ST6GAL1 sialyltransferase promotes acinar to ductal metaplasia and pancreatic cancer progression.

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 ß-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D had greatly accelerated PDAC progression compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1's role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.

Intrinsic STAT4 Expression Controls Effector CD4 T Cell Migration and Th17 Pathogenicity.

Effector CD4 T cells are central to the development of autoimmune chronic inflammatory diseases, yet factors that mediate pathogenicity remain ill-defined. Single-nucleotide polymorphisms in the human STAT4 locus are associated with susceptibility to multiple autoimmune disorders, and Stat4 is linked to the pathogenic Th17 gene signature; however, Th17 cells differentiate independently of STAT4. Hence the interplay between STAT4 and CD4 T cell function, especially Th17 cells, during autoimmune disease is unclear. In this article, we demonstrate that CD4 T cell-intrinsic STAT4 expression is essential for the induction of autoimmune CNS inflammation in mice, in part by regulating the migration of CD4 T cells to the inflamed CNS. Moreover, unbiased transcriptional profiling revealed that STAT4 controls the expression of >200 genes in Th17 cells and is important for the upregulation of genes associated with IL-23-stimulated, pathogenic Th17 cells. Importantly, we show that Th17 cells specifically require STAT4 to evoke autoimmune inflammation, highlighting, to our knowledge, a novel function for STAT4 in Th17 pathogenicity.

Protein Kinase CK2 Controls CD8+ T Cell Effector and Memory Function during Infection.

Protein kinase CK2 is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α') and two regulatory subunits (CK2ß). CK2 promotes cancer progression by activating the NF-κB, PI3K/AKT/mTOR, and JAK/STAT pathways, and also is critical for immune cell development and function. The potential involvement of CK2 in CD8+ T cell function has not been explored. We demonstrate that CK2 protein levels and kinase activity are enhanced upon mouse CD8+ T cell activation. CK2α deficiency results in impaired CD8+ T cell activation and proliferation upon TCR stimulation. Furthermore, CK2α is involved in CD8+ T cell metabolic reprogramming through regulating the AKT/mTOR pathway. Lastly, using a mouse Listeria monocytogenes infection model, we demonstrate that CK2α is required for CD8+ T cell expansion, maintenance, and effector function in both primary and memory immune responses. Collectively, our study implicates CK2α as an important regulator of mouse CD8+ T cell activation, metabolic reprogramming, and differentiation both in vitro and in vivo.

A Comprehensive Immune Cell Atlas of Cystic Kidney Disease Reveals the Involvement of Adaptive Immune Cells in Injury-Mediated Cyst Progression in Mice.

BACKGROUND: Inducible disruption of cilia-related genes in adult mice results in slowly progressive cystic disease, which can be greatly accelerated by renal injury. METHODS: To identify in an unbiased manner modifier cells that may be influencing the differential rate of cyst growth in injured versus non-injured cilia mutant kidneys at a time of similar cyst severity, we generated a single-cell atlas of cystic kidney disease. We conducted RNA-seq on 79,355 cells from control mice and adult-induced conditional Ift88 mice (hereafter referred to as cilia mutant mice) that were harvested approximately 7 months post-induction or 8 weeks post 30-minute unilateral ischemia reperfusion injury. RESULTS: Analyses of single-cell RNA-seq data of CD45+ immune cells revealed that adaptive immune cells differed more in cluster composition, cell proportion, and gene expression than cells of myeloid origin when comparing cystic models with one another and with non-cystic controls. Surprisingly, genetic deletion of adaptive immune cells significantly reduced injury-accelerated cystic disease but had no effect on cyst growth in non-injured cilia mutant mice, independent of the rate of cyst growth or underlying genetic mutation. Using NicheNet, we identified a list of candidate cell types and ligands that were enriched in injured cilia mutant mice compared with aged cilia mutant mice and non-cystic controls that may be responsible for the observed dependence on adaptive immune cells during injury-accelerated cystic disease. CONCLUSIONS: Collectively, these data highlight the diversity of immune cell involvement in cystic kidney disease.

Intrinsic IL-2 production by effector CD8 T cells affects IL-2 signaling and promotes fate decisions, stemness, and protection.

Here, we show that the capacity to manufacture IL-2 identifies constituents of the expanded CD8 T cell effector pool that display stem-like features, preferentially survive, rapidly attain memory traits, resist exhaustion, and control chronic viral challenges. The cell-intrinsic synthesis of IL-2 by CD8 T cells attenuates the ability to receive IL-2-dependent STAT5 signals, thereby limiting terminal effector formation, endowing the IL-2-producing effector subset with superior protective powers. In contrast, the non-IL-2-producing effector cells respond to IL-2 signals and gain effector traits at the expense of memory formation. Despite having distinct properties during the effector phase, IL-2-producing and nonproducing CD8 T cells appear to converge transcriptionally as memory matures to form populations with equal recall abilities. Therefore, the potential to produce IL-2 during the effector, but not memory stage, is a consequential feature that dictates the protective capabilities of the response.

Mitochondrial Oxidative Phosphorylation Regulates the Fate Decision between Pathogenic Th17 and Regulatory T Cells.

Understanding metabolic pathways that regulate Th17 development is important to broaden therapeutic options for Th17-mediated autoimmunity. Here, we report a pivotal role of mitochondrial oxidative phosphorylation (OXPHOS) for lineage specification toward pathogenic Th17 differentiation. Th17 cells rapidly increase mitochondrial respiration during development, and this is necessary for metabolic reprogramming following T cell activation. Surprisingly, specific inhibition of mitochondrial ATP synthase ablates Th17 pathogenicity in a mouse model of autoimmunity by preventing Th17 pathogenic signature gene expression. Notably, cells activated under OXPHOS-inhibited Th17 conditions preferentially express Foxp3, rather than Th17 genes, and become suppressive Treg cells. Mechanistically, OXPHOS promotes the Th17 pioneer transcription factor, BATF, and facilitates T cell receptor (TCR) and mTOR signaling. Correspondingly, overexpression of BATF rescues Th17 development when ATP synthase activity is restricted. Together, our data reveal a regulatory role of mitochondrial OXPHOS in dictating the fate decision between Th17 and Treg cells by supporting early molecular events necessary for Th17 commitment.

STAT4 Directs a Protective Innate Lymphoid Cell Response to Gastrointestinal Infection.

Innate lymphoid cells (ILCs) are strategically positioned at mucosal barrier surfaces where they respond quickly to infection or injury. Therefore, we hypothesized that ILCs are key contributors to the early immune response in the intestine against Listeria monocytogenes Using a modified strain of L. monocytogenes that mimics human gastrointestinal listeriosis in mice, we find ILCs to be essential for control of early replication of L. monocytogenes in the intestine as well as for restricted dissemination of bacteria to peripheral tissues. Specifically, group 1 ILCs (ILC1s) and group 3 ILCs (ILC3s) respond to infection with proliferation and IFN-γ and IL-22 production. Mechanistically, we show that the transcription factor STAT4 is required for the proliferative and IFN-γ effector response by ILC1s and ILC3s, and loss of STAT4 signaling in the innate immune compartment results in an inability to control bacterial growth and dissemination. Interestingly, STAT4 acts acutely as a transcription factor to promote IFN-γ production. Together, these data illustrate a critical role for ILCs in the early responses to gastrointestinal infection with L. monocytogenes and identify STAT4 as a central modulator of ILC-mediated protection.

IL-21 Controls ILC3 Cytokine Production and Promotes a Protective Phenotype in a Mouse Model of Colitis.

Group 3 innate lymphoid cells (ILC3s) have dual roles in intestinal health, acting in both protective and pathogenic capacities, and importantly, modulations in this population of innate lymphoid cells have been implicated in inflammatory bowel disease. Further, subpopulations of ILC3s have been described as serving specific functions in maintaining homeostasis or responding to infection, and aberrant activation of one or more of these subpopulations could exacerbate inflammatory bowel disease. However, the signals that enforce the protective and pathogenic features of ILC3s are not fully elucidated. In this article, we show that IL-21, a cytokine primarily produced by CD4 T cells, acts on a subpopulation of intestinal ILC3s to promote a protective phenotype. IL-21 signaling does not affect the MHC class II-expressing ILC3 subset but promotes ILC3s that express Tbet and are poised to produce IL-22. Consistent with a protective phenotype, IL-21 deficiency dampens cytokine-induced IL-17A production. We show that exacerbated colitis develops in mice lacking the IL-21 receptor, in agreement with a protective role for IL-21 signaling on ILC3s. To our knowledge, these data reveal a novel role for IL-21 in shaping innate lymphoid cell responses in the intestine and provide one mechanism by which effector CD4 T cells can influence innate immunity.

Urinary T cells correlate with rate of renal function loss in autosomal dominant polycystic kidney disease.

Several innate immune response components were recognized as outcome predictors in autosomal dominant polycystic kidney disease (ADPKD) and their causative role in disease pathogenesis was confirmed in animal models. In contrast, the role of adaptive immunity in ADPKD remains relatively unexplored. Therefore, we evaluated T cell populations in kidney and urine of ADPKD patients using flow cytometry and confocal immunofluorescence microscopy approaches. These analyses revealed ADPKD-associated overall increases in the number of intrarenal CD4 and CD8 T cells that were associated with a loss of polarity in distribution between the cortex and medulla (higher in medulla vs. cortex in controls). Also, the urinary T cell-based index correlated moderately with renal function decline in a small cohort of ADPKD patients. Together, these data suggest that similar to innate immune responses, T cells participate in ADPKD pathogenesis. They also point to urinary T cells as a novel candidate marker of the disease activity in ADPKD.

Effector CD4 T cells with progenitor potential mediate chronic intestinal inflammation.

Dysregulated CD4 T cell responses are causally linked to autoimmune and chronic inflammatory disorders, yet the cellular attributes responsible for maintaining the disease remain poorly understood. Herein, we identify a discrete population of effector CD4 T cells that is able to both sustain and confer intestinal inflammation. This subset of pathogenic CD4 T cells possesses a unique gene signature consistent with self-renewing T cells and hematopoietic progenitor cells, exhibits enhanced survival, and continually seeds the terminally differentiated IFNγ-producing cells in the inflamed intestine. Mechanistically, this population selectively expresses the glycosyltransferase ST6Gal-I, which is required for optimal expression of the stemness-associated molecule TCF1 by effector CD4 T cells. Our findings indicate that the chronicity of T cell-mediated inflammation is perpetuated by specific effector CD4 T cells with stem-like properties.

IL-10 Regulates Memory T Cell Development and the Balance between Th1 and Follicular Th Cell Responses during an Acute Viral Infection.

T cells provide protective immunity against infections by differentiating into effector cells that contribute to rapid pathogen control and by forming memory populations that survive over time and confer long-term protection. Thus, understanding the factors that regulate the development of effective T cell responses is beneficial for the design of vaccines and immune-based therapies against infectious diseases. Cytokines play important roles in shaping T cell responses, and IL-10 has been shown to modulate the differentiation of CD4 and CD8 T cells. In this study, we report that IL-10 functions in a cell-extrinsic manner early following acute lymphocytic choriomeningitis virus infection to suppress the magnitude of effector Th1 responses as well as the generation of memory CD4 and CD8 T cells. We further demonstrate that the blockade of IL-10 signaling during the priming phase refines the functional quality of memory CD4 and CD8 T cells. This inhibition strategy resulted in a lower frequency of virus-specific follicular Th (Tfh) cells and increased the Th1 to Tfh ratio. Nevertheless, neither germinal center B cells nor lymphocytic choriomeningitis virus-specific Ab levels were influenced by the blockade. Thus, our studies show that IL-10 influences the balance between Th1 and Tfh cell differentiation and negatively regulates the development of functionally mature memory T cells.

STAT4 controls GM-CSF production by both Th1 and Th17 cells during EAE.

BACKGROUND: In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, mice genetically deficient in the transcription factor signal transducer and activator of transcription 4 (STAT4) are resistant to disease. In contrast, deletion or inhibition of the Th1-associated cytokines IL-12 or IFNγ which act upstream and downstream of STAT4, respectively, does not ameliorate disease. These discordant findings imply that STAT4 may act in a non-canonical role during EAE. Recently, STAT4 has been shown to regulate GM-CSF production by CD4 T cells and this cytokine is necessary for the induction of EAE. However, it is not known if STAT4 controls GM-CSF production by both Th1 and Th17 effector CD4 T cells. METHODS: This study utilized the MOG(35-55) peptide immunization model of EAE. Intracellular cytokine staining and novel mixed bone marrow chimeric mice were used to study the CD4 T cell-intrinsic role of STAT4 during disease. STAT4 chromatin-immunoprecipitation (ChIP-PCR) experiments were performed to show STAT4 directly interacts with the Csf2 gene loci. RESULTS: Herein, we demonstrate that STAT4 controls CD4 T cell-intrinsic GM-CSF production by both Th1 and Th17 CD4 T cells during EAE as well as in vitro. Importantly, we show that STAT4 interacts with the Csf2 locus in MOG(35-55)-activated effector CD4 T cells demonstrating direct modulation of GM-CSF. CONCLUSIONS: Overall, these studies illustrate a previously unrecognized role of STAT4 to regulate GM-CSF production by not only Th1 cells, but also Th17 effector CD4 T cell subsets during EAE pathogenesis. Critically, these data highlight for the first time that STAT4 is able to modulate the effector profile of Th17 CD4 T cell subsets, which redefines our current understanding of STAT4 as a Th1-centric factor.

Astrocytes modulate the polarization of CD4+ T cells to Th1 cells.

T-cell characteristics are dynamic and influenced by multiple factors. To test whether cells and the environment in the central nervous system (CNS) can influence T-cells, we tested if culturing mouse CD4(+) T-cells on mouse primary astrocytes, compared with standard feeder cells, modified T-cell polarization to Th1 and Treg subtypes. Astrocytes supported the production of Th1 cells and Tregs, which was diminished by inflammatory activation of astrocytes, and glutamate accumulation that may result from impaired glutamate uptake by astrocytes strongly promoted Th1 production. These results demonstrate that astrocytes and the environment in the CNS have the capacity to regulate T-cell characteristics.

Virus-specific CD4 and CD8 T cell responses in the absence of Th1-associated transcription factors.

Effector and memory CD4 and CD8 T cell responses are critical for the control of many intracellular pathogens. The development of these populations is governed by transcription factors that molecularly control their differentiation, function, and maintenance. Two transcription factors known to be involved in these processes are Tbet and STAT4. Although Tbet has been shown to regulate CD8 T cell fate decisions and effector CD4 T cell choices, the contribution of STAT4 is less well understood. To address this, we examined the impact of STAT4 on T cell responses in the presence or absence of Tbet, following LCMV infection by using mice lacking Tbet, STAT4, or both transcription factors. STAT4 was not required for Tbet or Eomes expression; however, virus-specific effector CD8 T cells are skewed toward a memory-precursor phenotype in the absence of STAT4. This altered proportion of memory precursors did not result in an increase in memory CD8 T cells after the resolution of the infection. We also demonstrate that virus-specific effector and memory CD4 T cells formed independently of Tbet and STAT4, although a slight reduction in the number of antigen-specific CD4 T cells was apparent in mice lacking both transcription factors. Collectively, we have discovered distinct roles for Tbet and STAT4 in shaping the phenotype and function of virus-specific T cell responses.

IFNγ inhibits Th17 differentiation and function via Tbet-dependent and Tbet-independent mechanisms.

The transcription factor Tbet is critical for the differentiation of Th1 CD4 T cells and is associated with the induction of multiple autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE). Herein, we demonstrate that Tbet suppresses IL-17A and Th17 differentiation both in vitro and in vivo in a cell-intrinsic manner, and that in fact, Tbet is not necessary for EAE induction. Moreover, we find that IFNγ inhibits the production of IL-17A and IL-17F in a STAT1-dependent, Tbet-independent manner. These findings illustrate multiple mechanisms utilized by developing Th1 cells to silence the Th17 program.

Therapeutic efficacy of suppressing the Jak/STAT pathway in multiple models of experimental autoimmune encephalomyelitis.

Pathogenic Th cells and myeloid cells are involved in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The JAK/STAT pathway is used by numerous cytokines for signaling and is critical for development, regulation, and termination of immune responses. Dysregulation of the JAK/STAT pathway has pathological implications in autoimmune and neuroinflammatory diseases. Many of the cytokines involved in MS/EAE, including IL-6, IL-12, IL-23, IFN-γ, and GM-CSF, use the JAK/STAT pathway to induce biological responses. Thus, targeting JAKs has implications for treating autoimmune inflammation of the brain. We have used AZD1480, a JAK1/2 inhibitor, to investigate the therapeutic potential of inhibiting the JAK/STAT pathway in models of EAE. AZD1480 treatment inhibits disease severity in myelin oligodendrocyte glycoprotein-induced classical and atypical EAE models by preventing entry of immune cells into the brain, suppressing differentiation of Th1 and Th17 cells, deactivating myeloid cells, inhibiting STAT activation in the brain, and reducing expression of proinflammatory cytokines and chemokines. Treatment of SJL/J mice with AZD1480 delays disease onset of PLP-induced relapsing-remitting disease, reduces relapses and diminishes clinical severity. AZD1480 treatment was also effective in reducing ongoing paralysis induced by adoptive transfer of either pathogenic Th1 or Th17 cells. In vivo AZD1480 treatment impairs both the priming and expansion of T cells and attenuates Ag presentation functions of myeloid cells. Inhibition of the JAK/STAT pathway has clinical efficacy in multiple preclinical models of MS, suggesting the feasibility of the JAK/STAT pathway as a target for neuroinflammatory diseases.

Temporal requirements for B cells in the establishment of CD4 T cell memory.

CD4 T cell memory generation is shaped by a number of factors, including the strength and duration of TCR signaling, as well as the priming environment, all of which can be modified by B cells. Studies using B cell-deficient mice indicate B cells play a critical role in generating effector and memory CD4 T cells; however, when and how B cells are acting to promote these responses has not yet been ascertained. In this study, we use anti-CD20 Ab depletion of B cells at different times following Listeria monocytogenes infection to show that B cells are necessary for the induction of optimal CD4 T cell memory, but not for the transition and maintenance of this population. Importantly, the prerequisite of B cells early postinfection is partially dependent on their expression of MHC class II. B cells are not only required during the priming phase, but also necessary for the initiation of robust secondary responses by memory CD4 T cells. Interestingly, the requirement during the recall response is independent of B cell Ag presentation. Overall, these studies demonstrate the temporally and functionally distinct roles for B cells in regulating CD4 T cell responses.

Regulation of Th1 cells and experimental autoimmune encephalomyelitis by glycogen synthase kinase-3.

Experimental autoimmune encephalomyelitis (EAE) is a rodent model of multiple sclerosis (MS), a debilitating autoimmune disease of the CNS, for which only limited therapeutic interventions are available. Because MS is mediated in part by autoreactive T cells, particularly Th17 and Th1 cells, in the current study, we tested whether inhibitors of glycogen synthase kinase-3 (GSK3), previously reported to reduce Th17 cell generation, also alter Th1 cell production or alleviate EAE. GSK3 inhibitors were found to impede the production of Th1 cells by reducing STAT1 activation. Molecularly reducing the expression of either of the two GSK3 isoforms demonstrated that Th17 cell production was sensitive to reduced levels of GSK3ß and Th1 cell production was inhibited in GSK3α-deficient cells. Administration of the selective GSK3 inhibitors TDZD-8, VP2.51, VP0.7, or L803-mts significantly reduced the clinical symptoms of myelin oligodendrocyte glycoprotein35-55-induced EAE in mice, nearly eliminating the chronic progressive phase, and reduced the number of Th17 and Th1 cells in the spinal cord. Administration of TDZD-8 or L803-mts after the initial disease episode alleviated clinical symptoms in a relapsing-remitting model of proteolipid protein139-151-induced EAE. Furthermore, deletion of GSK3ß specifically in T cells was sufficient to alleviate myelin oligodendrocyte glycoprotein35-55-induced EAE. These results demonstrate the isoform-selective effects of GSK3 on T cell generation and the therapeutic effects of GSK3 inhibitors in EAE, as well as showing that GSK3 inhibition in T cells is sufficient to reduce the severity of EAE, suggesting that GSK3 may be a feasible target for developing new therapeutic interventions for MS.