Background Mass cytometry (CyTOF) previously revealed that T follicular helper (Tfh) cells, T peripheral helper (Tph) cells, and age-associated B cells (ABCs) are robustly expanded in patients with newly diagnosed SLE. However, how these and other immune cell populations change over time in SLE remains unclear.

Methods We employed CyTOF with two 39 marker panels (T and B cell) in cryopreserved PBMCs from 9 newly diagnosed SLE, 15 established SLE, and 14 non-inflammatory controls. FlowSOM and marker analysis by tSNE used to identify and quantify clusters based on their 39-parameter characterization. For the newly diagnosed cohort, PBMCs were analyzed at 3 time points (A=at diagnosis, B=6 months after the diagnosis, C=12 months after the diagnosis). Serum samples were also analyzed to quantify 65 cytokines by Luminex multiplex assay, and associations between cell types and cytokines assessed by Spearman correlation.

Results We first confirmed that among CD4 T cells, Tfh cells (PD-1^hi CXCR5⁺ CD4 T cells) and Tph cells (PD-1^hi CXCR5^- CD4 T cells) were significantly increased in SLE patients. A broad analysis of B cells identified CD11c⁺ CD21^- ABCs and CD19^int Ki67^hi B cell population significantly increased in the patients with SLE. This CD19^int Ki67^hi cluster was also CD21^lo, CD11c^low, CD27^hi, and CD38^hi, consistent with a Ki67hi plasmablast population (figure 1A). Among CD8 T cells, we identified one highly expanded cluster in SLE patients compared to controls, which expressed Ki67^hi, ICOS^hi, PD-1^int, CD57^low, and granzyme B^int (figure 1A). In longitudinal analyses, the frequency of Tfh cells decreased over the first year of SLE, while Tph cells, ABCs, CD19^int Ki67^hi plasmablasts, and Ki67⁺ ICOS⁺ CD8 T cells remained elevated at 12 months (figure 1B). Correlation analyses including both immune cell frequencies and cytokines revealed an association of Tph cells, Ki67⁺ ICOS⁺ CD8 T cells, ABCs, and CD19^int Ki67^hi plasmablasts. These associated populations, but not Tfh cells, were also significantly correlated with CXCL13 and TSLP (figure 1C).

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Abstract 602 Figure 1

Longitudinal CyTOF and cytokine analyses of newly diagnosed SLE. (A) Expanded two Ki67+ populations in PBMCs of SLE patients. (B) Longitudinal CyTOF analysis of PBMCs in SLE patients. (C) A hierarchical clustering heatmap with immune cell frequencies and cytokines in SLE.

Conclusions This longitudinal immunophenotyping and cytokine profiling approach highlights persistent activation of a Tph-CXCL13-ABC-plasmablasts axis in both early and established phases of SLE.

Acknowledgments We thank the patients who donated samples and medical staffs at the Hospital.