Deep Immunophenotyping of Circulating T and B Cells in Relapsing Adult-Onset Still’s Disease

Round 1

Reviewer 1 Report

The authors performed a flow cytometric analysis of circulating T and B cells in Relapsing Adult-Onset Still's Disease. The disease is classified as autoinflammation and is not fully understood. Within 7 months, 7 patients were included and their values were compared with 15 healthy controls. Material and methods are shown only briefly and are based on previously published protocols. The results are based on multiple comparisons and are interpreted with caution.

Unfortunately, the work was not continued after 7 months. Prospective confirmation of the differences found would possibly confirm the relevance of the findings. At this stage, the results remain purely speculative. 

Author Response

Reviewer #2 (Comments for the Author):

The authors performed a flow cytometric analysis of circulating T and B cells in Relapsing Adult-Onset Still's Disease. The disease is classified as autoinflammation and is not fully understood. Within 7 months, 7 patients were included and their values were compared with 15 healthy controls. Material and methods are shown only briefly and are based on previously published protocols. The results are based on multiple comparisons and are interpreted with caution.

Response. We thank the reviewer for finding our study interesting and for valuable comments. We understand the concern of the Reviewer#2 regarding the number of patients with adult-onset Still’s disease. However we need to emphasize that the disease is remarkably rare. The epidemiology of AOSD is not well-defined due to its rarity, and prevalence estimates vary across different populations. The annual incidence of AOSD is estimated to be around 0.16 cases per 100,000 persons. To be more clear, we modified our ‘Introduction’ section and added the information on the epidemiology of AOSD. We also added the description of adult-onset Still's disease ‘Introduction’ section to help our readers understand the etiology of this disease. Finally, to be more clear with ‘Material and methods‘ part of our manuscript, we added Supplementary Figures S1-S3 with flow cytometry gaiting strategies, that were used for T and B cell subsets determination.

Question. Unfortunately, the work was not continued after 7 months. Prospective confirmation of the differences found would possibly confirm the relevance of the findings. At this stage, the results remain purely speculative.

Response. We are fully agree that prospective investigation will be significant for the understanding of fine-tune pathogenesis of adult-onset Still’s disease. Nevertheless on this stage our aim was to find out if T- and B-lymphocytes subsets involved in Still’s disease manifestation and pathogenesis. Thus, we investigated patients with acute form of the illness and performed deep immunophenotyping of T and B cell subsets using previously described and validated by our group approach for research. We are concluding that our achieved results are look like very promising and may be interpreted as the first stage of investigation that need to be declared and discussed. Probably that is why our interpretations are delicate. Nevertheless, we are planning to continue our study performing follow up investigation with extended spectrum of methods to use.

Reviewer 2 Report

In this manuscript article, the authors investigated adult-onset Still's disease (AOSD), a complex autoinflammatory disorder. Using in-depth flow cytometry analysis, the authors studied peripheral blood T-cell and B-cell subpopulations in seven patients with relapsing Still's disease and fifteen healthy controls. The authors' findings showed changes in major circulating lymphocyte subpopulations, Th cell maturation and "polarization," CD8+ T cell maturation and "polarization," and CD19+ B cell maturation in patients with AOSD compared to healthy controls. Overall, this paper is an interesting study. However, I still have some concerns about the current form of the manuscript. The authors need to address several aspects before this can be published as follows:

Main concerns:

1.     Adult-onset Still's disease (AOSD) is a rare autoinflammatory systemic disease, so the authors need to describe it more in the introduction to help readers understand the etiology of this disease.

2.     In addition to describing the results of the deep flow cytometry analysis in the results section, the authors should further analyze the implications of the findings in adult-onset Still's disease as well as the role in developing prospective treatments.

Author Response

We thank the reviewer for finding our study interesting and for valuable comments.

1. Authors are to be congratulated as they addressed most of the previous comments raised by Adult-onset Still's disease (AOSD) is a rare autoinflammatory systemic disease, so the authors need to describe it more in the introduction to help readers understand the etiology of this disease.

Response: We thank the Reviewer for bringing up this important issue. As suggested, we have revised the introduction to be more concise and clear and reader understand the aim of that study.

‘Still's disease, initially described by Sir George Frederic Still in children [1], was renamed in 2001 as systemic juvenile idiopathic arthritis (sJIA) for standardized terminology. Eric Bywaters identified it in adults 75 years after the first description [2], labeling it adult-onset Still disease (AOSD). Until 2014, AOSD and sJIA were regarded as distinct nosologies, but this perception changed following gene expression analysis in 2014 [3]. This rare disorder has an annual incidence of 0.16 cases per 100,000 persons, marked by sterile inflammation, prolonged fever, musculoskeletal involvement, skin manifestations, serous membrane involvement, other variable symptoms, and prominent neutrophil-driven leukocytosis [4].

The systemic polycyclic course of AOSD is recognized for its distinct phases of relapse and remission. During a relapse, patients experience severe symptoms, marked by abnormal laboratory test results showcasing a diverse array of pro-inflammatory cytokines and acute-phase reactants, particularly elevated levels of ferritin. Elevated ferritin concentrations independently predict the emergence of secondary macrophage activating syndrome (MAS) or secondary hemophagocytic lymphohistiocytosis (HLH) [5]. Conversely, in remission, symptoms abate, and laboratory indicators revert to normal levels [6].

The etiology remains unknown, and the pathogenesis, considered a possible mixed (autoinflammatory > autoimmune) systemic inflammatory disease, involves dysregulation of both the innate and adaptive immune systems [7]. On the one hand, gathered data suggest dysregulation within the innate immune system, reflecting the autoinflammatory nature of the disease characterized by the activation of interleukin-1 beta, and interferon-gamma signaling pathways [8].

Studies have shown increased NLRP3 expression in PBMCs from AOSD patients that positively correlates with disease activity [9]. On the other hand, a number of patients exhibits a genetic association with MHC class II alleles [7], highlighting the potential influence of adaptive immunity (indicating an autoimmune origin of the disorder). This observation may elucidate why not all patients respond to therapy with interleukin-1 and interleukin-6 blockers, as well as the transition from a relapsing to a chronic course.

Limited studies explore adaptive immunity's role in Still's disease pathogenesis.’

2. In addition to describing the results of the deep flow cytometry analysis in the results section, the authors should further analyze the implications of the findings in adult-onset Still's disease as well as the role in developing prospective treatments.

Response. We appreciate for bringing this issue and we have now clarified this part of ‘Discussion’ section:

‘Finally, our findings provide new insights into the development of targeted therapies for patients with AOSD. Currently, the management of AOSD is predominantly based on anti-inflammatory therapy, rather than disease–modifying anti–rheumatic drugs (DMARDs). The concept of the "window of opportunity" for anti-cytokine therapy is extensively discussed, with a focus on IL-1 and IL-6 inhibitors, primarily as first-line agents. It has been observed that initiating this class of drugs early is more effective and leads to a more enduring response. In refractory cases and in the development of HLH, Janus kinases inhibitors as well as interferon-gamma inhibitor are also considered [7]. Furthermore, the efficacy of canakinumab therapy on patients with sJIA, both clinically and in the laboratory, was demonstrated in clinical trials, where gene expression analysis was conducted three days after drug administration. There was a distinctive pattern marked by the swift return to normal levels of transcripts related to sJIA overexpressed genes, including genes associated with IL-1 and IL-6 signaling pathways [48]. Another study assessing the dynamics of IL-1RA, IL-18, and IL-6, a reduction in these parameters was noted by the 6th week of canakinumab therapy [49]. Several cohort studies evaluating anakinra therapy have demonstrated consistent improvements in the mentioned cytokines, bringing them back to the normal range throughout the course of treatment [50, 51]. However, some studies have demonstrated that IL-18, unlike other pro-inflammatory cytokines and proteins, does not decrease during remission [6].

Round 2

Reviewer 1 Report

Thank you for additional information.