Cutting Edge: Systemic Autoimmunity in Murine STAT3 Gain-of-Function Syndrome Is Characterized by Effector T Cell Expansion in the Absence of Overt Regulatory T Cell Dysfunction Free

Patients with gain-of-function (GOF) mutations in the STAT3 gene develop a complex syndrome of early-onset autoimmunity. Among the diverse clinical manifestations are type 1 diabetes, autoimmune enteropathy, inflammatory lung disease, polyarthritis, autoimmune cytopenias, and postnatal short stature (1–3). The JAK/STAT signaling pathway regulates transcriptional responses to extracellular cytokines and growth factors. The complex phenotypes of STAT3 GOF syndrome can therefore be attributed both to STAT3 activation downstream of multiple cytokine receptors and to ubiquitous STAT3 expression in hematopoietic and nonhematopoietic lineages. Because of this complexity, our understanding of STAT3 GOF pathophysiology remains poor.

Several hypotheses have been proposed to explain STAT3 GOF autoimmunity. Chief among these, subjects with STAT3 GOF syndrome exhibit reduced regulatory T (Treg) cell numbers (1, 2), although immunophenotyping has been limited to peripheral blood, and at least one patient with STAT3 GOF developed multisystem autoimmunity despite normal Treg cell numbers and Treg cell suppressor function (2). Thus, additional immune mechanisms may facilitate STAT3 GOF disease, including expansion of pathogenic Th17 cells, dysregulation of Th17–Treg balance, enhanced proinflammatory cytokine signaling, or tissue-specific impacts on organ function (4, 5).

Ultimately, the rarity of STAT3 GOF syndrome and limited availability of human clinical samples have prevented a detailed interrogation of underlying immune mechanisms. To address these limitations, we generated and phenotyped a new transgenic strain expressing a pathogenic human STAT3 mutation (STAT3^K392R) within the endogenous murine Stat3 locus.

The STAT3^K392R knock-in murine model is described in Supplemental Fig. 1. Murine studies were performed in a specific pathogen-free environment in accordance with institutional animal care and use committee–approved protocols. To establish mixed bone marrow (BM) chimeras, 6 × 10⁶ CD45.1 wild-type (WT) and CD45.2 Stat3^WT/GOF BM (50%/50% ratio) were injected retro-orbitally into lethally irradiated (450 cGy × two doses) CD45.1 WT recipients. The resulting BM chimeras were sacrificed at 14 wk after transplant.

Formalin-fixed, paraffin-embedded tissue sections from 12-mo-old Stat3^WT/GOF and WT littermates were stained with H&E. Pathology was scored by a board-certified veterinary pathologist blinded to genotype.

Flow cytometry of splenocyte, lymph node (LN), and BM suspensions was performed as described (6). Antimurine Abs used included the following: CD19 (1D3), CD95 (Jo2), CD45.1 (A20), and CD45.2 (104) from BD Biosciences; B220 (RA3-6B2), CD4 (GK1.5 and RM4-4), CD8 (53-6.7), CD19 (6D5), CD21 (7E9), CD23 (B3B4), CD24 (M1/69), CD25 (PC61), CD44 (IM7), CTLA-4 (IC10-4B9), Helios (22F6), and TACI (transmembrane activator and calcium modulator and CAML interactor; 8F10) from BioLegend; CD8 (53-6.7), FoxP3 (FJK-16s), IFN-γ (XMG1.2), and IL-17A (17B7) from eBioscience; CD8 (53-6.7) from Life Technologies; Live/Dead (L34962) from Thermo Fisher Scientific; peanut lectin (agglutinin) (Fl-1071) from Vector Laboratories; and goat anti-mouse IgM-, IgG-, IgA-HRP–conjugated, unlabeled, or isotype IgG2c (1079-02) from SouthernBiotech.

ELISA was performed as described (6) using the following reagents: calf thymus dsDNA (Sigma-Aldrich, D3664); Sm/ribonucleoprotein (Arotec Diagnostic Limited, ATR01-10); IgM, IgA, or IgG (total Ig titers); and goat anti-mouse IgG-, IgG2c-, IgG3-HRP–conjugated Ab (SouthernBiotech).

WT and Stat3^WT/GOF naive CD4⁺ T cells were purified using the Naive CD4+ T Cell Isolation Kit (Miltenyi Biotec, catalog no. 130-104-453), cultured in supplemented RPMI (Th0, Th1, and Treg) or supplemented IMDM (Th17) media, and stimulated with plate-bound anti-CD3 (2.5 μg/ml; Bio X Cell, 145-2C11) and soluble anti-CD28 (1 μg/ml; SouthernBiotech, PV-1). T cell differentiation conditions were as follows: Th1, anti–IL-4 (10 μg/ml; Bio X Cell, 11B11), murine IL-2 (50 ng/ml; PeproTech, catalog no. 212-12), and murine IL-12 (10 ng/ml; R&D Systems, catalog no. 419-ML-010); Treg, anti–IL-4, anti–IFN-γ (10 μg/ml; Bio X Cell, R4-6A2), murine IL-2, and human TGF-β (2.5 ng/ml; PeproTech, catalog no. 100-21); classical Th17, anti–IL-4, anti–IFN-γ, human TGF-β, and murine IL-6 (30 ng/ml; PeproTech, catalog no. 216-16); pathogenic Th17, anti–IL-4, anti–IFN-γ, human TGF-β, murine IL-1β (20 ng/ml; Miltenyi Biotec, catalog no. 130-094-053), and murine IL-23 (20 ng/ml; R&D Systems, catalog no. 1887-ML-010); Th0, anti–IL-4, anti–IFN-γ, and IL-2. On day 5, cells were stimulated with PMA (50 ng/ml; Sigma-Aldrich, catalog no. 5.00582), ionomycin (1 μg/ml; MilliporeSigma, catalog no. 407950), and GolgiPlug Protein Transport Inhibitor (1:1000; BD Biosciences, catalog no. 555029) and then stained for flow cytometry.

CD45.1⁺CD25⁻CD4⁺ WT T cells and CD45.2⁺CD25⁺CD4⁺ Treg cells (WT and Stat3^WT/GOF) were purified from spleens and LNs using the CD4⁺CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi Biotec, catalog no. 130-091-041). CellTrace Violet–labeled CD45.1⁺CD25⁻CD4⁺ T cells (1 × 10⁵) were stimulated with 5 × 10⁵ irradiated CD4^neg cells and 0.5 μg/ml anti-CD3 mAbs (Bio X Cell, catalog no. BE0001-1) plus titrated ratios of Treg cells. Proliferation of CD45.1⁺CD25⁻CD4⁺ T cells was quantified by CellTrace Violet dilution on day 4.

Nine- to 11-wk-old WT and Stat3^WT/GOF mice were injected s.c. with 200 µl myelin oligodendrocyte glycoprotein (MOG)_35-55/CFA emulsion plus 100 ng pertussis toxin on days 0 and 1 (Hooke Laboratories, EK-2110) and monitored daily for weight loss and clinical score (scored 0–5). On day 17, CNS tissue was processed in digestion buffer (HBSS, 10% FBS, 1.5 mg/ml collagenase, 10 µg/ml DNase; 37°C for 45 min), followed by immune cell isolation on a 38%/70% Percoll gradient.

We generated a knock-in mouse model allowing conditional expression of pathogenic Stat3^K392R in the endogenous locus via Cre-mediated replacement of WT Stat3 exon 13 with the K392R-expressing mutant allele (Supplemental Fig. 1). In the present study, we focused our analyses on the impact of global Stat3^K392R expression by crossing Stat3^K392R and CMV-Cre animals (7) to induce germline recombination of the mutant allele (hereafter Stat3^GOF model).

When heterozygous Stat3^WT/GOF mice were intercrossed to generate experimental animals, homozygous Stat3^GOF/GOF offspring were born at frequencies below expected Mendelian ratios, suggesting embryonic or perinatal lethality (Fig. 1A). In addition, Stat3^GOF/GOF homozygous mice exhibited accelerated mortality, with only 50% of mice surviving beyond 12 wk (Fig. 1B). Although heterozygous Stat3^GOF expression did not result in reduced survival, male Stat3^WT/GOF mice manifested reduced weight gain with age, reflecting either Stat3^GOF-dependent growth failure (8) or the impact of chronic illness (Fig. 1C). Cohorts of heterozygous Stat3^WT/GOF mice, sacrificed at 6 and 12 mo, exhibited splenomegaly and progressive LN expansion (Fig. 1D, E). In addition, histopathologic evaluation of organ inflammation in 12-mo-old Stat3^WT/GOF heterozygous mice identified widespread inflammatory lesions within the salivary glands, lungs, liver, and pancreas of affected animals (Fig. 1F–J). In summary, murine expression of a pathogenic human STAT3 GOF variant promotes gene-dose-dependent systemic inflammation.

To gain a greater understanding of the cellular drivers of STAT3 GOF autoimmunity, we immunophenotyped aged Stat3^WT/GOF heterozygous mice. In parallel with widespread organ inflammation, we observed increased activated CD44-expressing CD4⁺ and CD8⁺ T cells in spleens and LNs of Stat3^WT/GOF animals (Fig. 2A, B). Surprisingly, no decrease in Foxp3⁺ Treg cells in Stat3^WT/GOF animals was noted. Rather, the number of Treg cells was modestly expanded in Stat3^WT/GOF mice in proportion to the expansion of CD44⁺ effector CD4⁺ T cells (Fig. 2C–E). In addition, CD25 and CTLA-4 expression was preserved on the surface of Stat3^WT/GOF Foxp3⁺ Treg cells, as was the ratio of thymus-derived versus induced Treg cells, defined by Helios expression (Fig. 2F, G) (9). Finally, in keeping with this unaltered surface phenotype, Stat3^WT/GOF Treg cells exhibited normal suppressive activity in vitro (Fig. 2H, I). These data were unexpected because STAT3-mediated induction of suppressor of cytokine signaling 3 (SOCS3) limits STAT5 phosphorylation, a known transcriptional regulator of Treg cell differentiation (10, 11). Consistent with this model, naive CD4⁺ T cells from Stat3^WT/GOF mice exhibited reduced differentiation into CD25⁺Foxp3⁺ Treg cells in vitro (Fig. 2J). To reconcile these data, we established mixed BM chimeras to assess whether the Stat3^GOF allele impacts Treg differentiation in competitive settings. Although Stat3^GOF expression did not block Treg cell development, WT CD4⁺ T cells exhibited a modest competitive advantage in vivo (Fig. 2K–M). Thus, despite these more subtle impacts on regulatory T cell biology, functional Treg cells can develop in murine STAT3 GOF syndrome, suggesting important contributions of additional immune lineages.

Humoral autoimmunity, in particular autoimmune cytopenia, is a frequent manifestation of STAT3 GOF syndrome. STAT3 functions downstream of several key cytokines impacting B cell activation, including IL-6, IL-10, and IL-21. On the basis of these data, we anticipated that Stat3^GOF expression would drive polyclonal B cell activation and possibly spontaneous germinal center formation and class-switched autoantibody production. However, patients with STAT3 GOF syndrome frequently exhibit hypogammaglobulinemia, highlighting the complex roles for STAT3 in regulating B cell function.

Onset of systemic autoimmunity in 12-mo-old Stat3^WT/GOF mice was accompanied by increased total splenic and LN B cells, although enhanced STAT3 activity exerted no major impact on splenic B cell development, except for a modest reduction in transitional T1 and T2 cells and expanded follicular mature B cells (Supplemental Fig. 2A–C). We also observed no spontaneous germinal center formation and no increase in spleen, LN, or BM plasma cells in aged Stat3^WT/GOF mice (Supplemental Fig. 2D, 2E). Moreover, although total serum IgM and IgG titers were modestly increased in aged animals, 12-mo-old Stat3^WT/GOF mice lacked anti-dsDNA and anti-Sm/ribonucleoprotein autoantibodies, despite multiorgan inflammation at this age (Supplemental Fig. 2F, 2G).

Coordinated cytokine signals promote differentiation of naive CD4⁺ T cells into distinct effector subsets. Th17 differentiation is facilitated by TGF-β and IL-6 cytokines, which induce STAT3-dependent activation of the lineage-defining transcription factor retinoic acid-related orphan receptor γt, implicating Th17 cell expansion in STAT3 GOF autoimmunity (1, 2). To validate that murine Stat3^K392R functions as a GOF allele, we performed in vitro CD4⁺ T cell differentiation assays and confirmed that Stat3^GOF drove increased Th17 differentiation while modestly reducing Th1 differentiation (Fig. 3A). To address the potential T cell lineages promoting systemic inflammation in vivo, we measured T cell cytokine production in aged Stat3^WT/GOF mice. Surprisingly, IL-17–producing CD4⁺ T cells were not markedly increased in diseased Stat3^WT/GOF animals. Rather, onset of autoimmunity in 12-mo-old Stat3^WT/GOF mice was characterized by a prominent accumulation of IFN-γ–producing CD4⁺ and CD8⁺ T cells (Fig. 3B–E).

Finally, we sought to reconcile the limited Th17 expansion in aged Stat3^WT/GOF mice with our murine T cell differentiation data and with increased Th17 differentiation in human STAT3 GOF syndrome (12). Th17 cells exhibit significant plasticity in vivo, including trans-differentiation into IFN-γ–producing cells during autoimmune inflammation (13, 14). For this reason, we quantified Th cell differentiation during acute autoimmune inflammation using the EAE model of multiple sclerosis. Although neurologic disease is not a common manifestation of human STAT3 GOF syndrome, we reasoned that EAE could be used to model the impact of the Stat3^GOF allele on T cell biology during autoimmunity. Following MOG immunization, WT and Stat3^WT/GOF mice developed similar disease severity (Fig. 4A). In keeping with reduced Treg cell differentiation in competitive settings, CNS Foxp3⁺ Treg cells were moderately reduced in Stat3^WT/GOF animals, although the intra-CNS T effector/Treg cell ratio was not altered (Fig. 4B), suggesting that Stat3^GOF does not prevent the differentiation and/or migration of Foxp3⁺ Treg cells to sites of organ inflammation. Notably, Stat3^WT/GOF animals exhibited an expansion of IL-17A–producing Th17 cells after MOG immunization, with no increase in Th1 or Th1/Th17 double-positive CD4⁺ T cells (Fig. 4C, D). Thus, despite the relative expansion of IFN-γ–producing Th1 cells in aged animals with spontaneous disease, enhanced STAT3 promotes Th17 differentiation during acute autoimmunity.

Targeted animal models carrying human disease–associated mutations are an important strategy in understanding the pathogenesis of Mendelian immune disorders. Here, we generated a new murine strain expressing a human pathogenic STAT3 GOF variant and confirmed that this mutation drives spontaneous, multisystem autoimmunity on the nonautoimmune C57BL/6 background.

STAT3 regulates the balance of Th17 and Treg cell differentiation. IL-6–dependent STAT3 phosphorylation, in combination with TGF-β, drives Th17 differentiation while inhibiting FOXP3 expression to theoretically shift the balance from Treg to pathogenic Th17 cells (15). In addition, increased STAT3 activation promotes SOCS3 expression, which inhibits STAT5 signals downstream of the IL-2 receptor. Multiple lines of evidence link STAT5 with normal Treg cell function, including reduced Treg cells in STAT5a/b-deficient mice (11), enhanced suppressive function of Treg cells expressing constitutively active STAT5b (16), and patients with STAT5b loss-of-function mutations exhibiting systemic inflammation reminiscent of individuals with FOXP3 mutations (17). The overlap in STAT3 GOF and STAT5b loss-of-function clinical phenotypes extends beyond immune dysregulation to include growth hormone–resistant short stature (17). Together, these data strongly supported an important role for Treg dysfunction in STAT3 GOF autoimmunity.

Despite this prevailing model, we observed no defect in Treg differentiation or in vitro suppressive activity in Stat3^WT/GOF mice. Rather, progressive STAT3-driven autoimmunity was accompanied by a parallel expansion of activated CD44⁺ Treg cells and restoration of T effector–Treg cell balance. An important caveat is that reduced Stat3^GOF Treg cell differentiation in competitive settings may implicate relative Treg cell dysfunction as contributing to organ-specific autoimmunity in patients with STAT3 GOF. However, although not precluding these more subtle impacts on Treg cell biology, our findings complement a recent study by the Anderson group in which the identical STAT3^K392R allele accelerated the development of type 1 diabetes on the NOD murine background (18). Consistent with our data, Treg cell development was normal in NOD.Stat3^K392R mice, and NOD.Stat3^K392R Treg cells were able to prevent diabetes in transfer models.

Finally, despite the increase in Th17 relative to Th1 differentiation in vitro, a notable feature of the Stat3^GOF model is the prominent accumulation of IFN-γ–producing Th1 cells. These data are strikingly consistent with a similar increase in Th1 cells, but modest Th17 expansion, in NOD.Stat3^K392R animals (18). Importantly, Th17 cells exhibit extensive plasticity in vivo with ex-Th17 cells forming a major source for CD4⁺ IFN-γ production during chronic inflammation. This trans-differentiation into unconventional Th1 cells is driven by STAT3-dependent IL-23 receptor signaling, suggesting a mechanism for Th1 expression during chronic Stat3^GOF inflammation (13, 14). Alternatively, IL-23R signaling can directly drive the differentiation of colitogenic Th1 cells during murine inflammatory bowel disease in the absence of initial Th17 differentiation (M. Pawlak, D. DeTomaso, G. M. zu Horste, Y. Lee, J. Nyman, D. Dionne, C. Wang, A. Wallrapp, P. R. Burkett, S. J. Riesenfeld, et al., manuscript posted on bioRxiv, DOI: 10.1101/2021.01.24.426445). Testing these models for Th1 expansion in STAT3 GOF syndrome will require additional IL-17A fate-mapping experiments. In addition, whether additional cellular lineages (such as CD8⁺ T cells or B cells) contribute to autoimmunity in STAT3 GOF syndrome remains to be determined but will be a major focus of future studies using this Cre-driven Stat3^WT/GOF model.

In summary, these findings highlight the complexity of STAT3 regulation of immune tolerance and emphasize the challenges in predicting in vivo biology from ex vivo human assays. The ability to model cytokine- and lineage-specific roles for STAT3 in autoimmunity using this new Stat3^GOF model holds the promise of advancing our understanding of both rare and common autoimmune syndromes.

The authors have no financial conflicts of interest.