Mass Cytometry Identifies Distinct Lung CD4+ T Cell Patterns in Löfgren's Syndrome and Non-Löfgren's Syndrome Sarcoidosis

Abstract

Sarcoidosis is a granulomatous disorder of unknown etiology, characterized by accumulation of activated CD4⁺ T cells in the lungs. Disease phenotypes Löfgren's syndrome (LS) and "non-LS" differ in terms of clinical manifestations, genetic background, HLA association, and prognosis, but the underlying inflammatory mechanisms largely remain unknown. Bronchoalveolar lavage fluid cells from four HLA-DRB1*03⁺ LS and four HLA-DRB1*03^- non-LS patients were analyzed by mass cytometry, using a panel of 33 unique markers. Differentially regulated CD4⁺ T cell populations were identified using the Citrus algorithm, and t-stochastic neighborhood embedding was applied for dimensionality reduction and single-cell data visualization. We identified 19 individual CD4⁺ T cell clusters differing significantly in abundance between LS and non-LS patients. Seven clusters more frequent in LS patients were characterized by significantly higher expression of regulatory receptors CTLA-4, PD-1, and ICOS, along with low expression of adhesion marker CD44. In contrast, 12 clusters primarily found in non-LS displayed elevated expression of activation and effector markers HLA-DR, CD127, CD39, as well as CD44. Hierarchical clustering further indicated functional heterogeneity and diverse origins of T cell receptor Vα2.3/Vβ22-restricted cells in LS. Finally, a near-complete overlap of CD8 and Ki-67 expression suggested larger influence of CD8⁺ T cell activity on sarcoid inflammation than previously appreciated. In this study, we provide detailed characterization of pulmonary T cells and immunological parameters that define separate disease pathways in LS and non-LS. With direct association to clinical parameters, such as granuloma persistence, resolution, or chronic inflammation, these results provide a valuable foundation for further exploration and potential clinical application.

Keywords: CD4+ T cells; Löfgren’s syndrome; bronchoalveolar lavage; disease phenotypes; granuloma; mass cytometry; sarcoidosis.

Figure 1

Significant differences in CD4⁺ T cell patterns between Löfgren’s syndrome (LS) and non-LS. (A) Citrus network tree visualizing the hierarchical relationship between identified bronchoalveolar lavage fluid CD4⁺ T cell populations in LS (n = 4) and non-LS (n = 4). Circle size reflects number of cells within a given cluster. Clusters differing significantly in abundance between the two conditions are divided into three main groups (highlighted). Populations more abundant in LS are indicated in blue, while those more abundant in non-LS are marked in red. (B) Citrus-generated box plots for four representative and differentially regulated populations in LS and non-LS, illustrative of all three cluster groups shown in panel (A). Cluster abundances are shown on a logarithmic scale for clusters 24990, 24987, 24992, and 24961, respectively, annotated according to panel (A). All differences in abundance are significant at FDR < 0.25.

Figure 2

Regulatory elements dominate in Löfgren’s syndrome (LS) CD4⁺ T cells. (A) t-stochastic neighborhood embedding (t-SNE) plots visualizing distribution and intensity of CTLA-4 and PD-1 expression in LS and non-LS CD4⁺ T cells, respectively, following clustering of all samples combined and subsequent separation by condition. (B) Citrus-generated histograms showing significant changes in marker distributions in individual clusters (red) compared to background total CD4⁺ T cells (blue). Representative LS- and non-LS-specific populations are exemplified by clusters 24987 and 24988, respectively. (C) Representative FlowJo contour plots manually gated for single, live (DNA⁺ Cisplatin⁻), CD3⁺, CD4⁺, CD8⁻ T cells in LS and non-LS patients, respectively, showing expression of CTLA-4 and PD-1 in the two patient groups, as well as in a bronchoalveolar lavage fluid sample from a healthy control (HC).

Figure 3

Non-Löfgren’s syndrome (non-LS) CD4⁺ T cells exhibit a pronounced effector profile. Enhanced influence of HLA-DR, CD127, and CD39 in non-LS CD4⁺ T cells as visualized by Citrus-generated histograms. A representative cluster (24971) abundant in non-LS patients is shown in panel (A), exemplifying the significantly higher expression of these effector markers in non-LS. Similarly, cluster 24966 illustrates elevated expression of CD57 in one of two clusters prominent in non-LS (B).

Figure 4

Adhesion marker CD44 is consistently reduced in Löfgren’s syndrome (LS). Expression of CD44 was significantly reduced in clusters more abundant in LS, as exemplified by cluster 24975 (A), compared to non-LS (B) (cluster 24988).

Figure 5

T cell receptor (TCR) Vα2.3⁺ cells in Löfgren’s syndrome (LS) constitute a functionally heterogeneous population. t-stochastic neighborhood embedding (t-SNE) plots filtered for expression of Vα2.3 (A), CXCR3 (B), CCR4 (C), CCR6 (D), HLA-DR (E), CD11c (F), CD31 (G), and CTLA-4 (H) in LS bronchoalveolar lavage fluid cells. The main Vα2.3⁺ cluster is highlighted in red. Citrus comparison of Vα2.3⁺Vβ22⁻ (I) and Vα2.3⁺Vβ22⁺ (J) clusters (24987 and 24983, respectively) reveals distribution of effector (e.g., CD127, CD28) and regulatory markers (e.g., PD-1, ICOS) to vary with TCR expression.

Figure 6

Near-complete overlap of CD8 and Ki-67 expression. t-stochastic neighborhood embedding (t-SNE) plots revealing an almost perfect overlap in expression of CD8 (A) and Ki-67 (B), the latter being a marker of actively proliferating cells, in both Löfgren’s syndrome (LS) and non-LS patients.