Spectral cytometry of rheumatoid arthritis patients implicates myeloid dendritic cells and granular HLA-DR+CD15+CD16+ cells in pro-inflammatory antigen presentation

Abstract

Introduction: Rheumatoid arthritis (RA) is a systemic autoimmune disease that leads to inflammation of synovial joints and other organs. Many RA patients "share" a common peptide sequence within the HLA-DR (MHC II) molecule expressed on antigen-presenting cells (APC), suggesting that HLA-DR+ cells are important in RA inflammation. We use HLA-DR positivity to comprehensively immunophenotype APC by spectral cytometry.

Methods: We measured mean fluorescence intensities (MFI) of HLA-DR and molecules associated with dendritic cells (CD141, CD1c, CD163, CD11c, CD123, and CD303), monocytes (CD14 and CD16), granulocytic markers (CD15 and CCR3), co-stimulatory molecules (CD86 and CD275), and chemokine receptors (CCR2, CCR3, and CCR7) from RA patients and healthy donors by spectral flow cytometry.

Results: DC2 (CD1c+) showed higher CD86, CD275 (ICOS-L), CD56, and CCR7 in RA (all p < 0.05). CD56 was also increased in (CD163+) DC3 (p = 0.0453). CD15 was increased throughout RA dendritic cell subsets and classical and intermediate monocytes (all p < 0.01). Except for B cells, HLA-DR was not different in RA. A distinct HLA-DR+CD15+CD16+ population appeared in RA (p = 0.0004), which contributed a mean of 1.3% (± SD 2.85%) to the overall HLA-DR+ APC compartment. This HLA-DR+CD15+CD16+ subset was positive for CD83, CD275, and, like plasmacytoid pDC, CD303+. However, in contrast to pDC, it formed distinct t-SNE clusters and differed from reference pDC (CD123+CD303+) by much less CD123 (p < 0.01). The HLA-DR+CD15+CD16+ phenotype correlated with clinical markers of systemic inflammation (p < 0.01).

Discussion: In conclusion, dendritic cell and monocyte alterations in RA include an increased co-stimulatory phenotype of CD1c+ DC2 and CD163+ DC3 with increased CD56 and CD15 in dendritic cells and monocytes. Moreover, the blood of RA patients contains HLA-DR+ cells with shared dendritic cell and granulocytic features. These phenotypic characterizations of RA patients implicate CD1c+ DC2 and CD163+ DC3 in the systemic autoimmune disease rheumatoid arthritis and suggest that increased HLA-DR+ phenotypes with shared granulocytic and dendritic cell features can contribute to RA, potentially by providing enhanced co-stimulatory presentation of self-antigen(s) to CD4+ T lymphocytes.

Keywords: CD1c+ dendritic cells; antigen-presenting cell; intermediate monocytes; low-density granulocytes; myeloid cells; rheumatoid arthritis.

Figure 1

Gating strategy (A–F) and immunophenotype (G–L) of CD1c+ dendritic cells (DC2; CD1c+). Mean fluorescence intensities (MFI) were measured by spectral cytometry (y-axis). RA DC2 are characterized by higher MFI for CD56 (A), CD15 (B), the co-stimulatory molecules CD86 and CD275 (ICOS-L) (C, D), and C–C chemokine receptor type 7 (E). HLA-DR MFI did not differ between RA and controls (F). Blue, healthy control donors; orange, RA patients. RA index patients are highlighted in red.

Figure 2

CD56 in APC and NK cell populations. Representative flow cytometry plots, gated on non-lymphoid live singlets (CD3-CD19-). CD56 (x-axis) and HLA-DR (y-axis). Gated are HLA-DR+ antigen-presenting cells (red gates; A, B). Distinct CD56+ NK cells are marked with gray circles. (C-F): NK cells (purple) and monocytes (blue) classified using the SPADE algorithm and dendritic cells (orange) were backgated as color-coded dot plots and manually overlayed with a contour view representation showing increased CD56 APC (solid arrows) and the APC gating strategy (dashed borders). CD56 (x-axis) and HLA-DR (y-axis). Left panels, healthy control donor. Right panels, RA patient.

Figure 3

Monocytes (A–F) and CD15 in APC subsets (G–M) in healthy control donors (blue) and RA patients (orange). Mean fluorescence intensities (MFI) were measured by spectral cytometry (y-axis). RA intermediate monocytes differed by higher MFI for CD275 (A), CD163 (B), CD15 (C), C–C chemokine receptor type 2 (D), and CD14 (E). Classical monocytes had higher CD15 (F). CD15 in DC1 (G), DC2 (H), DC3 (I), pDC (J), classical monocytes (K), non-classical monocytes (L), and intermediate monocytes (M). RA index patients are highlighted separately (red).

Figure 4

Quantification of CD15+CD16+ and CD15+CD16+HLA-DR+ in RA and healthy control donors. (A) Representative flow cytometry of CD15 (x-axis) and CD16 (y-axis). Left plot, index patient 005 at the initial visit at a time of severe disease. Right plot, the same patient after starting treatment. (B) Representative healthy control donor. (C) Quantification of CD15+CD16+ in healthy controls (blue) and RA patients (orange), in % live lymphocyte singlets. (D) Quantification of HLA-DR+CD15+CD16+ in healthy controls (blue) and RA patients (orange), in % live lymphocyte singlets. (E) Quantification of CD15+CD16+ in healthy controls (blue) and RA patients (orange), in cells/mL of blood. (F) Quantification of HLA-DR+CD15+CD16+ in healthy controls (blue) and RA patients (orange), in cells/mL of blood. For (C–F), index patients are highlighted in red.

Figure 5

HLA-DR+CD15+CD16+ (in % leukocytes; y-axis) and markers of inflammation (x-axis) in RA patients. RA index patients are highlighted separately. (A) ESR, Erythrocyte sedimentation rate. (B) CRP, C-reactive protein.

Figure 6

A model of how the changes in the blood APC (HLA-DR+) compartment can contribute to the pathophysiology of RA. Left: In healthy individuals, the blood APC compartment contains DC and monocytes that can present antigen to induce protective T cell responses. Right: In RA, there is (1) an increase in co-stimulatory molecules, adhesion and migratory molecules in dendritic cells, and monocytes and (2) appearance of an CD15+CD16+HLA-DR+ phenotype that is positive for CD83, CD275—consistent with pro-inflammatory co-stimulation, migration, and adhesive properties suggesting involvement by altered antigen presentation to T lymphocytes. Many additional cell types contribute to RA. Created using Biorender.