Significant Amounts Of IL-6 Research Articles

Blockade of inhibitory killer cell immunoglobulin-like receptors and IL-2 triggering reverses the functional hypoactivity of tumor-derived NK-cells in glioblastomas

Killer cell immunoglobulin-like receptors (KIRs) comprise a group of highly polymorphic inhibitory receptors which are specific for classical HLA class-I molecules. Peripheral blood and freshly prepared tumor cell suspensions (n = 60) as well as control samples (n = 32) were investigated for the distribution, phenotype, and functional relevance of CD158ab/KIR2DL1,-2/3 expressing NK-cells in glioblastoma (GBM) patients. We found that GBM were scarcely infiltrated by NK-cells that preferentially expressed CD158ab/KIR2DL1,-2/3 as inhibitory receptors, displayed reduced levels of the activating receptors CD335/NKp46, CD226/DNAM-1, CD159c/NKG2C, and showed diminished capacity to produce IFN-γ and perforin. Functional hypoactivity of GBM-derived NK-cells persisted despite IL-2 preactivation. Blockade with a specific KIR2DL-1,2/3 monoclonal antibody reversed NK-cell inhibition and significantly enhanced degranulation and IFN-γ production of IL-2 preactivated NK-cells in the presence of primary GBM cells and HLA-C expressing but not HLA class-I deficient K562 cells. Additional analysis revealed that significant amounts of IL-2 could be produced by tumor-derived CD4+ and CD8+CD45RA- memory T-cells after combined anti-CD3/anti-CD28 stimulation. Our data indicate that both blockade of inhibitory KIR and IL-2 triggering of tumor-derived NK-cells are necessary to enhance NK-cell responsiveness in GBM.

In Vitro Assessment of Cytokine Expression Profile of MCF-7 Cells in Response to hWJ-MSCs Secretome.

Purpose: Several attempts have been made to identify the mechanisms by which mesenchymal stem cells (MSCs)-derived secretome exert anti-tumor or tumorigenic effects, but still further investigations are needed to explore this subject. Thus, in this study we want to examine the expression of different cytokines in secretome of hWJ-MSCs and their effects on cytokine expression profile of the MCF-7 tumor cells. Methods: The hWJ-MSCs were isolated and characterized according to the International Society for Cellular Therapy criteria. Then, secretome of hWJ-MSCs was collected and freeze-dried, and 20 mg/mL of the freeze-dried secretome was used to treat MCF-7 cancer cells for 48 hours. Afterwards, the expression levels of 12 cytokines including IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17A, TNFα, IFNγ and GM-CSF in secretome of hWJ-MSCs alone as well as in supernatant of tumor cells before and after treatment with hWJ-MSCs secretome were evaluated. Results: Our results indicate that MCF-7 cells express significant amount of IL-6 and IL-8. Moreover, significant amounts of IL-1a, IL-1b, IL-8, IL-6 and GM-CSF were detected in secretome of hWJ-MSCs. Furthermore, IL-1a, IL-2 and IL-4 were expressed significantly by MCF-7 cells after their treatment with hWJ-MSCs-derived secretome. Conclusion: According to our findings, the hWJ-MSCs derived secretome contains different cytokines which can exert either anti-tumor or tumorigenic effects.

Cytokine production in whole-blood cultures following immunization with an influenza vaccine

ABSTRACTA clinical trial of a quadrivalent split influenza vaccine was performed in the 2014/15 season. Sixty-four subjects aged 6 months to 18 years were enrolled in order to investigate the relationship between cellular and humoral immune responses. Subjects were categorized into two groups by measuring neutralizing antibodies: non-primed naïve/primed or seroconverted/non-seroconverted groups. Whole-blood cultures were stimulated with the H1N1 split antigen before immunization and one month after the first and second immunizations for subjects < 13 years and before and one month after the first dose for those ≥ 13 years in order to investigate cytokine production. Significant amounts of IL-2, IL-12, IL-13, MCP-1, MIP-1β, and TNF-α were detected from one month after the first dose in the naïve group. In addition to these cytokines, the production of IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17, G-CSF, and IFN-γ was enhanced one month after the second dose. No significant increase was noted in the primed group, except in the production of IL-10. In seroconverted subjects, the production of IL-2, IL-4, IL-8, IL-10, G-CSF, MCP-1, TNF-α, and IFN-γ increased one month after the first dose, which was earlier than in the naïve group, whereas no significant cytokine response was noted in subjects without seroconversion. Subjects ≥ 13 years were primed and the production of G-CSF, IL-4, and IL-1β increased in subjects with seroconversion. Whole-blood cultures were also stimulated with the H3N2 split antigen and similar cytokine profiles were obtained. Many cytokines and chemokines, including inflammatory cytokines, were produced in seroconverted, but not non-seroconverted subjects.

A Dual-layered Microfluidic System for Long-term Controlled In Situ Delivery of Multiple Anti-inflammatory Factors for Chronic Neural Applications.

We report the development of a microfluidic system capable of repeated infusions of anti-inflammatory factors post-implantation for use as a coating for neural probes. This system consists of a microchannel in a thin gelatin methacryloyl (GelMA)-polyethylene glycol (PEG) composite hydrogel surrounded by a porous polydimethylsiloxane (PDMS) membrane, where the hydrogel can be dried to increase the stiffness for easy insertion. Reswelling allowed us to perfuse interleukin (IL)-4 and dexamethasone (DEX) as anti-inflammatory factors through the channel with minimal burst release and significant amounts of IL-4 were observed to release for up to 96 hr post-infusion. Repeated injections of IL-4 increased the ratio of prohealing M2 versus proinflammatory M1 phenotypes of macrophages encapsulated in the hydrogel by six fold compared with a single injection, over a 2-week period. These repeated infusions also significantly downregulated the expression of inflammatory markers tumor necrosis factor (TNF)-α and IL-6 in astrocytes encapsulated in hydrogel. To demonstrate the system as a coating of neural probe for in vivo applications, we further fabricated a prototype device, where a thin dual-layered microfluidic system was integrated onto a metal probe. Such a drug delivery system could help reduce the formation of a glial scar around neural probes.

IM-02 * A scFv-BASED CAR TO REDIRECT T CELLS TO IL13R 2-POSITIVE PEDIATRIC GLIOMA

The intent of this project is to develop antigen-specific T cells as an effective immunotherapy for diffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM), the most aggressive, uniform fatal, primary human brain tumors in children. IL13Rα2 is expressed at a high frequency in DIPG and GBM but not in normal brain, making it is a promising target for CAR T-cell immunotherapy. While other investigators have generated IL13Rα2-targeted CARs using mutated forms of IL13 as CAR binding domains, our studies indicate that these CARs also recognize IL13Rα1, raising significant toxicity concerns. To overcome this limitation we have developed a high affinity IL13Rα2-specific scFv that does not recognize IL13Rα1. The goal of this project was to generate scFv-based IL13Rα2-specific CAR (IL13Rα2-CAR T cells) and evaluate their function. We constructed a panel of IL13Rα2-CARs that contain our IL13Rα2-specific scFv as an ectodomain, a short hinge (SH) or long hinge (LH), a CD28 transmembrane domain, and endodomains that contain signaling domains derived from CD3ζ and co-stimulatory molecules. IL13Rα2-CAR T cells were generated by retroviral transduction. CAR cell surface expression varied depending on used hinge and endodomain. In cytotoxicity assays, IL13Rα2-CAR T cells only killed target cells that expressed IL13Rα2 and not IL13Rα1 confirming specificity. While all IL13Rα2-CAR T cells secreted significant levels of IFNγ in co-culture assays with the IL13Rα2+ glioma cell line U373, only short hinge CAR T cells secreted significant amounts of IL2. In vivo, injection of IL13Rα2.SH.CD28.ζ-CAR T cells into U373-bearing mice resulted in regression of glioma xenografts as judged by bioluminescence imaging. IL13Rα2.LH.CD28.ζ- or IL13Rα2.Δ-CAR T cells had no antitumor effects. In conclusion, for the first time we have generated a CAR that only recognizes IL13Rα2 and not IL13Rα1. Our results warrant further active exploration of IL13Rα2-specific scFv-based CAR T cells for the adoptive immunotherapy of pediatric brain tumors.

Tu2005 Epidermal Growth Factor Receptor Is Overexpressed in Esophageal Biopsies of Children With Eosinophilic Esophagitis: An Immunohistochemical Analysis

Figure 1. Characterization of different MDSCs subsets in healthy controls and RAC patients. (A) Flow cytometric profile of peripheral blood (upper and mid rows) and tumor cells (lower row) from healthy control and RAC patient. X100 light microscopy image of representative cell in each subset is presented. (B) Flow cytometric analysis of different circulating MDSCs subsets in healthy controls and RAC patients. The figure represents 3 different subjects in each group; *-P<.05, *-P<.01. (C) Sonographic evaluation of locally advanced RAC before (left) and after (right) neoadjuvant therapy. T-Tumor; P-Prostate Background: Interaction between tumor and microenvironment is an important factor in tumor progression. Cancer stem cells (CSCs) in tumor, known as contributing cells to tumor recurrence and metastasis, also interact with a niche in the microenvironment which controls their maintenance and differentiation. We investigated the CSC-promoting effect of factors released from myofibroblasts in microenvironment and its molecular mechanism. Methods: To investigate the effect of factors secreted from myofibroblast on CSC expansion, we performed a flow cytometric analysis of CD133+CD44+ cells in Caco2 cells which were cultured in conditioned media (CM) of 18CO(pericryptal myofibroblast) cells. To identify contributing gene expressions, we utilized mRNA expressional microarray for detection of differential gene expressions between Caco2 cells and 18CO-cocultured Caco2 cells. The secreted factors from the 18CO cells were analyzed using cytokine antibody array, and anti- IL6/IL-8 antibody and anti-HES1 antibody were used for neutralization and Western blot analysis. Results: In mRNA expressional microarray comparing Caco2 cells and 18CO- cocultured Caco2 cells, HES1 expression, the target of Notch signaling, significantly increased in 18CO-cocultured Caco2 cells, compared to Caco-2 cells. Caco2 cells cultured in 18CO CM showed a significant increase in CD133+CD44+ cells and HES1 expression, compared to Caco2 cells cultured in the control media. Significant amounts of IL6 and IL8 were detected in 18CO CM, compared to the control media. 18CO CM-induced increase of CD133+CD44+ cells was attenuated by neutralizing antibodies to IL6 and IL8. Furthermore, neutralizing antibodies to IL6 and IL8, and STAT3 inhibitor reduced the expression of HES1 induced in Caco2 cells cultured in 18CO CM. Conclusion: The increase of CSCs by myofibroblast could be mediated by IL6/IL8-induced HES1 activation in tumor microenviron- ment. The IL6/IL8-mediated Notch/HES1 pathway in interaction between the CSCs and their microenvironment might be a target in drug development for colorectal cancer treatment. Objectives/Aims: Hydrogen sulfide (H 2S) plays a role in angiogenesis and cell proliferation— key components of tumorigenesis and metastasis. In mammals, this gasotransmitter is pro- duced mainly by cystathionine gamma-lyase (CSE) and cystathionine-β-synthase (CBS). We have shown previously that colorectal cancer (CRC) cell lines express high levels of CBS, but not CSE, and as a result, have increased levels of H 2S when compared to the normal epithelial. Thus, our objective was to identify the major source of CBS in CRC tumors and understand its pathophysiological contribution to the tumor progression. Methods: Immunostaining followed by in-situ confocal analysis was performed on CRC derived tumor tissue and adjacent normal control tissue, fixed in 1% paraformaldehyde. Non-tumorigenic colonic epithelial cell line, NCM356, and colon cancer-derived epithelial cell lines, HCT116, were also used in this study. Results: Using in-situ confocal microscopy, we first confirm our previous findings—observed on mRNA and total protein (Western blot) levels—that CBS expression is increased within CRC tumor. The increase in CBS was mostly observed within the basolateral side of epithelium and tumor stroma. Furthermore, higher CBS expression was mostly found in cells co-expressing epithelial marker, EpCAM, and stem- like cell marker, Oct-3/4, suggesting that cancer stem-like cells are mostly responsible for the increase in CBS-mediated H 2S production. We also confirmed this observation using CRC cancer cell line, HCT116, and demonstrated that CBS is mainly found within the cytosol. In contrast, normal epithelial cell, NCM356, lacked expression of CBS. Interestingly, we observed that cells expressing CBS in tumor stroma in-situ, unlike the normal control, bear an EpCAM+/α-SMA+/Oct-3/4+ phenotype. This suggests that within the CRC tumor stroma, the major cells expressing CBS are cells undergoing epithethial-mesenchymal transi- tion (EMT), preserving their cancer stem-like properties. Conclusion: Taken together, our previous and current data suggest that increase in expression of CBS within the tumor, and

Induction of boosted immune response in mice by leptospiral surface proteins expressed in fusion with DnaK.

Leptospirosis is an important global disease of human and veterinary concern. Caused by pathogenic Leptospira, the illness was recently classified as an emerging infectious disease. Currently available veterinarian vaccines do not induce long-term protection against infection and do not provide cross-protective immunity. Several studies have suggested the use of DnaK as an antigen in vaccine formulation, due to an exceptional degree of immunogenicity. We focused on four surface proteins: rLIC10368 (Lsa21), rLIC10494, rLIC12690 (Lp95), and rLIC12730, previously shown to be involved in host-pathogen interactions. Our goal was to evaluate the immunogenicity of the proteins genetically fused with DnaK in animal model. The chosen genes were amplified by PCR methodology and cloned into pAE, an E. coli vector. The recombinant proteins were expressed alone or in fusion with DnaK at the N-terminus. Our results demonstrate that leptospiral proteins fused with DnaK have elicited an enhanced immune response in mice when compared to the effect promoted by the individual proteins. The boosted immune effect was demonstrated by the production of total IgG, lymphocyte proliferation, and significant amounts of IL-10 in supernatant of splenocyte cell cultures. We believe that this approach could be employed in vaccines to enhance presentation of antigens of Leptospira to professional immune cells.

Bifidobacteria BbC50 Fermentation Products Induce Human Cd4+ Regulatory T Cells with Antigen-Specific Activation and Bystander Suppression

Probiotic bacteria have been shown to have health benefits in various situations (inflammation, allergy, infection). We previously showed that a bacteria-free fermentation product of Bifidobacterium breve C50 (BbC50sn) induced high IL-10 secretion by human dendritic cells. As IL-10 is a regulatory cytokine, the aim of the present study was to examine whether DCs cultured in the presence of BbC50sn could induce regulatory T cells in an allogeneic context. Purified CD4+CD25− human T cells were co-cultured with allogeneic BbC50sn-treated dendritic cells for 4 weeks. The T cell population (BbC50sn-T) was analysed both at phenotypical and functional [ability to inhibit a mixed lymphocyte reaction (MLR)] levels. We showed that T lymphocytes acquired phenotype characteristics of regulatory T cells after 4 weeks of co-culture with BbC50sn-DCs, and inhibited in vitro T lymphocyte proliferation and IFN-γ production in an MLR. Transwell experiments demonstrated that this suppressive activity was not T cell contact-dependent but probably mediated by a soluble factor. Although BbC50sn-T cells secreted significant amounts of IL-10 and TGF-β, their suppressive effect is most likely not mediated through these cytokines. This is, to our knowledge, the first demonstration of in vitro regulatory T cell induction by a bacteria-free fermentation product in an allogeneic context.

CD40-Licensing Of Human Dendritic Cells Further Matures Tnfa-Treated Dcs But Does Not Mediate Resistance Against Inhibition Of T-Cell Proliferation By TREG

Vaccination with cytokine-matured dendritic cells (DCs), loaded with autologous tumor lysate, represents a promising strategy in cancer immunotherapy, however, in clinical trials vaccination with this kind of DCs has shown only limited efficacy so far. In animal experiments, CD40-licensing of DCs conveys resistance to tumor-induced immunosuppression including immunomodulation of regulatory T cells (Tregs). DCs were generated by differentiation of monocytes for 7 days with GM-CSF/IL-4 followed by 48h maturation with TFNα/IL-1ß ± CD40L. Then, DC were analyzed with respect to phenotype, cytokine production, and T-cell stimulatory capacity. CD40-licensed DCs showed higher expression of CD86 (MFI 3074±630 vs. 2433±359 with vs. without CD40L, respectively, p&lt;.05) and a trend towards higher CD80, CD83 expression. Coculture of DCs with Tregs during maturation led to a reduction of costimulatory molecules, presumably by transendocytosis of Tregs. This phenomenon could be partially abrogated by CD40-licensing. TNFα/IL1ß-matured DCs produced significant amounts of IL-6, IL-8, TNFα, and IL-1ß. CD40-licensing did further increase cytokine production, however, no IL-12 could be detected. In contrast to murine DCs, a second round of LPS-stimulation after TNFα/IL1ß could not trigger IL-12 production. Control DCs matured with LPS/IFNγ showed up to 16% IL-12+ DCs. Using Melan-A as a model tumor antigen, priming capacity of CD40-licensed DCs to induce Melan-A specific CD8+ CTLs was slightly but not significantly improved compared to nonlicensed DCs, as demonstrated by somewhat higher frequencies of Melan-A multimer+ and TNFα+IFNγ+ CTLs after 11 days of culture with CD40-licensed DCs and rechallenge, respectively. Again, T-cell priming was best with control DCs matured with LPS/IFNγ. In contrast to T-cell priming, CD40-licensed DCs did not show any improved capacity to stimulate CD4+ T-helper cell proliferation. Furthermore, in a classcial MLR-suppression assay Tregs inhibited CD4+ T-helper proliferation by approx. 40%, this suppression was not alleviated by CD40-licensing of DCs. Interestingly, Treg proliferation in combined MLR-assays was increased in all experimental settings. Treg-suppression of CD4+ T-helper proliferation as well as the increased Treg-proliferation in the combined MLR-assays could not be prevented by the lysosomal inhibitor Bafilomycin A. This suggests, that other mechanisms than transendocytosis of costimulatory molcules by Tregs mediate these effects. In summary, these data show that CD40-licensing is a feasible tool to improve maturity of cytokine-treated DCs. However, CD40-licensing cannot induce IL-12 production in human DCs without TLR-stimulation and was not able to confer resistance against Treg-mediated T-cell proliferation inhibition. Thus, in order to strengthen DCs for cancer immunotherapy, CD40-licensing should be further investigated in combination with TLR-triggering DC-maturation cocktails. Disclosures: No relevant conflicts of interest to declare.

The role of myofibroblasts in upregulation of S100A8 and S100A9 and the differentiation of myeloid cells in the colorectal cancer microenvironment

Background/aimS100A8/A9 and myeloid cells in the tumor microenvironment play an important role in cancer invasion and progression, and the effect of tumor-infiltrated myofibroblasts on myeloid cells in the tumor microenvironment is relatively unknown. Accordingly, we investigated the role of myofibroblasts in the upregulation of S100A8/A9 as well as in the differentiation of myeloid cells in the colorectal cancer (CRC) microenvironment. Materials and methodsTo investigate the interactions among cancer cells, myofibroblasts, and inflammatory cells in the microenvironment of CRC, we used 10 CRC cell lines, 18CO cells and THP-1 cells, which were co-cultured with each other or cultured in conditioned media (CM) of other cells. Expression of S100A8/A9 was evaluated via Western blot, immunohistochemical staining and immunofluorescence. The secreted factors from the cell lines were analyzed using cytokine antibody array. Flow cytometry analysis was performed to analyze the differentiation markers of myeloid cells. Results18CO CM induced increased expression of S100A8/A9 in THP-1 cells. Increased expression of S100A8/A9 was noted in inflammatory cells of the peri- and intra-tumoral areas, along with myofibroblasts in colon cancer tissue. S100A8/A9-expressing inflammatory cells also exhibited CD68 expression in colon cancer tissue, and 18CO CM induced differentiation of THP-1 cells into myeloid-derived suppressor cells (MDSCs) or M2 macrophages expressing S100A8/A9. Significant amounts of IL-6 and IL-8 were detected in 18CO CM, compared to those in both controls and THP-1 CM, and tumor-infiltrated myofibroblasts expressed IL-8 in colon cancer tissue. Finally, neutralizing antibodies to IL-6 and IL-8 attenuated 18CO CM-induced increased expression of S100A8/A9. ConclusionsThe upregulation of S100A8/A9 in tumor-infiltrated myeloid cells could be triggered by IL-6 and IL-8 released from myofibroblasts, and myofibroblasts might induce the differentiation of myeloid cells into S100A8/9-expressing MDSCs or M2 macrophages in the CRC microenvironment.

Human Dendritic Cells followingAspergillus fumigatusInfection Express the CCR7 Receptor and a Differential Pattern of Interleukin-12 (IL-12), IL-23, and IL-27 Cytokines, Which Lead to a Th1 Response

Aspergillus fumigatus is the most prevalent airborne fungal pathogen and causes fatal invasive aspergillosis in immunocompromised patients. Given the essential role of dendritic cells (DC) in initiating and regulating immune responses, we investigated the impact of A. fumigatus conidial infection on human DC. A. fumigatus conidia were rapidly internalized and induced the release of tumor necrosis factor alpha within the first 8 h. After A. fumigatus infection, the majority of DC underwent full maturation, although CCR7 expression was observed only in DC that had internalized the conidia. Additionally, the analysis of regulatory cytokines showed that infected DC simultaneously produced interleukin-12p70 (IL-12p70) and significant amounts of IL-10. IL-10 neutralization was not able to further increase IL-12p70 production from infected DC. Whereas the central role of IL-12 in the generation of Th1 cells has long been appreciated, recently two other members of the IL-12 family, IL-23 and IL-27, were reported to play important roles in the regulation of gamma interferon (IFN-gamma) production from naïve and memory T cells. A. fumigatus-infected DC were also able to express high levels of IL-23p19 and low levels of IL-27p28 at later stages of infection. According to this expression pattern, A. fumigatus-infected DC were able to prime IFN-gamma production of naïve T cells. Thus, this study on the expression of the new IL-12 family members controlling the Th1 response sheds light on a novel aspect of the contribution of DC to anti-Aspergillus immunity.

Legionella pneumophilaEvades Gamma Interferon-Mediated Growth Suppression through Interleukin-10 Induction in Bone Marrow-Derived Macrophages

We examined the roles of Th1-Th2 cytokine cross talk in Legionella pneumophila-infected bone marrow-derived (BM) macrophages in the presence of costimulation with interleukin-12 (IL-12) and IL-18. Treatment with gamma interferon (IFN-gamma) alone or treatment with IL-12 in combination with IL-18 resulted in a 3- or 2-log reduction in bacterial numbers, respectively, in BM macrophages, whereas treatment with IL-12 or IL-18 alone had no effect. Significant amounts of IFN-gamma were detected in the culture supernatants of infected macrophages stimulated with IL-12 and IL-18 in combination but not independently. Neutralization of IFN-gamma by antibody completely abolished the growth inhibitory effects of IL-12 and IL-18. Interestingly, higher infectivity ratios of L. pneumophila or the addition of increasing concentrations of heat-killed bacteria (HKB) suppressed the production of IFN-gamma, which resulted in the increased intracellular growth of bacteria. Significant amounts of IL-10 were detected in culture supernatants when Legionella-infected macrophages were cocultured with HKB. Furthermore, neutralization of IL-10 by antibody resulted in an increase in IFN-gamma production by infected BM macrophages when cocultured with HKB. Treatment of HKB with trypsin but not polymyxin B attenuated the growth-promoting effects of HKB, suggesting the involvement of a protein component(s) in regulation of the growth of L. pneumophila. These findings demonstrate a crucial role of Th1-Th2 cross talk in L. pneumophila-infected BM macrophages. Our results also suggest that L. pneumophila modulates the cytokine balance from IFN-gamma-driven Th1 to more Th2 responses, likely through the induction of IL-10 by a bacterial protein component(s). These data provide new insights not only into the cellular mechanisms of Th1-Th2 cross talk in Legionella-infected macrophages but also into the pathogenesis of L. pneumophila pneumonia in humans.

Experimental Autoimmune Thyroiditis (EAT) Induced by the Thyroglobulin Peptide (2596–2608): Influence of H-2 and Non H-2 Genes

We have previously identified five thyroglobulin (Tg) peptides with Ak-binding motifs that induce experimental autoimmune thyroiditis (EAT) in CBA/J (H-2k) mice. In this study, we have examined whether H-2 or non H-2 genes can influence the immunopathogenicity of peptide p2596 (a.a. 2596–2608), which earlier elicited considerable pathology in CBA/J hosts. The p2596 peptide induced mild EAT—(infiltration index range=1–2)— in H-2-compatible AKR/J, B10.BR, and C3H/HeJ mice. Moreover, p2596-primed LNC from these mice exhibited peptide-specific proliferative responses and secreted significant amounts of IL-2 and IFN-γ in recall in vitro assays. Priming and boosting of these strains with p2596 resulted in the generation of specific IgG responses five weeks after the initial challenge. In contrast, s.c. challenge of H-2-incompatible strains such as DBA/1J (H-2q), SJL (H-2s), DBA/2J (H-2d) and C57BL/6 (H-2b) with the same peptide dose did not elicit EAT pathology and peptide-specific B- or T-cell responses. These data demonstrate the thyroiditogenic potential of p2596 in H-2k strains of diverse non-H-2 backgrounds but not in mice carrying H-2b, d, q or s haplotypes.

Mechanism of TCDD-induced suppression of antibody production: effect on T cell-derived cytokine production in the primary immune reaction of mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to suppress antigen-specific antibody production in humoral immune reactions, but the precise mechanism remains unclear. Since T cell activation and subsequent production of type 2 helper T (Th2) cell-derived cytokines are required for antigen-specific antibody production in humoral immunity, we examined the effects of TCDD on splenic T cell numbers, T cell growth factor IL-2 production, and Th2 cell-derived cytokine production. C57BL/6N mice were orally given TCDD (20 microg/kg) or vehicle, and immediately intraperitoneally immunized with ovalbumin (OVA) adsorbed to alum, and cellular changes in the spleen and cytokine production by spleen cells were investigated from Day 1 to Day 14. In vehicle-control mice the numbers of splenic CD3(+) T cells increased from Day 7 onward, but no increase was observed in the TCDD-exposed mice. When spleen cells from control mice were cultured and restimulated with OVA ex vivo, a significant amount of IL-2 was found from Day 1, but it decreased on Day 7, whereas TCDD exposure promptly suppressed the increase on Day 4. TCDD exposure significantly suppressed the production of Th2 cell-derived cytokines IL-4, IL-5, and IL-6, which were prominently increased from Day 4 onward in control mice. The dose-dependent study showed that IL-5 production was significantly suppressed in a dose-dependent manner starting at 1 microg/kg TCDD. Moreover, separation and reconstitution studies showed that the TCDD-induced suppression of IL-5 production was due to the impaired function of T cells rather than that of antigen-presenting cells. The results of this study suggest that TCDD-induced suppression of T cell activation and Th2-type cytokine production is involved in the impairment of antigen-specific antibody production.

INHIBITION OF THE ALLOGRAFT RESPONSE BY DONOR SPECIFIC BLOOD TRANSFUSION: ASSOCIATION WITH REDUCED LOCAL TH1 CYTOKINES AND NITRIC OXIDE BUT ENHANCED PROSTAGLANDIN E 2 PRODUCTION1

Donor-specific blood transfusion (DST) may improve allograft survival in human and animal models, but the mechanisms for this graft protective effect are incompletely understood. The sponge matrix allograft model was used to determine if DST induces regulatory factors within the allograft. C57BL/6 (H-2b) recipients received donor-specific (DBA/2J, H-2d) or syngeneic (C57BL/6) blood 7 days before sponge matrix allograft (DBA/2J) implantation. Fourteen days postgrafting, the sponge infiltrating cells (SIC) were examined for cytotoxic T cell (CTL) and natural killer (NK) activity, and sponge exudate fluid (SEF) was assessed for nitric oxide (.N=O) and prostaglandin E2 (PGE2) content. Interleukin- (IL) 2, IL-4, IL-10, and interferon-gamma (IFN-gamma) production by SIC was also determined. Recipient splenocytes were simultaneously assessed for anti-donor cytotoxic and proliferative responses and .N=O production. SIC from mice receiving syngeneic transfusions (ST) acquired both CTL and NK activity postgrafting, with maximal activity by day 14. DST suppressed both CTL and NK activity throughout the postgrafting period. Limiting dilution analysis (LDA) of SIC to determine precursor and native CTL frequency showed significantly lower responder cell frequency after DST compared with ST. SEF .N=O levels and SIC production of IL-2 and IFN-gamma in grafted DST mice were significantly lower than in grafted mice receiving ST. No significant amounts of IL-4 and very low levels of IL-10 were produced by SIC from grafted mice after either ST or DST. Conversely, PGE2 content of sponge fluid and serum from DST mice was higher than in mice receiving ST. Antigen stimulated splenocyte proliferation and CTL development assessed by LDA were also inhibited by DST. Reduction in local TH1 cytokines, absence of detectable TH2 cytokines, with enhanced PGE2 and depressed .N=O were observed in the local graft environment after DST. These data support the hypothesis that DST induces donor-specific intragraft suppressor factors, accompanied by reduced local and systemic immune activation.

Spectrum of gut immunologic reactions: selective induction of distinct responses to Vibrio cholerae WO7 and its toxin.

Past studies with Vibrio cholerae have shown that cholera toxin (CT) is mainly responsible for inducing T helper type 2 (Th2) responses with systemic IgG1, IgE and mucosal secretory IgA (sIgA) antibodies. In this study, V. cholerae WO7, which produces novel toxin unrelated to CT, was given orally to mice in order to determine whether the strain V. cholerae WO7 differs from V. cholerae 569B, which produces CT, in the nature of responses generated at the gut and splenic level. The analysis of immune responses evoked by V. cholerae WO7 in the gut of mice revealed striking differences as compared to those elicited by V. cholerae 569B infection. To assess the T helper cell type responses, lymphocytes from Peyer's patches and the spleen were stimulated in vitro for studying the cytokine patterns. PP and SP lymphoid cells from V. cholerae WO7 infected animals elaborated significant amounts of IL-2, IFN-gamma and IL-12 by 7 days p.i., suggesting a Th1 type of response. However by 15 days p.i., the PP and SP lymphoid cells secreted only IL-6 and IL-10 with traces of IFN-gamma. On the other hand, infection with V. cholerae 569B yielded mainly Th2 type responses at Peyer's patches as well as the splenic level. Infection with both V. cholerae WO7 and 569B induced toxin-specific IgA secreting cells at the gut and splenic level along with IgG1 secreting cells, indicating that both V. cholerae WO7 and 569B evoke an antigen-specific Th2 type of response in the gut as well as spleen. The persistence of IgA along with Th1-type cytokines indicates an alternate induction mechanism since mucosal IgA responses are usually associated with Th2-type responses. These observations are suggestive of a common mechanism employed by the host to clear different strains of V. cholerae infection (569B and WO7 in this case), while the nature of toxins elaborated failed to modulate the net outcome of the infection caused by V. cholerae.

Alterations in Cytokine Production Following Intraocular Injection of Soluble Protein Antigen: Impairment in IFN-γ and Induction of TGF-β and IL-4 Production

AbstractImmune deviation induced by intraocular injection of soluble protein Ag, referred to as anterior chamber-associated immune deviation (ACAID), is characterized by impairment of delayed hypersensitivity (DH). Two populations of splenic regulatory cells that impair the induction and expression phases of DH are involved in the ACAID response and may mediate their effects through cytokines. The purpose of the present study was to evaluate the role that cytokines play in ACAID. IFN-γ production in draining lymph nodes induced by conventional immunization with protein Ag and adjuvant was suppressed after intraocular injection of protein Ag administered either before or after sensitization; IL-12 production in these mice was not decreased, suggesting that suppression of IL-12 may not be the mechanism involved in the impairment in IFN-γ production. Surprisingly, although significant amounts of IL-4 (but not IL-10) were produced by spleen and lymph node cells from several different strains of mice, experiments in IL-4 knockout mice showed that impairment of neither DH nor IFN-γ production required IL-4. Interestingly, significant levels of TGF-β were detected in cultures of spleen cells from mice with ACAID. As determined by quantitative RT-PCR, TGF-β was produced primarily by the splenic CD4 and non-T cells and was of the TGF-β1 type. These results suggest that the Th1 response is impaired in ACAID by a mechanism(s) that does not require Th2-type cytokines, but may involve TGF-β at several different (including the effector) phases during the response.

Murine Schistosoma bovis infection: analysis of parasitic and immune parameters.

Humoral and cellular responses to Schistosoma bovis antigens have been evaluated over a period of 11 weeks in mice exposed to S. bovis cercariae and data analysed in the context of the parasitic parameters (worm and egg loads) recorded at days 30, 60 and 80 of the ongoing infection. Results revealed a decrease of worm burden, particularly marked for female worms, between day 60 and day 80 of infection suggesting a higher susceptibility of female schistosomes to attrition mechanisms. The B-cell response, studied by measuring the production of different isotypes, was directed against different stage specific antigens, with a predominance of IgG1 antibodies associated with a significant increase of IgA and IgE antibodies after egg deposition. The T-cell response, assessed after in vitro stimulation of splenocytes, showed a predominant production of Th-2 cytokines (IL-4, IL-5 and IL-10) occurring after egg laying. Interestingly in contrast to S. mansoni infection the Th-2 polarization did not seem to be exclusively triggered by egg-associated antigens since significant amounts of IL-10 were produced after stimulation with adult worm antigen preparation (SWAP) before the beginning of egg deposition.

Studies on the mechanism and specificity of the effect of the synthetic random copolymer GLAT on graft-versus-host disease

Graft-versus-host disease (GVHD), which occurs when donor T-cells recognize multiple minor host histocompatibility antigens as non-self, presents the major limitation to successful allogeneic bone-marrow transplantation. The synthetic random copolymer of the amino acids, l-Glu, l-Lys, l-Ala and l-Tyr, termed GLAT, with promiscuous binding to multipleMHC class II alleles, reduces the incidence, onset and severity of disease in the B10.D2 → BALB c model of lethal GVHD. GLAT inhibited the proliferative response towards host of both spleen cells from mice with GVHD and also of the effector T cell line established from these mice. Administration of GLAT for a limited period after transplantation completely abolished the cytotoxic activity toward host cells exerted by spleen cells from mice with GVHD. Whereas spleen and bone marrow cells from control mice with GVHD secreted IL-2 and INF-γ when cocultured with host cells, these inflammatory cytokines could not be detected in supernatants of cells from GLAT treated mice. Moreover spleens and bone marrow cells from GLAT treated mice secreted small but significant amounts of IL-4 and IL-6 when cocultured with GLAT, suggesting that GLAT not only inhibits pro-GVHD cytokines but also causes a beneficial effect by inducing secretion of Th2 type cytokines. GLAT binds strongly to MHC molecules of host as well as donor haplotype. d-GLAT, identical to GLAT but composed of d-amino acids is also effective in preventing GVHD. d-GLAT does not cross-react with l-GLAT, but still binds strongly to MHC-class II molecules. These findings indicate that MHC blocking is involved in the therapeutic effect of GLAT on GVHD. The cumulative data demonstrate that GLAT modulates the effector mechanisms involved in GVHD, and can be potentially used for the prevention of GVHD across minor histocompatibility barriers.

Expression of IL-6, IL-8, and RANTES on human bronchial epithelial cells, NCI-H292, induced by influenza virus A

Bronchial epithelial cells are primary sites of airway viral infection, and these cells may play an important role in the pathogenesis of respiratory diseases. It has recently been reported that bronchial epithelial cells express RANTES. RANTES attracts monocytes, T cells, eosinophils, and basophils; it can also activate eosinophils. To determine whether viral infection induces RANTES expression on bronchial epithelial cells, we infected a bronchial epithelial cell line, NCI-H292, with influenza virus A (H3N2). We then examined the concentration of RANTES in the culture medium of infected cells by ELISA and assessed expression of the gene for RANTES by the reverse-transcriptase polymerase chain reaction. We also investigated the concentrations of IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor in the medium of infected cells, because some virus infections have been reported to induce expression of these cytokines on bronchial epithelial cells, but there are few data concerning influenza virus infection. Small amounts of IL-6 and IL-8 were detected in the medium of uninfected cells. RANTES was not detected in the medium of uninfected cells. After influenza virus infection, significant amounts of IL-6, IL-8, and RANTES were released into the culture medium of infected cells, and RANTES messenger RNA was detected from infected cells. Granulocyte-macrophage colony-stimulating factor was not detected in the medium of uninfected and infected cells. These results suggest that influenza virus infection may stimulate production of IL-6, IL-8, and RANTES from human bronchial epithelial cells and that these cytokines may contribute to the pathogenesis of airway inflammatory diseases caused by influenza virus infection. (J Allergy Clin Immunol 1996;98:1080-7.)