Single-cell-level protein analysis revealing the roles of autoantigen-reactive B lymphocytes in autoimmune disease and the murine model

  1. Takemichi Fukasawa
  2. Ayumi Yoshizaki  Is a corresponding author
  3. Satoshi Ebata
  4. Asako Yoshizaki-Ogawa
  5. Yoshihide Asano
  6. Atsushi Enomoto
  7. Kiyoshi Miyagawa
  8. Yutaka Kazoe
  9. Kazuma Mawatari
  10. Takehiko Kitamori
  11. Shinichi Sato  Is a corresponding author
  1. Department of Dermatology, The University of Tokyo Graduate School of Medicine, Japan
  2. Laboratory of Radiology and Biomedical Engineering, The University of Tokyo Graduate School of Medicine, Japan
  3. Department of System Design Engineering, Keio university, Faculty of Science and technology, Japan
  4. Department of Applied Chemistry, The University of Tokyo Graduate School of Engineering, Japan
  5. Department of Mechanical Engineering, The University of Tokyo Graduate School of Engineering, Japan
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Frequencies and cytokine production ability of topo I-reactive B cells in systemic sclerosis (SSc) patients.

(A) Frequencies of topo I-reactive cells in CD27+ CD19+ cells obtained from the peripheral blood of 50 healthy controls (HC), 50 anti-CENP antibody-positive SSc patients, and 111 anti-topo I …

Figure 1—source data 1

Source file for frequencies and cytokine production ability of topo I-reactive B cells in systemic sclerosis (SSc) patients.

This archive contains all data used for the quantitative analysis shown in Figure 1.

https://cdn.elifesciences.org/articles/67209/elife-67209-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Blocking effect of topo I to frequencies of topo I-reactive B cells in anti-topo I antibody-positive systemic sclerosis (SSc) patients.

Topo I-reactive B cells in CD27+CD19+ cells obtained from anti-topo I antibody-positive SSc patients were flow cytometric pre-blocked with excess topo I.

Figure 2 with 3 supplements
The relationship between the affinity for topo I in topo I-reactive B cells and their ability to produce cytokines in systemic sclerosis (SSc) patients.

(A) Topo I-non-reactive B cells and topo I-reactive B cells were isolated from the peripheral blood of anti-topo I antibody-positive SSc patients, and topo I titers were measured at the single B …

Figure 2—source data 1

Source file for the relationship between the affinity for topo I in topo I-reactive B cells and their ability to produce cytokines in systemic sclerosis (SSc) patients.

This archive contains all data used for the quantitative analysis shown in Figure 2.

https://cdn.elifesciences.org/articles/67209/elife-67209-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Affinity analysis and correlation of affinities for topo I in topo I-reactive B cells with topo I-IgG(OD)/total IgG(OD).

Topo I-non-reactive B cells and topo I-reactive B cells in CD19+CD27+ B cells were isolated from the peripheral blood of anti-topo I antibody-positive systemic sclerosis (SSc) patients, and topo I …

Figure 2—figure supplement 1—source data 1

Source file for affinity analysis and correlation of affinities for topo I in topo I-reactive B cells with topo I-IgG(OD)/total IgG(OD).

This archive contains all data used for the quantitative analysis shown in Figure 2—figure supplement 1.

https://cdn.elifesciences.org/articles/67209/elife-67209-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Frequencies of CD38-CD95+ cells in topo I-PE+Topo I-APC+CD19+CD27+ B cells in anti-topo I antibody-positive systemic sclerosis (SSc) patients.

Frequencies of CD38-CD95+ cells in topo I-PE+Topo I-APC+CD19+CD27+ B cells in anti-topo I antibody-positive SSc patients were analyzed by flow cytometry (n = 10). The bar graphs show the mean + SD. …

Figure 2—figure supplement 2—source data 1

Source file for frequencies of CD38-CD95+ cells in topo I-PE+Topo I-APC+CD19+CD27+ B cells in anti-topo I antibody-positive systemic sclerosis (SSc) patients.

This archive contains all data used for the quantitative analysis shown in Figure 2—figure supplement 2.

https://cdn.elifesciences.org/articles/67209/elife-67209-fig2-figsupp2-data1-v1.xlsx
Figure 2—figure supplement 3
Expression of co-stimulatory molecules in topo I-PE+Topo I-APC+CD19+CD27+ B cells in anti-topo I antibody-positive systemic sclerosis (SSc) patients.

Expression of co-stimulatory molecules (CD80/86, ICOSL, and CD40) in topo I-non-reactive B cells and topo I-reactive B cells (topo I-PE+Topo I-APC+CD19+CD27+) with low or high affinity for topo I in …

Figure 2—figure supplement 3—source data 1

Source file for expression of co-stimulatory molecules in topo I-PE+Topo I-APC+CD19+CD27+ B cells in anti-topo I antibody-positive systemic sclerosis (SSc) patients.

This archive contains all data used for the quantitative analysis shown in Figure 2—figure supplement 3.

https://cdn.elifesciences.org/articles/67209/elife-67209-fig2-figsupp3-data1-v1.xlsx
Effects of topo I-reactive B cells on the differentiation of CD4+ T cells in systemic sclerosis (SSc) patients.

B cells with low affinity for topo I and those with high affinity for topo I as well as topo I-non-reactive B cells were obtained from anti-topo I antibody-positive SSc patients (n = 111). These B …

Figure 3—source data 1

Source file for the effects of topo I-reactive B cells on the differentiation of CD4+ T cells in systemic sclerosis (SSc) patients.

This archive contains all data used for the quantitative analysis shown in Figure 3.

https://cdn.elifesciences.org/articles/67209/elife-67209-fig3-data1-v1.xlsx
Figure 4 with 1 supplement
The development of fibrosis and cytokine production by topo I-reactive B cells in the topo I-induced systemic sclerosis (SSc) model mice.

(A) Topo I-induced SSc model mice were generated by immunizing topo I up to four times every 2 weeks. At days 0, 14, and 56, mice were sacrificed and used for study as non-immunized, first …

Figure 4—source data 1

Source file for the development of fibrosis and cytokine production by topo I-reactive B cells in the topo I-induced systemic sclerosis (SSc) model mice.

This archive contains all data used for the quantitative analysis shown in Figure 4.

https://cdn.elifesciences.org/articles/67209/elife-67209-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
The relationship between the affinity for topo I in topo I-reactive B cells and their ability to produce cytokines in complete systemic sclerosis (SSc) model mice.

Topo I-induced complete SSc model (complete model) mice were generated by immunizing topo I for four times every 2 weeks. At day 56, mice were sacrificed and used for study as complete model mice. …

Figure 4—figure supplement 1—source data 1

Source file for the relationship between the affinity for topo I in topo I-reactive B cells and their ability to produce cytokines in complete systemic sclerosis (SSc) model mice.

This archive contains all data used for the quantitative analysis shown in Figure 4—figure supplement 1.

https://cdn.elifesciences.org/articles/67209/elife-67209-fig4-figsupp1-data1-v1.xlsx
Correlation of affinities for topo I in topo I-reactive B cell with clinical parameters for skin and lung fibrosis in systemic sclerosis (SSc) patients.

In anti-topo I antibody-positive SSc patients (n = 111), the ratio of B cells with high affinity for topo I (high-affinity B cells) to B cells with low affinity for topo I (low-affinity B cells) was …

Figure 5—source data 1

Source file for correlation of affinities for topo I in topo I-reactive B cell with clinical parameters for skin and lung fibrosis in systemic sclerosis (SSc) patients.

This archive contains all data used for the quantitative analysis shown in Figure 5.

https://cdn.elifesciences.org/articles/67209/elife-67209-fig5-data1-v1.xlsx
Figure 6 with 2 supplements
Effect of the affinity for topo I in topo I-reactive B cells on the development of fibrosis in the topo I-induced systemic sclerosis (SSc) model mice.

Topo I-reactive B cells (104 cells) from mice immunized once or four times with topo I were adoptively transferred into wild-type (WT) mice, and 14 days later, these WT mice were immunized with topo …

Figure 6—source data 1

Source file for the effect of the affinity for topo I in topo I-reactive B cells on the development of fibrosis in the topo I-induced systemic sclerosis (SSc) model mice.

This archive contains all data used for the quantitative analysis shown in Figure 6.

https://cdn.elifesciences.org/articles/67209/elife-67209-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
The effect of fibrosis and cytokine production by topo I-reactive B cells in the topo I-induced incomplete systemic sclerosis (SSc) model mice.

Topo I-induced incomplete SSc model (Incomplete model) mice were generated by immunizing topo I for one time, followed by adjuvant immunization up to three times every 2 weeks (A). At days 0 and 56, …

Figure 6—figure supplement 1—source data 1

Source file for the effect of fibrosis and cytokine production by topo I-reactive B cells in the topo I-induced incomplete systemic sclerosis (SSc) model mice.

This archive contains all data used for the quantitative analysis shown in Figure 6—figure supplement 1.

https://cdn.elifesciences.org/articles/67209/elife-67209-fig6-figsupp1-data1-v1.xlsx
Figure 6—figure supplement 2
Effect of B cell-specific Blimp1 or class II in topo I-reactive B cells on the development of fibrosis in the topo I-induced systemic sclerosis (SSc) model mice.

Wild-type (WT) mice, B cell-specific Blimp1 CKO mice (PRDM1 CKO), and B cell-specific class II CKO mice (Class II CKO) were immunized four times with topo I. Then, 104 cells of topo I-reactive B …

Figure 6—figure supplement 2—source data 1

Source file for the effect of B cell-specific Blimp1 or class II in topo I-reactive B cells on the development of fibrosis in the topo I-induced systemic sclerosis (SSc) model mice.

This archive contains all data used for the quantitative analysis shown in Figure 6—figure supplement 2.

https://cdn.elifesciences.org/articles/67209/elife-67209-fig6-figsupp2-data1-v1.xlsx
Figure 7 with 1 supplement
Effect of Bruton’s tyrosine kinase (BTK) inhibition on the affinity for topo I of topo I-reactive B cells and the development of fibrosis in the topo I-induced systemic sclerosis (SSc) model mice.

(A) Frequencies of topo I-reactive B cells in splenic B cells from mice immunized four times with topo I and from mice treated with a BTK inhibitor along with topo I were examined. Ibrutinib (12.5 …

Figure 7—source data 1

Source file for the effect of Bruton’s tyrosine kinase (BTK) inhibition on the affinity for topo I of topo I-reactive B cells and the development of fibrosis in the topo I-induced systemic sclerosis (SSc) model mice.

This archive contains all data used for the quantitative analysis shown in Figure 7.

https://cdn.elifesciences.org/articles/67209/elife-67209-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Change of modified Rodnan skin score (mRSS) and anti-topo I antibody from baseline after rituximab (RTX) treatment.

For the 10 patients who received RTX treatment, data at 1 year after RTX showed that mRSS improved by an average of about six points, while titer remained the same.

Figure 7—figure supplement 1—source data 1

Source file for change of modified Rodnan skin score (mRSS) and anti-topo I antibody from baseline after rituximab treatment.

This archive contains all data used for the quantitative analysis shown in Figure 7—figure supplement 1.

https://cdn.elifesciences.org/articles/67209/elife-67209-fig7-figsupp1-data1-v1.xlsx

Tables

Table 1
Clinical correlation of the affinity for topo I of topo I-reactive B cells in anti-topo I antibody-positive SSc patients.
CharacteristicsHigh-affinity B cell-dominant group*Low-affinity B cell-dominant group*p-Value
Sex, number of males/females4/545/48N.S.
Age at onset, mean ± SD (age)52 ± 949 ± 7N.S.
Disease duration, mean ± SD (years)5 ± 22 ± 1<0.001
Number with dcSSc/lcSSc50/847/6N.S.
Clinical features
mRTSS, mean ± SD23 ± 516 ± 4<0.001
Pitting scars or ulcers (%)6213<0.01
Lung involvement
Interstitial lung disease (%)7868N.S.
%FVC, mean ± SD (%)70 ± 991 ± 11<0.001
%DLco, mean ± SD (%)57 ± 1183 ± 10<0.001
Laboratory findings
Serum IgG levels, mean ± SD (mg/dl)1512 ± 3711398 ± 353N.S.
Serum IgM levels, mean ± SD (mg/dl)159 ± 16179 ± 22N.S.
CRP levels, mean ± SD (mg/dl)0.28 ± 0.190.21 ± 0.14N.S.
Anti-topo I antibody levels, mean ± SD (U/ml)144 ± 32147 ± 43N.S.
  1. Unless noted otherwise, values are the number of observations.

  2. *The high-affinity B cell-dominant group had a higher frequency (>50%) of B cells with high affinity for topo I among CD27+ B cells, while the low-affinity B cell-dominant group had a higher frequency (>50%) of B cells with low affinity for topo I.

  3. dcSSc, diffuse cutaneous systemic sclerosis; lcSSc, limited cutaneous systemic sclerosis; CRP, C-reactive protein; N.S., not significant; mRTSS, modified Rodnan total skin thickness score; %FVC, percent predicted values of forced vital capacity; %DLco, percent predicted values of diffusion capacity of the lung for carbon monoxide; SSC, systemic sclerosis.

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