Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a genetic predisposition that results from the combined effects of variants in a large number of susceptibility genes (Tsokos, 2011). One of the leading factors driving lupus pathogenesis is the biased differentiation of pro-inflammatory effector T cells relative to FOXP3+ regulatory T (Treg) cells. Therefore, it is important to understand the mechanisms that regulate T cell effector functions in SLE. A linkage analysis in lupus-prone NZM2410 mice identified the Sle1 locus as strongly associated with glomerulonephritis, a major end-organ lesion in SLE (Morel et al., 1994). Functional studies established that Sle1 intrinsically regulates CD4+ T cell functions (Sobel et al., 2002). Fine mapping identified at least three clusters of genes in Sle1 (Morel et al., 2001), among which Sle1a induces the production of activated nucleosome-reactive CD4+ T cells that provide help to chromatin-specific B cells (Chen, Cuda and Morel, 2005). Sle1a expression also reduces the number and suppressive functions of Treg cells (Cuda et al., 2007). The main Sle1a locus to regulate Treg cell expansion and function is Sle1a1, which contains only one protein-coding gene, Pbx1 (Cuda et al., 2010).

Pre-B-cell leukemia homeobox 1 (Pbx1) encodes for a TALE (three amino acid extension) homeodomain protein that penetrates repressive chromatin marking genes for activation or repression by integrating signals through many cofactors, including MEIS1 and Pbx-regulating protein-1 (PREP1) (Berthelsen et al., 1999, Sagerstrom, 2004). Human and murine PBX1 are identical in exon and splice isoform structure as well as in protein sequence. PBX1 functions in various cell types, including in the maintenance of hematopoietic stem cells and the development of B cells from common lymphocyte progenitors (Ficara et al., 2008, Sanyal et al., 2007). It has recently been shown that lupus susceptibility is associated with a variant leading to low PBX1 expression in human immune cells (Gu et al., 2023). Furthermore, the deletion of Pbx1 in B cells accelerated autoimmune phenotypes in mice (Gu et al., 2023).

We have reported that Pbx1-D, a splice isoform that lacks the DNA-binding and Hox-binding domains, is overexpressed in the T cells of lupus-prone mice and SLE patients (Cuda et al., 2012). Pbx1-D transduction in MC3T3-E1 osteoblast progenitors mimicked the effects of short hairpin RNA silencing of Pbx1, showing that it functions as a dominant negative isoform (Sengupta et al., 2012). Pbx1-D overexpression in T cells promoted the expansion of follicular helper T (TFH) cells at the expense of Treg cells (Choi et al., 2016), a phenotype found in SLE patients and lupus-prone mice (Suarez-Fueyo, Bradley and Tsokos, 2016). This suggested that PBX1 functions as a novel regulator of CD4+ T cell effector function. We have also shown that PBX1-D directly transactivates the expression of CD44, a maker of CD4+ T cells activation and memory (Niu et al., 2017). However, the mechanisms by which PBX1 regulates T cell functions are largely unknown.

In some cancers, PBX1 promotes tumor cell proliferation and radio-resistance through the JAK2/STAT3 signaling pathway by directly transactivating STAT3 expression (Jung et al., 2016; Wei, Yu and Li 2028; Yu et al., 2020; Cao et al., 2022). STAT3 plays a critical role in regulating the effector functions of CD4+ T and B cells (Deenick et al., 2018), with a gain of function mutation leading to early onset autoimmunity (Woods et al., 2022). In immune cells, STAT3 is activated by IL-6, IL-10, and IL-21, which are cytokines found at high levels in mouse models of lupus as well as in patients with active SLE (Deng et al., 2015, Ding et al., 2020, Geginat et al., 2019). These increased cytokine levels may account for the high level of phosphorylated STAT3 (pSTAT3) and total STAT3 found in the T cells of SLE patients (Harada et al., 2007, Talaat et al., 2015). Additionally, activation of the STAT3 pathway is required for the development of the TH17 and TFH cell subsets (Ma et al., 2012, Yang et al., 2007), both of which are expanded in SLE patients and lupus-prone mouse models in correlation with disease activity (Kim et al., 2018, Koga et al., 2020). T cell specific deletion of Stat3 reduced the expression of IL-17 and IL-21, the cytokines produced by TH17 and TFH cells respectively, reducing disease severity in several autoimmune models (Harris et al., 2007, Liu et al., 2008). IL-21 production is induced in TFH cells by IL-6 through STAT3, and both cytokines decrease Foxp3 expression leading to Treg cell impairment (Bettelli et al., 2006, Nurieva et al., 2007). Patients with SLE display dysregulated numbers and functions in Treg cells (Giang and La Cava, 2016). These studies therefore provide evidence of a mutually exclusive relationship between TH17/TFH cells and Tregs and show the central contribution of STAT3 to this relationship.

In this study, we investigated whether PBX1 regulates CD4+ T cells through the STAT3 pathway. We confirmed that PBX1 binds directly to the STAT3 promoter controlling its transcriptional activity and showed that the overexpression of the dominant negative isoform Pbx1-D or Pbx1 deficiency decreased STAT3 expression. Accordingly, pSTAT3 expression was decreased in primary mouse CD4+ T cells with either Pbx1 deficiency or Pbx1-D overexpression, which was associated with a reduced TH17 induction in vitro. Pbx1 deficiency also decreased the frequency of CD4+ T cells and increased their apoptosis. The contribution of the PBX1 / STAT3 axis to lupus T cell phenotypes through effector functions relative to proliferation and apoptosis is discussed.