Serum, but Not Saliva, CXCL13 Levels Associate With Infiltrating CXCL13+ Cells in the Minor Salivary Gland Lesions and Other Histologic Parameters in Patients With Sjögren's Syndrome.

Recent studies suggest that elevated CXCL13 serum levels in patients with primary Sjögren's syndrome (pSS) associate with minor salivary gland (MSG) histologic features, disease severity, as well as high-risk status for non-Hodgkin lymphoma (NHL) development and NHL itself. In contrast, limited discriminative value of CXCL13 saliva levels has been reported. Prompt by these reports, we sought to validate the clinical utility of CXCL13 by investigating potential correlations of serum and saliva levels with MSG histopathologic [including CXCL13+-cell number, severity of infiltrates and germinal center (GC) formation], serologic and clinical parameters, as well as NHL. CXCL13 levels were evaluated in paired serum and saliva specimens of 45 pSS patients (15 with NHL; pSS-associated NHL: SSL), 11 sicca-controls (sicca-complaining individuals with negative MSG biopsy and negative autoantibody profile), 10 healthy individuals (healthy-controls) and 6 non-SS-NHLs. CXCL13+-cells were measured in paired MSG-tissues of 22 of pSS patients studied (including 7 SSLs) and all sicca-controls. CXCL13 serum levels were significantly increased in pSS and SSL patients compared to sicca- and healthy-controls and were positively correlated with the CXCL13+-cell number and biopsy focus-score. Serum CXCL13 was significantly higher in pSS patients with GCs, rheumatoid factor, hypocomplementemia, high disease activity, NHL and in high-risk patients for NHL development. CXCL13 saliva levels were significantly increased in SSL patients (compared to non-SS-NHLs), patients with GCs and in high-risk for NHL patients. Univariate analysis revealed that CXCL13 serum, but not saliva, levels were associated with lymphoma, an association that did not survive multivariate analysis. Conclusively, our findings confirm that serum, but not saliva, levels of CXCL13 are associated with histologic, serologic and clinical features indicative of more severe pSS.

Role of epstein-barr virus in the severity of recurrent respiratory papillomatosis.

OBJECTIVE: The objective was to investigate the prevalence of the Epstein-Barr virus (EBV) and its association with human papilloma virus (HPV) detection, clinicopathological features, and the severity of recurrent respiratory papillomatosis (RRP). METHODS: Cases of juvenile recurrent respiratory papillomatosis (JRRP) (n = 36) and adult recurrent respiratory papillomatosis (ARRP) (n = 44) were collected retrospectively and subdivided into low- and high-risk severity groups based on the Derkay score. We performed HPV detection and genotyping using a reverse hybridization protocol and investigated the presence of EBV by polymerase chain reaction (PCR) and in situ hybridization. CD21 levels were accessed by immunohistochemistry. RESULTS: All samples were HPV-positive, including 49 cases of HPV 6, 26 cases of HPV 11, four cases of HPV 6 and 11 coinfections, and one case of HPV 16. EBV-DNA was detected in nine samples by PCR, although none of the cases were positive by means of in situ hybridization. CD21 immunoexpression was not statistically associated with any of the variables analyzed. HPV 6 detection was significantly higher in ARRP cases (P = 0.03), whereas HPV 11 was more prevalent in JRRP cases (P = 0.02) and was even more prevalent in JRRP cases of greater severity (Derkay laryngoscopic scale ≥20) (P = 0.04). CONCLUSION: The presence of EBV does not seem to play an important role in the progression/severity of RRP. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:E611-E618, 2020.

Divergent immunohistochemical expression of CD21 and CD23 by follicular dendritic cells with increasing grade of follicular lymphoma.

BACKGROUND: Ultrastructural and immunohistochemical differences have been described in FDCs of primary and secondary follicles, illustrating the highly compartmentalized structure of lymph follicles. Differences in FDC immunophenotype in different grades of FL may reflect some parallelism between reactive and neoplastic conditions in terms of FDC-B cell interaction and may be used as a valuable additional tool for grading FL. METHODS: A total of 60 paraffin blocks from patients with follicular lymphoma, 30 cases each of grade 1 and 3, were retrieved from our archive. Immunohistochemical analysis was carried out for CD21, CD23, cyclin A, and Ki-67. RESULTS: Our study demonstrates that during evaluation, six patterns of FDC distribution were distinguished. The intensity of stain for CD21 was not statistically significant in grade 1 and grade 3 FL (p = 0.340). In contrast, grade 3 FLs exhibited a significant decrease of CD23 expression by the FDCs (p < 0.001). By CD21 stain, there was no significant difference in the distribution of pattern 1 in grades 1 and 3 (p = 0.098). In contrast, in grade 3, this pattern was significantly less observed by CD23 stain (p = 0.016). The same was observed for pattern 2 for CD21 (p = 0.940) and CD23 (p = 0.010) and pattern 4 for CD21 (p = 0.305) and CD23 (p = 0.005), respectively. Distribution of pattern 5 was significantly different between grades 1 and 3 both for CD21 (p = 0.005) and CD23 (p < 0.001). Distribution of patterns 2 and 6 was not significantly different between grades 1 and 3 for CD21 and CD23. The values of cyclin A and Mib-1 were also significantly different between grades 1 and 3 (p < 0.001). CONCLUSIONS: The observed patterns of FDCs lead us to believe that similar to reactive lymph node follicles, neoplastic follicles in FL, at least in early stages, have an organized structure. Hypothetically, with CD21, CD23, and cyclin A immunohistochemistry, the sequence of events in FL progression may be traced.

Comprehensive B Cell Phenotyping Profile for Chronic Graft-versus-Host Disease Diagnosis.

Previous studies have reported single B cell-related chronic graft-versus-host disease diagnostic (cGVHD) biomarkers, such as B cell-activating factor (BAFF), CD21low, and immature B cells, but research on the performance of biomarker combinations and the covariate effect of steroids is lacking. The primary objective of this study was to determine the most accurate combination of B cell populations using cell surface staining flow cytometry in an independent cohort of patients with cGVHD. Secondary objectives included assessing the effect of corticosteroid use at sample collection on the makeup and accuracy of the diagnostic panel and identifying the mechanism underlying low surface expression of BAFF receptor (BAFF-R) on B cells in cGVHD. Flow cytometry analysis was performed in an adult cohort of post-HCT patients with cGVHD onset (n = 44) and time-matched recipients without cGVHD (n = 63). We confirmed that the onset of cGVHD was associated with higher soluble BAFF (sBAFF) levels, elevated CD27-CD10-CD21low CD19+ B cell and classical switched memory B cell counts, and reduced transitional and naïve B cell counts. The highest single B cell population area under the receiver operating characteristic (ROC) curve (AUC) was .72 for transitional type 1 CD21low B cells. We also showed a significant inverse relationship between sBAFF and surface BAFF-R expression caused by sBAFF modulation of BAFF-R. Steroid use at sample collection influenced the significance of the sBAFF:B cell ratio, naïve and marginal zone-like B cells. The optimal combination of B cell subsets most significantly associated with cGVHD onset with or without concurrent corticosteroid use resulted in ROC AUCs of .87 and .84, respectively. Transitional and CD21low B cells were the only populations present in both panels; however, analyzing only these populations resulted in ROC AUCs of .79 and .78, respectively. This suggests that the inclusion of other populations and use of different panels depending on steroid use is necessary to achieve better accuracy. sBAFF was not a component of either panel. These novel B cell profiles could be tested prospectively in patients post-HSCT and could lead to focused mechanistic studies.

Investigação da presença e da influência do Epstein-Barr vírus na severidade da papilomatose laríngea / Investigation of the presence and influence of Epstein-Barr virus on the severity of laryngeal papilomatosis

A papilomatose laríngea é uma neoplasia benigna causada pelo papilomavírus humano (HPV), sendo os tipos 6 e 11 os mais comuns, e que ocorre em dois grupos etários, juvenil e adulto. A possível coinfecção viral tem sido sugerida em lesões de cabeça e pescoço; nesse sentido, o Epstein Barr vírus (EBV), que também apresenta tropismo por células epiteliais vem sendo estudado neste grupo de lesões. Os objetivos deste estudo foram genotipar os HPVs, investigar a presença de EBV-DNA por PCR e EBV-RNA por hibridização in situ. Além disso, associar a presença de EBV com a imunoexpressão de CD21, os resultados obtidos com a escala laringoscópica de Derkay et al. (1998) e com os dados clinicopatológicos. Oitenta casos de papilomatose laríngea, juvenil (n=36) e adulta (n=44), foram retrospectivamente analisados e subdivididos em grupos de menor e maior severidade, baseando-se na escala de Derkay. Todas as amostras foram HPV posivitas, com 49 casos HPV 6, 26 casos HPV 11, 4 casos HPV 6 e 11, e 1 caso HPV 16. A presença de EBV-DNA foi detectada em 9 amostras, entretanto EBV-RNA não foi não foi identificado em nenhuma amostra. Assim como a presença do EBV-DNA, a imunoexpressão de CD21 não se associou estatisticamente com quaisquer variáveis. A presença de HPV 6 foi mais comum em PLA e, o HPV 11 foi mais comum (p=0,02) e maior em casos de maior severidade (p=0,04), no grupo juvenil. A presença do EBV provavelmente não desempenha papel importante na progressão/severidade desta patologia(AU)

Laryngeal papillomatosis is a benign neoplasm caused by the human papillomavirus (HPV), been types 6 and 11 the most commonly related, and is divided into two groups: juvenile and adult. Viral coinfection has been suggested in head and neck lesions; in this sense, Epstein Barr virus (EBV), which also presents tropism for epithelial cells, has been studied in this group of lesions. The aims of this study are to perform HPV genotyping, investigate EBVDNA presence by PCR and EBV-RNA by in situ hybridization; and associate EBV presence with CD21 immunoexpression. Finally, the results were associated with Derkay laryngoscopic score. Eighty cases of laryngeal papillomatosis, juvenile (n = 36) and adult (n = 44) were retrospectively subdivided into low-risk and high-risk of severity based on the Derkay index. All samples were HPV-positive, with 49 cases of HPV 6, 26 cases of HPV 11, 4 cases of HPV 6 and 11, and 1 case of HPV 16. The presence of EBV-DNA was detected in 9 samples, however EBV-RNA was not identified in any sample. As the presence of EBV-DNA, the CD21 immunoexpression was not statistically associated with any variables. The presence of HPV 6 was more common in ALP, HPV 11 was more common (p = 0.02) and higher in cases of higher severity (p = 0.04) in juvenile group. The presence of EBV probably does not play an important role in the progression/severity of this pathology(AU)

Age-related alterations of the CD19 complex and memory B cells in children with Down syndrome.

Children with Down syndrome (DS) have a high incidence of recurrent respiratory tract infections, leukaemia and autoimmune disorders, suggesting immune dysfunction. The present study evaluated the role of the CD19 complex and memory B cells in the pathogenesis of immunodeficiency in children with DS. The expression levels (median fluorescein intensity-MFI) of CD19, CD21 and CD81 molecules on the surface of B cells and memory B cell subsets were studied in 37 patients and 39 healthy controls. Twenty-nine of the DS group had congenital cardiac disease. The B cell count was significantly low in children with DS compared with healthy age-matched controls for all three age groups (under 2 years; 2-6 years and older than 6 years). The MFI of CD19 was reduced in all the age groups, whereas that of CD21 was increased in those older than 2 years with DS. The expression level of CD81 was significantly increased in those older than 6 years. Age-related changes were also detected in memory B cell subsets. The frequency of CD27+IgD+IgM+ natural effector B cells was reduced in children with DS who had needed hospitalisation admission due to infections. The observed intrinsic defects in B cells may be responsible for the increased susceptibility of children with DS to severe respiratory tract infections.

Characteristics of germinal center-like structures in patients with Sjögren's syndrome.

AIM: To analyze the relationship between ectopic germinal centers (GCs) in the salivary glands and the clinical/laboratory characteristics of patients with Sjögren'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.

p16 expression in follicular dendritic cell sarcoma: a potential mimicker of human papillomavirus-related oropharyngeal squamous cell carcinoma.

Follicular dendritic cell sarcoma is a rare mesenchymal neoplasm that most commonly occurs in cervical lymph nodes. It has histologic and clinical overlap with the much more common p16-positive human papillomavirus (HPV)-related squamous cell carcinoma of the oropharynx, which characteristically has nonkeratinizing morphology and often presents as an isolated neck mass. Not surprisingly, follicular dendritic cell sarcomas are commonly misdiagnosed as squamous cell carcinoma. Immunohistochemistry is helpful in separating the 2 entities. Follicular dendritic cell sarcoma expresses dendritic markers such as CD21 and CD23 and is almost always cytokeratin negative. However, in many cases of HPV-related oropharyngeal carcinoma, only p16 immunohistochemistry as a prognostic and surrogate marker for HPV is performed. p16 expression in follicular dendritic cell sarcoma has not been characterized. Here, we investigate the expression of p16 in follicular dendritic cell sarcoma and correlate it with retinoblastoma protein expression. A pilot study of dendritic marker expression in HPV-related oropharyngeal squamous cell carcinoma was also performed. We found that 4 of 8 sarcomas expressed p16 with strong and diffuse staining in 2 cases. In 2 of the 4 cases, p16 expression corresponded to loss of retinoblastoma protein expression. Dendritic marker expression (CD21 and CD23) was not found in HPV-related oropharyngeal squamous cell carcinomas. As such, positive p16 immunohistochemistry cannot be used as supportive evidence for the diagnosis of squamous cell carcinoma as strong and diffuse p16 expression may also occur in follicular dendritic cell sarcoma. Cytokeratins and dendritic markers are critical in separating the two tumor types.

Efficacy and Safety of Sofosbuvir Plus Daclatasvir for Treatment of HCV-Associated Cryoglobulinemia Vasculitis.

Circulating mixed cryoglobulins are detected in 40%-60% of patients with hepatitis C virus (HCV) infection, and overt cryoglobulinemia vasculitis (CryoVas) develops in approximately 15% of patients. Remission of vasculitis has been associated with viral clearance, but few studies have reported the effectiveness of direct-acting antiviral drugs in these patients. We performed an open-label, prospective, multicenter study of the effectiveness and tolerance of an all-oral, interferon- and ribavirin-free regimen of sofosbuvir plus daclatasvir in patients with HCV-associated CryoVas. Forty-one consecutive patients with active HCV-associated CryoVas (median age, 56 y; 53.6% women) were recruited from hospitals in Paris, France, from 2014 through 2016. They received sofosbuvir (400 mg/day) plus daclatasvir (60 mg/day) for 12 weeks (n = 32) or 24 weeks (n = 9), and were evaluated every 4 weeks until week 24 and at week 36. Blood samples were analyzed for complete blood count, serum chemistry profile, level of alanine aminotransferase, rheumatoid factor activity, C4 fraction of complement, and cryoglobulin; peripheral blood mononuclear cells were isolated for flow cytometry analysis. Thirty-seven patients (90.2%) had a complete clinical response (defined by improvement of all the affected organs involved at baseline and no clinical relapse) after a median time of 12 weeks of therapy; all had a sustained virologic response (no detectable serum HCV RNA 12 weeks after the end of antiviral therapy). Patients' mean cryoglobulin level decreased from 0.56 ± 0.18 at baseline to 0.21 ± 0.14 g/L at week 36, and no cryoglobulin was detected in 50% of patients at this time point. After antiviral therapy, patients had increased numbers of T-regulatory cells, IgM+CD21-/low-memory B cells, CD4+CXCR5+ interleukin 21+ cells, and T-helper 17 cells, compared with before therapy. After a median follow-up period of 26 months (interquartile range, 20-30 mo), no patients had a serious adverse event or relapse of vasculitis.

An Epstein-Barr virus susceptible immature T-cell line, WILL4, established from a patient with T-lymphoblastic lymphoma bearing CD21 and a clonal EBV genome.

We managed a patient with an Epstein-Barr virus-associated T-cell lymphoblastic lymphoma. Mediastinal tumor cells at initial admission were positive for CD4, CD8, and TdT. Interestingly, a lymph node at necropsy was compatible for a CD4-positive peripheral T-cell lymphoma without CD8 and TdT expression, suggesting a different phenotype from the mediastinal tumor. Tumor cells in pleural effusion continued to proliferate in in vitro and were designated as WILL4. WILL4 cells were positive for CD3, CD4, CD8, CD21, T-cell receptor (TcR) αß, and TdT, indicating a similar phenotype to thymocytes. Southern blot analyses showed that the pleural tumor and WILL4 cells shared a TcR gene rearrangement, and that both contained a clonal EBV genome in an episomal form. RT-PCR showed that EBNA1 and LMP1 were expressed in the fresh tumor and WILL4 cells. Southern blot analyses revealed that WILL4 cells were susceptible to EBV infection in vitro using B95-8 supernatant. Anti-CD21 antibody inhibited in vitro infection of EBV, suggesting that CD21 plays a role in EBV infection into WILL4 cells. In vitro infection of EBV did not affect latent gene expression in WILL4 cells. WILL4 is a useful tool for analyzing the roles of EBV in onocogenesis in immature T-lymphoid malignancies.

Applied Protein and Molecular Techniques for Characterization of B Cell Neoplasms in Horses.

Mature B cell neoplasms cover a spectrum of diseases involving lymphoid tissues (lymphoma) or blood (leukemia), with an overlap between these two presentations. Previous studies describing equine lymphoid neoplasias have not included analyses of clonality using molecular techniques. The objective of this study was to use molecular techniques to advance the classification of B cell lymphoproliferative diseases in five adult equine patients with a rare condition of monoclonal gammopathy, B cell leukemia, and concurrent lymphadenopathy (lymphoma/leukemia). The B cell neoplasms were phenotypically characterized by gene and cell surface molecule expression, secreted immunoglobulin (Ig) isotype concentrations, Ig heavy-chain variable (IGHV) region domain sequencing, and spectratyping. All five patients had hyperglobulinemia due to IgG1 or IgG4/7 monoclonal gammopathy. Peripheral blood leukocyte immunophenotyping revealed high proportions of IgG1- or IgG4/7-positive cells and relative T cell lymphopenia. Most leukemic cells lacked the surface B cell markers CD19 and CD21. IGHG1 or IGHG4/7 gene expression was consistent with surface protein expression, and secreted isotype and Ig spectratyping revealed one dominant monoclonal peak. The mRNA expression of the B cell-associated developmental genes EBF1, PAX5, and CD19 was high compared to that of the plasma cell-associated marker CD38. Sequence analysis of the IGHV domain of leukemic cells revealed mutated Igs. In conclusion, the protein and molecular techniques used in this study identified neoplastic cells compatible with a developmental transition between B cell and plasma cell stages, and they can be used for the classification of equine B cell lymphoproliferative disease.

Expansion of Activated Peripheral Blood Memory B Cells in Rheumatoid Arthritis, Impact of B Cell Depletion Therapy, and Biomarkers of Response.

Although B cell depletion therapy (BCDT) is effective in a subset of rheumatoid arthritis (RA) patients, both mechanisms and biomarkers of response are poorly defined. Here we characterized abnormalities in B cell populations in RA and the impact of BCDT in order to elucidate B cell roles in the disease and response biomarkers. In active RA patients both CD27+IgD- switched memory (SM) and CD27-IgD- double negative memory (DN) peripheral blood B cells contained significantly higher fractions of CD95+ and CD21- activated cells compared to healthy controls. After BCD the predominant B cell populations were memory, and residual memory B cells displayed a high fraction of CD21- and CD95+ compared to pre-depletion indicating some resistance of these activated populations to anti-CD20. The residual memory populations also expressed more Ki-67 compared to pre-treatment, suggesting homeostatic proliferation in the B cell depleted state. Biomarkers of clinical response included lower CD95+ activated memory B cells at depletion time points and a higher ratio of transitional B cells to memory at reconstitution. B cell function in terms of cytokine secretion was dependent on B cell subset and changed with BCD. Thus, SM B cells produced pro-inflammatory (TNF) over regulatory (IL10) cytokines as compared to naïve/transitional. Notably, B cell TNF production decreased after BCDT and reconstitution compared to untreated RA. Our results support the hypothesis that the clinical and immunological outcome of BCDT depends on the relative balance of protective and pathogenic B cell subsets established after B cell depletion and repopulation.

Association between human papilloma virus/Epstein-Barr virus coinfection and oral carcinogenesis.

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 < 0.01). CONCLUSIONS: There is a high prevalence of HPV/EBV infection and coinfection in BOT and tonsil cancers, possibly reflecting their origins in lymphoid-rich tissue. In vitro, cells modeling coinfection have an increased invasive potential.

Antiretroviral therapy restores age-dependent loss of resting memory B cells in young HIV-infected Zambian children.

BACKGROUND: Antiretroviral therapy (ART) is associated with incomplete restoration of resting memory B (RMB) cell percentages in adults infected with HIV, but the effects on RMB cells in children are less well defined, in part because changes in RMB cell percentages are confounded by the development and maturation of the RMB cell pool. The objective of this study was to assess the effect of age at ART initiation on RMB cell percentages over time in HIV-infected Zambian children. METHODS: RMB cell percentages (CD19CD21CD27) were measured by flow cytometry in 146 HIV-infected Zambian children (9-120 months old) at baseline and at 3-month intervals after ART initiation and in 34 control children at a single study visit. RESULTS: RMB cell percentages among untreated HIV-infected children younger than 24 months did not differ from those of control children (P = 0.97). Among HIV-infected children older than 24 months of age, however, each 12-month increase in age at ART initiation was associated with a 1.8% decrease in RMB cell percentage. In contrast, RMB cell percentages in control children up to 48 months increased 4.4% with each 12-month increase in age. After 12 months of ART, children aged 24-60 months had a significant increase in RMB cell percentages that no longer differed from those of control children. CONCLUSIONS: Initiation of ART in 2- to 5-year-old HIV-infected children resulted in reconstitution of RMB cell percentages to levels similar to control children and may help restore normal development and maintenance of B-cell immunity.

Primary cutaneous follicular helper T-cell lymphoma treated with allogeneic bone marrow transplantation: immunohistochemical comparison with angioimmunoblastic T-cell lymphoma.

Primary cutaneous follicular helper T (TFH)-cell lymphoma has recently been proposed, and is characterized by proliferation of malignant T cells expressing TFH-cell markers, such as CXCL13, accompanied by numerous reactive B cells. We report here a patient whose skin histology showed massive infiltration of both T and B cells, with a proliferation of arborizing high endothelial venules and follicular dendritic cells. Infiltrating T cells were positive for CXCL13, programmed death (PD)-1, inducible T-cell co-stimulator, and BCL-6. Southern blot analyses using DNA from the skin revealed monoclonality of both T and B cells. The patient had marked resistance to treatments, and complete remission was achieved only after allogeneic stem cell transplantation. The present case showed overlapping features with angio-immunoblastic T-cell lymphoma (AITL), although systemic symptoms were not observed. Further study is needed to define the criteria of this provisional entity, representing the cutaneous counterpart of the nodal follicular peripheral T-cell lymphoma or AITL.

Hsp70 vaccination-induced primary immune responses in efferent lymph of the draining lymph node.

Bovine paratuberculosis is a highly prevalent chronic infection of the small intestine in cattle, caused by Mycobacterium avium subspecies paratuberculosis (MAP). In earlier studies we showed the protective effect of Hsp70/DDA subunit vaccination against paratuberculosis. In the current study we set out to measure primary immune responses generated at the site of Hsp70 vaccination. Lymph vessel cannulation was performed to obtain efferent lymph from the prescapular lymph node draining the neck area where the vaccine was applied. Hsp70 vaccination induced a significant increase of CD21(+) B cells in efferent lymph, accounting for up to 40% of efferent cells post-vaccination. Proliferation (Ki67(+)) within the CD21(+) B cell and CD4(+) T cell populations peaked between day 3 and day 5 post-vaccination. From day 7, Hsp70-specific antibody secreting cells (ASCs) could be detected in efferent lymph. Hsp70-specific antibodies, mainly of the IgG1 isotype, were also detected from this time point onwards. However, post-vaccination IFN-γ production in efferent lymph was non-sustained. In conclusion, Hsp70-vaccination induces only limited Th1 type immune responsiveness as reflected in efferent lymph draining the vaccination site. This is in line with our previous observations in peripheral blood. The main primary immunological outcome of the Hsp70/DDA subunit vaccination is B cell activation and abundant Hsp70-specific IgG1 production. This warrants the question whether Hsp70-specific antibodies contribute to the observed protective effect of Hsp70 vaccination in calves.

Reduced CD19 expression and decreased memory B cell numbers in transient hypogammaglobulinemia of infancy.

We aimed to evaluate the role of the CD19 complex in the pathogenesis of transient hypogammaglobulinemia of infancy (THI) and to better characterize the subsets of memory B cells. The study population consisted of 22 male and 14 female patients with a mean age at presentation of 20 ± 9.9 months. The CD19 complex and B cell subsets were evaluated by flow cytometry. While the CD19 median fluorescence index (MFI) in patients with THI was significantly lower than controls (122.9 ± 66.7 in patients; 184.2 ± 39 in controls, p < 0.01), expression of CD21 and CD81 was increased (94.4 ± 3, 96.8 ± 2.5 % in patients; 91 ± 3.9; 94.7 ± 3.5 % in controls, p < 0.01 vs. p < 0.05, respectively). The expressions of switched memory B cells and IgM memory B cells were found to be reduced in THI. Considering that the CD19 complex regulates the events following antigen stimulation, the change in CD19 complex detected in THI may be related to insufficiency of antibody production.

Clonal B cells of HCV-associated mixed cryoglobulinemia patients contain exhausted marginal zone-like and CD21 low cells overexpressing Stra13.

A clonal population of B cells expressing a V(H) 1-69-encoded idiotype accumulates in hepatitis C virus (HCV) associated mixed cryoglobulinemia (MC). These cells are phenotypically heterogeneous, resembling either typical marginal zone (MZ) B cells (IgM(+) IgD(+) CD27(+) CD21(+) ) or the exhausted CD21(low) B cells that accumulate in HIV infection or in common variable immunodeficiency. We show that both the MZ-like and the CD21(low) V(H) 1-69(+) B cells of MC patients are functionally exhausted, since they fail to respond to TLR and BCR ligands. The proliferative defect of V(H) 1-69(+) B cells can be overcome by co-stimulation of TLR9 and BCR in the presence of interleukin(IL)-2 and IL-10. The MZ-like V(H) 1-69(+) B cells do not express the inhibitory receptors distinctive of CD21(low) B cells, but display constitutive activation of extracellular signal regulated kinase (ERK) and attenuated BCR/ERK signaling. These cells also express abundant transcripts of Stra13 (DEC1, Bhlhb2, Sharp2, Clast5), a basic helix-loop-helix transcription factor that acts as a powerful negative regulator of B-cell proliferation and homeostasis. Our findings suggest that MZ B cells activated by HCV undergo functional exhaustion associated with BCR signaling defects and overexpression of a key antiproliferative gene, and may subsequently become terminally spent CD21(low) B cells. Premature exhaustion may serve to prevent the outgrowth of chronically stimulated MZ B cells.

Persistence of a large population of exhausted monoclonal B cells in mixed cryoglobuliemia after the eradication of hepatitis C virus infection.

PURPOSE: Functionally exhausted and mostly autoreactive B-cells with a peculiar CD21(low)CD11c(+) phenotype accumulate in several human immunological disorders including common variable immunodeficiency, HIV infection and rheumatoid arthritis. In HCV-associated mixed cryoglobulinemia (MC) there is accumulation of exhausted clonal B cells expressing a V(H)1-69-encoded cross-reactive idiotype; these cells are phenotypically heterogeneous, displaying either a CD21(low)CD11c(+) or a marginal zone (MZ)-like (IgM(+)CD27(+)CD21(+)CD11c(-)) phenotype. Irrespective of their phenotype, V(H)1-69(+) B-cells are unresponsive to the stimulation of Toll-like receptor 9 (TLR9). We investigated the fate of these cells after the eradication of HCV. METHODS: Fourteen MC patients were studied before and after antiviral therapy. V(H)1-69(+) B-cells were identified using the G6 monoclonal antibody and their phenotype and responsiveness to the stimulation of TLR9 were investigated. RESULTS: In seven virological non-responders, cryoglobulin levels and the number and phenotype of V(H)1-69(+) B cells remained substantially unchanged. By contrast, in sustained viral responders cryoglobulinemia subsided and the number of V(H)1-69(+) B cells declined. However, high proportions of MZ-like V(H)1-69(+) B cells retaining unresponsiveness to TLR9 stimulation persisted for several months in these patients. CONCLUSIONS: Clonal expansion of CD21(low) V(H)1-69(+) B cells may depend on continual stimulation by HCV, whereas their MZ-like counterparts may persist for years after the eradication of infection. Prolonged survival of exhausted MZ-like B cells after withdrawal of the initial inciting stimulus may contribute to the accumulation of autoreactive B cells in immunological disorders.

Role of Toll-like receptors in primary Sjögren's syndrome with a special emphasis on B-cell maturation within exocrine tissues.

Be they follicular cells within the germinal centers (GCs) or marginal zone (MZ), all naïve mature B lymphocytes need tonic signaling to stay alive. We reasoned that the same holds true for those B lymphocytes that proliferate in the salivary glands (SGs) of patients with primary Sjögren's syndrome. Based on B cell infiltration, 11 SGs and three tonsil samples were selected for further examination. Tissue sections were stained using CD20 combined with CD10, CD21, CD27, CD38 or IgD. They were also laser-microdissected for quantitative RT-PCR of transcription factors, GC-specific activation-induced cytidine deaminase (AID) and TLR9. Some B cell aggregates proved to be real GCs according to their membrane markers, whereas others were clusters of transitional type II B cells. These contained mRNAs for Notch-2 and Blimp-1, but not for Pax-5, Bcl-6 and AID. Unanticipated was the finding of mRNAs for TLR9 in these clusters of MZ B-cells, but not in the real GCs. Not only do TLR9 deliver sufficiency of tonic signaling to keep B cells alive, but they also confer autoreactive B cells with an MZ-like phenotype. Thus, TLRs might be targets for forthcoming biotherapies.