Distribution and functional characteristics of antigen-specific helper T cells arising after Peyer's patch immunization.

Antigen-specific T-helper cells for IgA responses arise in Peyer's patches (PP) following their immunization by subserosal injection of keyhole limpet haemocyanin (KLH). These are of the W3/25 phenotype and the W3/25 receptor is shown here to be involved in their helper function. These cells originate in PP and migrate via mesenteric lymph nodes (MLN) to thoracic duct lymph, although the MLN appear to be unnecessary for the induction or maturation of antigen-specific helper cells collected in thoracic duct lymph after intra-Peyer's patch (i.p.p.) immunization. KLH-specific helper cells can be detected subsequently in the intraepithelial lymphocyte population and also among lamina propria lymphocytes. The helper cells also relocate to PP distant to their site of origin where they are retained only when antigen is present. While i.p.p. immunization is an efficient route for the induction of IgA helper cells in gut-associated lymphoid tissue, it differs from oral immunization in that concomitant induction of antigen-specific splenic suppressor cells does not occur, indicating a role for epithelial antigen processing in this phenomenon.     

Expression of recombination-activating gene in mature peripheral T cells in Peyer's patch

Recombination-activating gene (RAG) 1 and 2 are essential for the gene rearrangement of antigen receptors of both T and B cells. To investigate RAG gene expression in peripheral lymphoid organs other than the thymus and bone marrow, we established mice in which a green fluorescent protein (GFP) gene is knocked-in the RAG2 gene locus (RAG2-GFP mice). In the thymus and bone marrow of heterozygous RAG2-GFP mice, as expected, GFP expression was detected in the appropriate stages of developing T and B cells. Interestingly, only a fraction of Thy-1.2(+) cells in the Peyer's patch were found to be GFP(+) amongst the peripheral lymphoid organs. The GFP(+) cells expressed high levels of surface TCRbeta and CD3, suggesting mature T cells with rearranged TCRalphabeta. However, they showed activated/memory phenotypes, i.e. CD45RB(low), CD69(high), CD44(high) and CD62L(low), and belonged to a CD4(+)CD8(+) population expressing c-kit, IL-7R and pTalpha characteristic of immature developing lymphocytes. Moreover, RAG(+) Peyer's patch T cells seem to be of thymic origin as judged by their expression of CD8alphabeta. These results show that there exists a fraction of mature T cells expressing RAG genes in the Peyer's patch, implying a potential for a secondary rearrangement of TCR in extrathymic tissues.     

Detection and quantitation of interleukin-2 from individual cells

In this report we present the use of cell blotting for the detection of interleukin-2 (IL-2)-producing lymphocytes. This is a rapid and sensitive immunochemical method analogous to Western blotting of proteins. When combined with image analysis one can determine the percentages of IL-2 positive cells as well as quantitate the amount of IL-2 surrounding each cell. When bovine lymph node cells (bLNC) were stimulated with the combination of concanavalin A (ConA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h, 46.4 +/- 0.6% stained positive for IL-2 and, on average, each cell produced 0.92 +/- 0.6 pg of IL-2 in 24 h. Phytohemagglutinin (PHA) and TPA-stimulated human peripheral blood mononuclear cells (hPBMC) produced approximately the same amount. 0.86 +/- 0.4 pg of IL-2 per cell in 24 h; 45.6 +/- 3.6% stained positive for IL-2.     

Systematic characterization of porcine ileal Peyer's patch, II. A role for CD154 on T cells in the positive selection of immature porcine ileal Peyer's patch B cells

We previously demonstrated that the majority (>/= 90%) of porcine ileal Peyer's patch (IPP) follicular cells are immature B cells destined to die by apoptosis, when incubated at 37 degrees. In this paper we approached the mechanisms responsible for positive selection of porcine IPP follicular immature B-cell selection, by screening for various cell types, cytokines and polyclonal and monoclonal antibodies for promoting the survival of IPP B cells. Of these reagents, only CD3 cross-linked purified T cells from mesenteric lymph nodes were able to rescue IPP follicular B cells from apoptosis, although polyclonal anti-IPP lymphocyte antibodies delayed apoptosis. This survival effect could be reproduced simply by incubating IPP follicular B cells with soluble and cell membrane-expressed CD154, an observation consistent with the demonstrated presence of CD40 and CD154 on porcine IPP follicular B cells and activated T cells, respectively. The IPP follicular B cells rescued in this manner expressed a more mature surface marker phenotype. Immunohistology and fluorescence-activated cell sorter analysis demonstrated that subpopulations of IPP follicular T cells (less than 0.5%) express CD154. Thus, perhaps unexpectedly, CD154 on T cells may play a role in the positive selection of immature B cells in the porcine IPP. The origin and control of the activated T cells identified within the porcine IPP remains to be investigated.     

Spontaneous proliferation of Peyer's patch cells in vitro

Under normal circumstances most lymphold cell populations donot exhibit strong proliferatlve reactions in culture unlessprovoked by antigen or mltogen. The autologous mixed lymphocytereaction (AMLR) mediated by adult T cells is a relatively weakproliferatlve response that occurs in the absence of known heterologousstimuli. In this investigation we demonstrate that Peyer's patch(PP) cells possess an inherent capacity to commence dividingIn vitro and to display an exceptionally vigorous AMLR. Themagnitude and kinetics of this spontaneous proliferation resemblethat of a secondary response to a strong mucosal immunogen suchas reovlrus type 1/Lang. Analysis of the cellular componentsof the PP cultures implicates CD4⁺CD8^– T cells as themajor responding population and dendritic cells (DC) as stimulators.Mixing experiments indicate that spleen contains a cell populationwhich can stimulate PP T cells, albeit to a lesser extent thanPP cells. Similarly, splenic T cells have a reduced but significantcapacity to respond to PP DC, In comparison to PP T cells. Thesedifferences suggest the possibility that there may be a decreasinggradient of antlgeniclty between the gut and the spleen whichis reflected in the spontaneous activity of PP versus splenicT cells In vitro. We propose that PP cells are in fact respondingIn vitro to heterologous antigens derived from food, entericmicrobes and other environmental sources. This notion is supportedby the observation that PP cells from antlgen-mlnlmized germ-freemice fall to proliferate spontaneously in culture.     

Effects of phorbol myristate and ionomycin on in vitro growth of aged Peyer's patch T and B cells.

The proliferative responses of Peyer's patch (PP) T cells from aged BALB/c mice to concanavalin A (Con A) are considerably reduced, as compared to those of the young (P < 0.001). This reduced reactivity of aged T cells could be partly, but not entirely, corrected by interleukin 2 (IL-2) (P < 0.001). PP T cells from aged mice responded synergistically to a protein kinase C (PKC) activator, phorbol myristate acetate (PHA), plus a calcium ionophore, ionomycin, at much lower concentrations than to Con A (P < 0.001); however, the maximal proliferative response still remained nearly at 8/10th of the young (P < 0.01) and higher levels of PMA (but not of ionomycin) were required (P < 0.001). Addition of IL-2 restored the diminished response to the levels of the young T cells (P < 0.05), but that of Con A did not (P > 0.05). The proliferative responses of PP B cells to lipopolysaccharide (LPS) do not differ from those of the young (P > 0.05), but the spontaneous proliferation of aged (unstimulated) B cells is enhanced nearly twofold versus that of the young (P < 0.001). Like the PP T cells, PP B cells from aged mice also responded synergistically to PMA plus ionomycin but to a lesser degree than those of the young under the same stimulation (P < 0.01). Their maximal proliferation required higher levels of PMA, but not of ionomycin and was also diminished (P < 0.01), compared to that of the young. B cell stimulatory co-factors, IL-4 and IL-6, failed to affect the response of aged and young B cells to PMA plus ionomycin (P > 0.05), whereas LPS remediates the reduced response of aged B cells to PMA plus ionomycin. Thus, T and B cells from senescent PP demonstrate an impaired proliferative responsiveness via the Ca-dependent PKC pathway. A T cell mitogen and B cell stimulatory cytokines did not alter this activation pathway, once optimally stimulated. Whereas, T cell stimulatory cytokine IL-2 and B cell mitogen LPS could restore the age-associated decline of the corresponding lymphocyte subsets, T and B cells, in activation of the Ca-dependent pathway. The altered transmembrane signal transduction appears to be intrinsically defective in these aged PP T and B cells.     

The role of Peyer's patch cells in antibody formation

The role of Peyer's patches in primary and secondary immune response was studied with Vibrio cholerae as the antigen. V. cholerae E1 Tor ME 7 is a typical enteric antigen which was shown to be suitable for the demonstration of antibody forming cells by localized lysis in agar. No plaque-forming cells (PFC) against this antigen were found in the organs of non-immunized mice. Consequently a primary immune response could be studied. In vitro an immune response against this antigen could only be obtained with spleen cells when the donor animal had been primed.

After primary intraperitoneal or enteral immunization many PFC were found in the spleen but none in Peyer's patches. This cannot be explained by insufficient penetration of the antigen into Peyer's patches as secondary responses were obtained with both routes of immunization.

After local injection of antigen directly into Peyer's patches, PFC only appeared after 5 days. This is an indication that an essential cell type for the immune response is lacking in Peyer's patches. The cells from Peyer's patches were unable to restore the immune response in lethally irradiated mice, unless they were injected together with thymus cells or cells from peripheral lymph nodes. This suggests the absence of active T cells and indicates the presence of B cells in Peyer's patches. The combination of thymus cells and bone marrow cells was inactive in restoring the immune response. This is an indication that the B cells in Peyer's patches are immunologically more mature than bone marrow cells.

     

Agents that activate protein kinase C rescue sheep ileal Peyer's patch B cells from apoptosis

The ileal Peyer's patch (PP) is the major site of B cell production and is a site of immunoglobulin gene diversification in the sheep. Within the ileal PP follicles there is both intense proliferation and death of B cells. We have previously demonstrated that most, if not all of this death can be attributed to apoptosis. Likewise, ileal PP B cells die rapidly by apoptosis in culture - after 6 h many cells appear pyknotic and about 50% of cellular DNA is fragmented. We now show that the DNA fragmentation and cell death of ileal PP B cells can be almost completely abrogated during the first 12 h of culture by the addition of the phorbol esters, phorbol dibutyrate (PBu₂) or phorbol myristate acetate. This inhibition of apoptosis could be sustained for greater than 24 h by the concomitant addition of both PBu and the Ca²⁺ ionophore A23187. However, the rescue of B cells from apoptosis by PBu, with or without Ca²⁺ ionophore, was prevented by macromolecular synthesis inhibitors or inhibitors of protein kinase C activation. Furthermore, treatment of cultures with PBu, with or without Ca²⁺ ionophore, resulted in an activated B cell phenotype and a three- to fourfold increase in cell proliferation. We conclude that protein kinase C activation in conjunction with an increase in intracellular [Ca²⁺] can provide the signals necessary to rescue ileal PP B cells from apoptosis, and speculate that these ileal PP B cells are destined to die unless they receive a signal that rescues them from the death pathway.     

Aging-associated intrinsic defects in IgA production by murine Peyer's patch B cells stimulated by autoreactive Peyer's patch T cell hybridoma-derived B cell stimulatory factors (BSF)

Immune functions deteriorate with age, primarily as a result of alterations in the number and subpopulations of T cells of the immune system. In contrast, the B cell component of the immune system is generally affected by senescence only to a minor extent. In the present report, we stimulated murine Peyer's patch (PP) B cells by nonspecific multifunctional B cell stimulatory factors (BSF) secreted by one of several autoreactive (self-major histocompatibility complex (MHC)-class II antigen-responsive) T cell hybridoma clones derived from PP of syngeneic mature adult mice, and then determined in vitro whether aging-associated intrinsic defects could be demonstrated in the proliferation of, and the synthesis and secretion of mucosal IgA by, the BSF-activated B cells. This approach could be a useful new in vitro method for assessing the effect of senescence on B cell Ig production, especially that of IgA, in the gut-associated lymphoid tissue (GALT). Aged PP B cells stimulated by the autoreactive PP T cell-derived BSF proliferated more (P less than 0.05), contained larger amounts of IgA (nearly 10 times) and also secreted considerably more IgA (nearly 4.5 times) than did mature adult PP B cells. However, the ratio of intracellular dimeric (d) IgA to total IgA in the aged B cell lysates was significantly reduced (by approx. 44%) as was also the secreted dIgA (by approximately 50%). The augumentation of not only the proliferation, but also the synthesis and secretion of IgA in vitro along with reduced dIgA/total IgA ratios of BSF-stimulated aged PP B cells appears to be due to aging-related intrinsic defects. Alterations in intracellular regulatory mechanisms of B cells, mediated by B cell receptors for autoreactive T cell-derived BSF, could be largely responsible for the observed polyclonal B cell hyperreactivity, associated with senescence.     

Gamma interferon induces Fas-dependent apoptosis of Peyer's patch T cells in mice following peroral infection with Toxoplasma gondii.

Since we previously observed a remarkable decrease in the numbers of T cells in the Peyer's patches of the small intestines in C57BL/6 mice following peroral infection with Toxoplasma gondii, we performed studies to examine the mechanism(s) whereby this decrease in numbers of the T cells occurs. We found that apoptotic cell death of CD4+ and CD8+ alphabeta T cells occurred in Peyer's patches following infection. Upregulation of Fas expression was observed in these T cells. C57BL/6-background mutant mice which lack functional Fas antigen did not develop apoptosis in their Peyer's patches following infection. Treatment of infected C57BL/6 mice with anti-gamma interferon (IFN-gamma) monoclonal antibodies prevented the upregulation of Fas on their Peyer's patch T cells and inhibited the occurrence of apoptosis of these T cells. These results indicate that IFN-gamma induces Fas-dependent apoptosis in CD4+ and CD8+ alphabeta T cells in Peyer's patches in C57BL/6 mice following peroral infection with T. gondii.     

Monoclonal antibody-directed targeting of fluorescent polystyrene microspheres to Peyer's patch M cells.

The ability to deliver particulates to Peyer's patch M cells for uptake into gut-associated lymphoid tissue was examined by administering simultaneously fluorescent green and red polystyrene microspheres into NZW rabbit intestinal loops containing Peyer's patches. Whereas green and red microspheres were taken up by M cells at equivalent concentrations (120 +/- 17 versus 125 +/- 18/mm length of dome), particles conjugated to the anti-M-cell monoclonal antibody 5B11 (IgM, kappa) were internalized by M cells 3-3.5 times more efficiently than conjugates displaying IgM of unrelated specificity (TEPC 183) or native particles of the reciprocal colour inoculated into the same loop at a comparable load. The microspheres formed a concentration gradient from lumen to subepithelial dome, and localized on M-cell apical membranes, M-cell pockets, and subepithelial domes. The transport rate across M cells of 5B11 or TEPC 183 conjugates was similar to that of untreated microspheres. These observations show that intestinal uptake into Peyer's patches can be upregulated by targeting M-cell luminal membrane structures.     

CD40 signalling in ileal Peyer's patch B cells: implications for T cell-dependent antigen selection

The ileal Peyer's patch (PP) plays a central role in B celldevelopment in young sheep and it is hypothesized that thisB cell development occurs independent of extrinsic antigen andT cells. Therefore, it was of interest to examine ileal PP folllcular(iPf) B cell responses to CD40 ligand, a molecule integral toT cell-dependent B cell development. A variable level of CD40expression was detected on a subpopulation of iPfB cells andJ558L cells, expressing a membrane form of mouse CD40 ligand(mCD40L), interacted specifically with the CD40 molecule oniPfB cells. In response to mCD40L the non-S phase iPfB cellswere rescued from apoptotic cell death and there was a markedproliferative response but viable cell number remained relativelyconstant. The mCD40L also induced decreased cytoplasmic cAMPlevels, blocked anti-Ig-induced iPfB cell death and inducedfunctional IL-2 receptor expression on a subpopulation of iPfBcells. Many of the mCD40L-induced responses of iPfB cells weresimilar to those reported for germinal centre and immature Bcells, and indicated that a cognate T cell-B cell interactioncould influence iPfB cell proliferation and differentiation.Finally, that mCD40L induced iPfB cell activation and differentiationwas evident as increased expression of CD5, the BAQ44A molecule,the CACT65A molecule and the expansion of surface IgG1⁺ B cells.These mCD40L-induced phenotypic changes were also observed onsubpopulations of freshly isolated iPfB cells and jejunal PPfollicular B cells. However, few iPfB cells had a phenotypesimilar to that observed in co-culture with mCD40L and thissuggested that T cell-dependent B cell development may playa minor role in ileal PP B cell development. The possible significanceof CD40 signalling is discussed in terms of the selection ofiPfB cells during development.     

Robo4 contributes to the turnover of Peyer's patch B cells

All leukocytes can get entrance into the draining lymph nodes via the afferent lymphatics but only lymphoid cells can leave the nodes. The molecular mechanisms behind this phenomenon have remained unknown. We employed genome wide microarray analyses of the subcapsular sinus and lymphatic sinus (LS) endothelial cells and found Robo4 to be selectively expressed on LS lymphatics. Further analyses showed high Robo4 expression in lymphatic vessels of Peyer's patches, which only have efferent lymphatic vessels. In functional assays, Robo4-deficient animals showed accumulation of naïve B cells (CD19⁺/CD62L^hi/CD44^lo) in Peyer's patches, whereas no difference was seen within other lymphocyte subtypes. Short-term lymphocyte homing via high endothelial venules to peripheral and mesenteric lymph nodes and Peyer's patches was also slightly impaired in Robo4 knockout animals. These results show for the first time, selective expression of Robo4 in the efferent arm of the lymphatics and its role in controlling the turnover of a subset of B lymphocytes from Peyer's patches.     

Immunosuppression caused by antigen feeding II. Suppressor T cells mask Peyer's patch B cell priming to orally administered antigen

Peyer's patch (PP) cells transferred into sublethally irradiated recipients generated substantial IgM, IgG and IgA anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) responses in the recipient spleen. If donor mice were given SRBC orally for 4–5 weeks prior to transfer, the adoptively transferred PP IgG and IgA responses were considerably suppressed, although the IgM responses were often unaffected. Co-injection of PP cells from antigen-fed mice with PP cells from normal mice resulted in marked suppression of the normal PP IgG and IgA response. However, treatment of PP cells from antigen-fed mice with anti-Thy-1.2 plus complement prior to cotransfer completely abrogated suppression of the IgG PFC response and partially abrogated the suppressed IgA response. B cells from the PP of antigen-fed mice, when transferred into SRBC-primed irradiated recipients (to provide T cell help) generated 2–3 times more IgG and IgA PFC than comparable numbers of B cells from the PP of normal mice. Thus antigen feeding generates suppressor T cells in PP which can mask the expression of B cell priming to orally administered antigen.     

Confocal analysis of fluorescent bead uptake by mouse Peyer's patch follicle-associated M cells.

Latex beads coated with secretory immunoglobulin (IgA) instilled into mouse intestinal loops are taken up by M cells present in Peyer's patch follicle-associated epithelial tissue. Bead adsorption and uptake is greater at the edge compared with the apex of follicle domes. Coating beads with bovine serum albumin (BSA) causes a fourfold reduction in adsorption and a twentyfold reduction in uptake. Results demonstrate selectivity between adsorption and uptake and between the ability of different proteins to facilitate uptake.     

Abnormal clones of T cells producing interleukin-5 in idiopathic eosinophilia.

BACKGROUND: The cause of persistent eosinophilia and the hypereosinophilic syndrome is unknown. Recent work suggests that in some patients with the hypereosinophilic syndrome, a clone of abnormal T cells produces large amounts of interleukin-5, a cytokine required for the growth and differentiation of eosinophils. We examined T-cell surface markers, rearranged T-cell-receptor genes, and in vitro production of cytokines by T cells from patients with idiopathic eosinophilia. METHODS: The expression of surface molecules on T cells was measured by flow cytometry. Cytokine expression was measured by enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemical analysis. To identify dominant (clonal) rearrangements of the T-cell receptor within the lymphocyte population, Southern blot analysis (beta chain) and the polymerase chain reaction (gamma chain) were performed according to standard protocols. RESULTS: Among 60 patients with idiopathic eosinophilia, 16 had circulating T cells with an aberrant immunophenotype. In each of these patients, the abnormal immunophenotype was unique. Evidence of clonal rearrangements of the T-cell receptor was obtained in 8 of the 16 patients. In most instances, the abnormal T cells expressed large amounts of surface proteins associated with T-cell activation (the alpha chain of the interleukin-2 receptor and the HLA-DR antigen). Moreover, the aberrant T cells produced large amounts of interleukin-5 in vitro. CONCLUSIONS: Clonal populations of abnormal T cells producing interleukin-5 occur in some patients with idiopathic eosinophilia.