SLE patients (13.9 +/- 6.4\microL) than in healthy persons (24.1 +/- 12.6\microL) (P < 0.001). The number of pDC was also significantly lower in SLE (6.6 +/- 3.6\microL) than in the control group (12.0 +/- 8.3\microL) (P < 0.02). Moreover, the mean pDC count was lower in active than in inactive disease (5.5 +/- 3.6\microL vs 7.6 +/- 3.4\microL; P < 0.04). The mean serum levels of IFN-alpha and IFN-gamma were significantly higher in SLE patients (63.8 pg\mL and 6.6 pg\mL, respectively) than in the control group (2.7 pg\mL and 0.5 pg\mL, respectively) (P < 0.008 and P < 0.001, respectively). Serum levels of TNF-alpha and IL-6 were also higher in SLE patients (mean 7.3 pg\mL and 18.4 pg\mL, respectively) than in healthy controls (4.2 pg\mL and 0.5 pg\mL, respectively) (P < 0.02 and P < 0.001, respectively). The mean serum IL-4 concentrations were similar in SLE and healthy persons (0.2 pg\mL and 0.31 pg\mL, respectively; P -/+ 0.119). A negative correlation was found between pDC number and the serum level of IFN-alpha (rho -/+ -- 0.386, P -/+ 0.02) and between mDC and IFN-gamma (rho -/+ -- 0.377, P -/+ 0.024). In conclusion, the correlation between peripheral blood DC subsets and serum levels of IFN-alpha and IFN-gamma suggests a possible relationship between these cytokines in the pathogenesis of SLE.     

Systemic immune activation in HIV infection is associated with decreased MDC responsiveness to TLR ligand and inability to activate naive CD4 T-cells

Background

HIV infection is characterized by ineffective anti-viral T-cell responses and impaired dendritic cell (DC) functions, including response to Toll-Like Receptor (TLR) ligands. Because TLR responsiveness may affect a host''s response to virus, we examined TLR ligand induced Myeloid and Plasmacytoid DC (MDC and PDC) activation of naïve T-cells in HIV+ subjects.

Methods

Freshly purified MDC and PDC obtained from HIV+ subjects and healthy controls were cultured in the presence and absence of TLR ligands (poly I∶C or R-848). We evaluated indices of maturation/activation (CD83, CD86, and HLA-DR expression), cytokine secretion (IFN-alpha and IL-6), and ability to activate allogeneic naïve CD4 T-cells to secrete IFN-gamma and IL-2.

Results

MDC from HIV+ subjects had increased spontaneous IL-6 production and increased CD83 and CD86 expression when compared to MDC of controls. MDC IL-6 expression was associated with plasma HIV level. At the same time, poly I∶C induced HLA-DR up-regulation on MDC was reduced in HIV+ persons when compared to controls. The latter finding was associated with impaired ability of MDC from HIV+ subjects to activate allogeneic naïve CD4 T-cells. PDC from HIV+ persons had increased spontaneous and TLR ligand induced IL-6 expression, and increased HLA-DR expression at baseline. The latter was associated with an intact ability of HIV PDC to activate allogeneic naïve CD4 T-cells.

Conclusion

These results have implications for the ability of the HIV+ host to form innate and adaptive responses to HIV and other pathogens.     

Cooperative induction of a tolerogenic dendritic cell phenotype by cytokines secreted by pancreatic carcinoma cells

Ag presentation by dendritic cells (DC) is essential to effective antitumor T cell responses in cancer patients. Depending on their origin, maturation state, and the ambient cytokine milieu, DC can differentiate into distinct subpopulations, which preferentially either induce Th1 cell activation (CD11c+,CD123- myeloid DC (MDC)) or immunosuppressive T cell development (CD11c-,CD123+ plasmacytoid DC (PDC)). The present study was undertaken to characterize the effects of pancreatic carcinoma cell-derived cytokines on immature monocyte-derived DC (iMo-DC) in vitro and in vivo. Medium conditioned by human pancreatic carcinoma cells inhibited iMo-DC proliferation, expression of costimulatory molecules (CD80 and CD40) and of HLA-DR, and functional activity as assessed by MLR and IL-12p70 production. iMo-DC generated from pancreatic carcinoma patients in advanced stages of the disease similarly showed decreased levels of HLA-DR expression and reduced ability to stimulate MLR in response to CD40L and IFN-gamma. Moreover, in tumor-patient peripheral blood, the ratio of MDC to PDC cells was lower than in healthy controls due to reduced numbers of MDC CD11c+ cells. Importantly, rather than a single cytokine, a combination of tumor-derived cytokines was responsible for these effects; these were primarily TGF-beta, IL-10, and IL-6, but not vascular endothelial growth factor. In summary, we have identified an array of pancreatic carcinoma-derived cytokines that cooperatively affect iMo-DC activation in a manner consistent with ineffective antitumor immune responses.     

TLR ligand-dependent activation of naive CD4 T cells by plasmacytoid dendritic cells is impaired in hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is characterized by diminished numbers and function of HCV-reactive T cells and impaired responses to immunization. Because host response to viral infection likely involves TLR signaling, we examined whether chronic HCV infection impairs APC response to TLR ligand and contributes to the origin of dysfunctional T cells. Freshly purified myeloid dendritic cells (MDC) and plasmacytoid DC (PDC) obtained from subjects with chronic HCV infection and healthy controls were exposed to TLR ligands (poly(I:C), R-848, or CpG), in the presence or absence of cytokine (TNF-alpha or IL-3), and examined for indices of maturation and for their ability to activate allogeneic naive CD4 T cells to proliferate and secrete IFN-gamma. TLR ligand was observed to enhance both MDC and PDC activation of naive CD4 T cells. Although there was increased CD83 and CD86 expression on MDC from HCV-infected persons, the ability of MDC to activate naive CD4 T cells in the presence or absence of poly(I:C) or TNF-alpha did not differ between HCV-infected and healthy control subjects. In contrast, PDC from HCV-infected persons had reduced activation marker (HLA-DR) and cytokine (IFN-alpha) expression upon R-848 stimulation, and these were associated with impaired activation of naive CD4 T cells. These data indicate that an impaired PDC responsiveness to TLR ligation may play an important role in the fundamental and unexplained failure to induce new T cell responses to HCV Ags and to other new Ags as a consequence of HCV infection.     

Flow cytometric detection of circulating dendritic cells in healthy subjects

Dendritic cells (DCs) are the key antigen-presenting cells controlling the initiation of the T cell- dependent immune response. Currently, two peripheral blood DC subsets have been identified on the basis of their CD11c expression. The CD11c-negative (CD11c-) DCs (expressing high levels of CD123) are designated as lymphoid-derived DCs (DC2), whereas the CD11c+/CD123- cells, do identify the myeloid-derived DCs (DC1). A growing number of studies have been conducted in recent years on both the quantitative and functional alterations of DCs and their subsets in different pathological conditions. In the present study we assessed, using two different flow cytometric (FCM) techniques, the normal profile of blood DCs in 50 italian adult healthy subjects (M/F: 25/25, median age 42.5 years, range 20-65). The percentage and the absolute number of DCs and their subsets, were obtained starting from whole blood samples in two ways: 1) by calculating the number of DCs when gated as lineage-negative/ HLA-DR+ and identifing the two subsets as CD11c+ (DC1) and CD123+ (DC2) and 2) by using three specific markers: BDCA.1 (CD11c+ high/CD123+ low, myeloid DCs); BDCA.2 (CD11c-/ CD123+high, lymphoid DCs); BDCA.3 (CD11c+low /CD123-, myeloid DCs). Six parameters, 4-color FCM analysis were perfomed with a BD FACSCanto equipment. The mean values of the percentage and of the absolute number were: 0.5+/-0.2% and 30+/-11 cells/microL for DCs; 0.2+/-0.1% and 15+/-6 cells/microL for DC1; 0.2+/-0.1% and 15+/-7 cells/microL for DC2. The same values were: 0.2+/-0.1% and 16+/-7 cells/microL for BDCA.1; 0.2+/-0.1% and 12+/-7 cells/microL for BDCA.2; 0.02+/-0.01% and 2+/-1 cells/microL for BDCA.3, respectively. Our study confirmes that the two types of FCM analysis are able to identify the DC population. We also provides the first reference values on normal rates and counts of blood DCs in italian adult healthy subjects.     

CpG-C immunostimulatory oligodeoxyribonucleotide activation of plasmacytoid dendritic cells in rhesus macaques to augment the activation of IFN-gamma-secreting simian immunodeficiency virus-specific T cells

There are two principle subsets of dendritic cells (DCs); CD11c(+)CD123(-) myeloid DCs (MDCs) and CD11c(-)CD123(+) plasmacytoid DCs (PDCs). DC activation via TNF-TNFRs (e.g., CD40L) and TLRs (e.g., immunostimulatory oligodeoxyribonucleotides (ISS-ODNs)) is crucial for maximal stimulation of innate and adaptive immunity. Macaque DC biology is being studied to improve HIV vaccines using the SIV macaque model. Using lineage (Lin) markers to exclude non-DCs, Lin(-)HLA-DR(+)CD11c(+)CD123(-) MDCs and Lin(-)HLA-DR(+)CD11c(-)CD123(+) PDCs were identified in the blood of uninfected macaques and healthy macaques infected with SIV or simian-human immunodeficiency virus. Overnight culture of DC-enriched Lin-depleted cells increased CD80 and CD86 expression. IL-12 production and CD80/CD86 expression by MDC/PDC mixtures was further enhanced by CD40L and ISS-ODN treatment. A CpG-B ISS-ODN increased CD80/CD86 expression by PDCs, but resulted in little IFN-alpha secretion unless IL-3 was added. In contrast, a CpG-C ISS-ODN and aldrithiol-2-inactivated (AT-2) SIV induced considerable PDC activation and IFN-alpha release without needing exogenous IL-3. The CpG-C ISS-ODN also stimulated IL-12 release (unlike AT-2 SIV) and augmented DC immunostimulatory activity, increasing SIV-specific T cell IFN-gamma production induced by AT-2 SIV-presenting MDC/PDC-enriched mixtures. These data highlight the functional capacities of MDCs and PDCs in naive as well as healthy, infected macaques, revealing a promising CpG-C ISS-ODN-driven DC activation strategy that boosts immune function to augment preventative and therapeutic vaccine efficacy.     

Impaired plasmacytoid dendritic cell innate immune responses in patients with herpes virus-associated acute retinal necrosis

Plasmacytoid dendritic cells (PDC), the main producers of type I IFNs in the blood, are important for the recognition and control of viral and bacterial infections. Because several viruses induce IFN-alpha production, severe courses of herpes virus infections in nonimmunocompromised patients may be related to numerical or functional PDC deficits. To evaluate this hypothesis, PBMC and PDC were repeatedly isolated from nine patients with acute retinal necrosis (ARN), caused by herpes simplex or varicella zoster virus. The patients experienced meningitis/encephalitis and frequent infections in childhood (n = 2), recurrent herpes virus infections at unusual localizations (n = 2), ocular surgery (n = 1), infections (n = 4), and stress around ARN (n = 6). The median percentage of isolated PDC was significantly lower in patients compared with 18 age-matched healthy controls (p < 0.001), confirmed by FACS analysis using peripheral blood, and was extremely low during acute disease. PDC counts dropped in five controls suffering from respiratory infections or diarrhea. IFN-alpha production in PDC and PBMC exposed to different stimuli was significantly lower in patients than in controls (p < 0.05). Anergy to these stimuli was observed on four occasions, in particular during acute disease. PDC of patients showed up-regulated IFN regulatory factor-7 mRNA levels and evidence of in vivo activation (CD80) and maturation (CD83) (p < 0.05). CD8+ cell responses were significantly lower in patients vs controls (p = 0.04). These data support a risk factor model in which numerical and functional deficits in PDC-mediated innate immune responses contribute to an impaired control of latent herpes virus infections and subsequent development of ARN.     

BDCA-2 signaling inhibits TLR-9-agonist-induced plasmacytoid dendritic cell activation and antigen presentation

Plasmacytoid dendritic cells (PDCs) express Toll-like receptor (TLR) 9, which mediates recognition of microbial DNA during infection or self-DNA in autoimmune diseases. Triggering TLR-9 in PDC induces either maturation (lysosomal TLR-9 triggering) or type I interferon (IFN-I) production (endosomal TLR-9 triggering). PDCs also express BDCA-2 (CD303), a C-type lectin receptor (CLR) unique to these cells. CLRs appear to function in innate immunity and microbial recognition, and may cooperate with TLRs to fine-tune inflammatory responses.It has been shown that anti-BDCA-2 monoclonal antibody is internalized by PDC for antigen presentation and inhibits TLR-9 induced IFN-I expression. Here we investigated the cross-talk between BDCA-2 and TLR-9-signaling during PDC maturation and antigen presentation. We found that BDCA-2-induced signaling in PDCs inhibits up-regulation of CD86 and CD40 molecules in CpG-activated PDCs, but not in CD40L-activated PDCs. Furthermore, triggering of BDCA-2 diminished the ability of CpG- and CD40L-stimulated PDCs to process and present antigen to antigen-specific autologous memory T cells. This study demonstrates that BDCA-2 represents an attractive target for clinical immunotherapy of IFN-I dependent autoimmune diseases influencing both, IFN-I production and antigen-specific T-cell stimulation by PDC.     

HIV replication leads to skewed maturation of CD8-positive T-cell responses in infected children

HIV-1 infection causes a severe T-cell impairment with alteration of immune response. However, in children the natural decline of lymphocytes and CD4 cells in early life makes it more difficult to monitor immunocompetence and progression of HIV-infection. Aim of this study was to characterize the CD8 response in non-vertically HIV-infected children exposed persistently to viremia and in HIV-infected children controlling efficiently viremia by ART, by analysing the effect of persistent viremia on CD4 and CD8 T-cells count, HIV-specific immune-response and naive/memory pattern of CD8 T-cell. Whereas, no differences of CD4 count between viremic patients and viral controllers were observed (1046.9 +/- 472.1 cells/microl vs 1101.3 +/- 415.4 cells/microl; p > 0.05), CD8 count was higher in the viremic patients (1080.6 +/- 652.1 cells/microl vs 747.5 +/- 389.9 cells/microl, p < 0.05). In viremic patients, HIV-specific CD8 T-cells correlated with viral load. However, in this group a loss of HIV-specific CD8 response was associated with a 7 fold decrease of na?ve and increase of pre-effector CD8 T-cells (62.8% +/- 10.21% vs 10.37% +/- 7.91%, p < 0.03). Persistent exposure to viremia alters HIV-specific CD8 response possibly through a persistent immune activation process leading to exhaustion of naive CD8 T-cells and skewed maturation of memory subset. Therefore, memory CD8 T-cells might lose the ability to respond correctly and efficiently to HIV-antigen exposure.     

Isolation of myeloid and plasmacytoid dendritic cells from human bronchoalveolar lavage fluid

Studies of bronchoalveolar lavage fluid (BALF) dendritic cells (DC) have been hampered by the scarcity of DC and the lack of DC-specific surface markers. Four surface Ag have been recently described as specific markers for distinct subsets of DC and have been used for the isolation and characterization of fresh noncultured DC from lung resection specimens: BDCA-1 (CD1c) and BDCA-3 for myeloid DC type 1 and type 2, respectively, and BDCA-2 and BDCA-4 for plasmacytoid DC. The aim of this study was to develop a new method for the isolation of BALF DC, using immunomagnetic separation of BDCA+ cells. Mononuclear cells were obtained from BALF after Ficoll-Paque density gradient centrifugation. Monocytes, T cells and B cells were magnetically labelled and depleted. The unlabelled cell fraction was incubated with BDCA-1, BDCA-3 and BDCA-4 beads and the total BDCA+ DC were retained. The ability of isolated DC to induce T-cell responses was evaluated by coculturing the isolated DC with immunomagnetically sorted naive T cells. The above procedure resulted in a population of viable DC that showed a strong capacity in induce T-cell responses. Functionally intact human BALF myeloid DC types 1 and 2 as well as plasmacytoid DC can be easily obtained by immunomagnetic isolation. Considering that bronchoalveolar lavage is a minimally invasive procedure, these cells are optimal candidates with which to elucidate the properties and capabilities of pulmonary DC.     

Fc gamma RIIa is expressed on natural IFN-alpha-producing cells (plasmacytoid dendritic cells) and is required for the IFN-alpha production induced by apoptotic cells combined with lupus IgG

An ongoing production of IFN-alpha may be of etiopathogenic significance in systemic lupus erythematosus (SLE). It may be due to the natural IFN-producing cells (NIPC), also termed plasmacytoid dendritic cells (PDC), activated by immune complexes that contain nucleic acids derived from apoptotic cells. We here examined the role of FcgammaR in the IFN-alpha production in vitro by PBMC induced by the combination of apoptotic U937 cells and autoantibody-containing IgG from SLE patients (SLE-IgG). The Fc portion of the SLE-IgG was essential to induce IFN-alpha production, because Fab fragments or F(ab')(2) were ineffective. Normal, especially heat-aggregated, IgG inhibited the IFN-alpha production, suggesting a role for FcgammaR on PBMC. Using blocking anti-FcgammaR Abs, the FcgammaRIIa,c (CD32) but not FcgammaRI or FcgammaRIII were shown to be involved in the IFN-alpha induction by apoptotic cells combined with SLE-IgG, but not by HSV or CpG DNA. In contrast, the action of all of these inducers was inhibited by the anti-FcgammaRIIa,b,c mAb AT10 or heat-aggregated IgG. Flow cytometric analysis revealed that approximately 50% of the BDCA-2-positive PBMC, i.e., NIPC/PDC, expressed low but significant levels of FcgammaRII, as did most of the actual IFN-alpha producers activated by HSV. RT-PCR applied to NIPC/PDC purified by FACS demonstrated expression of FcgammaRIIa, but not of FcgammaRIIb or FcgammaRIIc. We conclude that FcgammaRIIa on NIPC/PDC is involved in the activation of IFN-alpha production by interferogenic immune complexes, but may also mediate inhibitory signals. The FcgammaRIIa could therefore have a key function in NIPC/PDC and be a potential therapeutic target in SLE.     

Characterization of CD8(+) effector T cell responses in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine

Salmonella enterica serovar Typhi (S. typhi) strain Ty21a remains the only licensed attenuated typhoid vaccine. Despite years of research, the identity of the protective immunological mechanisms elicited by immunization with the Ty21a typhoid vaccine remains elusive. The present study was designed to characterize effector T cell responses in volunteers immunized with S. typhi strain Ty21a typhoid vaccine. We determined whether immunization with Ty21a induced specific CTL able to lyse S. typhi-infected cells and secrete IFN-gamma, a key effector molecule against intracellular pathogens. We measured the functional activity of these CTL by a (51)Cr-release assay using 8-day restimulated PBMC from Ty21a vaccinees as effector cells and S. Typhi-infected autologous PHA-activated PBMC as target cells. Most vaccinees exhibited consistently increased CD8-mediated lysis of targets by postimmunization PBMC when compared with preimmunization levels. We also developed an IFN-gamma ELISPOT assay to quantify the frequency of IFN-gamma spot-forming cells (SFC) in PBMC from Ty21a vaccinees using an ex vivo system. Significant increases in the frequency of IFN-gamma SFC following immunization (mean +/- SD, 393 +/- 172; range 185-548 SFC/10(6) PBMC; p = 0.010), as compared with preimmunization levels, were observed. IFN-gamma was secreted predominantly by CD8(+) T cells. A strong correlation was recorded between the cytolytic activity of CTL lines and the frequency of IFN-gamma SFC (r(2) = 0.910, p < 0.001). In conclusion, this work constitutes the first evidence that immunization of volunteers with Ty21a elicits specific CD8(+) CTL and provides an estimate of the frequency of CD8(+) IFN-gamma-secreting cells induced by vaccination.     

Thrombin regulates the function of human blood dendritic cells

Thrombin is the key enzyme in the coagulation cascade and activates endothelial cells, neutrophils and monocytes via protease-activated receptors (PARs). At the inflammatory site, immune cells have an opportunity to encounter thrombin. However little is known about the effect of thrombin for dendritic cells (DC), which are efficient antigen-presenting cells and play important roles in initiating and regulating immune responses. The present study revealed that thrombin has the ability to stimulate blood DC. Plasmacytoid DC (PDC) and myeloid DC (MDC) isolated from PBMC expressed PAR-1 and released MCP-1, IL-10, and IL-12 after thrombin stimulation. Unlike blood DC, monocyte-derived DC (MoDC), differentiated in vitro did not express PAR-1 and were unresponsive to thrombin. Effects of thrombin on blood DC were significantly diminished by the addition of anti-PAR-1 Ab or hirudin, serine protease inhibitor. Moreover, thrombin induced HLA-DR and CD86 expression on DC and the thrombin-treated DC induced allogenic T cell proliferation. These findings indicate that thrombin plays a role in the regulation of blood DC functions.     

Interaction of rotavirus with human myeloid dendritic cells

We have previously shown that very few rotavirus (RV)-specific T cells that secrete gamma interferon circulate in recently infected and seropositive adults and children. Here, we have studied the interaction of RV with myeloid immature (IDC) and mature dendritic cells (MDC) in vitro. RV did not induce cell death of IDC or MDC and induced maturation of between 12 and 48% of IDC. Nonetheless, RV did not inhibit the maturation of IDC or change the expression of maturation markers on MDC. After treatment with RV, few IDC expressed the nonstructural viral protein NSP4. In contrast, a discrete productive viral infection was shown in MDC of a subset of volunteers, and between 3 and 46% of these cells expressed NSP4. RV-treated IDC secreted interleukin 6 (IL-6) (but not IL-1beta, IL-8, IL-10, IL-12, tumor necrosis factor alpha, or transforming growth factor beta), and MDC released IL-6 and small amounts of IL-10 and IL-12p70. The patterns of cytokines secreted by T cells stimulated by staphylococcal enterotoxin B presented by MDC infected with RV or uninfected were comparable. The frequencies and patterns of cytokines secreted by memory RV-specific T cells evidenced after stimulation of peripheral blood mononuclear cells (PBMC) with RV were similar to those evidenced after stimulation of PBMC with RV-infected MDC. Finally, IDC treated with RV strongly stimulated naive allogeneic CD4+ T cells to secrete Th1 cytokines. Thus, although RV does not seem to be a strong maturing stimulus for DC, it promotes their capacity to prime Th1 cells.     

Expression of the T cell receptor αβ on a CD123+ BDCA2+ HLA-DR+ subpopulation in head and neck squamous cell carcinoma

Human Plasmacytoid Dendritic Cells (PDCs) infiltrating solid tumor tissues and draining lymph nodes of Head and Neck Squamous Cell Carcinoma (HNSCC) show an impaired immune response. In addition to an attenuated secretion of IFN-α little is known about other HNSCC-induced functional alterations in PDCs. Particular objectives in this project were to gain new insights regarding tumor-induced phenotypical and functional alterations in the PDC population. We showed by FACS analysis and RT-PCR that HNSCC orchestrates an as yet unknown subpopulation exhibiting functional autonomy in-vitro and in-vivo besides bearing phenotypical resemblance to PDCs and T cells. A subset, positive for the PDC markers CD123, BDCA-2, HLA-DR and the T cell receptor αβ (TCR-αβ) was significantly induced subsequent to stimulation with HNSCC in-vitro (p = 0.009) and also present in metastatic lymph nodes in-vivo. This subgroup could be functionally distinguished due to an enhanced production of IL-2 (p = 0.02), IL-6 (p = 0.0007) and TGF-β (not significant). Furthermore, after exposure to HNSCC cells, mRNA levels revealed a D-J-beta rearrangement of the TCR-beta chain besides a strong enhancement of the CD3ε chain in the PDC population. Our data indicate an interface between the PDC and T cell lineage. These findings will improve our understanding of phenotypical and functional intricacies concerning the very heterogeneous PDC population in-vivo.     

Reduced ecto-5'-nucleotidase activity and enhanced OKT10 and HLA-DR expression on CD8 (T suppressor/cytotoxic) lymphocytes in the acquired immune deficiency syndrome: evidence of CD8 cell immaturity

Markedly reduced ecto-5'-nucleotidase activity was found in peripheral blood lymphocytes from 27 out of 30 homosexual men with the acquired immune deficiency syndrome (AIDS) in association with Kaposi's sarcoma (AIDS-KS; 2.67 +/- 1.70 U/10(6) cells; n = 13), opportunistic infections (AIDS-OI; 9.29 +/- 7.32; n = 7), or the AIDS-related complex (ARC; 9.82 +/- 6.12; n = 10). These values were significantly different from healthy controls (22.70 +/- 4.58; p less than 0.001). In AIDS-KS patients, both T cells and non-T cells exhibited significantly reduced ecto-5'-NT activity (p less than 0.001). AIDS-KS CD8 cells contained 20% of the mean ecto-5'-NT activity (7.04 +/- 3.53) displayed by control CD8 cells (34.07 +/- 4.86; p less than 0.001). No significant difference in enzyme level was observed between control and AIDS-KS CD4 cells (11.93 +/- 4.98 vs 7.98 +/- 3.28, respectively). In AIDS patients, lymphocyte ecto-5'-NT activity was inversely related (r = -0.518; p less than 0.01) to the absolute number of OKT10+ cells, but no correlation was found with the number of HLA-DR+ cells (r =-0.224). Two-color analysis of lymphocytes from AIDS-KS patients revealed that 75 +/- 12% of circulating CD8 cells expressed the OKT10 antigen, whereas only 10 +/- 6% of control CD8 cells did. HLA-DR antigens, which are not normally found on circulating resting T cells, were expressed in AIDS-KS CD8 cells, although to a lesser extent than OKT10. These data demonstrate that most AIDS CD8 cells differ from control CD8 cells. Although it has been suggested that these cells are activated cytotoxic or suppressor cells, the data presented here support the hypothesis they are immature. Reduced T cell ecto-5'-NT activity and enhanced expression of OKT10 and HLA-DR antigens on circulating CD8 cells, in conjunction with lack of transferrin receptor-(OKT9) and IL 2 receptor-(Tac) bearing lymphocytes, sustain this latter hypothesis. The correlation of the numerical reduction of CD4 cells with the reduced levels of ecto-5'-NT (r = 0.606; p less than 0.01) suggests that the abnormal maturation of CD8 cells seen in AIDS might be a consequence of the CD4 deficiency characteristic of this syndrome.