Technological advances in fluorescence flow cytometry and an ever‐expanding understanding of the complexity of the immune system have led to the development of large (20+ parameters) flow cytometry panels. However, as panel complexity and size increase, so does the difficulty involved in designing a high‐quality panel, accessing the instrumentation capable of accommodating large numbers of parameters, and analyzing such high‐dimensional data. A recent advancement is spectral flow cytometry, which in contrast to conventional flow cytometry distinguishes the full emission spectrum of each fluorophore across all lasers, rather than identifying only the peak of emission. Fluorophores with a similar emission maximum but distinct off‐peak signatures can therefore be accommodated within the same flow cytometry panel, allowing greater flexibility in terms of panel design and fluorophore detection. Here, we highlight the specific characteristics of spectral flow cytometry and aim to guide users through the process of building, designing, and optimizing high‐dimensional spectral flow cytometry panels using a comprehensive step‐by‐step protocol. Special considerations are also given for using highly overlapping dyes, and a logical selection process for optimal marker‐fluorophore assignment is provided. © 2020 by John Wiley & Sons, Inc.

The presence of autoantibodies in systemic lupus erythematosus, particularly those of the IgG subclass, have long been associated with disease onset and activity. Here we explored the prevalence of autoreactive IgE in SLE and its relevance to disease in French (n = 79) and United States (US) (n = 117) cohorts with a mean age of 41.5±12.7 and 43.6±15.3 years and disease duration of 13.5±8.5 and 16.6±11.9 years, respectively. Our findings show that approximately 65% of all SLE subjects studied produced IgE antibodies to the seven autoantigens tested. This positivity was increased to almost 83% when only those subjects with active disease were considered. SLE subjects who were positive for anti-dsDNA, -Sm, and -SSB/La -specific IgE showed a highly significant association in the levels of these antibodies with disease activity similar to that of the corresponding IgG's. A strong association of IgE autoantibodies with active nephritis was also found in the combined cohort analysis. A test of the predictive value of autoreactive IgE’s and IgGs for disease activity (SLE Disease Activity Index (SLEDAI) ≥4) revealed that the best predictors were dsDNA-specific IgE and IgG, and that the age of an SLE subject influenced this predictive model. The finding argue that the overall levels of IgE autoantibodies, independently or in combination with IgG autoantibodies, may serve as indicators of active disease.

The (patho)physiological role of IgE in nonallergic inflammatory diseases is not well understood. Here, we explored the effect of IgE deficiency on the inflammatory response in FcγRIIB-deficient mice as well as in mice carrying both a deletion of FcγRIIB and the chromosomal translocation of Y-linked autoimmune acceleration (Yaa) that hastens and results in a more aggressive lupuslike disease in these mice. The findings show that deficiency of IgE delays disease development and severity as demonstrated by reduced autoantibody production and amelioration of organ pathologies. This was associated with decreased numbers of plasma cells and reduced levels of IgG2b and IgG3. Unexpectedly, the loss of IgE also caused a striking decrease of immune cell infiltration in secondary lymphoid organs with a marked effect on the presence of dendritic cells, monocytes, neutrophils, and eosinophils in these organs and decreased activation of basophils. The presence of autoreactive IgE in human systemic lupus erythematosus subjects was also associated with increased basophil activation and enhanced disease activity. These findings argue that IgE facilitates the amplification of autoimmune inflammation.

In systemic lupus erythematosus (SLE), autoantibody production can lead to kidney damage and failure, known as lupus nephritis. Basophils amplify the synthesis of autoantibodies by accumulating in secondary lymphoid organs. Here, we show a role for prostaglandin D₂(PGD₂) in the pathophysiology of SLE. Patients with SLE have increased expression of PGD₂receptors (PTGDR) on blood basophils and increased concentration of PGD₂metabolites in plasma. Through an autocrine mechanism dependent on both PTGDRs, PGD₂induces the externalization of CXCR4 on basophils, both in humans and mice, driving accumulation in secondary lymphoid organs. Although PGD₂can accelerate basophil-dependent disease, antagonizing PTGDRs in mice reduces lupus-like disease in spontaneous and induced mouse models. Our study identifies the PGD₂/PTGDR axis as a ready-to-use therapeutic modality in SLE.

The dendritic cell signals required for the in vivo priming of IL-4–producing T cells are unknown. We used RNA sequencing to characterize DCs from skin LN of mice exposed to two different Th2 stimuli: the helminth parasite Nippostrongylus brasiliensis (Nb) and the contact sensitizer dibutyl phthalate (DBP)-FITC. Both Nb and DBP-FITC induced extensive transcriptional changes that involved multiple DC subsets. Surprisingly, these transcriptional changes were highly distinct in the two models, with only a small number of genes being similarly regulated in both conditions. Pathway analysis of expressed genes identified no shared pathways between Nb and DBP-FITC, but revealed a type-I IFN (IFN-I) signature unique to DCs from Nb-primed mice. Blocking the IFN-I receptor at the time of Nb treatment had little effect on DC migration and antigen transport to the LN, but inhibited the up-regulation of IFN-I–induced markers on DCs and effectively blunted Th2 development. In contrast, the response to DBP-FITC was not affected by IFN-I receptor blockade, a finding consistent with the known dependence of this response on the innate cytokine TSLP. Thus, the priming of Th2 responses is associated with distinct transcriptional signatures in DCs in vivo, reflecting the diverse environments in which Th2 immune responses are initiated.