Background/Purpose:

Autoantibody production is a hallmark of Systemic Lupus Erythematosus (SLE), supporting a central role for B cells in disease pathogenesis. Prior studies have demonstrated augmented B cell receptor (BCR) signaling in a subset of SLE patients relative to healthy controls (HC). Both B cell intrinsic factors such as aberrant signal network wiring and B cell extrinsic factors such as excessive cytokines have been implicated in loss of B cell tolerance and B cell hyperactivity. However, previous studies have generally examined these parameters in total peripheral blood B cells rather than in well defined subpopulations. This is an important distinction because BCR signaling in mature naive B-cells leads to proliferation and survival, while these same signals lead to receptor editing, depletion or anergy in immature cells. We hypothesize that dysregulation of these signaling networks could contribute to SLE pathogenesis.

Methods:

Peripheral blood was obtained from 12 patients satisfying ACR SLE criteria and 12 HC that lacked a family history of autoimmune disease. To minimize confounding influences of cytokines, patients had low disease activity (SLEADI<5). B cells were enriched with RosettespTM and ficoll, rested at 37°C for 1 hr, stimulated with 10 µg/ml (low dose) or 50 µg/ml (high dose) of F(ab’)2 goat anti-human IgM or phorbal 12-myristate 13-acetae (PMA), immediately fixed, and processed for flow cytometry. Phosphorylation of intracellular signaling proteins (pSyk, pPLCg, pERK, pAKT, pS6) in transitional (CD20⁺CD27^–CD24^HiCD38^Hi) and naïve (CD20⁺CD27^–CD24^IntCD38^Int) B cells was measured using FlowJo 8.8.7 and Cytobank software.

Results:

The basal phosphorylation states of Syk, ERK, PLCg, and S6 were increased in both transitional and naïve B cells of SLE patients relative to HCs. Upon BCR ligation, both transitional and naïve SLE B cells demonstrated augmented phosphorylation of SYK, ERK, and PLCg relative to HC. Surprisingly, these same patients displayed hypo-phosphorylation of S6 in response to the same stimuli. Interestingly, hypo-activation of S6 was observed in mature naïve but not transitional B cells. The response to stimulation with PMA, which bypasses proximal signaling machinery, was equivalent in SLE and HC B cell subsets, indicating that the difference in signaling was upstream of S6.

Conclusion:

We find that both transitional and naïve B cells obtained from SLE patients with well-controlled disease have increased activation of the MAPK pathway in the basal state and in response to BCR stimulation. Phosphorylation of S6, which is downstream of the PI3K/AKT/mTOR pathway, is also elevated in the basal state in SLE B cell subsets, but surprisingly hypophosphorylated in naïve mature but not transitional B cells in response to BCR ligation in these same patients. While S6 is a target of the PI3K pathways after BCR ligation, it also receives inputs through growth hormone and cytokine receptors as well as the mTOR complex, which plays a key role in sensing energy and nutritional levels. Current investigations are under way to identify the mechanisms mediating these observations and their relation to SLE pathogenesis.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/dichotomous-responses-of-human-systemic-lupus-erythematosus-b-cell-subsets-to-b-cell-receptor-stimulation/