1735: An Unorthodox HLA-DR^hiCD15⁺ ‘Hybrid’ Population in Rheumatoid Arthritis Characterized Using Spectral Cytometry

Christian Geier¹, Haani Qudsi², Jihad BenGabr¹, Robert Winchester³ and Andras Perl⁴, ¹SUNY Upstate Medical University, Syracuse, NY, ²Norton College of Medicine, SUNY Upstate Medical University, Syracuse, NY, ³Columbia University, New York, NY, ⁴SUNY, Syracuse, NY

Background/Purpose: A variant of HLA-DR confers the strongest genetic risk for rheumatoid arthritis (RA) suggesting that DR^hi cells are important in RA. We previously found that RA blood contains 'unorthodox' DR^hi immune cells (non-lymphoid cells that do not conform to bona fide definitions of monocytesnor dendritic cells).The purpose of the study was to determine whether DR^hi immune cells expressing granulocyte associated molecules (CD15, CCR3)contribute to these alterations of the DR^hi pool in RA.

Methods: We studied RA patients (n=12) satisfying the 2010 ACR classification criteria and matched healthy donors by flow cytometry. PBMC and other low-density cells were isolated from blood by Ficoll density gradient centrifugation. To gate non-lymphoid DR^hi cells we excluded lymphocytes and non-viable cells based onforward and side scatter, CD3/CD19 dump and viability gates (Fig. 1A). We quantified the contribution of CD15⁺ cells to the non-lymphoid DR^hi pool and their expression (MFI) of co-stimulatory and co-inhibitory molecules. We used t-SNE on index patients (debilitating polyarticular synovitis)to clarify the higher-dimensional structure of the CD15positive DR^hi subpopulation followed by bi-axial gating to validate anyt-SNE guided observation and quantify distinctive features of DR^hiCD15⁺. Kruskal-Wallis testing with a threshold of p< 0.05 was performed to assess for significant differences.

Results: In RA we found a non-lymphoid DR^hi CD15⁺ population that was virtually absent in healthy donors (HD)(RA 0.98% vs. HD 0.05% of non-lymphoid DR^hi; p<0.01, Fig. 1B, 2B); these cells have near uniform CD16 co-expression (Fig 1A, gated in last plot).DR^hi CD15⁺ showed—as expected from granulocytic cells — high side scatter but differed from granulocytes by a striking co-expression of plasma cytoid (pDC) marker CD303 (Fig. 2A, B; circled);CD123, highly expressed by pDC, was not expressed. Given the shared features with both granulocytes and pDC we refer to this population as DR^hi 'Hybrid' cells. DR^hi Hybrids formed a separate cluster in RA which, along with CD303, co-expressed CD83 and CD275 (ICOS-L) (Fig. 2A, circled population).Lack of CD45RAseparatedCD303⁺DR^hi Hybrids from their apparent bone fide CD303⁺ CD45RA^int pDC counterparts (Fig. 3A and B, p< 0.001), CD45RA expression of RA pDC and HD pDC did not differ (Fig 3B, left). CCR3 expression within non-lymphoid DR^hi was negligible (not shown).

Conclusion: RA blood contains DR^hiCD15⁺ cells, contributing to the non-lymphoid DR^hi pool in RA. Because these low-density cells share features with both granulocytes and pDCs we refer to them as DR^hi Hybrids; their expression of CD303 challenges the notion that this molecule is specific for plasmacytoid DC. Co-expression of CD83, CD275 suggests an inflammatory potential whereas their lack of CD45RA (as opposed to CD303⁺pDCs) is reminiscent of the altered CD45RA expression we found previously in a DC2-like DR^hi subset. Joint expression of CD303 and DR suggests that DR^hi Hybrids maybe functionally involved in the capture and ultimate presentation of RA self-peptides to T lymphocytes. These apparently new DR^hi Hybrids and other unorthodox DR^hi populations are potential treatment targets in RA.








C. Geier: None; H. Qudsi: None; J. BenGabr: None; R. Winchester: None; A. Perl: None.