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The interactions of complement receptor 2 (CR2) and the degradation fragment C3d of complement component C3 mediate the innate and adaptive immune systems. Due to the importance of C3d-CR2 interaction in the design of vaccines, many studies have indicated the interactions are pH-dependent. Moreover, C3d-CR2 interactions at pH 5.0 are unknown. To investigate the molecular mechanism of pH-regulating C3d-CR2 interaction, molecular dynamics simulations for C3d-CR2 complex in different pH are performed. Our results revealed that the protonation of His9 in C3d at pH 6.0 slightly weakens C3d-CR2 association as reducing pH from 7.4 to 6.0, initiated from a key hydrogen bond formed between Gly270 and His9 in C3d at pH 6.0. When reducing pH from 6.0 to 5.0, the protonation of His33 in C3d weakens C3d-SCR1 association by changing the hydrogen-bond network of Asp36, Glu37, and Glu39 in C3d with Arg13 in CR2. In addition, the protonation of His90 significantly enhances C3d-SCR2 association. This is because the enhanced hydrogen-bond interactions of His90 with Glu63 and Ser69 of the linker change the conformations of the linker, Cys112-Asn116 and Pro87-Gly91 regions. This study uncovers the molecular mechanism of the mediation of pH on C3d-CR2 interaction, which is valuable for vaccine design.

Aim To analyze the relationship between ectopic germinal centers (GCs) in the salivary glands and the clinical/laboratory characteristics of patients with Sjogren's syndrome (SS). Methods Retrospectively, 126 patients with primary SS (pSS) and 16 patients with secondary SS (sSS) were analyzed. Minor salivary gland biopsies were evaluated for the presence of GC-like morphology by hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining for CD21. Clinical and serological data were obtained from medical records. Results GC-like structures were observed in 36/126 (28.6%) pSS patients and 4/16 (25.0%) sSS patients. The mean inflammatory focus score of the gland was significantly higher in GC-positive samples than in GC-negative ones in both pSS and sSS patients (P = 0.007 and 0.024, respectively). In pSS, significantly elevated titers of rheumatoid factor (RF)-IgM (P = 0.023) and antinuclear antibodies (ANA) (P = 0.036), increased levels of IgA (P = 0.012) and IgG (P = 0.017) were encountered in GC-positive patients. The GC-positive group also presented higher prevalence of anti-SSA antibodies, lower levels of white blood cells, higher levels of erythrocyte sedimentation rate and γ-globulin, although not statistically significant. In sSS patients with ectopic GC formation, ANA titers were remarkably elevated. The anticyclic citrullinated peptide (anti-CCP)-IgG titers and the prevalence of antikeratin antibody (AKA)-IgG, antiperinuclear factor (APF)-IgG were also increased, yet not significantly. GCs were found to be associated with antibody and immunoglobulin production. Conclusion This study indicates that SS patients with ectopic GCs have distinct features. Ectopic GC structures were particularly noted in patients with higher focus scores, and might play an essential role in sustaining antibody production as well as B cell activation.

B cells represent one of the cellular components of the immune system that protects the individual from invading pathogens. In response to the invader, these cells differentiate into plasma cells and produce large amounts of antibodies that bind to and eliminate the pathogen. A hallmark of autoimmune diseases is the production of autoantibodies i.e. antibodies that recognize self. Those that are considered pathogenic can damage tissues and organs, either by direct binding or when deposited as immune complexes. For decades, B cells have been considered to play a major role in autoimmune diseases by antibody production. However, as pathogenic autoantibodies appear to derive mainly from T cell dependent responses, T cells have been the focus for many years. The successful treatment of patients with autoimmune diseases with either B cell depletion therapy (rituximab) or inhibition of B cell survival (belimumab), suggested that not only the autoantibodies but also other B cell features are important. This has caused a surge of interest in B cells and their biology resulting in the identification of various subsets e.g. regulatory B cells, several memory B cell subsets etc. Also, in other conditions such as chronic viral infections and primary immunodeficiency, several B cell subsets with unique characteristics have been identified. In this review, we will discuss one of these subsets, a subset that is expanded in conditions characterized by chronic immune stimulation. This B cell subset lacks, or expresses low, surface levels of the complement receptor 2 (CD21) and has therefore been termed CD21(-/low) B cells.

Background The recent epidemic of head and neck squamous cell carcinomas associated with human papilloma virus (HPV) has not addressed its association with lymphoid tissue in the oropharynx or the potential role of Epstein–Barr virus (EBV)/HPV coinfection. Methods The prevalence of HPV and EBV infection/coinfection and CD21 mRNA expression were determined in normal and cancerous tissues from the oropharynx using in situ hybridization (ISH), p16, and quantitative reverse transcriptase PCR (qRT-PCR). The effects of coinfection on tumorigenicity were evaluated using proliferation and invasion assays. Results Normal oropharynx, tonsil, non-cancer base of tongue (BOT), and BOT from sleep apnea patients demonstrated EBV positivity ranging from 7% to 36% depending on the site and methods of detection used (qRT-PCR or ISH). Among non-malignant BOT samples, HPV positivity was noted only in 20%. The percent of tonsil and BOT cancers positive for HPV (up to 63% and 80%, respectively) or coinfected with HPV/EBV (up to 25% and 70%, respectively) were both significantly associated with cancer status. Notably, HPV/EBV coinfection was observed only in malignant tissue originating in lymphoid-rich oropharynx sites (tonsil, BOT). CD21 mRNA (the major EBV attachment receptor) was detected in tonsil and BOT epithelium, but not in soft-palate epithelium. Coinfected cell lines showed a significant increase in invasiveness (P

Background: The process of maturation of the immune system leads to generation of various lymphoid cell populations having the ability to react in specific way and expressing various markers on the cell surface. The study was set up to establish reference values for B lymphocyte subpopulations in peripheral blood of children and young adults to find the spectrum of their physiological age-related variation. Methods: Blood samples were taken from 292 children and young adults aged 0–31 years and tested for distribution of B cell subsets. Relative and absolute sizes of non-memory and memory, transitional, naive, immature marginal zone-like/IgM-only memory, class-switched memory, double negative, activated, and plasmacytoid cell populations were determined by four-color flow cytometry, based on differential expression of CD19, IgM, IgD, CD21, CD27, and CD38. Significant variation both in relative, as well as in absolute numbers of individual cell populations in tested groups was observed. Results: The reference values for age-related B cell subsets in eleven age groups, established as result of this study, may be used in diagnostics of any pathology related to B cell maturation process, as well as in attempts of correlating laboratory results with clinical symptoms of many defects affecting antibody production in pediatric population. Conclusion: Determination of B cell subpopulations carried in patients with antibody deficiencies may help to understand the nature of the disease and prevent its complications. © 2010 International Clinical Cytometry Society

Groups of 5-month-old lambs which had been trickle infected with Teladorsagia circumcincta for 8 weeks then drenched, and worm-free control lambs were challenged with 50 000 T. circumcincta L3s. From 10 days later fewer parasites were recovered from the previously infected sheep, and secondary cellular and humoral responses were observed in the gastric lymph. Increases in CD4+ and CD25+ T lymphoblast traffic on day 3, followed by CD21+ and IgA+ lymphoblasts on day 5, and an increase in total and parasite specific IgA concentrations peaking on day 6 were observed in previously infected lambs. Similar peaks in lymphoblast output were not observed until days 10-12 in the control lambs. This data was highly comparable with that obtained recently from yearling sheep subjected to an identical infection-challenge regime, and contrasted with that obtained from similar experiments in the 1980s when 4(1/2)-month-old previously infected lambs were more susceptible to and had much weaker immune responses to challenge than 10-month-old sheep. The fact that 40% fewer larvae were given during the trickle infection regime in the four recent trials is offered as an explanation for this difference.

Epstein-Barr virus (EBV) infection has been postulated to be an early event involved in the pathogenesis of nasopharyngeal carcinomas (NPC). The lack of representative premalignant nasopharyngeal epithelial cell system for EBV infection has hampered research investigation into the regulation and involvement of EBV infection in NPC pathogenesis. We have compared the efficiency of EBV infection in nasopharyngeal epithelial cells with different biological properties including immortalized, primary and cancerous nasopharyngeal epithelial cells. EBV infection could be achieved in all the nasopharyngeal epithelial cells examined with variable infection rate. TGF-beta effectively enhanced EBV infection into nasopharyngeal epithelial cells both in the immortalized and primary nasopharyngeal epithelial cells. Stable infection of EBV was achieved in a telomerase-immortalized nasopharyngeal epithelial cell line, NP460hTert. The expression pattern of EBV-encoded genes and biological properties of this EBV infected cell line on long-term propagation were monitored. The EBV-infected nasopharyngeal epithelial cells acquired anchorage-independent growth and exhibited invasive growth properties on prolonged propagation. A distinguished feature of this EBV-infected nasopharyngeal epithelial cell model was its enhanced ability to survive under growth factor and nutrient starvation. This was evidenced by the suppressed activation of apoptotic markers and sustained activation of pAkt of EBV-infected cells compared to control cells under nutrient starvation. Examination of cytokine profiles of EBV-infected NP460hTert cells to nutrient and growth factor deprivation revealed upregulation of expression of MCP-1 and GRO-alpha. The establishment of a stable EBV infection model of premalignant nasopharyngeal epithelial cells will facilitate research investigation into the pathogenic role of EBV in NPC development.

Receptors for sheep erythrocytes, for the Fc part of IgG (Fc gamma R) and IgM (Fc mu R), and for components of activated human complement C3 (C3bR and C3dR) in thymus tissue from fetuses, infants and children were studied using haemadsorption to cryostat sections in a closed chamber. Sheep erythrocytes (SE) did not adhere to sections of thymus from fetuses at 10-11 weeks of gestation, adhered weakly between 14 and 20 weeks, while they adhered in a dense monolayer in older fetuses, in infants and in children, denser in the cortex than in the medulla. Ox erythrocytes (OE), sensitized with IgG to demonstrate Fc gamma R, adhered focally to both cortical and medullary areas. The adherence was more pronounced to the sections of fetal thymus than to the sections from infants and children. OE, sensitized with IgM to demonstrate Fc mu R, adhered focally to all thymus tissue sections, preferentially to the cortex. The adherence was most pronounced in fetal thymus. SE, sensitized with IgM and coated with complement, adhered to sections of fetal thymus, but the density decreased markedly during fetal life. Indicator cells demonstrating C3bR adhered focally also to sections from infants and children, while indicator cells demonstrating C3dR did not adhere to sections from individuals older than 38 weeks of gestation. Indicator cells demonstrating C3bR and C3dR adhered both to cortex and medulla.

Severely impaired Ab responses are seen in animals lacking C (complement) factors C2, C3 or C4 as well as CR1/2 (C receptors 1 and 2). The molecular mechanism behind this phenomenon is not understood. One possibility is that C-containing immune complexes are endocytosed via CR2 on B cells and presented to specific CD4+ T cells, which would then proliferate and provide efficient help to specific B cells. In vitro, B cells can endocytose immune complexes via CR1/2 and present the Ag to T cells. Whether absence of this Ag presenting function in Cr2(-/-) mice (mice lacking CR1/2) explains their low Ab response is unclear. To address this question, Cr2(-/-) and wild type mice were transferred with OVA-specific T cells, obtained from the DO11.10 strain which has a transgenic TCR recognizing an OVA peptide. The animals were subsequently immunized with sheep red blood cells (SRBC) conjugated to OVA. Interestingly, proliferation of the OVA-specific T cells was normal in Cr2(-/-) mice, although their Ab response to both SRBC and OVA was severely impaired. These observations suggest that the impaired Ab response in Cr2(-/-) mice cannot be explained by a lack of appropriate induction of T cell help.

The influence of complement receptor 1 and 2 (CR1/2) was investigated on the susceptibility to low-dose collagen-induced arthritis (CIA) in wild-type (WT) and CR1/2-deficient DBA/1 mice. Significantly enhanced CIA was observed in female CR1/2-deficient mice compared with WT female mice, while male mutant and WT mice showed similar arthritis development. The enhanced CIA was accompanied with higher complement levels and a prolonged IgM anti-collagen type II response. When investigating whether estrogen contributed to the different arthritis susceptibility, we found that ovariectomy rendered WT females more sensitive to low-dose CIA and to the same extent as CR1/2-deficient females, while CR1/2-deficient mice were unaffected by ovariectomy. Notably, the ovariectomized WT mice displayed reduced CR1(+) B220(+) B-cell numbers and CR1 expression compared with sham-operated WT mice, suggesting a stimulatory effect of estrogen on CR1. In accordance, a significant correlation was observed between reduced CR1 expression in B cells and increased age in healthy female blood donors but not in male donors. Our findings demonstrate an important role of CR1/2 in suppressing CIA in female mice under low-antigen conditions. The data suggest that estrogen promote CR1 expression in B cells. These findings provide insight to the increased frequency of rheumatoid arthritis in postmenopausal women.

Angioimmunoblastic T-cell lymphoma (AILT) is a malignant disease of peripheral T-cell origin that is characterized by a prominent proliferation of high endothelial venules in the lymph node. To investigate angiogenic mechanisms in AILT we measured the angiogenic mediator gene expression levels in the lymph nodes of 54 non-Hodgkin lymphoma patients, by immunostaining and quantitative reverse transcription polymerase chain reaction. Angiogenic mediators angiopoietin (Ang) 1 (ANGPT1), Ang2 (ANGPT2) and their receptor, Tie2 (TEK), vascular endothelial growth factor (VEGF; VEGFA) and its receptor, VEGFR2 (KDR), and hepatocyte growth factor (HGF) and its receptor, c-Met (MET) were all more highly expressed in AILT lymph nodes (16 cases) than in B-cell lymphomas (24 cases). Moreover, significantly higher Ang1 and Tie2 expression was detected in AILT cases with CD10-positive neoplastic T-cells by comparison with unspecified peripheral T-cell lymphoma (14 cases). Immunostaining confirmed the expression of Ang1 and VEGF by both neoplastic T-cells and follicular dendritic cells. These results suggest that the angiopoietin system may play an important role in the development of high vascularity in AILT lymph nodes. Consequently, as neoplastic T-cells and follicular dendritic cells are both increased in AILT and may represent an important source of angiogenic mediators, targeting these cells with anti-angiogenic strategies might represent a novel therapy for AILT.

Antisera against the products of the major histocompatibility system (MHS), produced by immunization between congenic mouse strains differing only at that segment of the 17th chromosome rosettes which bears the H-2gene complex, exert a reproducible blocking effect on formation of with EACm (C3d rosettes). In many instances this effect is probably due to specific antibodies reactive with the lymphocytes, as indicated by immunochemical and adsorption experiments. Purified antibodies from the C3H anti-C3H.B10 immune ascites failed to exert this blocking effect. The blocking capacity can be restored, however, by addition of fresh normal mouse, rabbit or fetal calf serum, but not by heat inactivated serum. These experiments show that the presently defined H-2K, H-2D and H-2L antigens as well as some Ia antigens are themselves not the C3d receptors. The blocking effects observed in the presence of complement are possibly due to the proximity of H-2 or Ia molecules and C3d receptors, or to a rearrangement of membrane components after reaction with anti-MHC anti-MHC antibodies and complement.

Described herein is a case of thymic extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue. Using immunohistochemical double staining it was found that most of the thymic lymphoid follicles in this case possessed cytokeratin-positive and follicular dendritic cell (FDC) marker-positive cells. Moreover, using immunoelectron microscopy it was confirmed that some of the double-positive cells were thymic epithelial cells. The candidate of cytokeratin subtype expressed on the double-positive cells was cytokeratin 1 (CK1), which was expressed only by the epithelium of Hassall's corpuscles in thymuses from age-matched patients with myasthenia gravis. The present case indicates a possibility that some thymic epithelial cells become FDC, although it was uncertain whether they were derived from the epithelia of Hassall's corpuscles or whether they were at the same differentiation stage as Hassall's corpuscles.

The majority of human immunodeficiency virus type 1 (HIV-1) infections occur via mucosal transmission through contact with genital secretions containing cell-associated and cell-free virus. However, few studies have assessed whether exposure to cells, HIV-1 infected or uninfected, plays a role in the sexual transmission of HIV-1. This study examined phenotypic changes in mucosal and systemic lymphoid tissue 24 hr after vaginal exposure to in vitro equilibrated infectious doses of cell-associated or cell-free feline immunodeficiency virus, uninfected heterologous cells, or medium alone. We found that even at this early time-point, mucosal exposure to virus induced substantial alterations in the phenotype and distribution of leucocytes, particularly in the tissues of the mucosal immune system. Second, we found that the type of virus inoculum directly influenced the phenotypic changes seen. Vaginal exposure to cell-free virus tended to induce more generalized phenotypic changes, typically in the peripheral immune system (blood and systemic lymph nodes). In contrast, exposure to cell-associated virus was primarily associated with phenotypic shifts in the mucosal immune system (gut and mucosal/draining lymph nodes). In addition, we found that exposure to uninfected heterologous cells also induced alterations in the mucosal immune system. These data suggest that significant immune changes occur within the first 24 hr of virus exposure, well before substantial replication would be anticipated. As the mucosal immune system, and particularly the gut, is an early and persistent target for lentiviral replication, these findings have substantial implications for HIV-1 pathogenesis and vaccine development.

We studied 44 cases of Hodgkin's disease for the presence of Epstein-Barr virus (EBV) DNA, its localization and the expression of the EBV receptor on the tumour cells. EBV DNA was found in 52% (16/31) of the Hodgkin's lymphomas using the polymerase chain reaction. With a very sensitive non-radioactive DNA in situ hybridization technique in combination with immunohistochemistry for CD 30 or CD 15 antigens, EBV DNA was localized to Reed-Sternberg cells and its mononuclear variants. The relationship between the presence of EBV DNA and the expression of the EBV-receptor CR2 (CD 21) on Reed-Sternberg cells was studied using the same techniques and two different monoclonal anti-CD 21 antibodies. CR2 could be detected on a substantial number of the Reed-Sternberg cells in EBV DNA positive Hodgkin's lymphomas (9/12; 75%), whereas in EBV negative cases positivity with anti-CD 21 was rare (1/13; 8%). The results indicate that CR2 expression on Reed-Sternberg cells and the presence of EBV DNA sequences are frequently associated in Hodgkin's lymphomas.

Distinguishing angioimmunoblastic T-cell lymphoma from peripheral T-cell lymphoma, unspecified, using morphology, immunophenotype and molecular genetics Aims: To identify distinguishing histological, immunophenotypic and molecular genetic features between angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma (PTL). Methods: Nodal T-cell lymphomas examined (n =137), included AITL (n = 89), PTL (n = 22), anaplastic large cell lymphoma (n = 16) and 'AITL/PTL indeterminate' (n = 10) with overlapping features between AITL and PTL, showing morphology typical of AITL but lacking follicular dendritic cell expansion. Immunohistochemistry for CD3, CD20, CD21 and CD10, in situ hybridization for Epstein-Barr virus encoded RNA (EBER) and polymerase chain reaction for T-cell and B-cell clonality analysis were performed. Results: Of the AITLs, 74/89 showed typical morphology, whereas 15/89 showed hyperplastic follicles. AITL and 'AITL/PTL indeterminate' showed a polymorphous infiltrate and prominent vascularity in all cases. In both groups, CD10 was present in the majority and clear cells and EBER positivity were specific (but not universal) features lacking in PTL. Detection of T-cell clonality was significantly higher in AITL (90%) compared with PTLu (59%). Conclusions: Clear cells and EBV infection (when present) are useful distinguishing features and CD10 a sensitive and specific marker of AITL. Hyperplastic follicles are present in a significant minority of AITL. AITL/PTL indeterminate probably falls within the spectrum of AITL rather than PTL.

Follicular dendritic cell (FDC) sarcoma is an exceedingly uncommon tumor of lymph nodes and extranodal tissues. The inflammatory pseudotumor (IPT)-like variant of FDC sarcoma of intraabdominal location is considered a separate entity, with different clinical and pathological features than those of the classic FDC tumor. There have been only 12 cytological reports of FDC sarcomas in the literature. Two of them were metastases to the liver and, like our case, had features of IPT. Fine-needle aspiration biopsy (FNAB) and imprint and scrape cytology from the surgically excised tumor here reported revealed spindle tumor cells with moderate pleomorphism, nuclear grooves, prominent nucleoli, and cytoplasmic processes, admixed with inflammatory cells. To the best of our knowledge, this is the first cytology report of a primary hepatic FDC tumor. The cytological findings permit the recognition of this tumor. However, confirmation by inmunohistochemistry (IHQ) is mandatory for a definitive diagnosis.

Mice lacking C3, C4 or complement receptor 1/2 (Cr) have defective germinal centers (GC). The requirement for C4 implicates complement fixation by immune complexes (IC) via the classical pathway. Yet, transgenic (Tg) mice that lack circulating antibody but still express membrane IgM (mIgM) have normal GC responses. We showed previously that cross-linking mIgM leads to the deposition of C3 on the B cell surface and that disruption of this pathway diminishes GC responses. Here, we investigate the role of Cr in this process by generating mIgM-Tg mice that lack Cr and serum Ig. These mIgM/Cr-/- mice have smaller, transient GC, with incomplete B cell receptor down-regulation and peanut agglutinin up-regulation, compared to mIgM/Crwt counterparts. BM chimera experiments showed that Cr on B cells is required for normal GC responses. These results establish that Cr ligands generated at the B cell surface are sufficient for normal GC responses and function by signaling Cr on B cells. Unexpectedly, chimera experiments also showed a critical role for Cr on follicular dendritic cells (FDC), even in the absence of IC, indicating novel functions for FDC-expressed Cr beyond the capture of C3-coated IC.

Microparticles are small vesicles released from the plasma membrane of various cell types independently of apoptosis or cell death, are transferred between cells, and carry membrane proteins from one cell to another. We have studied the mechanism of uptake of microparticles by monocytes and B cells. The transfer of microparticles to B cells was almost completely dependent on complement. Incubation of microparticles with serum resulted in opsonization of microparticles with the complement cleavage product iC3b. The subsequent transfer to B cells was mediated by the complement receptor CR2. The interaction between iC3b-opsonized microparticles and B cells reduced the activation of B cells as measured by expression of MHC class II, CD86 and CD25. In contrast, transfer of microparticles to monocytes was only partially complement dependent, but involved calcium and annexin V, and was found to change the cytokine profile of monocytes towards a reduced release of the pro-inflammatory cytokines GM-CSF and TNF-alpha and an increased release of the anti-inflammatory cytokine IL-10. These data show that microparticles are taken up by B cells and monocytes by different mechanisms and modulate the activation of monocytes and B cells towards an anti-inflammatory phenotype. Microparticles might be involved in counterbalancing pro-inflammatory signals arising from tissue injury or inflammation.

OBJECTIVE High levels of serum anti-Actinobacillus actinomycetemcomitans immunoglobulin G (IgG) correlate with reduced extent and severity of periodontal disease and the present study was undertaken to begin testing the hypothesis that proinflammatory cytokines are important in the induction of optimal anti-A. actinomycetemcomitans IgG responses. BACKGROUND Studies with pokeweed mitogen indicate that interleukin-1alpha (IL-1alpha) and IL-1beta are necessary for optimal IgG1 and IgG2 production and that prostaglandin E(2) (PGE(2)) and interferon-gamma (IFN-gamma) selectively promote IgG2, which is a major component of the anti-A. actinomycetemcomitans response in vivo. The pokeweed mitogen results suggest that these proinflammatory cytokines would also be necessary for optimal production of IgG specific for A. actinomycetemcomitans. METHODS Peripheral blood mononuclear cells from A. actinomycetemcomitans-seropositive subjects with localized aggressive periodontitis were stimulated with A. actinomycetemcomitans in immune complexes capable of binding follicular dendritic cells that participate in the induction of recall responses in vivo. Cultures were manipulated with anti-IL-1alpha, anti-IL-1beta, anti-IFN-gamma, anti-IL-12, anti-CD21, indomethacin, and PGE(2). Actinobacillus actinomycetemcomitans specific IgG production was monitored by enzyme-linked immunosorbent assay (ELISA). RESULTS Addition of follicular dendritic cells to peripheral blood mononuclear cells cultures resulted in follicular dendritic cell-lymphocyte clusters and increased anti-A. actinomycetemcomitans IgG responses (3-40-fold increases) compared with controls lacking follicular dendritic cells. Anti-IL-1alpha, anti-IL-1beta, anti-IFN-gamma, anti-IL-12, anti-CD21 and indomethacin suppressed anti-A. actinomycetemcomitans IgG production by half or more. PGE(2) restored IgG responses suppressed by indomethacin. CONCLUSIONS The cytokines IL-1alpha, IL-1beta, IFN-gamma, IL-12, and PGE(2) were all necessary for optimal production of human anti-A. actinomycetemcomitans and the need for proinflammatory cytokines including the T helper 1 (Th1) cytokines is consistent with a response with a significant IgG2 component.

OBJECTIVE To determine whether Epstein-Barr virus (EBV) infection is related to renal cell carcinoma (RCC) tissues. MATERIALS AND METHODS We investigated EBV infection and its genotypes in 73 cases of different types of RCC and 18 of non-neoplastic kidney. EBV infection and its genotypes were determined by EBV-encoded RNAs in situ hybridization (EBER-ISH) and polymerase chain reactions for EBV-encoded nuclear antigen 1 (EBNA-1) and EBNA-3C. The immunophenotype and EBV status of the EBV-infected cells were examined by double-labelling of EBER-ISH and/or immunohistochemistry for lymphoid cell markers, EBV proteins, and CD21. RESULTS EBER-ISH signals were detected in five of 73 RCC tissues (6.8%), but in none of 18 non-neoplastic kidneys. Interestingly, EBER-ISH was positive only in five of the 10 sarcomatoid RCCs, and of these, four also showed amplification of EBNA-1. EBV was located exclusively in the tumour-infiltrating B lymphocytes of sarcomatoid RCCs. The genotype of EBV was determined as type 1. A few EBV-infected B cells expressed BZLF1 (an EBV immediate-early gene product) while none expressed EBNA-2 or latent membrane protein 1. This indicates that the B cells are of EBV latency type I, often replicating EBV. EBV infection did not affect the survival rates of patients with sarcomatoid RCC (P = 0.635, Kaplan–Meier analysis, log-rank test). CONCLUSION EBV is present only in tumour-infiltrating B lymphocytes of sarcomatoid RCCs. The present study suggests that sarcomatoid RCC modulates a function of EBV-specific T cells controlling EBV replication, or stimulates differentiation of memory B cells into plasma cells.

Complement receptors type 1 and 2 (CR1 (CD35)/CR2 (CD21)) are known to enhance the adaptive immune response. In mice, CR1/CR2 are expressed on B cells, follicular dendritic cells, and activated granulocytes. Recently, we showed that a subset of CD44high and CD62Llow T cells also expresses CR1 and CR2. We now report that CR1/CR2 are detectable on both CD4+ and CD8+ subsets of T cells. Lipopolysaccharide (LPS) from Gram-negative bacteria causes polyclonal activation of B cells and stimulation of macrophages and other antigen-presenting cells. We further demonstrate that LPS induced marked up-regulation of CD25 and CD69 on T cells from CR1/CR2 sufficient (Cr+/+), but significantly lower up-regulation on T cells from CR1/CR2 deficient (Cr-/-) mice. These findings point to a novel mechanism by which CR1/CR2 modulates the activation of T cells by LPS.

A soluble form of the complement receptor CD21 (sCD21) is shed from the lymphocyte surface. The amount of sCD21 in serum may modulate immunity as sCD21 levels are correlated with several clinical conditions. We report here the serum levels of sCD21 in juvenile arthritis (JA), systemic lupus erythematosus (SLE) and Sjogren's syndrome (SS). Using enzyme-linked immunosorbent assay, we determined sCD21 levels in SLE, SS and JA patients. Mann-Whitney test for nonparametric two-tail P value was performed to obtain statistical significance. Cytometrical analysis of synovial fluid leucocytes of JA patients was done on a FACSsort. While sCD21 levels in SLE and SS are reduced to levels previously found in rheumatoid arthritis (RA), JA sCD21 levels were normal. sCD21 levels did not correlate with clinical parameters and immunophenotype of synovial cells. CD4 T cells in the synovium were almost all of the CD45RO memory type and 13 of 40 patients displayed synovial expansion of gammadeltaT cells. CD21 shedding in JA differs from RA/SS/SLE. JA sCD21 levels in synovial fluid are always lower compared to blood levels of the same patients. Analysis of JA synovial T cells indicates a T-cell driven response.

Allergic diseases are characterized by the presence of eosinophils, which are recruited to the affected tissues by chemoattractants produced by T cells, mast cells and epithelium. Our objective was to evaluate if allergens can directly activate human eosinophils. The capacity of purified allergen extracts to elicit eosinophil chemotaxis, respiratory burst, degranulation and up-regulation of the adhesion molecule complement receptor 3 (CR3) was determined in eosinophils isolated from healthy blood donors. Eosinophils stimulated with an extract from house dust mite (HDM) released the granule protein major basic protein (MBP) and up-regulated the surface expression of CR3. Cat allergen extracts also induced the up-regulation of CR3, but not the release of MBP; instead cat, as well as birch and grass allergens, elicited the release of eosinophil peroxidase (EPO). In addition, grass pollen extract caused the secretion of MBP. None of the allergens stimulated eosinophilic cationic protein release, nor production of free oxygen radicals. Both HDM and birch extracts were chemotactic for eosinophils. These findings establish that common aeroallergens can directly activate eosinophils in vitro. We propose that eosinophil activation in vivo is not exclusively mediated by cytokines and chemokines of the allergic inflammatory reaction, but could partly be the result of direct interaction between allergens and eosinophils.

The C3b-binding receptor, CR1/CD35, supports CR2/CD21-mediated activation of complement by human B lymphocytes, possibly by associating with CR2 to promote or stabilize the binding of hydrolyzed C3 (C3i), the primary component of the AP convertase, C3i-Bb. To evaluate this hypothesis, we examined the uptake kinetics and binding equilibria for C3i dimer interaction with human blood cells in the absence and presence of CR1- and CR2-blocking mAb. C3i displayed dual uptake kinetics to B lymphocytes, comprising of rapid binding to CR1 and slower binding to CR2. The forward rate constants (k(1)) for CR1 and CR2, operating independently, differed ca. 9-fold (k(1)=193+/-9.4 and 22.2+/-6.0 x 10(3) M(-1)s(-1), respectively). Equilibrium binding of C3i to B lymphocytes was also complex, varying in strength by ca. 13-fold over the C3i concentration range examined. The maximum association constant (K(a, max)=109+/-27.2 x 10(7) l/mole) was ca. 9- and 6-fold greater, respectively, than those for CR1 or CR2 acting alone (K(a)=13.2+/-5.3 and 18.5+/-3.5 x 10(7) l/mole). The high avidity of the CR1-CR2 complex for C3i is consistent with its rates of C3i uptake and release being determined by CR1 and CR2, respectively.

We previously demonstrated that CR2 activation on human B lymphocyte surface specifically triggered tyrosine phosphorylation of the 95-kDa nucleolin, this leading to its binding on SH2 domains of p85 sub-unit of PI 3-kinase and to activation of this enzyme. The specificity of CR2 pathway was clearly demonstrated as neither CD19 nor BCR could induce tyrosine phosphorylation of nucleolin in normal B lymphocytes. These data led us to investigate herein additional molecular events, which were triggered by CR2 activation, upstream and downstream to PI 3-kinase activation. Upstream, we demonstrated that pp60src, a tyrosine kinase of the src family, was involved in tyrosine phosphorylation of nucleolin, while syk tyrosine kinase was not. We also demonstrated a direct protein-protein interaction of pp60src with nucleolin in a CR2-dependent and CD19-independent pathway. Downstream, we demonstrated that CR2 activation also triggered Akt and GSK3 enzyme activation, this pathway being under the control of pp60src tyrosine kinase activation. These regulatory functions of activated CR2 were specific as independent of syk tyrosine kinase and of CD19 and BCR activation. Thus, CR2 activation recruits a specific mechanism to activate PI 3-kinase and its subsequent pathways, this mechanism being different to those recruited by CD19 and BCR.

The complement receptor type 2 (CR2) associates with other surface antigens and proteins and its redistribution and/or unmasking occurs through still unknown mechanism(s). The data presented demonstrate that high-density cultured CR2-positive cells undergo apoptosis and that the redistribution and unmasking of Annexin V binding sites occurs in a fashion similar to the redistribution and unmasking of CR2. Therefore, apoptotic and non apoptotic cells from the same lineage may share a similar mechanism for the exposition of neo-surface markers

The human complement receptor type 2 (CR2/CD21), a transmembrane glycoprotein, associates with a variety of surface antigens and proteins in the cell membrane. We examined the possibilities that the CR2 units of CR2 complexes are associated through internal covalent links reactive with nucleophilic agents, e.g. H(2)O or methylamine, and that CR2-positive cells process anti-CR2 monoclonal antibodies (MoAbs). Data from immunoblotting and cytofluorimetry with CR2-binding site-specific MoAbs show that: (i) CR2-positive Raji cells release soluble CR2 isoforms into the medium when incubated in phosphate buffered saline; (ii) despite affecting the detection of one soluble CR2 isoform, methylamine treatment of soluble CR2 allows the detection of another of its isoforms; (iii) limited pre-treatment of cells with methylamine reveals a more heterogeneous CR2-positive cell population or enhances the detection of CR2; (iv) cell treatment with CR2-binding site-specific MoAbs enhances the detection of CR2 isoform(s). The data suggest that CR2 is shed mainly as a soluble CR2 complex, in which the CR2 units link covalently and react with nucleophilic agents. Raji cells may process bound fragments (145 kDa) that are recognised by and become bound by anti-CR2 MoAb.

Follicular dendritic cells (FDCs) in reactive germinal centres (GCs) show modulated expression of antigens indicative of step-wise maturation from more undifferentiated stroma. The present study compared the expression of FDC markers CD21, CD23, CD35, and chemokine CXCL13 and the stromal markers low-affinity nerve growth factor receptor (LNGFR) and CNA.42 in 35 follicular lymphoma (FL) cases with reactive lymphoic tissue. CXCL13 was expressed by follicular stroma in all FLs but most cases showed either partial (11/35 cases, 31%) or complete (10/35 cases, 29%) absence of other FDC antigens, most commonly CD23, followed by CD21 and CD35, with variable patterns of LNGFR and CNA.42 immunostaining. Only a minority of FL cases (14/35, 40%) showed stroma that resembled mature FDCs (CD23+, CD21+, CD35+) and these tumours were always associated with numerous intrafollicular T-cells, similar to reactive GCs. In the 25 FL cases that had identifiable extrafollicular tumour cells, the immunophenotype of follicular stroma showed the same variability but the extrafollicular stroma showed an absence of FDC markers, with the exception of frequent strong LNGFR staining. Stromal phenotypic changes in FL were not correlated with mean follicle size, percentage of diffuse growth, tumour mitotic rate or the proliferation index as determined by PCNA immunostaining. Serial biopsy specimens analysed in a subset of 15 patients showed either a stable stromal phenotype (seven cases, 47%) or loss of FDC antigens in tumour-associated stroma over time (seven cases, 47%). The GC-like subset of FLs, having both abundant intrafollicular T-cells and fully differentiated CD23+ FDCs, comprises a minority of FL cases that likely have different growth requirements from FLs that lack these features. The pattern of FDC antigen loss in stroma of FL is a readily assessable biological feature that appears independent of architectural growth pattern and may serve as a useful surrogate marker of tumour progression.

Epitope density and organization have been shown to be important factors for B cell activation in many animal model systems. However, it has been difficult to separate the role of antigen organization from the role of local antigen concentrations because highly organized antigens are usually particulate whereas non-organized antigens are more soluble. Hence, highly organized and non-organized antigens may interact with different cell types and in different locations within lymphoid organs. In order to assess the role of antigen organization in regulating B cell responses, we immunized mice with highly repetitive virus-like particles, which exhibit different epitope densities covalently attached to them. Therefore, the same particulate structure was used to present identical epitopes that differed in their degree of organization. Induction of epitope-specific IgM titers, reflecting early B cell activation, were unaffected by the degree of epitope density. Furthermore, the absence of Th cells or CD21/CD35 did not reduce the IgM response. In contrast, the degree of organization was a critical factor influencing the magnitude of the epitope-specific IgG response. Moreover, the threshold for IgG responses was shifted in the absence of CD21/CD35, resulting in the requirement for higher epitope densities to allow efficient IgG responses. Thus, IgG but not IgM responses are regulated by epitope density and B cell costimulatory thresholds.

Use of the pig as an animal model in schistosomiasis research is increasing, but knowledge of the porcine immune response to schistosome infection is still very limited. We investigated the immunohistology of different maturation stages of the Schistosoma japonicum egg granuloma in pigs. Liver sections from pigs experimentally infected with S. japonicum for 9, 12 or 21 weeks were examined by immunohistochemistry using a three-step streptavidin-biotin-complexlimmunoperoxidase method or a two-step alkaline phosphatase-mediated system. All granulomas showed marked expression of major histocompatibility complex (MHC) class II in epithelioid macrophages and were dominated by T lymphocytes, comprising both CD4 + and CD8 + phenotypes, with consistently higher proportions noted for CD8 + cells. B lymphocytes, as identified by expression of CD21, were confined to lymphoid nodular structures primarily associated with mature granulomas. Early and mature granulomas contained numerous immunoglobulin (Ig) G + plasma cells. Significant differences in immunohistology related to duration of infection were not observed. The results indicate that all stages of the hepatic S. japonicum egg granuloma in the pig manifests MHC class II-dependent CD4 + T cell activity concomitant with infiltration of CD8 + T cells. B cell activity preceding the effector cell stage appears to occur in granuloma-associated lymphoid nodules, whereas antibody, mainly IgG, is produced within the granuloma.

Objectives/Hypothesis The aim of this study was to analyze the relevance of the CD21 membrane receptor in nasopharyngeal carcinoma (NPC). CD21 is implicated in the introduction of the Epstein-Barr virus (EBV) genome into epithelial cells and B lymphocytes. Study Design Immunohistochemical analysis of CD21 in NPC. Methods Paraffin-embedded samples of NPC of different histological types with demonstrated presence of EBV were analyzed for CD21 expression using immunohistochemistry. Results We detected EBV by non-isotopic in situ hybridization (NISH) and nested polymerase chain reaction (PCR) in 100% of samples, regardless of histological type, supporting the previous view that all the types of NPC are variants of an EBV-associated malignancy. CD21 was not detected in NPC, and this absence was a typical feature in our data group. Conclusions The loss of the CD21 membrane receptor could be one of the immunophenotypical changes of the neoplastic cells that occur in the evolution of the NPC malignancy.

A specialized subpopulation of T lymphocytes is reactive to the MHC class I-like molecule CD1d. It is not clear which cells are the major antigen-presenting cells in vivo in the activation of CD1-restricted immune responses. We have characterized a subset of B lymphocytes expressing six- to eightfold higher levels of CD1 than the bulk of B cells. The cells have a surface phenotype (CD21(high), CD23(low), IgM(high), IgD(low)) found previously to characterize B cells residing in the splenic marginal zones. CD1(high) B cells localize preferentially to the spleen, and appear late in ontogeny, at 3 - 4 weeks of age. The CD1(high) B cells were present in mice lacking conventional helper T cells, ruling out an exclusive origin from T cell-dependent immune responses. Still, some CD1(high) B cells had been involved in T cell-dependent immune responses as suggested by mutations in their rearranged immunoglobulin gene regions. The population could still be found in mice with severely reduced B cell reactivity to bacterial lipopplysaccharides (C3H / HeJ mice) and in mice unable to respond to thymus-independent type 2 antigens (NFR.Xid mice), as well as in germ-free mice, indicating that bacterial antigens are not major stimuli for the induction of CD1(high) B cells. In contrast, the CD1(high) B cell population was severely reduced in CD19-deficient mice. Taken together, the results imply that the CD1(high) population is heterogenous and of mixed origin, dependent for its development or maintenance on signaling through the CD19 molecule.

We investigated the interaction of HIV immune complexes (HIV IC) with mononuclear cells from lymph nodes and blood. While antibody alone did not affect binding of HIV IC to mononuclear cells, antibody plus complement increased binding by as much as 10-fold and complement alone also increased binding slightly. Most of the increased binding of HIV IC to mononuclear cells was blocked by heat-inactivation of complement and by OKB7 monoclonal antibody, indicating that virus binding was to CR2 on B cells. A similar pattern of antibody and complement dependence for binding of HIV IC was observed with two model systems; Raji and Arent B-cell lines. Most of the HIV IC that bound to lymph node cells were not internalized, but remained on the cell surface and were gradually released. However, even after 48 hr some HIV IC could be detected bound to cells. Under certain conditions, HIV IC were infectious for T cells if bound to B cells but not infectious if added directly to T cells. Additionally, HIV IC bound to B cells led to higher virus replication. These studies show that B lymphocytes from blood and lymph nodes can transfer infectious HIV IC to T cells.

The aim of the present study was to determine the effect of repeated tonsillitis on the development of lymphocyte subsets in the tonsils and among peripheral blood lymphocytes (PBL) of children. Subsets of T- and B cells were analyzed in the tonsils and in PBL of patients undergoing tonsillectomy for idiopathic tonsillar hypertrophy, recurrent tonsillitis, or tonsillar hypertrophy and tonsillitis. The majority of the CD4+ cells in the tonsils displayed the CD45RO+ phenotype, while the majority of those in the PBL displayed the CD45RA+ phenotype. Likewise, the proportion of CD45RO+CD8+ cells was higher in the tonsils than among PBL. The proportion of CD4 cells expressing the CD45RO marker increased with age among PBL, but not in the tonsils. B cells, detected by their CD19, CD20, and CD21 markers, were three times more abundant in the tonsils than in the PBL. The proportion of CD38+ cells showed a negative correlation with age, both in the tonsils and among PBL. Among PBL a striking age-related reduction was seen in the proportion of CD19+, CD21+ and CD38+CD21+ B cells. In contrast, in the tonsils age-related changes could be detected only in the proportion of CD21+CD38+ cells. No difference among patients with various clinical diagnoses was detectable in any of the T- and B cell subsets in the tonsils and PBL. Thus, lymphocyte subsets evolve independently in the tonsils and peripheral blood, with the repeated antigenic challenge of tonsillar lymphocytes not influencing circulating memory cells.

Follicular dendritic cell (FDC) sarcoma is an exceedingly rare neoplasm of unknown pathogenesis. A case of FDC sarcoma complicated by the hyaline-vascular type Castleman's disease occurring in a schizophrenic male is presented. Swelling of the left cervical lymph node appeared in a 44-year-old male schizophrenic who had been medicated with major tranquilizers for 20 years. He had had a history of cervical lymphadenopathy 14 years before, for which a diagnosis of hyaline-vascular type Castleman's disease had been made. The present specimen, obtained from the same site, was an enlarged lymph node heavily infiltrated with oval to spindle-shaped atypical cells but was uninvolved at the periphery. The infiltrating cells showed nodular or sheet-like growth, occasionally taking fascicular or storiform patterns. Follicular involvement was also common. Peculiarly, various amounts of small lymphocytes were intermingled with the neoplastic cells. The atypical cells expressed two FDC-specific antigens, DRC-1 and Ki-M4 antigen, together with a few other markers that are shared by FDC, including CD21 and HLA-DR. These findings clearly show the tumor to be FDC sarcoma. In addition, a peculiar fibro-hyalinous change in the lymph follicle, which is compatible with hyaline-vascular type Castleman's disease, was noted at the periphery of the lymph node where neoplastic cells had not infiltrated. Surprisingly, similar hyaline-vascular changes were observed in the previous biopsy taken 14 years ago. Meanwhile, Kaposi's sarcoma-associated herpesvirus, which is often identified from generalized Castleman's disease, was not identified in the present case by polymerase chain reaction study. Thus, this case is unique in two aspects: (i) the overlap of FDC sarcoma with hyaline-vascular type follicular changes; and (ii) its occurrence in a schizophrenic patient.

Determination of lactation stage-dependent changes in levels of lymphocyte subpopulations in milk.Flow cytometric assay was used to identify and assay lymphocyte subpopulations in bovine milk at different stages of lactation.Lymphocyte subpopulations in mammary secretions of dairy cows change during the lactation cycle. In involuting glands (dry gland), approximately 80-90% of lymphocytes were CD2+ T cells. The proportion of CD2+ T cells, however, decreased to approximately 50% at the colostral stage an fluctuated between 50 to 60% in normal (mature) milk. Throughout the lactation stages, less than 5% were B cells as identified by the monoclonal antibodies against CD21 and MHC class II antigens. Subset analysis showed, however, that the proportion of CD5+ T cells decreased from 90% in involuting gland secretions to 75% in colostrum (peripartum stage), and to approximately 40-50% in the normal (mature) milk, CD4+ T cells constituted between 45 to 55% of lymphocytes in the dry gland secretion but decreased drastically at parturition and maintained at the level below 20% throughout normal lactation. In contrast, the proportion of CD8+ T cells in the dry gland secretion was low, between 30 to 40%, but increased steadily, in an inversely-related manner with that of CD4+ T cells, to approximately 40-50% at parturition and maintained at approximately 30-40% during the normal lactation stage thereafter. Two-color immunofluorescence study revealed further that practically all of the CD8+ cells in dry gland secretions were CD2+, and approximately 40% of them were CD5-. Throughout the lactation cycle, WC1+ gamma delta T cells comprised only 2 to 5% of lymphocytes in mammary secretions.T lymphocyte subpopulations change dynamically during stages of the lactation cycle. The selective migration of T lymphocyte subpopulations to and from the mammary gland, and their functional roles in the immune competence and regulation of the dam and sucklings remain to be elucidated.

Der p I, a cysteine protease representing a major allergen of the house dust mite Dermatophagoides pteronyssinus, has recently been shown to cleave CD23 from the surface of cultured human B cells (RPMI 8866 B cell line). We have now undertaken a detailed investigation of CD23 cleavage by Der p I. We demonstrate that Der p I cleaves CD23 at two sites (Ser155-Ser156 and Glu298-Ser299) to produce a 17-kDa fragment containing the lectin domain and only part of the C-terminal tail. No such effect was demonstrable with mouse CD23, a finding which was anticipated based on its lack of the cleavage sites identified on human CD23. Based on the cleavage pattern and the model of CD23, we propose a sequence of events leading to the liberation of the 17-kDa soluble CD23 fragment. The biological significance of such cleavage is underlined by the demonstration that Der p I-treated B lymphocytes lose their ability to bind IgE, and that the 17-kDa fragment (amino acids 156-298) contains the minimum structural requirement (amino acids 156-288) for binding to both IgE and CD21.

CD21 (complement receptor 2, CR2) binds the C3 degradation products, iC3b and C3d, which are covalently linked to antigen or immune complexes in the process of complement activation. The ability of antigen-nonspecific B cells to present immune complexes containing high titers of acquired antibodies was tested. Influenza virus was incubated with serum from immune donors to create complement-containing complexes. These bound specifically to CD21 on transfected fibroblasts and B cell lines, as measured by microcytofluorimetry. Binding of immune complexes was ablated by inactivation of serum complement. In addition, the immunoglobulin in immune human serum blocked influenza binding to cells in the absence of complement, implying a minimal role for immunoglobulin-Fc receptor interactions in this system. Significant immune complex binding required a threshold level of CD21 expression, suggesting that only those cells with the highest levels of CD21 are likely to participate in the processing of macromolecular antigens. B cells pulsed with complement-influenza complexes elicited an augmented response from a panel of influenza-specific, class II-restricted T cell clones, as compared with those which had bound immunoglobulin-influenza complexes lacking complement. This enhanced response did not require CD35. In addition, B cell lines expressing higher levels of CD21 were more efficient in processing antigen than those with lower levels. These findings suggest that presentation of antigen by B cells in immune individuals is dependent on the binding of complement-antigen immune complexes to CD21.

Stimulation in vitro of murine splenic B cells by lipopolysaccharide, anti-kappa Sepharose, anti-CD40 or allo-reactive T helper cells all up-regulated CD21 and CD23 surface expression. Neither anti-CD21 nor anti-CD23 antibodies induced B cell growth or differentiation when added in soluble form or coupled to Sepharose. However, anti-CD40-stimulated B cells showed increased proliferation in the presence of anti-CD21 antibodies coupled to Sepharose; co-stimulation via CD21 also induced differentiation to immunoglobulin secretion in a fraction of anti-CD40-stimulated B cells. Furthermore, anti-CD40 antibodies inhibited differentiation to immunoglobulin secretion induced by lipopolysaccharide and, hence, appears to be a dominant negative signal for B cell differentiation.

We report on a soluble (s) form of CD21 (the C3dg/Epstein-Barr virus receptor, CR2) that is spontaneously released by B and T lymphocytes. Immunoprecipitation with anti-CD21 mAb of culture supernatants of surface and biosynthetically labeled B and T cell lines revealed a single band with an apparent molecular mass of 135 kDa. The molecule exhibited a molecular mass 10 kDa lower than that of membrane CD21. The release of soluble CD21 (sCD21) was time dependent and correlated with a parallel decrease in the expression of the membrane-associated molecule. The protein was also found in culture supernatants of tonsillar B cells and normal human thymocytes. Epitopic analysis using combinations of anti-CD21 monoclonal antibodies (mAb) indicated that sCD21 and membrane CD21 were similarly recognized by mAb directed against short consensus repeats (SCR) 1-2, SCR 4-5 and SCR 9-11. Affinity-purified sCD21 was capable of binding to purified human iC3b and to human recombinant CD23, as assessed by enzyme-linked immunosorbent assay and by using the BIAcore technology. In addition, normal human serum was found to contain a soluble form of CD21 that exhibited a similar molecular mass to that of the molecule shed by B and T cells in culture. The serum form of CD21 was recognized by all anti-CD21 mAb that we tested and showed a high reactivity with mAb directed against SCR 1-2. Our observations suggest that B and T cells shed the extracellular portion of CD21 and release a soluble molecule that retains the ligand-binding properties of CD21, thus having a potential role in immunoregulation.

We studied the physiological role of complement receptor type II (CR2, C3d/EBV receptor) expressed on T cells using MT-2 cells. First, we confirmed CR2 expression on MT-2 cells by flow cytometry and found that the MW of CR2 molecules on these cells and Raji B cells were the same by SDS-PAGE analysis. When MT-2 lysates were incubated with anti-CR2 mAb HB5 and thereafter with 32P-labeled ATP, 52- and 74-kDa proteins were phosphorylated, suggesting the activation of MT-2 cells through the complex of CR2 with these proteins. In this respect, we measured lymphotoxin production by MT-2 cells when incubated with C3d or EBV. The cytotoxicity of the MT-2 supernatant against L929 cells was elevated in a dose- and time-dependent manner. Next, we confirmed EBNA expression on EBV-infected MT-2 cells and attempted to establish an EBV-positive MT-2 clone by in vitro EBV infection. However, these clones disappeared during cloning. To clarify this mechanism, we examined the EBV genome in MT-2 cells. By Southern blot analysis, BamHI digestion of DNA extracts from MT-2 cells 3 days after EBV treatment gave a 3.0-kb signal which comigrated with the EBV BamHI-W probe. The 3.0-kb signal of genomic EBV-DNA was detected at 1, 2, 3, 5, and 7 days after EBV treatment, but could not be detected at 14 days. Thus, natural ligands of CR2 stimulate CR2-positive MT-2 cells through their functionally active CR2 molecules and in vitro EBV infection of MT-2 cells might be transient.

Previous studies have documented a role for membrane-bound CD23 (the low affinity Fc epsilon RII) in presentation of alloantigens by B cells. The aim of the present study was to examine the involvement of cell surface CD23 in presentation of more conventional soluble protein antigens to T cells. We show that antibodies to CD23 and to its lymphocyte-associated second ligand, CD21, inhibit presentation of the cow's milk allergen casein, by autologous CD23+CD21+ B-EBV cell lines to casein-specific HLA-DP-restricted CD4+ T cell clones obtained from patients with either reaginic or enterophatic forms of cow's milk protein intolerance. Maximal inhibition was achieved when the antibodies were added at the initiation of the culture. The absence of specific inhibition by an anti-DR alpha monoclonal antibody (mAb) argues against a steric hindrance phenomenon impeding access of the T cell receptor to major histocompatibility complex class II molecules. Rather, anti-CD23 and anti-CD21 mAb-induced inhibition of antigen presentation seems to affect at least partly, heterotypic conjugate formation through CD23/CD21 interaction. Double immunofluorescence labeling of the T cell clones and antibody inhibition of T/B conjugate formation shows that functional CD23 and CD21 molecules are induced on T cells following contact with B-EBV cell lines. Taken together, these data indicate that CD23/CD21 interactions between T and B cells are required for presentation of soluble protein antigens by B-EBV cell lines to specific CD4+ T cells. The potential implications of these findings for allergen-specific T cell activation are discussed.Previous studies have documented a role for membrane-bound CD23 (the low affinity Fc epsilon RII) in presentation of alloantigens by B cells. The aim of the present study was to examine the involvement of cell surface CD23 in presentation of more conventional soluble protein antigens to T cells. We show that antibodies to CD23 and to its lymphocyte-associated second ligand, CD21, inhibit presentation of the cow's milk allergen casein, by autologous CD23+CD21+ B-EBV cell lines to casein-specific HLA-DP-restricted CD4+ T cell clones obtained from patients with either reaginic or enterophatic forms of cow's milk protein intolerance. Maximal inhibition was achieved when the antibodies were added at the initiation of the culture. The absence of specific inhibition by an anti-DR alpha monoclonal antibody (mAb) argues against a steric hindrance phenomenon impeding access of the T cell receptor to major histocompatibility complex class II molecules. Rather, anti-CD23 and anti-CD21 mAb-induced inhibition of antigen presentation seems to affect at least partly, heterotypic conjugate formation through CD23/CD21 interaction. Double immunofluorescence labeling of the T cell clones and antibody inhibition of T/B conjugate formation shows that functional CD23 and CD21 molecules are induced on T cells following contact with B-EBV cell lines. Taken together, these data indicate that CD23/CD21 interactions between T and B cells are required for presentation of soluble protein antigens by B-EBV cell lines to specific CD4+ T cells. The potential implications of these findings for allergen-specific T cell activation are discussed.

The present study demonstrates that the C3b receptor CR1 (CD35) and the C3dg/Epstein-Barr virus receptor CR2 (CD21) are expressed by 25% and 70% of normal human thymocytes, respectively. The expression of CR2 extends to both CD1+ and CD1- cells in the thymus. Two subsets of CR2+ thymocytes were defined expressing low and high density of the receptor. The CR2++ subset represented 20% of CR2+ thymocytes and co-expressed the CR1 receptor. CR2++ thymocytes expressed an immature CD1dull, CD3-, CD4dull, CD8-, CD7++ phenotype and included a subpopulation of large cells expressing CD34. Twenty percent of thymocytes expressed the CD21 epitope defined by monoclonal antibody BU32, which is involved in the binding of CD23 to CD21. These observations provide a basis for a role for CD21 in the proliferation and differentiation of thymocytes at early stages of maturation. The functionality of CR1 and CR2 on thymocytes was evidenced by the ability of the receptors to mediate infection of cells with complement-opsonized human immunodeficiency virus (HIV). The results may be relevant to the immunopathogenesis of HIV infection.

Usual methods of chemical fixation preclude examination of cells with most monoclonal antibodies due to alteration or destruction of the surface antigen itself. A method of chemical stabilization and preservation of human B-cell-associated surface antigens is described which facilitates retrospective flow cytometric analysis. This method involves pretreatment of the cells with protease enzyme inhibitors, followed by chemical crosslinking of surface proteins with 2% formalin, and finally blockade of non-specific reactive groups with excess glycine. Once prepared, the expression of pertinent cellular antigens is stable on the cell surface for as long as 4 years. Such methodology could conceivably be used for preparation of cells for longitudinal quality control of monoclonal antibodies or archival storage of patient specimens for retrospective flow cytometric analysis. © 1994 Wiley-Liss, Inc.

Background. Clinical and experimental studies have suggested that complement activation may play a role in tumor cytotoxicity. Little information is available concerning the presence of complement activation and the localization of complement-regulatory factors in cells or tissues of malignant tumors. The aim of the present study was to examine, using immunohistochemistry and immunoelectron microscopy, whether the complement system is activated in tissues of thyroid carcinoma and whether thyroid carcinoma cells are protected from cell lysis by in situ complement activation. Methods. Fresh tissues were obtained by thyroidectomy from 15 patients with papillary carcinomas, 7 with follicular carcinomas, and 5 with follicular adenomas. In addition, five specimens of histologically normal thyroid tissue and five specimens of chronically inflamed tissue adjacent to thyroid neoplasms were studied. Immunohis-tochemical and immunoelectron microscopic localization of complement components, C3d and C5b-9, and the complement-regulatory factors, such as s-protein, decay-accelerating factor (CD55), membrane cofactor protein (CD46), complement receptor types 1 (CD35) and 2 (CD21), and protectin (CD59), were examined in these tissues. Results. The staining patterns of C3d, C5b-9, and s-protein were positive and homogeneous in the nonneo-plastic and most neoplastic thyroid tissues. Immunoelectron microscopy showed these antigens were localized mainly on the subepithelial and vascular basement membranes and attached to the cell surface of thyroid follicular cells. Decay-accelerating factor (CD55) was present homogeneously on the basement membranes, on the basal cell border of the thyroid follicular cells, and often on the luminal surface of carcinoma cells. Both membrane cofactor protein (CD46) and protectin (CD59) were expressed strongly on the cell surface of almost all benign and malignant thyroid follicular cells. Membrane cofactor protein was expressed on both the basal and lateral membrane, showing cell-to-cell interaction, but rarely on the luminal surface, whereas protectin was expressed strongly on the luminal surface and often on the basal cell border but rarely on the lateral membrane. Neither complement receptor type 1 (CD35) nor complement receptor type 2 (CD21) was expressed on any thyroid follicular cells. Conclusions. The present study confirmed the presence of complement activation with subsequent deposition of C3d and C5b-9 complexes in thyroid carcinomas. It also indicated that thyroid carcinoma cells are protected from cell lysis because of complement activation in multiple phases by complete coverage of the entire cell membrane surface with complement-regulatory factors. These findings were similar to those found in nonneo-plastic thyroid follicular cells. Cancer 1994; 73:2808–17.

The expression of CD21 antigen, a receptor for the C3d fragment of complement and Epstein-Barr vlrus (EBV), was Investigated in a total of 85 cases of neoplastic lymphoid cells Including 39 cases of T-lineage acute lymphoblastic leukemia (ALL)/lymphoblastic lymphoma (LBL), although CD21 antigen is usually regarded as a pan-B antigen. The CD21 antigen was expressed by one of the eight cases of neoplastic lymphoid cells expressing the CD7 antigen as a sole pan-T antigen, by three of the 20 cases of pro- or early thymic stage (CD7+ CD5+ CD2-, CD7+ CD5- CD2+, or CD7+ CD5+ CD2+), and ten of 11 cases of thymic stage (CD3r CD4+ CD8+), but not by one case of late thymic stage (CD3 ± CD4+ CD8-) T-ALULBL cells. The CD21 antigen was not expressed by any of the 11 cases of adult T-cell leukemia (ATL) or two cases of chronic T-lineage leukemia. At most 4% of the normal thymocytes obtained from seven infants or children expressed the CD21 antlgen. While only a very limited population of normal thymocytes expresses CD21 antigen, T-ALL/LBL cells at the thymic stage characteristically express CD21 antigen in contrast to pro- or early thymic ALL/LBL or peripheral-stage neoplastic T cells. The estimation of the expression of CD21 antigen is useful for delineating stages of differentiation in T-ALL/LBL. Furthermore, these observation are notable, considering the possibiilty that the reported EBV-carrying T-cell lymphomas result from the penetration of EBV into EBV-negative neoplastic T cells. © 1994 Wiley-Liss, Inc.

Murine macrophages have been described as possessing complement receptors for C3b and iC3b. These binding activities have been assumed to be due to the presence of the CR1 and CR3 proteins, respectively. The mouse Cr2 gene produces two distinct gene products of approximately 190,000 relative molecular mass (M(r)) (Cr2-190) and 145,000 M(r) (Cr2-145). Because of the similarity in size to human complement receptors, the Cr2-190 protein has been dubbed murine CR1 while the murine Cr2-145 product has been termed murine CR2. In order to define the complement receptor genes expressed by murine macrophages, we investigated the expression patterns of Cr2-190, Cr2-145 and another mouse complement receptor, Crry, in three different mouse macrophage populations: bone marrow-derived macrophages, thioglycollate-elicited peritoneal macrophages and the macrophage cell line, J774. Neither of the Cr2 gene transcripts encoding the Cr2-145 and Cr2-190 proteins could be detected in these populations by RT-RPCR analysis although Crry transcripts were evident. Cr2-145 and Cr2s-190 proteins could not be detected on the surface of thioglycollate-elicited macrophages using a monoclonal antibody that recognizes both proteins. Thus, contrary to previously published data, murine macrophages do not possess the Cr2 gene products.