INTERFERON-INDUCED PROTEIN EXPRESSION IN THE PERIPHERAL BLOOD IMMUNE POPULATIONS OF SLE PATIENTS AT A SINGLE-CELL LEVEL: ASSOCIATION WITH CELLULAR ACTIVATION, TRAFFICKING MOLECULES, AND DISEASE ACTIVITY

O021 / #624 Topic: AS22 - SLE Heterogeneity ABSTRACT CONCURRENT SESSION 03: INNATE AND ADAPTIVE IMMUNITY IN SLE 22-05-2025 1:40 PM - 2:40 PM Background/Purpose High levels of peripheral blood interferon (IFN)-induced gene (IIG) expression are a characteristic feature of SLE and associated with an increased risk of flare. However, how these global changes correlate with those in individual immune populations and act to promote flares remains unclear. To address this question, we examined the IFN-induced immune changes in SLE patients at a single-cell level. Methods A 40-marker CyTOF panel was used to measure IFN-induced protein (IIP) levels in the peripheral blood immune populations of 15 healthy controls (HC), 26 quiescent (clinical SLEDAI-2K = 0 for 1 year), and 42 recently flaring (clinical SLEDAI-2K ≥ 1 requiring an escalation of therapy) SLE patients. Results Twenty-nine immune populations were identified (Figure 1A). The mean IIP levels in all populations strongly correlated with global IIG expression, and were higher in flaring than quiescent patients (Figure 1B,C). Despite this correlation, there was significant heterogeneity in IIP expression between and within the cell subsets of individual patients, with the highest median levels of IIP seen in monocytes, plasmablast/plasma cells, and activated double positive T cells. These differences paralleled the response of these populations to exogenous IFN in HC cells in vitro. Within each cell subset of individual patients, there was a variably broad distribution of IIP expression, sometimes with distinct peaks (Figure 1D). To assess the factors contributing to this heterogeneity, we performed an analysis of extremes comparing the top and bottom 10% of IIP expressing cells in each subject (Figure 2A). Although the top IIP expressing cell subset of most populations had elevated levels of activation markers, such as Ki67, CD86, TLR7, TLR9, and HLA-DR, these molecules were induced by IFN in vitro, suggesting that IFN plays a direct role in their upregulation in vivo (Figure 2B). Notably, increased levels of the trafficking markers were also seen in the high IIP expressing cell subset, but with the exception of β7 (an integrin implicated in homing and retention in the gut), were not induced by IFN in vitro. Furthermore, these trafficking molecules demonstrated distinct patterns of expression, suggesting that these cells had transited different tissues. Longitudinal analysis of IIP expression over time revealed relatively stable levels despite changes in disease activity, and although the levels of IIP in the different cell subsets tended to correlate with each other, only the levels within B cells were associated with sustained or recurrent disease activity 1 year later. Figure 1. A) UMAP of the individual cell types showing their differential abundance and relatedness, as well as the average of 6 IIPs In HCs, quiescent and flaring patients : 29 cell types were identified based on their expression of the markers in our panel. There is a gradient in expression of average IIP expression in most cell types from low to high in HCs, quiescent patients, and flaring patients. B) Correlation matrix in all cells : Correlation between IIP expression (shown on the y axis) and IIG expression in individual cell populations (shown on the x axis). R values are denoted by colour, and p values by the size of the dots. C) Immunologic differences in IIP expression in immuno cell populations comparing flaring and quiescent patients : Waterfall plot showing the differential levels of IIP scores between flaring and quiescent, with bars above the line indicating increased expression in SLE patients. 27/29 immune cell subsets have significantly higher IIP scores in flaring patients relative to quiescent. D) Heterogeneity in IIP expression levels within the cell subsets of individual patients and HCs : Patients were separated into IIG high and low groups based on the top and bottom 15% of IIG score. Regardless of IIG group, there was heterogeneity in the IIP signature on a single cell level that was found within patient cells. Myeloid cells had the most marked heterogeneity, followed by T cells and then B colls. Figure 2. A) Analysis of extremes . Comparing the top and bottom 10% of IIP expressing cells within the same HCs and patients from ex vivo samples, it was found that certain activation and trafficking markers are upregulated in the IIP high cells, some of which are directly induced by IFN. R values are denoted by colour, and p values by the size of the dots. B) Incubation with IFNα and IFNβ induces several of the cellular markers that are associated with increased IIP expression in-vitro . PBMCs from healthy controls were stimulated with the indicated IFNs for 18 or 24 hours in the presence of Golgi-Stop for the last 2 hours. Shown are fold increases relative to unstimulated control. Conclusions Although the mean IIP expression in each immune population correlates strongly with the IFN signature, there is significant heterogeneity between and within the cell populations of each patient in IIP expression. This appears to result not only from variability in the cells capacity to respond to IFN, but also variable exposure to IFN as cells traffic through the body.

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