Allo-Specific Humoral Responses: New Methods for Screening Donor-Specific Antibody and Characterization of HLA-Specific Memory B Cells

Antibody-mediated allograft rejection (AMR) causes more kidney transplant failure than any other single cause. AMR is mediated by antibodies recognizing antigens expressed by the graft, and antibodies generated against major histocompatibility complex (MHC) mismatches are especially problematic. Most research directed towards the management of clinical AMR has focused on identifying and characterizing circulating donor-specific HLA antibody (DSA) and optimizing therapies that reduce B-cell activation and/or block antibody secretion by inhibiting plasmacyte survival. Here we describe a novel set of reagents and techniques to allow more specific measurements of MHC sensitization across different animal transplant models. Additionally, we have used these approaches to isolate and clone individual HLA-specific B cells from patients sensitized by pregnancy or transplantation. We have identified and characterized the phenotypes of individual HLA-specific B cells, determined the V(D)J rearrangements of their paired H and L chains, and generated recombinant antibodies to determine affinity and specificity. Knowledge of the BCR genes of individual HLA-specific B cells will allow identification of clonally related B cells by high-throughput sequence analysis of peripheral blood mononuclear cells and permit us to re-construct the origins of HLA-specific B cells and follow their somatic evolution by mutation and selection.

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Activation and Proliferation of B Cell Chronic Lymphocytic Leukaemia (B-CLL) Cells through Anti-CD180, Anti-CD40 and IL-4.

Blood ◽

10.1182/blood.v104.11.4775.4775 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4775-4775

Author(s):

Nino Porakishvili ◽

Maria Manoussaka ◽

Nino Kulikova ◽

James Walton ◽

Amit Nathwani ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Normal Control ◽

Cell Activation ◽

Mononuclear Cells ◽

Leukemic Cells ◽

B Cell Activation ◽

Cell Cycle Protein ◽

Cd40 Ligation ◽

Ki 67

Abstract Introduction: We have previously shown that Toll-like receptor RP105 (CD180) is heterogeneously expressed on B-CLL cells and that the ligation of CD180 by monoclonal antibodies (mAb) on CD180+ B-CLL cells resulted in delineation of responder and non-responder B-CLL clones [1]. In this study we have examined the role of IL-4 together with CD180 and CD40 as activation signals. Methods: Blood mononuclear cells were separated from 7 responder B-CLL patients with both mutated and unmutated Ig Vh genes and 11 controls and were cultured for 72 hours in optimum concentrations of anti-CD180 (G28-8) or anti-CD40 mAb or both in presence and absence of 15 ng/ml of IL-4. CD19+ B cells were stained with mAb to the activation marker CD86 or cell cycle protein Ki-67, measured by flow cytometry and expressed as Mean Fluorescence Intensity (MFI) or % of Ki-67+ cells. Results: B-CLL cells and normal control B cells responded to CD180-ligation by activation and proliferation (Table). Higher levels of CD86 and Ki67 were detected when both anti CD40 mAb and anti CD180 mAb were added (p<0.05) compared with either alone. IL4 alone induced both activation and proliferation of control cells and this was even higher with the leukemic cells (p<0.01) confirming that IL-4 also provides a strong survival/activatory stimulus for B-CLL cells. Addition of IL-4 had no significant enhancing effect on normal B-cells stimulated with both anti-CD180 and anti-CD40, although IL-4 synergised with anti-CD40 in B cell activation (p=0.026) and with CD180 in B cell proliferation (p=0.044). Conclusion: CD180 had an additive effect with CD40 ligation in activation and proliferation of both B-CLL cells and normal control B cells. IL-4 provides a strong additional stimulus for B-CLL cells. CD86 and Ki-67 expression by CD19+ cells CD86 Ki67 B-CLL Control B-CLL control Spontaneous −IL-4 7.2±4.1 4.0±1.0 8.1±2.7 17.4±2.3 +IL-4 22.4±11.8 11.2±4.2 17.0±12.8 23.6±14.8 CD180 −IL-4 14.7±5.8 26.9±13.0 17.1±10.9 28.1±12.0 +IL-4 35.4±14.5 30.0±14.6 26.9±25.6 48.3±9.7 CD40 −IL-4 19.4±8.8 14.8±5.2 14.9±9.7 34.0±5.1 +IL-4 286±145.5 44.0±19.4 52.7±22.8 41.0±19.8 CD180+CD40 −IL-4 32.5±12.6 97.0±26.2 28.0±10.4 65.5±26.6 +IL-4 299±163.2 63.5±27.4 49.3±14.6 55.5±26.4

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Hantavirus infection-induced B cell activation elevates free light chains levels in circulation

PLoS Pathogens ◽

10.1371/journal.ppat.1009843 ◽

2021 ◽

Vol 17 (8) ◽

pp. e1009843

Author(s):

Jussi Hepojoki ◽

Luz E. Cabrera ◽

Satu Hepojoki ◽

Carla Bellomo ◽

Lauri Kareinen ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Activation ◽

Mononuclear Cells ◽

Cell Clone ◽

Light Chains ◽

Hantavirus Infection ◽

Puumala Virus ◽

B Cell Activation ◽

Free Light Chains

In humans, orthohantaviruses can cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). An earlier study reported that acute Andes virus HPS caused a massive and transient elevation in the number of circulating plasmablasts with specificity towards both viral and host antigens suggestive of polyclonal B cell activation. Immunoglobulins (Igs), produced by different B cell populations, comprise heavy and light chains; however, a certain amount of free light chains (FLCs) is constantly present in serum. Upregulation of FLCs, especially clonal species, associates with renal pathogenesis by fibril or deposit formations affecting the glomeruli, induction of epithelial cell disorders, or cast formation in the tubular network. We report that acute orthohantavirus infection increases the level of Ig FLCs in serum of both HFRS and HPS patients, and that the increase correlates with the severity of acute kidney injury in HFRS. The fact that the kappa to lambda FLC ratio in the sera of HFRS and HPS patients remained within the normal range suggests polyclonal B cell activation rather than proliferation of a single B cell clone. HFRS patients demonstrated increased urinary excretion of FLCs, and we found plasma cell infiltration in archival patient kidney biopsies that we speculate to contribute to the observed FLC excreta. Analysis of hospitalized HFRS patients’ peripheral blood mononuclear cells showed elevated plasmablast levels, a fraction of which stained positive for Puumala virus antigen. Furthermore, B cells isolated from healthy donors were susceptible to Puumala virus in vitro, and the virus infection induced increased production of Igs and FLCs. The findings propose that hantaviruses directly activate B cells, and that the ensuing intense production of polyclonal Igs and FLCs may contribute to acute hantavirus infection-associated pathological findings.

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Intrathecal B-cell activation in LGI1 antibody encephalitis

Neurology Neuroimmunology & Neuroinflammation ◽

10.1212/nxi.0000000000000669 ◽

2020 ◽

Vol 7 (2) ◽

pp. e669 ◽

Cited By ~ 2

Author(s):

Klaus Lehmann-Horn ◽

Sarosh R. Irani ◽

Shengzhi Wang ◽

Arumugam Palanichamy ◽

Sarah Jahn ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Activation ◽

Mononuclear Cells ◽

Cell Activity ◽

Oligoclonal Bands ◽

B Cell Activation ◽

Cell Repertoire ◽

Antibody Titers ◽

B Cell Subsets

ObjectiveTo study intrathecal B-cell activity in leucine-rich, glioma-inactivated 1 (LGI1) antibody encephalitis. In patients with LGI1 antibodies, the lack of CSF lymphocytosis or oligoclonal bands and serum-predominant LGI1 antibodies suggests a peripherally initiated immune response. However, it is unknown whether B cells within the CNS contribute to the ongoing pathogenesis of LGI1 antibody encephalitis.MethodsPaired CSF and peripheral blood (PB) mononuclear cells were collected from 6 patients with LGI1 antibody encephalitis and 2 patients with other neurologic diseases. Deep B-cell immune repertoire sequencing was performed on immunoglobulin heavy chain transcripts from CSF B cells and sorted PB B-cell subsets. In addition, LGI1 antibody levels were determined in CSF and PB.ResultsSerum LGI1 antibody titers were on average 127-fold higher than CSF LGI1 antibody titers. Yet, deep B-cell repertoire analysis demonstrated a restricted CSF repertoire with frequent extensive clusters of clonally related B cells connected to mature PB B cells. These clusters showed intensive mutational activity of CSF B cells, providing strong evidence for an independent CNS-based antigen-driven response in patients with LGI1 antibody encephalitis but not in controls.ConclusionsOur results demonstrate that intrathecal immunoglobulin repertoire expansion is a feature of LGI1 antibody encephalitis and suggests a need for CNS-penetrant therapies.

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Rheumatoid Factors Induce Signaling from B Cells, Leading to Epstein-Barr Virus and B-Cell Activation

Journal of Virology ◽

10.1128/jvi.78.18.9918-9923.2004 ◽

2004 ◽

Vol 78 (18) ◽

pp. 9918-9923 ◽

Cited By ~ 12

Author(s):

Lixin Yang ◽

Masayuki Hakoda ◽

Kazuya Iwabuchi ◽

Tsuyoshi Takeda ◽

Takao Koike ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Cell Activation ◽

Mononuclear Cells ◽

Rheumatoid Factors ◽

B Cell Activation ◽

Barr Virus ◽

Membrane Bound ◽

Epstein Barr

ABSTRACT B-cell antigen receptor signaling is initiated upon binding of the antigen to membrane-bound immunoblobulin (Ig), and the anti-Ig antibody (Ab) mimics this signaling. In B cells latently infected with Epstein-Barr virus (EBV), the same signals induce virus activation. We examine here whether rheumatoid factors (RFs), autoantibodies directed against the Fc portion of IgG, induce EBV and B-cell activation. As a source of RFs, RF-producing lymphoblastoid cell line (LCL) clones were isolated from peripheral blood mononuclear cells (PBMC) and synovial cells from patients with rheumatoid arthritis (RA) by EBV transformation. Burkitt's lymphoma-derived Akata cells, which are highly responsive to EBV activation by anti-Ig Abs, were used for the assay of EBV activation. Akata cells expressed IgG3 as membrane-bound Ig. RFs from a synovium-derived LCL were directed to IgG3 and induced EBV activation in 16 to 18% of Akata cells, whereas RFs from another synovium-derived LCL were directed to IgG1 and did not induce EBV activation. Pretreatment of RFs with the purified Fc fragment of human IgG completely abolished EBV activation. Furthermore, B-cell activation was assessed by incorporation of [3H]thymidine. RFs from synovium-derived LCLs efficiently induced B-cell activation, and the addition of CD40 ligand had a synergistic effect. On the other hand, RFs from PBMC-derived LCLs were polyreactive, had a lower affinity to IgG, and did not induce EBV and B-cell activation. The present findings imply a possible role for RFs as EBV and B-cell activators.

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Cell type-specific immune dysregulation in severely ill COVID-19 patients

10.1101/2020.07.23.20161182 ◽

2020 ◽

Author(s):

Changfu Yao ◽

Stephanie A Bora ◽

Tanyalak Parimon ◽

Tanzira Zaman ◽

Oren A Friedman ◽

...

Keyword(s):

Immune Response ◽

Cell Activation ◽

Mononuclear Cells ◽

Severe Disease ◽

Ventilatory Support ◽

Adaptive Immune Response ◽

Disease Course ◽

B Cell Activation ◽

Peripheral Blood Mononuclear ◽

Mild Disease

Coronavirus disease 2019 (COVID-19) has quickly become the most serious pandemic since the 1918 flu pandemic. In extreme situations, patients develop a dysregulated inflammatory lung injury called acute respiratory distress syndrome (ARDS) that causes progressive respiratory failure requiring mechanical ventilatory support. Recent studies have demonstrated immunologic dysfunction in severely ill COVID-19 patients. To further delineate the dysregulated immune response driving more severe clinical course from SARS-CoV-2 infection, we used single-cell RNA sequencing (scRNAseq) to analyze the transcriptome of peripheral blood mononuclear cells (PBMC) from hospitalized COVID-19 patients having mild disease (n = 5), developing ARDS (n = 6), and recovering from ARDS (n = 6). Our data demonstrated an overwhelming inflammatory response with select immunodeficiencies within various immune populations in ARDS patients. Specifically, their monocytes had defects in antigen presentation and deficiencies in interferon responsiveness that contrasted the higher interferon signals in lymphocytes. Furthermore, cytotoxic activity was suppressed in both NK and CD8 lymphocytes whereas B cell activation was deficient, which is consistent with the delayed viral clearance in severely ill COVID-19 patients. Finally, we identified altered signaling pathways in the severe group that suggests immunosenescence and immunometabolic changes could be contributing to the dysfunctional immune response. Our study demonstrates that COVID-19 patients with ARDS have an immunologically distinct response when compared to those with a more innocuous disease course and show a state of immune imbalance in which deficiencies in both the innate and adaptive immune response may be contributing to a more severe disease course in COVID-19.

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IL-21 in Conjunction with Anti-CD40 and IL-4 Constitutes a Potent Polyclonal B Cell Stimulator for Monitoring Antigen-Specific Memory B Cells

Cells ◽

10.3390/cells9020433 ◽

2020 ◽

Vol 9 (2) ◽

pp. 433

Author(s):

Fridolin Franke ◽

Greg A. Kirchenbaum ◽

Stefanie Kuerten ◽

Paul V. Lehmann

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

Cell Activation ◽

Immune Monitoring ◽

Memory B Cells ◽

B Cell Activation ◽

Cell Stimulation ◽

Additional Advantage ◽

Specific Memory

Detection of antigen-specific memory B cells for immune monitoring requires their activation, and is commonly accomplished through stimulation with the TLR7/8 agonist R848 and IL-2. To this end, we evaluated whether addition of IL-21 would further enhance this TLR-driven stimulation approach; which it did not. More importantly, as most antigen-specific B cell responses are T cell-driven, we sought to devise a polyclonal B cell stimulation protocol that closely mimics T cell help. Herein, we report that the combination of agonistic anti-CD40, IL-4 and IL-21 affords polyclonal B cell stimulation that was comparable to R848 and IL-2 for detection of influenza-specific memory B cells. An additional advantage of anti-CD40, IL-4 and IL-21 stimulation is the selective activation of IgM+ memory B cells, as well as the elicitation of IgE+ ASC, which the former fails to do. Thereby, we introduce a protocol that mimics physiological B cell activation through helper T cells, including induction of all Ig classes, for immune monitoring of antigen-specific B cell memory.

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STAT3 phosphorylation mediates the stimulatory effects of interferon alpha on B cell differentiation and activation in SLE

Rheumatology ◽

10.1093/rheumatology/kez354 ◽

2019 ◽

Cited By ~ 1

Author(s):

Aurélie De Groof ◽

Julie Ducreux ◽

Floor Aleva ◽

Andrew J Long ◽

Alina Ferster ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Cell Activation ◽

Mononuclear Cells ◽

Type I ◽

B Cell Differentiation ◽

B Cell Activation ◽

Loss Of Function ◽

Stat3 Phosphorylation

Abstract Objective Type I IFNs play a well-known role in the pathogenesis of SLE, through activation of CD4 T and antigen-presenting cells. Here, we investigated the effects of IFN alpha (IFNα) on SLE B cell activation and differentiation. Methods Peripheral blood mononuclear cells (PBMCs) and purified total or naïve B cells were obtained from healthy controls and SLE patients. The effects of IFNα on B cell differentiation were studied by flow cytometry. The role of STAT3 in B cell responses to IFNα was studied using pharmacological inhibitors and PBMCs from STAT3-deficient individuals. Results Incubation of normal PBMCs with IFNα induces a B cell differentiation pattern as observed spontaneously in SLE PBMCs. IFNα displays direct stimulatory effects on purified naïve B cells from healthy individuals, as evidenced by a significant induction of cell surface CD38 and CD95 in the presence of the cytokine. In purified naïve B cells, IFNα also induces STAT3 phosphorylation. IFNα-induced naïve B cell differentiation in total PBMCs is significantly inhibited in the presence of STAT3 inhibitors, or in PBMCs from individuals with STAT3 loss of function mutations. Spontaneous levels of STAT3, but not STAT1, phosphorylation are significantly higher in total B cells from SLE patients compared with controls. Pharmacological STAT3 inhibition in SLE PBMCs inhibits naïve B cell activation and differentiation. Conclusion IFNα displays direct stimulatory effects on B cell differentiation and activation in SLE. STAT3 phosphorylation mediates the effects of IFNα stimulation in naïve B cells, an observation that opens new therapeutic perspectives in SLE.

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In Vitro Characterization of Human CD24hiCD38hi Regulatory B Cells Shows CD9 Is Not a Stable Breg Cell Marker

International Journal of Molecular Sciences ◽

10.3390/ijms22094583 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4583

Author(s):

Fatin N. Mohd Jaya ◽

Sergio G. Garcia ◽

Francesc E. Borras ◽

Dolores Guerrero ◽

Godfrey C. F. Chan ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Activation ◽

B Cell Activation ◽

Functional Studies ◽

In Vitro Characterization ◽

Reliable Marker ◽

The Stability

Regulatory B (Breg) cells are endowed with immune suppressive functions. Various human and murine Breg subtypes have been reported. While interleukin (IL)-10 intracellular staining remains the most reliable way to identify Breg cells, this technique hinders further essential functional studies. Recent findings suggest that CD9 is an effective surface marker of murine IL-10 competent Breg cells. However, the stability of CD9 and its relevance as a unique marker for human Breg cells, which have been widely characterized as CD24hiCD38hi, have not been investigated. Here, we demonstrate that CD9 expression is sensitive to in vitro B cell stimulations. CD9 expression could either be re-expressed or downregulated in purified CD9-negative B cells and CD9-positive B cells, respectively. We found no significant differences in the Breg differentiation capacity of the CD9-negative and CD9-positive B cells. Furthermore, CD9-positive B cells co-express CD40 and CD86, suggesting their nature as B cell activation or co-stimulatory molecules, rather than regulatory ones. Therefore, we report the relatively unstable CD9 as a distinct surface molecule, indicating the need for further research for a more reliable marker to purify human Breg cells.

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