Comparison of IgD+ and IgD- thoracic duct B lymphocytes as germinal center precursor cells in the rat.

Genetically marked thoracic duct B cell subpopulations rich in either IgD+ or IgD- B cells were transferred to non-irradiated, congenic rats in order to compare the capacities of IgD+ versus IgD- B cells to form germinal centers (GCs). This comparison was made quantitatively based on flow cytometric analyses of lymph node cells prepared from chimeric rats 7 days after s.c. immunization. Donor-origin and host-origin B cells were distinguished using anti-Igk antibodies, and GC B cells were distinguished from other B cells in suspension by their lack of labeling with the mAb HIS22. IgK+ HIS22- lymph node cells corresponded well to GC B cells: they contained many large cells, were IgM+ but mostly IgD-, expressed relatively lower levels of IgM than HIS22+ B cells, and increased in number and frequency in response to antigen. Results from flow cytometric analyses, corroborated by immunofluorescence histochemical studies, showed that cell-for-cell, IgD- B cells from GCs much more efficiently than IgD+ cells. B cell populations enriched for IgD- cells became relatively more distributed to GCs than to other lymph node B cell areas and gave rise to many more GC B cells of donor origin per transferred B cell than whole, unseparated thoracic duct B cells (for which greater than 97% were IgD+). IgD- B cells from rats primed deliberately with antigen also became relatively more distributed to GCs and gave rise to more GC B cells of donor origin than either IgD+ B cells from primed donors or IgD- B cells from unprimed donors.(ABSTRACT TRUNCATED AT 250 WORDS)     

ROLE OF GERMINAL CENTER IN IMMUNE RESPONSE ENZYME IMMUNO-HISTOCHEMICAL STUDY USING HORSERADISH PEROXIDASE

Using enzyme Immuno-hlstochemlcal method with horseradish peroxidase (HRP), a study on the relation between localization of antigen and antibody, and development of germinal center in lymph node after primary and secondary stimulation was made.
Blastic antibody forming cells and intercellular localization of the antibody were noticed in newly formed germinal centers of draining lymph nodes within 7 days after primary antigenic stimulation. With increase of circulating antibodies produced by the draining lymph nodes, it accumulated in ready made germinal centers of generalized non-draining lymph nodes. At the time of primary stimulation, the HRP antigen was never observed in solid follicles or newly formed germinal centers, and even in ready made germinal centers it became localized only transiently and was never retained. After secondary stimulation, the antigen accumulated quickly in the light zone of germinal centers, being trapped by the previously present antibody. Three days later, antibody forming cells began to be noticed in germinal centers as well as in medullae of non-draining lymph nodes where no antibody forming cells had been so far observed, and in medullae of draining lymph nodes they increased markedly. Passively injected anti-HRP antibody accumulated also in germinal centers of non-primed mice and successively injected antigen was easily trapped in these germinal centers. Passively injected soluble antigen-antibody-complexes were localized in germinal centers of non-primed mice. In both these cases, antibody forming cells appeared only in germinal centers and none in medullae. Blastic cells, proliferating in germinal center and producing 2-ME sensitive antibody were not considered to be directly related with antibody forming cells in the medulla of lymph node.     

Interactions between primed and unprimed cells in the regulation of in vitro antibody responses. I. Role of “plasma cells” as inducers of suppression

Specific immune suppression has been shown to be activated in culture by the interaction of primed and unprimed T cell subsets. The primed cell involved is found 8 days after immunization in spleen but not in lymph node or thymus cell populations. When the primed spleen cells were fractionated by nylon wool passage or anti-Thy-1 plus complement (C) treatment, prior to culture with unseparated unprimed cells, suppression was detectable only with primed B cells present in the co-cultures. Treatment of the primed spleen cells with anti-PC. 1 (an antiserum specific for plasma cells) plus C eliminated their ability to cooperate with either unseparated or T cell-enriched populations of unprimed cells in suppressing the antibody response of the co-cultures. These data are consistent with the hypothesis that antibody-secreting plasma cells activate suppressor T cell precursors in cell populations not previously exposed to antigen.     

Antigen-induced changes in B cell subsets in lymph nodes: analysis by dual fluorescence flow cytofluorometry

Changes in the representation and surface phenotype of defined B cell subsets in murine lymph nodes stimulated with keyhole limpet hemocyanin or sheep red blood cells have been analyzed by two-color immunofluorescence fluorocytometric analysis. Shortly after immunization with either antigen there is a dramatic increase in both the frequency and absolute number of IgM+, IgD+ B cells, which is followed by the formation of germinal centers. Germinal center cells, as soon as they appear on day 3 after primary immunization, bind high levels of peanut agglutinin, bear low levels of surface IgM but no detectable surface IgD, and are characterized by lack of staining with MEL-14, a monoclonal antibody which recognizes a lymphocyte surface receptor involved in lymphocyte homing. The level of I-A and H-2K region-encoded surface antigens on early germinal center cells is higher than on PNAlo B cells. During the first 7 days of the germinal centers there is a progressive decrease in the average level of H-2K but not of Ia antigens. A similar decrease was observed for ThB. It is confirmed that the germinal center cell population contains the majority of antigen-binding cells in the stimulated lymph node. These findings indicate that B cells are recruited nonspecifically to antigen-stimulated lymph nodes, and that the antigen-specific cells then selectively participate in the formation of germinal centers where they undergo specific differentiation events.     

Kinetics of germinal center development in lymph nodes of young and aging immune mice

Recent findings imply that germinal center paucity in old mice, at least in part, results from a defect in the mechanisms responsible for the transport of antigens to lymphoid nodules (follicles) and the consequent impairment of the antigen retaining reticulum (ARR) of follicular dendritic cells (FDCs). The present objective was to observe the kinetics of lymph node germinal center development in old mice having antigen transport and ARR deficits. Germinal center development was monitored in popliteal (PLN) and axillary (AXLN) lymph nodes of 6-8 wk and 23-mo-old horseradish peroxidase (HRP) immune C57BL/6 mice. Using the selective binding of germinal center B cells for peanut agglutinin (PNA), germinal centers were identified in serial vibratome sections following histochemical labeling with PNA-peroxidase conjugates at times 0, 15 min, 1, 3, 5, and 10 days after footpad challenge with 8 micrograms HRP. To follow the fate of preexisting (environmental antigen-induced) germinal centers and the development of de novo (HRP-induced) germinal centers, it was essential to distinguish between these germinal centers. Accordingly, PNA positive germinal centers associated with HRP-retaining (peroxidase positive) ARR were identified as de novo germinal centers and germinal centers not associated with a peroxidase positive ARR were classified as preexisting germinal centers. Kinetic analysis of PNA positive germinal centers showed the following: 1) Preexisting, environmentally-induced germinal centers dissociated and disappeared by day 3 as indicated by a decline in their numbers after antigen injection: the process of germinal center dissociation remained unaffected by aging. 2) The latency of de novo germinal center appearance was approximately equal in duration (approximately 3 days) to the disappearance of pre-existing germinal centers. 3) The number and size of de novo HRP-induced germinal centers increased through the experimental period in young lymph nodes, but in old mice these parameters were depressed, resulting in a significant germinal center deficit. 4) The ratio of HRP-retaining ARR to de novo induced germinal centers was 1:1 in young and responder old mice. This ratio was not affected by aging. This finding favored the concept that antigen retention in ARR is a requirement of germinal center development. The observations supported our hypothesis that germinal center development, at least in part, depends on a normal antigen transport by showing that in aged mice with defective antigen transport-related ARR and iccosome deficits there is an impaired development of germinal centers.     

Polyclonal activation of the murine immune system by an antibody to IgD V. Effect on germinal centers

Heterologous anti-mouse delta chain has been shown to induce T-independent polyclonal B cell proliferation by 24 h after injection into mice and a T-dependent polyclonal increase in the number of IgG-secreting cells in spleen and lymph nodes 6 days after injection. The effect of anti-delta on germinal center cells has been difficult to determine, however, since anti-delta-induced blast transformation of mantle zone cells makes it difficult to distinguish these cells from germinal center cells. To clarify the effect of anti-delta on germinal centers, we stained lymph node sections from control and anti-delta-injected mice with peanut agglutinin conjugated to horseradish peroxidase which clearly differentiates the germinal center cells from other large and small lymphoid cells. For the first 5 days after i.v. injection of goat anti-mouse delta, the germinal center volumes did not differ significantly from those of mice injected with control antibodies. On days 6 and 7 after injection, the lymph node germinal center volumes of anti-delta-treated mice increased on average over 10-fold as compared to those of mice injected with an equal amount of control antibody. Thus, while anti-delta antibody directly induced blastogenesis of mantle layer B cells, it appears to have little direct effect on germinal center cells. In contrast, rapid germinal center development is seen in anti-delta-injected mice during the T-dependent phase of B cell activation and occurs simultaneously with the appearance of large numbers of IgG-secreting cells.     

The germinal center response is impaired in the absence of T cell-expressed CXCR5

Germinal centers support the differentiation of memory B cells and long-lived antibody-secreting cells during infection or upon vaccination. Here, we constructed mice with T cells that selectively lack the chemokine receptor CXCR5 to determine if expression of this receptor by T cells is mandatory for germinal center formation and function. In these animals, germinal centers that are properly localized in B cell follicles and contain T cells do form after immunization with a thymus-dependent antigen. However, fewer and smaller germinal centers form, resulting in a significant reduction in the frequency of germinal center B cells. The defect in germinal center formation is paralleled by decreased frequencies of isotype-switched antibody-secreting cells in the spleen and bone marrow and reduced serum concentrations of total and high-affinity hapten-specific IgG1. The results demonstrate that although CXCR5-dependent T cell positioning is important for maximal induction and expansion of germinal centers, stimulation of isotype class switching, and development of antibody-secreting cells that seed the spleen and bone marrow, it is not absolutely required for the formation and function of follicular germinal centers.     

AN ULTRASTRUCTURAL STUDY ON ANTIBODY PRODUCTION OF THE LYMPH NODES OF RATS WITH SPECIAL REFERENCE TO THE ROLE OF GERMINAL CENTERS

Antibody-containing cells in lymph nodes, especially in germinal centers, and in thoracic duct of rats sensitized by Injection of horseradish peroxidase were studied with the immunoperoxidase method. In the primary response, the large germinal center cells began to produce antibody on the 9th day and predominated thereafter In germinal centers, added by intercellular antibody deposition in its late stage. The "ordinary" medium-sized germinal center cells were a minor component. In the secondary response, the intracellular antibody positivity disappeared immediately after the antigenic rechallenge but reappeared from 6 hours later In the large germinal center cells which further transformed from 48 to 72 hours into the "specifically differentiated" medium-sized germinal center cells that disappeared by 96 hours after the secondary injection to enter the thoracic duct. Antibody-containing cells outside the germinal centers appeared 5 to 6 days after the primary injection and were mainly comprised of plasmocytic cell series which was classified Into lnterfollicular large cells, proplasmocytes and plasmocytes. Some results conflicting with the cytologlcal identity of both cell series were presented.     

T lymphocytes in rat germinal centres belong to an ER3+ subpopulation of CD4+ cells.

Two-colour immunofluorescence histochemistry showed directly that greater than 90% of CD4+ germinal centre T cells in rat spleen or lymph node examined 7 days after immunization bear the antigen recognized by the monoclonal antibody (mAb) ER3. By contrast, only 30-40% of all thoracic duct or lymph node CD4+ cells were ER3+, as determined by two-colour flow cytometry. CD8+ cells were ER3+, but nearly all B cells were ER3-. Thus, germinal centre T cells belong to a subpopulation of CD4+ cells. Because only 25-30% of CD4+ cells that lack higher molecular weight forms of CD45 (i.e. mAb MRC OX32 cells, equivalent to MRC OX22 cells) express ER3, the CD4+ subpopulations defined by ER3 are neither identical nor complementary to the subsets defined by restricted expression of CD45 epitopes.     

Deterioration of B cell proliferation correlates with dendritic reticulum cell destruction in germinal centers of an AIDS patient. Case study

A lymph node from a bisexual Caucasian male infected with human immunodeficiency virus (HIV) and in the persistent generalized lymphadenopathy (PGL) stage was studied. Dendritic reticulum cells (DRCs) were well preserved in over half of the germinal centers (GCs), while in the rest, they showed marked destruction, producing patchy or rather wide DRC-depleted areas. Proliferation-associated antigens, i.e., PC antigen and DNA-polymerase alpha, were demonstrated in nuclei of germinal center B cells in areas where the DRC network was intact, while they were prominently depleted in areas where the DRC network was lost. The p-24 viral core antigen was shown to be localized in DRCs, especially those in the process of degeneration. These results suggest that the DRC in this patient, when infected with HIV, were destroyed, and that the resulting DRC depletion led to the suppression of B cell proliferation in GCs.     

DETERIORATION OF B CELL PROLIFERATION CORRELATES WITH DENDRITIC RETICULUM CELL DESTRUCTION IN GERMINAL CENTERS OF AN AIDS PATIENT. Case Study

A lymph node from a bisexual Caucasian male infected with human immunodeficiency virus (HIV) and in the persistent generalized lymphadenopathy (PGL) stage was studied. Dendritic reticulum cells (DRCs) were well preserved in over half of the germinal centers (GCs), while in the rest, they showed marked destruction, producing patchy or rather wide DRC-depleted areas. Proliferation-associated antigens, i.e., PC antigen and DNA-polymerase a, were demonstrated in nuclei of germinal center B cells in areas where the DRC network was intact, while they were prominently depleted in areas where the DRC network was lost. The p-24 viral core antigen was shown to be localized in DRCs, especially those in the process of degeneration. These results suggest that the DRC in this patient, when infected with HIV, were destroyed, and that the resulting DRC depletion led to the suppression of B cell proliferation in GCs. ACTA PATHOL JPN 38: 1205∼1214, 1988.     

Avian lymph node: Light and electron microscopic study

We have observed by light and transmission electron microscopy lymphoid accumulations (LA) in the chicken located along the posterior tibial-popliteal and lower femoral veins. Three types of LA were characterized: (1) LA on the wall of the lymphatic, (2) LA with germinal center, and (3) well-developed LA possessing germinal centers and an intricate lymphatic sinus system. The latter will be termed a lymph node and is perhaps the structure responding to foot-pad injection of antigen and/or phytohemagglutinin (PHA). After the injection of PHA into the foot-pad, the lymph node enlarged and revealed the intermingling of two distinct groups of cells consisting of either small lymphocytes or medium lymphocytes and lymphoblasts. Because our earlier immunological paper proved the presence of T and B cells in the node, the two histologically distinct groups of cells appearing after PHA injection could reflect compartmentalization of T and B cells in the avian lymph node. Lymphoid and adipose tissues are in the same compartment. After PHA or antigen injection into the foot pad, the lymphoid tissue proliferates and the amount of the adipose tissue rapidly decreases. This suggests that lymphoid and adipose tissue form a special complex which is separated from the surrounding tissue by delicate connective tissue capsule. The relationship of the lymphoid and adipose tissue is comparable with that of myeloid and adipose tissue in the bone marrow. The majority of the sinuses shows smooth endothelial lining while others contain “hairy” macrophages attached to the endothelium. The germinal centers are located at the periphery of the node, but a few occur inside. The cellular content of the germinal centers is not unusual except for the presence of plasma cells.     

Kinetics of the tingible body macrophage response in mouse germinal center development and its depression with age

Although tingible body macrophages (TBM) have been recognized in germinal centers for over 100 years, their role in the germinal center response is not clear. In this study, the kinetics of the TBM response was quantitatively assessed and correlated with the kinetics of germinal center development in young mice. The TBM response in old mice (which have an age-related depression of germinal center development; Szakal et al., 1990) was analyzed for comparison. Young and old immune mice were challenged with human serum albumin and 0, 1, 3, 5, 7, 10, and 14 days later the popliteal and axillary lymph nodes were evaluated. Germinal centers were localized histochemically in alternate serial sections using horseradish peroxidase conjugated peanut agglutinin. TBM numbers were determined per germinal center on adjacent sections by the presence of tingible bodies or histochemically by using the monoclonal antibody Mac-2. Analysis of lymph nodes from young mice showed that TBM numbers decreased with the dissociation of preexisting germinal centers. TBM reappeared 5 days after challenge and the TBM kinetics paralleled the increase in size of de novo germinal centers. In fact, a constant ratio of one TBM to every 350-450 B cells was maintained from day 5 to day 10. In old lymph nodes, TBM were generally absent throughout germinal center development. The lack of TBM prior to germinal center development and their absence in aged mice are inconsistent with the concept that TBM are required for the induction of the germinal center reaction. However, the data are consistent with a role for TBM in regulating the magnitude of the germinal center reaction.     

The effect of adjuvants and prior immunization on the rate and mode of uptake of antigen into afferent popliteal lymph from sheep

Experiments were carried out to determine the effect of a range of adjuvants on the antibody response of sheep to a subcutaneous injection of ovalbumin. Incomplete Freund's adjuvant (IFA) was the best of the repository type adjuvants. From a range of soluble immunopotentiating substances tested, the polyion DEAE-dextran, or dextran sulphate, produced the greatest enhancement of the response. These results were confirmed in a factorial experiment, but no interactions were found between various classes of adjuvants. Further experiments were carried out to determine the effect of superior adjuvants, prior immunization or intradermal injection on the rate and mode of uptake of antigen from the injection site to afferent lymph. These experiments showed that, with IFA, most antigen is retained at the injection site, less than 15% entering lymph by 3 days. There was some delay in antigen uptake with adjuvant 65, while for aluminium phosphate precipitated antigen, the rate of uptake was the same as following a saline injection. There was some delay in uptake of antigen following injection with DEAE-dextran or in primed sheep but, following intradermal injection, the rate of uptake into lymph was the same as aqueous antigen injected subcutaneously. Experiments in which the distribution of radioactive antigen between cells and plasma in lymph was examined showed that, in unprimed sheep, virtually all antigen is unassociated with cells. In primed sheep, however, about 0.5% of antigen in lymph was found to be cell-associated. Gel filtration of lymph plasma from primed sheep demonstrated that nearly all the antigen in lymph is carried to the node as high molecular weight material, presumably antigen-antibody complexes.     

The role of cell number and source in adoptive immunity

The use of bacteriophage and bovine serum albumin as antigens for adoptive immunization has been investigated. Cells were transplanted from normal or previously immunized (`primed cells') donor mice into isologous hosts, and then stimulated with antigen. The reactions to phage of normal and primed cells could be distinguished and measured; for BSA, only primed cells could be used without the help of adjuvant. Irradiation of the prospective host enhanced the activity of the transplanted cells. Saturation was easily reached by increasing the number of phage-primed cells in the transplant. BSA-primed cells saturated less easily, probably because BSA is a relatively poor immunogen. Primed cells were localized, to a limited extent, in the lymph nodes draining the sites of injection of antigen, but could usually also be found in the blood.     

Functional and histological studies of adoptive immunity in neonatally transplanted rabbits.

The adoptive transfer of immunity by means of spleen and lymph node cells has been analysed in rabbits of defined major histocompatibility (RLA) types and immunoglobulin (Ig) allotypes. Previous results had shown that chimaeras formed by transplanting adult cells into RLA-matched newborn hosts become stable B-lymphocyte chimaeras, as determined by their continuous production of donor type Ig. However, specific antibodies made by adult chimaeras were demonstrably only of recipient origin, unless the donor had been primed. The present report describes early events after the transfer of cells from donors primed with trinitrophenylated keyhole limpet haemocyanin (TNP-KLH). Double-staining with enzyme-labelled antigen or antibody conjugates was applied to identify lymphocytes of donor or recipient origin in spleen sections of transplanted rabbits and also to identify cells producing anti-TNP. Lymphocytes with membrane-bound Ig (mIg+ cells) of donor allotype were readily identified scattered singly or in small clusters throughout the recipients' B-lymphocyte areas within 5 days after transfer. Groups of donor-derived cells producing anti-TNP were demonstrable only in animals that had been specifically stimulated. Serological analysis and lymphocyte marker and function studies correlated well with results obtained by the histocytochemical approach. These results show that a systematic study of the role and importance of mature B lymphocytes in transplantation is now feasible.     

Development of antibody diversity in single germinal centers: selective expansion of high-affinity variants

In a T cell-dependent immune response the microenvironment of the germinal center plays a crucial role in the affinity maturation of the antigen-specific immunoglobulins. In order to look at the development of antibody diversity we have isolated single germinal centers and sequenced light chains characteristic of 2-phenyl-oxazolone (phOx)-specific antibodies. Fourteen days after immunization we can demonstrate various stages of intraclonal diversity. There are germinal centers where B cells are practically unmutated, suggesting that in these cases a substantial clonal expansion has taken place prior to the activation of the hypermutation mechanism. In other germinal centers, sequences with a low number of randomly distributed somatic mutations were observed, indicating that these changes have been introduced recently and/or that they fail to generate high-affinity variants and hence provide no basis for affinity selection. Finally, germinal centers are found in which practically all sequences carry the amino acid substitutions characteristic of the high affinity phOx antibodies. In these latter cases the high-affinity variants have been preferentially expanded. We conclude that affinity selection is a process that operates right from the beginning of germinal center development. Those B cells with a relative high affinity for the antigen gain a proliferative advantage over other cells and will dominate the response and these are the cells which will be selected to differentiate into memory cells.     

Germinal-center cell proliferation in response to T-independent antigens: A stathmokinetic,morphometric and immunohistochemical study in vivo

Although the nature of the germinal center reaction during responses to T-dependent antigens has been well documented, much less is known regarding the relationship between germinal centers and T-independent antigens. In this study, germinal-center cell proliferation was determined at specific time points in spleens of C3H/HeN mice following immunization with either the type-1, T-independent antigen dinitrophenol-lipopolysaccharide (DNP-LPS), or the type-2, T-independent antigen DNP-Ficoll. A stathmokinetic technique was employed to assess proliferation in terms of germinal center cell birth rate and morphometry was used to measure actual growth and regression of the germinal center cell population. An estimate of the absolute rate of germinal-center (GC) cell proliferation was derived from these two values. In addition, immunohisto-chemistry was performed to correlate changes in GC cell proliferation with the presence or absence of antigen within GC. Following immunization with both antigens, there was an initial reduction of proliferation within pre-existing germinal centers which manifested as either GC dissociation (DNP-LPS) or a suppression of birth rates (DNP-Ficoll). This was followed by a period of increased GC cell proliferation in animals immunized with DNP-LPS, but not in those exposed to DNP-Ficoll. GC cell proliferation was then measured in mice treated with cyclosporin A from 1 day before to 2 days after immunization with DNP-LPS. In these animals, the expected increase in GC cell birth rates did not take place. Immunohistochemistry showed that DNP-Ficoll and DNP-LPS were present in GC from 1 day after immunization until the end of the experiment on day 7. Treatment with cyclosporin A did not affect the deposition of DNP-LPS in GC. These results show that only some T-independent antigens are able to stimulate GC cell proliferation, and we propose that this is related to their ability to recruit precursors of GC B cells into the GC reaction. In addition, the results indicate that GC proliferation seen in response to a so-called T-independent antigen is at least partly driven by T cell-derived cytokines.     

A case report of angioimmunoblastic T cell lymphoma (AITL) with localization of neoplastic clear cells in the outer zone of germinal centers

Angioimmunoblastic T cell lymphoma (AITL) is characterized by the presence of atypical lymphocytes with clear cytoplasm and follicular dendritic cells, arborization of high endothelial blood vessels, and infiltration by inflammatory cells, such as epithelioid histiocytes, eosinophils, immunoblasts, and plasma cells. The neoplastic clear cells are localized around the high endothelial blood vessels or interfollicular areas. Recent reports have suggested that these neoplastic clear cells originate from helper T cells in germinal centers, based on their expression of CD10, PD-1, and CXCL13. We experienced a case of AITL which is histologically unique. A 61-year-old male presented to our hospital (Ogaki Municipal Hospital) with edema of his lower legs. Inguinal lymph node biopsy revealed that neoplastic clear T cells were mainly localized in the outer zone of germinal centers, specifically within the follicular dendritic cell (FDC) meshwork. Moreover, these cells were positive for CD3, CD4, CD10, CD43, CD45RO, PD-1, and weakly positive for CXCL-13. This is the first report showing that the neoplastic clear T cells were localized in the outer zone of germinal centers morphologically as well as immunohistochemically. In conclusion, this case report further supports the notion of germinal center helper T cell origin of neoplastic clear cells in AITL.