Regulation of Intrinsic and Bystander T Follicular Helper Cell Differentiation and Autoimmunity by Tsc1

T Follicular helper (Tfh) cells promote germinal center (GC) B cell responses to develop effective humoral immunity against pathogens. However, dysregulated Tfh cells can also trigger autoantibody production and the development of autoimmune diseases. We report here that Tsc1, a regulator for mTOR signaling, plays differential roles in Tfh cell/GC B cell responses in the steady state and in immune responses to antigen immunization. In the steady state, Tsc1 in T cells intrinsically suppresses spontaneous GC-Tfh cell differentiation and subsequent GC-B cell formation and autoantibody production. In immune responses to antigen immunization, Tsc1 in T cells is required for efficient GC-Tfh cell expansion, GC-B cell induction, and antigen-specific antibody responses, at least in part via promoting GC-Tfh cell mitochondrial integrity and survival. Interestingly, in mixed bone marrow chimeric mice reconstituted with both wild-type and T cell-specific Tsc1-deficient bone marrow cells, Tsc1 deficiency leads to enhanced GC-Tfh cell differentiation of wild-type CD4 T cells and increased accumulation of wild-type T regulatory cells and T follicular regulatory cells. Such bystander GC-Tfh cell differentiation suggests a potential mechanism that could trigger self-reactive GC-Tfh cell/GC responses and autoimmunity via neighboring GC-Tfh cells.


INTRODUCTION
T follicular helper (Tfh) cells are important players in both normal immune responses and autoimmune disease via contact-dependent and independent mechanisms to provide helping signals to B cells in the germinal centers (GCs). Tfh cells promote GC B cell proliferation and survival, Ig class switch and affinity maturation, and plasma cell and memory B cell formation (1,2). However, deregulated Tfh cells can trigger abnormal GC B and memory B cell responses to produce autoantibodies and contribute to autoimmune diseases. Abnormal Tfh cells have been associated with or are the causal factors for autoimmune diseases in human patients and/or in autoimmune mouse models (3)(4)(5)(6)(7)(8)(9). Tfh cells express Bcl6, a transcription factor critical for their differentiation (10)(11)(12)(13). Tfh cell differentiation is regulated via multiple mechanisms, including TCR signal strength and duration, costimulatory signaling such as CD28 and ICOS, and chemokine receptors such as CXCR5 (12,(14)(15)(16)(17)(18)(19)(20)(21)(22). Additionally, Tfh cells and GC B cells are suppressed by T follicular regulatory (Tfr) cells to prevent dysregulated antibody responses and autoimmunity (23)(24)(25). Recently, evidence has emerged that mTOR, which integrates TCR, costimulatory, cytokine, and metabolic signals (26)(27)(28), is crucial for Tfh cell differentiation, homeostasis, and function via signaling through both mTOR complexes 1 and 2 to regulate Bcl-6 expression and Tfh cell proliferation, survival, and metabolism (29)(30)(31)(32)(33), and it regulates Tfr cell differentiation and function (29,30). TSC1/2 are key regulators of mTOR signaling, inhibiting mTORC1 and, in certain instances, promoting mTORC2 activities. TSC1, TSC2, and TBC1D7 form the core of the TSC protein complex. TSC2 contains GAP activity for RheB to inhibit mTORC1 activation. TSC1 is crucial for TSC2 stability (34)(35)(36). Via tight control of mTOR, TSC1/2 regulate diverse processes such as cell metabolism, growth, proliferation, differentiation, quiescence, stemness, and autophagy and play important roles in many diseases (37,38). Recent studies have revealed significant impacts of Tsc1 deficiency on immune cell development and function using mouse models with tissuespecific Tsc1 ablation. These studies have demonstrated that Tsc1 deficiency greatly affects hematopoietic stem cells, conventional T cells, regulatory T cells, iNKT cells, B cells, NK cells, macrophages (including M1/2 polarization), dendritic cells, and mast cells to influence both adaptive and innate immune responses, self-tolerance, and diseases (39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52). In T cells, Tsc1 has been found to be important for T cell homeostasis, quiescence, anergy, and effector and memory responses (39)(40)(41)(42)(43)(44)(45). However, the role Tsc1 plays in Tfh cells regarding controlling antibody responses has been unknown. In this report, we demonstrate that Tsc1 performs differential roles in Tfh cell differentiation in the steady state and during immune responses to immunization. In the steady state, Tsc1 inhibits Tfh cell differentiation, and T cell-specific Tsc1 deficiency causes spontaneous Tfh cell differentiation, leading to the accumulation of GC-B cells and the production of autoantibodies. In contrast, Tsc1 positively contributes to Tfh cell differentiation and antigen-specific antibody responses after immunization-at least in part by promoting Tfh cell survival via maintaining mitochondrial integrity and reducing reactive oxygen species. Additionally, Tsc1 deficiency not only intrinsically promotes Tfh cell differentiation but also extrinsically leads to bystander Tfh cell differentiation of WT T cells in the steady state. The discovery of bystander Tfh cell differentiation suggests potential mechanisms for the development of autoantibody and autoimmune diseases.

Mice
Tsc1 f/f mice (53) and Cd4-Cre (54) mice were purchased from the Jackson Laboratory and Taconic Farms, respectively. Tsc1 f/f mice were backcrossed to the C57BL/6 background for nine generations. All mice were generated and used according to protocols approved by the Duke University Institute Animal Care and Use Committee.

Immunization and Measurement of Antibody Responses
Mice were immunized with a single i.p. injection of 20 mg of 4hydroxy-3-nitrophenylacetyl conjugated chicken gamma globulin (NP 17 -CGG, Biosearch Technologies) in alum, as previously described (55). Serum was collected preimmunization and on day 7, 14, and 21 post-immunization. Appropriately diluted sera were added into 96-well plates precoated with 50 ml 2 mg/ml NIP 4 -BSA or NIP 26 -BSA in 0.1 M carbonate buffer (pH 9.0) at 4°C overnight. After incubation and multiple washes, HRP-conjugated goat anti-mouse IgM, IgG, IgG1, IgG2b, and IgG3 were used to detect NIP-specific IgM, IgG, and IgG subtypes.

Serum Immunoglobulin Concentrations
One hundred ml of appropriately diluted sera from unimmunized mice was added into 96-well plates (Corning, New York, NY) precoated with anti-mouse Igk and Igl antibodies (2mg/ml; SouthernBiotech, Birmingham, AL) in 0.1 M carbonate buffer (pH 9.0) at 4°C overnight. IgM, IgG, IgG1, IgG2b, and IgG3 levels were detected with ELISA using HRP-conjugated goat anti-mouse total or Ig subtype antibodies. Relative levels of Ig were computed by OD450 values.

Statistical Analysis
Data were presented as mean ± SEM and analyzed for statistical differences using the Prism 5/GraphPad software. Data with each experiment that contained a pair of test and control mice that were the same sex and age, were hosted in the same cage, and in most cases were also littermates were analyzed with a two-tailed pairwise Student t-test. A connection line in the scatterplots indicates a pair of test and control mice in each experiment.
Data that did not fall into the aforementioned pairwise Student ttest criteria such as from experiments with two or more test or control mice in one experiment were analyzed by an unpaired Student t-test. P-values less than 0.05 were considered significant.
Together, these data suggest that TSC1 may negatively control mTORC1 and the expression of Bcl-6 and ICOS to prevent spontaneous GC-Tfh cell differentiation.
In Tsc1-T-KO mice, splenic B220 + B cell percentages and numbers increased ( Figure 1J). Within B cells, IgM + IgDand IgM -IgD + B cell percentages were not altered, but IgM -IgD -(DN) B cell percentages increased about 30%. Due to increased total B cells, their numbers all increased ( Figure 1K). Consistent with increased GC-Tfh and Tfh cells, Tsc1-T-KO mice also contained increased Fas + GL7 + GC B cells ( Figures 1L, M). Thus, Tsc1 inhibited spontaneous Tfh differentiation and GC-B cell formation in the steady state.

TSC1 Intrinsically Inhibited Tfh Cell Differentiation and Extrinsically Suppressed Bystander Tfh Cell Differentiation
Because Tsc1 was absent in both CD4 and CD8 T cells in Tsc1 f/f -Cd4Cre mice, we generated mixed bone marrow (BM) chimeric mice to determine whether Tsc1 intrinsically controlled Tfh cell differentiation. We reconstituted lethally irradiated CD45.1 + CD45.  WT and CD45.2 + Tsc1 f/f -Cd4Cre (WT/KO) BM cells at a 1:1 ratio. Sixeight weeks after reconstitution, WT/KO mice displayed enlarged spleens with increased total cell numbers compared with WT/WT mice ( Figure 2A). Moreover, both CD4 T cell and B cell but not CD8 T cell numbers were increased in WT/KO mice ( Figure 2B).

Tsc1 Deficiency Resulted in Abnormal Tfh Cell Properties
To examine how Tsc1 deficiency increased GC-Tfh cells, we examined GC-Tfh cell proliferation and survival in the mixed BM chimeric mice. Both CD45.1 + WT and CD45.2 + Tsc1-T-KO GC-Tfh cells in WT/KO mice and WT/WT mice expressed similar levels of Ki67, a marker of cell proliferation ( Figure  3A) and showed a similar death rate ( Figure 3B), suggesting that increased GC-Tfh cells in WT/KO mice was not due to increased proliferation or improved survival. Within CD45.2 + Tsc1-T-KO GC-Tfh cells, Bcl6, cMaf, ICOS, and SLAM levels increased

Elevated IgG1 + GC-B Cells and Serum IgG1 Autoantibodies in Tsc1-Deficient Mice
We next examined whether dysregulated Tfh/GC B cells in Tsc1-T-KO mice would lead to altered antibodies and enhanced autoimmunity. Tsc1-T-KO mice contained elevated serum IgG1 levels but normal serum IgG3 levels. Their IgG2b levels were slightly decreased, but such decreases were not statistically significant (p = 0.412, Figure 4A). Within splenic Tsc1-T-KO GC B cells, IgG1 + class-switched cells increased but IgG2 classswitched cells were not changed ( Figures 4B, C), leading to increased numbers of IgG1 + cells but normal numbers of IgG2b + cells ( Figure 4C). Moreover, Tsc1-T-KO mice contained increased anti-double strand (ds) DNA IgM and IgG1but not IgG2b or IgG3 autoantibodies ( Figure 4D). Interestingly, Tsc1-T-KO Tfh cells expressed elevated GATA3 levels ( Figure 4E) and contained increased Bcl6 hi GATA3 + Tfh2 cells ( Figures 4F, G). Thus, TSC1 deficiency appeared to cause skewing of Tfh cells toward the Tfh2 cell sublineage, leading to elevated IgG1 classswitched GC B cells and serum IgG1 levels and the development of IgG1-dominant autoantibody responses.

Intrinsic and Extrinsic Regulation of Treg and Tfr Cells by Tsc1
Regulatory T cells (Tregs), especially Tfr cells, suppress Tfh/GC B cell differentiation and GC responses (23)(24)(25). mTOR regulates Treg and Tfr cell differentiation and function (29,30,64,65). Although Tsc1 promotes Treg stability and function (51), its role in Tfr cells has been unknown. In Tsc1 f/f -Cd4Cre mice, the percentages and numbers of Foxp3 + Tregs within CD4 T cells and CXCR5 + PD1 + Tfr cells within Foxp3 + Tregs were not obviously different from WT mice ( Figures 5A, B). However, Tsc1-T-KO Tfr cells expressed increased levels of Bcl6 but similar levels of Gata3 compared with WT Tfr cells ( Figure  5C), suggesting that some properties of Tsc1-T-KO Tfr cells were altered. Given the dysregulated Tfh/GC B cell differentiation in Tsc1-T-KO mice, it is possible that Tsc1-deficient Tregs and Tfr cells might be functionally impaired to suppress GC-Tfh cell and GC B cell differentiation and/or that Tsc1-deficient GC-Tfh cells might be resistant to Treg/Tfr cell mediated suppression. Future studies should examine these possibilities. Interestingly, in CD45.1 + WT/CD45.2 + WT (WT/WT) and CD45.1 + WT/CD45.2 + Tsc1-T-KO (WT/KO) mixed BM mice, as described in Figure 2, Treg percentages and numbers in WT/KO mice increased compared with those in WT/WT mice ( Figure 5D). Within WT/WT chimeric mice, Treg percentages and numbers in the CD45.2 + WT CD4 T cells and in the CD45.1 + WT CD4 T cells were similar ( Figure 5E). In contrast, Treg percentages and numbers decreased within the CD45.2 + Tsc1-T-KO CD4 T cells but increased within the CD45.1 + WT CD4 T cells in WT/KO chimeric mice ( Figure 5E). Such differences were also observed when they were compared with their counterparts in WT/WT chimeric mice. These data suggest that Tsc1 deficiency not only intrinsically limits Treg accumulation but also extrinsically promotes WT Treg accumulation in a competitive setting.
Within CD4 + Foxp3 + Tregs, PD1 + CXCR5 + Tfr cells were increased in WT/KO chimeric mice compared with WT/WT chimeric mice (Figures 5F, G). Moreover, the CD45.1 + WT Tfr cells increased, whereas the CD45.2 + Tsc1-T-KO Tfr cells decreased in both percentages and numbers within WT/KO chimeric mice. Such trends were also true when compared with their counterparts in WT/WT chimeric mice ( Figures  5H, I). Thus, Tsc1 deficiency intrinsically inhibits Tfr cell differentiation/maintenance but appears to extrinsically promote bystander WT Tfr cell differentiation/maintenance.

Impairment of Antibody Responses to Antigen Immunization in Tsc1-Deficient Mice
We further asked whether Tsc1 deficiency in T cells would affect antigen-specific antibody responses after immunization. We immunized Tsc1 f/f -Cd4Cre and Tsc1 f/f or Tsc1 +/+ -Cd4Cre control mice with a T cell-dependent antigen NP-CGG (4-Hydroxy-3-nitrophenylacetyl hapten conjugated to chicken gamma globulin). In Tsc1-T-KO mice, both total and high affinity anti-NIP IgM titers decreased on days 7, 14, and 21 after immunization; although total and high affinity anti-NIP IgG titers displayed reduced trend, such decreases were not statistically significant ( Figure 6). Further analyses of IgG subtypes revealed similar levels of IgG1 anti-NIP antibodies but obviously decreased IgG2b and IgG3 anti-NIP antibodies in Tsc1-T-KO mice compared with WT mice. Thus, TSC1 deficiency selectively impaired antigen-induced IgM, IgG2b, and IgG3 antibody responses with IgG1 responses spared.

Impaired Tfh Cell Responses to Antigen Immunization in Tsc1-Deficient Mice
Given the observations of increased Tfh/GC B cells in Tsc1 f/f -Cd4Cre mice in the steady state, it was surprising that antigenspecific antibody responses were impaired in these mice. We further examined GC-Tfh cells in these mice after NP-CGG/ alum immunization. Splenic GC-Tfh cell percentages within CD44 + CD4 T cells decreased in Tsc1-T-KO mice compared with WT mice after immunization ( Figures 7A, B). Tsc1-T-KO GC-Tfh cells expressed increased Bcl6 and Ki67 ( Figures 7C-E), suggesting that their decreases were not caused by reduced Bcl6 or impaired proliferation. In contrast, the death rates of Tsc1-T-KO GC-Tfh cells and non-GC-Tfh CD4 + effector T cells were increased 2.9-and 2.4-fold, respectively, compared with WT controls (Figure 7F), suggesting that the increased death of  Tsc1-deficient GC-Tfh cells might contribute to impaired GC-Tfh cells and antibody responses after immunization.
In both GC-Tfh and non-GC-Tfh cells from Tsc1-T-KO mice, reactive oxygen species (ROS) and iNOS levels increased, but mitochondrial potential, as reflected by TMRM (Tetramethylrhodamine methyl ester perchlorate) staining, decreased ( Figures 7G, H). These data suggested that increased ROS, in the absence of Tsc1, might damage mitochondrial integrity and cause the death of GC-Tfh cells during immune responses to antigens.

DISCUSSION
Understanding the regulation of Tfh cell differentiation is important for developing new strategies to effectively elicit protective immunity and improve the treatment of autoimmune diseases. Previous reports have established the importance of Tsc1 for the tight control of mTOR in T cells to regulate multiple aspects of T cell functions. We demonstrate here that Tsc1 also plays differential roles in Tfh cell/GC-B cell differentiation in the steady state and in the response to immunization.
Our data indicate that Tsc1 intrinsically inhibits GC-Tfh cell and subsequent GC B cell differentiation and autoantibody production in the steady state. In Tsc1-deficient mice, GC-Tfh cells increased. Because Tsc1-deficient GC-Tfh cell proliferation and survival is similar to WT controls, Tsc1 deficiency is likely to augment CD4 T cell differentiation to GC-Tfh cells.
Our data also revealed that Tsc1 is required for optimal Tfh/ GC B cell responses and antibody production to T-dependent antigens after immunization. The impaired antibody responses of Tsc1-T-KO mice to T-dependent antigens after immunization suggest that the proper control of Tsc1-regulated pathways is important for Tfh cell differentiation and function during antigen-induced responses. Tsc1-deficient Tfh cells contain FIGURE 6 | Impaired antigen-induced antibody responses in TSC1-deficient mice after immunization. Tsc1 f/f -Cd4Cre and control mice were immunized with NP 17 -CGG in alum. Total and high affinity-anti-NIP antibodies before immunization and 7, 14, 21 days after immunization were measured via ELISA. Line plots show Mean ± SEM of serum concentrations of anti-NIP antibodies. Data shown are representative of two experiments. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001 determined by an unpaired two-tailed Student t-test. increased ROS and manifested impaired mitochondrial integrity and increased cell death. It has been reported that CD44 + Tsc1deficient CD4 T cells also contain high levels of ROS, and the treatment of these cells with a ROS scavenger improve their survival (39). It is plausible that Tsc1 prevents overproduction of ROS and subsequent mitochondrial damage to promote GC-Tfh Additionally, Tsc1-deficient CD4 T cells are hyper-activated in the steady state (39)(40)(41), and excessive stimulation upon immunization could also contribute to impaired Tfh cell responses. Similar to GC-Tfh cells, Tsc1-deficient CD8 T cells are also defective in responses to microbial infection (43,44). Of note, Tsc1 is deleted in naïve CD4 T cells in Tsc1 f/f -Cd4Cre mice, and our data do not illustrate whether a Tsc1 deficiency affects multiple stages from T cell activation to GC-Tfh cell differentiation and homeostasis-or selectively affects GC-Tfh cells.
Our observations that Tsc1-deficient T cells enhance the bystander Tfh cell differentiation of WT CD4 T cells in mixed BM chimeric mice reconstituted with WT and Tsc1 f/f -Cd4Cre BM cells are surprising and interesting because bystander Tfh cell differentiation has not been previously noted. Although the exact mechanisms that regulate bystander Tfh cell differentiation remain to be illustrated, there are multiple possibilities that Tsc1deficient T cells, particularly Tfh cells, could cause bystander Tfh cell differentiation. Tsc1-deficient Tfh cells could lead to increased Tfh cell-promoting cytokines such as IL21 (82,83) in the local environment to enhance bystander Tfh cell differentiation. They may also indirectly promote bystander Tfh cell differentiation via GC-B cells (84). Increased GC-B cell numbers and potentially altered properties associated with these B cells induced by Tsc1deficient Tfh cells could promote WT T cell differentiation to Tfh cells and/or expansion of Tfh cells already generated in the steady state. GC-B cells in the WT/Tsc1-T-KO mixed BM chimeric mice expressed increased levels of ICAM1 and SLAM compared with those in WT/WT mice, which could not only positively provide feedback to Tsc1-deficient Tfh cells but also enhance WT Tfh cell differentiation/expansion as ICAM1 engages LFA1 on T cells and SLAM forms homodimer or heterodimer with SLAM on T cells to promote Tfh cell differentiation (61)(62)(63). Bystander Tfh cell differentiation could have important implications for autoimmune diseases. It is conceivable that bystander differentiation of self-reactive Tfh cells could occur during immune responses against pathogens or commensal bacteria, which could contribute to increased self-reactive Tfh cells and subsequent GC-B cells and subsequent autoantibody production. This hypothesis should be tested in the future.
In addition to Tfh cells, our data also indicate that Tsc1 is involved in the intrinsic and extrinsic regulation of Tfr cells. In Tsc1 f/f -Cd4Cre mice, Tsc1 is not crucial for Tfr cell accumulation but negatively regulates Bcl6 expression. In mixed BM chimeric mice, Tsc1-deficient Tfr cells and Tregs are less competitive than their WT counterparts. The striking increases of WT Tregs, and especially Tfr cells in WT/KO mice, suggest that Tsc1 may extrinsically prevent bystander Treg and Tfr cell differentiation/ accumulation. The increases of WT Treg and Tfr cells could be a compensatory response to the increased Tfh and GC B cells in these mice due to changes of the local environment that promote Treg and Tfr cell differentiation or both. Additional studies are needed to understand the mechanisms by which Tsc1 intrinsically and extrinsically regulates Tfr/Treg and Tfh/GC B cells.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by Duke University Institute Animal Care and Use Committee.

AUTHOR CONTRIBUTIONS
SZ designed and performed experiments, analyzed data, and prepared the manuscript. LL participated in data analysis. DX and SR performed experiments and analyzed data. LM and JS participated in data interpretation and manuscript preparation. X-PZ conceived the project, designed experiments, and prepared the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
Research in this manuscript was supported by the Institute of Allergy and Infectious Diseases of the NIH (R01AI079088) and a Translating Duke Health Pilot Project Grant in Immunology.