Search

Background: Chronic lymphoid leukemia (CLL) is a hematological disorder characterized by the clonal expansion of small mature lymphocytes that accumulate in the blood and bone marrow. CLL can arise from B-, T-, or natural killer cell clones. The cytological evaluation of blood smears is often the simplest and least invasive method for diagnosing lymphoid leukemia. Immunophenotyping is used to further subclassify the type of lymphoid leukemia., Case Presentation: A 15-year-old, 4.4-kg spayed female Shih Tzu was presented to the veterinary medical teaching hospital of Kangwon National University. Despite having a normal appetite and activity level, cervical and inguinal lymph node enlargement was noted on physical examination. Complete blood count revealed severe leukocytosis, severe lymphocytosis, and monocytosis. Splenomegaly, hepatomegaly, and lymph node enlargement were detected on radiographic and ultrasonographic examination. Immunophenotyping was performed using peripheral blood mononuclear cells (PBMCs). The majority of lymphocytes exhibited the following profiles: CD3 - CD79a - (97.5%), CD4 - CD8 - (98.6%), CD21 - CD79a - (98.4%), CD34 - (0.1%), CD45 + (99.6%), major histocompatibility complex class II + (99.5%), and CD14 - (0.5%). Based on the immunophenotyping results, possible differentials considered included the following: the majority of lymphocytes may be natural killer (NK) cell clones, plasma cell clones, or show aberrant expression or loss of CD21 marker due to the neoplastic nature of the cells. Further flow cytometry was performed using antibodies against CD3, CD5, CD94, and granzyme B. The combined results indicated that the predominant lymphocyte subset in the PBMCs was CD3 - CD5 - CD21 - CD94 - granzyme B - . To confirm monoclonality and exclude the aberrant loss of CD markers, a polymerase chain reaction for antigen receptor rearrangement (PARR) assay was conducted. The PARR assay, using DNA from blood and lymph node samples, showed B-cell monoclonality. Immunocytochemistry using PBMCs showed that the plasma cell marker Multiple Myeloma Oncogene 1 (MUM1) was not expressed. Therefore, the diagnosis was confirmed to be B-cell CLL., Conclusion: Immunophenotyping can help subclassify the type of lymphoid leukemia; however, as tumor cells can show aberrant expression or loss of the CD21 marker, combining immunophenotyping with the PARR assay could yield a more accurate diagnosis., (© 2024. The Author(s).)

Effective inhibition of the complement system is needed to prevent the accelerated clearance of nanomaterials by complement cascade and inflammatory responses. Here we show that a fusion construct consisting of human complement receptor 2 (CR2) (which recognizes nanosurface-deposited complement 3 (C3)) and complement receptor 1 (CR1) (which blocks C3 convertases) inhibits complement activation with picomolar to low nanomolar efficacy on many types of nanomaterial. We demonstrate that only a small percentage of nanoparticles are randomly opsonized with C3 both in vitro and in vivo, and CR2-CR1 immediately homes in on this subpopulation. Despite rapid in vivo clearance, the co-injection of CR2-CR1 in rats, or its mouse orthologue CR2-Crry in mice, with superparamagnetic iron oxide nanoparticles nearly completely blocks complement opsonization and unwanted granulocyte/monocyte uptake. Furthermore, the inhibitor completely prevents lethargy caused by bolus-injected nanoparticles, without inducing long-lasting complement suppression. These findings suggest the potential of the targeted complement regulators for clinical evaluation., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The pathological outcome of dengue disease results from complex interactions between dengue virus (DENV) and host genetics and immune response. Complement receptor types 1 and 2 (CR1 and CR2) mediate complement activation through the alternative pathway. This study investigated the possible association of genetic polymorphisms and plasma levels of CR1 and CR2 with dengue disease. A total of 267 dengue patients and 133 healthy controls were recruited for this study. CR1 and CR2 gene polymorphisms were analyzed by Sanger sequencing, while plasma CR1 and CR2 levels were measured by ELISA. The frequency of the CR1 minor allele rs6691117G was lower in dengue patients and those with severe dengue compared to healthy controls. Plasma CR1 and CR2 levels were decreased in dengue patients compared to healthy controls (P < 0.0001) and were associated with platelet counts. CR1 levels were lower in dengue patients with warning signs (DWS) compared to those without DWS, while CR2 levels were decreased according to the severity of the disease and after 5 days (T1) and 8 days (T2) of follow-up. CR2 levels were decreased in dengue patients positive for anti-DENV IgG and IgM and patients with bleeding and could discriminate DWS and SD from dengue fever patients (AUC = 0.66). In conclusion, this study revealed a reduction in CR2 levels in dengue patients and that the CR1 SNP rs6691117A/G is associated with the dengue severity. The correlation of CR2 levels with platelet counts suggests that CR2 could be an additional biomarker for the prognosis of severe dengue disease., (© 2023. Springer Nature Limited.)

Autoreactive B cell subsets have been described in a variety of settings, using multiple classification schemes and cell surface markers also found on healthy cells. CD19 + CD21 lo B cells have been identified as an autoreactive-prone subset of B cells, although the downregulation of CD21 has been observed on a variety of B cell subsets in health and disease. This variation has led to confusion regarding the meaning and applicability of the loss or reduction of CD21 in peripheral B cells. To better understand the relationships between commonly used B cell markers and their associated characteristics, we analyzed human B cells from healthy participants using multiparameter flow cytometry and the visualization algorithm, tSNE. This approach revealed significant phenotypic overlap amongst five previously described autoimmune-prone B cell subsets, including CD19 + CD10 - CD27 - CD21 lo B cells. Interestingly, 12 different subpopulations of CD19 + CD21 lo B cells were identified, some of which mapped to previously described autoreactive populations, while others were consistent with healthy B cells. This suggests that CD21 is downregulated in a variety of circumstances involving B cell activation, all of which are present in low numbers even in healthy individuals. These findings describe the utility of unbiased multiparameter analysis using a relatively limited panel of flow cytometry markers to analyze autoreactive-prone and normal activated B cells., (© 2022 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

Down's syndrome (DS) presents with a constellation of cardiac, neurocognitive and growth impairments. Individuals with DS are also prone to severe infections and autoimmunity including thyroiditis, type 1 diabetes, coeliac disease and alopecia areata 1,2 . Here, to investigate the mechanisms underlying autoimmune susceptibility, we mapped the soluble and cellular immune landscape of individuals with DS. We found a persistent elevation of up to 22 cytokines at steady state (at levels often exceeding those in patients with acute infection) and detected basal cellular activation: chronic IL-6 signalling in CD4 T cells and a high proportion of plasmablasts and CD11c + Tbet high CD21 low B cells (Tbet is also known as TBX21). This subset is known to be autoimmune-prone and displayed even greater autoreactive features in DS including receptors with fewer non-reference nucleotides and higher IGHV4-34 utilization. In vitro, incubation of naive B cells in the plasma of individuals with DS or with IL-6-activated T cells resulted in increased plasmablast differentiation compared with control plasma or unstimulated T cells, respectively. Finally, we detected 365 auto-antibodies in the plasma of individuals with DS, which targeted the gastrointestinal tract, the pancreas, the thyroid, the central nervous system, and the immune system itself. Together, these data point to an autoimmunity-prone state in DS, in which a steady-state cytokinopathy, hyperactivated CD4 T cells and ongoing B cell activation all contribute to a breach in immune tolerance. Our findings also open therapeutic paths, as we demonstrate that T cell activation is resolved not only with broad immunosuppressants such as Jak inhibitors, but also with the more tailored approach of IL-6 inhibition., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Memory B cells (MBCs) are an essential part of our immunological memory. They respond fast upon re-encountering pathogens and can differentiate into plasma cells that secrete protective antibodies. The focus of this review is on MBCs that lack, or express low levels of, CD21, hereafter referred to as CD21-/low. These cells are expanded in peripheral blood with age and during chronic inflammatory conditions such as viral infections, malaria, common variable immunodeficiency, and autoimmune diseases. CD21-/low MBCs have gained significant attention; they produce disease-specific antibodies/autoantibodies and associate with key disease manifestations in some conditions. These cells can be divided into subsets based on classical B-cell and other markers, e.g. CD11c, FcRL4, and Tbet which, over the years, have become hallmarks to identify these cells. This has resulted in different names including age-associated, autoimmune-associated, atypical, tissue-like, tissue-resident, tissue-restricted, exhausted, or simply CD21-/low B cells. It is however unclear whether the expanded 'CD21-/low' cells in one condition are equivalent to those in another, whether they express an identical gene signature and whether they have a similar function. Here, we will discuss these issues with the goal to understand whether the CD21-/low B cells are comparable in different conditions., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Immunology.)

Background and Aims: Epstein-Barr virus (EBV) is associated with solid and hematopoietic malignancies. After allogeneic stem cell transplantation, EBV infection or reactivation represents a potentially life-threatening condition with no specific treatment available in clinical routine. In vitro expansion of naturally occurring EBV-specific T cells for adoptive transfer is time-consuming and influenced by the donor's T-cell receptor (TCR) repertoire and requires a specific memory compartment that is non-existent in seronegative individuals. The authors present highly efficient identification of EBV-specific TCRs that can be expressed on human T cells and recognize EBV-infected cells., Methods and Results: Mononuclear cells from six stem cell grafts were expanded in vitro with three HLA-B*35:01- or four HLA-A*02:01-presented peptides derived from six EBV proteins expressed during latent and lytic infection. Epitope-specific T cells expanded on average 42-fold and were single-cell-sorted and TCRαβ-sequenced. To confirm specificity, 11 HLA-B*35:01- and six HLA-A*02:01-restricted dominant TCRs were expressed on reporter cell lines, and 16 of 17 TCRs recognized their presumed target peptides. To confirm recognition of virus-infected cells and assess their value for adoptive therapy, three selected HLA-B*35:01- and four HLA-A*02:01-restricted TCRs were expressed on human peripheral blood lymphocytes. All TCR-transduced cells recognized EBV-infected lymphoblastoid cell lines., Conclusions: The authors' approach provides sets of EBV epitope-specific TCRs in two different HLA contexts. Resulting cellular products do not require EBV-seropositive donors, can be adjusted to cell subsets of choice with exactly defined proportions of target-specific T cells, can be tracked in vivo and will help to overcome unmet clinical needs in the treatment and prophylaxis of EBV reactivation and associated malignancies., (Copyright © 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Pre-existing pathogen-specific memory T cell responses can contribute to multiple adverse outcomes including autoimmunity and drug hypersensitivity. How the specificity of the T cell receptor (TCR) is subverted or seconded in many of these diseases remains unclear. Here, we apply abacavir hypersensitivity (AHS) as a model to address this question because the disease is linked to memory T cell responses and the HLA risk allele, HLA-B*57:01, and the initiating insult, abacavir, are known. To investigate the role of pathogen-specific TCR specificity in mediating AHS we performed a genome-wide screen for HLA-B*57:01 restricted T cell responses to Epstein-Barr virus (EBV), one of the most prevalent human pathogens. T cell epitope mapping revealed HLA-B*57:01 restricted responses to 17 EBV open reading frames and identified an epitope encoded by EBNA3C. Using these data, we cloned the dominant TCR for EBNA3C and a previously defined epitope within EBNA3B. TCR specificity to each epitope was confirmed, however, cloned TCRs did not cross-react with abacavir plus self-peptide. Nevertheless, abacavir inhibited TCR interactions with their cognate ligands, demonstrating that TCR specificity may be subverted by a drug molecule. These results provide an experimental road map for future studies addressing the heterologous immune responses of TCRs including T cell mediated adverse drug reactions., (© 2022. The Author(s).)

Epstein-Barr virus (EBV) infections in healthy individuals are usually cleared by immune cells, wherein CD8 + T lymphocytes play the most important role. However, in some immunocompromised individuals, EBV infections can lead to the development of cancer in B, T, natural killer (NK) cells and epithelial cells. Most EBV-associated cancers express a limited number of virus-specific antigens such as latent membrane proteins (LMP1 and LMP2) and nuclear proteins (EBNA1, -2, EBNA3A, -B, -C, and EBNA-LP). These antigens represent true tumor-specific antigens and can be considered useful targets for T cell receptor (TCR) gene therapy to treat EBV-associated diseases. We used a TCR isolation platform based on a single major histocompatibility complex class I (MHC I) K562 cell library for the detection, isolation, and re-expression of TCRs targeting immunodominant peptide MHC (pMHC). Mature dendritic cells (mDCs) were pulsed with in vitro -transcribed (ivt) RNA encoding for the selected antigen to stimulate autologous T cells. The procedure allowed the mDCs to select an immunogenic epitope of the antigen for processing and presentation on the cell surface in combination with the most suitable MHC I molecule. We isolated eight EBV-specific TCRs. They recognize various pMHCs of EBV antigens LMP1, LMP2A, and EBNA3C, some of them described previously and some newly identified in this study. The TCR genes were molecularly cloned into retroviral vectors and the resultant TCR-engineered T cells secreted interferon-γ after antigen contact and were able to lyse tumor cells. The EBV-specific TCRs can be used as a basis for the generation of a TCR library, which provides a valuable source of TCRs for the production of EBV-specific T cells to treat EBV-associated diseases in patients with different MHC I types.

Alloimmune responses driven by donor-specific antibodies (DSAs) can lead to antibody-mediated rejection (ABMR) in organ transplantation. Yet, the cellular states underlying alloreactive B cell responses and the molecular components controlling them remain unclear. Using high-dimensional profiling of B cells in a cohort of 96 kidney transplant recipients, we identified expanded numbers of CD27+CD21- activated memory (AM) B cells that expressed the transcription factor T-bet in patients who developed DSAs and progressed to ABMR. Notably, AM cells were less frequent in DSA+ABMR- patients and at baseline levels in DSA- patients. RNA-Seq analysis of AM cells in patients undergoing ABMR revealed these cells to be poised for plasma cell differentiation and to express restricted IGHV sequences reflective of clonal expansion. In addition to T-bet, AM cells manifested elevated expression of interferon regulatory factor 4 and Blimp1, and upon coculture with autologous T follicular helper cells, differentiated into DSA-producing plasma cells in an IL-21-dependent manner. The frequency of AM cells was correlated with the timing and severity of ABMR manifestations. Importantly, T-bet+ AM cells were detected within kidney allografts along with their restricted IGHV sequences. This study delineates a pivotal role for AM cells in promoting humoral responses and ABMR in organ transplantation and highlights them as important therapeutic targets.

An important channel of cell-to-cell communication is direct contact. The immune synapse is a paradigmatic example of such type of interaction: it forms upon engagement of antigen receptors in lymphocytes by antigen-presenting cells and allows the local exchange of molecules and information. Although mechanics has been shown to play an important role in this process, how forces organize and impact on synapse function is unknown. We find that mechanical forces are spatio-temporally patterned at the immune synapse: global pulsatile myosin II-driven tangential forces are observed at the synapse periphery while localised forces generated by invadosome-like F-actin protrusions are detected at its centre. Noticeably, we observe that these force-producing actin protrusions constitute the main site of antigen extraction and endocytosis and require myosin II contractility to form. The interplay between global and local forces dictated by the organization of the actomyosin cytoskeleton therefore controls endocytosis at the immune synapse.

Objectives: ACA-positive/primary Sjögren's syndrome (pSS) represents a distinct overlapping entity with intermediate features in between limited systemic sclerosis (lSSc) and pSS. Few data are available on their general risk for lymphoproliferative complications, specifically regarding adverse predictors at the level of minor salivary gland (MSG) histology. The objectives of this work are: a) to characterise, through a detailed immunohistochemistry study, the organisation of the lymphomonocitic infiltrates in ACA-positive/pSS patient vs. ACA-negative/pSS patients focusing on the presence of GC-like structures in minor salivary gland biopsies; b) to compare the frequency of traditional clinical and serological risk factors for lymphoma between the two subgroups., Methods: We analysed 28 MSG samples from ACA-positive/pSS patients and 43 consecutive MSGs from ACA-negative/pSS, using sequential IHC staining for CD3, CD20 and CD21 in order to define the T/B cell segregation within the periductal infiltrates and presence of ectopic GC-like on the detection of GC-like structures. Clinical and serological data of all the patients were retrieved and analysed., Results: Ectopic lymphoid structures (ELS) with GC-like structures were observed in 7 out of 28 ACA-positive/pSS patients (25%) and in 13 out of 43 ACA-negative/pSS patients (30.2%). Similarly, no statistical significant difference was found between the two groups as far as the classical pSS risk factors for lymphoproliferative complications was concerned (i.e. salivary gland enlargement, purpura, low C4, leukocytopenia, clonal gammopathy). Finally, the 3 cases of non-Hodgkin's lymphoma observed were equally distributed between the two subsets., Conclusions: Overall, this study indicates that ACA-positive/and ACA-negative pSS patients apparently present a similar risk for lymphoproliferative complications as suggested indirectly by the analogies between the two groups observed at the histopathology level.

Significant loss of RNA followed by severely reduced cellular protein pool, a phenomenon termed host shutoff, is associated with a number of lytic virus infections and is a critical player in viral pathogenesis. Until recently, viral DNA exonucleases were associated only with processing of viral genomic DNA and its encapsidation. However, recent observations have identified host shutoff and exonuclease function for the highly conserved viral exonucleases in γ-herpesviruses, which include Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus and the mouse model murine gammaherpesvirus-68, also referred to as MHV-68. In this study, we show that although ablation of the MHV-68 exonuclease ORF37 caused a restrictive phenotype in WT IFN-α/β receptor-positive cells such as NIH 3T3, lack of ORF37 was tolerated in cells lacking the IFN-α/β receptor: the ORF37Stop virus was capable of forming infectious particles and caused loss of mRNA in IFN-α/β receptor knockout cells. Moreover, ORF37Stop virus was able to establish lytic infection in the lungs of mice lacking the IFN-α/β receptor. These observations provide evidence that lytic MHV-68 infection and subsequent loss of mRNA can take place independently of ORF37. Moreover, efficient growth of ORF37Stop virus also identifies a role for this family of viral nucleases in providing a window of opportunity for virus growth by overcoming type I IFN-dependent responses.

Half a century has passed since Epstein-Barr virus (EBV) particles were isolated from the cultured lymphoblasts of Burkitt lymphoma. During the period, molecular biology, hematology/immunology, and transplantation medicine made amazing progress, that clarified the mode of infection and pathophysiology of the virus in human diseases. Research strategies on the relationship between EBV and human have expanded to the epidemiology, structures and functions of both genomes, regulatory genes including microRNA, and the nature of epigenetics. Although no animal models of EBV infection long hampered the completion of in vivo experiments, humanized mice have broken through a barrier of in vitro study on EBV-infected cell lines. Our understanding of the life cycle of EBV has continued to deepen about the infection via the CD21 receptor expressed on B cells, the latency, reactivation/reinfection, and transformation, and also the dynamics of T-cell immune response and the intracellular immunosurveillance beyond acquired and innate immunity. On the other hand, the disease entity of life-threatening lymphoproliferative disease of EBV-infected T cells or NK cells is on controversial. The other parts of this special issue include the recent topics of the basic and clinical researches of EBV as the oncogenic virus. Then, we herewith overview the research history of EBV with special reference to the infected cells and host immune responses in EBV-associated diseases.

One of the challenges of treating patients with glomerulonephritis is to accurately assess disease activity. As renal biopsies are routinely stained for deposits of C3 activation fragments and glomerular C3 deposits are found in most forms of glomerulonephritis, we sought to determine whether a relatively noninvasive measure of C3 fragment deposition in the kidney can serve as a good biomarker of disease onset and severity. We recently developed a magnetic resonance imaging (MRI)-based method of detecting glomerular C3 and used this to track the progression of renal disease in the MRL/lpr mouse model of lupus nephritis using superparamagnetic iron oxide nanoparticles conjugated to complement receptor type 2 as a targeting agent. Quantitative immunofluorescence showed that glomerular C3b/iC3b/C3d deposition progressively increased with disease activity, a finding replicated by the T2-weighted MRI. The T2 relaxation times decreased with disease activity in the cortex and medulla of the MRL/lpr but not in MRL/Mpj control mice. Thus, MRI contrast agents targeted to glomerular C3 fragments can be used to noninvasively monitor disease activity in glomerulonephritis. As therapeutic complement inhibitors are used in patients with renal disease, this method, should it become feasible in humans, may identify those likely to benefit from complement inhibition.

Charge plays an important role in protein-protein interactions. In the case of excessively charged proteins, their electrostatic potentials contribute to the processes of recognition and binding with other proteins or ligands. We present an automated computational framework for determining the contribution of each charged amino acid to the electrostatic properties of proteins, at atomic resolution level. This framework involves computational alanine scans, calculation of Poisson-Boltzmann electrostatic potentials, calculation of electrostatic similarity distances (ESDs), hierarchical clustering analysis of ESDs, calculation of solvation free energies of association, and visualization of the spatial distributions of electrostatic potentials. The framework is useful to classify families of mutants with similar electrostatic properties and to compare them with the parent proteins in the complex. The alanine scan mutants introduce perturbations in the local electrostatic properties of the proteins and aim in delineating the contribution of each mutated amino acid in the spatial distribution of electrostatic potential, and in biological function when electrostatics is a dominant contributing factor in protein-protein interactions. The framework can be used to design new proteins with tailored electrostatic properties, such as immune system regulators, inhibitors, and vaccines, and in guiding experimental studies. We present an example for the interaction of the immune system protein C3d (the d-fragment of complement protein C3) with its receptor CR2, and we discuss our data in view of a binding site controversy., (Copyright © 2011 American Institute of Chemical Engineers (AIChE).)

Human herpesvirus type 6 (HHV6) is known to reactivate after hematopoetic stem cell transplantation (HSCT) and has been suggested to be associated with increased mortality and severe clinical manifestations, including graft versus host disease (GvHD). The exact etiological role of HHV6 reactivation in increased morbidity and mortality after HSCT remains unclear. This review will focus on the current available evidence of HHV6 reactivation after HSCT and its immuno-modulatory capacities, with particular emphasis on the severe complication GvHD. At present, no effective specific antiviral treatment for HHV6 reactivation has been identified. The currently available antiviral agents are outlined, as well as possible future strategies for the treatment of HHV6 reactivation. Non-toxic, specific treatment or prevention of HHV6 reactivation might improve the safety and efficacy of the HSCT procedure.

A vaccine against canine visceral leishmaniasis (CVL), comprising Leishmania braziliensis promastigote protein, sand fly gland extract (SGE) and saponin adjuvant, was evaluated in dog model, in order to analyse the immunogenicity of the candidate vaccine. The vaccine candidate elicited strong antigenicity in dogs in respect of specific SGE and Leishmania humoral immune response. The major saliva proteins recognized by serum from immunized dogs exhibited molecular weights of 35 and 45 kDa, and were related to the resistance pattern against Leishmania infection. Immunophenotypic analysis revealed increased circulating CD21+ B-cells and CD5+ T-cells, reflected by higher counts of CD4+ and CD8+ T-cells. The observed interaction between potential antigen-presenting cells (evaluated as CD14+ monocytes) and lymphocyte activation status indicated a relationship between innate and adaptive immune responses. The higher frequency in L. chagasi antigen-specific CD8+ T-lymphocytes, and their positive association with intense cell proliferation, in addition to the progressively higher production of serum nitric oxide levels, showed a profile compatible with anti-CVL vaccine potential. Further studies on immunological response after challenge with L. chagasi may provide important information that will lead to a better understanding on vaccine trial and efficacy.

To investigate the effects of in vivo CD23 destabilization on CD23 shedding and IgE production, an anti-CD23 stalk monoclonal (19G5), previously shown to enhance proteolysis of CD23 in vitro, was utilized. Compared to isotype control-treated mice, BALB/cJ mice injected with 19G5 displayed significantly enhanced serum soluble CD23 and IgE. Soluble CD23 and IgE levels were also increased in 19G5-treated C57BL/6J mice (intermediate IgE responders); however, the kinetics of the responses differed between the high (BALB/cJ) and intermediate responder mice, suggesting a potential role for CD23 in regulating IgE responder status. The 19G5-induced IgE response was dependent on IL-4 and independent of CD21 as demonstrated through use of IL-4Ralpha and CD21/35-deficient mice, respectively. Overall, the data provide a direct demonstration for CD23's role in regulating IgE production in vivo and suggest that therapies aimed at stabilizing cell surface CD23 would be beneficial in controlling allergic disease.

Whether HAART allows complete recovery of humoral immune function in HIV-infected individuals is still controversial. Our objective was to study the effect of HAART on both B cell repopulation and hypergammaglobulinemia in 72 naïve patients, including 35 HCV-coinfected individuals, during 156 weeks on HAART. The possible role of HCV coinfection on the recovery of the humoral immune system was also investigated. At baseline, HCV-coinfected patients had greater circulant IgG levels than HIV-only-infected patients, while B cell count and CD21(low) B cell subpopulation were similar in both groups. During HAART, HIV-only-infected patients reached normal B cell counts and circulant IgG levels, while HCV-coinfected individuals did not. CD21(low) B cell subpopulation significantly decreased in both groups of patients at week 48 after the initiation of HAART compared to baseline. Thus, B cells remained continuously stimulated in HCV-coinfected patients and this stimulation seemed to be through a CD21-independent pathway.

Yaba-like disease virus (YLDV) is a yatapoxvirus, a group of slow-growing poxviruses from primates. Analysis of the growth cycle of YLDV in tissue culture showed that maximum virus titres were reached 3 days post-infection and at this time only 3.3 % of infectious progeny was extracellular. The intracellular and extracellular virions have different buoyant densities and are separable on CsCl density gradients. They are also distinguishable by electron microscopy with the extracellular virions having an additional lipid envelope. In YLDV-infected cells, thick actin bundles with virions at their tips were seen protruding from the cell surface, despite the fact that YLDV lacks a protein comparable to Vaccinia virus A36R, which is required for VV-induced actin tail formation. In addition to these observations, the YLDV gene Y144R was characterized. This gene is predicted to encode a transmembrane protein containing three short consensus repeat (SCR) motifs common to members of the complement control protein family. Antibody generated against recombinant Y144R recognized products of 36, 41 and 48-55 kDa in YLDV-infected cells and purified extracellular enveloped virus (EEV) but not intracellular mature virus (IMV). Y144R protein is a glycoprotein with type I membrane topology that is synthesized early and late during infection. By immunoblot, indirect immunofluorescence and immuno-cryoelectron microscopy the Y144R protein was detected on the intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and EEV. This represents the first study of a YLDV IEV, CEV and EEV protein at the molecular level.

We report a case of systemic B-cell lymphoma that presented as an isolated cutaneous lesion on the ear, mimicking a primary cutaneous B-cell lymphoma. Although there was no clinical evidence of systemic disease, bone marrow involvement was found on further investigation and subsequent immunoglobulin gene analysis revealed an identical clone in the skin lesion and bone marrow aspirate. Evidence of a t(14 : 18) translocation was not identified. This case is unusual for several reasons. First, involvement of the pinna as a presenting feature of systemic lymphoma has not been reported previously. Second, the cutaneous lesion had been present for 3 years prior to diagnosis and there has been no clinical progression of systemic lymphoma during 2 years of follow-up. Third, the lymphoma does not correspond exactly to any of the entities in the REAL classification of systemic B-cell lymphoma. This case underlines the indolent nature of some systemic B-cell lymphomas and the need to investigate thoroughly patients with disease apparently confined to the skin.

Follicular dendritic cells (FDC) provide a reservoir for HIV type 1 (HIV-1) that may reignite infection if highly active antiretroviral therapy (HAART) is withdrawn before virus on FDC is cleared. To estimate the treatment time required to eliminate HIV-1 on FDC, we develop deterministic and stochastic models for the reversible binding of HIV-1 to FDC via ligand-receptor interactions and examine the consequences of reducing the virus available for binding to FDC. Analysis of these models shows that the rate at which HIV-1 dissociates from FDC during HAART is biphasic, with an initial period of rapid decay followed by a period of slower exponential decay. The speed of the slower second stage of dissociation and the treatment time required to eradicate the FDC reservoir of HIV-1 are insensitive to the number of virions bound and their degree of attachment to FDC before treatment. In contrast, the expected time required for dissociation of an individual virion from FDC varies sensitively with the number of ligands attached to the virion that are available to interact with receptors on FDC. Although most virions may dissociate from FDC on the time scale of days to weeks, virions coupled to a higher-than-average number of ligands may persist on FDC for years. This result suggests that HAART may not be able to clear all HIV-1 trapped on FDC and that, even if clearance is possible, years of treatment will be required.

Two populations of B lymphocytes, B-1 (CD5+ and/or CD11b+) and B-2 (CD5- and CD11b-) cells have been described. In mice, which is the species of reference for B-1 and B-2 cell studies, these two subsets present different developmental schemes, phenotypes, antibody repertoires, localization and behaviours. Interestingly, in sheep, B cells rearrange their immunoglobulin (Ig) loci around the neonatal period, similarly to murine B-1 cells. However, the phenotype of the sheep B cells has not been characterized with regards to their developmental pathway. In this report, we show that two sheep B-cell subsets can be distinguished on the basis of CD11b expression. Relative to CD11b- B cells, the CD11b+ B cells frequently co-express CD5, CD11c, higher levels of surface IgM (sIgM), show larger cell size and higher cell-cycling activity, and thus present a B-1-like phenotype. However, unlike murine B-1 cells, sheep B-1 like cells mainly localize in blood, display a higher propensity to spontaneous apoptosis relative to B-2-like cells, and proliferate after sIgM stimulation. Our data show that despite neonatal immunoglobulin loci rearrangements, sheep B cells do not all express a B-1-like phenotype. However, B-1-and B-2-like cells co-exist and present phenotypic and behavioural specificities. Nevertheless, sheep B-1-and B-2-like cells differ from the murine B-1 and B-2 cells in their cell behaviour. These subsets can thus not be considered as true homologues among species.

The human herpes virus Epstein-Barr (EBV) is clearly associated with African Burkitt's lymphoma and the undifferentiated form of nasopharyngeal carcinoma (NPC). EBV has been implicated in other types of lymphoma, as well as in some human breast cancers. However, its route of entry into epithelial cells is incompletely understood. We report here evidence that there is no gene alteration in the SCR 1 and 2 exons of EBVR/CR2 in human embryonic nasopharyngeal epithelial (HENE) cells and NPC cells and that SCR 1 and 2 mRNA could be detected in HENE cells, different differentiated NPC cell lines and well-differentiated NPC biopsies. None of 15 cases of poorly differentiated NPC cryosections has SCR 1 and 2 mRNA. We also provide evidence that transformation of HENE cells results from exposure to infectious EBV and that transformation is dependent on the presence of phorbol ester. These data suggest that expression of SCR 1 and 2 of EBVR/CR2 may be associated with replication of EBV and support the notion of direct infection and transformation of human nasopharyngeal epithelial cells destined to evolve into the carcinoma by EBV through EBVR/CR2.

It is difficult to study how Epstein-Barr virus (EBV) causes transformation of human epithelial cells. The major difficulty is that cultured human epithelial cells do not express EBV receptor (complement receptor 2, CR2), hence EBV cannot infect such epithelial cells directly. In order to investigate the role of EBV in the transformation of human epithelial cells, pSG-CR2-Hyg carrier was transfected into immortalized human epithelial cells (293 cells) to express EBV receptor. EBV could infect these CR2-positive cells directly, and expressed EBV antigens. EBV-infected epithelial cells grew in piles with multiple cellular layers and lost contact inhibition in vitro. In soft-agar culture containing 12-0-tetradecanoylphorbol 13-acetate (TPA), EBV-infected 293 cells formed more and larger colonies. When EBV-infected 293 cells were transplanted subcutaneously into nude mice, and treated with TPA, poorly differentiated carcinoma was induced. These results suggest that EBV could induce the malignant transformation of immortalized human epithelial cells in synergy with TPA.

A functionally intact spleen with a marginal zone, containing B cells with high density of surface C3d-receptors (CD21), is essential for the ability to induce a primary immune response to thymus-independent type 2 (TI-2) antigens. Main representatives of natural TI-2 antigens are capsular pneumococcal polysaccharides (PPSs). In this study the localization of different types of PPS antigen is determined in human spleen tissue. Our findings indicate that a main type of TI-2 antigen, PPS, localizes preferentially in the marginal zone. PPSs show co-localization with C3, presumably C3d, at the surface of strongly CD21+ B cells equipped for rapid activation. This enables a rapid primary humoral response. The other main PPS localization at follicular dendritic cells in germinal centers, relevant for isotype switching of anti-PPS antibodies, does not seem to be dependent on the presence of specific immunoglobulin. This may explain the finding of specific IgG in an early stage after antigenic challenge. It seems likely that complement C3 fragments (likely C3d), bound to PPSs, enable PPS localization at B-cell and follicular dendritic cell surfaces by binding to CD21, the C3d receptor.

Epithelial cell lines transfected with the Epstein-Barr virus (EBV) receptor CR2 are susceptible to infection by EBV. Following infection with certain EBV strains we found that these cells became positive for MHC class II. The class II was confirmed as being of viral and not target cell origin by immunostaining with HLA-specific monoclonal antibodies. Electron microscopic immunogold staining confirmed the presence of MHC class II on the surface of the virion. While some MHC class I was also found on the EB virion, other cell surface molecules were absent. Dual color immunofluorescence and confocal microscopy analysis demonstrated colocalization of class II with EBV-encoded structural proteins (MA and VCA) in infected epithelial cells. However, preincubation of EBV with antibodies against either MHC class I or MHC class II failed to affect either EBV binding or EBV infection. The presence of MHC on the surface of the EB virion may be a consequence of the intracellular pathways through which productive virus exits from the cell and may influence the target cell tropism of EBV.

Human long-term bone marrow cultures (HLTBMCs) are a valuable in vitro model for studying the role of the haemopoietic microenvironment. Here we report the spontaneous appearance of EBV-positive B cells in 6/40 HLTBMCs from patients with various haematological diseases after 3-5 months of culture. After subcultivation of these cells, a novel type of cell line could be characterized, which displayed surface markers and morphological features typical for EBV transformed B-cell lines. As the deproteinized and ultrafiltrated culture supernatants of these cell lines were found to contain an agent with stroma toxic properties, they were termed SSB lines (stroma-toxic-agent-secreting B-cell lines). This agent also exhibited a colony-inhibitory activity on in vitro myelopoiesis and erythropoiesis. These properties are typical for the two polyamines spermine and spermidine which were detected at elevated levels in the culture supernatants of SSB lines. The hypothesis that latent presence of EBV in bone marrow may induce an increased synthesis of spermine and spermidine, which are known to be associated with malignant haematological diseases and bone marrow aplasia, is discussed.

Labeling the EBV membrane with octadecylrhodamine-b-chloride (R18) we were able to monitor spectrofluorometrically the early events of EBV fusion, under conditions in which we could affect PKC activity. Binding of EBV to Raji cells induces PKC translocation from the cytosol to the plasma membrane and 32P incorporation into its cellular receptor CR2. CR2 phosphorylation is completely inhibited when cells are preincubated with the PKC inhibitor calphostin c. This treatment also generates a strong inhibition of EBV fusion. Taken together this result suggests a key role of CR2 phosphorylation in the EBV entry into Raji cells.

Epstein-Barr virus (EBV) receptors (EBV/C3d receptors) were detected, using the monoclonal antibody HB5, on 23 ectocervical and 5 endocervical biopsies of the uterine cervix. Elevated IgA titers against the viral capsid antigen and early antigen of EBV were also found in the cervical secretions from cervical carcinoma patients (83%), compared with samples from patients with cervical intraepithelial neoplasia (75%), herpes simplex virus-infected patients (0%), and gynecologic patients with nonmalignant conditions (0%). EBV DNA was present in 63% of cervical carcinoma biopsies detected by in situ hybridization. These observations suggest a positive association between EBV and carcinoma of the cervix.

Epstein-Barr virus, the causative agent of mononucleosis and several human cancers, infects cells via complement receptor type 2 (CR2). Expression of this receptor is restricted to B lymphocytes, some epithelial cells and immature thymocytes; expression of CR2-like proteins has been also found on T cells. In the present report, we identified the presence, on the membrane of Li7A cells, of a novel EBV receptor distinct from CR2 capable of triggering fusion with EBV virions with more rapid kinetics than that found with lymphoblastoid cells (Raji).

Sixty-five unselected cases of gastric cancer have been analysed for EBV DNA by polymerase chain reaction, in situ hybridization and immunohistochemistry for CD21 antigen expression. Four cases were found EBV-positive by PCR, while ISH yielded positive results in 3 of these cases, demonstrating EBV in the nuclei of cancerous cells. CD21 antigen was expressed in cancerous cells in all 3 ISH-positive cases. All the EBV-positive cancers of the present series were poorly to moderately differentiated adenocarcinomas with prominent lymphoid infiltration. These results are discussed also on the basis of the literature.

Epstein-Barr virus/C3d receptor (CR2) is a glycoprotein of mol. wt 140,000 expressed on the surface of Raji cells. We previously isolated phosphorylated CR2 from purified Raji cell nuclei. We have analyzed the nuclear localization of CR2 by electron microscope immunochemistry of thin sections of Raji cells and we have compared the binding properties of CR2 expressed on purified plasma membranes or nuclei. Anti-CR2 mAb immunogold labeling of thin sections of Raji cells identified CR2 at the nuclear surface and also within the nucleus. Nuclear envelope associated CR2 was localized mainly at nuclear pores. Within the nucleus, CR2 was associated with ribonucleoprotein (RNP) interchromatin fibrils. This labeling was preserved in nuclear matrix preparations. CR2 expressed on the surfaces of purified nuclei or on the cell surface interacted with soluble and particle-bound C3bi/C3d. Monoclonal anti-CR2 antibodies, which recognized extracellular domains of CR2, reacted differently with CR2 depending on its subcellular localization. The presence of CR2 in nuclei may be due to translocation of the cell surface CR2 and/or the presence of two distinct intracellular pathways for mature CR2.

Severe combined immunodeficiency (scid) mice develop EBV (+)B cell tumors after infusion of EBV(+)B cells or of B cells and EBV. In this study, scid mice were infused with B cell lines derived from three patients who developed a B lymphocyte proliferative disorder after bone marrow or organ transplantation. Intraperitoneal injection of 5 x 10(6) B cells induced tumor growth in all mice, leading to death within 60 d. Human B cells were identified in spleen and bone marrow by means of immunofluorescence or EBV genome amplification, and human IgM was detected in serum. Infusion of murine monoclonal antibodies specific for human B cell membrane antigens CD21, CD24, and CD23 was effective in 80% of animals, against two of the three cell lines preventing tumor development or inducing remission according to the time of treatment. The effect was antibody dose dependent and was optimal with four intravenous infusions of at least 0.1 mg 4 d apart. Human IgM in serum and human B cells in spleen and bone marrow became undetectable when peritoneal tumors regressed completely. Infusions of IgG1 isotype-matched anti-CD4 antibody or anti-CD3 antibody had no effect. Tumors developed or recurred in 50% of these animals injected with one of the B cell line 3 mo after treatment was stopped. The same anti-CD21 and anti-CD24 antibodies had been used to treat the three patients, and shown similar degrees of effectiveness as in the scid mouse model. These results indicate that scid mice may be suitable for assessing therapeutic approaches to human B cell proliferation.

The molecule CD23, a low-affinity receptor for IgE (Fc epsilon R2), is a type II transmembrane molecule expressed on many haemopoietic cell types. CD23 has pleiotropic roles in the control of lymphocyte behaviour, suggesting that CD23 may interact with another ligand in addition to IgE. To identify such a CD23 ligand, we expressed and purified full-length recombinant CD23, incorporated it into fluorescent liposomes and used these as a probe. We report here that fluorescent liposomes carrying CD23 interact specifically with the cell-surface protein CD21, identified as the receptor for Epstein-Barr virus and the complement receptor-2 on B cells, some T cells and follicular dendritic cells. In addition, fluorescent CD23-liposomes were shown to bind to hamster kidney cells (BHK-21) transfected with CD21 complementary DNA. The interaction between fluorescent CD23-liposomes and B cells or CD21-transfected BHK-21 cells was specifically inhibited by anti-CD21 and anti-CD23 monoclonal antibodies. Western blotting analysis revealed that 14C-labelled liposomes carrying CD23, in contrast to anti-CD21 antibodies, reacted with a subtype of CD21 molecules. Triggering of CD21 either with an anti-CD21 antibody or with recombinant soluble CD23 was shown to increase specifically interleukin-4-induced IgE production from blood mononuclear cells. These results demonstrate that the cell-surface protein CD21 is a ligand for CD23 and that the pairing of these molecules may participate in the control of IgE production.

We have investigated the expression, molecular association, ligand binding properties, and ability to transduce intracellular signals of CR1 and CR2 C3 receptors on cells of the human HPB-ALL T cell line. CR1 and CR2 on HPB-ALL cells bound polymeric C3b and C3dg and several anti-CR1 and anti-CR2 mAb recognizing different epitopes of the receptors on normal peripheral blood cells. Immunoprecipitated CR1 and CR2 exhibited similar m.w. to those of the receptors on normal peripheral blood T and B lymphocytes. CR1 and CR2 were partially associated in the form of CR1/CR2 complexes in the cell membrane as assessed by the ability of the receptors to cocap and cointernalize and to form a detergent-sensitive complex upon immunoprecipitation analysis. Triggering of CR2 with mAb OKB7 that recognizes an epitope associated with the ligand binding site of the receptor induced an increase in intracellular free calcium concentration in HPB-ALL cells. The signal provided by mAb OKB7 did not synergize with that triggered by anti-CD3 mAb UCHT1. Triggering of CR1 did not result in changes in intracellular free calcium concentration. Our observations have significance for the biology of normal human T cells because the majority of peripheral blood T cells that express CR1 also expressed CR2 and because a change in (Ca2+)i was induced by mAb OKB7 in purified normal T cells. These functions may be relevant for the regulatory role of C3 fragments on the immune response to T-dependent Ag and for the penetration into T cells of lymphocytotropic viruses.

A number of studies have indicated that the complement receptor type 2 (CR2), which is the receptor for C3d, a degradation fragment of the complement component C3, regulates B lymphocyte activation and growth. Early reports have described that C3 regulates T cell-dependent (TD) antibody responses. The involvement of CR2 in the antibody response to T cell-independent type 2(TI-2) antigens was investigated because neonatal B cells, which are unresponsive to TI-2 antigens both in vivo and in vitro, express a significantly decreased level of CR2 as compared to B cells of adult donors. We utilized type 4 pneumococcal polysaccharide (PS4) as a model TI-2 antigen. In order to study the relationship between CR2 and the response to PS4, B cells were costimulated with PS4 and monoclonal antibodies (MAb) to CR2. HB5 and OKB7 anti-CR2 monoclonal antibodies enhanced the in vitro response of adult B cells to PS4, as measured in a PS4-specific spot-forming cell assay. Neonatal B cells could only be induced to respond to PS4 using high concentrations of OKB7 anti-CR2 MAb. The 8-mercaptoguanosine (8MGuo), an agent that can overcome the in vitro unresponsiveness to PS4 of neonatal B cells, increased CR2 expression on adult and neonatal B cells. Furthermore, 8MGuo synergizes strongly with anti-CR2 antibodies in augmenting the anti-PS4 antibody response. Data presented in this report provide evidence of CR2 involvement in the antibody response to PS4 and that the neonatal B cell unresponsiveness to TI-2 antigens may be due to the decreased expression of CR2.

Cell extracts of Candida albicans were fractionated by concanavalin A affinity chromatography. Eluted mannosylated proteins (fraction II) and nonbinding, nonmannosylated proteins (fraction I) were collected and assayed directly for inhibition of adherence of C. albicans to endothelium. Fraction II blocked blastospore adherence to endothelial cells. Fraction I blocked both blastospore and germ tube adherence to endothelial cells. Monoclonal antibody OKM-1 (anti-CR3) and an anti-C. albicans monoclonal antibody, CA-A (anti-CR2), reacted in Western blots with proteins from fraction I, suggesting the presence of the CR2- and CR3-like proteins that have been previously identified on C. albicans germ tubes.

A 9-month-old infant developed Epstein-Barr virus-induced lymphoproliferative syndrome with mediastinal and central nervous system localizations, associated with mediastinal tuberculosis, 5 months after heart transplantation. As a combination of anti-B cell antibodies (CD21- and CD24-specific) and recombinant interferon alpha 2b, given intravenously, was not effective on the central nervous system disease, the anti-CD21 antibody was infused intrathecally via an Ommaya reservoir. High local concentrations of monoclonal antibodies were achieved, with no adverse effects. A dramatic clinical response was obtained, with clearance of abnormal cells from the cerebrospinal fluid and a clear reduction in the abnormalities on the brain images. The patient is well 7 months later. This observation indicates that treatment of B lymphoproliferative syndrome with central nervous system localization is feasible using a nontoxic, local B cell-specific approach.

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.

On a panel of nine human B cell lines we showed that the expression of the complement regulatory factors complement receptor type 2 (CR2; CD21), decay-accelerating factor, (DAF; CD55) and homologous restriction factor (HRF20, CD59) is not correlated. All lines expressed DAF, six lines carried detectable amounts of CR2 and three carried HRF20. Upon incubation in human serum, under conditions which allowed the activation of complement through the alternative pathway, the CR2-carrying lines bound C3 fragments and two of them (Ramos and one of its two sublines) were damaged. These two lines had the lowest DAF expression, less than 50% of the cells reacted with the IA10 monoclonal antibody. By modulating the expression of the complement regulatory molecules, the lytic sensitivity of the B cell lines could be altered. Blockade of DAF on the HRF20-, CR2+ lines with the specific monoclonal antibodies increased their sensitivity to lysis by human serum. With the DAF- and HRF20+ cells significant lytic effect was obtained only when they were pretreated with both of the specific antibodies. Interferon-gamma or tumor necrosis factor-alpha treatment elevated the amount of CR2 on the low-CR2 expressor line (Ramos/HR1K) which thereafter bound higher amounts of C3 fragments and was lysed when incubated in human serum. This line had relatively low DAF level and lacked HRF20. The cytokine treatment did not alter the expression of these molecules. The CR2+ Ramos and the CR2- Rael cells were treated with 5-azacytidine which induced HRF20 and increased DAF expression. In parallel with this change Ramos cells became resistant to C-mediated lysis. The experiments with the panel of human B cell lines showed thus that cytolysis through activation of complement in homologous serum can be regulated at several steps by cell surface molecules. While expression of CR2 was required for C3 fixation, DAF and HRF20 inhibited lysis. By independent modulation of the quantities of these molecules, cells acquired or lost their sensitivity.

A comparative immunohistochemical study of the distribution pattern of complement components and regulatory proteins within secondary lymphoid follicles was performed by the immunoperoxidase technique. Fifteen lymphoid tissues including appendices. Peyer's patches and tonsils were analysed. Sixty secondary lymphoid follicles with evident polarity, that is, the distinct coexistence of a light zone, dark zone and mantle zone in the same lymphoid follicle, were tested with single antibodies. The light zones were consistently immunostained in a dendritic meshwork pattern with all antibodies. The immunostaining patterns were classified into two major groups based on the immunoreactivity of the dark zone. One immunostaining pattern was characterized by no immunostaining of the dark zone to the majority of the antigens. The second group was characterized by a diffusely weak to moderate dendritic meshwork pattern of the dark zone to some of the immunostainings of C9 (monoclonal), S-protein, and DF-DRC1, and all immunostainings of CR1 (CD35), Ber-Mac-DRC (CD35), CR2 (CD21), and R4/23. All four complement regulatory proteins were localized by immunoelectron microscopy attached to the cell surface of the cells, including follicular dendritic cells, in the light zone. Our data indicate that there is an evident functional difference between the light zone and the dark zone, and that complete activation of the complement system occurs only in the light zone.

Despite the fact that the early components of the mouse and human complement cascades are very similar, there are marked differences between the two species in the structure of C3 receptors and the molecules that control homologous lysis. Here, Michael Holers, Taroh Kinoshita and Hector Molina compare and contrast the mouse and human RCA region products and conclude that the receptor and regulatory roles are conserved despite the structural variation.

Epstein-Barr virus (EBV), a ubiquitous human B-lymphotropic virus, is associated with certain lymphoproliferative diseases of T-cell lineage. To understand the mechanism by which EBV infects T cells, we have tested the susceptibility of various human T-cell lines to the virus. We report here that the HTLV-I-harbouring T-lymphoblastoid line MT-2 carries a high level of CD21/EBV receptors on their surface, adsorbs fluorescein isothiocyanate-labeled EBV, and synthesizes virus latent antigens (EBNA-1 and LMP) following EBV infection. Pretreatment of MT-2 cells with anti-CD21 monoclonal antibody OKB7 inhibited the virus binding as well as the synthesis of virus latent antigen. These data suggest that human T-cells can be infected with EBV via functionally active virus receptors.

Human peripheral blood neutrophils (PMN) treated with granulocyte-macrophage CSF (GM-CSF) increase the amount of class I 42-kDa H chain and 12-kDa L chain, beta 2-microglobulin (beta 2m), that they synthesize by 2.1- and 2.6-fold, respectively. To determine whether the increase in translation was associated with an increase in levels of class I H chain transcript, RNA blot analysis was performed on PMN that had been cultured in the presence of GM-CSF. Under no conditions were there increased levels of class I H chain transcript when class I heterodimer protein synthesis was increased. In addition, there was neither an increase in the synthesis of H chain mRNA, as measured by transcription assay, nor an alteration in the degradation rates of class I H chain transcript in PMN cultured with GM-CSF. In situ hybridization demonstrated that both the percentage of PMN that expressed class I transcript and the relative amounts of transcript per cell in GM-CSF-cultured PMN were the same as those in control PMN. Although there is increased translation of class I heterodimer in PMN treated with GM-CSF, there is no increase in its expression on the plasma membrane. The maintenance of constant levels of class I on the plasma membrane is dependent on continued protein synthesis and is maintained by release of class I heterodimer and free beta 2m into the medium. Heterodimer is released in the context of plasma membrane-derived vesicles, whereas beta 2m is released as a soluble protein. Maintenance of constant levels of class I heterodimer on the plasma membrane is also regulated by constitutive internalization. Up to 30% of class I molecules bearing 125I-Fab-labeled mAb to class I are internalized over 2 h at 37 degrees C. Therefore, inducible synthesis of class I by PMN is likely a consequence of post-transcriptional regulation, whereas the continued synthesis of class I heterodimer is required for maintenance of its expression. Furthermore, there is no increase in class I expression, in spite of increased synthesis, due to the release of class I heterodimer and beta 2m and the internalization of class I heterodimer from the plasma membrane. Thus, PMN are capable of post-transcriptional regulation of protein synthesis and are able to modulate the expression of plasma membrane proteins by regulated expression, release, and internalization.