scholarly journals Circulating Exosomes Inhibit B Cell Proliferation and Activity

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2110
Author(s):  
Jan C. Schroeder ◽  
Lisa Puntigam ◽  
Linda Hofmann ◽  
Sandra S. Jeske ◽  
Inga J. Beccard ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Surface Expression ◽  
Receptor Expression ◽  
Healthy Individuals ◽  
Inhibitory Effects ◽  
Healthy Donors ◽  
Checkpoint Receptors

(1) Background: Head and neck squamous cell carcinoma (HNSCC) is characterized by a distinctive suppression of the anti-tumor immunity, both locally in the tumor microenvironment (TME) and the periphery. Tumor-derived exosomes mediate this immune suppression by directly suppressing T effector function and by inducing differentiation of regulatory T cells. However, little is known about the effects of exosomes on B cells. (2) Methods: Peripheral B cells from healthy donors and HNSCC patients were isolated and checkpoint receptor expression was analyzed by flow cytometry. Circulating exosomes were isolated from the plasma of HNSCC patients (n = 21) and healthy individuals (n = 10) by mini size-exclusion chromatography. B cells from healthy individuals were co-cultured with isolated exosomes for up to 4 days. Proliferation, viability, surface expression of checkpoint receptors, and intracellular signaling were analyzed in B cells by flow cytometry. (3) Results: Expression of the checkpoint receptors PD-1 and LAG3 was increased on B cells from HNSCC patients. The protein concentration of circulating exosomes was increased in HNSCC patients as compared to healthy donors. Both exosomes from healthy individuals and HNSCC patients inhibited B cell proliferation and survival, in vitro. Surface expression of inhibitory and stimulatory checkpoint receptors on B cells was modulated in co-culture with exosomes. In addition, an inhibitory effect of exosomes on B cell receptor (BCR) signaling was demonstrated in B cells. (4) Conclusions: Plasma-derived exosomes show inhibitory effects on the function of healthy B cells. Interestingly, these inhibitory effects are similar between exosomes from healthy individuals and HNSCC patients, suggesting a physiological B cell inhibitory role of circulating exosomes.

Is Zap-70 Protein Detection by Flow Cytometry a Reliable Tool in CLL Prognostic Evaluation?.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4771-4771
Author(s):  
Magali Le Garff-Tavernier ◽  
Michel Ticchioni ◽  
Rémi Letestu ◽  
Martine Brissard ◽  
Frédéric Davi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Flow Cytometric Analysis ◽  
Protein Detection ◽  
Flow Cytometric ◽  
Mutational Status ◽  
Healthy Donors

Abstract Background : Expression of ZAP-70 protein has been shown to be correlated with mutational status of immunoglobulin heavy-chain variable region (IgVH) genes, a major prognostic factor in CLL. We investigated whether the detection of ZAP-70 protein by flow cytometric analysis using unconjugated and conjugated monoclonal antibodies (mAbs) could be applied securely in the workup of patients with CLL. Methods: Flow cytometric analysis of ZAP-70 protein was performed using the method described by Crespo et al (N Engl J Med2003;348:1764) with minor modifications. Both fresh and cryopreserved mononuclear cells from CLL patients and healthy donors were fixed and permeabilized using Fix and Perm kit (Caltag Laboratories), incubated with anti-ZAP-70 mAb (clone 2F3.2, Upstate Biotechnology) and then revealed with goat antimouse FITC mAb (Immunotech). Finally cells were incubated with CD3-APC, CD56-APC and CD19-PC5. We also tested 3 mAbs conjugated to various fluorochromes: 2F3.2-FITC (Upstate), 1E7.2-PE (eBioscience), 1E7.2-PE or -Alexa 488 (Caltag). ZAP-70 protein detection in B-cells was expressed either as a percentage of its expression in the T and NK-cells or as a ratio (R) of T-cell mean cell fluorescence (MCF) to B-cell MCF. Western blotting of protein lysates from purified B-cells was carried out to control results obtained by cytometry in 55 samples. Mutational status was defined using a cutoff of 98%. Results: In 13 healthy donors, the mean percentage of ZAP-70 protein expression obtained by flow cytometry with unconjugated mAb (clone 2F3.2) was 4.69% ± 1.93 [range 2–9%] and the R ratio was 6.64 ± 1.54 and > 4.8. In 83 B-CLL samples, ZAP-70 expression was determined using the same method and compared to IgVH mutational status. Results in table below show a 75% concordance between gene mutations and ZAP-70 expression when considering a percentage of positive B-cells > 20%. A better concordance (81%) is obtained with a threshold T-cell MCF/ B-cell MCF at 4 determined by Youden’s index. To note the high concordance (90%) between unmutated status and ZAP-70 + expression (19/21). Comparison with at least 1 of the 3 conjugated mAbs has been performed for 63 samples, with discordant results in our laboratories. 62 mutated IgVH samples 21 unmutated IgVH samples ZAP-70 B-Cells + ≤ 20 % : 43 > 20% : 19 T-cell MCF/B-cell MCF ≥ 4 : 48 < 4 : 19 Conclusions: Our data document the concordance between IgVH gene mutational status and ZAP-70 protein expression measured by flow cytometry, particularly in ZAP-70 negative samples. We found that the indirect method of labelling with unconjugated anti-ZAP-70 mAb remains until now, in our hands, the gold standard method compared to the available dyes conjugate mAbs.

Analysis of PRAME Protein Expression in Normal Lymphoid Tissues and during B Ontogenesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3924-3924
Author(s):  
Lorena L. de Figueiredo-Pontes ◽  
Fabio M. do Nascimento ◽  
Rodrigo S. de Abreu e Lima ◽  
Rodrigo Proto-Siqueira ◽  
Aglair B. Garcia ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Mononuclear Cells ◽  
Hematologic Malignancies ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Healthy Donors ◽  
Chronic Lymphoproliferative Diseases

Abstract PRAME (Preferentially Expressed Antigen in Melanoma) gene was originally isolated in melanoma. A significant increase in the number of PRAME transcripts has been demonstrated in hematologic malignancies such as acute myeloid and lymphoid leukemias, multiple myeloma and chronic lymphoproliferative diseases. Furthermore, our group generated an anti-PRAME monoclonal antibody (MoAb) and by quantitative flow cytometry has demonstrated that PRAME protein was aberrantly expressed in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma. However, the expression of this antigen in normal lymphoid tissues and during B cells ontogeneis has not been characterized. To address this question, PRAME protein expression was studied by flow cytometry in peripheral blood (PB, n=15) and bone marrow (BM, n=6) from healthy donors, lymphonodes (n=4) and spleen (n=4) from patients submitted to lymphonode excision or splenectomy for non malignant diseases. First, we determined in which hematopoietic lineage PRAME was expressed by concomitantly staining PB, BM, lymphonode and spleen mononuclear cells (MCs) with anti-PRAME and a panel of MoAbs specific to B(CD19)/ T(CD3)/ NK(CD16/56), monocytic(CD14) and granulocytic(CD33) markers. PRAME was detected exclusively in CD19+ cells. The median percenatge of PRAME positive cells was 5,31% (2,55–12,34%), 13,01% (8,47–38,15%), 12,79% (3,15–23,06%) and 17,5% (12,67–27,43%) in PB, BM, lymphonode and spleen MCs, respectively. Amongst CD19+ cells, we have observed that PRAME was expressed by 42,39% (16,16–75,72%), 16% (13–69,5%), 15,16% (5,49–41,20%) and 48,82%(12,67–58,89%) in PB, BM, lymphonode and spleen, respectively. To establish in which stage of B ontogenesis PRAME was expressed on, cell suspensions stained with anti-CD19 were submitted to positive magnetic separation and labeled with anti-PRAME, CD5, CD27, CD38, CD34, CD10 and IgD MoAbs. PRAME+/CD19+ cells were CD5−, CD27+, CD38+, CD34−, CD10− and IgD+, thus suggesting that PRAME is expressed by the memory B cell compartment of the normal lymphoid tissues. This study defines PRAME as a B cell antigen that may accompany the neoplastic clone proliferation of mature B cell neoplasms. Although PRAME is mainly an embryonic antigen, expressed by carcinomas of immature phenotype, it is expressed by mature B cells in normal and pathological lymphoid tissues. Our findings suggest that maturational events occurring at the germinal center of lymphoid follicles affects PRAME expression.

Bone Marrow Derived Mesenchymal Stem Cells from Splenic Marginal Zone Lymphoma Patients Exhibit Altered Proliferative Potential and B Lymphocyte Immunomodulatory Properties

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2396-2396
Author(s):  
Athanasia Kalyva ◽  
Charalampos Pontikoglou ◽  
Christina Kalpadakis ◽  
Athina Trakaki ◽  
Nikitas Zorzos ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Ex Vivo ◽  
Proliferative Potential ◽  
Splenic Marginal Zone Lymphoma ◽  
Healthy Individuals ◽  
Healthy Donors ◽  
Normal Controls

Abstract Splenic marginal zone lymphoma (SMZL) originates from the neoplastic transformation of mature B-lymphocytes. However, there is a concurrent high prevalence of bone marrow (BM) infiltration, suggesting that BM microenvironment dynamics could have a potential involvement in disease pathology. In this regard, we aim to characterise BM derived mesenchymal stem cells (MSCs), since they comprise key components of the BM hematopoietic stroma, in order to investigate if MSCs show altered properties in SMZL patients compared to healthy controls. BM MSCs were isolated from 8 SMZL patients and 10 age- and sex-matched healthy controls. MSCs were in vitro expanded and re-seeded for a total of 5 passages (P). The colony forming unit-fibroblast (CFU-F) assay was used for the estimation of MSC frequency within the BM mononuclear cell (BMMC) fraction. Ex-vivo expanded MSCs were phenotypically characterized by flow cytometry (FC) using appropriate markers. In vitro differentiation to adipocytes and osteoblasts was assessed by cytochemical stains. The proliferative potential of ex vivo expanded MSCs was evaluated by Methyl Triazolyl Tetrazolium (MTT)-based assay and survival characteristics were studied using FC and 7-Aminoactinomycin D (7-AAD) staining. To assess the effect of patient MSCs on B cell growth, B cells were immunomagnetically isolated (Miltenyi Biotec GmbH, Germany) from peripheral blood (PB) of normal individuals, labeled with carboxy fluorescein succinimidyl ester (CFSE; Gibco Invitrogen, Paisley, Scotland) and subsequently cultured in the absence or presence of confluent layers of allogeneic BM-MSCs from SMZL patients or normal controls in the presence of CpG oligonucleotide 2006 (Invivogen, France) and IL-2 (R&D Systems, Minneapolis, MN). In a separate set of experiments, B cell survival was evaluated via FC and 7-AAD staining, after co-culturing with BM-MSCs from patients or healthy donors. Finally, to study BM-MSC capacity to chemotactically attract B-cells, transwell migration assays were set. In the bottom chambers MSCs from patients or healthy individuals were grown until confluency and then isolated B cells from PB of either patients or controls were added into the upper chamber. Twelve hours later migrated cells were enumerated. Grouped data are expressed as means± 1 standard error of the mean (SEM). MSCs were successfully expanded from all participants in the study. Adherent cells from both study groups displayed the typical spindle-shape morphology and immunophenotypic analysis at the end of P2-P3-P4 demonstrated that cultures constituted of a homogeneous cell population, typically expressing CD29, CD44, CD73, CD90 and CD105 while being negative for CD14, CD34 and CD45. SMZL-derived MSCs were similar to their normal counterparts in the capacity to differentiate towards adipocytes and osteocytes as evidenced by Oil Red O and Alizarin Red staining, respectively. The frequency of MSCs within the BMMC compartment was significantly lower in patients as compared to healthy individuals (2.5±0.68/105 ΒΜΜCs and 7.23±0.6/105 ΒΜΜCs, respectively; P=0.0032) apparently due to the predominance of the lymphoma cells within patient BMMCs. SMZL MSCs displayed defective proliferative potential as compared to their normal counterparts at P2, as evidenced by the MTT assay (P<0.0001). To explore the influence of SMZL BM-MSCs in B cells survival we compared the viability of B cells isolated from the PB of healthy individuals cultured in medium alone to that of such cells co-cultured with either BM-MSCs derived from patients or normal controls. 43.85±1.46% of B cells cultured alone were apoptotic, while only 20.8±2.63% and 12±0.77% of B cells co-cultured with either normal MSCs or SMZL MSCs were apoptotic (P<0.0001 and P<0.0001, respectively). Notably patient MSCs confer a survival advantage in B cell viability over their normal counterparts (P=0.0374). Finally SMZL MSCs had a more potent chemotactic activity on B cells from healthy donors, as compared to MSCs from normal controls ( P<0.05). In conclusion we have shown for the first time that SMZL lymphoma MSCs are intrinsically defective in terms of proliferative potential and exert an altered modulation of B cell apoptosis and B cell chemotaxis. These preliminary results concerning the properties of SMZL MSCs merit further investigation and provide the theoretical background for exploring their potential implication in lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

Requirement for MD-1 in cell surface expression of RP105/CD180 and B-cell responsiveness to lipopolysaccharide

Blood ◽  
2002 ◽  
Vol 99 (5) ◽  
pp. 1699-1705 ◽  
Author(s):  
Yoshinori Nagai ◽  
Rintaro Shimazu ◽  
Hirotaka Ogata ◽  
Sachiko Akashi ◽  
Katsuko Sudo ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Cell Surface ◽  
B Cell ◽  
Cell Activation ◽  
Costimulatory Molecule ◽  
Structural Similarity ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Cell Surface Molecule

RP105 is a B-cell surface molecule that has been recently assigned as CD180. RP105 ligation with an antibody induces B-cell activation in humans and mice, leading to proliferation and up-regulation of a costimulatory molecule, B7.2/CD86. RP105 is associated with an extracellular molecule, MD-1. RP105/MD-1 has structural similarity to Toll-like receptor 4 (TLR4)/MD-2. TLR4 signals a membrane constituent of Gram-negative bacteria, lipopolysaccharide (LPS). MD-2 is indispensable for TLR4-dependent LPS responses because cells expressing TLR4/MD-2, but not TLR4 alone, respond to LPS. RP105 also has a role in LPS responses because B cells lacking RP105 show hyporesponsiveness to LPS. Little is known, however, regarding whether MD-1 is important for RP105-dependent LPS responses, as MD-2 is for TLR4. To address the issue, we developed mice lacking MD-1 and generated monoclonal antibodies (mAbs) to the protein. MD-1–null mice showed impairment in LPS-induced B-cell proliferation, antibody production, and B7.2/CD86 up-regulation. These phenotypes are similar to those of RP105-null mice. The similarity was attributed to the absence of cell surface RP105 on MD-1–null B cells. MD-1 is indispensable for cell surface expression of RP105. A role for MD-1 in LPS responses was further studied with anti–mouse MD-1 mAbs. In contrast to highly mitogenic anti-RP105 mAbs, the mAbs to MD-1 were not mitogenic but antagonistic on LPS-induced B-cell proliferation and on B7.2 up-regulation. Collectively, MD-1 is important for RP105 with respect to B-cell surface expression and LPS recognition and signaling.

Sustained Functional T Cell Persistence and B Cell Aplasia Following CD19-Targeting Adoptive T Cell Immunotherapy for Relapsed, Refractory CD19+ Malignacy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 756-756
Author(s):  
Michael Kalos ◽  
Bruce L. Levine ◽  
Timothy L Macatee ◽  
Irina Kulikovskaya ◽  
Erica Suppa ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Surface Expression ◽  
University Of Pennsylvania ◽  
Cell And Gene Therapy

Abstract Abstract 756 Background Advances in ex vivo T cell engineering have facilitated clinical trials to evaluate the potential for adoptive T cell transfer to target malignancy. Gene-modified T cells have the potential to expand, functionally persist and mediate long-term anti-tumor activity. Most clinical studies have shown only limited persistence of infused cells, with modest and often temporary anti-tumor activity. We reported initial clinical data on CAR T cells targeting CD19 expressed on normal and malignant B cells (CART19 cells) (Porter, et al NEJM 2011; Kalos et al Sci Trans Med 2011). The CAR included signaling domains from CD3zeta and CD137 (4-1BB) that mediated effector and proliferation/persistence signals. Here we report functional persistence of CART19 cells from the initial cohort at approximately 2 years post-infusion, and data from a more recently treated cohort of CLL patients and a pediatric patient with advanced, treatment refractory ALL. Methods Persistence of gene-modified T cells was assessed by quantitative ABI-Taqman based PCR and a qualified assay that detects the CD137-TcR-zeta junction in the anti-CD19 CAR. CAR19 surface expression was detected by flow cytometry with an anti-CAR-19 idiotype specific antibody. B cell aplasia was evaluated by multiparametric flow cytometry. Multiplex cytokine analyses were performed using Luminex™assays. Results 10 patients with relapsed, refractory disease have been treated to date: 9 adults w/CLL and one child w/pre- B cell ALL. Each had been extensively pre-treated and had active disease at the time of CART19 infusion. All CLL patients received lymphodepleting chemotherapy 4–6 days before infusions, while the ALL patient did not get further lymphodepletion. Patients were infused with an average total of 1.7–50 × 108 total T cells which corresponded to 0.14–5.9 x108CART-19 cells. 9/10 treated patients were evaluable on 8.14.12. Detailed clinical outcomes will be reported separately at this meeting (Porter, D.L., Grupp S. et al). 4 pts (3 CLL, 1 ALL) had achieved CR at the primary endpoint (30 days post infusion) which is sustained and ongoing in all patients (range 1–24 months). Two CLL patients had a partial response (PR) lasting 3 and 5 months, while 3 patients did not respond (NR). In all patients with CR, robust in vivo expansion of CART19 cells was observed. By molecular analysis, CART19 cells demonstrated in vivo expansion, followed by contraction and an ongoing stable persistence at all evaluated timepoints. Expansion kinetics were unique for each patient; in all cases maximal expansion was observed by day +30 post CART-19 infusion. In patients with CR, observed peak marking for CART-19 ranged from 1 × 102-1 × 103 CART-19 cells/uL blood. Patients with PR demonstrated less robust in vivo expansion, with peak observed marking ∼1 × 101 CART-19 cells/uL blood. In NR patients, peak marking was <1 × 101CART-19 cells/uL blood. Long term peripheral blood persistence of CART19 cells and CAR19 surface expression was observed in all patients with CR in both CD3+/CD8+ and CD3+/CD4+ subsets. In patients with CR, elimination of peripheral B cells was observed at the time of CART19 in vivo expansion. Ongoing B cell aplasia has been documented in each CR patient in both peripheral blood and marrow by flow cytometry. Patients with PR showed transient elimination of malignant and normal B cells. Multiplex-cytokine analysis of serum samples from CR patients revealed a broad pro-inflammatory signature with significant elevation in a subset of soluble immune modulators including IL-6, IL-8, IFN-g, MIP1b, and IL2ra. In contrast, NR patients did not have elevated serum cytokines. In CR patients, elevation of cytokines tracked with expansion of CART19 cells and elimination of B cells, suggesting the potential for a cytokine-based diagnostic signature to monitor CART19 treatment efficacy. Conclusions Adoptive transfer of CART19 cells engineered to express CD137 and TCR-zeta signaling domains can result in in vivo expansion, homing to disease sites, and long-term functional persistence of CART19 cells, accompanied by ongoing complete clinical responses and long-term B cell aplasia in a substantial fraction of patients with advanced, refractory and high risk CLL and relapsed refractory ALL. A detailed cytokine profile and persistent B cell aplasia has been identified that may correlate with treatment efficacy. Disclosures: Kalos: University of Pennsylvania: Patents & Royalties. Levine:TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. June:Novartis: Research Funding, institution owned patents have been licensed by Novartis, institution owned patents have been licensed by Novartis Patents & Royalties.

scholarly journals Prevalence of Allelic Variants and Clonality of IGHV1-69 Expressing B-Cells in Patients with Different Severity of COVID 19 Disease

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 994-994
Author(s):  
Irina Panovska-Stavridis ◽  
Nevenka Ridova ◽  
Simona Stojanovska ◽  
Milena Stevanovic ◽  
Tatjana Stojanoska ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Critically Ill ◽  
Critically Ill Patients ◽  
Cell Populations ◽  
Healthy Individuals ◽  
Allelic Variants ◽  
Healthy Donors ◽  
Significant Difference ◽  
Severity Of The Disease

Abstract Introduction: Since the first months of the COVID-19 pandemic, efforts have been made to understand the importance of broadly neutralising natural antibodies in determining the response to SARS-CoV-2. Previous studies have shown that allelic variants of the IGHV1-69 gene play a dominant role in protective natural antibody responses to several other viral pathogens, including influenza virus, hepatitis C virus, human immunodeficiency virus and, most notably, the SARS-CoV-2-related viruses SARS-CoV and MERS-CoV. These allelic variants are commonly known as 51p1-related and differ from the other IGHV1-69 alleles (known as hv1263-related) in the presence of a Phe54 residue in the CDR2 region. Importantly, crystallographic studies have shown that the Phe54 residue is critical for the binding of IGHV1-69 antibodies to the SARS-CoV and MERS-CoV spike proteins. In this study, we evaluated the prevalence of 51p1 and hv1263 alleles and the clonality of 51p1- and hv1263-expressing B cells in a large cohort of healthy individuals and COVID-19 patients and correlated the findings with the severity of the disease. Мaterials and methods: A total of 419 samples were included in the study, of which 78 asymptomatic/mildly symptomatic individuals, 200 hospitalized patients with severe disease, 94 critically ill patients and 47 healthy donors. Peripheral blood was collected 8-20 days after the onset of symptoms and total cellular RNA was extracted from whole blood using an automated procedure. Аllelle-specific Ig-gene fingerprinting of IgM heavy chain transcripts was used to simultaneously analyse the clonality of the IgM+ B-cell population and the clonality of the 51p1- and hv1263-expressing B cell populations. The significance of the differences in the prevalence of clonal B-cell populations between healthy donors and patients and between patients with different severity of the disease was calculated with the Chi-Square test. Results: Analysis of the clonality of the IgM+ B-cell population showed a polyclonal pattern in most of the investigated healthy individuals (33/47, 70%) but in only 20% of all SARS-CoV-2 infected individuals (75/372, p&lt;0.001). A significant difference was also observed between mildly affected and severely/critically ill patients [31/78 (39.7%) vs. 44/294 (15%), respectively) (p&lt;0.001)], but not between severely and critically ill patients [28/200 (14,%) vs. 16/94 (17,1%), (p=n.s.)]. No 51p1 transcripts were detected in 74/372 (19.9%) of SARS-CoV-2 infected individuals and in 14/47 (29,8%) of the control group (p&gt;0,01), while hv1263 transcripts were not detected in 155/289 (53,6%) and in 27/47 (68,6%) tasted patients and controls, respectively (p&gt;0,05). We did not find a statistically significant difference in the prevalence of 51p1 and hv1263 alleles between patients with different disease severity. However, a significantly higher number of patients displayed clonal expansions of 51p1- or hv1263-expressing B cells (219/372(58.9%) and 118/244 (48,4%), respectively in comparison to healthy donors [5/47(10.6%) and 7/47(14.9%), respectively]. There was no statistically significant difference between mildly affected and severely/critically ill patients in the clonallity status of 51p1- 38/61 (62,3%) and 182/237 (76,7%) respectively or between hv1263- expressing B cells in the same two groups of patients [20/25 (80%) and 98/109 (89,9%), p&gt;0.05]. Conclusions: Our results show that SARS-CoV-2 infection stimulates clonal expansions of IGHV1-69 -expressing B-cells, but this is independent of the severity of the disease. In addition, no difference in the prevalence of IGHV1-69 alleles was observed between patients at different stages of the disease, indicating that natural neutralizing antibodies encoded by this gene are not an important determinant of COVID-19 severity and progression. Disclosures No relevant conflicts of interest to declare.

scholarly journals Anti-CD40 antibody KPL-404 inhibits T cell-mediated activation of B cells from healthy donors and autoimmune patients

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
John Marken ◽  
Sujatha Muralidharan ◽  
Natalia V. Giltiay
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Cytokine Production ◽  
B Cell Activation ◽  
Cell Responses ◽  
Healthy Donors ◽  
B Cell Responses

Abstract Background CD40-CD40L is a key co-stimulatory pathway for B cell activation. As such, its blockade can inhibit pathogenic B cell responses in autoimmune diseases, such as Sjogren’s syndrome (SjS) and systemic lupus erythematosus (SLE). In this study, we examined the in vitro effects of KPL-404, a humanized anti-CD40 monoclonal antibody (Ab), on primary human B cells derived from either healthy donors (HD) or autoimmune patients and compared them to the effects of G28-5, a partially antagonistic anti-CD40 antibody. Methods PBMCs from HD or SjS and SLE patients were cultured in high-density cell cultures in the presence of IgG4 isotype control or anti-CD40 Abs KPL-404 or G28-5. Cells were stimulated with anti-CD3/CD28 cross-linking reagent ImmunoCult (IC) to induce CD40L-CD40-mediated B cell responses. B cell proliferation and activation, measured by dilution of proliferation tracker dye and the upregulation of CD69 and CD86, respectively, were assessed by flow cytometry. Anti-CD40 Ab cell-internalization was examined by imaging flow cytometry. Cytokine release in the PBMC cultures was quantified by bead-based multiplex assay. Results KPL-404 binds to CD40 expressed on different subsets of B cells without inducing cell depletion, or B cell proliferation and activation in in vitro culture. Under the same conditions, G28-5 promoted proliferation of and increased CD69 expression on otherwise unstimulated B cells. KPL-404 efficiently blocked the CD40L-CD40-mediated activation of B cells from HD at concentrations between 1 and 10 μg/ml. Treatment with KPL-404 alone did not promote cytokine production and blocked the production of IFNβ in healthy PBMC cultures. KPL-404 efficiently blocked CD40L-CD40-mediated activation of B cells from patients with SjS and SLE, without affecting their anti-IgM responses or affecting their cytokine production. Consistent with the differences of their effects on B cell responses, KPL-404 was not internalized by cells, whereas G28-5 showed partial internalization upon CD40 binding. Conclusions Anti-CD40 mAb KPL-404 showed purely antagonistic effects on B cells and total PBMCs. KPL-404 inhibited CD40L-CD40-mediated B cell activation in PBMC cultures from both healthy controls and autoimmune patients. These data support the therapeutic potential of CD40 targeting by KPL-404 Ab for inhibiting B cell responses in SjS and SLE.

scholarly journals AB0024 IN VITRO CHARACTERIZATION OF CENERIMOD, A POTENT AND SELECTIVE SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 (S1P1) MODULATOR IN PREVENTING MIGRATION OF NON-ACTIVATED AND ACTIVATED PRIMARY HUMAN B CELLS IN THE PRESENCE OR ABSENCE OF GLUCOCORTICOIDS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1046.1-1046
Author(s):  
L. Schlicher ◽  
P. Kulig ◽  
M. Murphy ◽  
M. Keller
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Activation ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
B Cell Activation ◽  
Human B Cells ◽  
Circulating Lymphocytes

Background:Cenerimod is a potent, selective, and orally active sphingosine 1-phosphate receptor 1 (S1P1) modulator that is currently being evaluated in a Phase 2b study in patients with systemic lupus erythematosus (SLE) (NCT03742037). S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including B lymphocytes) in the blood stream and in inflamed tissues. Extensive clinical experience has become available for the nonselective S1P receptor modulator fingolimod in relapsing forms of multiple sclerosis, supporting this therapeutic concept for the treatment of autoimmune disorders.Objectives:Although the effect of S1P-receptor modulators in reducing peripheral B cells is well documented1,2, the role of the S1P1 receptor on this cell type is only incompletely understood. In this study, the mode of action of cenerimod on primary human B cells was investigated in a series of in vitro experiments, including S1P1 receptor cell surface expression and chemotaxis towards S1P. Moreover, S1P1 expression following B cell activation in vitro was studied. As glucocorticoids (GC) are frequently used in the treatment of patients with autoimmune disorders including SLE, the potential influence of GC on the mode of action of cenerimod was evaluated.Methods:Primary human B lymphocytes from healthy donors were isolated from whole blood. In one set of experiments, cells were treated with different concentrations of cenerimod to measure S1P1 receptor internalization by flow cytometry. In a second set of experiments, isolated B cells were activated using different stimuli or left untreated. Cells were then analysed for S1P1 and CD69 cell surface expression and tested in a novel real-time S1P-mediated migration assay. In addition, the effect of physiological concentrations of GCs (prednisolone and prednisone) on cenerimod activity in preventing S1P mediated migration was tested.Results:In vitro, cenerimod led to a dose-dependent internalization of the S1P1 receptor on primary human B lymphocytes. Cenerimod also blocked migration of nonactivated and activated B lymphocytes towards S1P in a concentration-dependent manner, which is in line with the retention of lymphocytes in the lymph node and the reduction of circulating lymphocytes observed in the clinical setting. Upon B cell activation, which was monitored by CD69 upregulation, a simultaneous downregulation of S1P1 expression was detected, leading to less efficient S1P-directed cell migration. Importantly, physiological concentrations of GC did not affect the inhibitory activity of cenerimod on B cell migration.Conclusion:These results show that cenerimod, by modulating S1P1, blocks B lymphocyte migration towards its natural chemoattractant S1P and demonstrate compatibility of cenerimod with GC. These results are consistent with results of comparable experiments done previously using primary human T lymphocytes.References:[1]Nakamura M et al., Mult Scler. 2014 Sep; 20(10):1371-80.[2]Strasser DS et al., RMD Open 2020;6:e001261.Disclosure of Interests:None declared

scholarly journals OP0186 CHANGES IN CIRCULATING B CELL LEVELS AND IMMUNOPHENOTYPE ARE ASSOCIATED WITH DEVELOPMENT OF ARTHRITIS FOLLOWING TREATMENT WITH CHECKPOINT INHIBITORS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 112.2-113
Author(s):  
M. Gatto ◽  
S. Bjursten ◽  
C. Jonell ◽  
C. Jonsson ◽  
S. Mcgrath ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Adverse Events ◽  
B Cell ◽  
Mononuclear Cells ◽  
Checkpoint Inhibitors ◽  
Therapy Monitoring ◽  
Cell Subset ◽  
Peripheral Blood Mononuclear ◽  
Transitional B Cells

Background:Inflammatory arthritis (IA) is frequent among rheumatic side effects induced by checkpoint inhibitor (CPI) therapy for metastatic malignancies1. While T cells are likely to sustain the inflammatory process2, fewer data are available concerning the role of B cells3.Objectives:To investigate the phenotype of circulating B cells in patients who develop CPI-induced IA (CPI-IA) and to compare it with features of B cells in patients not developing immune-related adverse events (irAE) upon CPI treatment.Methods:B cell subsets at baseline (before CPI initiation) and during CPI treatment were analyzed in CPI-IA patients and in patients receiving CPI but who did not develop irAE (non-irAE). Peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry and B cells were identified as CD19+ and divided into naïve (CD27-IgD+), memory (CD27+IgD+/-), double negative (CD27-IgD-) and transitional (CD10+CD24+CD38+/hi) B cells. Levels of CD21, an activation marker on transitional B cells, were also analyzed. Non-parametric tests were used for analysis of differences between groups.Results:Six CPI-IA and 7 non-irAE patients matched for age, gender and CPI treatment were included, who had received CPI treatment due to metastatic melanoma. Flow cytometry revealed a significant increase of circulating B cells (p=0.002) (Figure 1A) and especially of transitional B cells in CPI-IA patients vs. non-irAE (median %, range: 7.8 (4.5-11.4) vs. 3.2 (1.6-4.3),p=0.007) (Figure 1B), while no remarkable changes were seen across other subsets. Transitional B cell levels significantly decreased from active to quiescent CPI-IA in all patients (p=0.008). In two CPI-IA patients for whom baseline sampling was available, the increase of transitional levels occurred early after CPI treatment and before CPI-IA onset. Levels of expression of CD21 on transitional B cells were increased in CPI-IA vs. non-irAE (p=0.01).Conclusion:Transitional B cells are expanded in CPI-IA patients and seem to increase early after start of CPI therapy. Monitoring this B cell subset might lead to closer follow-up and earlier diagnosis of CPI-IA.References:[1]Ramos-Casals M, Brahmer JR, Callahan MK, et al. Immune-related adverse events of checkpoint inhibitors. Nat Rev Dis Primers 2020;6:38[2]Murray-Brown W, Wilsdon TD, Weedon H, et al. Nivolumab-induced synovitis is characterized by florid T cell infiltration and rapid resolution with synovial biopsy-guided therapy. J Immunother Cancer 2020;8:e000281[3]Das R, Bar N, Ferreira M, et al. Early B cell changes predict autoimmunity following combination immune checkpoint blockade. J Clin Invest. 2018;128:715-2Disclosure of Interests:None declared

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