Sarcoidosis is a multisystem granulomatous disorder of unknown etiology, characterized by accumulation of activated CD4⁺ T cells in the lungs (1). Löfgren’s syndrome (LS) is distinguished by an acute disease onset with fever, bilateral hilar lymphadenopathy (BHL), erythema nodosum, and/or ankle arthritis, as well as a good prognosis and typically self-limiting disease course (2). In contrast, “non-LS” sarcoidosis constitutes a more heterogeneous patient group that is usually characterized by an insidious onset and disease progression, with substantial risk of developing chronic disease and pulmonary fibrosis. LS is associated with carriage of the HLA-DRB1*03 allele (3), which also strongly influences spontaneous disease resolution (4). The presence of clonal T cell receptor (TCR)-restricted cells in HLA-DRB1*03⁺ patients further implicates specific antigen recognition in the lungs (5, 6).

Detailed genetic profiling indicates that LS and non-LS share much fewer features than previously acknowledged (7), and arguments have been made for LS to be considered a separate disease entity. However, understanding of pulmonary immune pathways underlying the observed clinical heterogeneity remains incomplete and, until recently, methodology for adequately addressing this question has been lacking.

In this study, we, therefore, subjected bronchoalveolar lavage fluid (BALF) cells from four HLA-DRB1*03⁺ LS and four HLA-DRB1*03⁻ non-LS patients (Table 1), which are believed to represent two clinical opposites, to mass cytometry, utilizing a T cell panel of 33 unique markers. Unbiased data analysis was performed using two different clustering algorithms to identify differentially regulated cell populations. Specific attention was paid to the recently identified subpopulation preferentially expressing TCR variable segments Vα2.3 and Vβ22 in HLA-DRB1*03⁺ LS patients (8). With the aim of identifying novel cell populations and pathways that associate with disease resolution or progression, this study demonstrates the sensitivity of mass cytometry, its ability to detect significant differences between patient groups despite limited sample sizes, and the benefit of its application in complex diseases. Albeit in a small study population, we here present novel data on immunological markers that differentiate between sarcoidosis subtypes at the molecular level, and that can be targeted by future exploration in larger patient cohorts.

While marked differences in clinical presentation and outcome have long defined LS and non-LS, comprehensive genetic disparities were only recently demonstrated (7), and the immunological mechanisms driving these two forms of sarcoidosis are still incompletely understood. To address key characteristics of CD4⁺ T cells in LS and non-LS in an unbiased fashion, we subjected BALF cells from two genetically and clinically distinct patient groups to mass cytometry and cluster analysis, identifying cell populations of significantly different abundance in the two disease phenotypes. On the individual marker level, significant changes were most notably observed for inhibitory receptors CTLA-4 and PD-1, adhesion marker CD44, effector and activation markers HLA-DR, CD127 and CD39, as well as a range of markers associated with cells expressing TCR variable segments Vα2.3 and/or Vβ22. These findings connect immunological profiles to established clinical characteristics of disease, and the identified novel markers may allow for both prognostic evaluation and more precise differentiation between sarcoidosis subtypes. Despite sample size being limited by methodological requirements and patient availability, these statistically robust differences further argue for LS and non-LS to be considered as immunologically distinct conditions.

Believed to influence tolerance induction and long-term tolerance maintenance, respectively, inhibitory receptors CTLA-4and PD-1 act to constrain development of autoimmunity. Notably, anti-CTLA-4 and anti-PD-1 treatments have the potential to initiate or exacerbate sarcoid-like inflammation (21, 22), reinforcing the notion of altered immune activation and possibly an autoimmune component in sarcoidosis. Broos et al. also previously found reduced CTLA-4 expression on sarcoidosis T cells compared to healthy individuals, suggestive of a shift in balance between immune activation and regulation that results in exacerbated T cell activity and, at length, chronic inflammation, and tissue damage (23). In this study, a similar reduction in CTLA-4 and PD-1 expression could be observed in sarcoidosis compared to a healthy individual. Among sarcoidosis patients, however, expression of both receptors was primarily found in LS CD4⁺ T cells, indicating a higher degree of immune self-restriction compared to non-LS patients. In addition, ICOS expression was significantly increased in some, though not all, LS clusters compared to background CD4⁺ T cells. Elevated ICOS expression has previously been demonstrated in LS (24) and could associate with the higher IL-10 production seen in these patients (25, 26). From a clinical perspective, LS patients in general and HLA-DRB1*03⁺ individuals in particular have a favorable prognosis, and in our opinion, it is reasonable to believe that superior regulatory capacity of CD4⁺ T cells in LS compared to non-LS influences the characteristic resolving disease course. Accordingly, it is interesting to speculate that stimulation of regulatory elements could have therapeutic potential in non-LS patients prior to progression to a chronic disease state.

In contrast, compared to background BALF CD4⁺ T cells from non-LS patients displayed an elevated expression of activation markers HLA-DR, CD127, and CD39 consistent across all clusters. Polymorphisms in the CD127 (IL-7R) gene are known risk factors for sarcoidosis development (27), and the IL-7 receptor is crucial during T cell activation, homeostasis, and differentiation. Importantly, it is differentially expressed on effector and regulatory T cells (Tregs), with the latter being defined by low levels of this receptor. The lack of CD127 in LS-dominated clusters, combined with the expression of CTLA-4 and PD-1, suggests immune regulation to be maintained in LS CD4⁺ T cells, while the balance is shifted in favor of effector functions in non-LS CD4⁺ T cells. The role of CD39 in this context is less established, but CD39⁺CD4⁺ T cells have been proposed as inducers of T cell proliferation and cytokine production (28). Moreover, a recent study showed reduced suppressive function of CD39⁺ compared to CD39⁻ Tregs (29), implicating dysfunctional regulatory mechanisms particularly in the pathogenesis of non-LS. Notably, CD39⁺ cells have also been found in sarcoid granulomas (30).

While expression of CD57 is most commonly associated with NK cells, its presence has been documented in T cells with a more advanced differentiation state, reduced replicative capacity but retained cytokine production, particularly of IFNγ (31–33), and possibly cytotoxic functions (34). In the present study, two clusters, both of which more abundant in non-LS patients, demonstrated elevated CD57 expression compared to background CD4⁺ T cells. Higher IFNγ production in non-LS patients has been demonstrated previously (25, 26), and could possibly be at least partly attributed to such terminally differentiated cells with cytotoxic potential.

The adhesion molecule CD44 has been detected on lymphocytes in granulomas of both Crohn’s disease and pulmonary sarcoidosis, and suggested to affect their homing and activation, as well as macrophage differentiation (35). Interestingly, in the present study, CD44 was consistently reduced in LS CD4⁺ T cell clusters compared to background, while the opposite was observed in non-LS clusters. As T cells are essential for maintenance of granulomatous structures, reduced adhesive capacity of T cells may well play a role in spontaneous resolution of granulomas, thereby influencing disease resolution in LS. Of note, any clinically relevant differences between LS and non-LS granulomas have yet to be investigated, but the prospect that alternate granulomatous development might ultimately affect disease outcome is worth considering. Recently, involvement of the mTORC1 pathway was proposed to promote disease progression in sarcoidosis (36). Intriguingly, mTORC1 complexes can be inhibited by CD44 blockade (37), suggesting a dual role for CD44 in granuloma persistence, acting both during cell–cell adhesion and signal transduction.

The recently noted clonality of Vα2.3⁺Vβ22⁺CD4⁺ T cells (8) indicates them to be a driving force in HLA-DRB1*03⁺-mediated antigen recognition. The current study further suggests that cells expressing Vα2.3 can initiate and/or act within different immune pathways. Moreover, expression of Vα2.3 together with Vβ22 appear to associate with effector-like markers, while Vα2.3⁺ populations with other Vβ segment pairings preferentially expressed markers associated with immune regulation, such as PD-1 and ICOS. As hierarchical clustering suggests these clusters to arise from separate origins, it is plausible that several oligoclonal Vα2.3⁺ populations expand in the lungs in response to different epitopes derived from the same antigen, and that these cells serve discrete purposes during antigen recognition and clearance. Previous observations of both an effector phenotype of Vα2.3⁺ cells (5, 6, 8, 38), as well as reduced IFNγ and Th1 responses in LS patients (25, 26), could possibly be at least partly attributed to divergent TCRβ chain preferences. Expression of multiple chemokine receptors (CXCR3, CCR4, and CCR6) suggests that Vα2.3⁺CD4⁺ T cells might follow different migratory patterns compared to Vα2.3⁻CD4⁺ T cells, possibly allowing for more efficient interaction with other immune cells. The concurrent high expression of HLA-DR and CD31 might also signify their recent activation and ongoing homing to the site of antigen presentation. Activated, antigen-specific T cells expressing CD11c have been found to associate with both effector and regulatory profiles (39–41), and in this study, we show that CD11c⁺Vα2.3⁺ cells can express either co-stimulatory or co-inhibitory molecules. Alternatively, this functional discrepancy may reflect a transitional state of activated T cells following antigen recognition, implying that multiple stages of differentiation occur throughout the disease process. A gradual shift toward a more regulatory profile may thus be an inherent characteristic of LS lung CD4⁺ T cells, ultimately contributing to a better prognosis.

In both patient groups, the near-complete overlap of Ki-67 and CD8 expression conceivably points to a more pronounced activation of CD8⁺ T cells in sarcoidosis than previously appreciated. It is intriguing to speculate that lack of regulatory capacity in non-LS CD4⁺ T cells may result in unrestrained proliferation and increased cytotoxic activity of CD8⁺ T cells, which could promote tissue damage and chronic inflammation. In line with this hypothesis, a stronger cytotoxic response in non-LS CD8⁺ T cells was recently observed by Brighenti et al. (in preparation). Accordingly, tighter regulation of the CD8⁺ response by CTLA-4- and PD-1-expressing CD4⁺ T cells in LS patients could contribute to spontaneous resolution and absence of permanent tissue scarring.

Despite the differences observed, the low sample number is an obvious limitation to the study, as is the inability to perform validation experiments in the same cohort. Mass cytometry is a powerful method for detecting robust differences in small sample sizes, thus making it suitable for unbiased discovery studies. However, the cell number requirement is higher than for flow cytometry, which is a restraint when using clinical samples, and due to sample vaporization, no additional sorting or functional characterization experiments can be performed on the same cells. Moreover, established techniques such as flow cytometry or western blot have lower sensitivity and are, therefore, not ideal means of validation of mass cytometry data. Nevertheless, the current study demonstrates a novel approach to the study of CD4⁺ T cells in the lung and lays a foundation for future studies, ideally comprising larger groups of patients. The deliberately high FDR threshold for Citrus analysis enables identification of subpopulations that can be further explored in more stringent and comprehensive studies. Of note, several differentially regulated clusters in this study are derived from the same “parent” cluster and, therefore, exhibit similarities in marker expression, thus strengthening the accuracy of identified differences at the individual marker level despite a higher FDR. Future analyses should also aim to expand the current T cell panel to include markers of interest when investigating tissue-resident T cells, such as CD69, or for detailed delineation of Tregs, e.g., CD73 and CD25. Targeting of additional cytokine receptors and a broad range of transcription factors could also contribute to a more comprehensive subclassification of T cells and their role in different disease phenotypes.

Together, these mass cytometry-based findings contribute new information on lung T cell differentiation and activation in two forms of sarcoidosis that may in time be considered as separate diseases. In LS, a stronger regulatory profile along with decreased adhesive capacity of CD4⁺ T cells, offer a stark contrast to the non-LS effector profile and a direct link to characteristic clinical symptoms. The demonstrated ability to detect significant differences at the molecular level, despite low sample numbers and limited availability of cells from the lungs, could be employed to further distinguish between clinical subtypes, e.g., based on presence of extra-pulmonary symptoms.

In conclusion, mass cytometry provides a powerful and sensitive means of understanding cellular mechanisms of disease that could prove especially valuable in heterogeneous conditions such as non-LS sarcoidosis, and markers that differentiate disease phenotypes could potentially be exploited for future therapeutic purposes in non-resolving disease.

This study was carried out in accordance with the recommendations of Karolinska Institutet, Karolinska University Hospital and the Stockholm County Regional Ethical Committee with written informed consent from all subjects in accordance with the Declaration of Helsinki. The protocol was approved by the Stockholm County Regional Ethical Committee.

Mass cytometry data in the form of FCS files can be accessed from FlowRepository (https://flowrepository.org/) via accession number FR-FCM-ZY9W.

Conception and study design: YK, AA, and JG. Sample collection: YK and AE. Mass cytometry panel development, optimization, and implementation: TL, JM, and PB. Data pre-processing and cluster analysis (Citrus, ACCENSE): YC and PB. Data visualization (t-SNE): YK. Interpretation and assessment of clinical parameters: YK, AA, and JG. Manuscript preparation: YK, AA, and JG. Critical reading and intellectual assessment of manuscript: YK, TL, AE, PB, AA, and JG. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.