The TYK2-P1104A Autoimmune Protective Variant Limits Coordinate Signals Required to Generate Specialized T Cell Subsets

TYK2 is a JAK family member that functions downstream of multiple cytokine receptors. Genome wide association studies have linked a SNP (rs34536443) within TYK2 encoding a Proline to Alanine substitution at amino acid 1104, to protection from multiple autoimmune diseases including systemic lupus erythematosus (SLE) and multiple sclerosis (MS). The protective role of this SNP in autoimmune pathogenesis, however, remains incompletely understood. Here we found that T follicular helper (Tfh) cells, switched memory B cells, and IFNAR signaling were decreased in healthy individuals that expressed the protective variant TYK2A1104 (TYK2P). To study this variant in vivo, we developed a knock-in murine model of this allele. Murine Tyk2P expressing T cells homozygous for the protective allele, but not cells heterozygous for this change, manifest decreased IL-12 receptor signaling, important for Tfh lineage commitment. Further, homozygous Tyk2P T cells exhibited diminished in vitro Th1 skewing. Surprisingly, despite these signaling changes, in vivo formation of Tfh and GC B cells was unaffected in two models of T cell dependent immune responses and in two alternative SLE models. TYK2 is also activated downstream of IL-23 receptor engagement. Here, we found that Tyk2P expressing T cells had reduced IL-23 dependent signaling as well as a diminished ability to skew toward Th17 in vitro. Consistent with these findings, homozygous, but not heterozygous, Tyk2P mice were fully protected in a murine model of MS. Homozygous Tyk2P mice had fewer infiltrating CD4+ T cells within the CNS. Most strikingly, homozygous mice had a decreased proportion of IL-17+/IFNγ+, double positive, pathogenic CD4+ T cells in both the draining lymph nodes (LN) and CNS. Thus, in an autoimmune model, such as EAE, impacted by both altered Th1 and Th17 signaling, the Tyk2P allele can effectively shield animals from disease. Taken together, our findings suggest that TYK2P diminishes IL-12, IL-23, and IFN I signaling and that its protective effect is most likely manifest in the setting of autoimmune triggers that concurrently dysregulate at least two of these important signaling cascades.

TYK2 is a JAK family member that functions downstream of multiple cytokine receptors. Genome wide association studies have linked a SNP (rs34536443) within TYK2 encoding a Proline to Alanine substitution at amino acid 1104, to protection from multiple autoimmune diseases including systemic lupus erythematosus (SLE) and multiple sclerosis (MS). The protective role of this SNP in autoimmune pathogenesis, however, remains incompletely understood. Here we found that T follicular helper (Tfh) cells, switched memory B cells, and IFNAR signaling were decreased in healthy individuals that expressed the protective variant TYK2 A1104 (TYK2 P ). To study this variant in vivo, we developed a knock-in murine model of this allele. Murine Tyk2 P expressing T cells homozygous for the protective allele, but not cells heterozygous for this change, manifest decreased IL-12 receptor signaling, important for Tfh lineage commitment. Further, homozygous Tyk2 P T cells exhibited diminished in vitro Th1 skewing. Surprisingly, despite these signaling changes, in vivo formation of Tfh and GC B cells was unaffected in two models of T cell dependent immune responses and in two alternative SLE models. TYK2 is also activated downstream of IL-23 receptor engagement. Here, we found that Tyk2 P expressing T cells had reduced IL-23 dependent signaling as well as a diminished ability to skew toward Th17 in vitro. Consistent with these findings, homozygous, but not heterozygous, Tyk2 P mice were fully protected in a murine model of MS. Homozygous Tyk2 P mice had fewer infiltrating CD4 + T cells within the CNS. Most strikingly, homozygous mice had a decreased proportion of IL-17 + /IFNγ + , double positive, pathogenic CD4 + T cells in both the draining lymph nodes (LN) and CNS. Thus, in an autoimmune model, such as EAE, impacted by both altered Th1 and Th17 signaling, the Tyk2 P allele can effectively shield animals from disease. Taken together, our findings suggest that TYK2 P diminishes IL-12, IL-23, and IFN I signaling and that its protective effect is most likely manifest in the setting of autoimmune triggers that concurrently dysregulate at least two of these important signaling cascades.

INTRODUCTION
Systemic lupus erythematosus (SLE) comprises a group of heterogenous disorders classified under a broad clinical phenotype of systemic autoimmunity (1,2). Loss of tolerance and sustained autoantibodies are key factors in the SLE pathogenesis (1). T cells play a critical role in SLE pathogenesis and previous work has identified alterations in CD4 + T cell subsets in patients with lupus (1). This reflects differentiation of naïve CD4 + T cells into alternative specialized T helper (Th) subtypes, including Th1, Th2, Th17, and T follicular helper (Tfh) cells. Differentiation is dependent on the cytokine milieu that the T cell encounters, and appropriate signaling through multiple cytokine pathways is required for lineage commitment. In SLE, heightened percentages of Tfh-like cells are present in both germinal centers and peripheral blood and correlate with serum autoantibody titers (3,4). Tfh cells are key components of the adaptive immune response, providing the T cell help necessary for the development and maintenance of germinal center (GC) B cells and a robust antibody response (3,(5)(6)(7). Commitment to the Tfh lineage is driven by expression of transcription factor Bcl-6 expression (8). A number of cytokine signals have been implicated in the regulation of Bcl-6 expression, including IL-6, IL-21, IL-12, IL-2, IL-23, TGF-β, and IFN-γ through the Janus Kinase (JAK)-STAT pathways (9)(10)(11)(12)(13). Not surprisingly, dysregulation of these cytokine programs can contribute to disease through preferential expansion or depletion of particular Th lineages (3,14).
Consistent with the altered T cell subsets observed in SLE, IL-12, and IL-23 levels have been found to be increased in SLE patients (3,15,16). Further, a positive correlation between levels of IL-12 and SLEDAI were seen in these lupus patients and active lupus nephritis had even higher levels of IL-12 compared to inactive SLE patients (15,16). Type I interferons (IFN I) are also frequently upregulated in SLE subjects. IFN I impacts T cell subset commitment by promoting Bcl-6 expression, independent of STAT3 signaling and IL-21 production (17). However, IFN I has also been shown to be a corepressor of Tfh in the absence of STAT3 while augmenting interferon stimulated genes (ISGs) and Th1-like commitment (18). Given the complexity of T cell subset generation and the genetic heterogeneity of human autoimmunity, further work is needed to define the interplay of signals that control Tfh development and survival, and the role of T cell subsets in the pathogenesis of SLE and other autoimmune disorders.
Genome wide association studies (GWAS) have identified a single nucleotide polymorphism (SNP; rs34536443) in the TYK2 gene associated with several autoimmune diseases (28)(29)(30)(31)(32)(33). This SNP results in a proline to alanine substitution at amino acid 1,104 in the kinase domain of the protein (P1104A; A1104 referred to hereafter as TYK2 P ) (31). Strikingly, the TYK2 P variant has been associated with protection from multiple autoimmune diseases including: SLE, type 1 diabetes (T1D), multiple sclerosis (MS), rheumatoid arthritis, psoriasis, Crohn's disease, inflammatory bowel disease, and ulcerative colitis (28)(29)(30)(31)(32)(33)(34). Early studies suggested that TYK2 P was a hypomorphic allele (35,36). However, these studies reported conflicting results using alternative cell lineages suggesting that the signaling activity of the variant might depend on context and cell type (35,36). More recent work has shown that TYK2 P leads to hypomorphic signaling including reduced IFN I responses in all cell types and reduced IL-12/IL-23 signaling in human and murine T cells (33). The precise role(s) for TYK2 P in altering autoimmune pathogenesis, however, remains poorly elucidated.
In the current study, we utilized cells from healthy human subjects with the variant and knock-in mice to assess the impact of TYK2 P on T cell subsets and cytokine signaling and on normal and autoimmune responses in vivo. First, we demonstrate that healthy individuals with the protective variant exhibit decreased IFN I signaling and have a decreased frequency of circulating Tfh cells and switched memory B cells. We established a knock-in murine model of this allele and show that homozygous Tyk2 P T cells exhibit decreased IL-12 receptor signaling and diminished in vitro Th1 skewing. Surprisingly, in vivo formation of Tfh and GC B cells was unaffected by Tyk2 P expression in alternative murine models of T cell dependent immune responses. Further, expression of the protective variant did not protect against murine lupus in alternative murine SLE models. Additionally, we found that Tyk2 P expressing T cells had reduced IL-23 dependent signaling and diminished ability to skew toward Th17 in vitro. Unlike lupus murine models, homozygous Tyk2 P mice were fully protected from EAE, and infiltrating CD4 + T cells within the CNS. Moreover, homozygous variant mice had a markedly decreased population of pathogenic IL-17 + /IFNγ + CD4 + T cells in both the draining lymph nodes (LN) and CNS. Thus, our data suggest that TYK2 P reduces IFN I, IL-12, and IL-23 signaling in T cells, and that only when autoimmune disease synchronously dysregulates multiple cytokine signaling programs will the protective phenotype be observed.

Human Samples and Genotyping
Cryopreserved PBMCs were obtained from adult participants in the Benaroya Research Institute (BRI) Immune Mediated Diseases Registry and Repository. Subjects were selected based on TYK2 genotype and the absence of autoimmune disease or any family history of autoimmunity. Study groups were designated as follows: subjects homozygous for the non-protective (NP) allele "C" at rs34536443: "NP/NP"; subjects homozygous for the protective (P) allele "G" at rs34536443: "P/P", and heterozygous subjects: "NP/P". TYK2 SNP rs2304256 was held constant "C/A" as far as possible (all NP/NP and NP/P subjects). The "P/P" group was homozygous "A/A" at rs2304256 in all cases. Subjects were age matched (mean age: NP/NP group, 37.7 ± 12.6 years; NP/P group, 37.7 ± 14.3 years; P/P group, 45.3 ± 18.1 years) and sex matched as far as possible (NP/NP group, 21 males and 20 females; NP/P group, 15 males and 17 females; P/P group 3 male and 1 female). All experiments were performed in a blinded manner with respect to TYK2 genotype. Genomic DNA was genotyped for the TYK2 SNPs rs34536443 (C/G) (P1104A) and rs2304256 (C/A) (V362F) using a Taqman SNP genotyping assay (Applied Biosciences) or were genotyped using the Illumina ImmunoChip by the University of Virginia Center for Public Health Genomics. The Taqman genotyping assay was validated using HapMap DNAs of known genotype, and controls of each genotype were included in every genotyping experiment. Results were checked for adherence to Hardy-Weinberg equilibrium. The research protocols were approved by the Institutional Review Board at BRI (#07109-148).

Mice
A construct designed to generate a P1124A mutation in exon 21 of Tyk2 by homologous recombination in C57BL/6J mice was generated and injected by Biocytogen as previously described (37). After successful recombination, two FRT sequences with a neomycin-resistance selection cassette were inserted into intron 21. To create lineage-specific deletion, loxP sites were also present in intron 19 and 21. C57BL/6J embryonic stem (ES) cells had the introduction of the construct and clones were obtained by limited dilution. G418 selection was used to select clones. Clones that contained successful integration of the knock-in template into the locus were confirmed by Southern blot and PCR analysis of genomic DNA. Successfully targeted clones were injected into BALB/c blastocysts and were subsequently transferred into pseudopregnant females. One clone gave rise to a line with germline transmission of the allele. The mutation was confirmed by sequencing of Exon 21 (Supplementary Figure 1), and PCR was used to genotype all litters (using the following primers: 5 ′ -CCACTCCTAACCTTGTAGAGCAC-3 ′ and 5 ′ -AACGCAAATCTCTACAACAGTGG-3 ′ ). Mice were crossed with B6.Cg-Tg(ACTFLPe)9205Dym/J (Jackson Laboratory) mice to delete the neomycin-resistance selection cassette. In Th1 skewing assays, Tyk2 knockout mice were created by Dr. Mathias Müller and were kindly provided by Dr. George Yap (23). Tyk2 knockout mice were also generated by crossing TYK2 P mice with B6.C-Tg(CMV-cre)1Cgn/J (Jackson Laboratory) strain to make a global knockout of Tyk2. Deletion was confirmed by sequencing loxP sites (Supplementary Figure 1), and PCR was used to genotype all litters (using the following primers: 5 ′ -CCACTCCTAACCTTGTAGAGCAC-3 ′ and 5 ′ -CCTCCCTGTGTGTGATGTGG-3 ′ ). WAS-/-mice are on a C57BL/6J background (38). All strains were maintained in a specific-pathogen-free facility, and studies were performed in accordance with procedures approved by the Institutional Animal Care and Use Committees of Seattle Children's Research Institute.

In vitro Stimulation and Th Skewing Assays
For IL-12 signaling, thawed PBMCs were washed and resuspended in complete medium (RPMI, 10% human serum, 1% PenStrep) at 4 × 10 6 cells/ml. Cells were activated with anti-CD3/CD28 Dynabeads (ThermoFisher) at a bead to cell ratio of 1:10 for 72 h. Following removal of the magnetic beads, cells were rested in X-vivo 15 medium (Lonza) for 2 h, washed with PBS and stimulated with 2.5 ng/ml of recombinant human IL-12 (BD Pharmingen) for 30 min. For IFN-α signaling, thawed PBMCs were washed and rested in X-vivo 15 medium for 45 min. Cells were washed and stimulated with 2,000 IU/ml of recombinant IFN-α (PBL) for 12 min.

In vitro Tfh Generation
Splenic CD4 + T cells were isolated as described above. All cells were stimulated with anti-CD3/CD28 coated beads (Thermo Fisher), with IL-2 (Peprotech) and supplemented with the following for Th0 and Tfh, respectively: anti-IFN-γ (30 µg/ml; BioXcell) and anti-IL-4 (20 µg/ml; BioXcell); IL-12 alone (20 ng/ml). Beads were removed after 48 h of stimulation and fresh media was added to each condition with the respective cytokines described above. Cells were harvested six days after initial stimulation to assess for Tfh surface markers.

In vivo Immunizations
VLPs were made with bacteriophage Qβ capsid protein that contain single-stranded RNA which were kindly provided by Dr. Baidong Hou (39). Mice were injected with 2 µg of VLPs i.p. Twelve days post-immunization, spleens and serum were harvested from the mice. Cells were stained with surface markers for Tfh and GC B cells. Serum was analyzed for VLP-specific antibodies as previously described (40).
Mice were immunized by i.p. with 200 µl of PBS containing 20% sheep red blood cells (SRBCs). Spleens and serum were harvested on day 5 to assess surface markers and total IgG1 by ELISA.

Flow Cytometry
PBMCs were thawed, washed with PBS and rested in X-vivo 15 medium (Lonza) at 37 • C and 5% CO 2 for 45 min. Cells were washed with PBS and 1 × 10 6 cells were stained in FACS buffer (PBS/0.5% BSA/0.1 NaN 3 ) with a cocktail of fluorophore-conjugated antibodies at RT for 20 min. For human IL-12/pSTAT signaling, cells were fixed and permeabilized using Fix buffer I (BD Biosciences) and Perm buffer III (BD Biosciences), respectively, according to the manufacturer's instructions. Cells were washed and stained simultaneously for surface markers and intracellular pSTAT3 and pSTAT4 at RT for 45 min. For human IFN-α, cells were washed and stained simultaneously for surface markers and intracellular pSTAT1 at RT for 45 min. IFNAR surface levels were determined in unfixed, non-permeabilized cells. The following antibodies were used for the detection of proteins in human samples:  (39,41). FlowJo (version 10) was used for data analysis.

Statistical Analysis
All statistical analysis was performed using GraphPad Prism (version 7). All statistical tests and P-values are specified in the figure legends.

Healthy Subjects With the TYK2 P Variant Exhibit a Decrease in Both Tfh Cells and Switched Memory B Cells
To evaluate the effect of TYK2 P on lymphocyte populations, we examined peripheral blood mononuclear cells (PBMC) in healthy individuals with no family history of autoimmunity.
Specifically, we assessed adaptive immune cells which require TYK2-dependent pathways for development and activation (27). Thawed PBMCs were stained with fluorophore-conjugated antibodies for a panel of T and B cell subset markers and analyzed by flow cytometry. We observed no effect of TYK2 P on the frequency of CD4 + naïve (RA + ) and memory (RA − ) T cells or of total CD3 − CD19 + or memory CD3 − CD19 + CD27 + CD10 − B cells. In contrast, we found that individuals expressing the TYK2 P allele have decreased circulating CD4 + CD45RA − PD1 + CXCR5 + Tfh cells (Figures 1A,B). Consistent with the role for Tfh cells in promoting B cell GC responses, we also observed a reduced frequency of CD3 − CD19 + CD27 + CD10-IgM − switched memory B cells (Figures 1C,D) in individuals with the protective allele. Thus, individuals expressing TYK2 P exhibited low frequencies of Tfh cells, essential for germinal center formation, and switched memory B cells, products of germinal centers, suggesting that TYK2 plays a role in cytokine pathways important for regulation of germinal centers and immune activation.
In vitro IL-12 Driven Tfh Generation and B Cell IL-6 Production Is Decreased Using Murine Tyk2 P Cells To gain better understanding of the function of TYK2 P in cytokine signaling and autoimmune disease, we generated a knock-in mouse strain containing the identical amino-acid substitution in the murine TYK2 protein (Tyk2-P1124A), hereafter referred to as Tyk2 P mice or as Tyk2 NP/P and Tyk2 P/P for heterozygous and homozygous animals, respectively (further detailed explanation of genotypes, please see Supplementary Table 1). To generate founder mice, we used homologous recombination on the non-autoimmune prone C57BL/6 genetic background (Supplementary Figures 1A-C). Gene targeting produced the variant coding change (encoding the substitution P1124A) in exon 21 of Tyk2 (Supplementary Figure 1B). Based upon our targeting strategy, we also crossed Tyk2 P mice with a murine line ubiquitously expressing CRE to create Tyk2 knockout (Tyk2 −/− ) animals of an identical genetic background for use in some studies (Supplementary Figures 1A,C).
The TYK2-dependent IL-12 cytokine pathway is important for Tfh generation by promoting phosphorylation of STAT3 (pSTAT3) (12). To test pSTAT3 levels in murine cells, we isolated CD4 + T cells from littermate control (Tyk2 NP/NP ), heterozygous (Tyk2 NP/P ) or homozygous (Tyk2 P/P ) and assessed for IL-12induced pSTAT3. We found that homozygous Tyk2 P/P cells exhibited diminished pSTAT3 (Figure 2A). Further, Tyk2 P/P CD4 + T cells were also unable to skew toward a Th1 phenotype in vitro, a process also dependent on IL-12 signaling ( Figure 2B). Similar to previously published findings, Tyk2 −/− CD4 + T cells exhibited a similar decrease in the capacity to skew toward a Th1 phenotype (24,42). These data mirrored our findings using Tyk2 P/P CD4 + T cells implying that the protective allele encoded for a TYK2 protein with reduced functional activity. Despite the decreased Tfh cells in the circulation in human subjects heterozygous for the protective variant, we could not discern significant differences in IL-12-induced pSTAT3 or pSTAT4 using primary human CD4 + T cells from a cohort of heterozygous healthy subjects ( Supplementary Figures 2A-H).
To explore the role of TYK2 P in IL-12-induced Tfh cell generation, we used an in vitro assay to examine this question. Tyk2 P/P CD4 + T cells were not able to generate Tfh-like cells as efficiently as Tyk2 NP/NP cells in response to IL-12 alone (Figure 2C). Based on the diminished switched memory population in healthy donors with the protective variant (Figure 1D), we assessed the role of IL-12 signaling in modulating the activation of Tyk2 P murine B cells. Previous work has implicated IL-12 in promoting B cell activation and antibody production (43,44). We used an in vitro "GC-like" stimulation with and without the addition of IL-12 and monitored the production of IL-6. IL-6 is produced by activated B cells and promotes GC B and Tfh cell development (45), and B cell intrinsic IL-6 production is required for autoimmune GC B cell responses (46). Under all conditions, Tyk2 P/P B cells exhibited a trend for diminished IL-6 production compared to control Tyk2 NP/NP or heterozygous (Tyk2 NP/P ) B cells ( Figure 2D) but these differences did not reach statistical significance. In summary, diminished in vitro Tfh-like and Th1 generated T cells from Tyk2 P/P mice were most likely secondary to diminished IL-12 signaling.

Tyk2 P Does Not Impact Tfh and GC B Cell Formation Following T-Dependent Immunization
Next, based on its impact on Tfh cells in vitro and in human subjects, we examined the effect of Tyk2 P on generation of Tfh and GC B cells in vivo. We first assessed T cell-dependent immunization using TLR7-loaded virus-like particles (VLP) in control (Tyk2 NP/NP ) mice, mice heterozygous (Tyk2 NP/P ), or homozygous (Tyk2 P/P ) for the protective variant. At the peak of the immune response, there was no difference in the proportion or number of Tfh cells or GC B cells generated by these strains (Figures 3A-C). Additionally, we saw no differences in VLP-specific GC B cells or in highaffinity anti-VLP IgG2c antibodies (Figures 3D,E). We expanded upon this result by using a second immunization strategy designed to promote a more sustained GC response triggered via delivery of sheep red blood cells (SRBCs) and also included cohorts of Tyk2 −/− animals. Again, all strains exhibited equivalent production of GC B cells, Tfh cells, and antibodies (Supplemental Figures 3A-D). Taken together, Tyk2 P appears to have little or no impact on T-dependent GC and antibody formation in response to immunization strategies that rely on the formation and function of Th1/Th2 cells.
Tyk2 P Does Not Affect Tfh and GC B Cell Formation in Murine Lupus Models TYK2 P has been associated with protection from multiple autoimmune diseases including SLE (32). Therefore, we next directly assessed the role of the protective variant in disease development using alternative murine lupus models utilizing Tyk2 P mice. As an initial test, we used the BM12 T cell adoptive transfer model of lupus. The BM12 strain was derived from C57BL/6 mice and contains a three-amino-acid change in the major histocompatibility complex class II molecule H2-AB1 b (47). An autoimmune GC response is generated when BM12 CD4 + T cells are adoptively transferred into C57BL/6 recipients leading to production of autoantibodies directed against dsDNA within ∼3 weeks following the cell transfer (48,49). Therefore, to assess the impact of Tyk2 P in this setting, we transferred BM12 CD4 + T cells into control (Tyk2 NP/NP ), heterozygous (Tyk2 NP/P ), or homozygous (Tyk2 P/P ) recipient mice. Following CD4 + T cell transfer, there was no difference in the proportion of Tfh and GC B cells in any strain (data not shown). We also observed no differences in autoantibody levels at disease peak (Figures 4A-C).
Tyk2 NP/P , or Tyk2 P/P , respectively. As shown schematically in Figure 4D, cohorts of animals for each of these µMT −/− strains were lethally irradiated and reconstituted by BM transplantation using a mixture of 80% µMT −/− BM (expressing Tyk2 NP/NP , Tyk2 NP/P , or Tyk2 P/P , respectively) and 20% WAS −/− BM (co-expressing Tyk2 NP/NP , Tyk2 NP/P , or Tyk2 P/P , respectively; Figure 4D). As an additional control to assess the impact of IL-12 receptor signaling in disease development, we utilized µMT −/− recipient strains and donor BM cells both deficient for IL-12Rβ2 ( Figure 4D). Strikingly, all recipients of WAS −/− BM developed high-titer class-switched IgG2c anti-dsDNA and anti-smRNP antibodies within 4 months post-transplant. We also observed no differences in relative levels of autoantibody production, GC B cells, or Tfh cells between recipients with alternative Tyk2 P alleles (Figures 4D-H). Moreover, despite the anticipated role for IL-12 in modulating T and B cell activation, IL-12Rβ2 deficiency exerted no appreciable impact on disease within this model with recipients developing autoantibodies, GC B cells, and Tfh cells as efficiently as WAS −/− chimeras (Figures 4D-H). Taken together, these findings suggest that Tyk2 P does not play a major role in development of autoimmune GC responses or in modulating autoantibody production in murine SLE.

Type I Interferon Signaling Is Reduced in T Cells From TYK2 P Healthy Subjects
Another pathway with a requirement for TYK2 is type I interferon (IFN I) signaling (18). This pathway may also impact Tfh generation. To test the role of TYK2 P in type I interferon receptor (IFNAR) signaling, we stimulated PBMCs from healthy control subjects and subjects heterozygous for the protective variant using IFN-α and examined phosphorylated STAT1 (pSTAT1) levels following activation. Naïve TYK2 NP/P CD4 + and CD8 + T cells exhibited a decrease in IFN-α induced pSTAT1 levels compared to cells from control TYK2 NP/NP subjects (Figures 5A-D), a difference that was not due to altered IFNAR surface expression. These findings were consistent with a previous report showing diminished pSTAT1 and pSTAT3 levels following IFN-α stimulation in subjects with the protective allele (33). Thus, IFNAR signaling is reduced by the expression of TYK2 P .

TYK2 P Is Involved in IL-23 Signaling, Th17 Skewing, and Tfh-17 Formation
Circulating human Tfh cells are comprised of three distinct developmental subsets that can be discriminated based on relative surface expression levels of CXCR3, CCR6, and CCR7 (14). Therefore, we next investigated whether a specific Tfh lineage was preferentially impacted by expression of TYK2 P . Though not statistically different, we discovered that individuals expressing the protective variant exhibited a trend for a decrease in the relative proportion Tfh-17 cells (p = 0.123) but exhibited no changes in the proportion of Tfh-1 or Tfh-2 cells (Figures 6A-C). Consistent with this data, TYK2 is activated downstream of the IL-23 receptor engagement (19). To further study the role of TYK2 P in Th17 commitment and in Tfh-17 cells, we investigated IL-23 signaling in the murine CD4 + T cells derived from control (Tyk2 NP/NP ), heterozygous (Tyk2 NP/P ) or homozygous (Tyk2 P/P ) mice and from Tyk2 −/− animals. Both Tyk2 P/P and Tyk2 −/− T cells exhibited decreased IL-23 dependent pSTAT3 and heterozygous Tyk2 NP/P T cells exhibited a trend consistent with an intermediate phenotype ( Figure 6D). Further, both Tyk2 P/P and Tyk2 −/− T cells displayed a diminished Th17 skewing in vitro (Figure 6E). In summary, TYK2 P plays a role in IL-23 signaling mostly likely contributing to the observed decrease in Tfh-17 cells in subjects expressing the protective variant. Each symbol represents an individual biological replicate;Tyk2 NP/NP n = 3, Tyk2 NP/P n = 4,Tyk2 P/P n = 3 or PBS n = 2 (A-C); WT n = 4,Tyk2 NP/NP n = 9, Tyk2 NP/P n = 12,Tyk2 P/P n = 7 or IL-12R −/− n = 8 (E,F); WT n = 8,Tyk2 NP/NP n = 9, Tyk2 NP/P n = 12,Tyk2 P/P n = 7 or IL-12R −/− n = 8 (G,H). Tyk2 P Mice Are Protected From EAE and Exhibit Reduced Numbers of IFN-γ + /IL-17 + Pathogenic CD4 + T Cells TYK2 P has also been associated with protection in MS (31). We used a murine model of MS, experimental autoimmune encephalomyelitis (EAE), to test the role of Tyk2 P in modulating disease. As shown schematically in Figure 7A, control (Tyk2 NP/NP ), heterozygous Tyk2 NP/P and homozygous Tyk2 P/P mice were immunized with MOG peptide in complete Freund's adjuvant (CFA) and also treated with pertussis toxin to increase permeability of the blood brain barrier. While both control and heterozygous Tyk2 NP/P animals developed disease manifestations beginning at ∼10 days post-immunization, mice expressing Tyk2 P/P were completely protected from EAE ( Figure 7A, lower panel). Both Th1 and Th17 cells have been shown to be important for EAE disease development (54). Notably, the proportion of draining LN T cells expressing IL-17 + was similar in Tyk2 NP/NP , Tyk2 NP/P and Tyk2 P/P animals and there was only a trend toward a reduced proportion of IFN-γ + cells in Tyk2 P/P animals (Figures 7B-E). In contrast, the proportion of double-positive IFN-γ + /IL-17 + pathogenic CD4 + T cells was specifically decreased in Tyk2 P/P mice (Figure 7E). The number of CD4 + T infiltrating the central nervous system (CNS) was markedly reduced in Tyk2 P/P mice and included reduction in both IFN-γ + or IFN-γ + /IL-17 + double positive T cells (Figures 7F-I). Altogether, these data demonstrate that Tyk2 P protects from EAE by decreasing pathogenic CD4 + T cells which depend on both IL-12 and IL-17 signaling to promote disease development.

DISCUSSION
While TYK2 P has been shown to be a hypomorphic allele, its protective role in autoimmunity still remains largely unexplored. Here we show that TYK2 P limits signaling in response to IL-12, IL-23, and IFN I cytokines. Despite these cytokine defects, Tyk2 P mice were not protected in two independent lupus models and exhibited no difference in the response toward two different T dependent immunization models. Yet healthy individuals expressing TYK2 P displayed diminished Tfh and switched memory B cells, and homozygous Tyk2 P mice were fully protected in a murine model of MS. Our findings highlight the complexity of the cytokine milieu that regulate immune responses in both man and mouse, and the likely requirement for concurrent alterations in multiple cytokine signals in order for this variant to manifest a disease protective phenotype.

Functional Role of TYK2 P in Cytokine Signaling
In our murine model, we found a deficiency in IL-12 induced pSTAT3 in homozygous Tyk2 P expressing CD4 + T cells. This was complimentary to a recent study that developed a similar mouse model of Tyk2 P1104A and showed decreased IL-12 induced pSTAT4 (33). Similar to the murine data, Dendrou et al. found diminished IL-12 induced pSTAT4 in human CD4 + T cells expressing TYK2 P/P compared to TYK2 NP/NP T cells (33). In contrast, we did not identify differences in pSTAT3 or pSTAT4 following IL-12 stimulation in homozygous non-protective vs. heterozygous protective individuals. Our findings are consistent with a recent data set comparing TYK2 NP/NP to TYK2 NP/P participants (55). Differences between our studies likely reflect the large number of homozygous TYK2 P individuals (7 vs. 2) studied by Dendrou et al. and/or differences in stimulation conditions. Further, we found that IL-23 signaling and IL-17 + cells were decreased in murine homozygous Tyk2 P CD4 + Th17 populations consistent with previous mouse and human data (33). Lastly, we demonstrate that TYK2 P also limits type I interferon signaling in humans, and in the Tyk2 P murine model (data not shown) as observed in human TYK2 P T cells (33). Of note, while Tyk2 NP/NP and Tyk2 NP/P T and B cells did not exhibit statistically significant differences in IL-12 mediated signals (Figures 2A-D), in each assay, Tyk2 NP/P cells showed a slight decrease in pSTAT3 and Th skewing (and in IL-12 triggered B cell IL-6 production) compared to Tyk2 NP/NP cells suggesting a potential dose-dependent effect on in vitro IL-12 signaling. The findings mimicked the impact of heterozygous dosage of the protective variant in human cells in various settings. Taken together, our observations support the conclusion that TYK2 P exerts an allele-dose dependent limiting effect on in vitro responses to IL-12, IL-23, and IFN I signaling.
Individuals that are TYK2-deficient manifest impaired cytokine responses to IL-12, IL-23, IFN-α, and IL-10 (21). Moreover, these patients exhibit an increased risk for mycobacterial and viral infections (21). Consistent with this phenotype, TYK2 P individuals also exhibit signaling defects in IL-12, IL-23, and IFN-α. However, based upon the clinical data within our biorepository, the small number of homozygous protective variant-expressing subjects have not displayed increased infections similar to the TYK2-deficient patients (21) and other studies to date also have not reported an increase in infectious risk for such individuals; suggesting that larger populations studies are likely required to address this question (33). Differences between complete TYK2 deficiency versus a hypomorphic allele may reflect retention of a protein scaffold function. This idea may also be consistent with observations that individuals or mice heterozygous for the protective allele exhibit subtle alterations in lymphocyte subsets and signaling activity, implying a possible dominant negative effect of the protective allele. Studies have also linked loss of TYK2 expression to altered stability of STAT proteins in murine cells and TYK2 associated receptor surface expression on human cells (20,21,23,33). Similar findings have not been previously reported or observed in our Tyk2 P murine model (data not shown). TYK2 P expression was shown not to affect IFNAR surface expression (Figures 5C,D) and IL-12R (33). More work is needed to fully elucidate the TYK2 interactome in various cell lineages and its impact(s) in modulation of cytokine signaling.
Dysregulation of the IL-12, IL-23, or IFN signaling pathways may also contribute to SLE disease (3,15,16). However, signaling molecules within these pathways seem to compensate for each other. Hence, there is the need for multiple aberrant pathways to lead to complex autoimmune diseases such as SLE. There is evidence that these pathways are on a fine axis. When one is dysregulated, it throws off the balance of the other pathways leading to further abnormal signaling, irregular activation and ultimately autoimmune disease. One example of this is deficiency in STAT3 which causes a decrease in Tfh and GC B cells, leading Th cells to take on the Th1-like phenotype. However, in STAT3 deficient cells the normal populations are rescued when IFNAR is blocked (18). Together these cytokine pathways are dependent on one another in vivo and must be studied collectively to get a complete picture of how such signals contribute to disease.

TYK2 P and T Helper Subsets
IL-12 and IL-23 represent critical cytokines for generation of Th1 and Th17 cells, respectively. Herein we show that IL-12 and IL-23 signaling and their respective Th subsets are diminished in an in vitro setting when TYK2 P is expressed. Importantly, these cytokines are also involved in Tfh cell generation. We show for the first time that Tfh cells, specifically the Tfh-17 cell subset which has superior ability to provide B cell help (56), are reduced in healthy human subjects with the TYK2 P allele. Further, we show that naïve murine Tyk2 P CD4 + T cells exhibit a defect in in vitro Tfh generation. Consistent with these findings, individuals lacking IL-12Rβ1 exhibit diminished circulating memory Tfh and memory B cells (57). IL-23 also signals through STAT3 and can to contribute to Tfh generation (13). Our combined observations support a model wherein combined reduction in IL-12 and IL-23 signals leads to a reduced number of Tfh-17 cells in healthy TYK2 P donors. Thus, TYK2 P is a critical regulator for Tfh populations by reducing IL-12 and IL-23 signaling cascades.
Herein we also found healthy individuals expressing TYK2 P to have diminished switched memory B cells. This is consistent with IL-12Rβ1 deficient subjects who exhibited both reduced switched and unswitched memory B cells (57). This reduction is most likely due to defective GC responses from diminished IL-12 signaling in T cells. IL-12 is an efficient inducer of IL-21 production from Tfh cells, a cytokine critical for the activation of human GC B cells (14,58). Additionally, IL-12Rβ1-, TYK2-, or STAT3-deficient CD4 + T cells display reduced IL-12 induced IL-21 production in vitro (12). However, Tyk2 P mice did not display any differences in GC responses post-immunization. Further investigation is needed to assess GC formation and its link to memory B cells in Tyk2 P mice.

TYK2 P in Autoimmune Disease
TYK2 P has been associated with protection from MS (31) and Tyk2 −/− mice are fully protected from EAE (26). In our study, we found that homozygous Tyk2 P/P mice are completely protected from EAE. Infiltrating T cells within the CNS were markedly reduced in Tyk2 P/P mice and protection correlated most strongly with a reduction in double-positive IFN-γ + /IL-17 + CD4 + T cells within both the draining lymph nodes and the CNS. Of note, consistent with the partial in vitro phenotype in response to cytokine stimulation, heterozygous Tyk2 NP/P mice exhibited a trend toward reduced single IFN-γ + and double-positive IFNγ + /IL-17 + CD4 + T cells in the draining lymph nodes. However, heterozygous animals were not protected from EAE in vivo. Our combined findings are consistent with and expand upon previous data from Dendrou et al. (33). Both the IL-12 and IL-23 signaling programs contribute to EAE disease (59). Protection for EAE in Tyk2 P/P mice aligns with the reduced IL-12 and IL-23 signaling and reduced Th1 and Th17 in vitro skewing described above. MS patients also exhibit populations of Tfh-1 and Tfh-17 cells and the relative proportions of these effectors varies among MS cohorts, with IL-23 signaling playing a more dominant role in some subjects (3,60). More work is required to determine whether protection from MS in TYK2 P carriers might be predicted based upon the proportion of Tfh-17 cells and/or dual-positive IFN-γ + /IL-17 + CD4 + effector T cells.
In contrast to the EAE data, we show that Tyk2 P does not shield mice from autoantibody production and disease progression in two separate lupus models even though GWAS has linked this variant to protection from SLE (28)(29)(30)33). SLE is a heterogeneous disorder that reflects both variable genetic and environmental contributions. The lack of protection observed in our studies may reflect the specific disease models studied. We observed no impact of Tyk2 P in the BM12 adoptive transfer lupus model where autoantibody production is driven by self-reactive T cell triggered autoimmune GC responses that are characterized by expanded Tfh populations. Despite our findings of altered Tfh and memory B cell populations in healthy TYK2 P subjects, we did not observe alterations in Tfh or autoantibody generation in this model. We also showed no impact of Tyk2 P in the WAS B cell chimera lupus model. This latter model leads to spontaneous autoimmune GC responses driven by altered B cell receptor (BCR) and TLR7 signaling. Autoimmune GC production is also dependent upon B cell intrinsic antigen presenting cell (APC) activity, IFNγ1-R1 signaling, and IL-6 production (46,51). Surprisingly, in the current study, we also show that the WAS chimera model is not impacted by global Il12rb2 deficiency and our previous work has shown that B cell-intrinsic IFNAR is also dispensable for lupus development in this model (51). Thus, two key programs modulated by Tyk2 P play a limited role in this model. As noted above, TYK2 P can function to limit IFN I signaling. IFN I signaling is increased in a subset of SLE subjects and IFN I blockade has provided partial benefit in some patients (61)(62)(63). Thus, the potential protective impact of Tyk2 P may be most relevant in lupus models that are driven or accelerated by an altered IFN I program. Future studies using co-modeling with other relevant SLE GWAS risk alleles, including the common IFIH1 risk variant (37), may provide insight into the impact of TYK2 P in SLE disease pathogenesis.
TYK2 A1104 allele is a rare variant at ∼2.7% overall allelic frequency (64). Thus, the association with protection in multiple autoimmune disorders is predominantly within heterozygous individuals. As noted above, while we observed alterations in key lymphocyte populations in healthy subjects with the protective allele, we observed only trends toward reduced signaling activity using heterozygous Tyk2 NP/P murine and human cells in our in vitro studies. Disease protection in vivo, when present, was only evident in Tyk2 P/P animals. The requirement for homozygous TYK2 P expression to manifest differences in our assays suggests that protection likely involves a more complex process than simply altering a single cytokine program. Instead, the variant appears to provide protection by modestly altering multiple pathways, thereby subtly diminishing immune responses that lead to autoimmunity. This complex role for TYK2 P in protection from autoimmune pathogenesis highlights the value of our combinatorial studies using both murine models and healthy human subjects to assess its impact on human disease. Whether protection primarily reflects diminished Tfh cell populations or another cell type remains to be fully defined the ability of TYK2 to impact a subset of key cytokine pathways highlights its potential utility as a therapeutic target. Consistent with this concept, recent findings using an oral TYK2 inhibitor have demonstrated beneficial effects in treatment of adult subjects with psoriasis (65). Taken together, our findings suggest that targeting TYK2 kinase activity may provide a relatively broad therapeutic window for protection from autoimmune disease while limiting the potential risk for immunosuppression.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available for the corresponding author upon request.

AUTHOR CONTRIBUTIONS
JG designed and performed experiments, analyzed data, and wrote the manuscript. CH designed and performed experiments, analyzed data, and edited manuscript. MK, TA, EA, CC, SW, KT, AE, SK, MH, and MO developed required models/strains or reagents and/or performed experiments and/or edited manuscript. SWJ designed and interpreted WASp mouse studies. KC genotyped human subjects, interpreted data, and edited manuscript. JHB and DJR conceived and supervised the study, interpreted data, and edited the manuscript.

FUNDING
This work was supported by grants from the NIH: DP3-