Immunohistological characterization of proximal colonic lymphoid tissue in the rat

Proximal colonic lymphoid tissue (PCLT) is a lymphoid structure located in the proximal colon of the mouse and the rat. In the present investigation we studied the immunomorphology and cytology of PCLT in the rat. We also studied sites of lymphocyte proliferation using the BrdU-anti BrdU technique. Results demonstrated no evident phenotypical differences between the lymphocyte populations of PCLT and either jejunal or ileal Peyer's patches (PP). The majority of the lymphocytes within PCLT were B cells localized in follicles, which were separated from each other by interfollicular T cell areas. Germinal centers (GC), containing ED5⁺ follicular dendritic cells, are found within PCLT follicles. The T cell areas contained both MHC Class II⁺ interdigitating cells and high endothelial venules. Studies using BrdU-anti BrdU indicated that lymphocyte proliferation within PCLT taken place mainly in germinal centers. Together the data show that the organization, lymphoid constituents, and sites of lymphocyte production are very similar in PCLT and PP. We therefore conclude that PCLT in the rats is not a Bursa equivalent, but more likely a PP with some special characteristics. © 1992 Wiley-Liss, Inc.     

Histological studies on the ontogeny of bovine gut-associated lymphoid tissue: appearance of T cells and development of IgG+ and IgA+ cells in lymphoid follicles

The development and distribution of lymphocyte subsets in bovine gut-associated lymphoid tissues (ileal and jejunal Peyer's patches (PP)) were examined. Before birth, the composition of lymphocyte subsets in both PP follicles did not differ except for the dimensions of the interfollicular area and the dome region. Many IgM+ cells were observed in these follicles, but very few CD3+, IgG+, and IgA+ cells could be found. At neonatal period, the IgG+ cells, which did not produce IgG mRNA, were dominant within both PP follicles. From 1 month after birth, many CD3+ cells, IgG mRNA expression, and IgA mRNA expression were detected within the jejunal PP follicles, but very few were in the ileal PP follicles. These data suggest that the characteristics of the jejunal PP follicles metamorphose into secondary lymphoid tissue such as germinal centers at around 1 month after birth, whereas the characteristics of ileal PP follicles were distinct from those of germinal centers.     

Analysis of cell division among subpopulations of lymphoid cells in sheep. II. Peripheral lymphocytes

The number, distribution and surface phenotype of dividing cells in lymph nodes and blood and differences between the cell-cycle status of lymphocyte subpopulations were studied in lambs using double-labelling techniques. Dividing cells were labelled in vivo for various time periods with 5-bromo-2-deoxyuridine (BrdU). After removal of lymphoid tissues, the proportions of constituent lymphocyte subpopulations which had synthesized DNA during the labelling period were measured by flow cytometry or immunohistochemistry using a panel of monoclonal antibodies (mAbs) specific for sheep lymphocyte differentiation antigens and MHC class I and class II antigens in conjunction with an anti-BrdU mAb. There was a higher overall level of cell division in the ileocaecal lymph node than in either the prescapular or parathymic lymph nodes. In all three lymph nodes, the majority of lymphocytes which incorporated BrdU occurred in B-cell follicles or germinal centers. CD4+ and CD8+ T cells had a higher level of cell division (LI 5-14%) than those recognized by mAb 197 (CD4- CD8- subset) (LI less than or equal to 3%).     

Immunohistochemical analysis of ageing human B and T cell populations reveals an age-related decline of CD8 T cells in spleen but not gut-associated lymphoid tissue (GALT)

It is thought that senescence of the immune system is responsible, at least in part, for many health problems associated with ageing. Previous studies on changes in lymphocyte composition have used flow cytometry to study peripheral blood lymphocytes (PBL's), or cells isolated from rodent tissue, and have yielded conflicting results. We have used immunohistochemistry to determine whether the B and T cells in human tissue from spleen and gut are affected by age. Areas of germinal centre, mantle zone and marginal zone of B cell follicles were measured. In addition, CD4 and CD8 T cells in T cell areas and in B cell follicles were counted. We observed a striking age-related decrease in the proportion of CD8+ T cells in the T cell zones of the spleen. This decrease was not apparent in the T cell population that occupies splenic B cell areas, or in GALT. Further differences, in CD4+ cells, were seen between T cell populations in the T cell zones and those in B cell areas. These findings highlight differences between lymphocyte populations in different lymphoid tissues, and different compartments within each tissue, which may be of importance in future studies of the ageing immune system.     

The changing preference of T and B cells for partners as T-dependent antibody responses develop

Summary: Recirculating virgin CD4⁺ T cells spend their life migrating between the T zones of secondary lymphoid tissues where they screen the surface of interdigitating dendritic cells. T-cell priming starts when processed of interdigitating dendritic cells. T-cell priming starts when processed peptides or superantigen associated with class II MHC molecules are recognised. Those primed T cells that remain within the lymphoid tissue move of the outer T zone, where they interact with B cells that have taken up and processed antigen. Cognate interaction between these cells initiates immunoglobulin (Ig) class swith-recombination and proliferation of both B and T cells; much of this growth occurs outside the T zones. B cells both B and T cells; much of this growth occurs outside the T zones. B cells migrate to follicles. Where they form germinal centres, and to extrafollicular sites of B-cell growth, where they differentiate into mainly short-lived plasma cells. T cells do not move to the extrafollicular foci but to the follicles; there they proliferate and are subsequently involved in the selection of B cell that have mutated their Ig variable-region genes. During primary antibody responses T-cell proliferation in follicles produces many times the peak number of T cell found in that site; a substantial proportion of the CD4⁺ memory T-cell pool may originate from growth in follicles.     

Evidence for a stromal cell-dependent,self-renewing B cell population in lymphoid follicles of the ileal Peyer's patch of sheep

Lymphoid follicles of the ileal Peyer's patch (PP) of young sheep function as the major source of B cells and a site of immunoglobulin (Ig) receptor diversification. However, extensive cell death in culture has restricted investigations of ileal PP follicular (iPf)B cell biology. We investigated the possibility that sustained iPfB cell proliferation may require an interaction with mesenchymal stromal cells (SC). Four SC lines, cloned from lymphoid follicles of the ileal PP, and various sheep and xenogeneic mesenchymal cells were used to characterize the nature of iPfB cell-SC interactions. A sustained proliferative response was unique to iPfB cells, required iPfB cell-SC contact, and SC membranes functioned as intact SC to either enhance or inhibit iPfB cell proliferative responses. The iPfB cell proliferation in SC co-cultures was accompanied by extensive cell death and a slow decline in viable cell number. Flow cytometric analysis confirmed that viable lymphocytes, present in SC co-cultures, were immature B cells that expressed surface IgM, with either λ or χ Ig light chain, and that SC co-culture inhibited iPfB cell differentiation. Finally, addition of soluble anti-sheep Ig to iPfB cell-SC co-cultures did not inhibit SC-dependent iPfB cell proliferation or iPfB cell binding to SC. These data indicate that an interaction between specific SC membrane molecules and non-Ig molecules of iPfB cells either supported or inhibited a self-renewing proliferative response by immature (sIgM^Lo, BAQ44A^?) iPfB cells. Finally, SC-dependent iPfB cell proliferation was independent of T cells and extrinsic antigen which further suggests that a functionally distinct B cell population resides in lymphoid follicles of the ileal PP.     

Ontogeny of pig discrete Peyer's patches: distribution and morphometric analysis.

We investigated the development of lymphoid and non-lymphoid cells in discrete Peyer's patches (PP) of the pig using immuno-histology and image analysis. In newborn piglets discrete PP were mainly populated by CD2+, CD3+ T cells, and major histocompatibility complex class II+ cells, many of which were of macrophage and dendritic cell lineage. Four days after birth, cells were localized in defined regions: the follicle; the inter-follicular area and the dome region. Compartmentalization within the follicle started about 6 days after birth. The first signs of secondary follicles were seen from about 14 days. The pig discrete PP attained their mature structure at about 3 weeks after birth. Here we show that despite the demonstration at birth of the cell types that support antigen processing and presentation, PP did not fully differentiate morphologically until at least this time when antigen can be handled in an efficient manner.     

Suitability of cell metabolic colorimetric assays for assessment of CD4+ T cell proliferation: comparison to 5-bromo-2-deoxyuridine (BrdU) ELISA

The conventional tritiated thymidine (3H-TdR) incorporation assay is considered as the 'gold standard' for the assessment of cell growth. However, the 3H-TdR incorporation assay has several disadvantages which have prompted the development of nonradiolabelling proliferation assays such as 5-bromo-2-deoxyuridine (BrdU) ELISA, tetrazolium microplate assay and acid phosphatase assay. In studies, these three proliferation assays have shown equivalent sensitivity and reproducibility to the 3H-TdR incorporation assay. However the results may be affected by the cell type studied. In the present study, we have used these three proliferation assays for the assessment of rat lymph node CD4 + T lymphocyte growth in response to polyclonal antigen stimulation. The proliferation assays were compared on the basis of four criteria: sensitivity, reproducibility, stimulation index and insensitivity of the assay to the cell number. Our results indicated that the BrdU ELISA demonstrated the highest sensitivity, reproducibility and stimulation index but had a limited linear range for cellular growth. The tetrazolium microplate assay also had a relatively good sensitivity, reproducibility, stimulation index and a wider linear response range for cell growth in comparison to the BrdU ELISA. The acid phosphatase assay showed the lowest reproducibility and stimulation index. Because BrdU incorporation in DNA of proliferating cells has been reported to block cell division, we have investigated this possibility in sequential assays. Our results indicated that in our experimental conditions no evidence of an anti-mitogenic action of BrdU was observed. We also compared the performance of the MTS assay and BrdU ELISA in measuring substance P-induced CD4 + T cell proliferation. The results indicated that the MTS assay may reflect change in cell activation leading to an overestimate of cell growth. In conclusion, our results indicate that the BrdU ELISA is the most sensitive of the three proliferation assays used for the assessment of CD4 + T lymphocyte growth and is the preferred assay when small changes in cell growth are expected.     

Distribution of lymphocyte subsets in a case of angiofollicular lymph node hyperplasia

A series of monoclonal and polyclonal antibodies was used to determine the distribution of lymphocyte subsets in frozen tissue sections of a case of angiofollicular lymph node hyperplasia (ALNH), intermediate type. Primary follicles and mantle zones of secondary follicles consisted of BA1+OKIa1+-C3RTo5+sIgM+sIgD+ lymphocytes. Poorly developed follicular centers reacted with OKIa1, C3RTo5, DRC-1 and anti-IgM but contained no OKT4+Leu-3a+ or Leu-7+ cells. Mantle zones and primary follicles contained more OKT4+Leu-3a+ and less OKT8+ cells than normally observed. The interfollicular area consisted mainly of OKT4+Leu-3a+OKIa1- helper/inducer T-cells, admixed with some OKT8+ suppressor/cytotoxic T-cells and OKIa1+ interdigitating reticulum cells. Polyclonal plasma cells were found both in follicular centers and interfollicular areas. Based on the analogy to the peripheral lymphoid tissue in nude mice and in children with variable thymic dysfunction, it is suggested that the abnormal distribution of immunoregulatory T-cells may be of pathogenetic significance and may account for the morphology of the lymphoid follicles in ALNH.     

In situ immune complexes, lymphocyte subpopulations, and HLA-DR-positive epithelial cells in Hashimoto thyroiditis

Surgical specimens from thyroid glands from seven patients with Hashimoto thyroiditis and two patients with non-autoimmune colloid goiter were analyzed by immunohistologic techniques (direct and indirect immunofluorescence and immunoperoxidase tests) using polyclonal antisera against total immunoglobulin, Ig classes (IgM, IgD, IgG, and IgA), and complement component C3 and monoclonal antibodies specific for B cells, T cell subpopulation, macrophages, natural killer cells, granulocytes, and HLA-DR antigen. Complement-fixing immune complexes (IgG+, C3+) were noted predominantly in areas with only slight destruction and only moderate lymphoid infiltration of thyroid follicles. In areas with intense lymphoid infiltration of thyroid follicles, where many well-developed germinal centers and significant perivascular lymphoid infiltration were seen, immune complexes were scarce. In these latter areas T helper cells (OKT4+, Leu3a+), were more abundant than T cytotoxic/suppressor cells (OKT8+), macrophages (OKM1+), and plasma cells (IgG+); only a few B lymphocytes (smIgM+, smIgD+), granulocytes (ViMD5+), and natural killer cells (VEP13+, Leu7+) were noted in the interstitium between thyroid follicles, intruding between thyroid follicular epithelial cells and merging into the thyroid follicular lumen. Many activated T cells (OKT10+, HLA-DR+) were present in these areas of advanced destruction. HLA-DR antigen expression was seen on macrophages, tissue reticulum cells, vascular endothelial cells, lymphoid cells, and, most interestingly, on thyroid epithelial cells. Normal thyroid epithelial cells did not express HLA-DR. Only a few epithelial cells in the vicinity of lymphoid infiltrations were HLA-DR+ in early stages of Hashimoto thyroiditis, and the number of HLA-DR+ epithelial cells was significantly increased in advanced stages of the disease. In our present report the potential role of HLA-DR+ thyroid epithelial cells for the in situ stimulation of the immune system within the thyroid gland of patients with Hashimoto thyroiditis is discussed, and it is hypothesized that HLA-DR+ thyroid epithelial cells may be an important factor for the progression and self-perpetuation of the disease, which is probably initiated by humoral components of the immune system but further propagated by cellular immunopathologic mechanisms.     

The incidence of thymic B lymphoid follicles in healthy beagle dogs

The incidence of thymic B cell lymphoid follicles was retrospectively studied in 62 male and 58 female healthy control beagle dogs (age 11.3 +/- 4.8, range 6 to 23 months). The animals were selected from toxicological studies performed in the period 1990-2001 at the Organon labs. The animals had received vehicle treatment. Thorough microscopic examination of the thymus in hematoxylin & eosin (H&E)-stained sections resulted in an unexpectedly high overall incidence of 70% of medullary lymphoid follicles. Occasionally, these lymphoid follicles contained germinal centers. With the use of a T- and B cell marker (respectively CD3 and CD79alpha) we confirmed that the lymphoid follicles exclusively contained large numbers of B lymphocytes. Moreover, with the use of the B cell marker, almost all animals (97%) prove to have B cell rich medullary areas. The study also confirmed that the dog thymus underwent progressive involution during the period from 6 to 23 months of age. As a consequence of the involution, B cell areas and lymphoid follicles may be obscured in some H&E sections. Results of this study indicated that dense B lymphocyte aggregates and/or B lymphoid follicles are a normal constituent of the canine thymus.     

Local proliferation contributes to lymphocyte numbers in normal lungs.

Lymphocytes in the bronchoalveolar lavage fluid (BAL) are increased in many lung diseases, which might be an indicator for protective reactions or pathology. Higher lymphocyte numbers at distinct organ sites may be due to a number of reasons such as increased entry, increased proliferation, reduced apoptosis or reduced exit. It is not known whether lymphocyte numbers are influenced by local proliferation in the healthy lung. Therefore, the proliferation of lymphocytes was studied in vivo in different lung compartments of healthy rats: the marginal vascular pool, the interstitial pool and the bronchoalveolar pool. Bromodeoxyuridine (BrdU) was used as an S-phase proliferation marker. The cells were obtained 1 h and 24 h after i.v. injection of BrdU. The labeled cells and their phenotypes were determined by immunocytochemistry, since it was not possible to use flow cytometry because of the low numbers. In the lung compartments 0.7-1.5% of all nucleated cells were found to be BrdU+, whereas 24 h later this increased to 3.2-5.7%. The frequency of BrdU+ T cells was significantly higher in the lung compartments compared to the blood, with the maximum in the marginal vascular pool. Local proliferation of lymphocytes involved mainly CD8+ T cells. Thus, local proliferation plays a role in the number and composition of lymphocytes in the healthy normal lung.     

Organizing a mucosal defense

Summary: Gastrointestinal associated lymphoid tissue can be divided into loosely organized effector sites, which include the lamina propria and intraepithelial lymphocytes, and more organized structures, such as mesenteric lymph nodes (LNs), Peyer's patches (PPs), isolated lymphoid follicles, and cryptopatches (CPs). These organized structures in the gastrointestinal tract have been hypothesized to play the role of primary lymphoid organ, supporting the extrathymic development of T lymphocytes (CPs), secondary lymphoid organs involved in the induction of the mucosal immune response (PPs), and tertiary lymphoid structures whose function is still under debate (isolated lymphoid follicles). The most widely studied lymphoid structure found in the small intestine is the PP. PPs are secondary lymphoid structures, and their development and function have been extensively investigated. However, single lymphoid aggregates resembling PPs have been also described in humans and in the murine small intestines. These isolated lymphoid follicles have both germinal centers and an overlying follicle‐associated epithelium, suggesting that they also can function as inductive sites for the mucosal immune response. This review compares and contrasts the development and function of the four main organized gastrointestinal lymphoid tissues: CPs, isolated lymphoid follicles, PPs, and mesenteric LNs.     

Negative signaling by surface IgM on B cells isolated from ileal Peyer's patch follicles of sheep

Lymphoid follicles from the sheep ileal Peyer's patch (PP) were used to prepare a cell suspension consisting of 98% surface IgM-positive (sIgM+) B cells and 1% T cells. Co-stimulation of follicular cells with pokeweed mitogen and either recombinant bovine interleukin 1 (IL 1) or IL 2 resulted in a marked proliferative response. In contrast, the addition of soluble F(ab')2 rabbit anti-sheep Ig completely inhibited the proliferative response induced by pokeweed mitogen and IL 1 or IL 2 co-stimulation. Anti-Ig inhibition of B cell proliferation was specific for ileal PP follicular cells and was not observed with mesenteric lymph node cells or splenocytes. Furthermore, suppression of ileal PP follicular B cell proliferation required at most divalent cross-linking of sIg was independent of Fc receptors, but was dependent on the concentration of anti-Ig and required 48 h for maximal effect. Negative signaling by sIgM indicates that ileal PP follicular B cells are functionally distinct from B cells in other secondary lymphoid tissues. Also, the present observations are consistent with previous reports indicating that B cell proliferation in ileal PP follicles is antigen independent.     

Microanatomical dissection of human intestinal T-cell immunity reveals site-specific changes in gut-associated lymphoid tissues over life

Defining adaptive immunity with the complex structures of the human gastrointestinal (GI) tract over life is essential for understanding immune responses to ingested antigens, commensal and pathogenic microorganisms, and dysfunctions in disease. We present here an analysis of lymphocyte localization and T cell subset composition across the human GI tract including mucosal sites (jejunum, ileum, colon), gut-associated lymphoid tissues (isolated lymphoid follicles (ILFs), Peyer's patches (PPs), appendix), and mesenteric lymph nodes (MLNs) from a total of 68 donors spanning eight decades of life. In pediatric donors, ILFs and PP containing naïve T cells and regulatory T cells (Tregs) are prevalent in the jejunum and ileum, respectively; these decline in frequency with age, contrasting stable frequencies of ILFs and T cell subsets in the colon. In the mucosa, tissue resident memory T cells develop during childhood, and persist in high frequencies into advanced ages, while T cell composition changes with age in GALT and MLN. These spatial and temporal features of human intestinal T cell immunity define signatures that can be used to train predictive machine learning algorithms. Our findings demonstrate an anatomic basis for age-associated alterations in immune responses, and establish a quantitative baseline for intestinal immunity to define disease pathologies.     

Peyer's patches are precocious to the appendix in human development

PP are first visible at approximately 15.5 wk gestation after which there is a rapid spurt in the development and maturation of lymphoid follicles so that at any given point of time new foci of PP development are continuously formed at a rapid rate. Addition of rows of follicles results in the formation of a PP. Immature PP of younger fetuses have a spongy structure in contrast with the compact lymphoid follicles of mature PP of older fetuses. Immunocytochemical studies reveal that there is a subtle gradation in the expression of lymphocyte surface markers with increasing fetal age. Expression of antigenic markers occurs in an ordered sequence viz. HLA - DR, CD19 (B cell population), CD9 (pre-B cells), CD3 T lymphocytes, CD4 helper / inducer lymphocytes, the CD8 suppressor / cytotoxic cells and lastly, the CD57 Natural Killer cells. The antigens are expressed first on lymphocytes of PP and thereafter in those of the appendix. Our findings clearly demonstrate that the approximately 5 wk fetal period from 17.5 wk to 22 wk represents a major growth phase in the development of surface markers of lymphocytes in the mucosal immune system of the gut.     

The development of gut associated lymphoid tissue in the terminal ileum of fetal human intestine.

Lymphoid tissue in formalin fixed and snap frozen human fetal ileum has been studied using immunohistochemistry. At 11 weeks gestation clusters of cells expressing CD4 (leu-3a positive) are present in fetal ileum but these do not express CD3 (UCHT1 negative) and are probably macrophages. Aggregates of lymphoid tissue are apparent from 14 weeks gestation which contain T cells of helper/inducer and suppressor/cytotoxic phenotype. Both B and T cells are present at 16 weeks but with no cellular zonation. By 19 weeks, distinct follicles of B cells are present surrounded by T cells of helper/inducer and suppressor/cytotoxic phenotype. Follicular dendritic cells are also present within the B cell areas. The B cells at this age express surface IgM and IgD, C3b- and C3d-receptors. They also express the antigen CD5 which has been shown by others to be present on some fetal B cells but which is almost exclusively associated with T cells in the adult. HLA-D region antigens are present on apparently all of the cells within the fetal lymphoid follicles. The antigen on activated B cells, CD23 (recognized by MHM6), was present on some cells scattered within the B cell follicle. This is indicative of antigen independent B cell proliferation.     

Structural and functional differences between putative mucosal inductive sites of the rat

Nasal-associated lymphoid tissue (NALT), bronchus-associated lymphoid tissue (BALT) and Peyer's patches (PP) were compared structurally and functionally using a model of local mucosal infection of rats with reovirus. Histological analyses showed that BALT lacks the typical lymphoid organization found in NALT and PP. After local reovirus infection, germinal centers developed in NALT with appearance of IgA+ cells, whereas no germinal centers or isotype-switched cells were found in BALT. Production of reovirus-specific IgA was observed in NALT and PP, but only small amounts of specific IgA were secreted by BALT. Both NALT and BALT showed considerable production of IgG2b, whereas this isotype was poorly produced by PP. These data reveal profound qualitative differences between these three mucosal sites, and strongly suggest that BALT is not a mucosal inductive site for reovirus-specific immune responses in the rat.     

Cellular choreography in the germinal center: new visions from in vivo imaging

Germinal centers (GC) are large aggregates of proliferating B lymphocytes within follicles of lymphoid tissue that form during adaptive immune responses. GCs are the source of long-lived B cells that form the basis for pathogen-specific lifelong B cell immunity. The complex architecture of these structures includes subdomains that differ significantly in their stromal cell and T lymphocyte subset composition. In part due to their structural complexity and potential to generate some lymphomas, much interest and many theories about GC dynamics have emerged. Here, we review recent research employing in vivo imaging that has begun to untangle some of the mysteries.     

Th17 cells induce ectopic lymphoid follicles in central nervous system tissue inflammation

Ectopic lymphoid follicles are hallmarks of chronic autoimmune inflammatory diseases such as multiple sclerosis (MS), rheumatoid arthritis, Sj?gren's syndrome, and myasthenia gravis. However, the effector cells and mechanisms that induce their development are unknown. Here we showed that in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, Th17 cells specifically induced ectopic lymphoid follicles in the central nervous system (CNS). Development of ectopic lymphoid follicles was partly dependent on the cytokine interleukin 17 (IL-17) and on the cell surface molecule Podoplanin (Pdp), which was expressed on Th17 cells, but not on other effector T cell subsets. Pdp was also crucial for the development of secondary lymphoid structures: Pdp-deficient mice lacked peripheral lymph nodes and had a defect in forming normal lymphoid follicles and germinal centers in spleen and lymph node remnants. Thus, Th17 cells are uniquely endowed to induce tissue inflammation, characterized by ectopic lymphoid follicles within the target organ.