Background/Purpose:

Interferons (IFN) reportedly are central to SLE pathogenesis and increased expression of IFN regulated genes (the ‘IFN signature’) is associated with active disease. Clinical utility of the IFN signature is unclear, and refinement to define further patient subgroups may improve disease management. Toll-like receptor (TLR) activation leads to IFNα induction. To increase understanding of the role of IFNs in SLE pathobiology, and connectivity between IFN and TLR signaling, functional profiling of immune signaling downstream of IFNα, IFNγ and TLR modulators in peripheral blood mononuclear cells (PBMC) of SLE donors was performed and compared with signaling in healthy donors (HD).

Methods:

Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry based technology that enables simultaneous analysis of signaling networks in multiple immune cell subsets. PBMC from 60 SLE patients (meeting ACR criteria (2007), SELENA SLEDAI ≥6) and 59 HD were profiled by SCNP, interrogating IFN modulated JAK-STAT signaling and TLR modulated signaling relevant to SLE. CD4+/- CD45RA+/- T cells, CD20+ B cells, CD14+ monocytes and CD11b+ myeloid dendritic cells were profiled (see Table 1). 

Table 1. Overview of nodes and cell subsets interrogated

Results:

IFNα and IFNγ modulated p-STAT1, -3 and -5 signaling was more heterogeneous in SLE vs HD. An SLE subgroup demonstrated low IFNα/high IFNγ signaling in lymphocytes and monocytes. Based on low IFNα->p-STAT5 /high IFNγ->p-STAT1 modulated signaling in B cells, the SLE-IFN subgroup was defined as outside the 95 percentile (z-score>+/-1.96) of HD, comprising 20 of 60 SLE samples.

The SLE-IFN subgroup was 9.4-fold more likely to be positive for anti-dsDNA antibodies (Fisher’s exact test p-val<0.001), consistent with published data on the IFN signature and its link to disease activity, and supporting the clinical relevance of this observation. Significant associations with ANA Ab positivity (p=0.04), report of a new rash (p=0.03) and age (p=0.04) were also identified. No significant associations with other clinical or demographic parameters were identified.

Strikingly, the members of the SLE-IFN subgroup displayed higher TLR7/8 modulated signaling in B cells (Wilcoxon test p=0.003-0.03, depending on the intracellular readout), and dendritic cells (p=0.03), but not in monocytes. Moreover, TLR9 signaling was lower in B cells (p=0.02), and TLR1/2 and TLR4 modulated signaling was lower in monocytes (p=0.003-0.01). 

Conclusion:

These data identify potential connectivity in immune signaling across cell subsets and signaling pathways that underlie disease pathobiology and further define SLE donor subgroups. Refinement of the IFN signature in SLE through SCNP may facilitate the clinical applicability of the signature to better inform patient stratification for treatment options.