17 research outputs found
Human CD27+ Memory B Cells Colonize a Superficial Follicular Zone in the Palatine Tonsils With Similarities to the Spleen. A Multicolor Immunofluorescence Study of Lymphoid Tissue
Background: Memory B cell (mBC) induction and maintenance is one of the keys to long-term protective humoral immunity. MBCs are fundamental to successful medical interventions such as vaccinations and therapy in autoimmunity. However, their lifestyle and anatomic residence remain enigmatic in humans. Extrapolation from animal studies serves as a conceptual basis but might be misleading due to major anatomical distinctions between species.
Methods and findings: Multicolor immunofluorescence stainings on fixed and unfixed frozen tissue sections were established using primary antibodies coupled to haptens and secondary signal amplification. The simultaneous detection of five different fluorescence signals enabled the localization and characterization of human CD27+CD20+Ki67- mBCs for the first time within one section using laser scanning microscopy. As a result, human tonsillar mBCs were initially identified within their complex microenvironment and their relative location to naĂŻve B cells, plasma cells and T cells could be directly studied and compared to the human splenic mBC niche. In all investigated tonsils (n = 15), mBCs appeared to be not only located in a so far subepithelial defined area but were also follicle associated with a previous undescribed gradual decline towards the follicular mantle comparable to human spleen. However, mBC areas around secondary follicles with large germinal centers (GCs) in tonsils showed interruptions and a general widening towards the epithelium while in spleen the mBC-containing marginal zones (MZ) around smaller GCs were relatively broad and symmetrical. Considerably fewer IgM+IgD+/- pre-switch compared to IgA+ or IgG+ post-switch mBCs were detected in tonsils in contrast to spleen.
Conclusions: This study extends existing insights into the anatomic residence of human mBCs showing structural similarities of the superficial follicular area in human spleen and tonsil. Our data support the debate of renaming the human splenic MZ to 'superficial zone' in order to be aware of the differences in rodents and, moreover, to consider this term equally for the human palatine tonsil
Cannabidivarin for HIVâAssociated Neuropathic Pain: A Randomized, Blinded, Controlled Clinical Trial
HIV remains a major burden to the health care system and neuropathic pain is the most common neurological complication of HIV infection. Because current treatment strategies often lack satisfying pain relief, cannabinoids (CBs) are discussed as a new option. We investigated cannabidivarin (CBDV) as treatment for HIV-associated neuropathic pain. We conducted a randomized, double-blind, placebo-controlled crossover study. Patients underwent two successive treatment phases (4 weeks each) and were treated with CBDV (400 mg/day) or placebo in a randomized order. A 3-week washout phase was designed to eliminate potential carry-over effects. Patients were followed up for 3 weeks after the end of the second treatment phase. The primary end point was pain intensity on an 11-point numeric rating scale, recorded in a diary. Secondary end points were additional pain medication, pain characteristics, and quality of life. We included 32 patients. The mean pain intensity under CBDV was 0.62 points higher compared with placebo (P = 0.16, 95% confidence interval -0.27 to 1.51). CBDV did not influence the amount of additional pain medication, pain characteristics, or quality of life. The incidence of adverse events was similar during both treatments. No suspected unexpected adverse reactions occurred during either treatment. CBDV was safe but failed to reduce neuropathic pain in patients with HIV. This may be explained by a lack of CB receptor activation, as indicated by preclinical experiments. Although a larger patient number might be desirable, we would not expect a change in the conclusions because the present differences are far from statistical significance. Therefore, we would currently not consider CBDV as a clinically meaningful treatment option for neuropathic pain
Human IgA-Expressing Bone Marrow Plasma Cells Characteristically Upregulate Programmed Cell Death Protein-1 Upon B Cell Receptor Stimulation
The functions of bone marrow plasma cells (BMPC) beyond antibody production are not fully elucidated and distinct subsets of BMPC suggest potential different functions. Phenotypic differences were identified for human BMPC depending on CD19 expression. Since CD19 is a co-stimulatory molecule of the B-cell-receptor (BCR), and IgA+ and IgM+ BMPC express the BCR on their surface, we here studied whether CD19 expression affects cellular responses, such as BCR signaling and the expression of checkpoint molecules. We analyzed 132 BM samples from individuals undergoing routine total hip arthroplasty. We found that both CD19+ and CD19- BMPC expressed BCR signaling molecules. Notably, the BCR-associated kinase spleen tyrosine kinase (SYK) including pSYK was higher expressed in CD19+ BMPC compared to CD19- BMPC. BCR stimulation also resulted in increased kinase phosphorylation downstream of the BCR while expression of CD19 remained stable afterwards. Interestingly, the BCR response was restricted to IgA+ BMPC independently of CD19 expression. With regard to the expression of checkpoint molecules, CD19- BMPC expressed higher levels of co-inhibitory molecule programmed cell death protein-1 (PD-1) than CD19+ BMPC. IgA+ BMPC characteristically upregulated PD-1 upon BCR stimulation in contrast to other PC subsets and inhibition of the kinase SYK abrogated PD-1 upregulation. In contrast, expression of PD-1 ligand, B and T lymphocyte attenuator (BTLA) and CD28 did not change upon BCR activation of IgA+ BMPC. Here, we identify a distinct characteristic of IgA+ BMPC that is independent of the phenotypic heterogeneity of the subsets according to their CD19 expression. The data suggest that IgA+ BMPC underlie different regulatory principles and/or exert distinct regulatory functions
Chitosan nanoparticles as antigen vehicles to induce effective tumor specific T cell responses
Cancer vaccinations sensitize the immune system to recognize tumor-specific antigens de novo or boosting preexisting immune responses. Dendritic cells (DCs) are regarded as the most potent antigen presenting cells (APCs) for induction of (cancer) antigen-specific CD8+ T cell responses. Chitosan nanoparticles (CNPs) used as delivery vehicle have been shown to improve anti-tumor responses. This study aimed at exploring the potential of CNPs as antigen delivery system by assessing activation and expansion of antigen-specific CD8+ T cells by DCs and subsequent T cell-mediated lysis of pancreatic ductal adenocarcinoma (PDAC) cells. As model antigen the ovalbumin-derived peptide SIINFEKL was chosen. Using imaging cytometry, intracellular uptake of FITC-labelled CNPs of three different sizes and qualities (90/10, 90/20 and 90/50) was demonstrated in DCs and in pro- and anti-inflammatory macrophages to different extents. While larger particles (90/50) impaired survival of all APCs, small CNPs (90/10) were not toxic for DCs. Internalization of SIINFEKL-loaded but not empty 90/10-CNPs promoted a pro-inflammatory phenotype of DCs indicated by elevated expression of pro-inflammatory cytokines. Treatment of murine DC2.4 cells with SIINFEKL-loaded 90/10-CNPs led to a marked MHC-related presentation of SIINFEKL and enabled DC2.4 cells to potently activate SIINFEKL-specific CD8+ OT-1 T cells finally leading to effective lysis of the PDAC cell line Panc-OVA. Overall, our study supports the suitability of CNPs as antigen vehicle to induce potent anti-tumor immune responses by activation and expansion of tumor antigen-specific CD8+ T cells
Deep Phenotyping of CD11c+ B Cells in Systemic Autoimmunity and Controls
Circulating CD11c+ B cells are a key phenomenon in certain types of autoimmunity but have also been described in the context of regular immune responses (i.e., infections, vaccination). Using mass cytometry to profile 46 different markers on individual immune cells, we systematically initially confirmed the presence of increased CD11c+ B cells in the blood of systemic lupus erythematosus (SLE) patients. Notably, significant differences in the expression of CD21, CD27, and CD38 became apparent between CD11c- and CD11c+ B cells. We observed direct correlation of the frequency of CD21-CD27- B cells and CD21-CD38- B cells with CD11c+ B cells, which were most pronounced in SLE compared to primary Sjögren's syndrome patients (pSS) and healthy donors (HD). Thus, CD11c+ B cells resided mainly within memory subsets and were enriched in CD27-IgD-, CD21-CD27-, and CD21-CD38- B cell phenotypes. CD11c+ B cells from all donor groups (SLE, pSS, and HD) showed enhanced CD69, Ki-67, CD45RO, CD45RA, and CD19 expression, whereas the membrane expression of CXCR5 and CD21 were diminished. Notably, SLE CD11c+ B cells showed enhanced expression of the checkpoint molecules CD86, PD1, PDL1, CD137, VISTA, and CTLA-4 compared to HD. The substantial increase of CD11c+ B cells with a CD21- phenotype co-expressing distinct activation and checkpoint markers, points to a quantitative increased alternate (extrafollicular) B cell activation route possibly related to abnormal immune regulation as seen under the striking inflammatory conditions of SLE which shows a characteristic PD-1/PD-L1 upregulation
Histone Deacetylase Inhibitor Modulates NKG2D Receptor Expression and Memory Phenotype of Human Gamma/Delta T Cells Upon Interaction With Tumor Cells
The functional plasticity and anti-tumor potential of human γΎ T cells have been widely studied. However, the epigenetic regulation of γΎ T-cell/tumor cell interactions has been poorly investigated. In the present study, we show that treatment with the histone deacetylase inhibitor Valproic acid (VPA) significantly enhanced the expression and/or release of the NKG2D ligands MICA, MICB and ULBP-2, but not ULBP-1 in the pancreatic carcinoma cell line Panc89 and the prostate carcinoma cell line PC-3. Under in vitro tumor co-culture conditions, the expression of full length and the truncated form of the NKG2D receptor in γΎ T cells was significantly downregulated. Furthermore, using a newly established flow cytometry-based method to analyze histone acetylation (H3K9ac) in γΎ T cells, we showed constitutive H3K9aclow and inducible H3K9achigh expression in VΎ2 T cells. The detailed analysis of H3K9aclow VΎ2 T cells revealed a significant reversion of TEMRA to TEM phenotype during in vitro co-culture with pancreatic ductal adenocarcinoma cells. Our study uncovers novel mechanisms of how epigenetic modifiers modulate γΎ T-cell differentiation during interaction with tumor cells. This information is important when considering combination therapy of VPA with the γΎ T-cell-based immunotherapy for the treatment of certain types of cancer
Establishment of immunohistochemical multicolor stainings for memory B cell localization studies within human lymphatic tissues
GedÀchtnis-B-Zellen zÀhlen zu den Hauptmediatoren unseres ImmungedÀchtnisses.
Das VerstÀndnis ihrer Physiologie bildet die Grundlage zahlreicher
medizinischer Interventionen, sowohl prophylaktisch, in Form iatrogener
ImmunitÀt durch Impfungen, als auch therapeutisch, in Form von
Immunmodulationen. Ăber die organspezifischen Nischen sowie das Wissen um
deren potentiellen Eigenschaften fĂŒr das Ăberleben und die Reifung dieser
Zellen ist jedoch weder fĂŒr die gesunden VerhĂ€ltnisse noch fĂŒr die
AutoimmunitÀt hinreichend aufgeklÀrt. Die Mehrzahl histologischer Studien in
humanen lymphatischen Geweben basiert auf konventionell lichtmikroskopisch
untersuchten ParaffinprÀparaten. Der erste Teil der vorliegenden Arbeit
widmete sich daher, zur methodischen Erweiterung und Verbesserung der
bisherigen Untersuchungen, der Etablierung immunhistochemischer
MultifluoreszenzfÀrbungen auf Gefrierschnitten humaner Milzen und Tonsillae
palatinae fĂŒr die Analyse mittels konfokalem Laser-Scanning-Mikroskop. Dies
gelang unter der parallelen Verwendung mehrerer durchflusszytometrisch
genutzter Mausantikörper unter Benutzung von Haptensystemen, wodurch die
Spezieslimitation in der Detektion umgangen wird und zusÀtzlich eine direkte
Visualisierungsoption durchflusszytometrisch erhobener Daten besteht. Unter
Anwendung dieser etablierten Methode war es durch die separate digitale
Information von fĂŒnf Fluoreszenzsignalen eines einzelnen Gewebeschnittes
erstmals möglich, die histologische Verteilung tonsillÀrer CD27+CD20+Ki67-
GedÀchtnis-B-Zellen im Kontext ihres Mikroenviroment zu beschreiben. In den
anschlieĂenden vergleichenden histologischen Analysen zwischen Milzen und
Tonsillae palatinae konnte als Gemeinsamkeit die dominierende
GedÀchtnis-B-Zellakkumulation, als unklar begrenztes follikelassoziiertes
Areal mit heterogener Zusammensetzung ihrer Immunglobulin-Isotypen,
herausgestellt werden. Die vorliegenden Ergebnisse unterstĂŒtzen dabei den
kĂŒrzlich durch Steiniger1 publizierten Vorschlag der Bezeichnung der humanen
marginalen Zone der Milz als âsuperfizielle Zoneâ, um von der Marginalzone der
Nagetiere abzugrenzen und sprechen darĂŒber hinaus fĂŒr die Verwendung dieses
Terms ebenso fĂŒr weitere humane sekundĂ€re lymphatische Organe. FĂŒr kĂŒnftige
Versuchstierstudien bedeutet dies, neben den vielen Vorteilen, kritischer mit
terminologischen Gleichsetzungen umzugehen. BezĂŒglich der nachgewiesenen
Unterschiede in der Immunglobulin-Subklassendominanz und der AusprÀgung der
GedÀchtnis-B-Zellareale bleibt allgemein zu klÀren, welche Bedeutung dabei dem
Anteil unterschiedlicher AdhĂ€renzmolekĂŒle zukommt und vor allem wie der
immunologische mit dem follikulÀren Status in Organismen langfristig zusammen
hÀngen.Memory B cells are major mediators of our immunological memory. The
understanding of their physiology is the basis for numerous medical
interventions like prophylaxes by iatrogenic immunity through vaccine as well
as therapy by immune modulation. However, the organ-specific niches and the
knowledge about their potential features for the survival and maturation of
these cells have not been sufficiently clarified yet, neither for healthiness
nor for autoimmunity. The majority of histological studies within human
lymphatic tissues are based on paraffin sections which are examined by
conventional light microscopy. To methodological extend and improve these
existing investigations, in the first part of this study immunohistochemical
multicolor stainings with frozen sections from human spleens and palatine
tonsils were established and analyzed with confocal microscopy. This succeeded
through the simultaneous usage of several mouse antibodies, also used in flow
cytometry, in combination with hapten systems, whereby the species limitation
in detection is circumvented and in addition a direct option to visualize flow
cytometric analyses is gained. The application of this established method
allowed the separate, digital information of five fluorescence signals within
a single tissue section and therefore the initial histological localization of
tonsillar CD27+CD20+Ki67- memory B cells in the context of their
microenvironment. In the subsequent comparative studies it was found, that the
commonality between the histological memory B cell localization in human
spleens and palatine tonsils is the general accumulation of these cells
directly around the follicular mantle in form of an unclear limited follicle
associated area with a heterogeneous composition of their immunoglobulin
isotypes. The present results support the recently published proposal by
Steiniger1 to rename the human splenic marginal zone to âsuperficial zoneâ to
distinguish it from the differing rodent spleen and, moreover, to use this
term equally for other secondary lymphoid organs. Besides many benefits, this
emphasizes for future animal studies, to be more careful with terminological
equations. Regarding the observed differences in the form of the memory B cell
areas including the proportion of their immunoglobulin isotype distribution,
the significance of various adherence molecules remains to be explored and
efforts should be undertaken to inform about the long term interrelationships
between the immunological and the follicular status in organisms
The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes
Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)-associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR-induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate-treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in-gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3É, WASP, and WIPF1) and identified several novel putative Nck-binding proteins. We subsequently verified the SH2 domain binding to the actin-binding protein HIP55 and to FYB/ADAP, and the SH3-mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR-to-cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes
BTLA Expression and Function Are Impaired on SLE B Cells
B- and T-lymphocyte attenuator (BTLA/CD272) is an inhibitory checkpoint molecule expressed on T and B cells. Prior studies reported defective function of BTLA by T cells in patients with systemic lupus erythematosus (SLE), whereas nothing is known about its role on B cells in SLE, a disease with various B cell abnormalities. Peripheral blood mononuclear cells (PBMCs) from 23 healthy donors (HD) and 34 SLE patients were stained for BTLA and its expression on B cells was assessed. PBMCs or CD27(-)IgD(+) naive B cells were stimulated together with an activating anti-BTLA antibody or an inhibitor of spleen tyrosine kinase (SYK) and differentiation as well as the expression of activation markers CD71, PD-1 and CD86 were analyzed. Our phenotypic and functional studies revealed reduced BTLA expression on CD27(-)IgD(+) naive B cells from SLE patients (p=0.0017) related to anti-dsDNA antibody titers (p=0.0394) and SIGLEC-1/CD169 expression on monocytes (p=0.0196), a type I interferon marker related to disease activity. BTLA engagement was found to control CpG/TLR9 activation limiting plasmablast (p=0.0156) and B cell memory induction (p=0.0078) in normal B cells in contrast to other B cell activation pathways (CD40, BCR). These BTLA functions were impaired in SLE B cells. Inhibition of SYK was found to mimic the effects of BTLA activity in vitro. Thus, is it possible that reduced BTLA expression and function of CD27-IgD+ antigen- and T cell-inexperienced SLE B cells could be overcome by SYK inhibition which should be tested in future studies as potential therapeutic principle