CD40 stimulation activates ‘post-activated’ B Cells which are hyporesponsive to B Cell receptor and toll-like receptor 9 stimulation in autoimmunity

Autoimmunity arise when the immune system attacks self-antigens and destroys body’s own cells and tissues. Chronic autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and the primary Sjögren’s syndrome (pSS) decrease life quality of patients and can lead to life threatening events such as lupus nephritis [1]. Recognition of self-antigens by the B cell receptor (BCR), development of autoimmune memory and beneficial therapeutic approaches by targeting B cells highlight the pathogenic role of B cells in development and maintenance of autoimmunity [2-10]. Genotyping and genome wide association studies (GWAS) of B cells from autoimmune patients and functional studies in human and mice underline the role of altered BCR signaling and its contribution to autoimmunity [11-16]. Strength of the BCR signal together with co-signals such as cytokines, toll-like receptor (TLR) signaling and interaction with T cells determines cell fate [17-21]. However, the literature is conflicting on whether BCR signaling is reduced or increased [14-16]. Recently, our group reported unbalanced BCR signaling with significantly reduced phosphorylation of the protein tyrosine kinase (PTK) spleen tyrosine kinase (Syk) and reduced intracellular Ca2+ release in SLE B cells [14]. Further studies reported reduced responsiveness of SLE B cells to TLR9 stimulation which recognizes nuclear antigens and is considered key in self-tolerance brake to nuclear antigens together with the BCR [22-25]. T cell help by CD40-CD40L binding drives germinal center (GC) reaction and the development of long term (auto)-immune B cell memory and is further known to augment BCR signaling via Syk [26, 27]. To draw a more general picture of B cell activation in autoimmunity, this study aimed at comparatively analyzing the nature of peripheral B cell activation in different autoimmune diseases, mainly SLE, RA and pSS, with emphasis on BCR, TLR9 and CD40 signaling. To do so, BCR signaling quality was assessed in peripheral and tissue resident CD27- B cells and CD27+ memory B cells by ex vivo analysis of tonic and anti-IgG/IgM induced tyrosine (Y or Tyr) or serine (S or Ser) phosphorylation of the kinases Syk, Bruton’s tyrosine kinase (Btk) and protein kinase B (Akt). This was supplemented by data on protein tyrosine phosphatase (PTP) and protein serine/threonine phosphatase (PSP) activities and recruitment of the PTP Src homology region 2 (SH2) domain-containing phosphatase-1 (SHP-1). The relevance of cytokines, epigenetic methylation and chronic stimulation of TLR9 and the BCR was assessed. The potential of TLR9 and TLR9 together with BCR activation to induce proliferation and differentiation into antibody secreting cells (ASCs) was comparatively analyzed and finally, the impact of CD40/CD40L with and without interleukin (IL)-4 or IL-21 interaction on BCR-induced Syk(Y352) phosphorylation and in vitro proliferation and differentiation of B cells was determined. This was completed by expression data of selected PTPs and PSPs upon CD40/IL-4 co-stimulation. As a commonality, phosphorylation of the PTKs Syk(Y352) and Btk(Y223) was reduced in peripheral CD27+ memory B cells from SLE, RA and pSS patients in comparison to healthy donor (HD) controls. Notably, Syk(Y352) phosphorylation was diminished in peripheral SLE CD27- B cells compared to HD controls and in tissue resident CD27+ B cells from patients with immune thrombocytopenia (ITP) and pSS compared to non-autoimmune patients, too. However, Akt(S473) phosphorylation kinetics were increased in SLE CD27- and CD27+ B cells and comparable to HDs in pSS and RA B cells. PTP/PSP activities in SLE and overall baseline recruitment of SHP-1 to the negative co-receptor CD22 in SLE, RA and pSS peripheral CD19+ B cells were increased compared to HDs. Signaling abnormalities of SLE, RA and pSS B cells did not correlate to differences in tonic phosphorylation or baseline expression of signaling molecules (Syk, Btk, 1 Phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2 (PLCγ2) and Akt), cytokine expression/stimulation and epigenetic programming. However, chronic in vitro stimulation of the BCR led to reduced Syk(Y352) phosphorylation upon re-stimulation after 24 h, 48 h and 72 h. Proliferation and differentiation into CD27+CD38+ ASCs of SLE, RA and pSS peripheral CD19+ B cells was reduced upon in vitro TLR9 stimulation compared to HD controls. Combined BCR and TLR9 activation enhanced proliferation and differentiation of SLE, RA and pSS CD19+ B cells only to a certain degree. CD40 in vitro co-stimulation with and without IL-4 or IL-21 increased anti-IgG/IgM induced Syk(Y352) phosphorylation in SLE, RA, pSS and HD peripheral B cells. Interestingly, SLE CD27- B cells increased their Syk(Y352) phosphorylation to HD values. Furthermore, CD40 co-stimulation led to high proliferation of SLE, RA, pSS and HD CD19+ B cells. Improvements of CD40 co-stimulation correspond to reduced gene expression of non-receptor-type PTPs (NRPTPs) such as tyrosine-protein phosphatase non-receptor type 2 (PTPN2) and PTPN22, and several receptor-type PTPs (RPTPs) in HD as well as SLE CD19+ B cells upon CD40L/IL-4 co-stimulation. Contrary to the general understanding of B cell hyperreactivity in autoimmune diseases, this study found peripheral and tissue resident B cell from autoimmune patients being functionally impaired towards BCR and TLR9 stimulation due to increased negative regulation by PTPs such as SHP-1. However, the strongest phenotype was observed in SLE B cells. This reduced responsiveness may be functionally related to B cell anergy and likely reflects a ‘post-activation’ status due to chronic B cell activation rather than a general signaling abnormality. The results underline that communication of B and T cells via CD40/CD40L interaction might be key for activating ‘post-activated’ B cells in autoimmune diseases via downmodulation of PTPs. Therefore, new therapeutic approaches may target B cell activation via CD40 or negative regulation of PTPs via the BCR.

Author dc:creator

• Weißenberg, Sarah Yasmin

Advisor dc:contributor.advisor

• Dörner, Thomas

Licence dc:rights.uri

• https://creativecommons.org/licenses/by/4.0/

Language dc:language.iso

en

Identifier URI

http://dx.doi.org/10.14279/depositonce-11054

OAI identifier oai:identifier

oai:depositonce.tu-berlin.de:11303/12180