Vincent K. Tsiagbe

Biology of germinal centers in lymphoid tissue.

Germinal centers in lymphoid tissue are the sites of generation of memory B cells undergoing isotype switching and somatic mutation in their Ig genes. Their formation cannot be induced by stimuli other than immunogenic ones. It seems likely that in the function and possibly also in the formation of germinal centers, one important factor is the localization of immune complexes with fixed complement on the surface of follicular dendritic cells. CD4+ T cells, located primarily in the “apical light zones” of the centers, are necessary for germinal center formation. However, their exact role in the process needs clarification, as both cell to cell contact and cytokine production could be involved at different stages of the germinal center generation. These T cells are usually specific for the antigen inducing the germinal center, but they may sometimes respond to other surface components on the B cell surface. In view of the possible stimulatory role of CD4+ T cells in follicular center‐derived lymphomas, the functional significance of these T cells in germinal center proliferation is important to unravel. The B cells in germinal centers proliferate extremely rapidly, especially those located in the “dark zones.” Many of them undergo apoptosis, particularly in the “basal light zones.” The microenvironment of these centers is well suited to the task of expanding and selecting memory B cells of high affinity for the inducing antigen. The interactions of the proliferating B cells with dendritic cells and T cells, unevenly distributed in the various zones of the germinal center, are thought to determine which cells deserve rescue from apoptosis and induction to differentiation into small resting memory B cells. The memory B cells that emerge from the germinal center bear sIg, usually of “switched” isotype, and exhibit somatic mutations in the variable regions of their rearranged Ig genes.— Thorbecke, G. J., Amin, A. R., Tsiagbe, V. K. Biology of germinal centers in lymphoid tissue. FASEB J. 8: 832‐840; 1994.

Linkage of superantigen-like stimulation of syngeneic T cells in a mouse model of follicular center B cell lymphoma to transcription of endogenous mammary tumor virus.

The MHC class II I‐A(s) positive B cell lymphomas reticulum cell sarcoma (RCS) that arise in > 90% of SJL mice by the age of 12 months have superantigen‐like stimulating properties. In the present study, therefore, RCS cell lines were examined for abnormal expression of endogenous mouse mammary tumor virus (MMTV) proviruses. Extraordinarily high expression of a 1.8 kb mRNA hybridizing with the long terminal repeat (LTR) of MMTV was found in both primary lymphomas and in vitro RCS lines, but not in an SJL B cell lymphoma, NJ101, that does not stimulate syngeneic T cells, or in LPS activated SJL B cells. A cDNA was cloned from cRCS‐2 and sequenced. A 31mer oligonucleotide probe, prepared based on the unique C‐terminal sequence of this RCS‐Mtv LTR, detected the 1.8 kb mRNA in all RCS lymphomas, while a similar probe for the C‐terminal sequence of Mtv‐8 LTR hybridized with the larger mRNA present in normal B cells and in NJ101. Preincubation with 19mer antisense S‐oligonucleotides, prepared based on the sequences of the first two potential translation initiation sites common to both Mtv‐8 and the RCS‐Mtv LTR, significantly reduced the ability of RCS cells to stimulate syngeneic T cells. Moreover, transfection of NJ101 cells with the cloned RCS‐MMTV cDNA conferred V beta 16 T cell stimulating properties on to these cells. It is concluded that expression of the product of this MMTV‐LTR mRNA provides RCS with the strong T cell stimulating properties that it needs for its growth. These results thus identify a novel oncogenic property of MMTV‐LTR.

The Path of Memory B‐Cell Development

Germinal centers (GC) form in lymphoid tissue draining a site of soluble antigen injection, starting approximately on day 4 and reaching peak development by d 10 after the injection. Many investigators have observed that the maximal deveiopment of GC after injection of soluble antigen comes later than the peak ofantibody production (White 1960, Langevoort et al. 1963. Coico et al. 1983). Sites ofantibody production in various tissues, but particularly in the spleen of the rabbit where it was examined in detail, are invariably associated with sites of accumulation of proliferating plasma blasts and plasma cells. In the spleen, these are found at the border of white and red pulp and along penicilli arterioles extending from the white into the red pulp (Fig. 1; Thorbecke & Keuning 1956, Langevoort et al. 1963). Both histologicai demonstration of the antibody by immunofluorescence (Leduc et al. 1955) or immunoperoxidase (Sordat et al. 1970). and production ofantibody in tissue culture have clearly identified plasma cell aggregates as the major antibody-producing foci. Compartmentalization of GC and plasma cell foci also occurs in lymph nodes draining sites of antigen injections. GC are in the outer part ofthe cortex, while maturing plasmabiasts, again arising earlier than the GC. move from the dense lymphoid areas of the cortex to the medullary cords during the response (Nieuwenhuis &. Keuning 1974, Jacobson et al. 1974).

Aggregatibacter actinomycetemcomitans Infection Enhances Apoptosis In Vivo through a Caspase-3-Dependent Mechanism in Experimental Periodontitis

ABSTRACT The purpose of this study was to test the hypothesis that diabetes aggravates periodontal destruction induced by Aggregatibacter actinomycetemcomitans infection. Thirty-eight diabetic and 33 normal rats were inoculated with A. actinomycetemcomitans and euthanized at baseline and at 4, 5, and 6 weeks after inoculation. Bone loss and the infiltration of polymorphonuclear leukocytes (PMNs) in gingival epithelium were measured in hematoxylin-eosin-stained sections. The induction of tumor necrosis factor alpha (TNF-α) was evaluated by immunohistochemistry and of apoptotic cells by a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay. After A. actinomycetemcomitans infection, the bone loss in diabetic rats was 1.7-fold and the PMN infiltration 1.6-fold higher than in normoglycemic rats (P < 0.05). The induction of TNF-α was 1.5-fold higher and of apoptotic cells was up to 3-fold higher in diabetic versus normoglycemic rats (P < 0.05). Treatment with a caspase-3 inhibitor significantly blocked noninflammatory cell apoptosis induced by A. actinomycetemcomitans infection in gingival epithelium and connective tissue (P < 0.05). These results provide new insight into how diabetes aggravates A. actinomycetemcomitans-induced periodontal destruction in rats by significantly increasing the inflammatory response, leading to increased bone loss and enhancing apoptosis of gingival epithelial and connective tissue cells through a caspase-3-dependent mechanism. Antibiotics had a more pronounced effect on many of these parameters in diabetic than in normoglycemic rats, suggesting a deficiency in the capacity of diabetic animals to resist infection.

Adaptive immune response in osteoclastic bone resorption induced by orally administered Aggregatibacter actinomycetemcomitans in a rat model of periodontal disease

There is mounting evidence that innate and adaptive immunity are critical for periodontal disease-mediated bone resorption. These studies examined the role of B and CD4 T cells in adaptive immunity of rats infected with Aggregatibacter actinomycetemcomitans (Aa). Sprague-Dawley male rats were fed Aa-containing mash or control-mash for 2 weeks. B and CD4 T cells were obtained from draining lymph nodes at 2, 4 and 12 weeks, postinoculation. Quantitative polymerase chain reaction-based messenger RNA expression was conducted for 89 cytokine family genes. Disease-relevance of the differentially expressed genes was assessed using a biological interaction pathway analysis software. B and CD4 T cells of Aa-infected rats increased and were activated, resulting in enhanced isotype-switched serum immunoglobulin G by 2 weeks postinoculation. Bone resorption was evident 12 weeks after Aa-feeding. In B cells, interleukin-2 (IL-2), macrophage-inhibiting factor, IL-19, IL-21, tumor necrosis factor (TNF), CD40 ligand (CD40L), CD70, bone morphogenetic protein 2 (BMP2), BMP3, and BMP10 were upregulated early; while IL-7, Fas ligand (FasL), small inducible cytokine subfamily E1, and growth differentiation factor 11 (GDF11; BMP11) were upregulated late (12 weeks). BMP10 was sustained throughout. In CD4 T cells, IL-10, IL-16, TNF, lymphotoxin-beta (LTbeta), APRIL, CD40L, FasL, RANKL and osteoprotegerin were upregulated early, whereas IL-1beta, IL-1RN, IL-1F8, IL-24, interferon-alpha1, GDF11 (BMP11), and GDF15 were upregulated late (12 weeks). Adaptive immunity appears crucial for bone resorption. Several of the deregulated genes are, for the first time, shown to be associated with bone resorption, and the results indicate that activated B cells produce BMP10. The study provides a rationale for a link between periodontal disease and other systemic diseases.

B cell lymphomas of C57l/J mice; the role of natural killer cells and T helper cells in lymphoma development and growth

The Hodgkins-like Type B neoplasms which arise spontaneously in aging C57L mice (25% incidence at 21 months of age) were first reported over 40 years ago, but since then relatively little has been published about these lymphomas. Based on previous studies in SJL mice, we investigated the phenotypic and functional properties of C57L-derived lymphomas in relation to Mtv29-encoded vSAg expression by the tumor cells, and their ability to stimulate TCR Vbeta-restricted T cells. The cell surface phenotype of the C57L lymphomas indicates a B cell origin (sIg(+), MHC II(+)). These B lymphoma cells also express co-stimulatory molecules [B7-1 (CD80) and HSA (CD24)], and stimulate marked proliferation of syngeneic CD4(+) T cells. C57L B lymphoma cells exhibit Mtv-encoded mRNA by northern analysis, and also stimulate IL-2 production from Vbeta16(+) T cell hybrids, suggesting a role for Mtv 29 in this syngeneic T cell response. After transfer to syngeneic recipients, primary C57L lymphomas grow slowly, if at all. However, tumor growth is greatly accelerated by pretreatment of C57L recipients with anti-asialo GM1 antibody (but not anti-CD8 mAb), suggesting that NK cells play a major role in inhibiting lymphoma growth. If, in addition to anti-asialo GM1, the mice are also pretreated with anti-CD4 mAb, tumor growth is markedly inhibited, indicating that the lymphoma-responsive syngeneic CD4(+) T cells promote tumor growth. Therefore, although the vSAg-induced response stimulated by vSAg29 expressing lymphoma cells in syngeneic TCR Vbeta-restricted CD4(+) T cells is an important etiologic factor in this type of B cell neoplasm both in C57L and in SJL mice, the final outcome of the spontaneous neoplastic process appears strongly influenced by endogenous NK activity in aging mice.

Regulatory T cells as central regulators of both autoimmunity and B cell malignancy in New Zealand Black mice.

Regulatory T cells (Tregs) play an important role in protection against autoimmune disease and are also known to be potent inhibitors of anti-tumor immune responses. The New Zealand Black (NZB) mouse is a murine model for both autoimmune diseases, since high levels of autoantibodies are present, and human CLL, due to the expansion of malignant B-1 cells. In this study, we examined the functional role of CD4(+)CD25(+) Foxp3(+) Tregs in these different manifestations. Flow cytometric analysis showed increased levels of Tregs in NZB mice compared to healthy C57Bl/6 controls. Aged NZB mice that have developed a B-1 cell malignancy identified as IgM(+)CD5(+), have the most pronounced increase in Tregs. Ex vivo treatment of splenocytes from NZB mice with IFN-alpha resulted in a decrease in the frequency of Tregs and malignant B-1 cells. In vivo treatment of both NZB and C57Bl/6 mice with poly (I:C), a potent inducer of IFN-alpha, also led to a decrease in the levels of Tregs and malignant B-1 cells (NZB only) while amplifying autoimmune manifestations. These results indicate that while high levels of Tregs found in NZB mice might suppress a more severe autoimmune disease, they may also contribute to the development of the B cell malignancy.

META-Controlled env-Initiated Transcripts Encoding Superantigens of Murine Mtv29 and Mtv7 and Their Possible Role in B Cell Lymphomagenesis

Spontaneous germinal center (GC)-derived B cell lymphomas of SJL mice (RCS) transcribe a 1.8-kb Mtv-29 mRNA under control of the META-env promoter. The encoded vSAg29 stimulates syngeneic Vβ16+ CD4+ T cells, thereby acquiring T cell help necessary for RCS growth. Other strains of B cell lymphoma-prone mice include Mtv29+ C57L and MA/MyJ, and the Mtv29− Mtv7+-recombinant inbred strain, SW × J-1. The lymphomas of these mice produce similar mouse mtv-vSAg-encoding mRNA, as characterized by Northern blotting, PCR, and RNase protection. A 1.8-kb mRNA in C57L/J and MA/MyJ lymphomas hybridized with an Mtv29-specific oligonucleotide, whereas SW × J-1 lymphomas produced 1.8-kb transcripts hybridizing with an Mtv7-specific oligonucleotide. Similar META-env-initiated transcripts were absent from LPS-activated B cells from any strain examined but were detected in Peyer’s patch RNA from SJL mice. Like typical SJL-derived RCS, all these lymphomas stimulated syngeneic CD4+ T cells and Vβ16+ T hybridoma cells. Immunohistochemical staining of primary tumors showed the presence of peanut agglutinin binding (PNA+) highly mitotic lymphoblasts, suggesting their GC derivation. The findings indicate that this novel mRNA for Mtv29 is present in B cell lymphomas from several Mtv29+ mouse strains. Additionally, this is the first description of the ability of Mtv7 to produce transcripts that are controlled and spliced identically to those of Mtv29 and that are expressed in SW × J-1, I-As+, lymphomas that also stimulate Vβ16+ T cells. Our results suggest an important role for mouse mtv-vSAgs and Vβ16 T cell stimulation in the development of GC-derived murine B cell lymphomas.

Superantigens related to B cell hyperplasia

ConclusionsIn both mouse and humans, B cell hyperplasia is seen in many situations that involve T cell stimulation. In the mouse, some of these conditions appear to involve a SAg-induced T cell response, which can result in chronic B cell hyperplasia and development of B cell lymphomas. From our investigation of the B cell lymphomas that characteristically arise in a majority (≃90%) of SJL/J mice, it is clear that a vSAg which is encoded by a unique endogenous mammary tumor provirus is involved in the neoplastic process. The tumor-associated vSAg (mtv-29-LTR) stimulates a TCR Vβ-restricted subset of T cells (Vβ16+) to produce cytokines which the lymphoma cells use as paracrine growth factors for progressive growth. We speculate that themtv-29-encoded vSAg may be expressed on germinal center B cells, even in young SJL mice, and that the characteristic chronic B cell hyperplasia seen in this strain is driven by the cytokines produced by responding Vβ16+ T cells. Moreover, the cytokine dependence of the SJL B cell lymphomas on IL-5 and IL-4 suggests that preferential stimulation of Th2 cells is involved. This scenario of B cell hyperplasia associated with SAg-induced Th2 cell stimulation may also be a major element in other murine models, such as chronic parent → F1 GvHD and MAIDS.Mechanisms similar to those described in these mouse models may also be operative in human diseases caused by infectious microorganisms, such asH. pylori, EBV, HIV,M. arthritidis, mycobacteria, malarial parasites, and others. However, the involvement of SAgs in these conditions, and their precise role in eliciting from T cells those eytokines which promote chronic B cell hyperplasia and lymphoma development remain to be clearly defined. The possible role ofγδ T cells in similar disease mechanisms will need to be explored in the light of overwhelming evidence that certain unconventional antigens such as HSP stimulate subpopulations of these T cells. In our discussion of these diseases we have, therefore, given consideration to the possibility that the definition of SAg may have to be expanded to include such antigens.

A Comparison of Aggregatibacter actinomycetemcomitans (Aa) Virulence Traits in a Rat Model for Periodontal Disease

Our aim was to explore the effects of Cytolethal Distending toxin (Cdt) in a well established rat model of periodontal disease where leukotoxin (LtxA) was thought to have no known effect. In vitro studies, were used to assess CdtB activity using Aa Leukotoxin as a negative control. These studies showed that both CdtB and LtxA (unexpectedly) exerted significant effects on CD4+ T cells. As a result we decided to compare the effects of these two prominent Aa virulence factors on bone loss using our rat model of Aa-induced periodontitis. In this model, Aa strains, mutant in cdtB and ltxA, were compared to their parent non-mutant strains and evaluated for colonization, antibody response to Aa, bone loss and disease. We found that bone loss/disease caused by the ltxA mutant strain, in which cdtB was expressed, was significantly less (p<0.05) than that due to the wild type strain. On the other hand, the disease caused by cdtB mutant strain, in which ltxA was expressed, was not significantly different from the wild type strain. This data indicates that Aa LtxA exerts a greater effect on bone loss than Cdt in this rat model of periodontal disease and supports the utility of this model to dissect specific virulence factors as they relate to immunopathology in studies of Aa-induced disease.