Cell Help For Production Research Articles

Biodegradable PLGA-b-PEG Nanoparticles Induce T Helper 2 (Th2) Immune Responses and Sustained Antibody Titers via TLR9 Stimulation.

Sustained immune responses, particularly antibody responses, are key for protection against many endemic infectious diseases. Antibody responses are often accompanied by T helper (Th) cell immunity. Herein we study small biodegradable poly (ethylene glycol)-b-poly (lactic-co-glycolic acid) nanoparticles (PEG-b-PLGA NPs, 25–50 nm) as antigen- or adjuvant-carriers. The antigen carrier function of PEG-b-PLGA NPs was compared against an experimental benchmark polystyrene nanoparticles (PS NPs, 40–50 nm), both conjugated with the model antigen ovalbumin (OVA-PS NPs, and OVA-PEG-b-PLGA NPs). The OVA-PEG-b-PLGA NPs induced sustained antibody responses to Day 120 after two immunizations. The OVA-PEG-b-PLGA NPs as a self-adjuvanting vaccine further induced IL-4 producing T-helper cells (Th2), but not IFN-γ producing T-cells (Th1). The PEG-b-PLGA NPs as a carrier for CpG adjuvant (CpG-PEG-b-PLGA NPs) were also tested as mix-in vaccine adjuvants comparatively for protein antigens, or for protein-conjugated to PS NPs or to PEG-b-PLGA NPs. While the addition of this adjuvant NP did not further increase T-cell responses, it improved the consistency of antibody responses across all immunization groups. Together these data support further development of PEG-b-PLGA NPs as a vaccine carrier, particularly where it is desired to induce Th2 immunity and achieve sustained antibody titers in the absence of affecting Th1 immunity.

Mathematical modeling of trypsin activation in acinar cell environment

s / Pancreatology 13 (2013) e1–e94 e79 Background: Endocrine insufficiency in chronic pancreatitis (CP) is believed to be a late manifestation occurring as a result of b cell apoptosis. It is now shown that significant b cell dysfunction exists even in early CP in the absence of clinical diabetes. Our earlier studies suggested that in CP, IFN-g is the predominant cytokine in pancreas responsible for b cell dysfunction. In this study we investigated the source of pancreatic IFN-g and its effect on b cell function and apoptosis. Methods: Pancreatic tissues were obtained from CP patients with and without diabetes (study group) and those undergoing surgery for non pancreatic malignancy (control). IFN-g producing T-helper cells were enumerated in pancreatic tissues and circulation on Flow cytometry and confirmed by immunoflorescence. Islets were sorted for b cells; markers of apoptosis and GSIR were evaluated from controls, CP patients and control islets exposed to IFN-g. Islet proteins from controls, CP patients and control islets exposed to IFN-g were probed on western blots for signal pathway intermediates of insulin gene transcription. Results: IFN-g producing T-helper cell subsets (Th1, Th17) were significantly higher (p<0.01) in circulation and in pancreatic tissues from CP patients as compared to controls. b cells from non-diabetic CP patients showed minimal apoptosis (2 0.8%) but significantly diminished GSIR (60 12%), while 40 9% b cells from diabetic CP patients were apoptotic and remaining 60 7.5% b cells had very minimal GSIR (2 1.5%). Similar results were seen on chronic exposure of normal islets to IFN-g. Preexposing b cells to EGCG (IFN-g inhibitor) reverted islet functions completely and reduced apoptosis significantly. Western blots of islet proteins revealed reduced phosphorylation of pdx1 (transcription factor) resulting in reduced insulin gene transcription and diminished GSIR. Conclusion: Our results suggest that arresting b cell dysfunction early in the course of CP can potentially prevent b cell apoptosis and diabetes.

Interleukin-17–Producing T-Helper Cells as New Potential Player Mediating Graft-Versus-Host Disease in Patients Undergoing Allogeneic Stem-Cell Transplantation

Graft-versus-host disease (GVHD) is a major obstacle to safe allogeneic hematopoietic stem-cell transplantation, leading to significant mortality. Recently, T-helper (TH)-17 cells have been shown to play a central role in mediating several autoimmune diseases. The aim of our study was to investigate the relationship between TH-17 cells and GVHD occurring in transplanted patients. Blood samples were collected from 51 hematopoietic stem-cell transplantation patients and 15 healthy donors. Patients with GVHD were monitored for the presence of TH-17 cells by ELISPOT or flow cytometry in the peripheral blood and by confocal microscopy in GVHD lesions. Cytokine plasma levels were detected by ELISA. An increased TH-17 population (up to 4.8% of peripheral blood CD4+T lymphocytes) was observed in patients with acute GVHD and (up to 2.4%) in patients with active chronic GVHD along with an inflammatory process. In contrast, the percentage of TH-17 cells drastically decreased in patients with inactive chronic GVHD. TH-17 cells consisted of both interleukin (IL)-17+/interferon (IFN)-gamma- and IL-17+/IFN-gamma+ subsets and expressed IL-23 receptor. Interestingly, IFN-gamma+ TH-17 cells were able to infiltrate GVHD lesions as observed in liver and skin sections. Moreover, the proportion of TH-17 was inversely correlated with the proportion of regulatory T cells observed in the peripheral blood and tissues affected by GVHD. Finally, we demonstrated a strong correlation between TH-17 levels and the clinical status of patients with GVHD. These findings support the hypothesis that TH-17 are involved in the active phases of GVHD and may represent a novel cellular target for developing new strategies for GVHD treatment.

Adhesive substrate-modulation of adaptive immune responses

While it is well-known that adsorbed proteins on implanted biomaterials modulate inflammatory responses, modulation of dendritic cells (DCs) via adhesion-dependent signaling has only been begun to be characterized. In this work, we demonstrate that adhesive substrates elicit differential DC maturation and adaptive immune responses. We find that adhesive substrates support similar levels of DC adhesion and expression of stimulatory and co-stimulatory molecules. Conversely, DC morphology and differential production of pro- and anti-inflammatory cytokines (IL-12p40 and IL-10, respectively) is adhesive substrate-dependent. For example, DCs cultured on collagen and vitronectin substrates generate higher levels of IL-12p40, whereas DCs cultured on albumin and serum-coated tissue culture-treated substrates produce the higher levels of IL-10 compared to other substrates. Additionally, our results suggest substrate-dependent trends in DC-mediated allogeneic CD4 + T-cell proliferation and T-helper cell type responses. Specifically, we show that substrate-dependent modulation of DC IL-12p40 cytokine production correlates with CD4 + T-cell proliferation and T h1 type response in terms of IFN-γ producing T-helper cells. Furthermore, our results suggest substrate-dependent trends in DC-mediated stimulation of IL-4 producing T-cells, but this T h2 type response is not dependent on DC production of IL-10 cytokine. This work has impact in the rational design of biomaterials for diverse applications such as tissue-engineered constructs, synthetic particle-based vaccines and the ex vivo culture of DCs for immunotherapies.

NK T cells provide lipid antigen-specific cognate help for B cells

The mechanisms of T cell help for production of antilipid antibodies are largely unknown. This study shows that invariant NK T cells (iNK T cells) and B cells cooperate in a model of antilipid antigen-specific antibody responses. We use a model haptenated lipid molecule, 4-hydroxy-3-nitrophenyl-alphaGalactosylCeramide (NP-alphaGalCer), to demonstrate that iNK T cells provide cognate help to lipid-antigen-presenting B cells. B cells proliferate and IgG anti-NP is produced from in vivo-immunized mice and in vitro cocultures of B and NK T cells after exposure to NP-alphaGalCer, but not closely related control glycolipids. This B cell response is absent in CD1d(-/-) and Jalpha18(-/-) mice but not CD4(-/-) mice. The antibody response to NP-alphaGalCer is dominated by the IgM, IgG3, and IgG2c isotypes, and marginal zone B cells stimulate better in vitro lipid antigen-driven proliferation than follicular B cells, suggesting an important role for this B cell subset. iNK T cell help for B cells is shown to involve cognate help from CD1d-instructed lipid-specific iNK T cells, with help provided via CD40L, B7-1/B7-2, and IFN-gamma, but not IL-4. This model provides evidence of iNK T cell help for antilipid antibody production, an important aspect of infections, autoimmune diseases, and vaccine development. Our findings also now allow prediction of those microbial antigens that would be expected to elicit cognate iNKT cell help for antibody production, namely those that can stimulate iNKT cells and at the same time have a polar moiety that can be recognized by antibodies.

CD4+CD25hi regulatory T‐cell frequency correlates with persistence of human papillomavirus type 16 and T helper cell responses in patients with cervical intraepithelial neoplasia

CD4(+)CD25(hi)CTLA4(+)FoxP3(+) regulatory T cells (Treg) have been shown to maintain immune tolerance against self antigens and increased circulating frequencies have been reported in various types of cancers. Circulating invariant natural killer T-cells (iNKT) are reduced in cancer patients and low iNKT frequency is related to poor prognosis. It is not yet clear whether high Treg numbers and low iNKT cell numbers pose an increased risk for the progression of premalignant lesions or whether Treg and iNKT cell numbers are influenced by dysplasia. We therefore studied prospectively the relation between iNKT cell and Treg frequencies and the natural course of human papillomavirus type 16 (HPV16) induced pre-malignant cervical dysplasia in 82 patients who participated in a nonintervention cohort study of women with abnormal cytology. Treg frequencies were significantly increased in women who had persistent HPV16 infection. Within the HPV16 persistence group there was no difference in Treg frequencies among patients who developed a CIN3 lesion and patients who did not progress to CIN3. Furthermore, Treg frequencies were increased in patients who had detectable HPV16 E7 specific IL-2 producing T-helper cells, which suggests a causal role of HPV infection in Treg development in parallel with HPV16 specific T helper cells. No evidence was found for a role for iNKT cells in persistence of HPV16 and progression of HPV16 induced CIN lesions. However, HPV-persistence-associated Tregs may explain the inefficacy of concomitant persistence associated immunity and may contribute to subsequent progression to neoplasia.

Interleukin 17-producing T-helper cells and autoimmune diseases: Time for a paradigm shift?

Introduction For almost 20 years, the pathogenic role of helper T cells has been intensively studied in autoimmune diseases. In multiple sclerosis (MS), an autoimmune disorder affecting the brain and spinal cord, overwhelming evidence has indicated that interferon (IFN)–secreting T-helper type 1 (Th1) cells are involved in the inflammatory process leading to the destruction of myelin sheath [1]. Adoptive transfer of Th1 cells reactive to myelin antigen cause an MS-like disease called experimental autoimmune encephalomyelitis (EAE), supporting this postulate. Notably, the role of Th1 cells in MS has been emphasized not only by experiments with laboratory animals but also by unwanted results of clinical trials. In past clinical trials, administration of IFNpromoted exacerbations of MS, whereas recombinant IFNhas been shown to suppress them [2]. In a more recent study, an altered form of myelin basic protein (MBP) peptide was evaluated for its therapeutic potential. However, it caused serious relapses and massive expansion of MBP-reactive Th1 cells [3]. The deleterious effects of IFNand the MBP analogue peptide have been regarded as evidence for the role of Th1 cells in the pathogenesis of MS. On the other hand, T-helper 2 (Th2) cells that secrete interleukin (IL)-4, IL-5, and IL-10 were shown to counterregulate Th1 cell functions via secretion of Th2 cytokines. This finding led to speculation that autoimmune diseases could be cured by shifting the functional balance between Th1 cells and Th2 cells toward Th2. The relevance of this idea has been verified vigorously in numerous studies, which have actually demonstrated that Th2-biasing agents are effective for downmodulating clinical and pathologic manifestations of EAE. Given such theoretic, empiric, and experimental evidence, our understanding of the pathogenesis of autoimmune diseases has been heavily influenced by the Th1/Th2 paradigm [4]. However, contradictory to the “Th1 disease” dogma, EAE and collagen-induced arthritis (CIA) became worse in mice deficient in those molecules that are critical for Th1 cell development (eg, IL-12 receptor 2 and IFN) [5]. Thus, the question has arisen as to whether Th1 cells are truly needed for induction of autoimmune diseases. The recent discovery of a new T-helper cell subset characterized by production of IL-17 has clarified that the IL-17–producing T-helper cells (Th17) would mediate autoimmune disease models including EAE, CIA, and experimentally induced colitis without help from Th1 cells [6–9]. Very recent papers have identified that a combination of proinflammatory cytokine (IL-6) and anti-inflammatory cytokine (transforming growth factor [TGF]) would cause differentiation of Th17 cells [10–12], unveiling a new principle governing the maintenance and disruption of immunologic tolerance.

148: The IL-23/IL-17 axis in human cardiac allograft vasculopathy

Purpose: Cardiac Allograft Vasculopathy (CAV) after heart-transplantation is a major factor limiting the long-term patient survival. CAV is characterized by a concentric intima proliferation, which is induced by a mononuclear cell infiltrate (MNC). This process is often compared with a proliferative autoimmune disease in which IL-17 producing T-helper cells (Th17) play a central role. In this study the role of Th17 in the development of CAV was analyzed. Methods and Materials: Coronary arteries (CA) were obtained from CAV patients (n 6) and compared to CA from non-CAV patients (n 5). Q-PCR analysis was performed using a low density array on cDNA prepared from 5 microdissected areas: 1) luminal layer of the intima (LL), 2) smooth muscle cell layer of the intima, 3) tunica media, 4) adventitia (AD), and 5) lymphoid clusters outside the vessel. Genes (50) were selected for Th1/Th2/Th17 characterization. Positive gene expression by Q-PCR analysis was confirmed and localized by in situ hybridization and immunohistochemistry. Results: Tand B-cells were mainly detected in the LL and the AD of CAV and were absent in control CA. Cytokine expression in CAV was generally low, probably due to the immunosuppression. IFN, known to inhibit Th17, was however, significantly expressed, mainly in LL and AD of CAV but not in control CA. TGF, which induces Th17, was detected in both CAV and control CA, and was localized in macrophages. Th1 markers were abundantly present, but Th17 markers such as IL17, IL-23, and IL23R were weakly expressed or absent. Likewise, regulatory T-cells (FoxP3) were not detected in CAV and control CA, and Th2 markers only on a minority of MNC. Conclusions: In autoimmunity, a dichotomy between inducing Th17 and inhibiting regulatory T-cells has been described. In CAV, both are absent. This is suggestive of a different mechanism driving the proliferative response, possibly due to the low cytokine response under immunosuppression. Therefore, we suggest that the Th1-cells, which are predominant in the MNC, are responsible for the direct or indirect induction of the proliferative response.

Deficient humoral responses underlie susceptibility to Toxoplasma gondii in CD4-deficient mice.

Resistance to infection with Toxoplasma gondii was studied in mice lacking CD4 expression. Such mice developed more brain cysts and survived for a shorter time than did wild-type controls after peroral infection with ME49 cysts. After immunization with the ts-4 strain of T. gondii, CD4-deficient mice exhibited impaired resistance to a challenge infection with virulent RH tachyzoites. Thus, deficient CD4 expression increases the susceptibility of mice to a primary peroral T. gondii infection with cysts and impairs their ability to be successfully vaccinated. CD8(+) T cells from blood or spleens of Toxoplasma-infected, CD4-deficient mice expressed markers of activation at frequencies similar to those of infected wild-type mice. Production of IFN-gamma in vitro was moderately depressed, and levels of Toxoplasma-specific immunoglobulin G2a in serum were substantially lower than in wild-type mice. Administration of Toxoplasma-immune serum to ts-4-vaccinated CD4-deficient mice significantly improved their resistance to RH challenge. Also, the survival of CD4-deficient mice chronically infected with ME49 was significantly prolonged by administration of immune serum. These results demonstrate that in addition to CD8(+) T cells and IFN-gamma, which are known to be critical for resistance, CD4(+) cells also contribute significantly to protection against chronic T. gondii infections and against challenge infections with highly virulent tachyzoites in immunized mice via their role as helper cells for production of isotype-switched antibodies.

IL-4 receptorα gene polymorphism in heart transplantation

IL-4 is a cytokine of the Th2 subtype, sectreted by lymphocytes and mast cells. The IL-4 receptor (IL-4R) consists of a common Γ-chain and the IL-4 specific α-chain. In transplantation Th2 cells are believed to be involved in induction of graft tolerance. Our previous studies indicated that patients with a high frequency of IL-4 producing T-helper cells had no or less rejection episodes compared to patients with low frequencies. Since cytokine release in the micronvironment of the transplant is critical for the incidence of rejection, the cytokine genotype of the donor might influence this. Previously, we showed a relationship between high IL-4 producing -590∗T positive promotor genotype in the donor heart and reduced graft rejection in the recipient. As IL-4 acts via its receptor, in this study we analysed the influence of 6 IL-4R single nucleotide polymorphisms (SNP) in the α-chain, that influence the signaling pathways and cytokine binding. Genotypes of 70 HTX patients (35 DCM, 29 IHD, 6 other causes), 62 cardiac donors and a control panel were determined. No relation between genotypes of the IL-4R SNP and the original heart failure was found. No relation between the patient SNP genotype and post-transplant rejection activities was detected as well. Interestingly, we did find a relation between the donor IL-4R genotype at positions 478 and 551. Patients that acquired a “wild-type” IL-4R in their donor heart suffered significantly more from rejection than patients receiving a polymorhic IL-4R in either one of the two SNP-sites. On position 478 there is a substitution from a serine to a proline (P= 0,006), and on position 551 a glutamine to arginine substitution (p=0,036). Immuunhistochemical analysis revealed that macrophages were the main cell type expressing the IL-4R. In Conclusion:: The presence of the polymorphic IL-4R on cells in the graft might lead to increased signalling and activity of the IL-4R upon binding of IL-4. This may lead to a higher Th2 response and reduced rejection incidence. Macrophages seem to play an important role in this immune regulation by IL-4.

FLOW CYTOMETRIC DETECTION OF TYPE 1 (IL-2, IFN-γ) AND TYPE 2 (IL-4, IL-5) CYTOKINES IN T-HELPER AND T-SUPPRESSOR/CYTOTOXIC CELLS IN RHEUMATOID ARTHRITIS, ALLERGIC ASTHMA AND ATOPIC DERMATITIS

Type 1 cytokines (a.o. IL-2 and IFN-γ) play an important role in the pathogenesis of rheumatoid arthritis. On the other hand, IgE-mediated diseases such as allergic asthma and atopic dermatitis show a type 2 cytokine (amongst others IL-4 and IL-5) profile.This study examined simultaneously the intracellular production of IL-2, IFN-γ, IL-4 and IL-5 in T-lymphocytes of patients with rheumatoid arthritis during treatment with methotrexate or salazopyrin, patients with allergic asthma or atopic dermatitis under stable treatment, compared to healthy controls.A three-colour flow cytometric analysis was used for cytokine detection in T-helper cells and T-suppressor/cytotoxic cells.Compared to controls, patients with symptomatic atopic dermatitis showed an increased number of IL-4-producing T-helper lymphocytes in basal circumstances (P=0.001), in contrast to asymptomatic allergic asthma patients. Compared to controls, rheumatoid arthritis patients, treated with salazopyrin, showed an increased number of IL-2-producing T-helper and T-suppressor/cytotoxic lymphocytes after in vitro stimulation with PMA and ionomycin (P=0.01). In contrast, rheumatoid arthritis patients, treated with methotrexate, a more potent disease modifying drug, did not show this type 1 cytokine profile. A positive correlation was found between the number of IFN-γ producing T-helper cells and disease activity (Ritchie Index and number of swollen joints) in both rheumatoid arthritis patient groups.Active atopic dermatitis patients showed a type 2 cytokine profile, whereas stable asthma patients with lower disease activity did not show a predominance of type 2 cytokines. Rheumatoid arthritis patients under treatment with salazopyrin had a type 1 cytokine profile, which could not be demonstrated in patients treated with methotrexate. This imbalance between type 1 and type 2 cytokines in different immune mediated disorders can be related with treatment and the grade of disease activity. These results stress the need for further investigation of the influence of therapy on cytokine profiles.

Why are we not all allergic: basic mechanisms for tolerance development

Harmless antigens encountered on the mucosal surface are normally tolerated in the sense that they do not induce inflammatory immune responses. Oral tolerance is the type of mucosal immune regulation that prevents inflammatory reactions to food proteins. However, parasites and invasive microorganisms at the mucosal surfaces must be recognised and dealt with in a proper manner. The immune system does so by cross-regulating the response where it either produces IgA to exclude invasion, IgE to fight parasites, or IgG to destroy the invasive organisms. Allergy is an anti-parasitic reaction to a misinterpreted but harmless antigen. This lack of tolerance induction is influenced by genetic factors controlling the amount of interleukin (IL)-4 produced initially in the immune response. IL-4 directs B-cells to produce IgE, induces naive T-cells to become IL-4 producing T-helper cells (Th2 cells) and prevent other T-cells from entering into the IFN-γ-producing Th1 pathway. Long lasting Th2 clones lose their IL-12 responsiveness and can no longer be induced to produce IFN-γ thus they are locked in an allergy inducing Th2 phenotype. Environmental factors play on the genetic background and influence the outcome of the immune response. Mucosal tolerance depends on an intact mucosal surface, is influenced by the age of the subject, and can be manipulated through antigen dose and place of entry. Immune-manipulating therapy may be more successful in primary than in secondary prevention of allergy.

Functional and phenotypic differences between CD4+ and CD4- T cell receptor-gamma delta clones from peripheral blood.

CD4+ TCR-gamma delta+ T cells comprise a very small subset of TCR-gamma delta+ T cells. CD4+ TCR gamma delta+ T cell clones were established to study the phenotypical and functional characteristics of these cells. Thirty-four CD4+ TCR-gamma delta+ T cell clones were established after sorting CD4+ T cells from a pre-expanded TCR-gamma delta+ T cell population. These clones as well as the CD4- TCR-gamma delta+ T cells from the same donor used V gamma 2 and V delta 2. In a second cloning experiment CD4+ TCR-gamma delta+ T cells were cloned directly from freshly isolated TCR-gamma delta+ T cells using a cloning device coupled to a FACS sorter. Forty-three clones were obtained, which all expressed CD4 and TCR-gamma delta. Eleven of these clones used V delta 1 and three of them coexpressed V gamma 2. The other CD4+ TCR-gamma delta+ T cell clones used both V delta 2 and V gamma 2. CD4+ TCR-gamma delta+ T cell clones expressed CD28 irrespective of the V gamma or V delta usage, and were CD11b negative. Three CD4-CD8+ TCR-gamma delta+ clones expressed CD8 alpha but not CD8 beta and were CD11b positive. CD28 expression among CD4-CD8+ and CD4-CD8- was variable but lower than on CD4+ T cell clones. CD4- TCR-gamma delta+ T cell clones using V gamma 2 and V delta 2 specifically lyse the Burkitt lymphoma cell line Daudi and secrete low levels of IFN-gamma and granulocyte-macrophage-CSF upon stimulation with Daudi. In contrast, most CD4+ T cell clones that use V gamma 2 and V delta 2 had a very low lytic activity against Daudi cells and secrete high levels of IFN-gamma and granulocyte-macrophage-CSF after stimulation with Daudi cells. The NK-sensitive cell line K562 was killed efficiently by the CD4- TCR-gamma delta+ T cell clones, but not by CD4+ TCR-gamma delta+ T cell clones, and could not induce cytokine secretion in CD4+ or CD4- T cell clones. CD4+ TCR-gamma delta+ T cell clones, but not the CD4- clones, could provide bystander cognate T cell help for production of IgG, IgM, and IgA in the presence of IL-2 and IgE in the presence of IL-4. Thus, CD4+ TCR-gamma delta+ T cells are similar to CD4+ TCR-alpha beta+ T cells in their abilities to secrete high levels of cytokines and to provide T cell help in antibody production.(ABSTRACT TRUNCATED AT 400 WORDS)