Abstract Rationale Sarcoidosis is a systemic disease of granulomatous inflammation with predominance of CD4+ T cells polarized to both Th1 and Th17 phenotypes, especially those producing interferongamma (IFN-γ). Although IFN-γ-related pathways are essential in granuloma biology, the mechanisms that contribute to on-going inflammation and related markers to assess disease prognosis have yet to be identified. We previously found that cells with high expression of the Th1-defining transcription factor T-bet had high capacity for IFN-γ protein expression and that these T-betHi cells are present in patients with longitudinal pulmonary function changes. Therefore, we further investigated the functional relevance of these cells in sarcoidosis by developing a novel protocol for performing intracellular single cell RNA CITE-Seq to identify unique transcriptional profiles. We also assessed their expression of the Th17-associated transcription factor protein RORγt. Methods We developed a protocol to perform single cell RNA sequencing combined with surface and intracellular CITE-seq to identify cells expressing the T-bet protein. We tested multiple fixation and permeabilization methods to optimize RNA quality and intracellular anti-T-bet antibody penetration. We performed single cell CITE-Seq experiments using this protocol on the 10X platform to identify the T-bet-expressing CD4+ T cells and compared gene expression in stimulated and unstimulated conditions. We also used multi-parameter flow cytometry to analyze PBMCs from sarcoidosis subjects who had longitudinal changes in pulmonary function to assess the relationships between T-betHi cells expressing unique markers, including RORγt, and clinical phenotype. Results We found that using our fixation and permeabilization CITE-seq protocol, we could recover similar amounts of RNA from live vs. fixed/permeabilized cells (RIN 9.5) while differentiating distinct Tbet-expressing populations by flow cytometry. Our 10X data showed that T-betHi cells had higher mean IFNG gene expression (p 0.001). Our flow cytometry data revealed that sarcoidosis subjects with PFT changes had higher frequencies of RORγt+T-betHi cells (of CD4+ cells) compared to those with stable PFTs or healthy controls (p = 0.004). In longitudinal mixed models, we found that those with PFT increases had a drop in %RORγt+T-betHi cells compared to those with PFT declines (p = 0.003) (Figure 1). Conclusions We developed a novel intracellular CITE-Seq protocol and identified increased IFNG gene expression in T-betHi CD4+ T cells. We also found that T-betHi cells expressing RORγt protein were associated with pulmonary function changes, especially with PFT declines. These data support the potential of these T-betHi to contribute to IFN-γ production and suggest a unique Th1/Th17-polarized phenotype important for sarcoidosis progression. This abstract is funded by: NIH, ATS Foundation
Arger et al. (Fri,) studied this question.