Background/Purpose: Lupus erythematous (LE) is a systemic autoimmune disease with a variety of cutaneous manifestations. Antimalarials are first-line systemic therapy, yet not all patients respond to hydroxychloroquine (HCQ), quinacrine (QC), or either (NR). Our group has previously shown that QC responders demonstrate increased conventional dendritic cells (cDC) and TNF relative to HCQ responders.

Methods: Here, we investigated the differences between these patients using imaging mass cytometry (IMC), an unbiased multiplexed technique. 12 HCQ, 11 QC, and 20 NR treatment-naïve FFPE samples were stained with two panels consisting of 37 metal-conjugated antibodies and ablated on the Hyperion Imaging System (Fluidigm). Images were segmented using a nuclear staining based algorithm in Visiopharm and imported into histoCAT where single cell mean pixel intensity data was obtained to cluster cells using the Phenograph algorithm. RNA fluorescent in situ hybridization (ISH) using RNAscope was performed to confirm certain findings. Kruskal-Wallis, and post-hoc Dunn’s tests were performed for cell percentages, and median with IQR are depicted. One-way ANOVA and post-hoc Tukey were performed for normally distributed intracellular markers with mean and SEM shown. Bivariate correlations were determined by Pearson’s r.

Results: We identified 12 unique cell types. T cells were then manually gated on CCR7, CD69, and CD45RA to identify effector and memory subsets. NR patients were found to have a decreased percentage of Tregs compared to QC responders (p< 0.05) (Figure 1A). There was a significant increase in CD4+ central memory T cells in NR patients compared to HCQ patients (p< 0.05) (Figure 1B). We compared epidermal staining and found a significant difference is pNFκB staining between the treatment groups (p< 0.05) (Figure 2A). The post hoc test was not significant, but there was a trend towards decreased pNFκB in the QC group compared to both HCQ and NR. In the dermis, HCQ responders had significantly more pJAK3 staining than the QC group (p< 0.05) (Figure 2B). QC responders had a higher expression of pSTING and dermal cell IFNκ compared to HCQ responders (p< 0.05) (Figure 2A). The total expression of pSTING and IFNκ was found to positively correlate (Figure 3A) and colocalize in the dermis (p< 0.0001, r=0.676). We identified conventional dendritic cells (cDCs) as the major producers of IFNκ in CLE and found that their production of IFNκ and phosphorylation of STING was also elevated in the QC group compared to HCQ (Figure 3B). Since keratinocytes have been shown to be important producers of IFNκ in CLE, we compared the mean pixel intensity (MPI) of IFNκ in the epidermis to the cDCs and found a higher MPI in cDCs in all treatment groups. We then performed RNA ISH on QC responder biopsies and found CD11c mRNA (ITGAX) and IFNκ mRNA colocalized, suggesting cDCs are producers of IFNκ in CLE. CD14+CD16+/CD68+ macrophages and cDCs were the predominant cell types found to express pSTING and IFNκ.

Conclusion: This analysis on treatment naïve biopsies may lead to further discovery of biomarkers that may predict patient response to therapy and direct targeted treatment.

Figure 1: Cell types identified in cutaneous lupus stratified according to patient responses to antimalarials (A). CD4 (B) and CD8 (C) T cell subsets in cutaneous lupus stratified by patient responses to antimalarials.

Figure 2: Mean pixel intensities of intracellular cytokines and activated cell signaling pathways in cutaneous lupus erythematosus.

Figure 3: Phosphorylated-STING (pSTING) and interferon kappa are correlated in cutaneous lupus (r=0.676) (A). Conventional dendritic cells (cDCs) express more pSTING (p < 0.01) and interferon kappa (p < 0.05) in quinacrine (QC) responders (B).

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/multiplexed-profiling-of-treatment-naive-cutaneous-lupus-skin-stratified-by-patient-response-to-antimalarials/