Eosinophils function as antigen-presenting cells

Eosinophils release lipid mediators, including leukotriene C4, platelet-activating factor, and liposins, and contain four distinct granule cationic proteins, major basic protein, eosinophil peroxidase, eosinophil cationic protein, and eosinophil-derived neurotoxin, which may cause dysfunction and destruction of other cells. Eosinophils are primarily thought of as terminal effectors of allergic responses and of parasite elimination. Eosinophils are characteristically present within the airway lumina of asthmatics, and these airway eosinophils have been induced in vivo to express major histocompatibility complex II (MHC-II) complexes and costimulatory molecules, which are required for T lymphocytes to be functionally activated. In in vitro experiments, eosinophils can process antigen and express the costimulatory molecules, and after cytokine-elicited induction of MHC-II, expression can function as antigen-presenting cells in stimulating T lymphocyte responses. Airway luminal eosinophils can migrate into draining paratracheal lymph nodes, localized to T cell-rich paracortical areas, and stimulate antigen-specific T cell proliferation in vivo within paratracheal lymph nodes, which was CD80- and CD86-dependent and limited to CD4+ T cells. Furthermore, eosinophils within the lumina of airways promote expansion of T helper cell type 2 (Th2) by presenting antigen, suggesting that eosinophils actively modulate immune responses by amplifying Th2 cell responses.     

Intestinal immune cells in Strongyloides stercoralis infection.

BACKGROUND: Strongyloides stercoralis can cause a wide spectrum of disease in man, ranging from a chronic asymptomatic infection to a hyperinfective, often fatal syndrome. In rodents, spontaneous expulsion of Strongyloides spp occurs after experimental infection. Mast cells, goblet cells, and eosinophils have been identified as possible effectors of this expulsion. AIMS: To investigate intestinal histopathology and mucosal immunity in immunocompetent patients with chronic S stercoralis infection. METHODS: Jejunal biopsies were performed in 19 immunocompetent patients with a positive stool examination for S stercoralis and few or no symptoms, and in seven healthy controls. Specimens were processed for histopathological analysis and stained by the immunoperoxidase technique, using the following monoclonal antibodies: CD2, CD3, CD4, CD8, anti-T cell receptor (TcR) gamma/delta, RFD1 and RFD7 (two different macrophage markers), Ki67+ (proliferating) cells, antihuman leucocyte antigen (HLA)-DR, and anticollagen IV. In addition, CD25+ cells, mast cells, IgE expressing cells, calprotectin containing cells, and neutrophil elastase positive cells were stained by the alkaline phosphatase method. RESULTS: Jejunal morphology and the numbers of different T cell subsets, mast cells, IgE expressing cells, eosinophils, and goblet cells were unaffected by S stercoralis infection. Conversely, the numbers of mature macrophages and dividing enterocytes in the crypts were reduced significantly. Crypt enterocytes did not express HLA-DR in both groups. The expression of HLA-DR by villus enterocytes was also comparable in patients and controls. There were no activated (CD25+) cells in the mucosa of either patients or controls. CONCLUSIONS: Compared with seven healthy uninfected volunteers, a group of 19 Brazilians with clinically mild strongyloides infection showed no abnormality of mucosal structure and no increase in non-specific inflammatory cells. Likewise, there was no increase in mucosal T cells or macrophages.     

Subcultured human endothelial cells can function independently as fully competent antigen-presenting cells

Recent evidence has suggested that dendritic cells, epidermal Langerhan's cells and endothelial cells (EC) as well as macrophages, fulfill the requirements of antigen-presenting cells. Despite a variety of controls, one weakness in the evidence that these latter cell types can independently serve as antigen-presenting cells is that the cell preparations may contain small numbers of contaminating macrophages or other cell types. The experiments described in this paper are directed towards providing firm evidence that human EC are independently capable of presenting antigen to T cells. EC were isolated from human umbilical veins and maintained continuously by serial subculture for periods of up to 8 months. The subcultured EC displayed classic EC morphology and uniform immunofluorescent staining for Factor VIII-related antigen. The subcultured EC (tested to the 18th subculture) presented both particulate and soluble antigens to macrophage-depleted T cells with an efficiency equivalent to freshly isolated cells. Monoclonal antibodies to HLA-DR and HLA-DS determinants inhibited antigen presentation by either autologous macrophages or EC. In addition, antigen presentation by the subcultured EC was not affected by the macrophage-specific monoclonal antibody Mac-120, which inhibited antigen presentation by autologous macrophages in the same experiments. These results are consistent with human EC being able to independently function as fully competent antigen-presenting cells.     

嗜酸性粒细胞可作为抗原呈递细胞

目的探讨ＨＬＡ－ＤＲ＋嗜酸性粒细胞（ＥＯＳ）在体外培养条件下作为抗原呈递细胞（ＡＰＣ）将破伤风类毒素抗原（ＴＴ）呈递给Ｔ淋巴细胞的能力。方法新分离的ＥＯＳ经人重组粒细胞－巨噬细胞集落刺激因子刺激２４小时,以诱导ＨＬＡ－ＤＲ的表达,然后将其暴露于不同浓度的ＴＴ,检测ＨＬＡ－ＤＲ＋ＥＯＳ对自身Ｔ细胞增殖反应的影响,以评价ＥＯＳ呈递抗原的能力。结果ＨＬＡ－ＤＲ＋ＥＯＳ于ＴＴ存在时可以明显促进Ｔ细胞的增殖反应,并与ＴＴ的浓度呈剂量相关性。而抗ＨＬＡ－ＤＲ单克隆抗体则可以明显地抑制ＥＯＳ的抗原呈递过程。结论人ＥＯＳ可以摄入和处理抗原并能将其传递给自身Ｔ细胞,而ＥＯＳ呈递抗原的过程具有明显的ＨＬＡ－ＤＲ依赖性。     

Murine epidermal antigen-presenting cells in primary and secondary T-cell proliferative responses to herpes simplex virus in vitro.

The role of epidermal Langerhans' cells in infection with herpes simplex virus (HSV) was investigated using a culture system that supports antigen-specific primary and secondary T-cell proliferative responses. Epidermal cell suspensions were capable of restimulating the response of in vivo primed T cells to UV-inactivated HSV. This capability was also present in cell suspensions enriched for Langerhans' cells, but was abrogated by the depletion of I-A-bearing cells. The magnitude, kinetics and phenotype of the responding cells were similar to those elicited when HSV was presented to primed T cells by antigen-presenting cells from the spleen. In marked contrast, whereas splenic antigen-presenting cells induced strong antigen-specific proliferation of unprimed T cells (primarily of the helper phenotype), Langerhans' cells failed to invoke any detectable reaction of such cells.     

Murine epidermal antigen-presenting cells in primary and secondary T-cell proliferative responses to a soluble protein antigen in vitro.

The capacity of epidermal cells (EC) to present antigen to primed and non-immune T cells was investigated using a culture system that supports antigen-specific primary and secondary proliferative responses. Although both naive and bovine serum albumin (BSA)-immune T cells reacted against BSA in the presence of either splenic or epidermal antigen-presenting cells (APC), important differences were noted in the kinetics and the magnitudes of the various responses. Most conspicuous was the relatively poor primary response supported by EC which evidently elicited very few BSA-immune T-helper cells. Despite this, the primed antigen-specific T cells recovered were phenotypically similar to those resulting from the stronger primary responses induced by spleen cells. In contrast to this disparity in the ability to prime, EC and spleen cells stimulated secondary reactions of comparable magnitude. We therefore consider that, in comparison with splenic APC, EC may require some additional stimulus to acquire the capacity to prime.     

Resting B cells can act as antigen presenting cells in vivo and induce antibody responses

Although it is well established that B lymphocytes are ableto present antigen in vitro, the ability of small resting Bcells to act as antigen presenting cells in vivo remains controversial.In this report we have studied the antigen presentation andthe antibody response induced by mouse B cells after in vivoor in vitro targeting antigens to membrane Ig (mIg), using ratmAbs. Our results show that injection of these mAbs coupledto 2,4-dinitrophenyl (DNP) strongly enhances the IgG1 antibodyresponse against DNP and rat Ig. T cell depleted spleen cellspulsedin vitro with rat Ig without specificity for B cells inducedan antibody response when re-injected into mice, this responsebeing much higher if the antigen was specific for mlg. Moreover,purified resting B cells were shown to induce a specific IgG1response in vivo only when they were cultured with rat mAb againstmIgM or mIgD but not with myeloma rat Ig of the same isotype.B cells do not need to be activated to present antigen sincethe induction of the specific antibody response does not correlatewith the mitogenic activity of rat mAb nor with the IgG1 polyclonalsynthesis in vivo. These data clearly show that resting B cellscan present antigen in vivo and induce an antibody response,and underline the importance of mIgM and mIgD as targets forantigens.     

Infection of mice with the helminth Strongyloides stercoralis suppresses pulmonary allergic responses to ovalbumin

Asthma and helminth infections induce similar immune responses characterized by the presence of peripheral blood eosinophilia and elevated serum IgE levels. Epidemiological surveys have reported either increases or decreases in the development of atopic diseases and asthma based on the prevalence of helminth infections in the population. The aim of this study was to determine if a pre-existing helminth infection would increase or decrease subsequent allergic responses to an unrelated allergen in the lungs. BALB/cByJ mice were infected with the nematode parasite Strongyloides stercoralis prior to ovalbumin (OVA) immunization and intratracheal challenge. Bronchoalveolar lavage (BAL) and fluid (BALF) were collected 3 days post-challenge and cellular and humoral immune responses were measured. Intracellular cytokine staining revealed increased IL-4 and IL-5 producing cells in BAL from mice infected with S. stercoralis before OVA sensitization. Increased IL-5 protein levels and decreased IFN-gamma protein levels were also observed in the BALF. There was, however, no increase in airway eosinophil accumulation in mice infectd with parasites before sensitization with OVA as compared to mice exposed to OVA alone. Furthermore, eotaxin levels in the lungs induced by OVA was suppressed in mice infected with the parasite before OVA sensitization. The development of OVA specific IgE responses in BALF was also impaired in mice infected with the parasite before sensitization with OVA. These results suggest that a pre-existing helminth infection may potentiate a systemic Type 2-type response yet simultaneously suppress in the lungs allergen-specific IgE responses and eotaxin levels in response to subsequent exposure to allergens.     

Murine keratocytes function as antigen-presenting cells.

Keratocytes express MHC class I molecules constitutively, and keratocytes stimulated with IFN-gamma express MHC class II molecules. Unstimulated keratocytes constitutively express B7-1 and ICAM-1, as well as low levels of CD40 and 4-1BBL. These findings indicate that keratocytes may deliver both antigen-specific and costimulatory signals to CD4(+) and CD8(+) T cells. To demonstrate that keratocytes expressing B7-1 provide a costimulatory signal to T cells, CD4(+) or CD8(+) mouse T cells were incubated with anti-CD3 mAb and irradiated keratocytes. Enhanced proliferation of both CD4(+) and CD8(+) T cells occurred, and could be inhibited by anti-B7-1 mAb, indicating T cell costimulatory activity by B7-1 on the keratocytes. To demonstrate that keratocytes can deliver an antigen-specific signal, CD4(+) and CD8(+) T cells from herpes-infected mice were incubated with HSV-1-infected, irradiated keratocytes. The resulting T cell proliferation and production of Th1 cytokines (IL-2, IFN-gamma) indicated T cell activation by antigens presented by the infected keratocytes. These results show that keratocytes in the corneal stroma of the mouse can function as antigen-presenting cells and, thus, may play a role in immune-mediated stromal inflammation such as herpetic stromal keratitis.     

Difference in capacity of Sendai virus envelope components to induce cytotoxic T lymphocytes in primary and secondary immune responses.

Studies were made on the abilities of Sendai virus envelope components to induce primary and secondary generations of virus-specific cytotoxic mouse T lymphocytes (CTL). The primary CTL response in BALB/c mice was induced by reassembled envelope particles that had fusion activity but not by envelope glycoproteins without fusion activity, although both preparations induced a humoral immune response. Reconstitution of membrane-bound envelope proteins from envelope glycoproteins with lipids restored the fusion activity and the capacity to induce CTL. Target cells susceptible to virus-specific CTL could be induced by reassembled envelope particles, but not by envelope glycoproteins or LLC-MK2 cell-grown Sendai virus, neither of which had fusion activity. On the other hand, all the viruses and envelope components tested were found to stimulate a virus-specific CTL response in the in vitro secondary generation of CTL from virus-primed spleen cells. These results suggest that Senaei virus fusion activity is involved in primary induction of the CTL response as well as in target cell formation, but that it is not essential for secondary stimulation of the CTL response.     

Visualization of granzyme B-expressing CD8 T cells during primary and secondary immune responses to Listeria monocytogenes

CD8 T cells contribute to long-term protection against Listeria monocytogenes infection by differentiating into memory T cells. These rapidly respond to antigen or inflammation upon secondary infection. In this study we used CD8 T cells from OT1 mice and CD4 T cells from OT2 mice expressing a fluorescent chimeric granzyme (GZMB-Tom) protein to monitor the primary response to infection with ovalbumin-expressing L. monocytogenes (Lm-OVA). We show that, unlike poorly responding CD4 T cells, CD8 T cells readily proliferated and expressed high levels of GZMB-Tom as early as 2 days after infection. FACS analysis showed GZMB-Tom expression in undivided CD8 T cells, with its level increasing over one to four divisions. OT1 T cells were visualized in the T-cell zone by confocal microscopy. This showed GZMB-Tom-containing granules oriented towards MHCII-positive cells. Twenty hours later, most OT1 T cells had divided but their level of GZMB-Tom expression was reduced. Recently divided OT1 cells failed to express GZMB-Tom. Fourteen hours after secondary infection, GZMB-Tom was re-expressed in memory OT1 T cells responding either to Lm-OVA or L. monocytogenes. Differences in the activation phenotype and in the splenic distribution of OT1 T cells were observed, depending on the challenge. Notably, OTI T cells with polarized granules were only observed after challenge with cognate antigen. This work showed that the GZMB-Tom knock-in mice in which GZMB-Tom faithfully reproduced GZMB expression, provide useful tools to dissect mechanisms leading to the development of anti-bacterial effector and memory CD8 T cells and reactivation of the memory response to cognate antigen or inflammatory signals.     

Murine dendritic cells generated under serum-free conditions have a mature phenotype and efficiently induce primary immune responses

Vaccination with in vitro-generated dendritic cells (DC) that present tumor-associated antigens is a promising approach for immunotherapy of malignant tumors. For optimization of DC-based vaccination protocols, preclinical tumor models that mimic the clinical situation closely are highly desirable. Strong non-specific T cell activation was observed in experimental immunization of mice with syngeneic DC generated in standard FCS-supplemented culture medium. To avoid deviation of the immune response to FCS-derived antigens, a serum-free culture protocol for in vitro generation of murine DC from bone marrow progenitor cells was developed. In comparison to DC differentiated with FCS supplementation, DC generated under serum-free conditions (sfDC) have a more homogeneous phenotype with higher expression of IL-12 and the differentiation and activation markers CD11c, CD40, CD80, CD83, CD86, DEC-205, and MHC class II. Demonstration of strong uptake of protein and carbohydrate antigens and analysis of the in vivo migration behaviour of sfDC also indicated excellent APC function. Vaccination of mice with peptide-pulsed sfDC efficiently induced an antigen-specific T cell response as assessed by MHC tetramer staining, IFN-γ ELISPOT and in vivo cytotoxicity assay. sfDC may therefore represent a valuable tool to improve active tumor immunotherapy in animal models.     

Mesenchyme cells can function to induce epithelial cell proliferation in starfish embryos

Here, we show that mesenchyme cells have a novel morphogenetic function in epithelial cell proliferation in starfish embryos. Blastula embryos were injected with pure populations of mesenchyme cells and the total cell numbers in the treated embryos were subsequently determined at different developmental stages. When a total of 40–50 mesenchyme cells was injected, total cells numbers in mid‐gastrula embryos and 3‐day‐old bipinnaria larvae increased significantly (by 1.3‐fold) compared with controls, with no indication of any mitotic activity in the injected mesenchyme cells. However, injection of more than 150 mesenchyme cells failed to induce proliferation of the epithelial cells and, moreover, interfered with normal morphogenesis. These developmental abnormalities occurred concomitantly with a severe condensation of the fibrous component of the extracellular matrix. Our data suggest that epithelial cell proliferation is induced by an appropriate number of mesenchyme cells in concert with the fibrous component of the extracellular matrix. Developmental Dynamics 239:818–827, 2010. © 2010 Wiley‐Liss, Inc.     

Naive human T cells can be a source of IL-4 during primary immune responses

IL-4 plays a key role in driving the differentiation of CD4+ Th precursors into Th2 cells, both in mice and in humans. The source of IL-4 during primary immune responses is, however, still debated. When IL-4 consumption in in vitro T cell cultures was blocked with a MoAb to the IL-4 receptor alpha-chain (IL-4Ralpha), it became evident that freshly isolated naive (CD45RO-) CD4+ T cells from adults or cord blood produce IL-4 upon activation with anti-CD3 and CD80. IL-4 production by naive T cells is strictly IL-2-dependent. Endogenous IL-4 activity in naive CD4+ T cell cultures modulates the production of interferon-gamma (IFN-gamma) on the one hand and IL-5 and IL-13 on the other hand in opposite directions, and it is partly responsible for the low IFN-gamma production by cord blood T cells. Comparison of the ratio of IL-4/IFN-gamma in supernatants of T cell cultures reveals a skewing towards IL-4 production by cord blood T cells, while naive T cells from (non-atopic) adults predominantly produce IFN-gamma. We conclude that CD4+ naive T cells can produce IL-4 without the need for Th2 differentiation, and therefore that they can be the initial source of IL-4 required at the time of priming for T cell differentiation into Th2 cells.     

Rapid immune responses to a botulinum neurotoxin Hc subunit vaccine through in vivo targeting to antigen-presenting cells

The clostridial botulinum neurotoxins (BoNTs) are the most potent protein toxins known. The carboxyl-terminal fragment of the toxin heavy chain (Hc) has been intensively investigated as a BoNT vaccine immunogen. We sought to determine whether targeting Hc to antigen-presenting cells (APCs) could accelerate the immune responses to vaccination with BoNT serotype A (BoNT/A) Hc. To test this hypothesis, we targeted Hc to the Fc receptors for IgG (FcγRs) expressed by dendritic cells (DCs) and other APCs. Hc was expressed as a fusion protein with a recombinant ligand for human FcγRs (R4) to produce HcR4 or a similar ligand for murine FcγRs to produce HcmR4. HcR4, HcmR4, and Hc were produced as secreted proteins using baculovirus-mediated expression in SF9 insect cells. In vitro receptor binding assays showed that HcR4 effectively targets Hc to all classes of FcγRs. APCs loaded with HcR4 or HcmR4 are substantially more effective at stimulating Hc-reactive T cells than APCs loaded with nontargeted Hc. Mice immunized with a single dose of HcmR4 or HcR4 had earlier and markedly higher Hc-reactive antibody titers than mice immunized with nontargeted Hc. These results extend to BoNT neutralizing antibody titers, which are substantially higher in mice immunized with HcmR4 than in mice immunized with Hc. Our results demonstrate that targeting Hc to FcγRs augments the pace and magnitude of immune responses to Hc.     

UVB irradiation modulates systemic immune responses by affecting cytokine production of antigen-presenting cells

The immunosuppressive effects of UVB irradiation have been well documented. The production of cytokines by keratinocytes is considered to play a major role in the induction of local as well as systemic immunosuppression. It is thought that partly due to the interaction of locally produced cytokines with antigen-presenting cells (APC) systemic effects, like antigen-specific tolerance, can be induced. In this study we examined the effect of UVB irradiation on cytokine profiles of peripheral APC as well as the functional consequences. Our results indicate that UVB irradiation impairs T(h)1-mediated immune responses in vivo by suppression of the systemic IL-12p70 production. Splenic APC from UVB-exposed mice showed an enhanced production of prostaglandin E(2), IL-1, IL-6 and tumor necrosis factor-alpha after in vitro stimulation. Also, spleen cells from UVB irradiated IL-4(-/-) mice showed increased IL-6 levels. These APC were less efficient in inducing IFN-gamma production by CD4(+) T cells and suppressed IgM production by B cells. We conclude that the altered cytokine profile of peripheral APC can be responsible for the systemic effects of UVB irradiation on the T(h)1/T(h)2 balance as well as on B cell responses.     

Melanoma-derived gangliosides impair migratory and antigen-presenting function of human epidermal Langerhans cells and induce their apoptosis

Gangliosides are ubiquitous, membrane-associated, glycosphingolipids, the composition and production of which is altered in many tumour cells. They have been shown to inhibit the in vitro generation and differentiation of dendritic cells (DCs) from progenitors, but their effect on human tissue-residing DCs is yet to be investigated. In the present study, we analysed the effect of GM3 and GD3 gangliosides purified from human melanoma tumours on the phenotypic and functional maturation of human epidermal Langerhans cells (LCs), the first immune barrier against the tumour cells. We showed that both gangliosides impaired spontaneous LC maturation induced by a short in vitro culture, as assessed by significant down-regulation of co-stimulation (CD40, CD54, CD80, CD86) and maturation markers (CD83, CCR7), which correlated to an impaired ability of the cells to mount allogeneic T cell proliferation. Furthermore, the ganglioside-treated cells displayed less ability to migrate towards CCL19/macrophage inflammatory protein 3 beta, the chemokine that specifically binds CCR7 and mediates LC migration to lymph nodes. Lastly, we showed that both GM3 and GD3 gangliosides enhance LC spontaneous apoptosis. Globally, these in vitro results might explain, at least in part, the altered number and distribution of LCs in melanoma-bearing patients. They underscore a new mechanism for gangliosides to impede the host immune response by inducing LC dysfunction in the tumour microenvironment.     

成纤维细胞作为有效的抗原提呈细胞诱导特异性CTL反应

目的;探讨利用癌症患者自体纤维细胞作为抗原提呈细胞,诱导抗肿瘤的细胞毒Ｔ淋巴细胞反应的可能性。方法：制备阳离子脂质体,并包大肠癌相关抗原ＣＡ－Ｈｂ３。制备自体成纤维细胞,肿瘤细胞和外周血淋巴细胞（ＰＢＬ）。借助脂质体将ＣＡ－Ｈｂ３抗原导入成纤维细胞浆内,采用此细胞抗原周期刺激法,体外诱导抗原特异性ＣＴＬ反应。     

Flagellin fusion proteins as adjuvants or vaccines induce specific immune responses

Vaccination is the most efficient prophylaxis against a variety of infectious diseases. New vaccination strategies rely on the incorporation of effective adjuvants, which stimulate the innate immune response and, in turn, activate the adaptive immune response. It is well established that flagellin induces inflammatory responses through the activation of antigen-presenting cells (APCs). In order to evaluate whether flagellin can serve as a carrier for the development of adjuvants or vaccines, we prepared a flagellin-enhanced green fluorescent protein (EGFP) fusion protein. Our results demonstrate that a flagellin-EGFP fusion protein is capable of stimulating APCs, resulting in the maturation of these cells and secretion of proinflammatory cytokines. Furthermore, APCs pulsed with the flagellin-EGFP fusion protein effectively process and present EGFP antigens. More importantly, animals immunized with the flagellin-EGFP fusion protein developed specific anti-EGFP T-cell responses. In contrast, recombinant EGFP was not able to stimulate APCs, nor did it induce a T-cell response. Thus, recombinant-flagellin fusion proteins may be suitable carriers as adjuvants or vaccines for the development of new vaccination strategies to induce and boost immune responses against infectious diseases and cancer.     

Correlations between immunoglobulin- and antibody-synthesizing cells during primary and secondary immune responses of rats immunized with peroxidase.

The development of cells synthesizing immunoglobulins without detectable antibody activity and of antibody-synthesizing cells was studied during primary and secondary immune responses of rats immunized with horseradish peroxidase. After primary immunization with peroxidase emulsified in Freund's complete or incomplete adjuvant, the first antibody-producing cells appeared 4 days after injection. They were preceded by cells synthesizing IgG and IgM without antibody function, appearing 3 days after giving antigen. The ratio between the latter and the former population of cells regularly decreased during the primary response. Seventy to 100 per cent of cells synthesizing immunoglobulins without antibody activity were induced by the antigen, the remainder being induced by the adjuvant. In both populations, the positive cells were always immature or mature plasmocytes. At various times after primary injection, animals received a booster inoculation of soluble peroxidase or of peroxidase emulsified in Freund's adjuvant. Antibody-producing cells, in early stages of differentiation, appeared between 2 and 3 days after challenge and were not preceded by cells synthesizing immunoglobulins without antibody function. These latter cells were reduced or absent after secondary challenge. Increasing the sensitivity of detection of active sites of antibodies, by using direct methods of staining with fixed or unfixed cells gave no increase of antibody-producing cells.