Lupus susceptibility gene Pbx1 controls the development, stability, and function of regulatory T cells via Rtkn2 expression

Abstract

The maintenance of regulatory T (T_reg) cells critically prevents autoimmunity. Pre-B cell leukemia transcription factor 1 (Pbx1) variants are associated with lupus susceptibility, particularly through the expression of a dominant negative isoform Pbx1-d in CD4⁺ T cells. Pbx1-d overexpression impaired T_reg cell homeostasis and promoted inflammatory CD4⁺ T cells. Here, we showed a high expression of Pbx1 in human and murine T_reg cells, which is decreased in lupus patients and mice. Pbx1 deficiency or Pbx1-d overexpression reduced the number, stability, and suppressive activity of T_reg cells, which increased murine responses to immunization and autoimmune induction. Mechanistically, Pbx1 deficiency altered the expression of genes implicated in cell cycle and apoptosis in T_reg cells. Intriguingly, Rtkn2, a Rho-GTPase previously associated with T_reg homeostasis, was directly transactivated by Pbx1. Our results suggest that the maintenance of T_reg cell homeostasis and stability by Pbx1 through cell cycle progression prevent the expansion of inflammatory T cells that otherwise exacerbates lupus progression in the hosts.

Fig. 1.. PBX1 is preferentially expressed in T_reg cells.

(A to C) Pbx1 expression in murine T_reg (A) and non-T_reg T (B) cells from B6 and the combination of three lupus-prone strains (SLE: TC, BWF1, and BXSB.Yaa). (C) Paired analysis between T_reg and non-T_reg cells for all strains combined (N = 5 per strain). (D to I) PBX1 (D to F) and PBX1-D [(G) to (I)] expression in human T_reg [(D) and (G)] and non-T_reg (E and H) T cells from patients with SLE (N = 10) and HCs (N = 7). (F) and (I) Paired analysis between T_reg and non-T_reg cells for all human samples combined. (J to L) PBX1-D/PBX1 ratios corresponding to the human T cells shown in (D) to (I). Mean + SEM compared with Mann-Whitney tests or paired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2.. Pbx1 deletion or Pbx1-d sole expression decreased the peripheral T_reg cell population and their suppressive function.

(A and B) Representative FACS plots (A) and frequency (B) of total splenic T_reg cells, CD4⁺FOXP3⁺NRP-1⁺ nT_reg cells, and CD4⁺FOXP3⁺NRP-1⁻ iT_reg cells. (C and D) WT non-T_reg cells were transferred into B6.Rag-1^−/− mice without or with T_reg cells from WT, KO, or KO-Tg mice. (C) Changes in body weight (BW) after transfer. (D) Representative histopathology of hematoxylin and eosin–stained colons (original magnification: ×60, scale bar: 200 μm) for the four groups and corresponding number of foci. (E and F) Representative fluorescence-activated cell sorting(FACS) plots and frequency of splenic CD69⁺CD4⁺ T cells. (G to J) Representative FACS plots and frequency of splenic CD4⁺CXCXR5⁺PD1⁺BCL6⁺FOXP3⁻ T_FH (H), CD4⁺CXCXR5⁺PD1⁺BCL6⁺FOXP3⁺ T_FR cells (I) in CD4⁺ T cells, and FOXP3 MFI in T_FR cells (J). Mean ± SEM of N = 5 to 14 3-month-old [graphs in (B) and young] mice and 8- to 10-month-old (old) mice compared with Dunnett’s multiple comparisons tests. (C) and (D) Mean + SEM of N = 6 mice per group compared with two-way analysis of variance (ANOVA) (C) or Dunnett’s multiple comparisons tests (D). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. PD-1, Programmed cell death protein 1.

Fig. 3.. Pbx1-d expression promotes T_reg instability.

Representative FACS plots (A) and frequency of CD4⁺dTomato⁺FOXP3⁻ ex-T_reg cells and CD4⁺dTomato⁺FOXP3⁺ T_reg cells analyzed in CD4⁺ T cells (B and C) in the spleen and mLN from 3-month-old B6.R26R^RFP and Tg.R26R^RFP mice. Frequency of IFN-γ⁺ (D) and IL-10⁺ (E) cells in ex-T_reg cells. Frequency of IFN-γ⁺ ex-T_reg (F) and IL-10⁺ ex-T_reg (G) in CD4⁺ T cells. Frequency of IFN-γ⁺ T_reg (H) and IL-10⁺ T_reg (I) in CD4⁺ T cells. Representative FACS plots (J) and frequency of ex-T_reg cells and T_reg cells in CD4⁺CD44⁺PD-1⁺CXCR5⁺ follicular T (T_FO) cells (K). The graphs on the left show distributions, and the graph on the right shows the comparison between ex-T_reg cells. Mean + SEM of N = 8 to 9 mice per group compared with t tests. *P < 0.05 and **P < 0.01.

Fig. 4.. T_regcell–specific Pbx1 deletion or Pbx1-d sole expression enhanced TD-humoral response.

Mice were immunized with NP-KLH in alum, boosted 6 weeks later, and analyzed at week 7. (A and B) Frequency (top) and absolute number (bottom) of CD4⁺CD69⁺ cells (A) and T_FH cells (B). (C and D) Frequency (top) and FOXP3 mean fluorescence intensity (MFI) (bottom) in T_FR (C) and T_reg (D) cells. (E) Serum levels of high-affinity anti-NP₄ and low-affinity anti-NP₂₅ IgG1, IgG2a, and IgG2b. Optical density (OD) values were normalized between three cohorts. Mean ± SEM of N = 5 to 10 mice per group performed in three cohorts, compared with Dunnett’s multiple comparisons tests. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 5.. Pbx1 deletion or Pbx1-d sole expression in T_reg cells enhanced CD4⁺ T cell autoimmune responses.

(A to D) Mice were treated with R848 for 1 week and sacrificed at day 10. (A) Frequency of CD4⁺CD69⁺ cells. (B) Representative FACS plot and frequency of CD4⁺CD44⁺CD62L⁻ T_EM cells. Frequency of T_FH cells (C) and IgD⁻CD138⁺ plasma cells (D). (E to H) cGVHD induced by transfer of B6.bm12 splenocytes into the four strains of recipient mice. Frequency of CD4⁺CD69⁺ cells (E) and T_EM cells (F). Levels of anti-dsDNA IgG (G) and anti-kinetoplast IgG positive Crithidia cells (H). The representative images in (H) show a Crithidia assay for a WT and a Tg recipient mouse with the low power showing the entire well, with magnification of the boxed areas. Arrows indicate anti-kinetoplast IgG-positive cells. Mean ± SEM of N = 4 to 12 mice per group compared with Dunnett’s multiple comparisons tests. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 6.. Transcriptional regulation of T_reg and T_eff cells in mice with Pbx1-deficient T_reg cells.

RNA-seq analysis was conducted on CD4⁺FOXP3⁺ T_reg cells and CD4⁺CD44⁺FOXP3⁻ T_eff cells from WT and KO mice (N = 4). (A) Scatterplot of DEGs in T_reg cells. (B) GO biological processes enriched in genes that were up-regulated (top) or down-regulated (bottom) in KO T_reg cells. (C) Heatmap of DEGs in the apoptosis and cell cycle/proliferation pathways between WT and KO T_reg cells. (D) Scatterplot of DEGs in T_eff cells from WT and KO mice. (E) Database for Annotation, Visualization and Integrated Discovery Gene Ontology (DAVID GO) biological processes that were up-regulated or down-regulated in KO T_eff cells compared to WT controls. cAMP, cyclic adenosine 3′,5′-monophosphate; MAP, mitogen-activated protein.

Fig. 7.. Pbx1 directly regulates Rtkn2 expression.

(A and B) Rtkn2 gene (A) and RTKN2 protein (B) expression in T_reg and T_eff cells. N = 4. (C) Dual-luciferase analysis of murine Rtkn2 expression in HEK293T cells in the presence of PBX1-B or PBX1-D, showing fold change relative to the control expression plasmid [empty vector (EV)]. (D) ChIP-qPCR analysis of the human RTKN2 promoter in PBX1-B or PBX1-D overexpressing Jurkat T cells. Results are shown relative to the IgG control. N = 3 to 4. (E) CDKN1A and CDKN1B expression normalized to HMBS in Jurkat T cells treated with siRTKN2 or si control. N = 3. Mean + SEM compared with t tests *P < 0.05 and **P < 0.01. Ab, antibody. WB, Western blot; RU, relative unit; BS, binding site.

Fig. 8.. Pbx1 regulates cell proliferation in T_reg cells.

Representative FACS plot (A) and frequency of Ki-67⁺ cells in T_reg (B) and non-T_reg (C) cells from WT, KO, KO-Tg, or Tg mice. Mean ± SEM of N = 4 to 9 3-month-old mice per group compared with Dunnett’s multiple comparison tests. (D) Cdkn1a and Cdkn1b expression in T_reg and Teff cells from WT and KO mice. Mean ± SEM of N = 3 to 8 3-month-old mice per strain compared with Mann-Whitney tests. Representative FACS histograms of propidium iodide staining relative to cell cycle phases (E) and corresponding frequency (F) in PBX1-B or PBX1-D overexpressing Jurkat T cells as compared to empty vector controls. Mean + SEM of N = 3 compared with t tests. *P < 0.05, **P < 0.01, and ***P < 0.001. RMSD, root mean square deviation.