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.

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.

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.

The Distribution of Human Stem Cell-like Memory T Cell in Lung Cancer.

Human stem cell-like memory T (Tscm) cells are long-lived, self-renewing memory lymphocytes that can differentiate into effector cells and mediate strong antitumour response in murine model. The distribution and function of Tscm cells in human lung cancer remain unknown. In this study, we investigated the properties of human Tscm cells in the blood and lymph node of non-small cell lung cancer (NSCLC) patients. There were more CD4 Tscm cells in blood from NSCLC patients than from healthy donors, fewer CD4 and CD8 TSCM cells in blood than in lymph node from NSCLC patients. To further analyze their properties, we stimulated peripheral blood mononuclear cells from NSCLC patients by mitogens to examine cytokine production. Our data suggest that both CD4 and CD8 Tscm cells in blood produced interferon-γ significantly increased in NSCLC patients compare with healthy subjects. In addition, fewer Tscm cells produced interferon-γ in lymph node than in blood from NSCLC patients. Our results strongly suggest that the distribution and function of CD4 Tscm cells in NSCLC patients is upregulated. Understanding of the properties of stem-like memory T cells will supply a good rationale for designing the new adoptive immunotherapy in cancer.

Functionally distinct subsets of CD4⁺ regulatory T cells in patients with laryngeal squamous cell carcinoma are indicative of immune deregulation and disease progression.

CD4+ regulatory T cells (Tregs) mediate immune tolerance in laryngeal squamous cell carcinoma (LSCC). However, Tregs are functionally heterogeneous. Recently, we reported that three distinct Treg subsets (resting Tregs, activated Tregs and cytokine-secreting CD45RA-Foxp3lowCD4+ T cells) vary in the peripheral circulation of patients with head and neck squamous cell carcinoma (HNSCC); however, the potential implication of these Treg subsets in LSCC immunity is unclear. Here, we report that activated Tregs and cytokine­secreting CD45RA-Foxp3lowCD4+ T cells were increased in LSCC patients compared with healthy donors (HD) (p<0.001, p<0.001), whereas resting Tregs were decreased (p<0.001). Activated Tregs inhibited the proliferation of CD4+CD25- T cells (p<0.001) and secreted lower levels of interleukin-2 (p<0.001), interferon-γ (p<0.001) and tumor necrosis factor-α (p<0.001) compared with the cytokine-secreting CD45RA-Foxp3lowCD4+ T cells. Importantly, activated Treg prevalence was correlated with tumor stage (p=0.001) and nodal status (p=0.007). The prevalence of naïve CD4+ (p<0.001), naïve CD8+ (p=0.002), and Th1 T-cell subsets (p<0.001, p<0.001) was decreased in the LSCC patients. In conclusion, our findings showed that activated Tregs with suppressive activity are a distinct subset of Tregs in LSCC, and correlate with disease progression. Several immune system abnormalities in LSCC patients are represented by expansion of functionally activated Tregs, both in the circulation and tumor microenvironment along with decreased frequencies of naïve T-cell populations and Th1-cell populations.

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.

CD45RA-Foxp3high but not CD45RA+Foxp3low suppressive T regulatory cells increased in the peripheral circulation of patients with head and neck squamous cell carcinoma and correlated with tumor progression.

BACKGROUND: T regulatory cells (Tregs) contribute to the progression of head and neck squamous cell carcinoma (HNSCC) by suppressing antitumor immunity. However, little is known regarding the functional heterogeneity of Tregs in HNSCC patients. METHODS: Using multicolor flow cytometry, the frequency of three Treg subsets, separated on the basis of CD45RA and Foxp3, from the peripheral circulation of newly-presenting HNSCC patients (19 oral cavity squamous cell carcinoma, 20 hypopharyngeal squamous cell carcinoma, 18 nasopharyngeal squamous cell carcinoma, 19 oropharyngeal squamous cell carcinoma, and 36 laryngeal squamous cell carcinoma) were assessed with regard to 31 healthy donors and clinicopathological features. Moreover, the functional capacity of each Treg subsets was evaluated based on CD45RA and CD25 expression. RESULTS: The frequency of Tregs in the peripheral circulation of HNSCC patients as a whole cohort was higher than in healthy donors (P < 0.0001). However, the frequency of Tregs was similar between patients with oral cavity squamous cell carcinoma and healthy donors (P = 0.269). Further dividing Tregs into three subsets based on Foxp3 and CD45RA expression revealed that the frequency of CD45RA-Foxp3high Tregs and CD45RA-Foxp3lowCD4+ T cells in patients with HNSCC developing from different subsites was higher than in healthy donors (P < 0.0001, P < 0.0001), whereas the frequency of CD45RA+Foxp3low Tregs was lower than in healthy donors (P < 0.0001). Functionally study revealed that CD45RA-CD25+++ Tregs significantly inhibit the proliferation of CD4+CD25- T cells (P < 0.001) and secrete lower levels of cytokines (P < 0.01) compared with CD45RA-CD25++CD4+ T cells. Importantly, the frequency of CD45RA-Foxp3high Tregs positively correlate with tumor stage (P < 0.0001) and nodal status (P < 0.0001). CONCLUSIONS: CD45RA-Foxp3high Tregs increase in the peripheral circulation of HNSCC patients, and correlate with tumor stage and nodal status; suggesting a role in tumor progression which may be manipulated by future immunotherapy.

[IL-12-induced expression of TRAIL enhances the cytotoxicity of NK cells against Jurkat cells].

AIM: To study the mechanism underlying the IL-12-induced cytotoxic function of NK cells to Jurkat cells. METHODS: NK cells from peripheral blood mononuclear cells (PBMCs) were purified by magnetic sorting and stimulated with or without IL-12. The expression of genes on IL-12-treated and non-IL-12-treated NK cells was analyzed by gene chips and the expression of cytolytic molecules was evaluated by flow cytometry. RESULTS: Seventeen genes were up- (5/17) or down-regulated (12/17) on IL-12-treated NK cells compared with non-IL-12-treated NK cells (fold change≥10). IL-12-induced expression of TRAIL on NK cells mediated the cytotoxicity to Jurkat cells. The expression of TRAIL on subsets of CD56(+);CD16(+); and CD56(-);CD16(+); NK cells significantly increased after the stimulation with IL-12 and Jurkat cells expressed high level of TRAIL receptor 2 (TRAIL-R2). Importantly, the neutralizing mAbs against TRAIL (RIK-2) significantly inhibited the cytotoxicity of NK cells induced by IL-12. CONCLUSION: The expression of TRAIL on human NK cells induced by IL-12 was one of the major mechanisms of cytotoxicity to Jurkat cells.

[Differences between CD3⁺ TCRvα24⁺ NKT cell and CD3⁺ TCRvß11⁺ NKT cell in PBMC].

AIM: Clarified the differences between CD3(+);TCRvα24(+); NKT cells and CD3(+);TCRvß11(+); NKT cells in their frequencies, subpopulations, phenotypes and biological functions, so as to fully understand the effects of NKT cells in immune responses. METHODS: PBMCs from blood donors were isolated and cell surface markers (CD3, TCRvα24, TCRvß11, CD4, CD8, CD45RA, CD62L, CCR7) and intracellular cytokines (IL-4, IFN-γ) were detected by flow cytometry directly or after stimulation with PMA plus Ionomycin. RESULTS: The mean frequencies of CD3(+);TCRvα24(+); NKT cells and CD3(+);TCRvß11(+); NKT cells in PBMCs were 0.63% and 0.43% and they varied according to individuals. A small population of NKT cells coexpressed TCRvα24 and TCRvß11. The subpopulations of CD4(+); NKT 64.35%, CD8(+); NKT 19.04%, CD4(-);CD8(-); NKT 17.18% in human CD3(+);TCRvα24(+); NKT cells and CD4(+); NKT 53.69%, CD8(+); NKT 18.99%, CD4(-);CD8(-); NKT 29.74% in CD3(+);TCRvß11(+); NKT cells could be identified based upon the expressions of CD4 and CD8 molecules. There were no significant differences between relative subtypes. The frequency of CD45RA(+);CD3(+);TCRvß11(+); NKT cells(71.14%) was higher than the frequency of CD45RA(+);CD3(+);TCRvα24(+); NKT cells and the differences between them were significant. The differences between the frequencies of CD62L(+);CD3(+);TCRvα24(+); NKT cells(46.26%) and CD62L(+);CD3(+);TCRvß11(+); NKT cells(42.36%), the frequencies of CCR7(+);CD3(+);TCRvα24(+); NKT cells(9.24%) and CCR7(+);CD3(+);TCRvß11(+); NKT cells(8.22%) were not significant. There were no significant differences in the secretions of IL-4 by CD3(+);TCRvα24(+); NKT cells(13.01%) and CD3(+);TCRvß11(+); NKT cells(6.62%), and IFN-γ by CD3(+);TCRvα24(+); NKT cells(38.12%) and CD3(+);TCRvß11(+); NKT cells(26.95%). However, there were significant differences between the mean frequency of IFN-γ(+);IL-4(+);CD3(+);TCRvα24(+); NKT cells(12.65%) and that of IFN-γ(+);IL-4(+);CD3(+);TCRvß11(+); NKT cells(3.02%). CONCLUSION: There were some differences between CD3(+);TCRvα24(+); NKT cells and CD3(+);TCRvß11(+); NKT cells in their frequencies, phenotypes and productions of cytokines. In all, although their frequencies were low, the complicated phenotypes and high secretions of cytokines(IL-4 and IFN-γ) assigned NKT cells immunoregulatory effects.

[Phenotypic analysis of BCG-specific effector memory CD4+ T cells in PBMCs by eight-color flow cytometry].

AIM: To evaluate cytokine production and subsets in PBMCs from PPD+ normal donors after stimulation with BCG. METHODS: PBMCs were isolated from PPD+ normal individuals, and cytokine production and BCG-specific T cell subsets were assessed by eight-color flow cytometry. RESULTS: Following stimulation with BCG, CD4+ but not CD8+ T cells expressed IFN-γ, IL-2 and TNF-α. Phenotypic analysis indicated that cytokine-producing cells were CD4+CD45RO+CD62L-CD27- and CD4+CD45RO+CD62L-CD27+. CONCLUSION: BCG predominantly induced CD4+ T cells to produce cytokines following stimulation with BCG. Further analysis indicated that these cells are CD4+CD45RO+CD62L- effector memory cells, suggesting that these cells probably played essential role in preventing TB infection.

Peripheral nerve injury leads to working memory deficits and dysfunction of the hippocampus by upregulation of TNF-α in rodents.

Patients with chronic pain usually suffer from working memory deficits, which may decrease their intellectual ability significantly. Despite intensive clinical studies, the mechanism underlying this form of memory impairment remains elusive. In this study, we investigated this issue in the spared nerve injury (SNI) model of neuropathic pain, a most common form of chronic pain. We found that SNI impaired working memory and short-term memory in rats and mice. To explore the potential mechanisms, we studied synaptic transmission/plasticity in hippocampus, a brain region critically involved in memory function. We found that frequency facilitation, a presynaptic form of short-term plasticity, and long-term potentiation at CA3-CA1 synapses were impaired after SNI. Structurally, density of presynaptic boutons in hippocampal CA1 synapses was reduced significantly. At the molecular level, we found that tumor necrosis factor-α (TNF-α) increased in cerebrospinal fluid, in hippocampal tissue and in plasma after SNI. Intracerebroventricular or intrahippocampal injection of recombinant rat TNF mimicked the effects of SNI in naive rats, whereas inhibition of TNF-α or genetic deletion of TNF receptor 1 prevented both memory deficits and synaptic dysfunction induced by SNI. As TNF-α is critical for development of neuropathic pain, we suggested that the over-production of TNF-α following peripheral nerve injury might lead to neuropathic pain and memory deficits, simultaneously.

[Analysis of ESAT-6-specific multifunctional CD4+; T cells in pleural fluid from patients with tuberculous pleurisy].

AIM: To evaluate cytokine production by CD4(+); T cells and frequency of multifunctional CD4(+); T cells after stimulation with ESAT-6 peptide pool. To understand the role of ESAT-6 specific CD4(+); T cells in the control of local TB infection. METHODS: PFCs were isolated from patients with tuberculous pleurisy, and assessed by flow cytometry for cytokine production, subpopulation, frequency and function of multifunctional CD4(+);T cells after stimulation with ESAT-6 peptide pool. RESULTS: Following stimulation with BCG, ESAT-6 peptide pool and recombinant ESAT-6 protein, CD4(+); but not CD8(+); T cells expressed IFN-γ, IL-2 and TNF-α. Distinct from BCG, ESAT-6 peptide pool and recombinant ESAT-6 protein induced similar frequencies of IFN-γ, IL-2 and TNF-α. High frequency of multifunctional CD4(+);T cells was observed. Analysis of mean fluorescence intensity (MFI) indicated that triple positive cells produced most amounts of cytokines on single cell level compared with double positive and single positive cells. CONCLUSION: ESAT-6 peptide pool predominantly induced Th1 cytokine production by CD4(+);T cells in PFCs. Multifunctional CD4(+); T cells were observed and secreted most amounts of cytokines on single cell level, suggesting that these cells probably played essential role in local TB infection.

[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 memory CD4+ IL-21+ T cells in human PBMC].

AIM: To characterize IL-21-producing T cells in human PBMCs. METHODS: PBMCs from healthy individuals were stimulated with or without anti-CD3 (OKT3), OKT3 coupled with anti-CD28, or PMA coupled with ionomycin. The cell subsets of IL-21-producing T cells were determined by FACS. PBMCs, CD4+, CD4+CD45RA⁻, CD4+ CD45RA+ or CBMCs were stimulated with PMA coupled with ionomycin. The phenotype of CD4+ IL-21+ T cells and the correlation of IL-21-producing CD4+; T cells with Th1, Th2, Th17 and Th22 cell populations were analyzed by FACS. RESULTS: PMA and ionomycin induced the highest level of IL-21 production among the stimuli tested. CD4+ T cells but not CD8+ T cells mainly expressed IL-21. CD4+ IL-21+ T cells expressed CD45RO instead of CD45RA. Some of the CD4+ IL-21+ T cells expressed CCR6, CCR7 or CXCR5. CD4+ CD45RA⁻ T cells expressed much more IL-21 than CD4+ CD45RA+ T cells. Furthermore, CD4+ T cells from PBMCs but not CBMCs, expressed IL-21. Approximately 24% of CD4+ IL-21+ cells expressed IFN-γ. Less than 10% of CD4+ IL-21+ cells expressed IL-4, IL-17 or IL-22. CONCLUSION: IL-21 is induced from human PBMCs following various polyclonal stimulations. The majority of IL-21-producing cells in PBMCs are memory CD4+ T cells. In addition, some of the CD4+ IL-21+ T cells are distinct from Th1, Th2 and Th17 cells.

Leptin signaling protects NK cells from apoptosis during development in mouse bone marrow.

Increasing evidence indicates a role of leptin in immune response, but it remains largely unclear whether leptin signaling is involved in regulating NK cell development in the bone marrow (BM). In this study, we have characterized NK cell differentiation and maturation in the BM of leptin-receptor deficient db/db mice at a prediabetic stage. Although the BM cellularity was similar to the control value, the total number of NK cells was severely reduced in mutant mice. Flow cytometric analysis of db/db BM cells revealed significantly decreased frequencies of developing NK cells at various stages of differentiation. BM db/db NK cells displayed markedly increased apoptosis but maintained normal cell cycling status and proliferative capacity. Moreover, recombinant leptin could significantly enhance the survival of NK cells from wild-type mice in cultures. Further examination on NK cell functional activity showed that db/db NK cells exhibited normal intrinsic cytotoxicity with significantly increased IL-10 production. Taken together, our findings suggest that leptin signaling regulates NK cell development via enhancing the survival of immature NK cells in mouse BM.

[Bacillus Calmette-Guérin enhances the function of human nature killer cells by inducing IL-12 production and IL-12R expression].

AIM: To study the effect and mechanism of BCG on human nature killer cells. METHODS: PBMC or purified NK cells were isolated from normal human peripheral blood with negative anti-Mycobacterium tuberculosis antibody and cultured with BCG, IL-12, BCG plus IL-12 and BCG plus anti-IL-12R beta 1 mAb (2B10), respectively. The levels of IFN-gamma and IL-12p40 in the culture supernatants were measured by ELISA. The frequency of IFN-gamma and granzyme B producing cells were analyzed by ELISpot. The cytolytic activity was detected by MTT reduction assay. The surface expression of IL-12R beta 1 on NK cells was detected by flow cytometry. RESULTS: BCG significantly induced IFN-gamma production by PBMC in a dose-dependent manner. When PBMC was stimulated with BCG, the frequency of granzyme B producing cells was higher than that in unstimulated PBMC (P<0.05). BCG enhanced the cytotoxic activity of PBMC. BCG alone didn't induce IFN-gamma production by purified NK cells, but it can augment IL-12-induced IFN-gamma production by purified NK cells. The cytotoxic activities of BCG-stimulated and unstimulated purified NK cells were not significantly different (P>0.05). BCG induced IL-12 production by PBMC in a dose-dependent manner and enhanced IL-12R beta 1 expression on different subsets of NK cells. Blocking the effect of IL-12 by anti-IL-12R beta 1 mAb (2B10) inhibited BCG-induced IFN-gamma production and granzyme B releasing by PBMC. CONCLUSION: BCG can indirectly promote biologic activity of NK cells and the production of endogenous IL-12 combined with up-regulation IL-12R beta 1 expression on the surface of NK cells is a part of the mechanisms of IL-12 on human NK cells.

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.