Increase in IL-17-positive cells in sinonasal inverted papilloma.

OBJECTIVE: Neutrophil infiltration in patients with sinonasal inverted papilloma (SNIP) is significantly high. Whether IL-17, which is a potent factor mediating neutrophilic inflammation, is involved in the neutrophilic phenotype of SNIP is investigated in the current study. STUDY DESIGN: Laboratorial study. PARTICIPANTS: Nasal papilloma and inferior turbinate were collected from patients with SNIP (n = 50) and control subjects with septal deviation (n = 15). METHODS: IL-17 + cells were evaluated in tissues obtained from patients with SNIP and control subjects with septal deviation, by immunohistochemistry and flow cytometry. MAIN OUTCOME MEASURES: The IL-17 + cells were mainly localised in mononuclear cells and neutrophils, and were up-regulated in the SNIP samples compared with those in the controls. The IL-17 + T-cell subsets mainly included CD4+ (Th17, 60.0%) and CD8+ (Tc17, 30.0%), and both subsets were enhanced in the SNIP samples than controls. The total level of IL-17 + cells was significantly correlated with neutrophil infiltration in the SNIP tissues. Furthermore, the SNIP homogenates could significantly promote IL-17 production in peripheral blood mononuclear cells. CONCLUSIONS: An increase in IL-17 + cells is evident in SNIP and may be involved in neutrophil infiltration in local tissues. IL-17 could be a potential therapeutic target to relieve the neutrophilic pathological change in SNIP.

A Novel Hypothesis on Excessive Activation of Residual B Lymphocytes in Common Variable Immunodeficiency Concurrent with Aseptic, Erosive Polyarthritis.

The aim of this study was to report aseptic, erosive polyarthritis in a patient with common variable immunodeficiency (CVID), which is quite different from the vastly more common nonerosive form. Peripheral blood mononuclear cells of the patient were isolated. Flow cytometry was used to analyze the proportion and function of lymphocytes. A Parker-Pearson needle biopsy was performed on the right knee. Four of her unaffected family members were enrolled as controls. A 21-year-old woman was admitted for recurrent polyarthritis of 3-year duration. The right knee, hip, wrist, proximal interphalangeal joints, and left elbow were involved, with progressive joint destruction. She was diagnosed as having CVID based on her recurrent infections, poor response to vaccines, and marked hypogammaglobulinemia. No bacterium or mycobacterium was detected in synovium or synovial fluid. The synovium was infiltrated by lymphocytes rather than neutrophils. Polyarthritis did not resolve by adequate intravenous immunoglobulin substitution and empirical antibiotic treatment, but resolved gradually after treatment with methylprednisolone and tacrolimus, supporting the diagnosis of aseptic polyarthritis. Further analyses showed that although only 0.5% of residual B lymphocytes were existent in peripheral blood of the patient, expressions of activation marker CD69 and production of IL-1ß, IL-6, and TNF-α were high. Marked infiltration with CD19+B lymphocytes (as well as CD4+ or CD8+ T lymphocytes) was detected in the synovium. The proportion of IL21+CD4+Th cells from peripheral blood of the patient was high. CD4+ Th cells from the patient secreted nearly 3 times more IL-21 than the same cell type analyzed from unaffected family members, perhaps due to excessive compensation to assist the function of residual B lymphocytes. A novel hypothesis in CVID concurrent with aseptic, erosive polyarthritis is that excessive activation of residual B lymphocytes infiltrate into the synovium of the involved joints and lead to polyarthritis and joint destruction.

Human memory, but not naive, CD4+ T cells expressing transcription factor T-bet might drive rapid cytokine production.

We found that after stimulation for a few hours, memory but not naive CD4(+) T cells produced a large amount of IFN-γ; however, the mechanism of rapid response of memory CD4(+) T cells remains undefined. We compared the expression of transcription factors in resting or activated naive and memory CD4(+) T cells and found that T-bet, but not pSTAT-1 or pSTAT-4, was highly expressed in resting memory CD4(+) T cells and that phenotypic characteristics of T-bet(+)CD4(+) T cells were CD45RA(low)CD62L(low) CCR7(low). After short-term stimulation, purified memory CD4(+) T cells rapidly produced effector cytokines that were closely associated with the pre-existence of T-bet. By contrast, resting naive CD4(+) T cells did not express T-bet, and they produced cytokines only after sustained stimulation. Our further studies indicated that T-bet was expressed in the nuclei of resting memory CD4(+) T cells, which might have important implications for rapid IFN-γ production. Our results indicate that the pre-existence and nuclear mobilization of T-bet in resting memory CD4(+) T cells might be a possible transcriptional mechanism for rapid production of cytokines by human memory CD4(+) T cells.

PD-1+CXCR5-CD4+ Th-CXCL13 cell subset drives B cells into tertiary lymphoid structures of nasopharyngeal carcinoma.

BACKGROUND: A major current challenge is to exploit tertiary lymphoid structures (TLSs) to promote the lymphocyte infiltration, activation and differentiation by tumor antigens to increase antitumor immune responses. The mechanisms that underlie the role of TLS formation in the adaptive immune responses against nasopharyngeal carcinoma (NPC) remain largely unknown. METHODS: Cell populations and the corresponding markers were identified by single-cell RNA sequencing and fluorescence-activated cell sorting analysis. In vitro differentiation experiments were used to simulate the generation, regulation and function of the Th-CXCL13 cell subset in the tumor microenvironment of NPC. These were followed by histological evaluation of the colocalization of tumor-associated B cells (TABs) and Th-CXCL13 cells within TLSs, and statistical analysis of the relationship between the cells in TLSs and overall survival. RESULTS: A PD-1+CXCR5-CD4+ Th-CXCL13 cell subset was identified in NPC. This subset was a major source of CXCL13, representing the majority of the CD4+ T cells at levels comparable with Th1 and Tfh cells present in the TLSs. Monocytes activated by toll-like receptor 4 agonists served as the antigen-presenting cells that most efficiently triggered the expansion of Th-CXCL13 cells. Transforming growth factor beta 1 (TGF-ß1) stimulation and activation of Sox4 were critical for the induction and polarization of Th-CXCL13 cells in this process. The potential functional contributions of TABs recruited by Th-CXCL13 cells which induced plasma cell differentiation and immunoglobulin production via interleukin-21 and CD84 interactions in the TLSs demonstrated improved survival. CONCLUSIONS: Induction of Th-CXCL13 cells links innate inflammation to immune privilege in tumor-associated TLSs and might predict better survival.

Innate Resistance to Leishmania amazonensis Infection in Rat Is Dependent on NOS2.

Leishmania infection causes diverse clinical manifestations in humans. The disease outcome is complicated by the combination of many host and parasite factors. Inbred mouse strains vary in resistance to Leishmania major but are highly susceptible to Leishmania amazonensis infection. However, rats are highly resistant to L. amazonensis infection due to unknown mechanisms. We use the inducible nitric oxide synthase (Nos2) gene knockout rat model (Nos2 -/- rat) to investigate the role of NOS2 against leishmania infection in rats. Our results demonstrated that diversion toward the NOS2 pathway is the key factor explaining the resistance of rats against L. amazonensis infection. Rats deficient in NOS2 are susceptible to L. amazonensis infection even though their immune response to infection is still strong. Moreover, adoptive transfer of NOS2 competent macrophages into Nos2 -/- rats significantly reduced disease development and parasite load. Thus, we conclude that the distinct L-arginine metabolism, observed in rat macrophages, is the basis of the strong innate resistance to Leishmania. These data highlight that macrophages from different hosts possess distinctive properties and produce different outcomes in innate immunity to Leishmania infections.

Dendritic cells are responsible for the capacity of CpG oligodeoxynucleotides to act as an adjuvant for protective vaccine immunity against Leishmania major in mice.

Vaccination with leishmanial Ag and CpG oligodeoxynucleotides (ODN) confers sustained cellular immunity and protection to infectious challenge up to 6 mo after immunization. To define the cellular mechanism by which CpG ODN mediate their adjuvant effects in vivo, the functional capacity of distinct dendritic cell (DC) subsets was assessed in the lymph nodes (LNs) of BALB/c mice, 36 h after immunization with the leishmanial antigen (LACK) and CpG ODN. After this immunization, there was a striking decrease in the frequency of the CD11c+B220+ plasmacytoid DCs with a proportionate increase in CD11c+CD8-B220- cells. CD11c+CD8+B220- cells were the most potent producers of interleukin (IL)-12 p70 and interferon (IFN)-gamma, while plasmacytoid DCs were the only subset capable of secreting IFN-alpha. In terms of antigen presenting capacity, plasmacytoid DCs were far less efficient compared with the other DC subsets. To certify that DCs were responsible for effective vaccination, we isolated CD11c+ and CD11c- cells 36 h after immunization and used such cells to elicit protective immunity after adoptive transfer in naive, Leishmania major susceptible BALB/c mice. CD11c+ cells but not 10-fold higher numbers of CD11c- cells from such immunized mice mediated protection. Therefore, the combination of LACK antigen and CpG ODN adjuvant leads to the presence of CD11c+ DCs in the draining LN that are capable of vaccinating naive mice in the absence of further antigen or adjuvant.

Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against leishmania major infection.

CpG oligodeoxynucleotides (ODN) have potent effects on innate and adaptive cellular immune responses. In this report, the ability of CpG ODN to confer long-term immunity and protection when used as a vaccine adjuvant with a clinical grade of leishmanial antigen, autoclaved Leishmania major (ALM), or a recombinant leishmanial protein was studied. In two different mouse models of L. major infection, vaccination with ALM plus CpG ODN was able to control infection and markedly reduce lesion development in susceptible BALB/c and resistant C57BL/6 (B6) mice, respectively, up to 12 wk after immunization. Moreover, B6 mice immunized with ALM plus CpG ODNs were still protected against infectious challenge even 6 mo after vaccination. In terms of immune correlates of protection, ALM plus CpG ODN-vaccinated mice displayed L. major-specific T helper cell 1 and CD8+ responses. In addition, complete protection was markedly abrogated in mice depleted of CD8+ T cells at the time of vaccination. Similarly, mice vaccinated with a recombinant leishmanial protein plus CpG ODN also had long-term protection that was dependent on CD8+ T cells in vivo. Together, these data demonstrate that CpG ODN, when used as a vaccine adjuvant with either a recombinant protein or heat-killed leishmanial antigen, can induce long-term protection against an intracellular infection in a CD8-dependent manner.

CD4+ CD25+ Treg cells inhibit human memory gammadelta T cells to produce IFN-gamma in response to M tuberculosis antigen ESAT-6.

Gammadelta T cells play an important role in innate immunity against infections; however, the regulation of these cells remains largely unknown. In the present study, we show that ESAT-6, an antigen of Mycobacterium tuberculosis, induces IFN-gamma secretion by human gammadelta T cells. In addition, ESAT-6 also induces the activation and proliferation of gammadelta T cells. Phenotypic analysis indicates that IFN-gamma-producing gammadelta T cells are mainly effector memory cells with the surface phenotype of CD45RA(-)CD62L(-)CCR7(-). These results were further confirmed by the fact that naive gammadelta T cells from cord blood did not produce IFN-gamma in response to ESAT-6. Further studies indicated that stimulation with ESAT-6 directly induced purified gammadelta T cells to produce IFN-gamma, independent of both antigen-presenting cells and CD4(+) T cells. Unexpectedly, depletion of CD4(+) T cells markedly enhanced IFN-gamma production by gammadelta T cells, indicating that CD4(+) T cells regulate the response of gammadelta T cells. Importantly, CD4(+)CD25(+) T regulatory (Treg) cells but not CD4(+)CD25(-) T cells significantly inhibited IFN-gamma production by gammadelta T cells. Taken together, these data demonstrate for the first time that Treg cells can play an important role in the regulation of immune responses of antigen-specific human memory gammadelta T cells.

[Cytokine production and clonal analysis of memory γδ T cells from patients with tuberculous pleurisy induced by bacille calmette guerin].

OBJECTIVE: To evaluate cytokine production and expression of γδ T cells within pleural fluid cells (PFCs) from patients with tuberculous pleurisy following bacille calmette guerin (BCG) stimulation. METHODS: PFCs were isolated from patients with tuberculous pleurisy, and assessed for cytokine production, cell subpopulation, phenotype and characterization of T cell receptors after stimulation with BCG. The positive PCR products were further labeled with fluorescence and analyzed by genescan technique to determine the CDR3 size and evaluate the clonality of the detectable TCR Vγ and Vδ T cells. RESULTS: Following stimulation with BCG, the positivity of interferon-γ (IFN-γ)-producing CD(4) T cells and γδ T cells were 0.38% and 5.35%, respectively. Phenotypic analysis indicated that the majority of IFN-γ(+)γδ(+) T cells expressed CD(45RO)(+) (73.5%). In addition, δ(2) T cells produced IFN-γ (11.1%) and TNF-α (25.5%). After expansion with BCG for 3 weeks, cells were harvested and mRNA extracted and RT-PCR conducted to amplify cDNA with 3 primers for Vγ and 8 primers for Vδ. The results indicated that BCG selectively expanded δ(2) T cells with oligoclonal peak in Vδ(2) cells. CONCLUSIONS: BCG induced memory γδ and δ(2) T cells to produce cytokines in PFCs. Genescan analysis showed that Vδ(2) displayed oligoclonality.

Characterization of SARS-CoV-specific memory T cells from recovered individuals 4 years after infection.

SARS-CoV infection of human results in antigen-specific cellular and humoral immune responses. However, it is critical to determine whether SARS-CoV-specific memory T cells can persist for long periods of time. In this study, we analyzed the cellular immune response from 21 SARS-recovered individuals who had been diagnosed with SARS in 2003 by using ELISA, CBA, ELISpot and multiparameter flow cytometry assays. Our results demonstrated that low levels of specific memory T cell responses to SARS-CoV S, M, E and N peptides were detected in a proportion of SARS-recovered patients, and IFN-gamma was the predominant cytokine produced by T cells after stimulation with peptides. Cytometry analysis indicated that the majority of memory CD8(+) T cells produced IFN-gamma, whereas memory CD4(+) T cells produced IFN-gamma, IL-2 or TNF-alpha. These results might provide valuable information on the cellular immune response in recovered SARS-CoV patients for the rational design of vaccines against SARS-CoV infection.

Natural killer cell degeneration exacerbates experimental arthritis in mice via enhanced interleukin-17 production.

OBJECTIVE: An altered phenotype and dysfunction of natural killer (NK) cells have been observed in patients with rheumatoid arthritis. The aim of this study was to determine whether dysregulated NK cells contribute to the pathogenesis of experimental arthritis. METHODS: For initiation of collagen-induced arthritis (CIA), DBA/1J mice were immunized with type II collagen in Freund's adjuvant. Control mice were immunized with adjuvant alone. NK cells from the blood, spleens, and bone marrow of immunized mice were analyzed by flow cytometry. Levels of interleukin-17 (IL-17) secretion and autoantibody production were measured by enzyme-linked immunosorbent assays. Immunized mice in which NK cells were depleted by anti-asialo G(M1) antibody treatment were assessed for the development of CIA. Moreover, sorting-purified NK cells from both mice with CIA and control mice were analyzed for cytokine gene expression. RESULTS: We observed markedly reduced frequencies of NK cells in the blood and spleens of mice with CIA compared with the frequencies in adjuvant-treated control mice. Upon NK cell depletion, immunized mice displayed an early onset of arthritis with more severe clinical symptoms, which correlated with increased plasma cell generation and autoantibody production. Moreover, a substantially increased number of IL-17-secreting cells in synovial tissue and more pronounced joint damage were observed. Freshly isolated NK cells from mice with CIA showed markedly reduced expression of interferon-gamma (IFNgamma). Furthermore, coculture of normal NK cells and CD4+ T cells revealed that NK cells strongly suppressed production of Th17 cells via their IFNgamma production. CONCLUSION: These results suggest that NK cells play a protective role in the development of experimental arthritis, an effect that is possibly mediated by suppressing Th17 cell generation via IFNgamma production.

A selective phosphodiesterase 4 (PDE4) inhibitor Zl-n-91 suppresses IL-17 production by human memory Th17 cells.

Th17 cells are highly proinflammatory and involved in the immunopathogenesis of severe autoimmune diseases. Selective phosphodiesterase 4 (PDE4) inhibitors, which elevate intracellular cAMP by inhibiting the hydrolysis of cAMP, have been demonstrated to be an effective anti-inflammatory agent in airway inflammatory diseases. In the present study, we assessed the effect of a selective PDE4 inhibitor Zl-n-91 on IL-17 production by PBMCs and by purified CD4(+) T cells following stimulation. The results for the first time demonstrated that the addition of Zl-n-91 into cell cultures of PBMCs and purified CD4(+) T cells could result in the suppression of IL-17 production at the protein and mRNA levels. Further analysis indicated that Zl-n-91 had a direct inhibitory effect on the IL-17 production by memory Th17 cells via the suppression of activation, proliferation and division of CD4(+) T cells. Our data suggested that Zl-n-91 might have beneficial effects in the treatment of IL-17-related autoimmune diseases.

Phenotypic and functional heterogeneity of natural killer cells from umbilical cord blood mononuclear cells.

Natural killer cells (NK) from umbilical cord blood (CB) play an important role in allogeneic stem cell transplantation and defending infections of newborn. Based on the surface expression of CD56 and CD16 or inhibitory and activatory receptors, NK cells could be subdivided into various subsets with distinct functions. To investigate the biological characterization of NK subsets, the phenotypes and intracellular proteins in freshly isolated CB NK subsets were analyzed at the single cell level by flow cytometry in current study. The production of IFN-gamma and cytotoxicity against K562 target cells were also evaluated after stimulation with IL-12. The results showed that NK cells from CB could be divided into four subsets on the basis of CD56 and CD16 expression. Interestingly, CB NK cells expressed CD45RA but not CD45RO molecules that is similar to the naïve T cells. Moreover, CD27, a memory T cell marker, highly expressed on CD56(hi)CD16- NK cells. The killing-associated molecules, NKG2A, NKG2D, CD95 and the intracellular granzyme B and perforin were heterogeneously expressed among the 4 subsets. Addition of IL-12 into cultures resulted in the induction of IFN-gamma expression by CD56(hi)CD16- and CD56(lo)CD16- subsets and the enhancement of NK cytolytic activity. Taken together, this study elucidated the heterogeneity in phenotypes and biological functions of CB NK cells.

[The role of CD4+ CD25+ T cells in the mechanism of myasthenia gravis in children and adults].

OBJECTIVE: To investigate the role of CD4+CD25+ regulatory T cells in the mechanism of myasthenia gravis (MG) in children and adults. METHODS: Peripheral blood samples were collected from 13 MG patients, 5 being aged > 14 and 8 being aged

Tumor necrosis factor-alpha induces long-term potentiation of C-fiber evoked field potentials in spinal dorsal horn in rats with nerve injury: the role of NF-kappa B, JNK and p38 MAPK.

Compelling evidence has shown that in hippocampus tumor necrosis factor alpha (TNF-alpha) at pathological concentration inhibits long-term potentiation (LTP), a synaptic model of learning and memory. In the present work we investigated the role of TNF-alpha in LTP of C-fiber evoked field potentials in spinal dorsal horn, which is relevant to pathological pain. We showed that spinal application of TNF-alpha affected neither basal synaptic transmission mediated by C-fibers nor spinal LTP of C-fiber evoked field potentials induced by tetanic stimulation in intact rats. However, in rats with neuropathic pain, produced by either lumbar 5 ventral root transection (L5 VRT) or spared nerve injury (SNI), spinal application of TNF-alpha induced LTP of C-fiber evoked field potentials. Spinal application of JNK inhibitor (SP600125) or p38 MAPK inhibitor (SB203580) did not affect the spinal LTP induced by tetanic stimulation in intact rats, but completely blocked LTP induced by TNF-alpha in L5 VRT rats. NF-kappa B (NF-kappaB) inhibitor (PDTC) also blocked LTP induced by TNF-alpha. These results suggest that TNF-alpha and its downstream molecules may have no acute effect on spinal synaptic transmission in intact animals and induce LTP in rats with neuropathic pain produced by nerve injury.

Increased frequency of CD4(+)CD25(high) Treg cells inhibit BCG-specific induction of IFN-gamma by CD4(+) T cells from TB patients.

Cell-mediated immunity plays a considerable role in the protection against Mycobacterium tuberculosis infection. The immune response to tuberculosis (TB) was dominated by both CD4(+) T cells with the T helper 1 type cytokines and CD8(+) T cells. Recent studies have suggested that the circumstances in which protective or tissue-damaging T cell responses to microbes are affected by the activity of Treg (CD4(+)CD25(high)) cells. In the present study, we demonstrated that the frequencies of CD4(+)CD25(+) and CD4(+)CD25(high) T cells in TB patients were significantly higher compared to normal individuals. These Treg cells expressed CTLA-4 and Foxp3 at protein level and displayed activation and memory phenotypes as assessed by flow cytometric analysis. The frequencies of CD4(+)CD25(high)CTLA-4(+) and CD4(+)CD25(high)Foxp3(+) T cells within the total CD4(+) T cell population were significantly increased in the blood of TB patients compared to healthy donors. Moreover, the expression of GITR on Treg cells was higher in TB patients than in normal donors. The phenotypic analysis demonstrated that CD4(+)CD25(high) Treg expressed higher levels of CD45RO and HLA-DR, and lower levels of CD45RA compared to CD4(+)CD25(low) and CD4(+)CD25(-) T cells. The addition of CD4(+)CD25(high) T cells back to cultures could significantly suppress the antigen-specific production of IFN-gamma induced by BCG-stimulated CD4(+)CD25(-) T cells, suggesting that Treg might play a key role in the control of cellular immune responses in TB infection.

IL-12 promotes HBV-specific central memory CD8+ T cell responses by PBMCs from chronic hepatitis B virus carriers.

It is very important to understand the mechanism of immune tolerance or hyporesponsiveness in hepatitis B virus (HBV) infected individuals so as to treat HBV. In the present study we detected the cellular immune states of chronic asymptomatic HBV carriers and tried to probe their mechanisms and the strategy to promote their specific cellular immune responses. HBV non-specific antigens and specific antigens alone or with IL-12 were used to stimulate peripheral blood mononuclear cells (PBMCs) from the HBV carriers and healthy controls for evaluation of cytokine responses. IFN-gamma, TNF-alpha, IL-12P40 and IL-12P70 in the supernatants were assayed by ELISA, and the frequency as well as phenotype of IFN-gamma-producing cells were detected by ELISpot and FACS, respectively. The results showed that PBMCs from the HBV carriers secreted less IFN-gamma and IL-12 than those from the healthy controls. Exogenous IL-12 in combination with HBV specific antigens promoted PBMCs from the HBV carriers to secret more IFN-gamma by augmenting the frequency of CD8(+) IFN-gamma(+) T cells. Further studies indicated that majority of IFN-gamma-producing cells belonged to central memory CD8(+) T cells. Thus, our study provided the evidence that insufficient production of IL-12 is involved in the mechanism of hyporesponsiveness in HBV infected persons. The addition of IL-12 plus HBV specific antigen into cultures of PBMCs could restore their specific cellular immune responses. These data may have an important implication in the possibility of designing effective vaccine against HBV infection.

Human memory T cell responses to SARS-CoV E protein.

E protein is a membrane component of severe acute respiratory syndrome coronavirus (SARS-CoV). Disruption of E protein may reduce viral infectivity. Thus, the SARS-CoV E protein is considered a potential target for the development of antiviral drugs. However, the cellular immune responses to E protein remain unclear in humans. In this study, we found that peripheral blood mononuclear cells (PBMCs) from fully recovered SARS individuals rapidly produced IFN-gamma and IL-2 following stimulation with a pool of 9 peptides overlapping the entire E protein sequence. Analysis of the immune responses by flow cytometry showed that both CD4+ and CD8+T cells were involved in the SARS-CoV E-specific immune responses after stimulation with SARS-CoV E peptides. Moreover, the majority of IFN-gamma+CD4+T cells were central memory cells expressing CD45RO+CCR7+CD62L-; whereas IFN-gamma+CD8+ memory T cells were mostly effector memory cells expressing CD45RO-CCR7-CD62L-. The results of T-cell responses to 9 individual peptides indicated that the E protein contained at least two major T cell epitopes (E2 amino acid [aa] 9-26 and E5-6: aa 33-57) which were important in eliciting cellular immune response to SARS-CoV E protein in humans.

Role of T-cell receptor V beta 8.3 peptide vaccine in the prevention of experimental autoimmune uveoretinitis.

BACKGROUND: T-cell receptor (TCR) plays an important role in the development of autoimmune diseases. Recently, it was reported that immunization of animals with TCR peptide derived from the pathogenic cells could prevent autoimmune diseases. The aim of this study was to investigate whether vaccination with a synthetic peptide from the hypervariable region of TCR V(beta) 8.3, an experimental autoimmune uveoretinitis (EAU)-associated gene, was able to prevent the disease. METHODS: EAU was induced in Lewis rats by immunization with IRBP R16 peptide emulsified in complete Freund's adjuvant (CFA). The clinical and histological appearances were scored. Delayed type hypersensitivity (DTH) and lymphocyte proliferation were detected. Cytokine levels of aqueous humour, supernatants of cells from spleen and draining lymph nodes were measured by enzyme linked immunosorbent assay (ELISA). Gene expression of TCR V(beta) 8.3 on CD(4)(+) T cells was examined by real time quantitative polymerase chain reaction (PCR). RESULTS: After vaccination, the intraocular inflammation was significantly mitigated, antigen specific DTH and lymphocyte proliferation responses were suppressed, interleukin (IL)-2 in aqueous humour, interferon (IFN)-gamma and IL-2 produced by the spleen and draining lymph node cells were significantly decreased, whereas the production of IL-4 and IL-10 were increased. The response of draining lymph node cells to TCR V(beta) 8.3 peptide was enhanced after vaccination. Inoculation with CFA alone did not affect the severity of EAU and the above parameters. The suppression of EAU was much stronger in the group of four fold inoculations than the group of two fold inoculations. The expression of TCR V(beta) 8.3 gene was significantly reduced in the group of fourfold inoculations. CONCLUSION: Vaccination with the synthetic TCR V(beta) 8.3 peptide could remarkably inhibit the development of EAU.

[Human peripheral blood CD56+ natural killer cell subsets and their phenotypic and biological properties].

OBJECTIVE: To characterize the phenotypic and biological properties of CD56(+) natural killer cells from human peripheral blood mononuclear cells (PBMCs). METHODS: Surface markers and intracellular cytotoxic molecules were stained with multi-color-labeled monoclonal antibodies and analyzed at the single cell level the relation between NK subsets and biological characteristics by flow cytometry. RESULTS: NK cells in PBMCs could be divided into two major populations, CD56(bright) and CD56(dim), based upon the expression of CD56 molecules. Both CD56(bright) and CD56(dim) expressed CD95 (Fas) with CD95(bright) and CD95(dim) subsets. CD56(dim) subsets had higher percentage of CD8, granzyme B and perforin expression compared to those of CD56(bright) subsets. In CD56(bright) and CD56(dim) subpopulations, CD95(bright) and CD8(+) subsets had higher percentage of granzyme B and perforin expression. CONCLUSION: CD56(+) NK cells in PBMCs are composed of distinct subpopulations, CD56(dim) and CD56(dim) CD8(+) NK subsets have higher percentage of granzyme B and perforin and may play an important role in the killing of target cells.