Sustained low-dose interleukin-2 therapy alleviates pathogenic humoral immunity via elevating the Tfr/Tfh ratio in lupus

Kaili Liang, Jing He, Yunbo Wei, Qunxiong Zeng, Dongcheng Gong, Jiahuan Qin, Huihua Ding, Zhian Chen, Ping Zhou, Peng Niu, Qian Chen, Chenguang Ding, Liangjing Lu, Xiao-Xiang Chen, Zhanguo Li, Nan Shen, Di Yu, Jun Deng, Kaili Liang, Jing He, Yunbo Wei, Qunxiong Zeng, Dongcheng Gong, Jiahuan Qin, Huihua Ding, Zhian Chen, Ping Zhou, Peng Niu, Qian Chen, Chenguang Ding, Liangjing Lu, Xiao-Xiang Chen, Zhanguo Li, Nan Shen, Di Yu, Jun Deng

Abstract

Objectives: Low-dose interleukin-2 (IL-2) has shown promising clinical benefits in the treatment of systemic lupus erythematosus (SLE), but how this therapy alleviates pathogenic humoral immunity remains not well understood. The dilemma is that IL-2 can suppress both follicular helper and regulatory T (Tfh and Tfr) cells, which counteract each other in regulating autoantibody production.

Methods: Female NZB/W F1 mice received recombinant human IL-2 (3 × 104 IU/dose) in three treatment regimens: (1) short, daily for 7 days; (2) medium, daily for 14 days, and (3) long, every second day for 28 days. Tfh (Foxp3-CXCR5+Bcl6+), Tfr (Foxp3+CXCR5+Bcl6+), germinal centre (GC, B220+GL-7+Fas+) and antibody-secreting cell (ASC, B220-CD138+TACI+) were analysed by flow cytometry. Serum anti-dsDNA level was determined by ELISA. Kidney pathology was evaluated by H&E and immunofluorescence staining. Circulating Tfh and Tfr cells in SLE patients treated with low-dose IL-2 from a previous clinical trial (NCT02084238) was analysed.

Results: Low-dose IL-2 treatment consistently increased Tfr/Tfh ratio in mice and SLE patients, because of a stronger suppression on Tfh cells than Tfr cells. Three treatment regimens revealed distinct immunological features. Tfh suppression was observed in all regimens, but Tfr suppression and GC reduction were recorded in just medium and long regimens. Only the long treatment regimen resulted in inhibited ASC differentiation in spleen, which was accompanied by reduced anti-dsDNA titres and ameliorated kidney pathology.

Conclusion: Low-dose IL-2 therapy increases the Tfr/Tfh ratio, and a less frequent and prolonged treatment can alleviate pathogenic humoral immunity and improve renal function.

Keywords: B cell; Tfr/Tfh ratio; humoral immunity; interleukin‐2; systemic lupus erythematosus.

Conflict of interest statement

The authors declare no conflict of interest.

© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.

Figures

Figure 1
Figure 1
7‐day low‐dose IL‐2 therapy suppressed Tfh but not Tfr cells in NZB/W F1 mice. (a) Representative FACS plots showing Treg cells (B220−CD4+Foxp3+), Teff cells (CD4+Foxp3−CD44+), Tfh cells (CD4+Foxp3−CD44+CXCR5+Bcl6+) and Tfr cells (CD4+Foxp3+CXCR5+Bcl6+) in spleens of female NZB/W F1 mice with short treatment regimen of low‐dose IL‐2 or PBS for 7 days in spleens of female NZB/W F1 mice with IL‐2 or PBS treatment. (b–e) Frequency statistics of Treg (b), Teff (c), Tfh in Teff (d), Tfh in CD4+ T cell (e) and the statistics of Tfh cell number (f) in spleens of female NZB/W F1 mice with IL‐2 or PBS treatment. (g–i) Frequency statistics of Tfr cells in Treg cells (g), Tfr cells in CD4+ T cell (h) and the statistics of Tfh cell numbers (i) in spleens of female NZB/W F1 mice with IL‐2 or PBS treatment. (j, k) The ratio of Tfr cell frequency in CD4+ T cells to Tfh cell frequency in CD4+ T cells [Tfr/Tfh ratio (CD4+)] (j) and the ratio of Tfr cell number to Tfh cell numbers (Tfr/Tfh ratio (number) (k) in spleens of female NZB/W F1 mice with IL‐2 or PBS treatment. (l, m) Representative FACS plots and statistics showing the frequencies and the absolute numbers of GC B cells (B220+GL‐7+Fas+) (l) and ASC (B220− CD138+TACI+) (m) in spleens of female NZB/W F1 mice with IL‐2 or PBS treatment. Data are shown for individual (dots, n = 8) and mean (bars) values. Data are representative of three independent experiments and analysed by the Mann–Whitney U‐test. *P < 0.05 and **P < 0.01. ASC, antibody‐secreting cell; GC, germinal centre; NZB/W F1, F1 hybrid between the New Zealand Black (NZB) and New Zealand White (NZW) strains; Teff, effector T cell; Tfh, T follicular helper cell; Tfr, T follicular regulatory cell; Treg, regulatory T cell.
Figure 2
Figure 2
14‐day low‐dose IL‐2 therapy mitigated spontaneous GC responses in NZB/W F1 mice. (a–g) Representative FACS plots and statistics showing Tfh cells in Teff cells (b), Tfh in CD4+ T cells (c), Tfh cell numbers (d), Tfr cells in Treg cells (e), Tfr in CD4+ T cells (f), Tfr cell numbers (g) in spleens of NZB/W F1 mice with PBS or IL‐2 treatment. (h, i) The ratio of Tfr cell frequency in CD4+ T cells to Tfh cell frequency in CD4+ T cells [Tfr/Tfh ratio (CD4+)] (h) and the ratio of Tfr cell number to Tfh cell numbers [Tfr/Tfh ratio (number)] (i) in spleens of NZB/W F1 mice with medium treatment regimen of low‐dose IL‐2 or PBS for 14 days. (j) Representative FACS plots and statistics showing the frequencies and the absolute numbers of GC B cells (B220+GL‐7+Fas+) in spleens of NZB/W F1 mice with medium treatment regimen of low‐dose IL‐2 or PBS for 14 days. (k) Immunofluorescence staining of GL‐7 (green), CD4 (red) and B220 (blue) GC B cells in spleens of NZB/W F1 mice with PBS or IL‐2 treatment. Scale bar: 100 μm. Data are shown for individual (dots, n = 8) and mean (bars) values. Data are representative of three independent experiments and analysed by the Student's unpaired t‐test or the Mann–Whitney U‐test. **P < 0.01. GC, germinal centre; NZB/W F1, F1 hybrid between the New Zealand Black (NZB) and New Zealand White (NZW) strains; Teff, effector T cell; Tfh, T follicular helper cell; Tfr, T follicular regulatory cell; Treg, regulatory T cell.
Figure 3
Figure 3
28‐day low‐dose IL‐2 therapy inhibited Tfr cells but upregulated the Tfr/Tfh ratio in NZB/W F1 mice. (a–j) Representative FACS plots (a) and statistics showing Teff cells (b), Treg (c), Tfh cells in Teff cells (d), Tfh cells in CD4+ T cells (e), the numbers of Tfh cells (f), Tfr cells in Treg cells (g), Tfr cells in CD4+ T cells (h), the numbers of Tfr cells (i) and the ratio of Tfr/Tfh cells (CD4+) (j) in spleens of NZB/W F1 mice with long treatment regimen of low‐dose IL‐2 or PBS with every second day for 28 days. Data are shown for individual (dots, n = 6) and mean (bars) values. Data are representative of three independent experiments and analysed by the Student's unpaired t‐test or the Mann–Whitney U‐test. **P < 0.01. NZB/W F1, F1 hybrid between the New Zealand Black (NZB) and New Zealand White (NZW) strains; Teff, effector T cell; Tfh, T follicular helper cell; Tfr, T follicular regulatory cell; Treg, regulatory T cell.
Figure 4
Figure 4
28‐day low‐dose IL‐2 therapy attenuated pathogenic humoral immunity. (a, b) Representative FACS plots and statistics showing the ratio and cell numbers of GC B cells (a) and ASCs (b) in spleens of NZB/W F1 mice with long treatment regimen of low‐dose IL‐2 or PBS with every second day for 28 days. (c) ELISA measurement of anti‐dsDNA IgG titre in the serum of NZB/W F1 mice with PBS or IL‐2 treatment. (d, e) Immunofluorescence staining of deposits C3 (green), IgG (red) (d), IgG1 (white), IgG2a (red), IgG2b (pink) and IgG3 (yellow) (e) in kidneys of NZB/W F1 mice with PBS or IL‐2 treatment. Nucleus were stained with DAPI (blue). Scale bar: 30 μm. Data are shown for individual (dots, n = 6) and mean (bars) values. Data are representative of three independent experiments and analysed by the Student's unpaired t‐test or the Mann–Whitney U‐test. **P < 0.01. ASC, antibody‐secreting cell; C3, complement component 3; GC, germinal centre; and NZB/W F1, F1 hybrid between the New Zealand Black (NZB) and New Zealand White (NZW) strains.
Figure 5
Figure 5
28‐day low‐dose IL‐2 therapy ameliorated renal pathology and improved renal function. (a) H&E staining and histological scores of renal tissues from C57BL/6 mice and NZB/W F1 mice with long treatment regimen of low‐dose IL‐2 or PBS with every second day for 28 days. Scale bar: glomerular 25 μm, tubular 150 μm and perivascular 60 μm. Data are expressed as means ± SE. (b) Representative images of PAS staining of renal sections and quantification of mesangial matrix index of glomerulus in C57BL/6 mice with PBS treatment and NZB/W F1 mice with long treatment regimen of low‐dose IL‐2 therapy with every second day for 28 days. Scale bar: 25 μm. Data are expressed as means ± SE. (c–e) Urinary albumin (c), urinary creatinine (d) and urinary albumin‐to‐creatinine ratio (UACR) (e) in NZB/W F1 mice with PBS or IL‐2 treatment. Data are shown for individual (dots, n = 6) and mean (bars) values. Data are representative of three independent experiments. The Student's unpaired t‐test was employed for comparisons between two groups; one‐way ANOVA followed by the post hoc Tukey test for multiple comparisons was used for groups of three or more. *P < 0.05 and **P < 0.01. NZB/W F1, F1 hybrid between the New Zealand Black (NZB) and New Zealand White (NZW) strains and PAS, periodic acid–Schiff.
Figure 6
Figure 6
Low‐dose IL‐2 therapy elevated the Tfr/Tfh ratio in SLE patients. (a) Representative FACS plots showing circulating TCRαβ+CD3+CD4+CXCR5+CD25+CD127− Tfr cells in the PBMC of SLE patients at baseline (0w) and after low‐dose IL‐2 therapy (12w). (b–d) Statistics of CXCR5+ cells in total CD4+ T cells (b), Tfr cells in total CD4+ T cells (c) and the Tfr/Tfh ratio (d) at baseline (0w) and after IL‐2 therapy (12w). (e) Graphic model of the proposed low‐dose IL‐2 therapy mitigates pathogenic humoral immunity in NZB/W F1 mice via a step‐by‐step process. Short treatment regimen of low‐dose IL‐2 therapy inhibits Tfh cells and increases Tfr/Tfh ratio, medium treatment regimen of low‐dose IL‐2 therapy reduces Tfr cells and GC B cells, and long treatment regimen of low‐dose IL‐2 therapy mitigates pathogenic humoral immunity, alleviates renal histopathology and improved renal function. Data are shown for individual (dots, n = 23) and mean (bars) values and analysed by the Student's paired t‐test. PBMC, peripheral blood mononuclear cell and SLE, systemic lupus erythematosus.
Figure 7
Figure 7
Flow cytometric analysis of CD25 and CD122 expressions in Tnaive, Treg, Tfh and Tfr cells in spleens of NZB/W F1 mice and in PBMC of SLE patients. (a, b) Representative dot plots and percentage statistics of CD25 (a) and CD122 (b) expressions in Tnaive (B220−CD4+CD44−CD62L+), Treg (B220−CD4+Foxp3+), Tfh (B220−CD4+CD44+CXCR5+BCL6+) and Tfr (B220−CD4+Foxp3+CXCR5+BCL6+) cells in spleens of 5‐month‐old NZB/W F1 mice. Data are shown for individual (dots, n = 6). Data are representative of three independent experiments. (c–g) Representative dot plots and the statistics of the median fluorescence index (MFI) of CD122 (e), the frequency of CD122 positive portion (f) and the median fluorescence index (MFI) of CD25 (g) among Tnaive (CD3+CD4+CD25−CD45RA+), Treg (CD3+CD4+CD25+CD127−), Tfh (CD3+CD4+CXCR5+CD25−) and Tfr (CD3+CD4+CXCR5+CD25+CD127−) cells in PBMC from SLE patients. Data are shown for individual (dots, n = 16) and mean (bars) values and analysed by one‐way ANOVA followed by the post hoc Tukey test. **P < 0.01.

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