In this study, we aimed to comprehensively evaluate the proportions of CD4+ T cell and B cell subpopulations in peripheral blood collected from HCs and RA patients, and compared them between two locations; Tsukuba City and Karuizawa Town, which differ in altitude by 1000 m and, thus, have distinct differences in average air temperature and atmospheric pressure. Studying populations from these two locations, therefore, allows us to elucidate the effect of climate on the immunological features of RA. IPW adjustment with propensity scores was adopted to control for potential confounding factors such as age, sex, positivity of RF and anti-CCP antibody, dose of prednisolone, dose of methotrexate, use of bDMARDs or tsDMARDs, and DAS28-CRP in the RA cohorts, and age and sex in the HC cohorts between from Tsukuba and Karuizawa. Our analysis revealed that the proportion of T and B cells subpopulations were significantly different not only in RA patients but HCs between Tsukuba and Karuizawa, suggesting that climate variations might essentially affect immune cell phenotype regardless of background characteristics, like with or without RA. In addition, some of those differences of T and B cell subpopulations were observed only in the patients with RA, indicating that they might be a characteristic immunophenotype in RA.

In T cell subpopulations, a significant increase in cTh1 cells, cTfh1 cells, and Tph cells and significant decrease in cTh17 cells, cTh17.1 cells, and CD8+ Treg cells was observed in the patients with RA from Karuizawa compared with those of Tsukuba after IPW adjustment. Among these T cell subpopulations, Tph cells tended to be increased, and cTh17 cells and CD8+ Treg cells were significantly increased in RA patients compared to HCs from Karuizawa. However, there were no significant correlations between disease activity of RA and the T cell subpopulations in which a significant difference was found when Tsukuba and Karuizawa were compared. IFNγ secreting Th1 cell was identified in synovial fluids from RA patients9,10, and induce macrophage activation characterized by an increased capacity to produce pro-inflammatory cytokines such as TNF11, Past study reported that there was no significant difference in peripheral blood CXCR3+CCR6− Tfh1 cell between RA patients and HCs12. Tph cells have been defined as PD-1hiCXCR5CD4+ T cells, and reported to be uniquely poised to promote B cell response and antibody production within pathologically inflamed non-lymphoid tissue in RA13. Combining mass cytometry and transcriptomics also revealed expansion of Tph cells in RA synovia14. Our previous study and other reports have indicated the pathogenetic role of Th17 cells in RA15,16,17. Th17.1 cells are a subgroup of Th17 cell characterized by the expression of CXCR3 and the production of IFNγ, and have been reported as the most pathogenic among the Th17 cells and as the predictor of therapeutic response in patients with RA18,19. CD122+CD8+Treg cells have the capacity to inhibit T cell responses and suppress autoimmunity, however, their role in RA has remained unclear20. Hence, it has been speculated that climatic environment might affect the pathology of RA through alternation of T cell subpopulations.

Analyses of B cell subpopulations showed that, after IPW adjustment, DNB cells, DN1 B cells, DN2 B cells, and class-switched memory B cells were significantly increased, and unswitched memory B cells, naïve B cells, and ABCs were significantly decreased in the patients with RA from Karuizawa compared with those from Tsukuba. Among these B cell subpopulations, unswitched memory B cells were significantly decreased, but ABCs were significantly increased in RA patients compared to HCs from Karuizawa. However, there was no significant correlation between disease activity of RA and the B cell subpopulations in which significant difference was found when Tsukuba and Karuizawa were compared. DNB cells have been defined as IgD-CD27- B cells and are subclassified into DN1 cells or DN2 cells according to expression of CXCR5. DNB cells have garnered interest in the field of autoimmunity, especially in systemic lupus erythematosus (SLE); autoreactive DN2 B cells were expanded and differentiated into autoantibody-secreting plasmablast via hyper-responsiveness to Toll-like receptor 7 in extra-follicle21. With regards RA, several studies have reported that DNB cells were increased in RA, particularly in ACPA+ patients22,23. On the other hand, immunoglobulin class switching and further differentiation of memory B cells were mediated by T-B interaction in the germinal center, and the enhancement of this process was suggested by two findings: firstly, that citrullinated antigen-specific B cells displayed markers of class-switched memory B cells24 and, secondly, that the number of class-switched memory B cells was significantly increased in subjects carrying the risk haplotype B lymphoid kinase (BLK), which is a member of the Src family of tyrosine kinases and associated with RA25,26. ABC was newly identified B cells subset, and found to accumulate in the spleens of aged mouse and model mice of systemic lupus erythematosus27,28. Furthermore, the expansion of human ABCs has been observed in many autoimmune diseases including RA29. Accordingly, it was conjectured that climatic environment might also affect the pathology of RA through alternation of B cell subpopulations.

Although our analysis revealed some significant altered proportion of T and B cell subpopulations when comparing Tsukuba and Karuizawa populations, it is unclear how these cell alterations interact reciprocally and regulate the pathology of RA. As mentioned above, it was reported that Tph cells play an important role in promotion of B cell response and antibody production13,14, and that DNB cells and class-switched memory B cells are also related with autoantibody formation including ACPA formation in RA22,23,24. Consequently, increase of Tph cells, DNB cells, and class-switched memory B cells in the Karuizawa population raises the possibility that enhancement of autoantibody production might be one of the underlying mechanisms of RA related to climatic environment.

The question remains as to how the climatic factors such as air temperature and air pressure regulate the differentiation and the function of immune cells in RA. Significant relationships have been reported regarding the number and percentage of CD4+, CD8+ T cells, CD20+ B cells, and ambient temperature, sunlight duration, and air pressure in healthy volunteers30. The systematic effect of general cooling by 5-min exposure to cold air at a temperature of − 25 °C in healthy volunteers leads to decrease of T-lymphocytes count in venous blood, which indicated their functional insufficiency31. Although it has been reported that environmental factors such as oxygen concentration32,33, acidification34,35, salt concentration36, and glucose, amino acid, and lipid metabolism37,38,39 altered the differentiation and the function of immune cells, and contributed to the pathology of autoimmune diseases, it remains unclear how climatic factors such as air temperature and air pressure regulate immune cell function and the development of autoimmune diseases including RA.

The current study has some limitations. First, the effect of air temperature on the results of our study was inferred to be slight, because air temperature is almost completely controlled in the average Japanese living environment. Second, patients recruited for this study, from both Tsukuba and Karuizawa, were undergoing treatment and their RA was well-controlled with anti-rheumatic therapies including b/tsDMARDs, which may have substantially affected and modified the results of our study. Indeed, our results in HCs revealed that the difference in proportion of peripheral blood immune cells seemed to be more remarkable, and observed in more T and B cell subpopulations than RA patients. Third, as mentioned above, we were not able to clarify how climatic environment regulates immune cell function and disease state. Forth, it was required to freezing all blood samples for preservation. In addition, samples in Karuizawa were needed to be transported to Tsukuba to be analyzed in Tsukuba. The results of FACS were not statistical but slightly different in some immune cell subsets such as cTh17, cTfh17, and Breg cells (Supplement Figs. 3 and 4) between with and without freezing preservation, and thus it seemed to be difficult to completely exclude the possibility of the effect of freezing preservation and transportation on our results. Further studies that include more patients with high disease activity or without therapeutic intervention are needed to elucidate the exact and specific mechanism how climatic environment affects the immune cell-mediated pathology of RA.

In conclusion, our results suggest the possibility that climatic environment such as air temperature and air pressure has an effect on the proportion of T and B cell subpopulations and their function, and is related to the pathogenic mechanism of RA including autoantibody formation induced by T-B interaction.

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