Memory B Cells Activate Brain-Homing, Autoreactive CD4+ T Cells in Multiple Sclerosis

Background:Neutrophils have been described as potent antigen-presenting cells able to activate T cells by MHC/TCR interaction and costimulatory molecules in tumor immunity. However, little is known about the direct interaction between neutrophils and CD4 T cells with respect to systemic lupus erythematosus (SLE). We have previously shown that OX40L expressed by monocytes from SLE patients promote the differentiation of naïve and memory cells into IL21 secreting T cells that are able to help B cells1,2.Objectives:In this study, we investigate OX40L expression on neutrophils from SLE patients and contribution of these OX40L+neutrophils in SLE pathogenesis to modulation of the B cell helper role of CD4 T cells.Methods:Surface expression of co-stimulatory molecules (OX40L, ICOSL, GITRL, 4-1BBL) on neutrophils from SLE patients and healthy donors (HD) was measured by flow cytometry (FC). Neutrophils from HD were stimulated with TLR7 or TLR8 agonists and IFNα after 5 hours of culture, OX40L expression was measured by FC and Western Blotting. CD4 T cells were cultured with the stimulated neutrophils for 3 days. At the end of the co-culture, percentages of IL21-expressing T follicular (Tfh) and peripheral helper (Tph) cells measured by FC. These generated T cells were also cultured in the presence of memory B cells. After 5 days of co-culture, plasmablast generation and Ig levels were assessed by FC and ELISA, respectively. Inhibition of OX40-OX40L interaction in vitro was achieved using ISB 830, a novel anti-OX40 mAb currently used in clinical trials.Results:Among the co-stimulatory molecules tested, percentages of OX40L+neutrophils in SLE (n=54) were increased compared to HD (n=25)(mean + SD: HD = 1,34%±1.62 vs SLE = 4,53%±8.1; p=0.29). OX40L expression positively correlated with SLE disease activity score (SLEDAI) (p = 0,04; r = 0,31) and with anti-DNA antibodies (p= 0,04, r = 0,33). Of note, the percentage of OX40L+neutrophils was higher in anti-sm-RNP+patients (n=16, mean= 9%±9.8), compared to anti-sm-RNP-patients (n=27, mean = 1,4%±2.5; p = 0,02). The percentage of OX40L+neutrophils was higher in patients with class III or IV lupus nephritis, and inflammatory infiltrate within the kidney biopsy disclosed OX40L+neutrophils, in close contact with T cells. Neutrophils from HD express OX40L with TLR8 agonist, or IFNα priming followed by TLR7 agonist. When memory CD4 T cells were cultured in the presence of TLR8-stimulated neutrophils, the proportion of IL21-expressing Tfh (CXCR5+PD1+) and Tph (CXCR5-PD1hi) were increased, compared to culture with unstimulated neutrophils. This process was dependent on OX40-OX40L interactions, since in vitro treatment with the anti-OX40 blocking antibody ISB 830, inhibited the differentiation of memory T cells into Tfh and Tph. Both generated Tfh and Tph were able to promote the differentiation of memory B cells into Ig-secreting plasmablasts.Conclusion:Our results disclose an unprecedented phenomenon where cross-talk between TLR7/8-activated neutrophils and CD4 lymphocytes operates through OX40L-OX40 costimulation, and neutrophils promote the differentiation of pro-inflammatory Tfh and Tph, as well as IL21 production. Therefore, OX40L/OX40 should be considered as a potentially therapeutic axis in SLE patients.References:[1]Jacquemin et al. Immunity 2015;[2]Jacquemin et al. JCI Insight 2018Disclosure of Interests:Angela Pappalardo Grant/research support from: Ichnos Sciences, Elodie Wojciechowski: None declared, Itsaso Odriozola: None declared, Isabelle Douchet: None declared, Nathalie Merillon: None declared, Andrea Boizard-Moracchini: None declared, Pierre Duffau: None declared, Estibaliz Lazaro: None declared, Marie-Agnes Doucey Employee of: Ichnos Sciences, Lamine Mbow Employee of: Ichnos Sciences, Christophe Richez Consultant of: Abbvie, Amgen, Mylan, Pfizer, Sandoz and UCB., Patrick Blanco Grant/research support from: Ichnos Sciences

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B cells are capable of independently eliciting rapid reactivation of encephalitogenic CD4 T cells in a murine model of multiple sclerosis

PLoS ONE ◽

10.1371/journal.pone.0199694 ◽

2018 ◽

Vol 13 (6) ◽

pp. e0199694 ◽

Cited By ~ 7

Author(s):

Chelsea R. Parker Harp ◽

Angela S. Archambault ◽

Julia Sim ◽

Mark J. Shlomchik ◽

John H. Russell ◽

...

Keyword(s):

Multiple Sclerosis ◽

T Cells ◽

B Cells ◽

Murine Model ◽

Cd4 T Cells

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Vaccine-elicited CD4 T cells prevent the deletion of antiviral B cells in chronic infection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2108157118 ◽

2021 ◽

Vol 118 (46) ◽

pp. e2108157118

Author(s):

Kerstin Narr ◽

Yusuf I. Ertuna ◽

Benedict Fallet ◽

Karen Cornille ◽

Mirela Dimitrova ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cd4 T Cells ◽

Memory B Cells ◽

Type I ◽

Clonal Deletion ◽

Cell Immunity ◽

B Cell Immunity

Chronic viral infections subvert protective B cell immunity. An early type I interferon (IFN-I)–driven bias to short-lived plasmablast differentiation leads to clonal deletion, so-called “decimation,” of antiviral memory B cells. Therefore, prophylactic countermeasures against decimation remain an unmet need. We show that vaccination-induced CD4 T cells prevented the decimation of naïve and memory B cells in chronically lymphocytic choriomeningitis virus (LCMV)-infected mice. Although these B cell responses were largely T independent when IFN-I was blocked, preexisting T help assured their sustainability under conditions of IFN-I–driven inflammation by instructing a germinal center B cell transcriptional program. Prevention of decimation depended on T cell–intrinsic Bcl6 and Tfh progeny formation. Antigen presentation by B cells, interactions with antigen-specific T helper cells, and costimulation by CD40 and ICOS were also required. Importantly, B cell–mediated virus control averted Th1-driven immunopathology in LCMV-challenged animals with preexisting CD4 T cell immunity. Our findings show that vaccination-induced Tfh cells represent a cornerstone of effective B cell immunity to chronic virus challenge, pointing the way toward more effective B cell–based vaccination against persistent viral diseases.

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Ectopic Lymphoid Follicles in Multiple Sclerosis: Centers for Disease Control?

Frontiers in Neurology ◽

10.3389/fneur.2020.607766 ◽

2020 ◽

Vol 11 ◽

Author(s):

Austin Negron ◽

Olaf Stüve ◽

Thomas G. Forsthuber

Keyword(s):

Multiple Sclerosis ◽

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

T Cell Responses ◽

Memory B Cells ◽

Lymphoid Follicles ◽

Cell Responses ◽

B Cell Responses

While the contribution of autoreactive CD4+ T cells to the pathogenesis of Multiple Sclerosis (MS) is widely accepted, the advent of B cell-depleting monoclonal antibody (mAb) therapies has shed new light on the complex cellular mechanisms underlying MS pathogenesis. Evidence supports the involvement of B cells in both antibody-dependent and -independent capacities. T cell-dependent B cell responses originate and take shape in germinal centers (GCs), specialized microenvironments that regulate B cell activation and subsequent differentiation into antibody-secreting cells (ASCs) or memory B cells, a process for which CD4+ T cells, namely follicular T helper (TFH) cells, are indispensable. ASCs carry out their effector function primarily via secreted Ig but also through the secretion of both pro- and anti-inflammatory cytokines. Memory B cells, in addition to being capable of rapidly differentiating into ASCs, can function as potent antigen-presenting cells (APCs) to cognate memory CD4+ T cells. Aberrant B cell responses are prevented, at least in part, by follicular regulatory T (TFR) cells, which are key suppressors of GC-derived autoreactive B cell responses through the expression of inhibitory receptors and cytokines, such as CTLA4 and IL-10, respectively. Therefore, GCs represent a critical site of peripheral B cell tolerance, and their dysregulation has been implicated in the pathogenesis of several autoimmune diseases. In MS patients, the presence of GC-like leptomeningeal ectopic lymphoid follicles (eLFs) has prompted their investigation as potential sources of pathogenic B and T cell responses. This hypothesis is supported by elevated levels of CXCL13 and circulating TFH cells in the cerebrospinal fluid (CSF) of MS patients, both of which are required to initiate and maintain GC reactions. Additionally, eLFs in post-mortem MS patient samples are notably devoid of TFR cells. The ability of GCs to generate and perpetuate, but also regulate autoreactive B and T cell responses driving MS pathology makes them an attractive target for therapeutic intervention. In this review, we will summarize the evidence from both humans and animal models supporting B cells as drivers of MS, the role of GC-like eLFs in the pathogenesis of MS, and mechanisms controlling GC-derived autoreactive B cell responses in MS.

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Immune Tolerance Developed in Platelet-Targeted FVIII Gene Therapy in Hemophilia Mice Is CD4+ T Cell-Mediated

Blood ◽

10.1182/blood.v126.23.1071.1071 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1071-1071

Author(s):

Yingyu Chen ◽

Xiaofeng Luo ◽

Juan Chen ◽

Jocelyn Schroeder ◽

Christina K Baumgartner ◽

...

Keyword(s):

Gene Therapy ◽

T Cells ◽

T Cell ◽

B Cells ◽

Immune Tolerance ◽

Cd4 T Cells ◽

Memory B Cells ◽

Cd4 T Cell ◽

Control Group

Abstract Immune response to factor VIII (FVIII) is not only a severe complication in protein replacement therapy, but also a major concern in gene therapy of hemophilia A. Our previous studies have demonstrated that platelet-targeted FVIII (2bF8) gene therapy together with in vivo drug-selection of transduced cells can not only rescue the bleeding diathesis but also induce anti-FVIII specific immune tolerance in FVIIInull mice. In the current study, we investigated 1) whether our non-selectable lentiviral vector (LV) for the induction of platelet-FVIII expression is sufficient to induce immune tolerance and 2) which cell compartment is tolerized after platelet gene therapy. Platelet-specific FVIII expression was introduced by 2bF8LV-transduction of hematopoietic stem cells followed by syngeneic transplantation into FVIIInull mice preconditioned with 660 cGy total body irradiation (TBI) or Busulfan (Bu) plus ATG (anti-thymocyte globulin). After bone marrow transplantation and reconstitution, animals were analyzed by PCR, qPCR, FVIII:C assay, and tail clipping test to confirm the success of 2bF8 gene therapy. Sixteen weeks after transplantation, animals were challenged with recombinant human FVIII (rhF8) via retro-orbital venous administration at a dose of 50 U/kg weekly for 4 weeks. The titers of anti-FVIII inhibitory antibodies (inhibitors) were determined by Bethesda assay. The CFSE-labeled CD4 T cell proliferation assay and ELISPOT-based memory B cell maturation assay were used to determine which cell compartment is tolerized to FVIII after 2bF8 gene therapy. The level of platelet-FVIII expression was 1.44 ± 0.39 mU/108 platelets (n = 6) in the 660 cGy group, which is not significantly different from the level obtained from the Bu+ATG group [3.04 ± 1.19 mU/108 platelets (n = 6)]. Even after rhF8 challenge, no antibodies were detected in 2bF8LV-transduced recipients in either group. In contrast, all animals in the control group that did not undergo gene therapy developed various levels of inhibitors (204±97 BU/ml, n=7). The frequency of regulatory T cells in both 660 cGy TBI (11.01±0.52%) and Bu+ATG (11.02±0.68%) groups were significantly higher than the control group (8.05±0.57%). T cell proliferation assay demonstrated that CD4+ T cells from 2bF8 LV-transduced recipients that had been challenged with rhF8 did not proliferate when restimulated with rhF8 in vitro. The daughter CD4+ T cells in the group with 10 U/ml of rhF8 were 5.84 ± 2.49% (n = 6), which was not significantly different from the control group without no rhF8 stimulation (0 U/ml) (5.33 ± 1.72%). CD4+ T cells from primed FVIIInull mice did proliferate after rhF8 restimulation. The proliferated daughter cell was 13.12 ± 6.76% (n = 7) in the group with rhF8 (10 U/ml) re-stimulation, which is significantly higher than the group without rhF8 co-culture (4.99 ± 1.16%). Since FVIII-specific memory B cell maturation is CD4+ T cell dependent, we isolated CD4+ T and memory B cells from 2bF8LV-transduced or FVIIInull mice after rhF8 immunization and co-cultured with rhF8 followed by ELISPOT assay to examine the antibody secreting cells. No spots were detected when memory B cells from rhF8-primed FVIIInull mice were co-cultured with CD4+ T cells from 2bF8LV-transduced recipients. In contrast, when memory B cells from either rhF8 immunized 2bF8LV-transduced or untreated FVIIInull mice were cultured with CD4+ T cells from rhF8-primed FVIIInull mice, there were 142 and 205 anti-FVIII antibody secreting cells, respectively, detected per 106 cells seeded. These results indicate that CD4+ T cells from 2bF8LV-transduced mice are tolerized to rhF8 stimulation. In conclusion, 2bF8 lentiviral gene transfer without in vivo selection of genetically manipulated cells can introduce FVIII-specific immune tolerance in hemophilia A mice and this immune tolerance is CD4+ T cell-mediated. Disclosures Baumgartner: Novo Nordisk: Research Funding. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETTED GENE EXPRESSION..

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Single-Cell Analysis of FVIII-Specific Memory B Cells in Murine Hemophilia A.

Blood ◽

10.1182/blood.v110.11.1157.1157 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1157-1157 ◽

Cited By ~ 1

Author(s):

Christina Hausl ◽

Rafi U. Ahmad ◽

Bernhard Baumgartner ◽

Hans Peter Schwarz ◽

Hartmut Ehrlich ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Single Cell ◽

Cd4 T Cells ◽

Single Cell Analysis ◽

Memory B Cells ◽

Cell Analysis ◽

Memory B Cell ◽

Specific Memory

Abstract The elimination of FVIII-specific memory B cells is an essential step in the design of new therapeutic strategies for the induction of immune tolerance in hemophilia A with FVIII inhibitors. Using a mouse model of hemophilia A we recently reported that low dose FVIII stimulates the differentiation of FVIII-specific memory B cells into antibody-secreting plasma cells whereas high dose FVIII inhibits this process. The inhibition of memory-B-cell re-stimulation is irreversible and seems to be due to an induction of apoptosis. Further understanding of the complex interactions that lead to either re-stimulation and differentiation of memory B cells or inhibition and eradication of these cells requires appropriate technologies for single-cell analysis and functional studies. We established a new technology for single-cell analysis and cell sorting of FVIII-specific murine memory B cells. A combination of magnetic bead separation and multi-color flow cytometry enabled us to analyze and purify FVIII-specific memory B cells obtained from hemophilic mice treated with FVIII. In a first step, we depleted undesirable cell populations (IgM+, IgD+, CD11c+, F4/80+, Gr1+ and CD49b+ cells) from total spleen cells by magnetic bead separation. In a second step, we used multicolor flow cytometry to exclude CD4+ T cells and analyze the FVIII-specific memory B cell compartment. This compartment was specified by staining the specific B-cell receptor with FVIII and anti-IgG antibodies. Frequencies of cells in this compartment ranged from 0.1–0.5% of total spleen cells in animals treated with 4 intravenous doses of FVIII, given at weekly intervals. We could not detect any FVIII-specific memory B cells in naïve mice. By means of single cell sorting we isolated FVIII-specific memory B cells for further functional studies. We were able to cultivate FVIII-specific memory B cells in microwell cultures in vitro and differentiate them into antibody-secreting plasma cells. The re-stimulation and differentiation of single-cell sorted memory B cells was strictly dependent on the presence of activated CD4+ T cells. CD4+ T cells obtained from naïve mice did not support the memory response. Furthermore, the re-stimulation and differentiation of memory B cells in the presence of activated CD4+ T cells did not require additional dendritic cells for antigen presentation. Obviously, memory B cells provide sufficient antigen presentation to CD4+ T cells to enable them to trigger the memory response. Our approach for single-cell analysis and purification of FVIII-specific memory B cells provides a new tool for tracking memory B cell populations in vivo and for directly analyzing the regulation of memory B cell function. It opens the field for future studies which should elucidate signals and molecules involved in activation or inhibition and eradication of FVIII-specific memory B cells. These activities will eventually lead to the identification of targets for the design of new treatment strategies for patients with FVIII inhibitors.

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Induction of Plasmodium falciparum-Specific CD4+ T Cells and Memory B Cells in Gabonese Children Vaccinated with RTS,S/AS01E and RTS,S/AS02D

PLoS ONE ◽

10.1371/journal.pone.0018559 ◽

2011 ◽

Vol 6 (4) ◽

pp. e18559 ◽

Cited By ~ 33

Author(s):

Selidji T. Agnandji ◽

Rolf Fendel ◽

Michaël Mestré ◽

Michel Janssens ◽

Johan Vekemans ◽

...

Keyword(s):

T Cells ◽

Plasmodium Falciparum ◽

B Cells ◽

Cd4 T Cells ◽

Memory B Cells

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CD4+T Cells Promote Antibody Production but Not Sustained Affinity Maturation during Borrelia burgdorferi Infection

Infection and Immunity ◽

10.1128/iai.02471-14 ◽

2014 ◽

Vol 83 (1) ◽

pp. 48-56 ◽

Cited By ~ 18

Author(s):

Rebecca A. Elsner ◽

Christine J. Hastey ◽

Nicole Baumgarth

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Cd4 T Cells ◽

Plasma Cells ◽

Affinity Maturation ◽

Memory B Cells ◽

Antibody Responses ◽

Content Type ◽

High Affinity

CD4 T cells are crucial for enhancing B cell-mediated immunity, supporting the induction of high-affinity, class-switched antibody responses, long-lived plasma cells, and memory B cells. Previous studies showed that the immune response toBorrelia burgdorferiappears to lack robust T-dependent B cell responses, as neither long-lived plasma cells nor memory B cells form for months after infection, and nonswitched IgM antibodies are produced continuously during this chronic disease. These data prompted us to evaluate the induction and functionality ofB. burgdorferiinfection-induced CD4 TFHcells. We report that CD4 T cells were effectively primed and TFHcells induced afterB. burgdorferiinfection. These CD4 T cells contributed to the control ofB. burgdorferiburden and supported the induction ofB. burgdorferi-specific IgG responses. However, while affinity maturation of antibodies against a prototypic T-dependentB. burgdorferiprotein, Arthritis-related protein (Arp), were initiated, these increases were reversed later, coinciding with the previously observed involution of germinal centers. The cessation of affinity maturation was not due to the appearance of inhibitory or exhausted CD4 T cells or a strong induction of regulatory T cells.In vitroT-B cocultures demonstrated that T cells isolated fromB. burgdorferi-infected but notB. burgdorferi-immunized mice supported the rapid differentiation of B cells into antibody-secreting plasma cells rather than continued proliferation, mirroring the induction of rapid short-lived instead of long-lived T-dependent antibody responsesin vivo. The data further suggest thatB. burgdorferiinfection drives the humoral response away from protective, high-affinity, and long-lived antibody responses and toward the rapid induction of strongly induced, short-lived antibodies of limited efficacy.

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