0514: Transitional Monocytes and Innate T Cell Populations Help Distinguish Ro Seropositive vs Ro Seronegative Sjögren’s Disease Using Whole Blood Immunophenotyping

Background/Purpose: Blood immunophenotyping reveals systemic immune alterations and therapeutically actionable molecular endotypes in Sjögren’s Disease (SjD). Reported changes include reduced memory B cells, plasmacytoid dendritic cells (pDC), and myeloid dendritic cells (mDC). Prior studies focused on Ro seropositive (Ro^Pos) SjD and utilized peripheral blood mononuclear cells, which are prone to processing artifacts. To avoid this limitation, we used mass cytometry with a point-of-care whole blood assay to compare immune cell frequencies in both Ro^Pos and Ro seronegative (Ro^Neg) SjD participants.

Methods: Blood samples from multiple sites were stained using the 30-marker Maxpar Direct Immune Profiling Assay (MDIPA) for broad immune phenotyping, then shipped to a central lab for CyTOF XT acquisition. Data were processed with OMIQ software, using FlowCut for quality control. Manual gating followed manufacturer guidelines to quantify total and lineage-specific immune cell subsets. Absolute cell counts were derived using individual white blood cell (WBC) counts. This study included 10 HC, 12 Ro^Neg SjD, and 31 Ro^Pos SjD participants, with cases meeting 2016 ACR/EULAR criteria. Principal component analysis (PCA) and heatmaps were used for dimension reduction and clustering.

Results: Novel findings included increased frequencies of transitional monocytes within the monocyte lineage in Ro^Pos vs. Ro^Neg SjD (p=0.022), with selective increases in Ro^Pos SjD cases with elevated disease activity (ESSDAI≥5) (p=0.032). Absolute counts of transitional monocytes trended higher in Ro^Pos SjD (p=0.13) despite reduced WBC counts in Ro^Pos vs. Ro^Neg SjD (p=0.025). In addition, absolute numbers of TCRγδ+ T cells were selectively increased in Ro^Neg SjD compared to both HC (p=0.009) and Ro^Pos SjD (p=0.025), while numbers of CD28⁺CD161^hi MAIT/NKT cells were selectively reduced in Ro^Pos SjD (p=0.013). Based on PCA, Ro^Pos participants with moderate to high ESSDAI scores clustered together at least in part due to increased transitional monocyte frequencies and decreased MAIT/NKT and γδT cells. In agreement with previous studies, we also found that frequencies, but not absolute numbers of memory and naïve B cells were reduced (p=0.018) and increased (p=0.015), respectively, in Ro^Pos SjD compared to HC. Absolute numbers of pDC were reduced in Ro^Pos SjD compared to both Ro^Neg SjD (p=0.02) and HC (p=0.024), and these reductions were driven by Ro^Pos cases with ESSDAI≥5 (p=0.013). Myeloid DC showed similar results, with reductions occurring in Ro^Pos patients with ESSDAI≥5 (p=0.012).

Conclusion: Point-of-contact whole blood immunophenotyping reveals transitional monocytes as a novel marker of systemic disease activity in Ro^Pos SjD. The differential abundance of transitional monocytes, γδ T cells, and MAIT/NKT cells between Ro^Pos and Ro^Neg patients suggests distinct underlying pathogenic mechanisms. These findings enable more precise patient selection for immuomodulatory therapies targeting monocyte- and innate T cell-driven dysregulation, Planned studies aim to validate these signatures longitudinally and assess their utility in predicting treatment response or disease progression.