Spleen Cell Suspensions Research Articles

Effects of a fusion protein LTβR-Fc on ovalbumin-induced dermatitis in a mouse model

Objective To evaluate effects of a fusion protein LTβR-Fc, which can block the herpesvirus entry mediator ligand (LIGHT-HVEM) signaling pathway, on ovalbumin-induced dermatitis in a mouse model. Methods Thirty BALB/c mice were randomly and equally divided into 3 groups: blank control group treated with 100 μl of sodium chloride physiological solution, model group sensitized with 100 μl of sodium chloride physiological solution containing 100 μg ovalbumin, blocker group firstly blocked with 100 μl of sodium chloride physiological solution containing 100 μg LTβR-Fc followed by sensitization with 100 μl of sodium chloride physiological solution containing 100 μg ovalbumin at 24 hours after the blocking. Disease severity was evaluated by eczema area and severity index (EASI) score, and lesional size was measured on day 0, 4, 8, 12, 15, 20, 23, 27, 31 and 34 after the first sensitization. A total of three sessions of sensitization were carried out. At the end of treatment, all the mice were sacrificed after serum was obtained from their orbital cavities. Thereafter, tissue specimens were obtained from skin lesions, and single cell suspensions of the spleen were prepared. RT-PCR was performed to detect mRNA expressions of interferon γ (IFN-γ) , interleukin 4 (IL-4) and IL-5 in murine lesions, ELISA to measure IFN-γ, IL-4 and IL-5 levels in culture supernatants of murine splenocytes, as well as ovalbumin-specific and total IgE and IgG1 levels in murine sera. Results LTβR-Fc significantly suppressed inflammatory response in the mouse model of dermatitis induced by ovalbumin. Compared with the model group, the blocker group showed significantly decreased lesion area and EASI score (both P<0.05). In addition, a significant decrease was observed in the mRNA expressions of IL-4 (0.88 ± 0.25 vs. 1.81 ± 0.25, P<0.05) , IL-5 (0.75 ± 0.15 vs.1.24 ± 0.26, P<0.05) and IFN-γ (0.62 ± 0.09 vs. 1.11 ± 0.19, P<0.05) in murine lesions, and in supernatant levels of IL-4 (9.58 ± 1.44 ng/L vs. 20.12 ± 5.39 ng/L, P<0.05) , IL-5 (11.37 ± 2.02 ng/L vs. 22.77 ± 4.07 ng/L, P<0.05) and IFN-γ (16 167 ± 950.40 ng/L vs. 23 930 ± 44.20 ng/L, P<0.05) in the blocker group compared with the model group. The serum levels of both total IgE and ovalbumin-specific IgE were significantly lower in the blocker group than in the model group (total IgE: 27 466.67 ± 2 052.64 μg/L vs. 32 277 ± 407.53 μg/L, P<0.05; ovalbumin-specific IgE: 1 296.33 ± 32.72 μg/L vs. 2 323.33 ± 502.43 μg/L, P<0.05) , so were those of total IgG1 (0.46 ± 0.11 μg/L vs. 0.84 ± 0.11 μg/L, P<0.05) and ovalbumin-specific IgG1 (0.62 ± 0.11 μg/L vs. 0.86 ± 0.07 μg/L, P<0.05). Conclusion The fusion protein LTβR-Fc can alleviate symptoms of ovalbumin-induced dermatitis in the mouse model likely by suppressing the LIGHT-HVEM signaling pathway, suggesting that this signaling pathway may serve as a target for the treatment of dermatitis (such as atopic dermatitis). Key words: Dermatitis, atopic; Models, animal; Ovalbumin; T-lymphocytes, helper-inducer; Interleukins; Interferon-gamma; LIGHT-HVEM

Boron Induces Lymphocyte Proliferation and Modulates the Priming Effects of Lipopolysaccharide on Macrophages.

Chemical mediators of inflammation (CMI) are important in host defense against infection. The reduced capacity of host to induce the secretion of these mediators following infection is one of the factors in host susceptibility to infection. Boron, which has been suggested for its role in infection, is reported in this study to increase lymphocyte proliferation and the secretion of CMI by the lipopolysaccharide (LPS)-stimulated peritoneal macrophages in BALB/c mice. Boron was administered to mice orally as borax at different doses for 10 consecutive days, followed by the stimulation of animals with ovalbumin and isolation of splenocytes for proliferation assay. The lymphocyte subsets were determined by flow cytometry in spleen cell suspension. The mediators of inflammation, TNF-α, IL-6, IL-1β and nitric oxide (NO), were measured in culture supernatant of LPS-primed macrophages isolated from borax treated mice. TNF and ILs were measured by ELISA. NO was determined by Griess test. The expression of inducible nitric oxide synthase (iNOS) in macrophages was studied by confocal microscopy. Results showed a significant increase in T and B cell populations, as indicated by an increase in CD4 and CD19, but not CD8, cells. Boron further stimulated the secretion of TNF-α, IL-6, IL-1β, NO and the expression of iNOS by the LPS-primed macrophages. The effect was dose dependent and most significant at a dose level of 4.6 mg/kg b. wt. Taken together, the study concludes that boron at physiological concentration induces lymphocyte proliferation and increases the synthesis and secretion of pro-inflammatory mediators by the LPS-primed macrophages, more specifically the M1 macrophages, possibly acting through Toll-like receptor. The study implicates boron as a regulator of the immune and inflammatory reactions and macrophage polarization, thus playing an important role in augmenting host defense against infection, with possible role in cancer and other diseases.

IL-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4+ T Cells

ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4+ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4+ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4+ T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4+ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4+ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production of IL-17 by CD4+ T cells in two major ways: (I) through the induction of IL-23 and IL-1 by APCs and (II) through the direct interaction with CD3 on the CD4+ T cells. This study contributes to elucidation of mechanisms accounting for the property of ArtinM in inducing Th17 immunity and opens new perspectives in designing strategies for modulating immunity by using carbohydrate recognition agents.

Detection of cross-sex chimerism in the common marmoset monkey (Callithrix jacchus) in interphase cells using fluorescence in situ hybridisation probes specific for the marmoset X and Y chromosomes.

Chimerism associated with placental sharing in marmosets has been traditionally analysed using conventional chromosome staining on metaphase spreads or polymerase chain reaction. However, the former technique requires the presence of proliferating cells, whereas the latter may be associated with possible blood cell contamination. Therefore, we aimed to develop a single-cell analysis technique for sexing marmoset cells. We applied fluorescent in situ hybridisation (FISH) to cell nuclei using differentially labelled X and Y chromosome-specific probes. Herein we present the validation of this method in metaphase cells from a marmoset lymphoblastoid cell line, as well as application of the method for evaluation of cross-sex chimerism in interphase blood lymphocytes and haematopoietic bone marrow cells from marmosets of same- and mixed-sex litters. The results show conclusively that haematopoietic cells of bone marrow and leucocytes from blood are cross-sex chimeric when the litter is mixed sex. In addition, single samples of liver and spleen cell suspensions from one individual were tested. Cross-sex chimerism was observed in the spleen but not in liver cells. We conclude that FISH is the method of choice to identify cross-sex chimerism, especially when combined with morphological identification of nuclei of different cell types, which will allow a targeted tissue-specific analysis.

High-dose immunosuppressant alters the immunological status of New Zealand white rabbits following skin transplantation.

The aim of this study was to investigate the effect of an immunosuppressant on the immunological status of New Zealand white rabbits after skin grafting, and to evaluate a method for monitoring the immunological status of subjects with skin transplants. The rabbits were randomly divided into allograft rejection, autograft tolerance, nontransplant, allograft low-dose immunosuppressant and allograft high-dose immunosuppressant groups. The rabbits in the low- and high-dose immunosuppressant groups were treated with cyclosporine A intravenously 8 h prior to skin transplantation and once daily following transplantation at doses of 2 and 25 mg/kg, respectively. At 12 days after skin transplantation, the spleens of donor (female) rabbits and recipient (male) rabbits were harvested for the preparation of single-cell suspensions. The splenocytes from recipient and donor rabbits were labeled with 0.3 or 6 µM carboxy fluorescein diacetate succinimidyl ester, respectively, and a mixed cell suspension was prepared. The final preparation was intravenously injected into recipient New Zealand white rabbits. The ratio of the two fluorescently labeled cell populations in the peripheral blood was measured using flow cytometry at 1, 2, 4 and 8 h after the injection, and the cell death rate was calculated. Histological analysis was also performed on samples collected at the time of splenectomy. The cell death rates of the allograft rejection and low-dose immunosuppressant groups reached their highest levels 8 h after the injection of spleen cell suspension. Allogeneic spleen cells from donor male rabbits were almost completely removed within 8 h of injection. The cell death rate increased slowly in the nontransplant, autograft and high-dose immunosuppressant groups without specificity. This study provides a specific method for the in vivo monitoring of the immunological status of patients after skin grafting. This method can quickly and accurately detect the immunological status of recipients following the injection of a mixed splenocyte suspension, thereby indicating the strength of immune rejection by the immune systems of the recipients.

What's in a Name? Some Early and Current Issues in Dendritic Cell Nomenclature.

The name dendritic cell (DC) was given by Steinman to describe the unusual cell type he saw in spleen cell suspensions. This morphological description is not sufficient to specify the cell of so much interest to immunologists; many cells can adopt a similar form. A useful functional definition evolved as Steinman and colleagues explored the immunological properties of this novel cell type (1). DCs were considered as antigen collecting and processing cells able to present antigen on MHC molecules and efficiently activate even primary T-cells. Nowadays, immunologists would likely add to this definition, a capacity to sense the context in which the antigen was collected, via receptors for pathogen or damaged cell-derived material. Why might we need to go beyond the name “dendritic cell” for cells with these well-understood functions? Some limitations of this single name arose early in DC research. This article surveys some problems of definition encountered in past work from our own laboratory. The problems we encountered arose from two sources, the first the discovery of different DC subsets and the need to determine whether these represented different maturation states or separate sub-lineages. The second was the difficulty in distinguishing these DC subsets from macrophages.

Abstract T P231: B- Cell Depletion Decreases Lesion Burden in a Murine Model of Cerebral Cavernous Malformation Disease

Introduction: The cerebral cavernous malformation (CCM), a common cerebrovascular anomaly, predisposes patients to a lifetime risk of seizures, hemorrhagic stroke, and other neurological sequelae. Previous studies revealed robust inflammatory cell infiltration and selective synthesis of IgG within the lesions. Recently, in situ antigen-driven B- cell clonal expansion and immune complex deposits were demonstrated in CCM lesions. However, we do not know whether B cells indeed could play a role in CCM genesis and maturation. Therefore we hypothesize B-cell depletion could inhibit CCM development in a murine model of CCMs. Methods: Based on the two-hit hypothesis, we crossed Ccm3 heterozygotes into a Trp53 background, a mouse model that demonstrated 100% penetrance with severe lesion burden. Three weeks after birth, 5 CCM3+/-Trp53-/- mice were assigned into treatment group, receiving anti-mouse BR3 (Genentech) to deplete B cells with IP injection once a week (5mg/kg/week) for a total of 12-15 weeks. At 4 months of age, the mouse brains were harvested and fixed. B cell depletion was demonstrated by FACS analysis of peripheral blood and spleen cell suspensions, further confirmed in the spleens via immunohistochemistry. CCM lesion burden was assessed using either poisson regression (number) or student t test (area). Results: CD19+, CD22+, and CD45R/B220+ B cells of peripheral blood and spleen cell suspensions was significantly decreased in all 5 CCM3+/-Trp53-/- mice treated with anti-mouse BR3, as compared with those in the control group. B cell depletion was further confirmed in spleen. Anti-mouse BR3-treated mice exhibited a significantly lower CCM burden, including the number of ballooned capillaries (stage 1) and multicavernous lesions (stage 2), and the areas of stage 2 lesions per animal, as compared to 15 placebo mice. Infiltrated B cells in CCM3+/-Trp53-/- CCM lesions were also depleted. Conclusions: This represented the first report of therapeutic benefit of B- cell depletion therapy in the development and progression of CCMs and provide proof of principle that B cells is a critical step in CCM lesion genesis and maturation.

Aberrant PD-L1 Expression in CLL As a Result of Adaptive Immune Resistance Mediated By Tumor-Secreted Circulating miRNA Binding to Toll-like Receptor 7

Background: Chronic lymphocytic leukaemia (CLL) is a model cancer to study immune evasion via PD-L1/PD-1 signalling: aberrantly expressed PD-L1 on CLL and PD-1 on CD8 T cells are key mediators of poor anti-tumor immune responses, which are a major hallmark of CLL. Several preclinical studies suggest that aberrant PD-L1 expression is a result of adaptive immune resistance and is induced during immune responses within the tumor microenvironment. It has recently been proposed that specific circulating microRNAs (miRNAs) shed by malignant cells participate in the complex crosstalk between cancer cells and microenvironment, and that they activate immune cells via Toll-Like Receptor (TLR) 7 due to their structural similarity to its natural ligand. In addition, TLR signaling has been demonstrated to result in the upregulation of costimulatory molecules such as CD80, CD86 and PD-L1/2. In CLL, aberrant circulating miRNA and TLR expression patterns have been well characterized. Therefore, we hypothesized that aberrant PD-L1 expression in CLL is a result of continuous TLR7 signaling mediated by circulating miRNAs. Our aims were to demonstrate that (1) specific circulating miRNAs induce PD-L1 expression and have functional consequences, (2) miRNA/PD-L1 associations are mediated via TLR7, and (3) miR/TLR/PD-L1 interactions are subject to the dynamics of CLL development.Methods and Materials: Mononuclear cells from spleen cell suspensions from 3 month old TCL1 transgenic, wild-type (WT) or TLR7-/- mice (total n=13) were treated ex vivo for 18hours with liposomal formulations of synthetic scrambled miRNA or miRNAs -16 (negative control), -21, -29, -150 and -155, which are reported to have an effect on immune cells and to be released by CLL cells. Specific TLR7 and TLR9 agonists were included for comparison. Primary human CLL cells and healthy B cells were treated with specific TLR2/6, TLR7 and TLR9 agonists. In adoptive transfer experiments, young WT mice (n=15) were injected with 4x107CLL cells from TCL1 transgenic mice. Mice were sacrificed at days 3, 6, 9, 12 and 15, and spleen cells were treated ex vivo as above. Changes in surface PD-L1, CD69 and CD86 expression on DAPI-negative CD19+ B cells/CLL cells were determined by flow cytometry. Supernatant cytokines were screened by multiplex ELISA.Results: PD-L1 surface expression on spleen B cells from both WT and non-leukemic TCL1 mice was strongly induced by miRs -21 and -29, and moderately by -150 and -155, but not by miR-16 negative control. The degree of PD-L1 upregulation by miRs -21 and -29 was comparable to the effect of direct TLR7 and TLR9 binding by specific agonists. Similar patterns were seen for CD69 and CD86 expression. Across treatment conditions, PD-L1 expression was highly correlated with the expression of CD69 (r .7777, p<.0001) and CD86 (r .7516, p<.0001). This observation strongly suggests that PD-L1 expression after TLR engagement is a marker of activation/costimulation, and therefore a physiological adaptive immune response to TLR binding in healthy B cells. Functionally, miR treatments resulted in increased IL-6, IL-10 and TNFα, with miR-29 having the strongest effect. PD-L1, CD69 and CD86 could also be induced by TLR engagement in healthy B cells, but not in CLL patient cells, where PD-L1 was confirmed to be already aberrantly expressed. To elucidate when in the course of CLL development PD-L1 expression becomes aberrant and if it ceases to be inducible by miR treatment, we sacrificed adoptively transferred mice every 3 days to simulate tumor development. With increasing CLL the magnitude of the fold-change of PD-L1 expression following miR treatment decreased substantially, and the baseline expression of miR-untreated B cells increased consistently until day 15. Interestingly, although the PD-L1 response was substantially decreased with tumor load, it was not completely abrogated, even on day 15 when mice had a median CLL load of 71%. Importantly, miR treatment did not result in increased PD-L1, CD69 or CD86 expression in B cells from TLR7-/- mice, indicating that the miR/PD-L1 interactions are indeed mediated by TLR7.Conclusions: Our findings support that PD-L1 expression on B cells can be induced by specific miRNAs known to be produced by CLL cells, and that this effect is mediated via TLR7. Therefore, aberrant PD-L1 expression on CLL is likely to be a result of adaptive immune resistance mediated by tumor cell-produced circulating miRNAs. DisclosuresGribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

On discovering thymus-marrow synergism.

In the 1960s, the thymus was an organ of mystery. Although it was full of lymphocytes, they made no antibodies. Furthermore, thymectomy (in mature animals) failed to produce immunological inadequacy. This situation changed when J.F.A.P. Miller did neonatal thymectomy, which was indeed followed by a syndrome including crippling of the immune response (1–3). The possible role of the thymus was the focus of a several-day symposium organized by Robert A. Good in November, 1962 in Minneapolis. Its proceedings, The Thymus in Immunobiology, summarized the clinical and experimental data (4). This mystery organ intrigued me. Systemically, immunized animals did not make antibody in the thymus as they did in lymph nodes or spleen. Maybe, we thought, there was some kind of blood–thymus barrier, which prevented systemic antigen from interacting with thymocytes in the thymic parenchyma. We used adoptive transfer of syngeneic cells into irradiated recipients. In this model, spleen cell suspensions responded to sheep erythrocyte (sheep red blood cells, SRBC) antigens by making hemolytic antibody in the recipient spleens and serum. Would thymus cell suspensions similarly prepared (so as to break any blood–thymus barrier) do the same? I was a young investigator working in the late David W. Talmage’s lab at the University of Colorado Medical School in Denver. It was a stimulating environment. Edward A. Chaperon and R. Faser Triplett were post-doctoral fellows. On day 0, the mice were irradiated and then injected i.v. with spleen cells or thymus cells. On day 1, we injected the SRBC antigen IV. On day 5, we sacrificed the mice and looked for anti-SRBC-producing cells in the recipient spleens. The results were clear-cut. Recipients of donor spleen cells made many antibody-producing cells while recipients of donor thymus cells did not. Perhaps, we thought, 4-days of exposure to antigen after transfer might have been sufficient to get the mature spleen cells to make antibody but insufficient for the (putatively) immature thymocytes to do the same. We needed to lengthen the protocol. The next experiments were identical on days 0 and 1, but on day-4 recipients got a booster injection of SRBC antigen, and we planned to sacrifice on day 8. This worked well in the group that received spleen cells but the recipients of thymus cells (the test group) were all dead by day 8. We figured that this represented radiation death in the thymus recipients, whereas the spleen recipients survived because of the hematopoietic stem cells in the inoculum. We knew of the radio-protective effects of bone marrow cells, so it made sense to add an aliquot of such cells to the thymus inoculum. Indeed, the recipients of thymus-plus-bone marrow survived until day 8, but to our surprise, these recipients produced almost as much antibody as did spleen recipients. (Later we added a new group as another control, i.e., bone marrow cells only. They caused no significant antibody production.) We called this phenomenon “thymus–marrow synergism,” and it was the first demonstration that two (presumably lymphoid) cell populations were needed for significant antibody production. We speculated that one sort of cell (the “effector”) made the antibody while another variety of cell from the other inoculum performed in an “auxiliary” mode. On the basis of indirect evidence, we postulated that the bone marrow provided the effector cells and the thymus cells were “auxiliary.” Support for this view had to await the definitive experiments by others. Our experiments were published in 1966 in Proc. Soc. Exptl. Biol. Med. (5). The paper was widely acknowledged to have demonstrated cell–cell interaction in the antibody response. Additional findings by the three of us were considered important enough many years later when they were chosen as the first article to be mentioned in the Journal of Immunology’s new historical series, Pillars of Immunology. This discovery was unexpected – almost representing serendipity. Not everyone was convinced. However, others used the paradigm to provide further elucidation of the mechanism of thymus–marrow synergism. Mitchell and Miller made great progress by identifying the antibody-forming cell as originating in the bone marrow (6). Additionally, Avrion Mitchison added the brilliant insight that the carrier effect was an example of T–B collaboration where the anti-hapten antibody was made by bone marrow-derived cells while the thymus-derived cells provided “help” (7–9).

Trafficking of Intra-Graft Donor T Cells After Skin Transplantation in Mice.

It is firmly established that the skin contains T lymphocytes including significant numbers of FoxP3+ regulatory T cells (Tregs). However, their role in skin transplantation is still unknown. Recent evidence has been provided by W. Burlingham's laboratory suggesting that intra-graft T cells may influence the alloresponse and rejection process (bi-directional alloimmunity). In this study, we investigated the trafficking of intra-graft donor T cells to the recipient's lymphoid organs after skin transplantation in mice. To test this C57BL/6 (B6) mice received a skin transplant from a double transgenic B6 donor whose T cells expressed DSred while FoxP3 expression was associated with GFP (Tregs being double positive). At different time points after transplantation, cell suspensions of recipients' lymph nodes, spleen and thymus were prepared. The presence and characterization of fluorescent donor cells was investigated using Image Stream (Amnis) technology. First, the presence of T cells (DSred+ FoxP3-) was detected only at d4-d7 posttransplantation i.e. after skin graft revascularization and essentially in the host's thymus. On the other hand, double positive (DSred+ FoxP3+) Tregs were found in all recipient's lymphoid organs. Surprisingly, high frequencies of single positive GFP+ cells were found in both ipsilateral lymph nodes and thymus. The nature of these cells is being investigated. In summary, our results show that most T cells leaving the graft are Tregs spreading through all recipient's lymphoid organs while intra-graft effector T cells migrate primarily to the host's thymus.

Exploring the innate immunological response of an alternative nonhuman primate model of infectious disease; the common marmoset.

The common marmoset (Callithrix jacchus) is increasingly being utilised as a nonhuman primate model for human disease, ranging from autoimmune to infectious disease. In order to fully exploit these models, meaningful comparison to the human host response is necessary. Commercially available reagents, primarily targeted to human cells, were utilised to assess the phenotype and activation status of key immune cell types and cytokines in naive and infected animals. Single cell suspensions of blood, spleen, and lung were examined. Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values. Comparison of the activation status of cells following experimental systemic or inhalational infection exhibited different trends in different tissues, most obvious in cell types active in the innate immune response. This work significantly enhances the ability to understand the immune response in these animals and fortifies their use as models of infectious disease.

Intrinsic predisposition of naïve cystic fibrosis T cells to differentiate towards a Th17 phenotype

BackgroundCystic fibrosis (CF) is a complex, multi-system, life-shortening, autosomal recessive disease most common among Caucasians. Pulmonary pathology, the major cause of morbidity and mortality in CF, is characterized by dysregulation of cytokines and a vicious cycle of infection and inflammation. This cycle causes a progressive decline in lung function, eventually resulting in respiratory failure and death. The Th17 immune response plays an active role in the pathogenesis of CF pulmonary pathology, but it is not known whether the pathophysiology of CF disease contributes to a heightened Th17 response or whether CF naïve CD4+ T lymphocytes (Th0 cells) intrinsically have a heightened predisposition to Th17 differentiation.MethodsTo address this question, Th0 cells were isolated from the peripheral blood of CF mice, human CF subjects and corresponding controls. Murine Th0 cells were isolated from single spleen cell suspensions using fluorescence-activated cell sorting. Lymphocytes from human buffy coats were isolated by gradient centrifugation and Th0 cells were further isolated using a human naïve T cell isolation kit. Th0 cells were then assessed for their capacity to differentiate along Th17, Th1 or Treg lineages in response to corresponding cytokine stimulation. The T cell responses of human peripheral blood cells were also assessed ex vivo using flow cytometry.ResultsHere we identify in both mouse and human CF an intrinsically enhanced predisposition of Th0 cells to differentiate towards a Th17 phenotype, while having a normal propensity for differentiation into Th1 and Treg lineages. Furthermore, we identify an active Th17 response in the peripheral blood of human CF subjects.ConclusionsWe propose that these novel observations offer an explanation, at least in part, for the known increased Th17-associated inflammation of CF and the early signs of inflammation in CF lungs before any evidence of infection. Moreover, these findings point towards direct modulation of T cell responses as a novel potential therapeutic strategy for combating excessive inflammation in CF.

Structural studies of an immunoenhancing glucan of an ectomycorrhizal fungus Ramaria botrytis

A water-soluble glucan was isolated from the alkaline extract of an ectomycorrhizal fungus, Ramaria botrytis. On the basis of sugar analysis, methylation analysis, Smith degradation, partial hydrolysis, and 1D/2D NMR studies, the structure of the repeating unit of the glucan was established as: [Display omitted] This glucan showed immunostimulating activity by NO production on RAW 264.7, a murine macrophage cell line. Splenocyte and thymocyte proliferation were measured using, respectively, single cell suspensions of spleen and thymus obtained from normal mice.

Study on inhibitory effect of combined administration of bear bile powder and cyclophosphamide on colorectal cancer liver metastasis by regulating tumor microenvironment

To explore the inhibitory effect of combined administration of bear bile powder (BBP) and cyclophosphamide (Cytoxan, CTX) on colorectal cancer liver metastasis by regulating tumor promotion inflammation microenvironment. The CRC liver metastasis mode in mice was established through in situ spleenic injection of SL4 tumor cells into spleens. The mice were randomly divided into 5 groups: the model group, the CTX (80 mg x kg(-1)) treatment group, the CTX + BBP high dose (300 mg x kg(-1)) group, the CTX + BBP middle dose (150 mg x kg(-1)) group and the CTX + BBP low dose (75 mg x kg(-1)) group. Mice were orally administered with drugs for 12 days, and sacrificed on the 13'h day for weighing their spleens and lives, HE staining, and immunofluorescence analysis. Their peripheral blood, and metastatic tumor in spleens and lives were analyzed with flow cytometry. Spleen and liver weights of the: CTX treatment group and other doses groups were significantly lower than that of the model group. HE staining and immunofluorescence analysis showed that lymphocyte infiltration was detected in normal tissues, and macrophages infiltration was observed around the tumor tissues. Flow cytometry analysis showed that the number of T-lymphocytes in peripheral blood of different doses groups were much higher than that of the CTX treatment group (P < 0.05), with the rise in the ratio of CD4/CD8; the total number of lymphocytes in spleen cell suspension increased in different doses groups, compared to the CTX treatment group, with notable increase in B cells (P < 0.05) and significant decrease in CD11b, F4/80 cells (P < 0.05). The combined treatment showed less monocyte macrophages in liver metastasis than that of the CTX treatment group. The combined treatment of bear bile powder and cyclophosphamide has the effect in not only protecting liver and increase immunity, but also in anti-inflammation and antitumor by regulating tumor microenvironment and reducing the collection of mononuclear macrophages. Particularly, the combined administration of low dose of bear bile powder and CTX shows the most significant effect in reducing inflammatory cell infiltration.

Single-Cell Analysis Reveals Isotype-Specific Autoreactive B Cell Repertoires in Sjögren’s Syndrome

Microengraving is a novel technology that uses an array of microfabricated subnanoliter wells to isolate and characterize secreted proteins from larger number of single cells. This printing technique permits the capture and characterization of secreted antibodies on glass slides. Here, we profiled the antigenic repertoires of B cells reacting against salivary gland tissues in Sjögren’s syndrome (SjS), an autoimmune disease targeting the exocrine glands. Single-cell suspensions of spleen and cervical lymph node cells prepared from normal C57BL/6 and SjS-susceptible (SjSs) C57BL/6.NOD-AecAec2 mice were dispersed into subnanoliter wells (nanowells). Capture slides preincubated with mouse immunoglobulins were used for printing. Detection antibodies included fluorescence conjugated anti-IgG1, salivary gland lysates of C57BL/6 and SjSs mice. Results indicate an increase in the frequency of IgG1-secreting cells in the spleen of SjSs mice compared to C57BL/6 mice. Cells from the lymph node of SjSs mice yield higher instances of IgG1 reactive against salivary gland antigens than cells from the lymph nodes of C57BL/6 mice. These data demonstrate the isotype-specific reactivity of antibodies during the autoimmune process, and further reveals significant differences in the non-autoimmune and autoimmune antibody repertoires. These results support the generation of self-reactive B cell repertoires during the autoimmune process, at the same time, verifying that microengraving of single cells might allow for identification of novel biomarkers in SjS.

Replication ability of three highly protective Marek's disease vaccines: implications in lymphoid organ atrophy and protection

The present work is a chronological study of the pathogenesis of three attenuated serotype 1 Marek's disease (MD) virus strains (RM1, CVI988 and 648A80) that provide high protection against MD but have been attenuated by different procedures and induce different degrees of lymphoid organ atrophy. All studied strains replicated in the lymphoid organs (bursa,x thymus and spleen) and a peak of replication was detected at 6 days post inoculation (d.p.i.). Differences, however, were observed among vaccine strains. RM1 strain replicates more in all lymphoid organs compared with CVI988 and 648A80 strains. In addition, replication of RM1 in the thymus did not decrease after 6 d.p.i. but continued at high levels at 14 d.p.i. and until the thymus was completely destroyed. Lung infection occurred very early after infection with all of the three vaccines and the level of replication was similar to that found in the lymphoid organs. Infected cells were very large and appeared scattered in the lung parenchyma and in the parabronchial lining. The study of the target cells for the early infection in cell suspensions of blood and spleen showed that both non-adherent cell populations (enriched in lymphoid cells) and adherent cells (enriched in monocytes/macrophages) supported MD virus infection. Infection in adherent cells was especially high at very early stages of the infection (3 to 6 d.p.i.). Atrophy of lymphoid organs is a major drawback in the production of highly protective vaccines against MD. A better understanding of the mechanisms associated with lymphoid organ atrophy will aid in overcoming this problem.

Emergence of Donor T Cells That Recognize Nonpolymorphic Antigens During Graft Versus Host Disease,

Abstract 4013Chronic graft versus host disease (GVHD) is a major cause of morbidity and mortality in allogeneic stem cell transplant recipients and typically develops from antecedent acute GVHD. In contrast to acute GVHD which is characterized by a restricted set of organ involvement, chronic GVHD has more protean manifestations with much broader tissue involvement that can affect nearly all organ sites. Moreover, clinical manifestations in chronic GVHD often bear striking similarity to what is observed in autoimmune diseases. How GVHD evolves from an acute inflammatory syndrome in which donor T cells recognize polymorphic antigens expressed by host APCs to one that is characterized by autoimmune manifestations is not well understood. More specifically, it is not clear whether T cells that mediate autoimmune manifestations during chronic GVHD continue to respond to polymorphic antigens or whether T cells that recognize nonpolymorphic antigens emerge in these recipients. To address this issue, we performed a comprehensive analysis of the clonotypic T cell response in mice that developed autoimmune-mediated GVHD. To conduct these studies, lethally irradiated Balb/c [H-2d] mice were transplanted with C57BL/6 [H-2b] bone marrow and spleen cells to induce acute GVHD. At 19–21 days post transplantation, spleen cells or purified T cells from completely donor-engrafted mice were transferred into non irradiated syngeneic B6 Rag animals. In this model, animals develop pathological damage in the colon 60–70 days post transfer characterized by lamina propria inflammation, goblet cell depletion, and crypt cell destruction. This is attributed to the presence of autoreactive donor T cells in the original spleen cell inoculum that expand in syngeneic recipients due to loss of effective T cell regulation. To examine the clonotypic T cell response, we performed T cell receptor beta spectratyping on pathologically involved colonic tissue to identify over represented, skewed bands that were shared by replicate mice within the 21 Vβ families. These bands within a given Vβ family were sequenced to define the specific T cell clonotypes within colitic tissue. In the vast majority of families across multiple experiments, there were high frequency clonotypes that were present in all replicate mice and comprised 50–90% of all sequences. Notably, these shared clonotypes between replicate animals had the same CDR3 nucleotide sequence, indicating that they were the same T cell clones. The presence of high frequency clonotypes was also evidence that autoimmunity was characterized by antigen-driven expansion of a limited number of clones. During the progression from acute to chronic GVHD in humans, host APCs are eliminated and presentation of host peptides is by donor APCs through the indirect alloreactive pathway. To simulate this condition in our experimental model, we created chimeric animals by transplanting B6 Rag BM cells into lethally irradiated Balb/c Rag mice. Spleen cells from primary B6→Balb/c GVHD mice were then transferred into these fully donor-engrafted chimeric animals. Clonotypic analysis of T cells obtained from replicate mice with colitis revealed that dominant clonotypes were observed and comprised 65–80% of all sequences, similar to what was observed during conditions of autoimmunity. Given these similar results in models of autoimmunity and indirect alloimmunity, we then determined whether T cells that were capable of responding to B6 and Balb/c antigens when presented by B6 APCs could be identified. To address this question, the same pooled spleen cell suspension from primary (B6→Balb/c) GVHD mice was transferred into B6 Rag and chimeric (B6 Rag→Balb/c Rag) animals. We observed that T cell clonotypes with the same nucleotide sequences could be identified in pathologically involved colon tissue from both cohorts of mice indicating the emergence of T cells that were capable of recognizing both B6 and Balb/c antigens presented by B6 APCs. These results demonstrate that the loss of self tolerance which occurs during acute GVHD leads to the emergence of broadly reactive donor T cells that are capable of recognizing nonpolymorphic antigens that are shared between donor and host. These data also provide a mechanistic explanation for how autoimmunity develops as a consequence of GVHD and help explain the progression of tissue involvement that characterizes the transition from acute to chronic GVHD. Disclosures:No relevant conflicts of interest to declare.

Th17 and non-Th17 interleukin-17-expressing cells in chronic lymphocytic leukemia: delineation, distribution, and clinical relevance

The levels and clinical relevance of Th17 cells and other interleukin-17-producing cells have not been analyzed in chronic lymphocytic leukemia. The objective of this study was to quantify blood and tissue levels of Th17 and other interleukin-17-producing cells in patients with this disease and correlate blood levels with clinical outcome. Intracellular interleukin-17A was assessed in blood and splenic mononuclear cells from patients with chronic lymphocytic leukemia and healthy subjects using flow cytometry. Interleukin-17A-producing cells were analyzed in formalin-fixed, paraffin-embedded spleen and lymph node sections using immunohistochemistry and immunofluorescence. The absolute numbers of Th17 cells in peripheral blood mononuclear cells and the percentages of Th17 cells in spleen cell suspensions were higher in patients with chronic lymphocytic leukemia than in healthy subjects; in six out of eight paired chronic lymphocytic leukemia blood and spleen sample comparisons, Th17 cells were enriched in spleen suspensions. Circulating Th17 levels correlated with better prognostic markers and longer overall survival of the patients. Two "non-Th17" interleukin-17-expressing cells were identified in chronic lymphocytic leukemia spleens: proliferating cells of the granulocytic lineage and mature mast cells. Granulocytes and mast cells in normal spleens did not express interleukin-17. Conversely, both chronic lymphocytic leukemia and healthy lymph nodes contained similar numbers of interleukin-17+ mast cells as well as Th17 cells. Th17 cells are elevated in chronic lymphocytic leukemia patients with better prognostic markers and correlate with longer survival. Furthermore, non-Th17 interleukin-17A-expressing cells exist in chronic lymphocytic leukemia spleens as maturing granulocytes and mature mast cells, suggesting that the microenvironmental milieu in leukemic spleens promotes the recruitment and/or expansion of Th17 and other IL-17-expressing cells. The pathophysiology of Th17 and non-Th17-interleukin-producing cells in chronic lymphocytic leukemia and their distributions and roles in this disease merit further study.

Maternal background strain influences fetal–maternal trafficking more than maternal immune competence in mice

The objective of this study was to determine if fetal-maternal cell trafficking is affected by maternal immune competence and/or parental background strain using fluorescence-activated cell sorting (FACS). In our experience the sensitivity of FACS allows for the detection of 5 fetal in 10(7) maternal cells and assessment of cell surface phenotype. Wild-type C57BL/6J (n=18), FVB/NJ (n=15), and immunodeficient B6129S7-Rag1(tm1Mom)/J (n=16) female mice were mated to C57BL/6J males homozygous for the green fluorescent protein (GFP) transgene. Single cell suspensions of maternal lung, liver, spleen, bone marrow, and blood were analyzed between late gestation (day e16-18) and 1 day post-partum for the number of GFP-positive fetal cells in relation to 10(7) maternal cells and the percentage of GFP-positive cells that expressed the surface markers CD11b, CD29, CD34, CD44, or CD105. The highest relative proportions of GFP-positive fetal cells were observed in maternal lungs and livers from immunocompetent allogenic females. Among congenic matings, fetal cell microchimerism was higher in immunodeficient compared with immunocompetent females. Maternal strain and strain differences between the mother and father statistically significantly affected both the numbers of fetal cells and the relative distribution of cell types in maternal organs. The highest relative proportion of fetal cells was observed in allogenic matings with immunocompetent females. Since allogenic matings are more similar to those that occur in humans, future studies using animal models of microchimerism should consider incorporating this type of experimental design.

414 VACCINATION WITH RAD5 CARRYING A FUSION CONSTRUCT OF PROSTATE-SPECIFIC ANTIGENS GENERATES EFFECTOR T CELLS

INTRODUCTION AND OBJECTIVES: Immunotherapy has emerged as a promising approach in the development of novel therapies for prostate cancer. While most currently approved vaccines are targeted toward a single antigen, the heterogeneity of prostate cancer may create a limitation toward achieving a maximal effector response with this approach. Our goal is to expand the range of anti-tumor responses by creating a multi-antigenic T cell response utilizing a single immunization with a recombinant viral construct containing a fusion of two known prostate cancer antigens. METHODS: A fusion construct (PSPA) containing prostatespecific antigen (PSA) and prostate stem cell antigen (PSCA) was created and combined with a recombinant adenovirus type 5 (rAd5) vector and injected into mice. Following immunization, spleen cell suspensions were processed to analyze the production of IFNin the vaccinated mice compared to control. In vivo cytotoxic T cell assays and tumor growth challenge studies were compared in the vaccinated group compared to control mice. Student’s t-test was used to determine levels of significance among groups studied. RESULTS: Average immune responses of anti-PSA (p 0.037) and anti-PSCA (p 0.026) CD8 T cells following Ad5PSPA immunization were significantly higher as compared to controls. A reduced level of anti-PSA IFNwas noted in comparison to anti-PSCA following immunization (p 0.056). Immunized mice showed a strong cytotoxic response, being able to lyse 80% and 100% of PSA or PSCA coated target cells, respectively (p 0.034 and 0.005, respectively). Immunized mice demonstrated a significant inhibition of tumor outgrowth derived from PSA-expressing murine tumor cell lines with a total survival rate of 60%. CONCLUSIONS: Immunization of mice with rAd5 vector carrying a fusion construct of PSA and PSCA simultaneously induces the expansion of anti-PSA and anti-PSCA CD8 T cells. The developed antigen-specific T cell responses were efficient in eliminating target cells expressing cognate antigens, and strong anti-tumor immunity was elicited when challenged with prostate tumor cell lines. The effector response caused by the use of multiple antigens may have a synergistic effect to significantly reduce tumor burden and have important implications for human clinical trials targeted at prostate cancer.