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OPEN Effect of climatic environment

on immunological features
of rheumatoid arthritis
Yuya Kondo 1, Saori Abe 1, Hirofumi Toko 1,2, Tomoya Hirota 1,2, Hiroyuki Takahashi 1,2,
Masaru Shimizu 1,2, Hisashi Noma 3, Hiroto Tsuboi 1, Isao Matsumoto 1, Toshiro Inaba 2 &
Takayuki Sumida 1*
The aim of this study was to clarify the effect of climatic environment on the immunological features
of rheumatoid arthritis (RA). Blood samples were collected from patients with RA and healthy
controls (HCs), matched by age and sex, living in two locations, Tsukuba and Karuizawa, which differ
in their altitude and average air temperature and atmospheric pressure. Analysis of peripheral blood
mononuclear cells (PBMCs) revealed that the proportion of T and B cell subpopulations in HCs and
RA patients were significantly different between two sites. Inverse probability weighting adjustment
with propensity scores was used to control for potential confounding factors. The results revealed
that, in comparison with RA patients in Tsukuba, those in Karuizawa showed a significant increase
in cTh1, cTfh1, and Tph cells, and significant decrease in cTh17, cTh17.1, and CD8+ Treg in T cell
subpopulations, and a significant increase in DNB, DN1, DN2, and class-switched memory B cells,
and a significant decrease in unswitched memory B, naïve B cells, and ABCs in B cell subpopulations.
Our results suggest the possibility that climatic environment might have an effect on immune cell
proportion and function, and be related to the pathogenic mechanism of RA.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder characterized by autoimmunity, infiltration of

activated inflammatory cells into the joint synovium, synovial hyperplasia, neoangiogenesis, and progressive
destruction of the cartilage and bone. RA is thought to be caused by breakdown of immune tolerance, and, thus,
shows some characteristic features of abnormality in acquired immune systems formed by T and B cells. Previous
studies have shown that subpopulations of CD4+ T helper (Th) cells and B cells coordinately induce autoimmune
synovial inflammation; Th17 cells, for example, infiltrate and induce neutrophilic inflammation in the joints, and
Tfh cells not only promote autoantibody formation but also enhances their ­pathogenicity1.
It is well known that the development of RA is associated with genetic and environmental factors. Environ-
mental risk factors include smoking and periodontal diseases mainly related with Porphyromonas gingivalis. These
factors are hypothesized to promote aberrant citrullination and provoke local breach of tolerance to citrullinated
peptides via the expression of peptidylarginine deiminase, which results in the initiation of autoimmune response
and the aggravation of joint inflammation in ­RA2–5. The assumption that climatic environment influences the
signs and symptoms of RA is widely believed. Indeed, anecdotally, we noticed that many patients with RA com-
plained of fluctuation in joint symptoms according to climatic factors such as air temperature, humidity, and
atmospheric pressure; however, there is a paucity of scientific evidence supporting this generally-held assumption.
Patberg et al. reviewed and evaluated evidence that suggested the signs and symptoms of RA were influenced
by the weather, and revealed that temperature and humidity appeared to have effects on the symptoms of ­RA6.
In addition, Savage, et al. showed that the disease activity of RA was significantly lower in both sunnier and less
humid ­conditions7. Terao, et al. also reported that atmospheric pressure was inversely associated with synovitis
in patients with RA, and these associations were independent from temperature and h ­ umidity8. Hence, the
reported effects of climatic environment vary depending on the methods applied and geographic location of the
studies. Moreover, it remains unclear whether climate affects the immunological pathology in patients with RA.
To elucidate the effect of climatic environment on the immunological features of RA, we collected blood
samples from patients with RA and healthy controls (HC) in two distinct geographic locations: Tsukuba City,
located in the flat Kanto region of Japan, at an elevation of only 33 m above sea level, and Karuizawa Town, in the
mountainous prefecture of Nagano, at an average elevation of 1000 m above sea level. This contrast in altitude

1
Department of Rheumatology, Faculty of Medicine, University of Tsukuba, 1‑1‑1 Tennodai, Tsukuba,
Ibaraki 305‑8575, Japan. 2Karuizawa Municipal Hospital, Karuizawa, Nagano, Japan. 3Department of Data Science,
The Institute of Statistical Mathematics, Tachikawa, Tokyo, Japan. *email: tsumida@md.tsukuba.ac.jp

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brings differences in average air temperature of approximately 5 °C and atmospheric pressure of approximately
100 hPa. In this study, the proportion of subpopulation in T cells and B cells were comprehensively evaluated
using multi-color flow cytometry, and compared between RA patients and HCs, disease activity of RA, and by
the two locations.

Results
Participant characteristics.  A total of 80 participants were recruited for this study. Twenty patients with
RA and 20 HCs were recruited from both the University of Tsukuba Hospital and from Karuizawa Municipal
Hospital. The participants were matched by age and sex (Table). Among the RA patients, average of CRP and
disease activity index were slightly high because a few patients were classified into moderate disease activity in
patients of Tsukuba while all of the patients in Karuizawa achieved less that low disease activity. However, there
were no significant differences in RF titer, positivity of anti-CCP antibody, disease activity shown by DAS28-
CRP, CDAI, and SDAI, and treatment such as use or dosage of corticosteroid, methotrexate, bDMARDs, and
tsDMARDs.

Comprehensive evaluation of T cell and B cell subpopulations in RA and HCs by location.  The
proportion of T cell and B cell subpopulations in peripheral blood mononuclear cells (PBMCs) were compre-
hensively evaluated using multi-color flow cytometry, and compared between RA patients and HCs and by the
two geographic locations, respectively.
Regarding T cells, the percentages of cTh1 cells, cTh17 cells, cTh17.1 cell, cTfh1 cells, cTfh2 cells, cTfh17 cells,
and Tph cells in memory ­CD4+ T cells, Treg cells in naïve and memory ­CD4+ T cells, and ­CD8+ T cells, and ­CD8+
­ D8+ T cells were analyzed. The results showed a significantly increased proportion of cTfh1 cells,
Treg cells in C
memory Treg cells, and CD8+ T cells and a significantly decreased proportion of naïve Treg cells were observed
only in HCs from Karuizawa compared with those from Tsukuba (Fig. 1). A significant increase in cTfh2 cells
and a significant decrease in cTh17.1 cells and CD8+ Treg cells were observed in both HCs and RA patients
from Karuizawa compared with those from Tsukuba (Fig. 1). Interestingly, Tph cells were significantly increased
and cTh17 cells were significantly decreased only in RA patients from Karuizawa compared with those from
Tsukuba (Fig. 1). Moreover, cTh17 cells, cTfh2 cells, cTfh17 cells, Tph cells, and CD8+ Treg cells were significantly
increased, and cTh1 cells and memory Treg cells were significantly decreased and in patients with RA compared
HCs from both Tsukuba and Karuizawa, and there was no difference between Tsukuba and Karuizawa regarding
the fluctuations of the proportions of these cells (Fig. 1). A significant increased proportion of naïve Treg cells was
observed in patients with RA compared to HCs from both Tsukuba and Karuizawa (Fig. 1). Collectively, there
were some significant differences between Tsukuba and Karuizawa regarding T cell subpopulations, whereas
their fluctuations between HCs and RA patients were comparable between Tsukuba and Karuizawa.
Regarding B cells, the percentage of double negative (DN) B cells, DN1 cells, DN2 cell, naïve B cells,
unswitched memory B cells, class switched B cells, and plasmablasts in CD19+CD20+ B cells, and B regulatory
­ D19+ B cells was analyzed. A significant increased proportion of unswitched memory B cells and
(Breg) cells in C
significant decreased proportion of ABCs were observed in the HCs from Karuizawa compared to those from
Tsukuba (Fig. 2). A significant increase in DNB cells, DN2 B cells, and class-switched memory B cells and a sig-
nificant decrease in naïve B cells and Breg cells were observed in both HCs and RA patients from Karuizawa com-
pared with those from Tsukuba (Fig. 2). Moreover, DN2 B cells were significantly increased in RA patients from
Karuizawa compared with those in Tsukuba (Fig. 2). Unswitched memory B cells and class switched memory B
cells were significantly increased, and naïve B cells were significantly decreased in RA patients compared to HCs
in Tsukuba, while plasmablast and ABC were significantly increased, and unswitched memory B cells, DN2 cells,
and Breg cells were significantly decreased in RA patients compared to HCs in Karuizawa (Fig. 2). Interestingly,
our data showed discordance between Tsukuba and Karuizawa regarding the fluctuations in the proportions of
these cells, especially in unswitched memory B cells (Fig. 2). Collectively, comparing Tsukuba and Karuizawa,
there were some significant differences and inconsistencies regarding B cell subpopulations of HCs and/or RA
patients. Accordingly, the proportion of the subpopulations of T cells and B cells were significantly different
between Tsukuba and Karuizawa, and some differences were only observed in the patients with RA, indicating
that the climate variations might affect immune function in both the normal and pathogenic mechanisms of RA.

Comparison of T and B cell subpopulations in the patients with RA and HCs after propensity
score weighting.  Although there were no significant differences in baseline characteristics of RA patients
and HCs between Tsukuba and Karuizawa (Table 1), inverse probability weighting (IPW) adjustments with pro-
pensity scores were performed to control for any bias caused by the imbalance of potential confounding factors.
In T cell subpopulations, a significant increase in cTh1 cells, cTfh1 cells, and Tph cells, and a significant
decrease in cTh17 cells, cTh17.1 cells, and CD8+ Treg cells were observed in the Karuizawa patients with RA
compared those from Tsukuba after the IPW adjustments (Fig. 3A). In addition, analyses of B cell subpopulations
showed that 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 Karuizawa patients
with RA compared with those from Tsukuba (Fig. 3B). Accordingly, the differences in T and B cell subpopulations
between Karuizawa and Tsukuba RA patients were confirmed even after IPW adjustment.
IPW adjustments in HCs revealed that a significant increase in cTfh1 cells, cTfh2 cells, Tph cells, and CD8+ T
cells, and a significant decrease in cTh17 cells, cTh17.1 cells, memory and naïve Treg cells, and CD8+ Treg
cells in the Karuizawa patients with RA compared those from Tsukuba (Fig. 4A). Moreover, analyses of B cell
subpopulations showed that DNB cells, DN1 B cells, DN2 B cells, and unswitched and class-switched memory
B cells were significantly increased, and naïve B cells, ABCs, and Breg cells were significantly decreased in the

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Figure 1.  Differences in the proportion of subpopulations in peripheral blood T cells between RA patients

and HCs from two distinct geographic locations. The proportion of T cell subpopulations in PBMC collected
from RA patients and HCs in two hospitals was analyzed using flow cytometry. Histograms show percentage
of cTh1 cells, cTh17 cells, cTh17.1 cells, cTfh1 cells, cTfh2 cells, cTfh17 cells, and cTph cells in memory C­ D4+
T cells, cTreg cells in naïve and memory C ­ D4+ T cells, ­CD8+ T cells, and C
­ D8+ Treg cells in ­CD8+ T cells. Data
are presented as mean ± SEM, and circle and square shows samples collected from Tsukuba and Karuizawa,
respectively. Unpaired t-test was performed to analyze statistical interactions between blood samples collected
from HCs and RA patients in Tsukuba or Karuizawa. Statistical significance was defined as *P < 0.05, **P < 0.01,
***P < 0.001, and ****P < 0.0001.

Karuizawa patients with RA compared with those from Tsukuba (Fig. 4B). These observations showed that the
proportion of the subpopulations of T cells and B cells were significantly different in both RA patients and HCs
between Karuizawa and Tsukuba after the control for the imbalance of potential confounding factors with IPW
adjustment, and suggesting that the climate variations might essentially affect immune cell phenotype regardless
of with or without RA.

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Figure 2.  Differences in the proportion of subpopulations of peripheral blood B cells between RA patients and HCs from
two distinct geographic locations. The proportion of subpopulation in B cells in PBMC collected from RA patients and HC
in two hospitals was analyzed by flow cytometry. Histograms show percentage of class switched memory B cells, unswitched
memory B cells, memory B cells, and double negative (DN) B cells in C ­ D19+CD20+ cells, DN1 cells and DN2 cells in DNB
­ D19+CD20− cells, ABC and Breg cells in ­CD19+ cells. Data are presented as mean ± SEM. Unpaired t-test
cells, plasmablast in C
was performed to analyze a statistical interaction between blood samples collected from HC or RA patients in Tsukuba or
Karuizawa. Statistical significance was defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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University of Tsukuba Hospital Karuizawa Municipal Hospital

HC RA HC RA
(N = 20) (N = 20) (N = 20) (N = 20)
Age 58 ± 12 62 ± 12 60 ± 12 65 ± 11
Female, n (%) 20 (100) 18 (90) 15 (75) 16 (80)
CRP (mg/dL) – 0.62 ± 1.43 – 0.29 ± 0.53
RF (U/ml) – 55 ± 63 – 71 ± 126
Anti-CCP antibody, n (%) – 15 ­(88a) – 14 ­(70a)
DAS28-CRP – 2.00 ± 0.76 – 1.79 ± 0.84
CDAI – 4.3 ± 3.5 – 2.9 ± 2.5
SDAI – 4.6 ± 3.6 – 3.2 ± 2.8
PSL use, n (%) – 6 (30) – 8 (40)
PSL dose, (mg/day) – 4.1 ± 2.8 – 3.6 ± 1.1
MTX use, n (%) – 14 (70) – 17 (85)
MTX dose, (mg/week) 7.4 ± 3.2 – 8.0 ± 4.0
b/tsDMARDs use, n (%) – 6 (30) – 4 (20)

Table 1.  Demographics and clinical characteristics of HCs and RA patients. Anti-CCP antibody, anti-cyclic
citrullinated peptide antibody; b/tsDMARDs, biologic or targeted synthesized disease modifying anti-
rheumatic drugs; CDAI, Clinical Disease Activity Index; CRP, C-reactive protein; MTX, methotrexate; PSL,
prednisolone; RF, rheumatoid factor; SDAI, Simplified Disease Activity Index. Continuous variables are
showed by Mean ± standard deviation (SD). a Anti-CCP antibody was measured in 20 RA patients from the
University of Tsukuba Hospital and Karuizawa Municipal Hospital, respectively (N = 40).

Correlation between disease activity of RA and subpopulations of T cells and B cells.  The rela-
tionship between disease activity of RA and subpopulation in T cells and B cells, respectively, were assessed. As
described above, there were no significant difference in disease activity of RA between Tsukuba and Karuizawa,
and most of patients achieved low disease activity (Table 1 and Fig. 5). Among T cell subpopulations, the propor-
tion of cTfh1 cells negatively correlated with DAS28-CRP in Tsukuba but not in Karuizawa (Fig. 5A). Proportion
of Tph cells also tended to be positively correlated with DAS28-CRP only in Tsukuba (Fig. 5A). On the other
hand, no significant correlations were observed between disease activity of RA and subpopulations in B cells
for both Tsukuba and Karuizawa (Fig. 5B). Taken together, these findings suggest that the disease activity of RA
might have less of an effect on T and B cell subpopulations in patients with RA achieving low disease activity
regardless of climate.

Discussion
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 anti-
body, 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 differ-
ences 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 com-
pared 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 char-
acterized by an increased capacity to produce pro-inflammatory cytokines such as T ­ NF11, 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-1hiCXCR5-CD4+ T cells, and reported to be uniquely poised to promote
B cell response and antibody production within pathologically inflamed non-lymphoid tissue in R ­ A13. Combining
mass cytometry and transcriptomics also revealed expansion of Tph cells in RA s­ ynovia14. Our previous study
and other reports have indicated the pathogenetic role of Th17 cells in R ­ A15–17. 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

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Figure 3.  Comparison of subpopulations of peripheral blood T and B cells in RA patients after adjustment with
propensity score weighting. Inverse probability weighting adjustments with propensity scores were performed
to control for biases caused by the imbalance of potential confounding factors in comparison of subpopulation
in T cells (A) and B cells (B) of PBMC collected from RA patients in two hospitals. The graph shows the mean
difference with 95% confidence intervals for each subpopulation after IPW adjustment. Positive or negative
numbers represent increase or decrease of the subpopulations in the patients in Karuizawa, respectively.
Statistical significance was defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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 u ­ nclear20. 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 subclas-
sified 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 R ­ A25,26. ABC was newly identified B cells subset, and found to accumulate in the spleens of

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A Decreased in Karuizawa Increased in Karuizawa

T cell subpopulations
cTh1
cTh17 *
cTh17.1 **
cTfh1 **
cTfh2 ***
cTfh17
Tph ***
Memory Treg ***
Naive Treg ***
CD8+ **
CD8+ Treg ***

-10 -5 0 5 10 15
Mean difference

B Decreased in Karuizawa Increased in Karuizawa

DNB ***
B cell subpopulations

DN1 **
DN2 ***
Unswitched Memory B ***
Class- switched memory B ***
Naive B ***
Plasmablast
ABC *
Breg ***

- 100 - 50 0 50
Mean difference
Figure 4.  Comparison of subpopulations of peripheral blood T and B cells in HCs after adjustment with
propensity score weighting. Inverse probability weighting adjustments with propensity scores were performed to
control for biases caused by the imbalance of potential confounding factors in comparison of subpopulation in
T cells (A) and B cells (B) of PBMC collected from HCs in two hospitals. The graph shows the mean difference
with 95% confidence intervals for each subpopulation after IPW adjustment. Positive or negative numbers
represent increase or decrease of the subpopulations in the patients in Karuizawa, respectively. Statistical
significance was defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

aged mouse and model mice of systemic lupus e­ rythematosus27,28. Furthermore, the expansion of human ABCs
has been observed in many autoimmune diseases including R ­ A29. 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 R ­ A22–24. 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 dura-
tion, and air pressure in healthy v­ olunteers30. 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 i­ nsufficiency31. Although it has been reported that environmental factors

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Figure 5.  Correlation between T cell or B cell subpopulations and disease activity in RA patients. Correlation
between disease activity of RA and proportion of the subpopulations in T cells (A) and B cells (B) in PBMCs
collected from Tsukuba and Karuizawa were analyzed. Correlation analysis was performed using Spearman rank
correlation coefficient.

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such as oxygen c­ oncentration32,33, ­acidification34,35, salt c­ oncentration36, and glucose, amino acid, and lipid
­metabolism37–39 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 cli-
matic 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.

Material and methods
Study participants.  In this study, patients with RA and HCs were recruited from the University of Tsukuba
Hospital and Karuizawa Municipal Hospital, respectively. Blood samples were collected from 20 RA patients
receiving treatment in the Department of Rheumatology and a further 20 samples from HC volunteers were
provided by Tsukuba Human-Tissue Biobank Center in the University of Tsukuba Hospital. Likewise, blood
samples were collected from 20 patients with RA and 20 HC volunteers in Karuizawa Municipal Hospital.
Although we collected blood samples without restriction of the time of year, there was no seasonal bias in both
Karuizawa and Tsukuba, and thus the effect of time of sample collection was considered to be a minimum. All
patients with RA fulfilled either the 1987 revised criteria of the American College of Rheumatology (ACR) for
the classification of RA or the 2010 ACR/European League Against Rheumatism (EULAR) classification criteria.
All RA patients were evaluated for age, sex, tender joint count (TJC), swollen joint count (SJC), patient global
assessment (patient visual analogue scale [Pt-VAS]), physician global assessment (doctor’s visual analogue scale
[D-VAS]), C-reactive protein (CRP) level, and disease activity index of RA such as Disease Activity Score 28
(DAS28) -CRP, Clinical Disease Activity Index (CDAI), and Simplified Disease Activity Index (SDAI) were cal-
culated based on above data. Rheumatoid factor (RF) value, positivity of anti-cyclic citrullinated peptide (CCP)
antibody, and medication at the time when the blood samples were collected were assessed using electronic
medical records.
The study was approved by the ethics committees of the University of Tsukuba Hospital and was carried
out in accordance with the Declaration of Helsinki. Patients recruited for the study were enrolled after written
informed consent was received (the reference number: H30-134).

Cell preparation.  After obtaining whole blood samples, collected using heparin tubes, peripheral blood
mononuclear cells (PBMCs) were isolated by density gradient using Ficol-Paque Plus (GE Healthcare). To evalu-
ate several samples simultaneously, PBMCs were cryopreserved in CELLBANKER (TakaraBio) at − 80 °C and
stored in a deep freezer. The process for preserving blood samples was standardized and not different between
Karuizawa and Tsukuba. Frozen samples of Karuizawa were transported to Tsukuba, and all of the samples
were analyzed in Tsukuba University Hospital. Before evaluation, PBMCs were thawed, rested for at least 1 h
to allow removal of cell debris as recommended in the protocol (catalog 3520-2A, MABTECH), washed, and
resuspended in RPMI Medium 1640 containing 10% FBS and 1% penicillin.

Flow cytometry analysis.  Before superficial antigen staining, cells were stained with 7-Amino-Actino-
mycin D (7-AAD) for 5 min at room temperature for the exclusion of nonviable cells in the flow cytometry
analysis. Surface staining of the subpopulations in T and B cells was conducted for 30 min on ice under dark-
ened conditions with the following antibodies for the analysis of T cells: anti-CD4-APC (BioLegend), anti-
CD8-APC-Alexa700 (BIoLegend), anti-CD25-BV711 (BioLegend), anti-CD45RA-APC-Cy7 (BD), anti-CD122-
BV421 (BioLegend), anti-CD127-BV605 (BioLegend), anti-CC chemokine receptor 6 (CCR6)-PE (BioLegend),
anti-CXC chemokine receptor 3 (CXCR3)-Alexa Fluor (AF) 488 (BioLegend), anti-CXCR5-PE-Cv7 (BioLeg-
end), and anti-PD-1-BV510 (BioLegend); and for the analysis of B cell: anti-CD11c-BV711 (BioLegend), anti-
CD19-FITC (BioLegend), anti-CD20-APC-Cy7 (BioLegend), anti-CD24-BV510 (BioLegend), anti-CD27-APC
(BioLegend), anti-CD38-PE-Cy7 (BioLegend), anti-CXCR5-Pacific Blue (BioLegend), and anti-IgD-PE (BioLe-
gend). FACS analysis was performed using LSRFortessa X-20 Flow Cytometer (BD Bioscience), and analyzed
with FlowJo software (Tree Star, Ashland, OR, USA).
In this study, the subpopulations of T cells and B cells were defined using cell surface markers reported in
previous studies. Definitions of each subpopulation are summarized in the Supplemental Table. Representative
plots and the gating strategy for evaluating T cells and B cells are shown in Supplement Figs. 1 and 2. We also

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confirmed that there was no difference of the results of FACS analysis of blood sample with or without freezing
preservation by exclusion of nonviable cells stained with 7-AAD. Results are shown in Supplement Figs. 3 and 4.

Statistical analysis.  Data are summarized as mean ± standard deviation (SD). Statistical differences in
baseline characteristics were evaluated using the Mann–Whitney U test or the Kruskal–Wallis test for continu-
ous variables and by Fisher’s exact test or Chi-squared test for categorical variables. Correlation analysis was
performed using Spearman’s rank correlation coefficient.
Student’s t-tests were performed to assess the differences between blood samples collected from HCs or RA
patients in Tsukuba and Karuizawa. Wilcoxon signed rank tests were performed to assess the differences between
blood samples before and after freezing preservation. It was anticipated that the background characteristics
of RA patients and HCs would differ substantially between the Tsukuba and Karuizawa groups, thus, inverse
probability weighting (IPW) adjustments with propensity scores were performed to control for biases caused
by potential confounding factors. We considered the following variables as potential confounding factors: age,
sex, positivity of RF and anti-CCP antibody, dose of prednisolone, dose of methotrexate, use of biologic disease-
modifying anti-rheumatic drugs (bDMARDs) or targeted disease-modifying anti-rheumatic drugs (tsDMARDs),
and DAS28-CRP in RA patients, and age and sex in HCs. We used the multiple imputation by chained e­ quation40
to treat missing data. We created 200 imputation data and used ordinary Rubin’s synthesis rule. We used the
R package m ­ ice41 for the computation. Since this study is an observational study, and there are no families of
statistical tests that multiplicity adjustments should be ­considered42. We did not adopt multiplicity adjustments
for all statistical tests. All P values quoted are 2-sided and the significant levels were set to 0.05.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding
author on reasonable request.

Received: 21 September 2022; Accepted: 27 December 2022

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Acknowledgements
We would like to thank Thomas Mayers, Medical English Communications Center, University of Tsukuba for
English language editing.

Author contributions
Y.K., I.M., T.I., and T.S. designed research. Y.K., S.A., H.T., T.H., H.T., and M.S. performed research. Y.K., S.A.
H.N., and H.T. analyzed data. Y.K., H.N. and T.S. wrote the paper. All authors reviewed the manuscript.

Competing interests 
The authors declare no competing interests.

Additional information
Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​022-​27153-3.
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