Measles virus infection in adults induces production of IL-10 and is associated with increased CD4+ CD25+ regulatory T cells

Despite steady progress in elimination of measles virus globally, measles infection still causes 500,000 annual deaths, mostly in developing countries where endemic measles strains still circulate. Many adults are infected every year in China, with symptoms more severe than those observed in children. In this study, we have used blood samples from adult measles patients in Shanghai and age-matched healthy controls to gain an understanding of the immune status of adult measles patients. IFN-alpha mRNA was reduced in patient PBMC compared with healthy controls. In contrast, gene expression and plasma production of IL-2, IL-10, and IFN-gamma were elevated in patient blood. A similar cytokine profile was observed at early times when cultured PBMC were infected with a clinical isolate of measles virus. In contrast to previous studies in pediatric patients, we did not find a reduction in total CD4(+) and CD8(+) T cells in patient PBMC. Interestingly, we found that CD4(+)CD25(+)CD127(low) regulatory T cells were significantly increased in patient PBMC compared with controls. Using intracellular cytokine staining we also show that the measles virus induces IL-10-producing CD14(+) and CD4(+)CD25(+) cells in PBMC. Our results show that adult measles patients in the acute phase of the disease have a mixed Th1/Th2 type response, accompanied with severe immunosuppression of both innate and adaptive responses including suppression of type I IFN. Both regulatory T cells and plasma IL-10 may contribute to the immunosuppression.     

Hepatitis C virus-specific Th17 cells are suppressed by virus-induced TGF-beta

IL-17-secreting T (Th17) cells play a protective role in certain bacterial infections, but they are major mediators of inflammation and are pathogenic in organ-specific autoimmune diseases. However, human Th17 cells appear to be resistant to suppression by CD4(+)CD25(+)FoxP3(+) regulatory T cells, suggesting that they may be regulated by alternative mechanisms. Herein we show that IL-10 and TGF-beta suppressed IL-17 production by anti-CD3-stimulated PBMC from normal individuals. TGF-beta also suppressed IL-17 production by purified CD4(+) T cells, whereas the inhibitory effect of IL-10 on IL-17 production appears to be mediated predominantly by its effect on APC. An examination of patients infected with hepatitis C virus (HCV) demonstrated that Ag-specific Th17 cells are induced during infection and that these cells are regulated by IL-10 and TGF-beta. PBMC from HCV Ab-positive donors secreted IL-17, IFN-gamma, IL-10, and TGF-beta in response to stimulation with the HCV nonstructural protein 4 (NS4). Furthermore, NS4 induced innate TGF-beta and IL-10 expression by monocytes from normal donors and at higher levels from HCV-infected patients. Neutralization of TGF-beta, and to a lesser extent IL-10, significantly enhanced NS4-specific IL-17 and IFN-gamma production by T cells from HCV-infected donors. Our findings suggest that both HCV-specific Th1 and Th17 cells are suppressed by NS4-induced production of the innate anti-inflammatory cytokines IL-10 and TGF-beta. This may represent a novel immune subversion mechanism by the virus to evade host-protective immune responses. Our findings also suggest that TGF-beta and IL-10 play important roles in constraining the function of Th17 cells in general.     

T lymphocyte cytokine profile at a single cell level in alveolar Echinococcosis.

The metacestode Echinococcus multilocularis causes a life-threatening disease in humans, named alveolar echinococcosis (AE). A comparative analysis of the early activation marker CD69 on peripheral blood mononuclear cells (PBMC) of patients with AE and healthy controls after in vitro culture with crude E. multilocularis antigen revealed that specific expression of CD69 was induced in CD4(+)T lymphocytes as well as in CD8(+)T lymphocytes. Using a protocol for intracellular staining of cytokines followed by fluorescence activating cell sorting (FACS) analysis, production of interleukin (IL)-2, IL-5 and IL-10 was detected in CD4(+)as well as in CD8(+)lymphocytes. Most notably, there was a definite increase in the expression of IL-10 in CD8(+) lymphocytes from patients with alveolar echinococcosis. The data support an important role of CD8(+) lymphocytes in the long persistence of the metacestode.     

Transgenic expression of Theiler's murine encephalomyelitis virus genes in H-2(b) mice inhibits resistance to virus-induced demyelination

We investigated the role of the immune system in protecting against virus-induced demyelination by generating lines of transgenic B10 (H-2(b)) congenic mice expressing three independent contiguous coding regions of the Theiler's murine encephalomyelitis virus (TMEV) under the control of a class I major histocompatibility complex (MHC) promoter. TMEV infection of normally resistant B10 mice results in virus clearance and development of inflammatory demyelination in the spinal cord. Transgenic expression of the viral capsid genes resulted in inactivation of virus-specific CD8(+) T lymphocytes (class I MHC immune function) directed against the relevant peptides, but it did not affect production of virus capsid-specific antibodies or lymphocyte proliferation to the virus antigen (class II MHC immune functions). Following intracerebral infection with TMEV, all three lines of mice survived the acute encephalitis but transgenic mice expressing VP1 (or the cluster of virus capsid proteins [VP4, VP2, and VP3] mapping to the left of VP1 in the TMEV genome) developed virus persistence and subsequent demyelination in spinal cord white matter. Transgenic mice expressing noncapsid proteins mapping to the right of VP1 (2A, 2B, 2C, 3A, 3B, 3C, and 3D) cleared the virus and did not develop demyelination. These results are consistent with the hypothesis that virus capsid gene products of TMEV stimulate class I-restricted CD8(+) T-cell immune responses, which are important for virus clearance and for protection against myelin destruction. Presented within the context of self-antigens, inactivation of these cells by ubiquitous expression of relevant virus capsid peptides partially inhibited resistance to virus-induced demyelination.     

Distinctive response of thyroid-infiltrating mononuclear cells to B cell activation through CD40 and interleukin-4 in Graves' patients

The possible role of abnormal T cell-dependent B-cell activation in Graves' disease was investigated by comparing lymphocyte subset distribution and the production of soluble CD8 (sCD8), sCD23, IL-10 and IL-12 by peripheral blood cells (PBMC) and thyroid-infiltrating lymphocytes (TL) in vitro. In TL, the percentage of CD8(+) cells was slightly higher and the sCD8 concentration was significantly higher than in PBMC. The ratio CD23(+) cells to CD20(+) cells (activated B/pan B cells) was increased in TL compared to PBMC from Graves' or normal controls, although the percentage of CD20(+) cells was decreased. Compared to PBMC in Graves' disease, the relative ratio of IL-10 to IL-12 release (IL-10/IL-12) by unstimulated TL was increased, despite a lack of significant difference between PBMC and TL in mean values for either IL-10 or IL-12 secretion. Incubating PBMC with a combination of anti-CD40 monoclonal antibodies and interleukin-4 (IL-4) resulted in B cell activation, reflected in an increase in the sCD23 level in both controls and Graves' patients, but especially prominent in the latter. Stimulation with anti-CD40 antibody and IL-4 also decreased the percentage of CD8(+) cells in PBMC but not TL from both Graves' disease and normal controls, and the percentage of CD8(+) cells in TL was higher than PBMC after the stimulation. The sCD23 concentration in TL was decreased compared to PBMC both in patients with Graves' disease and normal controls. However, in contrast to the increased responses observed in Graves' PBMC or normal controls after stimulation, sCD23 levels remained the same in stimulated TL from Graves' patients. This combination of B cell stimulants increased production of IL-10 in PBMC but not in TL obtained from patients with Graves' disease, and the increased IL-10/IL-12 ratio declined to a value no different from that in PBMC group after stimulation. Thus, T cell-dependent B-cell activation via a CD40 pathway may cause a shift in the Th(1)/Th(2) balance to Th(2) dominance in Graves' disease, while increased CD8(+) cells in TL may suppress sCD23 production and IL-10-producing Th(2) cells.     

Comparison of adjuvant efficacy of herpes simplex virus type 1 recombinant viruses expressing TH1 and TH2 cytokine genes

The adjuvant effects of cytokines in humoral and cell-mediated immunity to herpes simplex virus type 1 (HSV-1) have been examined in mice using HSV-1 recombinant viruses expressing murine interleukin-2 (IL-2), IL-4, or gamma interferon (IFN-gamma) gene. Groups of naive BALB/c mice were immunized intraperitoneally with one or three doses of the HSV-1 recombinant viruses expressing IL-2, IL-4, or IFN-gamma or with parental control virus. Despite similar replication kinetics, these three recombinant viruses elicited different immune responses to HSV-1 on immunization. Immunization with the recombinant virus expressing IL-4 elicited a humoral response of greater magnitude than immunization with the recombinant viruses expressing IL-2 or IFN-gamma or with parental virus. In contrast, immunization with recombinant virus expressing IL-2 elicited a higher cytotoxic T-cell response than immunization with viruses expressing IL-4 or IFN-gamma. Stimulation in vitro of splenocytes obtained from the mice immunized with UV-inactivated HSV-1 McKrae resulted in a T(H)1 pattern of cytokine expression irrespective of the recombinant virus used in the immunization. As observed for the parental virus, both CD4(+) and CD8(+) T cells contributed equally to the production of IL-2 by the splenocytes of mice immunized with any of the three recombinant viruses. However, the pattern of IFN-gamma production by CD4(+) and CD8(+) T cells differed according to the recombinant virus used. After lethal ocular challenge, all immunized mice were protected completely against death and manifestations of eye disease caused by HSV-1, which are typical responses in unimmunized mice. Mice immunized with IL-4-expressing virus cleared the virus from their eyes more rapidly than mice immunized with IL-2- or IFN-gamma-expressing virus. Taken together, our results suggest that, in contrast to IFN-gamma which did not exhibit an adjuvant effect, both IL-4 and IL-2 act as adjuvants in immunization with HSV, with IL-4 showing greater efficacy.     

Two distinct T cell subsets, CD4+ and CD8+CD60+, and their cytokines are required for in vitro induction of human ragweed-specific memory IgE responses

CD8(+)CD60(+) T cells (80-98% CD45RO(+); 20% CD23(+)) are significantly increased in the blood of serum IgE(+) ragweed-sensitized (RS) compared with serum IgE-nonatopic humans (p = 0.001). CD8(+)CD60(+) T cells of the RS patients produced IL-2, IL-4, IL-10, IL-12, IFN-alpha. and IFN-gamma, but not IL-6 or IL-13. When their PBMC were cultured with ragweed Ag (RA), peak IgE responses occurred on day 10; none was induced with non-cross-reacting or without Ag; nonatopic PBMC did not respond to any stimulant. When either CD4(+) or CD8(+)CD60(+) T cells were depleted from RS PBMC before culture with RA, no IgE responses were induced. If purified CD4(+) T cells or low numbers of CD8(+)CD60(+) T cells were added back to the depleted PBMC, IgE responses were restored. However, higher numbers of CD8(+)CD60(+) T cells totally suppressed IgE responses. Total suppression also was obtained when RS PBMC were cultured with RA and either anti-IL-2, IL-4, IL-10, IL-12, IFN-gamma (all concentrations), or IFN-alpha (low concentrations), but not anti-IL-6 or IL-13. Higher concentrations of anti-IFN-alpha potentiated IgE responses.     

Flow-cytometric analysis of cytokine production in human paracoccidioidomycosis

Human infection with Paracoccidioides brasiliensis may result in three major outcomes: paracoccidioidomycosis-infection (PI), which is observed in healthy carriers living in endemic areas and the adult form (AF) and juvenile form (JF) of the disease. In this study we proposed to examine the intracellular expression of IFN-gamma, TNF-alpha, IL-2, IL-10, IL-12, CXCL8, CXCL9 and CXCL10 by peripheral blood mononuclear cells (PBMC) of patients with the JF and AF of the disease, as well as of PI individuals stimulated with PMA plus ionomycin, LPS or anti-CD3 plus anti-CD28, by flow cytometry. The results showed that PI individuals present a higher percentage of cells producing IFN-gamma, TNF-alpha, IL-2, CXCL9 and CXCL10 when compared to AF and JF patients. IFN-gamma was predominantly detected in CD3(+)CD8(+) T cells, whereas IL-2 and TNF-alpha were mainly expressed in CD3(+)CD4(+) cells. Monocytes of PI individuals also presented higher expression of CD80 and lower expression of CD86 when compared to JF and AF patients, and higher expression of HLA-DR, only when compared to JF patients. These results indicate that the differential production of cytokines and chemokines, as well as the expression of co-stimulatory molecules involved in antigen presentation, may influence the outcome of PCM infection.     

Induction of Tc2 cells with specificity for prostate-specific antigen from patients with hormone-refractory prostate cancer

Prostate-specific antigen (PSA) is a potentially useful antigen for targeted T-cell immunotherapy of prostate cancer (CaP). Our laboratory has identified a synthetic nonamer peptide (PSA 146-154) homologue of PSA, which binds to the prevalent human leukocyte antigen, HLA-A2, and elicits specific cytotoxic T-lymphocyte (CTL) responses from normal individuals of the HLA-A2 phenotype. In the present study, we report on the induction of CTL from peripheral blood mononuclear cells (PBMC) of patients with hormone-refractory CaP, which exhibit the same specificity. T-cell lines were established from two patients by stimulation of PBMC with PSA 146-154 peptide in vitro. The T-cell lines exhibited specific cytolytic activity against T2 cells pulsed with PSA 146-154 peptide, but not a control HLA-A2 binding peptide (HIV-RT 476-484) via chromium release assay (CRA). The T-cell lines also showed PSA 146-154 peptide-specific IL-4 responses, but no detectable interferon-gamma (IFN-gamma) responses via enzyme-linked immuno-spot assays. Magnetic immuno-selection studies of one of the T-cell lines demonstrated that both cytolytic and interleukin-4 (IL-4) responses were mediated by CD8(+), but not by CD4(+) T cells. This Tc2 line was further characterized for the ability to recognize endogenously processed PSA epitopes. The line specifically secreted IL-4 in response to HLA-A2(+) target cells transfected to express PSA and specifically lysed the PSA(+) target cells, but not control transfected cells. The results indicate that the PSA 146-154 peptide emulates a naturally processed and presented peptide epitope of PSA that is within the T-cell repertoire of HLA-A2(+)patients with CaP.     

Parallel expansion of human virus-specific FoxP3- effector memory and de novo-generated FoxP3+ regulatory CD8+ T cells upon antigen recognition in vitro

Although FoxP3 has been shown to be the most specific marker for regulatory CD4(+) T cells, its significance in the CD8(+) T cell population is not well understood. In this study, we show that the in vitro stimulation of human PBMC with hepatitis C virus or Flu virus-specific peptides gives rise to two distinct Ag-specific T cell populations: FoxP3(-) and FoxP3(+)CD8(+) T cells. The FoxP3(+) virus-specific CD8(+) T cells share phenotypical markers of regulatory T cells, such as CTLA-4 and glucocorticoid-induced TNFR family-related gene, and do produce moderate amounts of IFN-gamma but not IL-2 or IL-10. IL-2 and IL-10 are critical cytokines, however, because the expansion of virus-specific FoxP3(+)CD8(+) T cells is blocked by IL-2- or IL-10-neutralizing mAbs. The virus-specific FoxP3(+)CD8(+) T cells have a reduced proliferative capacity, indicating anergy, and display a cell-cell contact-dependent suppressive activity. Taken together, our results indicate that stimulation with a defined viral Ag leads to the expansion of two different cell populations: FoxP3(-) memory/effector as well as FoxP3(+) regulatory virus-specific CD8(+) T cells.     

Regulatory T cells secreting IL-10 dominate the immune response to EBV latent membrane protein 1

Viruses exploit a number of strategies to evade immune recognition. In this study, we describe a novel mechanism by which EBV, rather than avoiding detection, subverts the immune response by stimulating regulatory T cells that secrete IL-10. Human PBMC from all EBV-seropositive, but not -seronegative, donors responded to both purified latent membrane protein 1 and the corresponding immunodominant peptides with high levels of IL-10 secretion by CD4(+) T cells. These IL-10 responses, characteristic of T regulatory 1 cells, inhibited T cell proliferation and IFN-gamma secretion induced by both mitogen and recall Ag. It was confirmed that the inhibition was IL-10 dependent by the use of neutralizing Ab. The deviation of the immune response toward suppression is likely to be important in maintaining latency and EBV-associated tumors.     

TH(1)- and TH(2)-TYPE cytokine expression by activated t lymphocytes from the lung and spleen during the inflammatory response to respiratory syncytial virus

RSV is an important cause of lower respiratory tract illness in infants and the elderly worldwide. The components involved in immunity and those that contribute to inflammation of RSV-induced disease are not clearly understood. To address the relationship between activation antigen and cytokine expression, intracellular levels of IL-2, IL-4, IL-5 and IFN-gamma were determined for CD3, CD44, CD49d, CD54, CD62L and CD102 lymphocytes from the bronchoalveolar lavage and spleen. To examine activation at the DNA level, lymphocytes expressing IL-2, IL-4, IL-5 or IFN-gamma were analysed for G2+M DNA content or phosphatidylserine expression (apoptosis). Trafficking of lymphocytes to the BAL was detected at day 5 p.i., peaked day 7 p.i., and predominately involved CD54(+)and CD102(+)lymphocytes expressing high levels of IL-2, IL-4, IL-5 and IFN-gamma. Lymphocytes expressing CD44(+), CD49d(+)and CD62L(lo)were also observed, however they expressed these cytokines to a lesser extent. DNA analysis of lymphocytes expressing IL-2 or IFN-gamma revealed higher G2'M levels compared to lymphocytes expressing IL-4 or IL-5, suggesting greater activation of Th(1)-type lymphocytes in the lung. These data demonstrate that RSV-induced pulmonary inflammation involves extensive cellular activation and cytokine expression, particularly by CD54(+)and CD102(+)lymphocytes in the lung.     

Phagocytosis, a potential mechanism for myeloid-derived suppressor cell regulation of CD8+ T cell function mediated through programmed cell death-1 and programmed cell death-1 ligand interaction

CD8(+) T cells become exhausted, inducing cell surface protein programmed cell death-1 (PD-1) as chronic virus diseases or tumors progress, but underlying mechanisms of this are unclear. We previously showed that M-CSF is important for developing tolerogenic dendritic cells (DCs) from human CD14(+) monocytes. In this article, we identify M-CSF-derived DCs (M-DCs) after stimulation with IL-10 as myeloid-derived suppressor cells with additional tolerogenic activities to CD8(+) T cells. IL-10 increased PD-1 ligand expression on M-DC, and IL-10-stimulated M-DCs (M-DC/IL-10) induced expression of PD-1 on, and apoptosis of, CD8(+) T cells and phagocytosed CD8(+) T cells. Enhanced phagocytic activity of M-DC/IL-10 required IFN-γ, which further increased PD-1 ligand and PD-2 ligand expression on M-DC/IL-10. IFN-γ-stimulated M-DC/IL-10 cells were phenotypically macrophage-like cells with little or no expression of CD86, a costimulatory molecule, but with high expression levels of CD14, CD200R, and CD80. No phagocytic activity was detected with GM-CSF-derived DCs. We propose that phagocytosis by IFN-γ-stimulated M-DC/IL-10 cells, which may be DCs or, alternatively, a unique subset of macrophages, may be a mechanism by which IFN-γ-producing CD8(+) T cells are tolerized after type 1 immune responses to chronic virus or tumor, and that IFN-γ links effector CD8(+) T cells to their phagocytic clearance.     

Multifunctional human immunodeficiency virus (HIV) gag-specific CD8+ T-cell responses in rectal mucosa and peripheral blood mononuclear cells during chronic HIV type 1 infection

The intestinal tract is a lymphocyte-rich site that undergoes severe depletion of memory CD4(+) T cells within days of simian immunodeficiency virus or human immunodeficiency virus type 1 (HIV-1) infection. An ensuing influx of virus-specific CD8(+) T cells, which persist throughout the chronic phase of infection, has also been documented in the gastrointestinal tract. However, little is known of the functionality of these effector cells or their relationship to the disease course. In this study, we measured CD8(+) T-cell responses to HIV-1 peptides in paired rectal and blood samples from chronically infected patients. In both blood and rectum, there was an immunodominant CD8(+) T-cell response to HIV Gag compared to Pol and Env (P < 0.01). In contrast, cytomegalovirus pp65 peptides elicited gamma interferon (IFN-gamma) secretion strongly in peripheral blood mononuclear cells (PBMC) but weakly in rectal CD8(+) T cells (P = 0.015). Upon stimulation with HIV peptides, CD8(+) T cells from both sites were capable of mounting complex responses including degranulation (CD107 expression) and IFN-gamma and tumor necrosis factor alpha (TNF-alpha) production. In rectal tissue, CD107 release was frequently coupled with production of IFN-gamma or TNF-alpha. In patients not on antiretroviral therapy, the magnitude of Gag-specific responses, as a percentage of CD8(+) T cells, was greater in the rectal mucosa than in PBMC (P = 0.054); however, the breakdown of responding cells into specific functional categories was similar in both sites. These findings demonstrate that rectal CD8(+) T cells are capable of robust and varied HIV-1-specific responses and therefore likely play an active role in eliminating infected cells during chronic infection.     

Cross-reactivity of T lymphocytes recognizing a human cytotoxic T-lymphocyte epitope within BK and JC virus VP1 polypeptides

A transgenic mouse model was used to identify an HLA-A*02-restricted epitope within the VP1 polypeptide of a human polyomavirus, BK virus (BKV), which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Peptide stimulation of splenocytes from mice immunized with recombinant modified vaccinia virus Ankara expressing BKV VP1 resulted in expansion of cytotoxic T lymphocytes (CTLs) recognizing the sequence LLMWEAVTV corresponding to amino acid residues 108 to 116 (BKV VP1p108). These effector T-cell populations represented functional CTLs as assessed by cytotoxicity and cytokine production and were cross-reactive against antigen-presenting cells pulsed with a peptide corresponding to the previously described JC virus (JCV) VP1 homolog sequence ILMWEAVTL (JCV VP1p100) (I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). A panel of 10 healthy HLA-A*02 human volunteers and two kidney transplant recipients were screened for T-cell immunity to this BK virus VP1 epitope by in vitro stimulation of their peripheral blood mononuclear cells (PBMC) with the BKV VP1p108 peptide, followed by tetramer labeling combined with simultaneous assays to detect intracellular cytokine production and degranulation. PBMC from 4/10 subjects harbored CTL populations that recognized both the BKV VP1p108 and the JCV VP1p100 peptides with comparable efficiencies as measured by tetramer binding, gamma interferon production, and degranulation. CTL responses to the JCV VP1p100 epitope have been associated with prolonged survival in progressive multifocal leukoencephalopathy patients (R. A. Du Pasquier et al., Brain 127:1970-1978, 2004; I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). Given that both human polyomaviruses are resident in a high proportion of healthy individuals and that coinfection occurs (W. A. Knowles et al., J. Med. Virol. 71:115-123, 2003), our findings suggest a reinterpretation of this protective T-cell immunity, suggesting that the same VP1 epitope is recognized in HLA-A*02 persons in response to either BK or JC virus infection.     

Interferon-gamma secretion and perforin expression are impaired in CD8+ T lymphocytes from patients with undifferentiated carcinoma of nasopharyngeal type

An efficent antitumor and antiviral cellular immune response requires optimal interferon-gamma (IFN-gamma) secretion and perforin expression in CD8(+) T cells. The aim of this study was to define whether CD4(+) and CD8(+) T cells from patients with undifferentiated carcinoma of nasopharyngeal type (UCNT), a tumor regularly associated with the Epstein-Barr virus (EBV), have abnormal phenotype profiles, cytokine production, perforin and CD3-zeta expressions. Our data showed that CD4 and CD8 subset distribution was not grossly altered in the peripheral blood of UCNT patients, while tumor biopsies contained an increased proportion of CD8(+) T cells. The analysis of the CD4(+) subset showed a defect in interleukin-2 (IL-2) production and a moderate increase of IL-10 production, a situation consistent with a Th1/Th2 imbalance. We have also demonstrated that CD8(+) lymphocytes from UCNT patients had a marked impairment of IFN-gamma secretion and perforin expression. This impairment was not related to the presence of detectable EBV DNA in the plasma. In UCNT patients, the blockade of the perforin pathway and of IFN-gamma production may constitute important mechanisms for immune escape by the tumor and for impaired control of EBV replication.     

Induction of CD83+CD14+ nondendritic antigen-presenting cells by exposure of monocytes to IFN-alpha

IFN-alpha is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-alpha effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-alpha levels during treatment of infections and cancers. We show that in vitro IFN-alpha exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-alpha induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-alpha concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-alpha-challenged CD83(+) cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-alpha-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-alpha-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4(+) T cells. Notably, autologous memory CD4(+) T cells proliferated when exposed to tetanus toxoid-pulsed IFN-alpha-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-alpha showed long-lasting STAT-1 phosphorylation. Remarkably, CD83(+)CD14(+) cells were present in varicella skin lesions in close contact with IFN-alpha-producing cells. The present findings suggest that IFN-alpha alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-alpha in vivo.     

Human immunodeficiency virus type 1 gp120 induces anergy in human peripheral blood lymphocytes by inducing interleukin-10 production.

The effects of recombinant gp120 on the proliferative responses and cytokine production by normal peripheral blood mononuclear cells (PBMC) were investigated. gp120 inhibited in a dose-dependent fashion the anti-CD3 monoclonal antibody (MAb)- and concanavalin A-induced proliferative responses. The production of interleukin-2 (IL-2) and IL-4 was diminished by gp120 in the anti-CD3- and concanavalin A-stimulated cultures. In unstimulated PBMC, gp120 induced the production of considerable amounts of IL-10, gamma interferon, and tumor necrosis factor alpha. The gp120-induced reduction in the proliferative responses of PBMC was at least partially reversed by the addition of IL-2, anti-CD28 MAb, or transfectants expressing CD80, CD86, or CD40 but not with exogenous IL-4. Also, a neutralizing anti-IL-10 MAb reversed the inhibitory effect of gp120 on the proliferative responses whereas exogenous IL-10 further enhanced this inhibitory effect. These findings indicate that IL-10 plays an important role in the inhibitory effect of gp120 on PBMC proliferation. The ratio of CD3+CD4+ to CD3+CD8+ T cells was the same in gp120-treated and untreated cell cultures. No apoptosis in these two T-cell populations was observed. However, the number of activated CD3+CD4+ T cells and CD3+CD8+ T cells, as judged by CD25, CD69, and HLA-DR expression, was consistently reduced. gp120 induced the expression of IL-10 in the monocyte/macrophage population, and therefore gp120 also reduced the proliferative responses of CD4+ T-cell-depleted PBMC. Taken together, our observations point to the importance of the cytokine pattern changes and, in particular, the role of IL-10 (produced by the monocytes) in the inhibitory effect of gp120. This mechanism of gp120-induced immunosuppression, if operative in vivo, could contribute to the depressed immune responses associated with human immunodeficiency virus infection and thus have important implications for immunotherapeutic strategies to slow down disease progression in AIDS.     

Newcastle disease virus expressing a dendritic cell-targeted HIV gag protein induces a potent gag-specific immune response in mice

Viral vaccine vectors have emerged as an attractive strategy for the development of a human immunodeficiency virus (HIV) vaccine. Recombinant Newcastle disease virus (rNDV) stands out as a vaccine vector since it has a proven safety profile in humans, it is a potent inducer of both alpha interferon (IFN-α) and IFN-β) production, and it is a potent inducer of dendritic cell (DC) maturation. Our group has previously generated an rNDV vector expressing a codon-optimized HIV Gag protein and demonstrated its ability to induce a Gag-specific CD8(+) T cell response in mice. In this report we demonstrate that the Gag-specific immune response can be further enhanced by the targeting of the rNDV-encoded HIV Gag antigen to DCs. Targeting of the HIV Gag antigen was achieved by the addition of a single-chain Fv (scFv) antibody specific for the DC-restricted antigen uptake receptor DEC205 such that the DEC205 scFv-Gag molecule was encoded for expression as a fusion protein. The vaccination of mice with rNDV coding for the DC-targeted Gag antigen induced an enhanced Gag-specific CD8(+) T cell response and enhanced numbers of CD4(+) T cells and CD8(+) T cells in the spleen relative to vaccination with rNDV coding for a nontargeted Gag antigen. Importantly, mice vaccinated with the DEC205-targeted vaccine were better protected from challenge with a recombinant vaccinia virus expressing the HIV Gag protein. Here we demonstrate that the targeting of the HIV Gag antigen to DCs via the DEC205 receptor enhances the ability of an rNDV vector to induce a potent antigen-specific immune response.     

Identification of Mycobacterium tuberculosis-specific Th1, Th17 and Th22 cells using the expression of CD40L in tuberculous pleurisy

Important advances have been made in the immunodiagnosis of tuberculosis (TB) based on the detection of Mycobacterium tuberculosis (MTB)-specific T cells. However, the sensitivity and specificity of the immunological approach are relatively low because there are no specific markers for antigen-specific Th cells, and some of the Th cells that do not produce cytokines can be overlooked using this approach. In this study, we found that MTB-specific peptides of ESAT-6/CFP-10 can stimulate the expression of CD40L specifically in CD4(+) T cells but not other cells from pleural fluid cells (PFCs) in patients with tuberculous pleurisy (TBP). CD4(+)CD40L(+) but not CD4(+)CD40L(-) T cells express IFN-γ, IL-2, TNF-α, IL-17 or IL-22 after stimulation with MTB-specific peptides. In addition, CD4(+)CD40L(+) T cells were found to be mostly polyfunctional T cells that simultaneously produce IFN-γ, IL-2 and TNF-α and display an effector or effector memory phenotype (CD45RA(-)CD45RO(+)CCR7(-)CD62L(-)ICOS(-)). To determine the specificity of CD4(+)CD40L(+) T cells, we incubated PFCs with ESTA-6/CFP-10 peptides and sorted live CD4(+)CD40L(+) and CD4(+)CD40L(-) T cells by flow cytometry. We further demonstrated that sorted CD4(+)CD40L(+), but not CD4(+)CD40L(-) fractions, principally produced IFN-γ, IL-2, TNF-α, IL-17 and IL-22 following restimulation with ESTA-6/CFP-10 peptides. Taken together, our data indicate that the expression of CD40L on MTB-specific CD4(+) T cells could be a good marker for the evaluation and isolation of MTB-specific Th cells and might also be useful in the diagnosis of TB.