Expression of cutaneous lymphocyte-associated antigen on human CD4(+) and CD8(+) Th2 cells

The cutaneous lymphocyte-associated antigen (CLA) represents the homing receptor involved in selective migration of memory/effector T cells to the skin. Numerous reports demonstrated distinct CLA expression on Th1 cells. However, T cells isolated from skin lesions and CLA(+) T cells circulating in peripheral blood of atopic dermatitis patients expressed high IL-5 and IL-13. Accordingly, we investigated the regulation of CLA on human type 1 and type 2 T cells. CLA was induced on freshly generated Th1 and Tc1 cells only, but not on those of type 2. Anti-CD3 stimulation was sufficient to induce CLA on Th2 cells in the absence of serum in the culture medium. In serum containing medium, IL-4 inhibited CLA and related alpha-fucosyltransferase mRNA expression. IL-12 and/or staphylococcal enterotoxin B (SEB) stimulation up-regulated CLA expression on either Th2 and Tc2 cells. On stimulation with IL-12, CLA was expressed on the surface of bee venom phospholipase A(2)-specific Th1, Th2, Th0 and T regulatory 1 clones, representing non-skin-related antigen-specific T cells. In addition, CLA could be re-induced on T cells that had lost CLA expression upon resting. These results suggest that skin-selective homing is not restricted to functional and phenotypic T cell subsets.     

Distinctive homing profile of pathogen-specific activated lymphocytes in human urinary tract infection

In contrast to other mucosal sites, information on migration/homing of lymphocytes activated in the human urinary tract is lacking. The expression of lymphocyte homing receptors (HR) on pathogen-specific antibody-secreting cells (ASC) originating from the urinary tract (patients with pyelonephritis, PN) was compared to that on antigen-specific ASC originating from the intestine (patients with gastroenteritis) or from a parenteral site (tetanus toxoid-immunized volunteers). In the PN group, 61% of ASC expressed the gut HR, alpha(4)beta(7,) 52% the peripheral lymph node HR, L-selectin, and 13% the skin HR, CLA. This homing profile of urinary tract-originating lymphocytes was found to differ from both of the two major vaccination routes, intestinal (less gut-targeting) or parenteral (more gut-targeting, less targeting to parenteral sites). This information on targeting of the immune response may prove useful when developing vaccines against urinary tract infection (UTI).     

Specific-Antibody-Secreting Cells in the Rectums and Genital Tracts of Nonhuman Primates following Vaccination

To determine optimal strategies to induce specific-antibody-secreting cells (specific ASC) in the rectal and vaginal mucosae, we immunized monkeys with a prototype mucosal immunogen, cholera toxin (CT), given locally or via gastric or parenteral administration. Repeated rectal or vaginal CT immunizations induced strong mucosal and systemic ASC responses. The mucosal responses were, however, confined to the immunization sites and comprised high levels of both specific antitoxin immunoglobulin A (IgA) and IgG. Large numbers of specific IgA and IgG ASC were detected in cell suspensions from dissociated genital and rectal tissues, demonstrating local accumulation of effector B cells at these sites. Intragastric immunization with CT did not per se give rise to cervicovaginal or rectal ASC responses but did prime for a rectal IgA ASC response to local booster immunization. Both rectal and vaginal immunizations also induced circulating blood IgG ASC and IgA ASC. In conclusion, these results show that local administration of antigen to the rectal or vaginal mucosa results in higher ASC responses than systemic or distant mucosal delivery. Furthermore, both the vaginal and the rectal mucosae can serve as inductive sites for systemic ASC responses. These observations should be relevant to the development of vaccines against sexually transmitted diseases such as that caused by human immunodeficiency virus.     

Quantification of antigen-specific antibody-secreting cells in the small intestine and other lymphoid organs of mice after oral booster immunization.

The intestinal immune response of mice against ovalbumin (OVA) was quantified by isolating lymphoid cells from the small intestine (SI) and testing them for antigen-specific immunoglobulin (Ig) secretion. The isolation procedure for functionally active lymphoid cells from the SI, originally developed to quantify the number of 'background' Ig-secreting cells in the SI, proved to be a useful method for evaluating antigen-specific intestinal immune responses quantitatively. The method was able to detect antigen-specific antibody-secreting cells (ASC) in the SI even when these cells occurred at a minimum frequency of only 0.006%. When mice were primed intraperitoneally (i.p.) with polymerized OVA and given an oral OVA booster immunization, OVA-specific ASC appeared in the SI from Day 3 after booster. After i.p. priming and an i.p. booster these cells could not be detected in the SI. The OVA-specific IgA-ASC responses in various organs after oral booster immunization were compared. From Day 5 after booster, when the response peaked, most OVA-specific IgA-ASC occurred in the SI. This suggested that these cells are mainly responsible for the OVA-specific antibodies demonstrated by ELISA in intestinal secretions from Day 6 after oral booster immunization. It is concluded that the quantitative method used in this study detects antigen-specific ASC in the SI with great sensitivity and could be used to evaluate immunization regimes aimed at inducing intestinal mucosal immune responses.     

Interleukin-4 induced down-regulation of skin homing receptor expression by human viral-specific CD8 T cells may contribute to atopic risk of cutaneous infection

Factors controlling the expression of cutaneous lymphocyte-associated antigen (CLA) by T cells are poorly understood, but data from murine and human CD4(+) T cell systems have suggested that cytokines play an important role. However, there are no data examining the influence of cytokines on the expression of CLA by human antigen-specific CD8(+) T cells. Peripheral blood mononuclear cells (PBMC) were isolated from 10 HLA-A*0201-positive healthy individuals. Using HLA-peptide tetrameric complexes refolded with immunodominant peptides from Epstein-Barr virus (EBV), cytomegalovirus (CMV) and influenza A virus, we investigated the temporal associations of CLA expression by viral-specific CD8(+) T cells following stimulation with antigen. Ex vivo influenza matrix-specific CD8(+) T cells expressed significantly (P < 0.05) greater levels of CLA than EBV BMLF1 and CMV pp65-specific CD8(+) T cells (mean 9.7% influenza matrix versus 1.4% BMLF1 versus 1.1% pp65) and these differences were sustained on culture. However, regardless of viral specificity, interleukin (IL)-12 and IL-4 induced significant (P < 0.05) dose-dependent up-regulation and down-regulation of CLA expression, respectively, with IL-4 showing a dominant negative effect. In many cases, IL-4 resulted in complete abrogation of detectable CLA expression by the viral-specific CD8(+) T cells. Overall these data demonstrate that CLA expression by human viral-specific CD8(+) T cells is highly dynamic and that IL-4 causes significant down-regulation. Disorders associated with a type 2 cytokine shift may reduce the efficiency of skin homing by viral-specific CD8(+) T cells. Furthermore, the ability to modify the local and systemic microenvironment may offer novel therapeutic strategies that influence tissue-specific T cell homing.     

Interleukin-4 promotes human CD8 T cell expression of CCR7

Despite strong evidence supporting a pathway of human T cell differentiation characterized by changes in the expression of CCR7, CD28, CD27 and CD62L, few studies have addressed the mechanisms of pathway regulation. Cutaneous lymphocyte-associated antigen (CLA)-positive skin-homing CD8(+) T cells expressed significantly elevated levels of activation markers compared with CLA(-) CD8(+) T cells in individuals (n = 27) with cutaneous atopic disease. Despite such an activated phenotype, CLA(+) T cells expressed significantly higher levels of CCR7 than a CLA(-) T cell subset. Interleukin (IL)-4 was found to dramatically promote CCR7 expression by antigen-specific CD8(+) cells. Furthermore, skin-homing CD8(+) T cells from individuals with severe disease produced significantly less IL-10 than those derived from mildly affected atopic subjects. Thus in a T-helper 2 dominated disease, tissue-specific CD8(+) T cells show altered CCR7 expression and cytokine production, which may contribute to continued lymph node homing, antigen presentation and disease. IL-4 promotes expression of CCR7, a marker linked to existing models of CD8(+) T cell differentiation.     

Mucosal vaccination increases endothelial expression of mucosal addressin cell adhesion molecule 1 in the human gastrointestinal tract

Homing of leukocytes to various tissues is dependent on the interaction between homing receptors on leukocytes and their ligands, addressins, on endothelial cells. Mucosal immunization results in homing of antigen-specific lymphocytes back to the mucosa where they first encountered the antigen. However, it is unknown whether this homing of antigen-specific cells is mediated by an altered endothelial addressin expression after vaccination. Using different immunization routes with an oral cholera vaccine, we show that the endothelial expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) is increased in the gastric and upper small intestinal mucosae after immunization through various local routes in the upper gastrointestinal tract. In contrast, rectal immunization did not influence the levels of MAdCAM-1 in the gastric or duodenal mucosa. Furthermore, we show that MAdCAM-1 can be induced on human endothelial cells by tumor necrosis factor alpha (TNF-alpha) and gamma interferon. The vaccine component cholera toxin B subunit (CTB) increased MAdCAM-1 expression on endothelial cells in cultured human gastric explants, an effect that seemed to be mediated by TNF-alpha. In conclusion, MAdCAM-1 expression is increased in the upper gastrointestinal tract after local immunizations with a vaccine containing CTB. This strongly suggests the involvement of MAdCAM-1 in the preferential homing of mucosal lymphocytes to their original site of activation.     

Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system

In the interest of better understanding the role of human memory B cells in protection against disease, we developed an assay to quantitate antigen-specific memory B cells in human blood. This assay utilizes a 6-day polyclonal stimulation of PBMC followed by an antigen-specific ELISPOT for the detection of memory B cells that have differentiated into antibody secreting cells (ASC) in vitro. We have used this assay to demonstrate that the anthrax vaccine (AVA; BioThrax) elicits a substantial population of protective-antigen (PA) specific memory B cells, and these B cells satisfy the canonical surface phenotype of human memory B cells: CD19(+)CD20(+)Ig(+)CD27(+). These anti-PA antigen-specific memory B cells are IgG(+) and represent up to 2% of circulating IgG(+) B cells. Furthermore, these results confirm that vaccine-elicited memory B cells reside in the CD27(+) B cell population. This ELISPOT-based system has been designed in a generalized manner, such that the assay can be rapidly adapted to detect human antigen-specific memory B cells of any given specificity. This method should be useful for quantitatively assessing the potency of vaccines and the longevity of B cell immunological memory to various vaccines or infectious diseases.     

Intestinal and circulating antibody-forming cells in IgA-deficient individuals after oral cholera vaccination

In search for a possible explanation for the different susceptibility to mucosal infections in IgA-deficient (IgAd) individuals, the frequency of total immunoglobulin-secreting cells (ISC) and vaccine-specific antibody-secreting cells (ASC) in intestinal mucosa and peripheral blood was determined by the enzyme-linked immunospot (ELISPOT) assay before and after peroral vaccination with a B subunit-whole cell cholera vaccine. Two groups of IgAd individuals, frequently infected and non-infected respectively, and normal controls were studied. Before cholera vaccination there were significantly higher frequencies of total IgM and IgG ISC in the gut, but not in the blood, in the IgAd individuals than in the controls. However, there were no significant differences between healthy and infection-prone IgAd individuals in this respect. In response to oral cholera vaccination, intestinal cholera toxin (CT)-specific IgG and IgM ASC were significantly more abundant among the IgAd individuals with a history of frequent infections than among the healthy IgAd individuals and controls. A similar difference in IgG and IgM ASC, although not significant, was also noted in blood. In IgAd individuals with frequent infections the vaccine induced variable anti-CT IgM ASC responses in the gut, ranging from no increase to a few strikingly high responses. In the controls, the CT-specific responses were dominated by IgA ASC. The data show that oral cholera vaccination evoked strong CT-specific IgG ASC responses, and in some cases also strong IgM ASC responses in the intestinal mucosa of IgAd patients with a history of frequent infections. The healthy IgAd individuals unexpectedly responded with lower numbers of CT-specific IgG ASC and did not show any increase of CT-specific IgM ASC in the intestinal mucosa. Thus, inability to mount a mucosal immune response to an oral antigen cannot in itself explain recurrent infections among many IgAd individuals.     

Differences in Immune Responses Induced by Oral and Rectal Immunizations with Salmonella typhi Ty21a: Evidence for Compartmentalization within the Common Mucosal Immune System in Humans

Based on the concept of the common mucosal immune system, immunization at various inductive sites can induce an immune response at other, remote mucosal surfaces. The immune responses elicited through rectal and oral routes of antigen delivery were compared with respect to (i) measurement of antibody responses in serum and various external secretions of the vaccinees and (ii) characterization of the nature and homing potentials of circulating antibody-secreting cells (ASC). Specific ASC appeared in the circulation in 4 of 5 volunteers after oral and 9 of 11 volunteers after rectal immunization with Salmonella typhi Ty21a. The kinetics, magnitude, and immunoglobulin isotype distribution of the ASC responses were similar in the two groups. In both groups, almost all ASC (99 or 95% after oral or rectal immunization, respectively) expressed α4β7, the gut homing receptor (HR), whereas l-selectin, the peripheral lymph node HR, was expressed only on 22 or 38% of ASC, respectively. Oral immunization elicited a more pronounced immune response in saliva and vaginal secretion, while rectal immunization was more potent in inducing a response in nasal secretion, rectum, and tears. No major differences were found in the abilities of the two immunization routes to induce a response in serum or intestinal secretion. Thus, the rectal antigen delivery should be considered as an alternative to the oral immunization route. The different immune response profiles found in various secretions after oral versus rectal antigen administration provide evidence for a compartmentalization within the common mucosal immune system in humans.

Mucosal delivery of antigens is one of the main goals of current vaccine development. Mucosal immunization has several advantages over the conventional parenteral route: it is safer, less expensive, and easier to carry out in developing countries, and the antigen can be introduced to the body through the same routes as in a natural infection. It seems appealing to administer antigens through the gastrointestinal route, as the intestine contains a large accumulation of lymphoid tissues with lymphoepithelial structures involved in the induction of mucosal immune responses (4). Accordingly, the oral route of antigen delivery is the most common and most frequently explored among the mucosal immunization routes. However, oral antigen delivery poses some problems, such as the denaturation of stomach acid and digestion of antigens due to long exposure to gastrointestinal proteolytic enzymes. Alternative gastrointestinal routes include rectal antigen delivery, which so far has not been extensively explored in humans (9, 14, 29, 35, 36). However, the rectal mucosa is known to be rich in lymphoepithelial structures analogous to Peyer’s patches (37).The different mucosal surfaces in the body are believed to be interconnected via circulating lymphocytes, as recognized by the concept of the common mucosal immune system (CMIS) (32): immunization at one mucosal inductive site (e.g., intestinal Peyer’s patches) can lead to an immune response at another, anatomically remote mucosal effector site (e.g., saliva or genital tract secretions). Consistent with this concept, mucosal immunization is known to be followed by a transient appearance of antibody-secreting cells (ASC) in the peripheral blood (11, 22), and antibody responses have been found on mucosal surfaces distant from the original site of mucosal immunization (7, 16, 32, 33). However, recent data suggest that some degree of compartmentalization may exist within the CMIS (18, 34); therefore, the general routes of lymphocyte homing from each mucosal site need to be explored.Recently, it has become possible to investigate the homing potentials of circulating ASC by examining their expression of homing receptors (HR) (24–26, 40, 41). Homing of lymphocytes into tissues is currently understood as a multistep process in which a cascade of events described as initial contact and rolling, activation, arrest, and finally diapedesis follow each other (5, 38, 45, 46). Many different molecules participate in the process, yet the organ specificity is regarded to be contributed by a small selection of them. HR are cell surface receptors that specifically bind to their ligands, addressins on the endothelial cells of the target tissues: this binding is a prerequisite for the penetration of the cell through the endothelial cell wall. The organ specificity of lymphocyte homing is based on a differential expression of the addressins in the target tissues. Examination of HR expression on lymphocytes reveals the homing potentials of the cells. Among the HR contributing to the organ specificity of the homing process are α4β7 integrin (guiding cells to the gut mucosa) (3, 13, 17), l-selectin (guiding cells to the peripheral lymph node) (6, 19, 20, 28), and cutaneous lymphocyte antigen (guiding cells to the skin) (2, 39). It has been suggested that the respiratory tract may have its own, still unidentified HR (1).α4β7 integrin is known as a gut-specific HR, yet the homing mechanisms of cells to other mucosal surfaces are still obscure: this information is currently obtained by analyzing antibody responses in various secretions. To characterize the human immune responses elicited by oral versus rectal antigen administration in different compartments of the immune system, we studied the ASC response with special attention to the homing potentials of the cells and the induction of immune response in serum and various external secretions.     

Secretory IgA as a vaccine carrier for delivery of HIV antigen to M cells

HIV transmission and spread in the host are based on the survival of the virus or infected cells present in mucosal secretions, and the virus' ability to cross the epithelial barrier and access immune target cells, which leads to systemic infection. Therefore, HIV‐specific immunity at mucosal sites is critical for control of infection. Although mucosal delivery would ensure the best onset of protective immunity, most candidate vaccines are administered through the parenteral route. Remarkably, secretory IgA (SIgA) interacts specifically with mucosal microfold (M) cells present in gut‐associated lymphoid tissues. Here we evaluate the feasibility of delivering chemically bound p24HIV antigen via SIgA into the intestinal mucosae in mice. After oral administration, p24–SIgA complexes are quickly delivered into the tissue and selectively captured by CX3CR1⁺ dendritic cells. Oral immunization with p24gag linked to SIgA (p24–SIgA) adjuvanted with E. coli heat labile enterotoxin (HLT) elicits both humoral and cellular immune responses against p24 at the systemic and mucosal levels and induces efficient protection against rectal challenge with a recombinant vaccinia virus encoding gag. This is the first study which underscores the remarkable potential of SIgA to serve as a vaccine carrier for an HIV antigen in mucosal administration targeting the gastrointestinal environment.     

Oral priming with Salmonella Typhi vaccine strain CVD 909 followed by parenteral boost with the S. Typhi Vi capsular polysaccharide vaccine induces CD27+IgD-S. Typhi-specific IgA and IgG B memory cells in humans

Attenuated live oral typhoid vaccine candidate CVD 909 constitutively expresses Salmonella Typhi capsular polysaccharide antigen (Vi). A randomized, double-blind, heterologous prime-boost clinical study was conducted to determine whether immunity to licensed parenteral Vi vaccine could be enhanced by priming with CVD 909. Priming with CVD 909 elicited higher and persistent, albeit not significant, anti-Vi IgG and IgA following immunization with Vi, than placebo-primed recipients. Vi-specific IgA B memory (B(M)) cells were significantly increased in CVD 909-primed subjects. S. Typhi-specific LPS and flagella IgA B(M) cells were observed in subjects immunized with CVD 909 or with the licensed Vi-negative oral typhoid vaccine Ty21a. CVD 909-induced B(M) cells exhibited a classical B(M) phenotype (i.e., CD3(-)CD19(+)IgD(-)CD27(+)). This is the first demonstration of classical B(M) cells specific for bacterial polysaccharide or protein antigens following typhoid immunization. The persistent IgA B(M) responses demonstrate the capacity of oral typhoid vaccines to prime mucosally relevant immune memory.     

Functionally divergent T lymphocyte responses induced by modification of a self-peptide from a tumor-associated antigen

The N- and C-terminal flanking domains of the invariant chain peptide, CLIP, have remarkable immunological properties. Addition of these flanking domains to a foreign peptide antigen increases its immunologic potency. The present studies evaluated whether altering a peptide ligand from the tumor-associated antigen c-neu with the flanking domains of CLIP could modify the systemic immune response. The results indicate that the immunogenicity of an MHC class II restricted peptide (NEU) derived from c-neu was significantly altered by addition of the flanking domains from CLIP. Interestingly, selective modification of the peptide with either the N- or the C-terminal flanking domains resulted in functionally divergent systemic immune responses. Immunization of normal F344 rats with the NEU peptide modified with the N-terminal domain of CLIP (N-NEU) resulted in an immune response primarily consisting of type 1 (IL-2, IFNgamma) cytokine producing T cells. On the other hand, type 2 (IL-4) cytokine responses were largely predominant following immunization with the self-peptide modified with the C-terminal flanking domain (NEU-C). The functionally divergent responses elicited by the modified self-peptides were accompanied by significant changes in the expression of the CD28/CTLA4/B7 family of co-stimulatory molecules. Immunization with the N-NEU peptide led to enhanced expression of CD28 in the antigen-specific, CD4+ T cell compartment while expression of B7.1 was dramatically reduced in antigen-specific CD8+ T cells. Comparatively, expression of CTLA4 was down-regulated in the antigen-specific CD4+ T cell compartment following immunization with NEU-C peptide. The N-NEU peptide also had a direct effect on dendritic cells leading to the up-regulation of B7.1 expression. Taken together, functionally divergent systemic immune responses can be elicited by strategically altering a self-peptide ligand with the N- and C-terminal flanking domains of CLIP. Moreover, changes in expression of co-stimulatory molecules that are required for T cell activation and T cell-T cell communication may account for the polarization of the immune response elicited by the chimeric peptides.     

Regulation of human T-cell homing receptor expression in cutaneous bacterial infection

We investigated the regulation of T-cell homing receptors in infectious disease by evaluating the cutaneous lymphocyte antigen (CLA) in human leprosy. We found that CLA-positive cells were enriched in the infectious lesions associated with restricting the growth of the pathogen Mycobacterium leprae, as assessed by the clinical course of infection. Moreover, CLA expression on T cells isolated from the peripheral blood of antigen-responsive tuberculoid leprosy patients increased in the presence of M. leprae (2.4-fold median increase; range 0.8-6.1, n = 17), but not in unresponsive lepromatous leprosy patients (1.0-fold median increase; range 0.1-2.2, n = 10; P < 0.005). Mycobacterium leprae specifically up-regulated the skin homing receptor, CLA, but not alpha(4)/beta(7), the intestinal homing receptor, which decreased on T cells of patients with tuberculoid leprosy after antigen stimulation (2.2-fold median decrease; range 1.6-3.4, n = 3). Our data indicate that CLA expression is regulated during the course of leprosy infection and suggest that T-cell responsiveness to a microbial antigen directs antigen-specific T cells to the site of infection.     

Protection against experimental cholera by oral or parenteral immunization.

Comparisons were made between the antigenic potency and protective capacity of several cholera toxin derivatives. Rabbits were immunized parenterally with 50 microgram of cholera toxin, A subunit, B subunit, procholeragenoid, or Wyeth glutaraldehyde toxoid 20101. Examination of the antibody response curves revealed that cholera toxin elicited serum antitoxin responses that rose more quickly than in the subunit-immunized animals; however, antitoxin levels were of the same magnitude after 10 weeks. Parenteral immunization with procholeragenoid evoked antibody titers that were similar to the toxin, whereas Wyeth toxoid yielded only one-tenth the level of antitoxin. Oral immunization with procholeragenoid as well as Wyeth toxoid resulted in lower serum antitoxin titers than that achieved with parenteral immunization, despite the oral administration of 10 times the parenteral dose. Analysis of protection against live-cell challenge revealed that parenteral administration of procholeragenoid provided the best protection against fluid accumulation. Oral immunization with procholeragenoid also was very effective, whereas oral immunization with B subunit or Wyeth toxoid resulted in minimal protection. Also, the A subunit provided surprisingly more protection than did cholera toxin.     

Delayed and deficient establishment of the long-term bone marrow plasma cell pool during early life

Early life antibody responses are characterized by a rapid decline, such that antigen-specific IgG antibodies decline to baseline levels within months following infant immunization. This generic observation remains unexplained. Here, we have analyzed the induction and organ-localization of antigen-specific IgG antibody-secreting cells (ASC) following immunization of 1-week-old or adult BALB/c mice with tetanus toxoid (TT), a T-dependent antigen. Early life priming induced only slightly lower numbers of TT-specific IgG ASC in the spleen, and these reached adult levels following repeat immunization. In contrast, early life immunization generated much fewer bone marrow plasma cells than in adults, even after boosting. A similar limitation of the natural development of the bone marrow pool of ASC was observed. Transfer experiments with adult or early life spleen ASC indicated limited homing of TT-specific adult ASC to the bone marrow of 4-week-old mice as compared to adult recipients, whereas homing patterns were similar when early life or adult ASC were transferred into adult recipients. These observations suggest that a limited bone marrow B cell homing capacity and, as a result, relatively deficient bone marrow ASC responses, are critical factors which may explain the limited persistence of IgG antibodies to T-dependent antigens in early life.     

Kinetics of oral tolerance: study of variables affecting tolerance induced by oral administration of antigen

The induction of antigen-specific tolerance by oral administration of hen egg albumin (OVA) was shown to be time- and dose-dependent. The IgE response was the most effectively suppressed by antigen feeding prior to or after parenteral immunization. The effect on IgG response paralleled that observed on the IgE response, but the magnitude of suppression was less pronounced. The IgA response was enhanced rather than suppressed if the interval between feeding and parenteral immunization was short or if the animals were antigen-fed after the intraperitoneal priming.     

Stimulation of antigen-specific T- and B-cell memory in local as well as systemic lymphoid tissues following oral immunization with cholera toxin adjuvant.

In the present study we investigated immunological memory at the cellular level following oral immunization using cholera toxin (CT) as the mucosal adjuvant. We found that memory cells, isolated from mice orally primed with keyhole limpet haemocyanin (KLH) admixed with CT adjuvant 8 months earlier, responded by increased proliferation to antigen-challenge in vitro. In contrast, unstimulated memory cells or KLH-stimulated cells from naive mice did not respond. Memory cells were isolated from different lymphoid tissues; spleen (SP), mesenteric lymph nodes (MLN), Peyer's patches (PP) as well as the intestinal lamina propria (LP). Thus, oral immunization using CT adjuvant promoted the generation of memory cells that were present in both systemic and local intestinal lymphoid tissues. The demonstration of lymphokine production in the KLH-responsive cultures indicated the presence of antigen-specific memory T cells. Lymphokine production early in culture was dominated by interleukin-2 (IL-2), which peaked on day 2-3, followed by IL-5 and, in particular, interferon-gamma (IFN-gamma) which increased over time. Lamina propria memory cells were found to proliferate poorly to recall antigen in vitro compared to lymphocytes from SP or MLN. In contrast, very significant production of IL-5 and, in particular, IFN-gamma was demonstrable in LP cell cultures. The use of CT adjuvant also stimulated the generation of antigen-specific memory B cells following oral immunization. This was evidenced by KLH-specific antibody production in antigen-challenged memory lymphocyte cultures. The memory B cells produced IgM anti-KLH, while no detectable antigen-specific IgG or IgA was found. Unstimulated memory cells or naive cells failed to produce anti-KLH antibodies. These in vitro findings provide evidence that oral immunization using CT adjuvant stimulates both antigen-specific memory T and B cells. Furthermore, our data suggest the existence of memory B cells following oral CT adjuvant immunization which have retained the ability to produce IgM and which therefore probably have not undergone terminal isotype switch differentiation to other isotypes and thus have not deleted the mu constant heavy-chain gene. Finally, our data also suggest that memory T and B cells, either sessile in the various lymphoid tissues or recirculating, can be activated by antigen in situ in, for example, lymph nodes and spleen and, more importantly, in the intestinal LP itself.     

Antigen-specific regulatory T cells are detected in Peyer's patches after the interaction between T cells and dendritic cells loaded with orally administered antigen

Systemic immune tolerance is induced for orally administered antigen, and this phenomenon is called oral tolerance. However, the mechanism of oral tolerance has not been completely elucidated. It has been suggested that antigen presentation and generation of regulatory T cells in Peyer's patches (PPs) are important for induction of oral tolerance. Hence, we orally administered fluorescence-labelled antigen to mice and examined kinetics of the antigen and interaction between antigen-loaded dendritic cells and T cells. It was visualized that dendritic cells in PP rapidly take up antigen. We next transferred antigen-specific naïve T cells from T cell receptor transgenic mice and administered the antigen orally. Antigen-specific T cells accumulated in IFR in PP and DCs that have ingested antigen come in contact with antigen-specific T cells in IFR. The accumulated T cells were then collected and analyzed for the pattern of gene expression by real-time PCR, which revealed a gene expression pattern similar to that of FoxP3-positive regulatory T (T_reg) cells. CCR9, an intestinal homing marker, was also strongly expressed. These results suggest that DCs that have captured oral antigens in PPs locally induce antigen-specific naïve T cells to differentiate into T_reg cells with the intestinal homing phenotype.     

Varied levels of reactivity by different E-selectin/Fc constructs with cutaneous lymphocyte-associated antigen (CLA)(+) CD4(+) T cells

T cells utilize the vascular adhesion molecule E-selectin to enter inflamed skin. T cells identified by the mAb HECA-452 [cutaneous lymphocyte-associated antigen (CLA) T cells] are enriched in E-selectin ligand expressing cells. However, the proportion of CLA(+) T cells reactive with an E-selectin/Fc chimeric construct, as determined by flow cytometry, can vary considerably between studies. One important variable in these studies has been the E-selectin/Fc chimera used to assess ligand expression. We therefore compared the reactivity of mouse, rat, and human E-selectin/Fc from the same widely used commercial source with peripheral blood CLA(+) CD4(+) T cells, neutrophils, and the promyelocytic cell line HL-60 by flow cytometry and by shear flow assays. We observed that the binding activities of the different E-selectin/Fc chimeras were considerably different. Mouse E-selectin/Fc demonstrated the highest binding activity with human neutrophils and HL-60 cells by both assay approaches, whereas human E-selectin/Fc demonstrated the lowest binding activity. In addition, mouse E-selectin/Fc binding increased essentially in a linear manner with increasing expression of CLA by CD4(+) T cells, whereas human and rat E-selectin/Fc binding occurred with only a subset of CLA(+) CD4(+) T cells. We conclude that there is substantial variability in the reactivity of different E-selectin/Fc constructs, thus caution should be used when assessing E-selectin ligand expression with these reagents. For instance, the discordance in expression of CLA and E-selectin ligands by T cells may in part be due to the E-selectin/Fc construct being used.