Zoha Faheem1, Giselle Boukhaled2, Kieran Manion1, Carolina Munoz-Grajales3, Carol Nassar1, Michael Kim1, Dafna Gladman4, Murray Urowitz5, Zahi Touma6, David Brooks2 and Joan Wither7, 1Schroeder Arthritis Institute, Krembil Research Institute, Toronto, ON, Canada, 2Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada, 3UHN/TWH, Toronto, ON, Canada, 4Schroeder Arthritis Institute, Krembil Research Institute, Toronto Western Hospital, Department of Medicine, University of Toronto, Toronto, ON, Canada, 5Schroeder Arthritis Institute, Krembil Research Institute; University of Toronto Lupus Clinic; Division of Rheumatology, Toronto, ON, Canada, 6University of Toronto, Toronto, ON, Canada, 7University Health Network, Toronto, ON, Canada
Background/Purpose: Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with unpredictable flares interspersed with prolonged periods of disease quiescence. Interferon (IFN) is a hallmark of disease and has been linked to disease flares, but the precise mechanism by which this occurs remain unclear. To address this question, we examined the association between IFN and the immune changes in flaring and quiescent SLE patients at a single cell level.
Methods: A 41-marker panel was developed that enabled measurement of IFN-induced proteins (IIPs) in peripheral blood immune populations using CyTOF.Fifteen healthy controls (HCs), 26 quiescent (clinical SLEDAI-2K = 0 for one year) and 42 recently flaring (< 1 month, change in clinical SLEDAI-2K ≥ 1 requiring escalation of therapy) SLE patients were examined. Expression of individual IIPs normalized to healthy controls, as well as a composite IIP score incorporating the expression of all seven proteins was assessed.
Results: Twenty-six distinct immune populations were identified, all of which demonstrated a strong correlation between IIP levels and the IIG score in SLE (Figure 1).In all immune populations, the levels of IIPs were significantly elevated in SLE patients compared to HCs. In most cell populations, the levels of IIPs were elevated in flaring compared to quiescent SLE patients (Figure 2A) and correlated with the clinical SLEDAI-2K. Comparison of the abundance of the 26 immune populations between flaring and quiescent patients revealed that only classical monocytes, intermediate activated classical monocytes, and age-associated B cells (ABCs) were significantly elevated in flaring patients (Figure 2B). Very limited changes were seen between these 2 groups of patients for markers of cellular activation, such as CD86, TLR7, TLR9, FOXP3, HLA-DR, Ki67, and PD1. In contrast, in many relevant immune populations, elevations of these markers were associated with IIP levels suggesting that IFN, rather than disease status (i.e., flare vs quiescence), is driving this activation. In patients who were flaring at baseline, elevations of IIPs in pre-ABCs, ABCs, low density granulocytes (LDGs), and intermediate activated monocytes were associated with clinical disease activity one year later (Figure 3A).In contrast, quiescent patients that subsequently flared and were active at one year had elevations of IIPs in multiple T and B cell populations (Figure 3B).
Conclusion: The findings suggest that IFN initiates flares by promoting B and T cell activation, while sustained or recurrent disease activity appears to be more dependent upon its impact on ABCs, LDGs, and monocytes.
Figure 1. IFN induced protein (IIP) expression is significantly correlated with IFN induced gene (IIG) expression. Correlation between IIP expression (shown on the x axis) in individual cell populations (shown on the y axis) and IIG expression, as measured by a composite score calculated by summing the log2 transformed, normalized expression of 10 IFN-induced genes. R values are denoted by colour, and p values by the size of the dots.
Figure 2. Immunologic differences between flaring and quiescent patients. Waterfall plots showing the fold change differences between flaring and quiescent SLE patients. Bars above the x axis are increased and below the x axis are decreased in flaring relative to quiescent patients. A) Waterfall plot of IIP scores showing that flaring patients have significantly higher levels of IIP expression in 21 out of the 26 immune populations surveyed. B) Waterfall plot of cellular abundance showing that flaring patients have significantly increases in some monocyte subsets and ABCs as compared to quiescent patients.
Figure 3. Interferon-induced protein (IIP) levels in distinct immune populations at baseline inform disease status at one year follow up. A) Patients who were flaring at baseline and were active at one year had significantly higher expression of IIPs in their ABCs, LDGs, and some monocyte cell subsets when compared to those who had inactive disease activity at follow up. B) Patients who were quiescent at baseline and eventually experienced an increase in disease activity at 1 year had significantly higher expression of IIPs in their B and T cell subsets than those who had sustained disease quiescence at one year follow up.
Z. Faheem: None; G. Boukhaled: None; K. Manion: None; C. Munoz-Grajales: None; C. Nassar: None; M. Kim: None; D. Gladman: AbbVie, 2, 5, Amgen, 2, 5, Bristol Myers Squibb, 2, Celgene, 2, 5, Eli Lilly, 2, 5, Galapagos, 2, Gilead Sciences, 2, Janssen, 2, 5, Novartis, 2, 5, Pfizer Inc, 2, 5, UCB, 2, 5; M. Urowitz: None; Z. Touma: AstraZeneca, 2, GSK, 2; D. Brooks: None; J. Wither: AstraZeneca, 1, 6, Pfizer, 12, Indirect salary support through a Chair award to the Division of Rheumatology at the University of Toronto.
