Pre-B cell leukemia homeobox 1 is associated with lupus susceptibility in mice and humans

Abstract

Sle1a.1 is part of the Sle1 susceptibility locus, which has the strongest association with lupus nephritis in the NZM2410 mouse model. In this study, we show that Sle1a.1 results in the production of activated and autoreactive CD4(+) T cells. Additionally, Sle1a.1 expression reduces the peripheral regulatory T cell pool, as well as induces a defective response of CD4(+) T cells to the retinoic acid expansion of TGF-β-induced regulatory T cells. At the molecular level, Sle1a.1 corresponds to an increased expression of a novel splice isoform of Pbx1, Pbx1-d. Pbx1-d overexpression is sufficient to induce an activated/inflammatory phenotype in Jurkat T cells and to decrease their apoptotic response to retinoic acid. PBX1-d is expressed more frequently in the CD4(+) T cells from lupus patients than from healthy controls, and its presence correlates with an increased central memory T cell population. These findings indicate that Pbx1 is a novel lupus susceptibility gene that regulates T cell activation and tolerance.

Fig. 1

Sle1a.1 expression results in the production of autoreactive hyperproliferative T cells. IL-2 (A) and IFNγ (B) secretion by B6, B6.Sle1a.1 and B6.Sle1a T cells cultured in the presence of histone (black circles), after anti-CD3 stimulation (white circles), or media alone (squares). Values between B6 and B6.Sle1a T cells were all different at p < 0.01. B6, Sle1a.1- or Sle1a-expressing T cells were adoptively transferred into B6.Sle1.Tcrα^-/- mice to assess anti-chromatin IgG AFCs (C) and the ratio of splenic transitional T1 relative to follicular (FO) B cells (D) 3 months after transfer. E-G. CD4⁺ T cells incorporate more BrdU in B6.Sle1a.1 than in B6 mice after 18 h in vivo exposure. E: Representative FACS plots showing BrdU incorporation in CD4⁺ gated cells. Quantitation of BrdU incorporation in CD4⁺ T cells and CD19⁺ B cells from B6 and B6.Sle1a.1 mice (F) and in Vβ5⁺ CD4⁺ T cells from B6.OTII and B6.Sle1a.1.OTII mice (G). A-D. T cells were obtained from 6 month old mice. E-G: Mice were tested at 2 months of age. Mean and SEM, * : p ≤ 0.05 and ** : p ≤ 0.01.

Fig. 2

Sle1a.1 expression reduces the size of the Treg pool and increases EAE susceptibility. A. Splenic and thymic Treg levels in 3 week and 3 month old B6.Foxp3-eGFP and B6.Sle1a.1.Foxp3-eGFP mice, with representative FACS plots of 3 month old spleens. B. EAE average scores in B6.Sle1a.1 (closed symbols) and B6 (open symbols) mice in the days following induction. Square symbols correspond to RA-treated mice. C. Average day of onset and D. maximal scores over the 35 days following EAE induction. B-D show values for 10-15 mice per strain per treatment. MOG recall assays performed with splenocytes collected 10 d after EAE induction in mice (5 per strain per treatment) treated with RA or carrier control, measuring IFNγ in the supernatant (E) and the percentage of IL-17F⁺ CD4⁺ T cells (F). Experiments shown in B-F were conducted with 2-3 month old mice. Mean and SEM, p ≤ 0.05, ** : p ≤ 0.01, and *** : p ≤ 0.001.

Fig. 3

Sle1a.1 results in defective CD4⁺ T cell responses to RA. A-C. Foxp3 induction in B6 and B6.Sle1a.1 CD4⁺ CD25^- cells by IL-2 and TGFβ in the presence or absence of RA. A: Representative FACS plots showing intracellular Foxp3 expression with TGFβ alone or with RA in B6 and B6.Sle1a.1 CD4⁺ T cells. Fold induction of Foxp3 by RA obtained with cells from 5-6 month old mice (10 mice per strain, B) and 3 month old mice (4 mice per strain, C). B6.TC T cells were included as a Sle1a.1 expressing strain. D-E. Suppression of proliferation by CD4⁺ CD25^- cells obtained from 6 week old B6 or B6.Sle1a.1 mice and stimulated in the presence of IL-2, TGFβ and RA. The Treg cultures were added at a 2:1 ratio to CSFE-labeled B6 CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 for three days. D. Representative FACS plot showing proliferation of CSFE⁺ CD4⁺ T cells with B6 Treg cultures (filled grey), Sle1a.1 Treg cultures (thick black line), or no Treg (thin black line). The marker selected for the 2^nd round of replication is indicated. E. Comparison of suppression of proliferation by RA-induced Treg cultures from B6 and B6.Sle1a.1 mice. The percentage of suppression was calculated as 100 minus the percentage of the live CD4⁺ cells that have undergone 2 rounds of replications with Treg cultures relative to the same proliferation in the absence of Treg cultures. F-G. Foxp3 induction by RA in B6 and B6.Sle1a.1 CD4⁺ CD25^- cells stimulated with IL-6 and TGFβ in the presence or absence of RA. F. Representative FACS plots showing intracellular Foxp3 expression with TGFβ and IL-6 with or without RA in B6 and B6.Sle1a.1 CD4⁺ T cells. G. Fold induction of Foxp3 by RA obtained with cells from 5-6 month old mice (4 mice per strain). In A and F, the values indicate the percentage of CD4⁺ cells that express Foxp3. Mean and SEM, *: p ≤ 0.05, **: p ≤ 0.01.

Fig. 4

The NZW allele of Pbx1results in the differential expression of two novel isoforms. A. Map of known isoforms Pbx1-a and Pbx1-b, and novel isoform Pbx1-d. B. RT-PCR analysis performed on CD4⁺ T cells from 5 month old B6, B6.TC, and B6.Sle1a.1 mice, using primers in Pbx1 exons 5 and 8. Pbx1-b (278 bp) found in all three strains, Pbx1-c (167 bp), found only in B6.TC T cells, and Pbx1-d (118 bp), found in all three strains, but to a greater level in the Sle1a.1-expressing congenic strains. Cd4 expression is shown as a positive control. C. Pbx1-d to Pbx1-b expression ratios calculated from densitometric analysis of data shown in B. Representative results obtained from 5 separate experiments. D. RT-PCR analysis performed on CD4⁺ T cells from 6 weeks old B6 and B6.Sle1a.1 mice (3 mice per strain), performed as shown in B. E. IL-10 production from B6 or B6.Sle1a.1 peritoneal macrophages stimulated with B6 apoptotic cells. The graph is representative of 3 experiments with 4 mice per strain in each. F. Pbx1 isoforms expressed in B6 and B6.Sle1a.1 macrophages (5 mice per strain). Cd11b expression is shown as a positive control. G-H. Intracellular IL-10 expression in B6, B6.Sle1a.1 and B6.TC CD4⁺ T cells, with representative FACS plots and quantitation. Mean and SEM, **: p ≤ 0.01.

Fig. 5

Pbx1-d expression decreased activation induced cell death with and without RA. A. Percentages of late apoptotic (Annexin V⁺ 7-AAD⁺) and live (Annexin V^- 7-AAD^-) Jurkat cells transfected with EV control, LV-Pbx1-b or LV-Pbx1-d, and stimulated with anti-CD3 and anti-CD28 with or without RA for 48 h. Representative stains highlighting the percentage of Annexin-V⁺ 7-AAD⁺ late apoptotic cells are shown in B. C. EV control, LV-Pbx1-b or LV-Pbx1-d transfected Jurkat cell counts over a 72 h period of stimulation with anti-CD3 and anti-CD28. Data shown in A-C was obtained in three independent transfections. D. Fold induction by RA of early apoptosis in mouse CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 for 24 h. The graph shows the combination of 4 experiments with 3 mice per strain in each. Mean and SEM, *: p ≤ 0.05, **: p ≤ 0.01 in comparisons with B6 values.

Fig. 6

PBX1-d expression is more frequent in lupus patients and correlates with an increased percentage of central memory CD4⁺ T cells. A. RT-PCR displaying the PBX1-a and PBX1-d isoforms in CD4⁺ T cells from three representative patients. B. PBX1 isoform distribution in 58 HC and 95 SLE patient samples. The percentage of samples expressing PBX1-a is represented in black, PBX1-d in white, and the co-expression of both by a hatched pattern. C. Comparison of the presence (grey) or absence (black) of PBX1-d expression between samples from patients that were treated or non-treated with steroids D. PBX1 isoform distribution in samples from HCs and SLE patients non-treated with steroids. Percentage of CD3⁺ CD4⁺ in HC and SLE patient PBLs (E) and in the total cohort according to the PBX1 isoform expression (F). Relative levels of memory CD4⁺ T cells ([%CD45RA^- CD45RO⁺ CD4⁺ CD3⁺] - [%CD45RA⁺ CD45RO^- CD4⁺ CD3⁺]) / [%CD45RA^- CD45RO⁺ CD4⁺ CD3⁺] in HC and SLE patient PBLs (G), and in the total cohort according to the PBX1 isoform expression (H). Naïve CD45RO^- CD62L⁺ to central memory CD45RO⁺ CD62L⁺ CD4⁺ T cells ratios in HC and SLE patient PBLs (I), and in the total cohort according to the PBX1 isoform expression (J). B-D: p values correspond to chi-square tests. E-J: Medians and interquantile ranges, p values correspond to Mann-Whitney tests; *: p ≤ 0.05, ** : p ≤ 0.01.