Phase Separation of a PKA Regulatory Subunit Controls cAMP Compartmentation and Oncogenic Signaling.

The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.

First-line regorafenib with nivolumab and chemotherapy in advanced oesophageal, gastric, or gastro-oesophageal junction cancer in the USA: a single-arm, single-centre, phase 2 trial.

BACKGROUND: The addition of nivolumab to chemotherapy improves survival in patients with advanced oesophagogastric (oesophageal, gastric, or gastro-oesophageal junction) adenocarcinoma; however, outcomes remain poor. We assessed the safety and activity of regorafenib in combination with nivolumab and chemotherapy in the first-line treatment of advanced oesophagogastric adenocarcinoma. METHODS: This investigator-initiated, single-arm, phase 2 trial in adult patients (aged ≥18 years) with previously untreated, HER2-negative, metastatic oesophagogastric adenocarcinoma was done at the Memorial Sloan Kettering Cancer Center (New York, NY, USA). Eligible patients had measurable disease or non-measurable disease that was evaluable (defined by Response Evaluation Criteria in Solid Tumours [RECIST] version 1.1) and Eastern Cooperative Oncology Group performance status of 0 or 1. Patients received FOLFOX chemotherapy (fluorouracil [400 mg/m2 bolus followed by 2400 mg/m2 over 48 h], leucovorin [400 mg/m2], and oxaliplatin [85 mg/m2]) and nivolumab (240 mg) intravenously on days 1 and 15, and oral regorafenib (80 mg) on days 1-21 of a 28-day cycle. Treatment was continued until disease progression (defined by RECIST version 1.1), unacceptable toxicity, or withdrawal of consent. The primary endpoint was 6-month progression-free survival in the per-protocol population (ie, all participants who received a dose of all study treatments). The regimen would be considered worthy of further investigation if at least 24 of 35 patients were progression free at 6 months. Safety was assessed in all participants who received at least one dose of any study treatment. This trial is registered with ClinicalTrials.gov, NCT04757363, and is now complete. FINDINGS: Between Feb 11, 2021, and May 4, 2022, 39 patients were enrolled, received at least one dose of study drug, and were included in safety analyses. 35 patients were evaluable for 6-month progression-free survival. Median age was 57 years (IQR 52-66), nine (26%) patients were women, 26 (74%) were men, 28 (80%) were White, and seven (20%) were Asian. At data cutoff (March 3, 2023), median follow-up was 18·1 months (IQR 12·7-20·4). The primary endpoint was reached, with 25 (71%; 95% CI 54-85) of 35 patients progression free at 6 months. Nine (26%) of 35 patients had disease progression and one (3%) patient died; the death was unrelated to treatment. The most common adverse event of any grade was fatigue (36 [92%] of 39). The most common grade 3 or 4 adverse events were decreased neutrophil count (18 [46%]), hypertension (six [15%]), dry skin, pruritus, or rash (five [13%]), and anaemia (four [10%]). Serious treatment-related adverse events occurred in ten (26%) patients, which were acute kidney injury (three [8%]), hepatotoxicity (two [5%]), sepsis (two [5%]), dry skin, pruritus, or rash (one [3%]), nausea (one [3%]), and gastric perforation (one [3%]). There were no treatment-related deaths. INTERPRETATION: Regorafenib can be safely combined with nivolumab and chemotherapy and showed promising activity in HER2-negative metastatic oesophagogastric cancer. A randomised, phase 3 clinical trial is planned. FUNDING: Bristol Myers Squibb, Bayer and National Institutes of Health/National Cancer Institute.

miR-99b-5p, miR-380-3p, and miR-485-3p are novel chemosensitizing miRNAs in high-risk neuroblastoma.

Neuroblastoma is a deadly childhood cancer arising in the developing sympathetic nervous system. High-risk patients are currently treated with intensive chemotherapy, which is curative in only 50% of children and leaves some surviving patients with life-long side effects. microRNAs (miRNAs) are critical regulators of neural crest development and are deregulated during neuroblastoma tumorigenesis, making miRNA-based drugs an attractive therapeutic avenue. A functional screen of >1,200 miRNA mimics was conducted in neuroblastoma cell lines to discover miRNAs that sensitized cells to low doses (30% inhibitory concentration [IC30]) of doxorubicin and vincristine chemotherapy used in the treatment of the disease. Three miRNAs, miR-99b-5p, miR-380-3p, and miR-485-3p, had potent chemosensitizing activity with doxorubicin in multiple models of high-risk neuroblastoma. These miRNAs underwent genomic loss in a subset of neuroblastoma patients, and low expression predicted poor survival outcome. In vitro functional assays revealed each of these miRNAs enhanced the anti-proliferative and pro-apoptotic effects of doxorubicin. We used RNA sequencing (RNA-seq) to show that miR-99b-5p represses neuroblastoma dependency genes LIN28B and PHOX2B both in vitro and in patient-derived xenograft (PDX) tumors. Luciferase reporter assays demonstrate that PHOX2B is a direct target of miR-99b-5p. We anticipate that restoring the function of the tumor-suppressive miRNAs discovered here may be a valuable therapeutic strategy for the treatment of neuroblastoma patients.

Postpartum psychosis: a public involvement perspective across three continents.

Postpartum psychosis is a psychiatric emergency that is currently not represented in diagnostic systems, to the detriment of people with lived experience. Engaging with stakeholders offers an important avenue to improve clinical practice and make research more impactful, by providing perspectives based on first-hand, expert experience. There is a paucity of reports on stakeholders' engagement in psychiatry. Activities have thus far been limited to Western countries and there are few reports on postpartum psychosis. We report the results of public involvement activities (in the form of discussion groups) with key stakeholders in India, Malawi and the UK. These discussions centred around the clinical picture of postpartum psychosis and the terminologies used to describe these episodes. Seven major areas were highlighted: how postpartum psychosis is handled within services, common symptoms and characteristics, impact of episode, barriers to care, non-medical approaches, terminology and research areas of interest. According to the discussions, postpartum psychosis presents similarly across countries, although there are differences in access to services, approaches to mental health and terminologies used within and across countries. With this understanding comes the foundation for cross-cultural assessment, service improvement and a stakeholder-informed research agenda.

Noncontrast MRI for Hepatocellular Carcinoma Detection: A Systematic Review and Meta-analysis - A Potential Surveillance Tool?

BACKGROUND AND AIMS: This meta-analysis investigates the diagnostic performance of non-contrast magnetic resonance imaging (MRI) for the detection of hepatocellular carcinoma (HCC). METHODS: A systematic review was performed to May 2020 for studies which examined the diagnostic performance of non-contrast MRI (multi-sequence or diffusion-weighted imaging (DWI)- alone) for HCC detection in high risk patients. The primary outcome was accuracy for the detection of HCC. Random effects models were used to pool outcomes for sensitivity, specificity, positive likelihood ratio (LR) and negative LR. Subgroup analyses for cirrhosis and size of the lesion were performed. RESULTS: Twenty-two studies were included involving 1685 patients for per-patient analysis and 2128 lesions for per-lesion analysis. Multi-sequence non-contrast MRI (NC-MRI) using T2+DWI±T1 sequences had a pooled per-patient sensitivity of 86.8% (95%CI:83.9-89.4%), specificity of 90.3% (95%CI:87.3-92.7%), and negative LR of 0.17 (95%CI:0.14-0.20). DWI-only MRI (DW-MRI) had a pooled sensitivity of 79.2% (95%CI:71.8-85.4%), specificity of 96.5% (95%CI:94.3-98.1%) and negative LR of 0.24 (95%CI:1.62-0.34). In patients with cirrhosis, NC-MRI had a pooled per-patient sensitivity of 87.3% (95%CI:82.7-91.0%) and specificity of 81.6% (95%CI:75.3-86.8%), whilst DWI-MRI had a pooled sensitivity of 71.4% (95%CI:60.5-80.8%) and specificity of 97.1% (95%CI:91.9-99.4%). For lesions <2 cm, the pooled per-lesion sensitivity was 77.1% (95%CI:73.8-80.2%). For lesions >2 cm, pooled per-lesion sensitivity was 88.5% (95%CI:85.0-91.5%). CONCLUSION: Non-contrast MRI has a moderate negative LR and high specificity with acceptable sensitivity for the detection of HCC, even in patients with cirrhosis and with lesions <2 cm. Prospective trials to validate if non-contrast MRI can be used for HCC surveillance is warranted.

Environmental enrichment rescues survival and function of adult-born neurons following early life stress.

Adverse experiences early in life are associated with the development of psychiatric illnesses. The hippocampus is likely to play pivotal role in generating these effects: it undergoes significant development during childhood and is extremely reactive to stress. In rodent models, stress in the pre-pubertal period impairs adult hippocampal neurogenesis (AHN) and behaviours which rely on this process. In normal adult animals, environmental enrichment (EE) is a potent promoter of AHN and hippocampal function. Whether exposure to EE during adolescence can restore normal hippocampal function and AHN following pre-pubertal stress (PPS) is unknown. We investigated EE as a treatment for reduced AHN and hippocampal function following PPS in a rodent model. Stress was administered between post-natal days (PND) 25-27, EE from PND 35 to early adulthood, when behavioural testing and assessment of AHN took place. PPS enhanced fear reactions to a conditioned stimulus (CS) following a trace fear protocol and reduced the survival of 4-week-old adult-born neurons throughout the adult hippocampus. Furthermore, we show that fewer adult-born neurons were active during recall of the CS stimulus following PPS. All effects were reversed by EE. Our results demonstrate lasting effects of PPS on the hippocampus and highlight the utility of EE during adolescence for restoring normal hippocampal function. EE during adolescence is a promising method of enhancing impaired hippocampal function resulting from early life stress, and due to multiple benefits (low cost, few side effects, widespread availability) should be more thoroughly explored as a treatment option in human sufferers of childhood adversity.

Evaluation of FGFR targeting in breast cancer through interrogation of patient-derived models.

BACKGROUND: Particular breast cancer subtypes pose a clinical challenge due to limited targeted therapeutic options and/or poor responses to the existing targeted therapies. While cell lines provide useful pre-clinical models, patient-derived xenografts (PDX) and organoids (PDO) provide significant advantages, including maintenance of genetic and phenotypic heterogeneity, 3D architecture and for PDX, tumor-stroma interactions. In this study, we applied an integrated multi-omic approach across panels of breast cancer PDXs and PDOs in order to identify candidate therapeutic targets, with a major focus on specific FGFRs. METHODS: MS-based phosphoproteomics, RNAseq, WES and Western blotting were used to characterize aberrantly activated protein kinases and effects of specific FGFR inhibitors. PDX and PDO were treated with the selective tyrosine kinase inhibitors AZD4547 (FGFR1-3) and BLU9931 (FGFR4). FGFR4 expression in cancer tissue samples and PDOs was assessed by immunohistochemistry. METABRIC and TCGA datasets were interrogated to identify specific FGFR alterations and their association with breast cancer subtype and patient survival. RESULTS: Phosphoproteomic profiling across 18 triple-negative breast cancers (TNBC) and 1 luminal B PDX revealed considerable heterogeneity in kinase activation, but 1/3 of PDX exhibited enhanced phosphorylation of FGFR1, FGFR2 or FGFR4. One TNBC PDX with high FGFR2 activation was exquisitely sensitive to AZD4547. Integrated 'omic analysis revealed a novel FGFR2-SKI fusion that comprised the majority of FGFR2 joined to the C-terminal region of SKI containing the coiled-coil domains. High FGFR4 phosphorylation characterized a luminal B PDX model and treatment with BLU9931 significantly decreased tumor growth. Phosphoproteomic and transcriptomic analyses confirmed on-target action of the two anti-FGFR drugs and also revealed novel effects on the spliceosome, metabolism and extracellular matrix (AZD4547) and RIG-I-like and NOD-like receptor signaling (BLU9931). Interrogation of public datasets revealed FGFR2 amplification, fusion or mutation in TNBC and other breast cancer subtypes, while FGFR4 overexpression and amplification occurred in all breast cancer subtypes and were associated with poor prognosis. Characterization of a PDO panel identified a luminal A PDO with high FGFR4 expression that was sensitive to BLU9931 treatment, further highlighting FGFR4 as a potential therapeutic target. CONCLUSIONS: This work highlights how patient-derived models of human breast cancer provide powerful platforms for therapeutic target identification and analysis of drug action, and also the potential of specific FGFRs, including FGFR4, as targets for precision treatment.

High prevalence of asymptomatic internal hernias after laparoscopic anterior resection in a retrospective analysis of postoperative computed tomography.

PURPOSE: Internal hernia (IH) after laparoscopic left-sided colorectal resection (small bowel herniating underneath the neo-descending colon) can be a potentially devastating complication, resulting in acute small bowel obstruction or ischemia. IH has been described as a rare occurrence in a few retrospective case series; however, patients undergoing laparoscopic resection seem to be more prone to this complication. We assessed the prevalence of IH in a large cohort of patients who had undergone laparoscopic left-sided colorectal resection for colon or rectal cancer (CRC). METHODS: A database of consecutive patients at a single institution from 2012 to 2017 was reviewed. Postoperative abdominal computed tomography (CT) scans performed for routine cancer follow-up between 3 and 36 months after surgery were assessed retrospectively. RESULTS: During the study period, 276 patients had undergone anterior resection for CRC, with 206 (75%) having been performed laparoscopically. A total of 198 eligible patients were identified, and a follow-up CT scan was available in 105 (53%) of these patients (median time to CT 10 months, range 3-34). Only one of the 198 (0.5%) patients presented with an acute small bowel obstruction secondary to an IH during follow-up. However, the prevalence of asymptomatic IH was noted to be much higher in the postoperative CT scans occurring in 22 of 105 (21%) patients. CONCLUSION: Asymptomatic IH after laparoscopic left-sided colorectal resection is common. Given the potential risk of acute small bowel obstruction and ischemia, routine closure of the mesenteric defect should be considered.

Novel Targets for the Treatment of Melanoma.

PURPOSE OF REVIEW: In this article, we will briefly review the current treatment landscape for metastatic melanoma and provide a comprehensive update on emerging novel treatment strategies. RECENT FINDINGS: Over the past decade, remarkable advances in immunotherapy and targeted therapy have greatly improved outcomes for patients with advanced melanoma. Although a subset of patients is able to achieve durable responses, the majority experience eventual disease progression on existing therapies. Trials evaluating novel combinatorial strategies, checkpoint inhibitors, immune agonists, T cell-based therapies, intratumoral agents, and others are ongoing. While strides have been made in the treatment of advanced melanoma, further research is needed to identify alternative immune and molecular targets in order to overcome resistance and achieve better clinical outcomes.

Cutting Edge: Adenosine A2a Receptor Signals Inhibit Germinal Center T Follicular Helper Cell Differentiation during the Primary Response to Vaccination.

Adenosine A2a receptor (A2aR) signaling acts as a barrier to autoimmunity by promoting anergy, inducing regulatory T cells, and inhibiting effector T cells. However, in vivo effects of A2aR signaling on polyclonal CD4 T cells during a primary response to foreign Ag has yet to be determined. To address this problem, we immunized mice with peptide Ag 2W1S coupled to PE in CFA and treated with the selective A2aR agonist CGS-21680 (CGS). 2W1S:I-Ab-specific tetramer-binding CD4 T cells did not become anergic or differentiate into Foxp3+ regulatory T cells. Additionally, CGS treatment did not inhibit Th1 or Th17 differentiation. However, CGS did abrogate germinal center T follicular helper cells, and blunted PE-specific germinal center B cell responses. The use of A2aR-deficient CD4 T cells established that this CGS effect was T cell intrinsic. Therefore, this study has identified a unique role for A2aRs in regulating CD4 T cell differentiation during vaccination.

Oral and upper gastrointestinal Crohn's disease.

Crohn's disease is a heterogeneous, inflammatory condition that can affect any location of the gastrointestinal tract. Proximal gastrointestinal involvement occurs in 0.5-16% of patients, and it is usually diagnosed after recognition of intestinal disease. Symptoms are often mild and nonspecific; however, upper gastrointestinal disease predicts a more severe Crohn's phenotype with a greater frequency of complications such as obstruction and perforation. Gastroscopy and biopsy is the most sensitive diagnostic investigation. There is a paucity of data examining the treatment of this condition. Management principles are similar to those for intestinal disease, commencing with topical therapy where appropriate, progressing to systemic therapy such as glucocorticoids, 5-aminosalicylic acid, immunomodulators, and biologics. Acid suppression therapy has symptomatic but no anti-inflammatory benefit for gastroduodenal and esophageal involvement. Surgical intervention with bypass, strictureplasty, or less frequently, endoscopic balloon dilation may be required for complications or failed medical therapy.

Endogenous circadian system and circadian misalignment impact glucose tolerance via separate mechanisms in humans.

Glucose tolerance is lower in the evening and at night than in the morning. However, the relative contribution of the circadian system vs. the behavioral cycle (including the sleep/wake and fasting/feeding cycles) is unclear. Furthermore, although shift work is a diabetes risk factor, the separate impact on glucose tolerance of the behavioral cycle, circadian phase, and circadian disruption (i.e., misalignment between the central circadian pacemaker and the behavioral cycle) has not been systematically studied. Here we show--by using two 8-d laboratory protocols--in healthy adults that the circadian system and circadian misalignment have distinct influences on glucose tolerance, both separate from the behavioral cycle. First, postprandial glucose was 17% higher (i.e., lower glucose tolerance) in the biological evening (8:00 PM) than morning (8:00 AM; i.e., a circadian phase effect), independent of the behavioral cycle effect. Second, circadian misalignment itself (12-h behavioral cycle inversion) increased postprandial glucose by 6%. Third, these variations in glucose tolerance appeared to be explained, at least in part, by different mechanisms: during the biological evening by decreased pancreatic ß-cell function (27% lower early-phase insulin) and during circadian misalignment presumably by decreased insulin sensitivity (elevated postprandial glucose despite 14% higher late-phase insulin) without change in early-phase insulin. We explored possible contributing factors, including changes in polysomnographic sleep and 24-h hormonal profiles. We demonstrate that the circadian system importantly contributes to the reduced glucose tolerance observed in the evening compared with the morning. Separately, circadian misalignment reduces glucose tolerance, providing a mechanism to help explain the increased diabetes risk in shift workers.

Cutting edge: Bcl6-interacting corepressor contributes to germinal center T follicular helper cell formation and B cell helper function.

CD4(+) germinal center (GC)-T follicular helper (Tfh) cells help B cells become long-lived plasma cells and memory cells. The transcriptional repressor Bcl6 plays a key role in GC-Tfh formation by inhibiting the expression of genes that promote differentiation into other lineages. We determined whether BCOR, a component of a Polycomb repressive complex that interacts with the Bcl6 BTB domain, influences GC-Tfh differentiation. T cell-targeted BCOR deficiency led to a substantial loss of peptide:MHC class II-specific GC-Tfh cells following Listeria monocytogenes infection and a 2-fold decrease following immunization with a peptide in CFA. The reduction in GC-Tfh cells was associated with diminished plasma cell and GC B cell formation. Thus, T cell-expressed BCOR is critical for optimal GC-Tfh cell differentiation and humoral immunity.

Endostatin's emerging roles in angiogenesis, lymphangiogenesis, disease, and clinical applications.

BACKGROUND: Angiogenesis is the process of neovascularization from pre-existing vasculature and is involved in various physiological and pathological processes. Inhibitors of angiogenesis, administered either as individual drugs or in combination with other chemotherapy, have been shown to benefit patients with various cancers. Endostatin, a 20-kDa C-terminal fragment of type XVIII collagen, is one of the most potent inhibitors of angiogenesis. SCOPE OF REVIEW: We discuss the biology behind endostatin in the context of its endogenous production, the various receptors to which it binds, and the mechanisms by which it acts. We focus on its inhibitory role in angiogenesis, lymphangiogenesis, and cancer metastasis. We also present emerging clinical applications for endostatin and its potential as a therapeutic agent in the form a short peptide. MAJOR CONCLUSIONS: The delicate balance between pro- and anti-angiogenic factors can be modulated to result in physiological wound healing or pathological tumor metastasis. Research in the last decade has emphasized an emerging clinical potential for endostatin as a biomarker and as a therapeutic short peptide. Moreover, elevated or depressed endostatin levels in diseased states may help explain the pathophysiological mechanisms of the particular disease. GENERAL SIGNIFICANCE: Endostatin was once sought after as the 'be all and end all' for cancer treatment; however, research throughout the last decade has made it apparent that endostatin's effects are complex and involve multiple mechanisms. A better understanding of newly discovered mechanisms and clinical applications still has the potential to lead to future advances in the use of endostatin in the clinic.

Clinical Management of Uveal and Conjunctival Melanoma.

Ocular melanoma is a rare but potentially devastating malignancy arising from the melanocytes of the uveal tract, conjunctiva, or orbit; it represents less than 5% of all melanoma cases in the United States. The management of ocular melanoma varies depending on its anatomic origin, since uveal and conjunctival melanoma have distinct biologies and thus different treatment strategies. Uveal melanoma is the most common type of ocular melanoma and is characterized by activation of the mitogen-activated protein kinase (MAPK) pathway (among other signaling pathways) via mutations in GNAQ or GNA11. Despite primary radiation or surgical therapy, up to 50% of patients will eventually develop metastatic disease, for which there is no standard therapy and no treatment that has been shown to improve overall survival. The biology of conjunctival melanoma is less well characterized but has been associated with BRAF and NRAS mutations, and results in metastatic disease in 20% to 30% of cases. Clinical trials are currently ongoing to further evaluate and optimize the role of targeted therapies, as well as immunotherapies, as both adjuvant and metastatic treatment in uveal and conjunctival melanoma.

MicroRNA profiling of the pubertal mouse mammary gland identifies miR-184 as a candidate breast tumour suppressor gene.

INTRODUCTION: The study of mammalian development has offered many insights into the molecular aetiology of cancer. We previously used analysis of mammary morphogenesis to discover a critical role for GATA-3 in mammary developmental and carcinogenesis. In recent years an important role for microRNAs (miRNAs) in a myriad of cellular processes in development and in oncogenesis has emerged. METHODS: microRNA profiling was conducted on stromal and epithelial cellular subsets microdissected from the pubertal mouse mammary gland. miR-184 was reactivated by transient or stable overexpression in breast cancer cell lines and examined using a series of in vitro (proliferation, tumour-sphere and protein synthesis) assays. Orthotopic xenografts of breast cancer cells were used to assess the effect of miR-184 on tumourigenesis as well as distant metastasis. Interactions between miR-184 and its putative targets were assessed by quantitative PCR, microarray, bioinformatics and 3' untranslated region Luciferase reporter assay. The methylation status of primary patient samples was determined by MBD-Cap sequencing. Lastly, the clinical prognostic significance of miR-184 putative targets was assessed using publicly available datasets. RESULTS: A large number of microRNA were restricted in their expression to specific tissue subsets. MicroRNA-184 (miR-184) was exclusively expressed in epithelial cells and markedly upregulated during differentiation of the proliferative, invasive cells of the pubertal terminal end bud (TEB) into ductal epithelial cells in vivo. miR-184 expression was silenced in mouse tumour models compared to non-transformed epithelium and in a majority of breast cancer cell line models. Ectopic reactivation of miR-184 inhibited the proliferation and self-renewal of triple negative breast cancer (TNBC) cell lines in vitro and delayed primary tumour formation and reduced metastatic burden in vivo. Gene expression studies uncovered multi-factorial regulation of genes in the AKT/mTORC1 pathway by miR-184. In clinical breast cancer tissues, expression of miR-184 is lost in primary TNBCs while the miR-184 promoter is methylated in a subset of lymph node metastases from TNBC patients. CONCLUSIONS: These studies elucidate a new layer of regulation in the PI3K/AKT/mTOR pathway with relevance to mammary development and tumour progression and identify miR-184 as a putative breast tumour suppressor.

Cutting edge: STAT1 is required for IL-6-mediated Bcl6 induction for early follicular helper cell differentiation.

Bcl6 is required for CD4 T cell differentiation into T follicular helper cells (Tfh). In this study, we examined the role of IL-6 in early processes of in vivo Tfh differentiation, because the timing and mechanism of action of IL-6 in Tfh differentiation have been controversial in vivo. We found that early Bcl6(+)CXCR5(+) Tfh differentiation was severely impaired in the absence of IL-6; however, STAT3 deficiency failed to recapitulate that defect. IL-6R signaling activates the transcription factor STAT1 specifically in CD4 T cells. Strikingly, we found that STAT1 activity was required for Bcl6 induction and early Tfh differentiation in vivo. IL-6 mediated STAT3 activation is important for downregulation of IL-2Rα to limit Th1 cell differentiation in an acute viral infection. Thus, IL-6 signaling is a major early inducer of the Tfh differentiation program unexpectedly mediated by both STAT3 and STAT1 transcription factors.

Bcl6 expressing follicular helper CD4 T cells are fate committed early and have the capacity to form memory.

Follicular helper CD4 T (Tfh) cells are a distinct type of differentiated CD4 T cells uniquely specialized for B cell help. In this study, we examined Tfh cell fate commitment, including distinguishing features of Tfh versus Th1 proliferation and survival. Using cell transfer approaches at early time points after an acute viral infection, we demonstrate that early Tfh cells and Th1 cells are already strongly cell fate committed by day 3. Nevertheless, Tfh cell proliferation was tightly regulated in a TCR-dependent manner. The Tfh cells still depend on extrinsic cell fate cues from B cells in their physiological in vivo environment. Unexpectedly, we found that Tfh cells share a number of phenotypic parallels with memory precursor CD8 T cells, including selective upregulation of IL-7Rα and a collection of coregulated genes. As a consequence, the early Tfh cells can progress to robustly form memory cells. These data support the hypothesis that CD4 and CD8 T cells share core aspects of a memory cell precursor gene expression program involving Bcl6, and a strong relationship exists between Tfh cells and memory CD4 T cell development.