In vitro responses to purified protein derivate of caprine T lymphocytes following vaccination with live strains of Mycobacterium avium subsp paratuberculosis

Live attenuated vaccines provide protection against intestinal lesions in goats infected with Mycobacterium avium subsp. paratuberculosis. To examine the role of different T lymphocyte subsets in the development of this protective immunity, CD4(+), CD8(+) and gamma delta T cell receptor (TCR)(+) cells from peripheral blood of goat kids vaccinated with live attenuated strains of M. a. paratuberculosis were studied. After in vitro stimulation with purified protein derivate, the expression of gamma-interferon (IFN-gamma) and the activation marker interleukin-2 receptor (IL-2R) was analysed by flow cytometry. A depletion experiment was performed, where the phenotypes and IL-2R expression was studied after stimulation of cultures depleted of a T lymphocyte subpopulation. Close to all of the IFN-gamma producing cells were of the CD4(+) subset, while only a small number were CD8(+) cells. The gamma delta TCR(+) cells were highly activated, but did not produce IFN-gamma after in vitro stimulation. Depletion of CD4(+) cells lead to a decrease in the percentage of total gamma delta TCR(+) cells and gamma delta TCR(+)IL2-R(+) cells. Removing the gamma delta TCR(+) cells increased the relative numbers of CD4(+), but not the CD4(+)IL-2R(+) cells. Insight into the in vitro recall responses of T cell subsets from animals vaccinated with live paratuberculosis vaccines is essential in the development of more efficient vaccines.     

Morphological Considerations in the Pathogenesis of Paratuberculosis in Goats

Paratuberculosis is a chronic granulomatous enteritis and lymphadenitis in ruminants caused by Mycobacterium avium subsp. paratuberuclosis . To investigate the efficacy of assays for early diagnosis, experimental infections were performed in goat kids. Oral doses of M. a. paratuberculosis were administered to goat kids over extended periods and the resulting infection was followed for 12–24 months. Complementary experiments were performed on goat kids vaccinated against M. a. paratuberculosis . Immunological, pathological and morphological examinations were undertaken in both these experimental systems. The analysis of results from these experiment series provided novel insights into the influence of the biology of gut-associated lymphoid tissue on the progress of disease, into the character of the tissue granuloma and its dominance of the local tissue response and into the relationship between the local and systemic immune responses.     

In vivo depletion of CD8+ cells does not affect primary maedi visna virus infection in sheep

T-cells have been implicated both, in promoting and reducing viral replication during lentivirus infection. CD8+ lymphocytes are believed to be important in controlling viral load through direct killing of virus-infected cells and by secretion of inhibitory chemokines and cytokines. To evaluate the role of CD8+ T-cells in the induction and control of the primary phase of a lentivirus infection, we have used a non-T-cell tropic lentivirus, maedi-visna virus (MVV), to study the initial pathogenesis and subsequent immune responses in sheep depleted in vivo of CD8+ cells. Sheep were depleted of CD8+ cells in both blood and efferent lymph for up to 14 days. No difference in MVV replication was observed in either the draining efferent lymph or lymph node of these sheep. Surprisingly, these animals displayed a normal induction of pCTL whereas the virus-specific proliferative responses were reduced. This could reflect either that a proportion of functional CD8+ lymphocytes remained in these animals, as suggested by the appearance of pCTLs, or that CD8+ cells are not required for control of primary MVV infection.     

In vivo kinetics of peripheral cellular immune responses in Mycobacterium avium subspecies paratuberculosis (MAP) infected and vaccinated goats

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis (ParaTB) also known as Johne’s disease (JD) in ruminants, which is characterized by chronic intestinal inflammation. A similar counterpart has been observed in the form of Crohn's disease in humans. The present study is the first trail in goats to understand the peripheral cellular immune responses following experimental MAP infection and vaccination. Fifteen apparently healthy male kids (3–6 months old) of Barbari breed were included in this study. In the experimental study, 5 kids were infected with ‘S 5’ strain of MAP (“Indian Bison Type”), 5 were vaccinated (Indigenous Vaccine) against MAP infection (Singh et al., 2007) and the remaining 5 kids were uninfected and non-vaccinated controls. Kids were observed for a period of 180 days post exposure (infection and vaccination) and were tested for development of infection. Cellular immune responses (in blood) were recorded post-exposure by three assays. We measured the frequencies of CD4 and CD8T cells, estimated plasma IFNγ and TNα and in the third assay, in vitro cytokine production by peripheral blood mononuclear cells (PBMCs) from vaccinated, infected and controls were examined in response to polyclonal stimulation. The frequencies of peripheral CD4 and CD8T cells were comparable in control, infected and vaccinated animals except around day 49 post-infection where MAP infected animals showed a trend towards significantly reduced frequencies of CD4 T cells compared to apparently healthy controls. Significantly reduced plasma TNFα levels were also observed in infected animals compared to vaccinated animals,during the course of infection. Diminished levels (although non significant) of TNFα were observed in the supernatants from polyclonally stimulated PBMCs at around day 49 post infection. It is conceivable that the diminished cellular immune responses may coincide with an impairment (immune exhaustion) of perhaps antigen-specific CD4T cells that might, in the course of infection, contribute to the progressive nature of caprine paratuberculosis.     

CD4+CD25+ regulatory T cells are infected and activated during acute FIV infection

HIV-induced AIDS may be mediated by the activation of immunosuppressive CD4+CD25+ T regulatory cells (Treg cells). Treg cells have been shown to regulate CD4+ and CD8+ immune responses to HIV and FIV antigens in vitro. We tested the hypothesis that Treg cells become infected and activated during the acute infection with FIV leading to the suppression of CD4+ T helper cell responses. Cats were experimentally infected with FIV-NCSU1 and blood and lymph node cells were collected at weekly intervals following inoculation. Real-time RT-PCR was used to determine plasma viremia and the relative expression of FIV, FoxP3, TGF-beta, and GAPDH mRNA copies in CD4+CD25+ and CD4+CD25- T cell subsets. Flow cytometry was used to assess the absolute numbers of each cell type and the expression of surface TGF-beta and intracellular FoxP3 in CD4+CD25+ and CD4+CD25- T cells at each time-point. Treg suppression of IL-2 production in CD4+ T helper cells was assessed by ELISPOT assays. Our results showed that peak viremia occurred at 2 weeks post infection and correlated with maximal infectivity in CD4+CD25+ T cell populations. FIV-gag-mRNA levels were higher in CD4+CD25+ T cells than CD4+CD25- T cells throughout the acute phase of infection. Induction of FoxP3 and TGF-beta indicated activation of Treg cells during the acute stage infection, which was confirmed by Treg cell suppression of IL-2 production by CD4+ Th cells in an ELISPOT assay. Our findings support the hypothesis that early activation of Treg immunosuppressor function may limit an effective anti-FIV response, contributing to the establishment of chronic infection and the immunodeficiency caused by this virus.     

Investigation of antigen-specific T-cell responses and subcutaneous granuloma development during experimental sensitization of calves with Mycobacterium avium subsp paratuberculosis

OBJECTIVE: To characterize the early cellular immune response to Mycobacterium avium subsp paratuberculosis (MAP) infection and evaluate the development of granulomatous inflammation at the SC injection site in experimentally inoculated calves. ANIMALS: Forty-eight 4-week-old calves. PROCEDURE: Calves received an SC injection of MAP strain 19698 (n = 25), sterile saline (0.9% NaCl) solution (20), or a commercial paratuberculosis vaccine (3); the inoculation site tissue and associated draining lymph node were excised at postinoculation day (PID) 0 (n = 36), 7 (14), 14 (6), 21 (8), and 60 (32). Sections of inoculation site tissues were evaluated immunohistochemically for T-cell subsets; lymph node mononuclear cells (LNMCs) were assessed for T-cell surface markers and for intracellular interferon-gamma via flow cytometry. RESULTS: At MAP inoculation sites, calves developed mild, focal granulomatous inflammation by PID 7; by PID 60, areas of inflammation contained macrophages with numerous lymphocytes. Compared with control calves, there was increased antigen-specific LNMC proliferation in MAP- and vaccine-inoculated calves at PID 60, although proliferation among lymphocyte subsets was not significantly different between MAP-inoculated and control calves; in vaccine-inoculated calves, CD4+ T-cells predominated. In MAP-inoculated and control calves, antigen-specific interferon-gamma production by LNMCs did not differ significantly; vaccine-inoculated calves had marked interferon-gamma expression by CD4+ T-cells. CONCLUSIONS AND CLINICAL RELEVANCE: In calves, SC administration of MAP resulted in granulomatous inflammation at inoculation sites and an antigen-specific T-cell proliferative response. Results suggest that this experimental system can be used to reproducibly generate antigen-specific T-cells during MAP infection for functional analysis.     

Bacterial isolation, immunological response, and histopathological lesions during the early subclinical phase of experimental infection of goat kids with Mycobacterium avium subsp. paratuberculosis

The diagnosis of Mycobacterium avium subsp. paratuberculosis infection is difficult, especially in the early stages of disease. This is due to the long incubation period, the variable lag phase associated with bacterial proliferation, and the multifocal distribution of slowly developing lesions. There are few previous studies of the early stages of experimental paratuberculosis in goats. In the present study, the ability of conventional diagnostic methods to detect M. a. paratuberculosis infection during the early stages of infection was assessed. Eight goat kids were experimentally infected with M. a. paratuberculosis and subjected to a series of immunological and bacteriological tests before being euthanatized at various times postinfection. At postmortem examination, the ages of the kids ranged from 1 1/2 to 12 months. Of the eight goats infected, three had histopathological evidence of paratuberculosis. Two of these goats were positive with bacteriology, but only one was also positive with all immunological tests. One animal had a positive immunological response, but infection could not be demonstrated by bacteriologic or histopathologic examination. Histopathologic lesions were found in the jejunum, in the ileum, and in one mesenteric lymph node, but only the mesenteric lymph nodes and one retropharyngeal lymph node gave positive results following bacteriologic culture. The disparity between the localization of histopathologic lesions and bacteriologic results emphasizes the need for exhaustive sampling to confirm a diagnosis during the early phase of an infection. It also highlights the need for a better understanding of the biology of M. a. paratuberculosis and its interaction with the immune system of the host.     

A preliminary investigation into the efficacy of intraperitoneal vaccination of sheep and goat kids against heartwater

The intraperitoneal route was explored both as an alternative to the intravenous route for the vaccination of sheep and goat kids against heartwater and for the administration of an antibiotic to animals suffering from the disease. Only 8 (57%) of the 14 adult sheep, and none of the 7 goat kids, were found to be immune after intraperitoneal vaccination with infective blood. However, this route was found to be very suitable and effective for the administration of tetracyclines to reacting animals.     

Protective killed Ehrlichia ruminantium vaccine elicits IFN-gamma responses by CD4+ and CD8+ T lymphocytes in goats

Interferon gamma (IFN-gamma) is considered as a key mediator of protective cell-mediated immunity against intracellular pathogens in general, and against Ehrlichia ruminantium, the causative agent of tick-borne heartwater disease of ruminants, in particular. However, the source of this important cytokine in animals immunized against E. ruminantium remains largely unknown. We have analyzed in goats protected by vaccination with a killed E. ruminantium vaccine, the potential of individual, genuine (i.e., non-cloned), T cell subsets to produce IFN-gamma after antigenic recall in vitro. In all vaccinated but none control animals, E. ruminantium-induced IFN-gamma secretion was observed in 24 h stimulated blood. Flow cytometric analysis of stimulated peripheral blood mononuclear cells (PBMCs) collected after each vaccine inoculation indicated that immune CD4+ and CD8+ T cells contribute to the same extent to the production of IFN-gamma, while WC1+ T cells are less important. This was confirmed by blocking the secretion of IFN-gamma with anti-classes I and II major histocompatibility complex antibodies. Blocking experiments also suggest that CD8+ need the help of CD4+ T cells in order to produce IFN-gamma. Thus, this work underlines the key role of CD4+ T cells in the production of IFN-gamma by immune goat PBMC. It also describes, for the first time in ruminants, E. ruminantium-specific CD8+ effector T cells. Since CD4+ and CD8+ T cells collectively contribute to the production of IFN-gamma in most vaccinated animals, and since these responses are associated with protection, it may be that a recombinant vaccine will need to incorporate E. ruminantium antigens capable of driving both responses.     

Accumulation of CD25+ CD4+ T-cells in the draining lymph node during the elicitation phase of DNCB-induced contact hypersensitivity in lambs

The elicitation phase of DNCB induced contact hypersensitivity in lambs was studied, and the presence of CD25+ cells in the lymph nodes draining the contact site was measured. Confocal laser scanning microscopy was used to capture images of two sets of triple immunofluorescence labellings. One set labelled CD25+, CD4+ and CD3+ cells, while the other labelled CD25+, VPM30+ and CD4+ cells. The CD25+ subpopulation labellings were assessed by area measurements in a morphometric protocol. The CD25+CD4+CD3+ cells were found to be increased in the DNCB treated group. This subpopulation of CD25+ cells comprised 75% of all CD25+ cells measured. The CD25+VPM30+CD4+ cells were also found to be increased in the DNCB group, but comprised only 17% of the total CD25+ cells measured. Since the VPM30 antibody detects an antigen found on activated T-cells, it was concluded that a substantial proportion of the triple CD25+CD4+CD3+ cells could represent a regulatory phenotype that may be active in suppressing the formation of effector immune cells in CHS of sheep.     

Control of paratuberculosis (Johne's disease) in goats by vaccination

After several years of unsuccessful efforts to eradicate paratuberculosis in goats in Norway by conventional methods such as general hygienic precautions and the isolation and slaughtering of clinically affected and serologically positive animals, a vaccination programme was initiated in 1967. The vaccine used consists of two live attenuated strains of Mycobacterium paratuberculosis suspended in a mixture of liquid paraffin, olive oil and pumice powder. The vaccine may be stored at 4 degrees C for two weeks, the dose is 1 ml and the goat kids are vaccinated at the age of two to four weeks. The efficacy of the vaccine has been judged mainly by post mortem examination of vaccinated and unvaccinated goats in the period 1967-82. During this period about 131,000 goats were vaccinated and, based on the post mortem examination of 15,219 goats, the infection rate was reduced from 53 to 1 per cent. Moreover, infection occurred almost exclusively in goats which for some reason or other had not been vaccinated or which had been too old when vaccinated. The results of these examinations showed that the adjuvanted vaccine with live M paratuberculosis bacteria offers a high degree of protection against paratuberculosis in goats.     

Use of a toxoid vaccine to protect goats against intradermal challenge exposure to Corynebacterium pseudotuberculosis

Two groups of male, 9-week-old goats (5 goats/group) were vaccinated subcutaneously with formalized exotoxin of Corynebacterium pseudotuberculosis, with Freund's incomplete adjuvant. Each goat was given 2 vaccinations, 2 weeks apart. At each vaccination, each group 1 goat was given 0.5 ml of toxoid, and each group 2 goat was given 1 ml of toxoid. Twenty days after the 2nd vaccination, vaccinated goats and 5 nonvaccinated 12-week-old goats (controls) were inoculated intradermally (challenge exposed) with live C pseudotuberculosis, monitored for 13 weeks, and euthanatized. At necropsy, 5 of the 10 vaccinated goats did not have C pseudotuberculosis lesions, 3 had abscesses limited to the inoculation site and draining lymph node, and 2 had disseminated bacterial lesions. Of the 5 nonvaccinated controls, 4 had disseminated abscesses and 1 had a single abscess in an internal node. Serologically, 9 of the 10 vaccinated goats developed positive (greater than or equal to 1:8) antibody titers against the exotoxin within 1 week after inoculation; the 10th goat seroconverted 2 weeks after inoculation, whereas control goats required 3 weeks to develop a positive antibody response. Therefore, early during an infection with C pseudotuberculosis, antibodies against the exotoxin may protect a goat against spread of the organism. All goats were injected intradermally before challenge exposure, 10 days after challenge exposure, and at 4, 8, and 12 weeks after challenge exposure with a skin-test reagent composed of fragmented bacterial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the antigen-specific IFN-gamma producing T-cell subsets in cattle experimentally infected with Mycobacterium bovis

Three 10 months old cattle were infected by the intratracheal route with 10(6)cfu of a field strain of Mycobacterium bovis. Blood samples were regularly collected for in vitro IFN-gamma production after antigenic stimulation. Peripheral blood cells of infected animals produced IFN-gamma in response to crude M. bovis antigens (live and heat-inactivated BCG and protein-purified derivative (PPD)) 3-4 weeks after infection. The ratio of the response to bovine PPD versus avian PPD indicated a specific sensitisation for M. bovis antigens. Three months post-infection (PI), animals were culled and M. bovis was cultured from tubercle lesions. At different time points, the frequency of specific M. bovis IFN-gamma producing CD4+, CD8+ and WC1+ T-cells in the peripheral blood was examined by flow cytometry. Two colour immunofluorescence staining of intracellular IFN-gamma and bovine cell surface molecules showed that both CD4+ and CD8+, but not WC1+, T-cells produced IFN-gamma following stimulation with PPD, live or killed BCG.In two animals analysed, the relative percentage of circulating IFN-gamma producing CD8+ cells decreased between week 5 and week 9 PI. The same evolution was not observed for IFN-gamma secreting CD4+ cells. Magnetic positive selection of T-cells from infected animals showed that CD4+ T-cells produced specific IFN-gamma only in the presence of antigen presenting cells (APCs). Positively selected CD8+ T-cells secreted IFN-gamma only in the presence of recombinant human IL-2 and APCs. In vitro depletion of the CD4+ T-cells, but not the depletion of CD8+ or WC1+ T-cells, resulted in abrogation of the specific IFN-gamma production showing the key role of this cell population for the specific IFN-gamma production.     

Differential immune response between fundic and pyloric abomasal regions upon experimental ovine infection with Haemonchus contortus

The effect of experimental haemonchosis on the number of tissue eosinophils, plasma cells and lymphocyte subpopulations was evaluated in the fundic abomasal region, the pyloric abomasal region and the abomasal lymph node of Blackbelly lambs, which are resistant to infection, and Columbia lambs, which are susceptible to infection. An increase in the number of tissue eosinophils and CD4+ and WC1(+)γδ T-cells was observed in the pyloric abomasal region of Blackbelly lambs and correlated with lower worm burden and greater resistance to infection. Increases in IgA+ plasma cells from the pyloric abomasal region were observed in both infected groups, but there was no difference between the groups. Therefore, increases in IgA+ plasma cells did not explain the resistance observed. Infection caused a significant increase in tissue eosinophils in the abomasal lymph node of Blackbelly lambs and a decrease in the number of CD4+ T-cells in lambs of both breeds. CD8+ T-cells and IgG+ and IgM+ plasma cells were not associated with either infection or resistance. In this work, clear differences were observed in the numbers of CD4+ and WC1(+)γδ T-cells, tissue eosinophils and IgA+ plasma cells between the abomasal regions studied. These differences indicate that the immunological response is not homogenous in all abomasal mucosa and that evaluating the response from a single abomasal region may not be representative of the cellular response across the abomasum.     

Cellular profiles in the abomasal mucosa and lymph node during primary infection with Haemonchus contortus in sheep

Cellular changes in the abomasal tissue and draining abomasal lymph nodes were examined after primary infection of lambs with Haemonchus contortus for 3, 5 or 27-36 days.Infection with H. contortus larvae resulted in a rapid and selective increase in the percentage of CD4(+) T-cells in the abomasal lymph node at 3 days post-infection (PI). By 5 days PI, the lymph node weight had increased two-fold; however, the percentage of lymphocyte populations in the abomasal lymph node resembled that seen in uninfected sheep. Lymph node weights remained at increased levels in the adult nematode infected sheep and down-regulation of B-cell surface markers (sIg and MHC Class II) was apparent in this group. Significant increases in the percentage of CD4(+) T-cells co-expressing MHC Class II, but not CD25, were observed in the larval infected groups except in adult nematode infected sheep. Increased numbers of eosinophils, CD4(+), gamma delta(+) T-cells and B-cells were found in the abomasal tissue by 5 days PI, but no further increases in these cell populations were observed in the adult nematode infected group. In contrast, the level of both lamina propria and intraepithelial mast cells observed in the abomasal mucosa was highest in the sheep carrying an adult nematode burden. These findings indicate that sheep are able to generate an early immune response to infection with H. contortus larvae, characterised by the activation of CD4 T-cells and B-cells in the draining lymph nodes and recruitment of eosinophils, CD4(+) and gamma delta-TCR,WC1(+) T-cells and B-cells in larval infected tissues. However, these changes do not seem to be maintained during infection with the adult parasite where increases in mast cell numbers dominate the local response, indicating that different parasite stages may induce distinct and possibly counteractive immune responses.     

Kinetics of IL-2 receptor expression on lymphocyte subsets from goats infected with Mycobacterium avium subsp. paratuberculosis after specific in vitro stimulation

Quantification of surface IL-2R expression on activated lymphocytes by flow cytometry have recently been reported to be useful in measuring cellular immunity against Mycobacterium avium subsp. paratuberculosis in goats (Whist et al., 2000, Vet. Immunol. Immunopathol. 73, 207-218). To characterise the phenotype of the peripheral lymphocytes expressing IL-2R after in vitro stimulation with purified protein derivative (PPD) from M. a. paratuberculosis, cells were processed for dual or triple colour analysis by flow cytometry (CD4 and IL-2R or CD8, gammadelta-TcR and IL-2R). To distinguish the response of antigen-specific T cells from non-specific stimulation, we performed a time-course study of proliferating cells in a group of M. a. paratuberculosis-infected animals and a control group. Following in vitro stimulation with PPD of whole blood for three different periods of time, IL-2R expression was detected mainly not only in gammadelta-T cells, but also in CD4+ and CD8+ T cells. We found a specific response of gammadelta-T cells from infected animals after 24h of stimulation. Following 120h of stimulation, however, gammadelta-T cells from control animals up-regulated IL-2R to the same level as those from infected animals, indicating either a non-specific stimulation or activation due to a first line of defence against Mycobacterium antigens. The CD4+ cells showed a specific response to PPD stimulation at all three time points. A minor population of antigen reactive gammadelta+ cells also expressed CD8. The proliferative responses differed between alphabeta and gammadelta-T cells; the IL-2R+ alphabeta T cell population mainly comprised proliferating cells, while the gammadelta+ population showed less expansion.     

Levamisole synergizes experimental F4ac+ Escherichia coli oral vaccine in stimulating ileal Peyer's patch T cells in weaned pigs

Recent findings demonstrate that priming by levamisole of weaned pigs experimentally vaccinated against postweaning colibacillosis (PWC) contributes to immune protection from challenge-induced clinical disease through stimulation of the mesenteric lymph node cells that participate in cell-mediated immunity. With the objective of better understanding the mechanisms by which levamisole induces protective mucosal cell-mediated immune response to vaccination against PWC, it was tested whether the drug synergizes experimental F4ac+ Escherichia coli oral vaccine in stimulating T cells also in the jejunal lamina propria (JLP) and ileal Peyer's patch (IPP) upon virulent challenge. Commercial crossbred pigs weaned at 4 weeks were allocated into two equal groups. The experimental group was i.m. primed with levamisole at an immunostimulatory dose of 2.5 mg/kg once daily, for 3 consecutive days, and controls received saline. Both groups were vaccinated orally with the vaccinal E. coli strain on day 0 and challenged with the virulent E. coli strain 7 days later. All pigs were killed on postchallenge day 6. The results determined by immunophenotyping of isolated cells indicate that priming by levamisole of the vaccinated weaned pigs selectively recruited and activated T cells in the IPP, a lymphoid organ-generating B lymphocytes. The pig IPP is normally populated with up to 5% of CD3+ T cells and CD6 is an activation antigen expressed exclusively by T cells in swine. Therefore, a significantly higher number of CD3+ (P < 0.01) and CD6+ (P < 0.001) cells observed within the IPP follicles of the primed-vaccinated vs. unprimed-vaccinated challenge-infected pigs suggest enhanced T cell-mediated immunity in this B-cell compartment induced by the potentiating action of the drug and vaccine. The ability of levamisole to influence interaction between activated T cells and B cells in the IPP of primed-vaccinated weaned pigs, and the possibility that this interaction plays a role in regulating B-cell maturation within the IPP follicles, are discussed.     

Characterization of efferent lymph cells and their function following immunization of cattle with an allogenic Theileria annulata infected cell line

Immunization of cattle with in vitro propagated bovine mononuclear cells infected with Theileria annulata induces a protective immune response. Activation and effector function of T cells exiting the lymph node draining the site of cell line immunization were investigated to understand the mechanisms involved in the generation of immunity. Immunized animals exhibited a biphasic immune response in efferent lymph as well as peripheral blood. The first phase corresponded to allogenic responses against MHC antigens of the immunizing cell line and the second was associated with parasite specific responses. An increase in the output of CD2(+) cells and MHC class II(+) cells in efferent lymph was observed after cell line immunization with a corresponding decrease in WC1(+) cells. Although the percentage of CD4(+) T cells did not change significantly over the course of the experiment, they became activated. Both CD25 and MHC class II expressing CD4(+) T cells were detected from day 7 onwards, peaking around day 13. Efferent lymph leukocytes (ELL) exhibited sustained responses to IL-2 in vitro following cell line immunization. Antigen specific proliferation was also detected first to the immunizing cell line and then to parasite antigens. The two peaks of CD2(+) cells were observed, which corresponded to similar peaks of CD8(+) cells. The increase in CD8(+) cells was more pronounced during the second parasite specific phase than the first allogenic phase. Activated CD8(+) T cells mainly expressed MHC class II and some expressed CD25. Significantly the peak of activated CD4(+) T cells preceded the peak of activated CD8(+) T cells, highlighting the role of T. annulata specific CD4(+) T cells in inducing parasite specific CD8(+) cytotoxic responses. A biphasic cytotoxic response also appeared in efferent lymph and peripheral blood, the first directed against MHC antigens of the immunizing cell line followed by MHC class I restricted parasite specific cytotoxicity. The cytotoxic responses in efferent lymph appeared earlier than peripheral blood, suggesting that activated CD8(+) cells exiting the draining lymph node following immunization with T. annulata infected schizonts play an important role in the development of protective immune responses.     

Lymphocyte migration in the intestinal mucosa: entry, transit and emigration of lymphoid cells and the influence of antigen

Lymphocyte migration is important to transport immunological information between the different compartments of the intestinal immune system. Large numbers of lymphocytes emigrate from the Peyer's patches and reach the blood circulation after expansion and maturation within the mesenteric lymph nodes. So far the frequency of antigen specific lymphocytes emigrating from the Peyer's patches after oral stimulation is not known. After mesenteric lymph node resection those cells emigrating from the intestinal wall are accessible by calculating the major intestinal lymph duct. The first antigen specific cells draining from the intestines are obviously not lymphocytes but dendritic cells, thus the antigen is rapidly trapped in the parenchyma of the lymph nodes in vivo. When lymphocytes were taken from intestinal lymph, labeled in vitro and retransfused, marked numbers of B-cells were re-detected in intestinal lymph. Later preferentially T-cells recirculated through the gut wall. After immigration into the intestinal lamina propria the lymphocytes may enter the space between epithelial cells, where they are present as intraepithelial lymphocytes. Lymphoid cells in the S-phase of the cell cycle have been detected in all compartments of the intestinal wall. Apoptosis is probably a further important mechanism for the regulation of intestinal immunity in removing cells reacting against harmless dietary antigens to maintain oral tolerance.