Single-cell-level protein analysis revealing the roles of autoantigen-reactive B lymphocytes in autoimmune disease and the murine model
- Department of Dermatology, The University of Tokyo Graduate School of Medicine, Japan
- Laboratory of Radiology and Biomedical Engineering, The University of Tokyo Graduate School of Medicine, Japan
- Department of System Design Engineering, Keio university, Faculty of Science and technology, Japan
- Department of Applied Chemistry, The University of Tokyo Graduate School of Engineering, Japan
- Department of Mechanical Engineering, The University of Tokyo Graduate School of Engineering, Japan
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 …
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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 …
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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 …
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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. …
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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 …
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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
Figure 3
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 …
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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 …
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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. …
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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
Figure 5
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 …
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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 …
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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, …
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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 …
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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 …
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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.
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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.
| Characteristics | High-affinity B cell-dominant group* | Low-affinity B cell-dominant group* | p-Value |
|---|---|---|---|
| Sex, number of males/females | 4/54 | 5/48 | N.S. |
| Age at onset, mean ± SD (age) | 52 ± 9 | 49 ± 7 | N.S. |
| Disease duration, mean ± SD (years) | 5 ± 2 | 2 ± 1 | <0.001 |
| Number with dcSSc/lcSSc | 50/8 | 47/6 | N.S. |
| Clinical features | |||
| mRTSS, mean ± SD | 23 ± 5 | 16 ± 4 | <0.001 |
| Pitting scars or ulcers (%) | 62 | 13 | <0.01 |
| Lung involvement | |||
| Interstitial lung disease (%) | 78 | 68 | N.S. |
| %FVC, mean ± SD (%) | 70 ± 9 | 91 ± 11 | <0.001 |
| %DLco, mean ± SD (%) | 57 ± 11 | 83 ± 10 | <0.001 |
| Laboratory findings | |||
| Serum IgG levels, mean ± SD (mg/dl) | 1512 ± 371 | 1398 ± 353 | N.S. |
| Serum IgM levels, mean ± SD (mg/dl) | 159 ± 16 | 179 ± 22 | N.S. |
| CRP levels, mean ± SD (mg/dl) | 0.28 ± 0.19 | 0.21 ± 0.14 | N.S. |
| Anti-topo I antibody levels, mean ± SD (U/ml) | 144 ± 32 | 147 ± 43 | N.S. |
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Unless noted otherwise, values are the number of observations.
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*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.
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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.
