In vitro suppression of lymphocyte activation in patients with seasonal allergic rhinitis and pollen-related asthma by cetirizine or azelastine in combination with ginkgolide B or astaxanthin

Novel strategies are evaluated for management of allergic rhinitis and asthma in patients co-afflicted with both disorders. It is hypothesized that the platelet activating factor receptor antagonist ginkgolide B (GB) and the carotenoid antioxidant astaxanthin (ASX) interact with antihistamines cetirizine dihydrochloride (CTZ) and azelastine (AZE) to potentiate their ability to downregulate potentially pathological immune activation. Peripheral blood mononuclear cells from asthmatics and healthy subjects, cultured 24 hours with 50 μg/ml phytohemaglutinin (PHA) or PHA plus each drug are analyzed by flow cytometry for expression of CD25+ or HLA-DR+ by CD3+ (T cells). Results are reported as stimulation indices for CD3+CD25+ (SICD3+CD25+) and CD3+HLA-DR+ (SICD3+HLADR+) cells in cultures treated with PHA alone, versus cultures treated with both PHA and drugs. Optimal suppression of activated cells was observed in cultures stimulated with ASX 10-6 M + CTZ 10-6 M (SICD3+CD25+, p = 0.016; SICD3+HLADR, p = 0.012); ASX 10-6 M + AZE 10-6 M (SICD3+CD25+, p = 0.012; SICD3+HLADR, p = 0.015); GB 10-6 M + CTZ 10-6 M (SICD3+CD25+, p = 0.024, SICD3+HLADR+, p = 0.019). Results demonstrate improved activity of antihistamines by 2 phytochemicals, suggesting dosing strategies for animal trials of ASX- or GB-augmented formulations for seasonal allergic rhinitis and asthma.     

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.     

Mechanisms of the natural reactivity of lymphocytes from noninfected individuals to membrane-associated Leishmania infantum antigens

Membrane-associated Leishmania Ags (MLA) or soluble Leishmania Ags were used in vitro to stimulate cord blood or PBMC from healthy donors noninfected by Leishmania parasites. MLA, but not soluble Leishmania Ags, constantly induce strong proliferation of cord blood mononuclear cells and PBMC from noninfected individuals. Responding cells are CD3+, CD4+, TCRalphabeta+, CD45RO+, and CD45RA+ and secrete IFN-gamma and IL-10, but not IL-4. MLA do not activate NK cells nor NKT cells. Membrane Ags also induce purified macrophages from noninfected individuals to secrete IL-10 and TNF-alpha, but have no effect on IL-1alpha or IL-12 secretion. The effects of MLA are proteinase K-sensitive and resistant to lipid extraction. The lymphoproliferative responses are inhibited by anti-HLA-DR Abs and require Ag processing by APCs, excluding that the biological effect of MLA could be attributed to a superantigen. Finally, TCR repertoire analysis shows that the T cell expansion induced by MLA uses TCR with various variable beta segment rearrangements and CDR3 lengths, features much more characteristic to those observed with a polyclonal activator than with a conventional Ag. These results suggest a particular mechanism developed during the host's natural response to Leishmania parasites that allows direct activation of naive CD4 lymphocytes by parasite membrane-associated Ags.     

Large-scale expansion of CD3<Superscript>+</Superscript>CD56<Superscript>+</Superscript> lymphocytes capable of lysing autologous tumor cells with cytokine-rich supernatants

We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMC) by the timed addition of cytokine-rich supernatants collected from allogeneic PBMC cultures stimulated with anti-CD3 monoclonal antibody (mAb) (allogeneic CD3 supernatants; ACD3S). These cytotoxic effectors belonged primarily to CD56(+) natural killer (NK) cells, and the cell subset with the greatest cytotoxic activity was an otherwise rare population of CD3(+)CD56(+) cells (NKT cells) that expand dramatically under these conditions. CD3(+)CD56(+) cytotoxic effectors were generated from the PBMC of 16 patients with several types of cancer. The CD3(+)CD56(+) cell subset expanded significantly and efficiently lysed NK- as well as lymphokine-activated killer (LAK)-sensitive targets. More importantly, ACD3S-activated CD3(+)CD56(+) cells were capable of efficiently lysing autologous tumor cells including metastatic colorectal, ovarian, breast, lung and pancreatic tumor cells as well as melanoma cells. ACD3S-expanded CD3(+)CD56(+) cells exhibited increased levels of cytoplasmic interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), gamma-interferon (IFN-gamma) and perforin. CD3(+)CD56(+) cell-mediated cytotoxicity was not restricted by major histocompatibility complex (MHC) gene products, since it was not blocked by anti-MHC class I mAb but was highly inhibited in the presence of CD2- and CD18-specific mAb. These data suggest that CD3(+)CD56(+) cells expanded under the presence of ACD3S may be utilized in clinical protocols for cancer immunotherapy.     

Gamma delta T cell responses associated with the development of tuberculosis in health care workers

This study evaluated T cell immune responses to purified protein derivative (PPD) and Mycobacterium tuberculosis (Mtb) in health care workers who remained free of active tuberculosis (HCWs w/o TB), health care workers who went on to develop active TB (HCWs w/TB), non-health care workers who were TB free (Non-HCWs) and tuberculosis patients presenting with minimal (Min TB) or advanced (Adv TB) disease. Peripheral blood mononuclear cells (PBMC) were stimulated with Mtb and PPD and the expression of T cell activation markers CD25+ and HLA-DR+, intracellular IL-4 and IFN-gamma production and cytotoxic responses were evaluated. PBMC from HCWs who developed TB showed decreased percentages of cells expressing CD8+CD25+ in comparison to HCWs who remained healthy. HCWs who developed TB showed increased gammadelta TCR+ cell cytotoxicity and decreased CD3+gammadelta TCR- cell cytotoxicity in comparison to HCWs who remained healthy. PBMC from TB patients with advanced disease showed decreased percentages of CD25+CD4+ and CD25+CD8+ T cells that were associated with increased IL-4 production in CD8+ and gammadelta TCR+ phenotypes, in comparison with TB patients presenting minimal disease. TB patients with advanced disease showed increased gammadelta TCR+ cytotoxicity and reduced CD3+gammadelta TCR- cell cytotoxicity. Our results suggest that HCWs who developed TB show an early compensatory mechanism involving an increase in lytic responses of gammadelta TCR+ cells which did not prevent TB.     

Human CD4+ T cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of IL-12 to kill tumors in situ by indirect effects of IFN-gamma

By implanting nondisrupted pieces of human lung tumor biopsy tissues into SCID mice, it has been possible to establish viable grafts of the tumor, as well as the tumor-associated microenvironment, including inflammatory cells, fibroblasts, tumor vasculature, and the extracellular matrix. Using this xenograft model, we have evaluated and characterized the effects of a local and sustained release of human rIL-12 (rhIL-12) from biodegradable microspheres. In response to rhIL-12, the human CD45+ inflammatory cells present within the xenograft mediate the suppression or the complete arrest of tumor growth in SCID mice. Analysis of the cellular events reveals that human CD4+ and CD8+ T cells are induced by rhIL-12 to produce and secrete IFN-gamma. Serum levels of human IFN-gamma in mice bearing rhIL-12-treated tumor xenografts correlate directly with the degree of tumor suppression, while neutralizing Abs to human IFN-gamma abrogate the IL-12-mediated tumor suppression. Gene expression profiling of tumors responding to intratumoral rhIL-12 demonstrates an up-regulation of IFN-gamma and IFN-gamma-dependent genes not observed in control-treated tumors. Genes encoding a number of proinflammatory cytokines, chemokines (and their receptors), adhesion molecules, activation markers, and the inducible NO synthase are up-regulated following the introduction of rhIL-12, while genes associated with tumor growth, angiogenesis, and metastasis are decreased in expression. NO contributes to the tumor killing because an inhibitor of inducible NO synthase prevents IL-12-induced tumor suppression. Cell depletion studies reveal that the IL-12-induced tumor suppression, IFN-gamma production, and the associated changes in gene expression are all dependent upon CD4+ T cells.     

Study of peripheral blood lymphocyte subset distribution and IL-2 receptor (IL-2 R) p55–p75 subunit expression in patients with cancer of different sites

The aim of the study was to evaluate the subset distribution and the IL-2 R p55–p75 subunit expression on unstimulated and phytohemagglutinin (PHA)-stimulated (at 3-d) peripheral blood mononuclear cells (PBMC), of patients with solid cancers of different sites. Indeed the expression of the two subunits of IL-2R is an essential prerequisite for The action of the IL-2 on CD8⁺, CD16⁺ lymphocytes as effectors in antitumor activity (LAK-cells). The subset distribution (CD3, CD4, CD8, CD16, DR) was assessed by cytofluorometry with specific monoclonal antibodies (MAbs); the p55 (CD25) and p75 subunit expression was evaluated by specific MAb (OKT26a and anti-p75). Ninety patients with advanced cancer (mainly non-small cell lung cancer [NSCLC], head and neck cancer, and gynecological cancer; mean age 55 yr; range 27–80) were studied. Thirty-five age- and sex-matched healthy subjects were studied as controls. Our data show that there is no significant difference in the subset distribution between cancer patients and controls. Furthermore, no difference has been found in the expression of p55 subunits on unstimulated PBMC between cancer patients and controls. No difference has been found in the expression of both p55 and p75 subunits on PHA-stimulated PBMC between cancer patients and controls. Our results can support the rationale for further clinical trials with IL-2 in solid malignancies.     

Phenotypic analysis of a large number of normal human bone marrow sample by flow cytometry

Bone marrow aspirates from 48 healthy donors (34 adults, 14 children) were analyzed by flow cytometry (FACS Analyzer) after purification of low-density bone marrow cells (Ld BMC) on a density gradient (d = 1,077) and labelling with 23 anti-hematopoietic cell monoclonal antibodies. Based on physical properties, these Ld BMC could be divided into four different populations called E, My, Mo and L, which comprised 14% +/- 9%, 31% +/- 16%, 10% +/- 5% and 45% +/- 14% of these cells, respectively. The phenotypic analysis of these different populations enabled the identification in E, of erythrocytes (Glycophorin A+, Rhesus D+, but negative for early erythroid differentiation markers such as the transferrin receptor (Tf. R) and the FA6-152 antigen); in My of cells of the myeloid lineage (VIM2+, HLA DR-); in Mo of cells of the monocytic lineage (VIM2+, CD14+) plus some myeloblasts (VIM2+, CD14-, HLADR+) and finally in L of a heterogeneous population including: 1. T lymphocytes labelled to the same extent by CD2, CD3, CD5 and CD6 (28% +/- 10%), B lymphocytes assessed by CD19 and CD20 (12% +/- 8%), Pre-B cells (CD10+ = 8% +/- 7%), less than 5% of "natural killer" cells (CD16+ or Leu7+) and finally, less than 6% of myelomonocytes (CD14+ and/or VIM2+). 2. The erythroid lineage (rhesus D+ = 42% +/- 20%, Tf.R+ and FA6-152+ = 32% +/- 12%). 3. Undifferentiated cells or progenitor cells (CD34+ = 7% +/- 5%). 4. Cells unlabelled by any antibodies (approximately 6%). We observed no difference between bone marrow samples from adults or children, with respect to physical properties, and with all but four immunological markers. A significantly higher proportion of B cells (CD19+ and CD10+) (P less than 0.001) and undifferentiated cells (CD34+ and HLADR+) (P less than 0.02) was observed in children. These data, obtained from a large number of bone marrow samples, could be used to quantify the imbalance of some bone marrow disorders.     

Infection with Host-Range Mutant Adenovirus 5 Suppresses Innate Immunity and Induces Systemic CD4+ T Cell Activation in Rhesus Macaques

Ad5 is a common cause of respiratory disease and an occasional cause of gastroenteritis and conjunctivitis, and seroconversion before adolescence is common in humans. To gain some insight into how Ad5 infection affects the immune system of rhesus macaques (RM) 18 RM were infected with a host-range mutant Ad5 (Ad5hr) by 3 mucosal inoculations. There was a delay of 2 to 6 weeks after the first inoculation before plasmacytoid dendritic cell (pDC) frequency and function increased in peripheral blood. Primary Ad5hr infection suppressed IFN-γ mRNA expression, but the second Ad5hr exposure induced a rapid increase in IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC). Primary Ad5hr infection suppressed CCL20, TNF and IL-1 mRNA expression in PBMC, and subsequent virus exposures further dampened expression of these pro-inflammatory cytokines. Primary, but not secondary, Ad5hr inoculation increased the frequency of CXCR3+ CD4+ T cells in blood, while secondary, but not primary, Ad5hr infection transiently increased the frequencies of Ki67+, HLADR+ and CD95+/CCR5+ CD4+ T cells in blood. Ad5hr infection induced polyfunctional CD4 and CD8+ T cells specific for the Ad5 hexon protein in all of the animals. Thus, infection with Ad5hr induced a complex pattern of innate and adaptive immunity in RM that included transient systemic CD4+ T cell activation and suppressed innate immunity on re-exposure to the virus. The complex effects of adenovirus infection on the immune system may help to explain the unexpected results of testing Ad5 vector expressing HIV antigens in Ad5 seropositive people.     

In vitro analysis of interferon gamma (IFN-gamma) and interleukin-12 (IL-12) production and their effects in ileal Crohn's disease

Crohn's disease is an inflammatory disease of the gut in which tumour necrosis factor (TNF) and T helper 1 (Th1) cytokines (interleukin (IL)-12, interferon (IFN)-gamma) are thought to play a major role. After the successes obtained with neutralisation of TNF, interest is now growing for therapy aiming at neutralisation of Th1-associated cytokines. Since cytokines are linked in a delicate network, in vitro cultures of ileal lamina propria mononuclear cells (LPMC) were set up for evaluation of a) IFN-gamma and IL-12 production, b) effects of rhIFN-gamma and rhIL-12 and c) effects of anti-IFN-gamma and anti-IL-12 on pro-inflammatory cytokines and IL-10 production. LPMC were isolated from surgical specimens of a total of 27 Crohn's disease and 17 caecum carcinoma (control) patients. Cells were stimulated with CD40L (which triggers myeloid CD40-expressing cells) or anti-CD3 +CD80 (which triggers T cells). LPMC from involved ileal, Crohn's disease produced, in both non-stimulated and stimulated conditions, more IFN-gamma and IL-12p70 than LPMC from non-involved tissue or from control patients. rhIFN-gamma significantly enhanced TNF production in both controls and in ileal Crohn's disease patients, while rhIL-12 enhanced IFN-gamma but not TNF production. LPMC from involved tissue were more sensitive to IL-12 than control LPMC. LP-T cell-dependent activation of monocytes was then studied by co-culture of anti-CD3/CD80-stimulated LPMC with fresh monocytes, which resulted in high IL-12, IFN-gamma, TNF and IL-10 production. The data show that neutralisation of either IL-12 or IFN-gamma with mAb in these cultures also affects secretion of the reciprocal cytokine and (in the case of anti-IL-12) also that of the anti-inflammatory cytokine IL-10. However, no effect of anti-IL-12 or anti-IFN-gamma on production of TNF, a cytokine with an important pathogenic role in Crohn's disease, could be found. Therapies aiming at neutralisation of IFN-gamma or IL-12 are therefore unlikely to replace anti-TNF, but they might provide an additive or synergistic effect.     

Systemic inflammation and cell activation reflects morbidity in chronic heart failure

Chronic heart failure (CHF) leads to complex effects distant from the heart. As these changes may be reflected in the balance of systemic inflammatory and fibrotic immunomodulators we measured these potential biomarkers in ambulatory CHF patients. Using the New York Heart Association (NYHA; levels II-IV) functional classification, 30 CHF patients were compared with 21 age and gender matched controls. Peripheral blood levels of regulatory cytokines (TNF-α, TGF-β, KGF, IL-8, IL-10 and IL-12) and markers of cellular activation (CD11b, CD16, CD18, CD34, HLADR, CXCR1 and CCR5) were analysed by ELISA and flow cytometry, respectively. NYHA classification, which reflected increasing pulmonary microvascular pressure (E:E') but not ejection fraction, was positively associated with TGF-β and IL-10 (p≤0.03). Similarly, monocytes, as well as cell surface expression of the neutrophil adhesion molecule CD11b, and the macrophage complement receptor complex (CD11b/CD18), were increased in CHF patients (p≤0.03), while the chemokine receptor CXCR1 was decreased on cells of CHF patients. Twenty month follow-up of CHF subjects identified monocyte number as a powerful prognostic factor for cardio-pulmonary adverse events (p=0.001); however, no concurrent relationship with cellular activation marker expression was found. In subjects with CHF, monocytes, TGF-β, IL-10, CD11b/CD18 and CXCR1 expression in peripheral blood may act as novel biomarkers of immune activation and remodelling. Given the importance of dyspnea and the relationship of pulmonary microvascular pressure to the NYHA classification, we suggest these findings may reflect a contribution by the lung.     

Lipopolysaccharide stimulates the proliferation of human CD56+CD3- NK cells: a regulatory role of monocytes and IL-10

NK cells recognize and kill tumor cells and normal cells, and these play an important role in immune defense in cancer, infectious disease, and autoimmunity. NK killing is regulated by positive or negative signals derived from the interaction of surface receptors with ligands on the target cells. However, the mechanisms controlling the proliferation and maintenance of NK cells in normal human individuals are less clearly defined. In this study, using an entirely autologous system, we demonstrate that human peripheral blood CD3-CD56+, killer cell-inhibitory receptor (KIR)-expressing cells proliferate and expand in response to LPS. These responses are enhanced in the presence of anti-IL-10 receptor-blocking Abs or on the removal of CD14+ cells from the cultures. This enhancement is also reflected in substantial increases in cytolytic activity and IFN-gamma production. The negative effect of CD14+ cells may also be IL-10 mediated, IL-10 being lost from the culture supernatants of CD14-depleted PBMC and rIL-10 reversing the effect of this depletion. On the other hand, mRNA for the p35 and p40 subunits of IL-12 is still induced in CD14-depleted cultures. The expansion of CD3-CD56+ cells was also inhibited by CTLA4-Ig, indicating a role for CD80/86. B lymphocytes were not required for the expansion of CD3-CD56+ cells, whereas removal of MHC class II+ cells from CD14-depleted cultures resulted in a complete abrogation of these responses. Expansion of CD3-CD56+ cells was reconstituted in MHC class II-depleted cell cultures by adding back monocyte-derived dendritic cells. These results indicate that the responses of CD3-CD56+ NK cells to LPS may be driven by a MHC class II+ B7+ CD14- peripheral population, most likely blood dendritic cells.     

化疗在中晚期非小细胞肺癌患者中对淋巴细胞亚群影响分析

目的:探讨化疗在中晚期非小细胞肺癌患者中对淋巴细胞亚群的影响。方法:随机抽取本院收治的60例中晚期非小细胞肺癌患者编为实验组进行化疗,另选取同期体检的50例健康志愿者作为对照组。随访12月-15月,采用流式细胞仪技术分别对两组外周血淋巴细胞亚群进行检测计数。结果:两组间相比,实验组患者的CD3+、CD4+、NK细胞的数量以及CD4+/CD8+比值均低于对照组(P<0.05),而CD8+细胞的比例却高于对照组。化疗后CD3+、CD4+、CD4+/CD8+、NK均较化疗前升高(P<0.05),但CD8+不变(P>0.05)。结论:应用化疗治疗中晚期非小细胞肺癌,可明显改善患者的免疫功能。     

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

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

IL-18-induced expression of intercellular adhesion molecule-1 in human monocytes: involvement in IL-12 and IFN-gamma production in PBMC

IL-18 time- and concentration-dependently upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) in a monocyte population in human PBMC as determined by FACS analysis while the expression of CD11a, CD18, CD29, CD44, and CD62L in monocytes and that of ICAM-1, CD11a, CD18, CD29, CD44, and CD62L in T cells was not influenced by IL-18. IL-18 in the same concentration range stimulated the production of IL-12, TNF-alpha, and IFN-gamma in culture of PBMC; however, IL-18-induced expression of ICAM-1 in monocytes was not inhibited by anti-IL-12, anti-TNF-alpha, or anti-IFN-gamma Ab, suggesting the independence of the upregulating effect of IL-18 on endogenous IL-12, TNF-alpha, and IFN-gamma production. IL-18 also induced the aggregation of PBMC, which was prevented by anti-ICAM-1 and anti-LFA-1 Abs. On the other hand, anti-ICAM-1 and anti-LFA-1 Abs inhibited IL-18-induced production of three cytokines, IL-12, IFN-gamma, and TNF-alpha, by 60 and 40%, respectively. These results strongly suggested that the IL-18-induced upregulation of ICAM-1 and the subsequent adhesive interaction through ICAM-1 on monocytes and LFA-1 on T/NK cells generate an additional stimulatory signaling as well as an efficient paracrine environment for the IL-18-initiated cytokine cascade.     

Persistent cytomegalovirus-specific memory responses in the lung allograft and blood following primary infection in lung transplant recipients

Primary CMV infection in lung transplant recipients (LTRs) is associated with increased mortality. We studied 22 donor CMV-positive, recipient-negative (D(+)R(-)) LTRs for the development of posttransplant CMV-specific immunity. We found that 13 of 22 D(+)R(-) LTRs (59.1%) seroconverted (CMV IgG Ab(+)). Using pooled peptides of the immunodominant CMV Ags pp65 and IE1, we detected CMV-specific CD8(+)IFN-gamma(+) T cells in the PBMC of 90% of seroconverted individuals following primary infection by intracellular cytokine staining. In contrast, few seroconverters had detectable CMV-specific CD4(+)IFN-gamma(+) T cells during viral latency. However, the majority of IgG(+) LTRs demonstrated CMV-specific CD4(+) and CD8(+) T cell proliferative responses from PBMC, with CD4(+)IFN-gamma(+) T cells detectable upon re-expansion. Examination of lung allograft mononuclear cells obtained by bronchoalveolar lavage revealed both CMV-specific CD4(+) and CD8(+)IFN-gamma(+) T cells, including patients from whom CD4(+)IFN-gamma(+) T cells were simultaneously undetectable in the PBMC, suggesting differential effector memory populations between these compartments. Moreover, both responses in the PBMC and lung allograft were found to persist, despite substantial immunosuppression, long after primary infection. Clinical correlation in this cohort demonstrated that the acquisition of CMV immunity was associated with freedom from CMV disease (p < or = 0.009) and preservation of allograft function (p < or = 0.02) compared with those who failed to develop CMV immunity. Together, our data reveal immunologic heterogeneity in D(+)R(-) LTRs, with the development and persistence of primary CMV responses that may provide clinical benefit.     

CD3+/CD16+CD56+ cell numbers in peripheral blood are correlated with higher tumor burden in patients with diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma is the commonest histological type of malignant lymphoma, and remains incurable in many cases. Developing more efficient immunotherapy strategies will require better understanding of the disorders of immune responses in cancer patients. NKT (natural killer-like T) cells were originally described as a unique population of T cells with the co-expression of NK cell markers. Apart from their role in protecting against microbial pathogens and controlling autoimmune diseases, NKT cells have been recently revealed as one of the key players in the immune responses against tumors. The objective of this study was to evaluate the frequency of CD3(+)/CD16(+)CD56(+) cells in the peripheral blood of 28 diffuse large B-cell lymphoma (DLBCL) patients in correlation with clinical and laboratory parameters. Median percentages of CD3(+)/CD16(+)CD56(+) were significantly lower in patients with DLBCL compared to healthy donors (7.37% vs. 9.01%, p = 0.01; 4.60% vs. 5.81%, p = 0.03), although there were no differences in absolute counts. The frequency and the absolute numbers of CD3(+)/CD16(+)CD56(+) cells were lower in advanced clinical stages than in earlier ones. The median percentage of CD3(+)/CD16(+)CD56(+) cells in patients in Ann Arbor stages 1-2 was 5.55% vs. 3.15% in stages 3-4 (p = 0.02), with median absolute counts respectively 0.26 G/L vs. 0.41 G/L (p = = 0.02). The percentage and absolute numbers of CD3(+)/CD16(+)CD56(+) cells were significantly higher in DL -BCL patients without B-symptoms compared to the patients with B-symptoms, (5.51% vs. 2.46%, p = 0.04; 0.21 G/L vs. 0.44 G/L, p = 0.04). The percentage of CD3(+)/CD16(+)CD56(+) cells correlated adversely with serum lactate dehydrogenase (R= -445; p 〈 0.05) which might influence NKT count. These figures suggest a relationship between higher tumor burden and more aggressive disease and decreased NKT numbers. But it remains to be explained whether low NKT cell counts in the peripheral blood of patients with DLBCL are the result of their suppression by the tumor cells, or their migration to affected lymph nodes or organs.     

Blockade of TLR9 agonist-induced type I interferons promotes inflammatory cytokine IFN-gamma and IL-17 secretion by activated human PBMC

Type I interferons (IFN) (IFN-alpha/beta) are recognized as both inhibitors and effectors of autoimmune disease. In multiple sclerosis, IFN-beta therapy appears beneficial, in part, due to its suppression of autoimmune inflammatory Th cell responses. In contrast, in systemic lupus erythematosus (SLE) triggering of plasmacytoid DC (pDC) Toll-like receptors (TLRs) by autoimmune complexes (autoICs) results in circulating type I IFN that appear to promote disease by driving autoantigen presentation and autoantibody production. To investigate how pDC-derived type I IFN might regulate Th cells in SLE, we examined a model in which sustained pDC stimulation by autoICs is mimicked by pretreating normal human PBMC with TLR9 agonist, CpG-A. Subsequently, PBMC Th cells are activated with superantigen, and APC are activated with CD40L. The role of CpG-A/TLR9-induced type I IFN in regulating PBMC is determined by blocking with virus-derived soluble type I IFN receptor, B18R. In summary, pretreatment with either rhIFN-alpha/beta or CpG-A inhibits PBMC secretion of superantigen-induced IFN-gamma and IL-17, and CD40L-induced IL-12p70 and IL-23. B18R prevents these effects. Data indicate that CpG-A-induced type I IFN inhibit IL-12p70-dependent PBMC IFN-gamma secretion by enhancing IL-10. Our results suggest that in SLE, circulating type I IFN may potentially act to inhibit inflammatory cytokine secretion.     

Alloantigenicity of human endothelial cells. 1. Frequency and phenotype of human T helper lymphocytes that can react to allogeneic endothelial cells.

To determine the relative ability of allogeneic endothelial cells to stimulate helper T lymphocytes (HTL), human PBMC or purified T cells were incubated in conventional lymphocyte microcultures or in limiting dilution microcultures with allogeneic human umbilical vein endothelia (HUVE), with cytokine-treated allogeneic HUVE, or with allogeneic peripheral blood monocytes. These cultures were tested for IL-2 production as an index of HTL stimulation. Dose-response studies in conventional lymphocyte cultures indicated that allogeneic monocytes were better than allogeneic HUVE at stimulating IL-2 production. Limiting dilution analyses revealed that untreated HUVE and TNF-treated HUVE stimulated small numbers of HTL (approximately 1 HTL/30,000 PBMC), whereas 5 to 10 times more HTL were stimulated by IFN-gamma-treated HUVE and 10 to 20 times more HTL were stimulated by allogeneic monocytes. Serologic deletion studies revealed that most of the high frequency HTL responding to IFN-gamma-treated HUVE were CD4+, whereas most of the low frequency HTL responding to nontreated HUVE or to TNF-treated HUVE were CD8+. Interestingly, mAb to MHC class I and class II molecules, which significantly impaired HUVE-induced proliferation, caused little interference with HUVE-induced IL-2 production. Finally, polymerase chain reaction analysis demonstrated that untreated allogeneic HUVE cells could stimulate PBMC to produce mRNA for IFN-gamma, as well as for IL-2. These data demonstrate the following hierarchy of allogeneic stimulatory capacity for human HTL: monocytes greater than IFN-gamma-treated HUVE much greater than TNF-treated HUVE = nontreated HUVE. Further, these data suggest that non-activated allogeneic endothelial cells can initiate immune responses by inducing IL-2 and IFN-gamma. Because IFN-gamma can induce MHC class II expression by the endothelial cells, this could recruit large numbers of CD4+ T cells for IL-2 production.