A novel function for FOXP3 in humans: intrinsic regulation of conventional T cells.

The role of forkhead box P3 (FOXP3) is well-established in T-regulatory cells, but the function of transient FOXP3 expression in activated human conventional T (Tconv) cells is unknown. In the present study, we used 2 approaches to determine the role of FOXP3 in human Tconv cells. First, we obtained Tconv clones from a female subject who is hemizygous for a null mutation in FOXP3, allowing the comparison of autologous T-cell clones that do or do not express FOXP3. Second, we knocked down activation-induced FOXP3 in Tconv cells from healthy donors with small interfering RNAagainst FOXP3. We found that FOXP3-deficient Tconv cells proliferate more and produce more cytokines than wild-type Tconv cells and have differential expression of 274 genes. We also investigated the role of FOXP3 in Th1 and Th17 cells and found that the expression of activation-induced FOXP3 was higher and more sustained in Th17 cells compared with Th1 cells. Knocking down FOXP3 expression in Th17 cells significantly increased the production of IFN-γ and decreased the expression of CCR4, but had no effect on IL-17 expression. These data reveal a novel function of FOXP3 in Tconv cells and suggest that expression of this protein is important in the function of multiple CD4(+) T-cell lineages.

A Nationwide Study to Evaluate Accessibility to Specialized Pediatric Palliative Care in Italy-Patients, Needs, and Critical Issues: The PalliPed Study.

Background: PalliPed is the first Italian nationwide project aimed at describing the characteristics of patients accessing specialized pediatric palliative care (PPC) and their families, in the main care settings (hospice, home care, and hospital). The project's secondary aim is to assess the extent and quality of regional PPC networks/facilities and the number of dedicated resources. In this article, we present the results of the first part of the project. Methods: All Italian PPC centers/facilities were invited to participate in the project. Children and young adults in the care of the specialized PPC networks/structures as of 24 October, 2022, including prenatal care, were involved. Children's eligibility for specialized PPC was assessed according to the Assessment Form for Complex Clinical Needs in Pediatrics (ACCAPED Scale) and after a multidisciplinary assessment by the healthcare team. Data were collected through an online survey. Results: A total of 867 patients were described. The lack of adequate specialized PPC service emerged, according to the available estimate of specialized PPC needs, as well as the need for improved referral to PPC by pediatricians or territorial services, particularly for infants and oncological patients. More family support measures also seem necessary, particularly for the mothers. Healthcare providers' communication skills should be improved to ensure greater involvement of patients and families in care decisions. Conclusions: This analysis represents the first step toward defining a constantly updated database for the census and monitoring of specialized PPC activities at the national level. This research model can be extended to other realities in different countries, allowing comparison of different care models.

The specialized pediatric palliative care service in Italy: how is it working? Results of the nationwide PalliPed study.

BACKGROUND: Accurate estimation of the specialized pediatric palliative care (PPC) burden and the definition of the extent and quality of PPC service in Italy represent urgent needs to enable the proper allocation of PPC resources and the definition of prevention and educational plans. The PalliPed project aimed to provide the first comprehensive assessment of the characteristics of Italian patients requiring PPC, the quality and extent of regional PPC networks/facilities, and the number of dedicated resources. In this paper, we present the results of the second part of the project, regarding the implementation and quality of PPC services in Italy. METHODS: The PalliPed study had an observational cross-sectional design. All Italian specialized PPC centers/facilities were invited to participate in the project and complete a survey on the characteristics of PPC centers/facilities in different care settings, reporting data as of 24 October 2022. Data were collected online. RESULTS: 19 PPC specialized centers/facilities from 12 Italian regions and two autonomous provinces responded to the survey. Among them, 11 are regional referral centers. Seven Italian regions out of 20 reported no PPC centers/facilities, mainly in central-southern Italy. Less than half (45%) of the regional referral centers cover the entire regional territory, and three offer 24/7 service. Ten centers have a dedicated team. Half of the eight non-referral centers offer 24/7 service and have a dedicated team. A total of 1,092 patients were reported by 18 centers as of 24 October 2022. Over the years, an increasing number of patients has been reported, rising from 1,202 (2019) to 1,544 (2021). The dedicated staff is inadequate, and most healthcare providers are not recognized at an institutional level. A shortage of 'young' staff and a lack of specific training was reported, particularly among nurses (77% had no training in PPC). CONCLUSIONS: The results obtained show how training, information, and research interventions are still necessary for the reorganization of the available resources and definition of proper strategies to respond dynamically to the new emerging needs of these populations. At the same time, our study represents a first step in defining a national registry of PPC models, useful for monitoring evolutions, and critical issues and planning any new or corrective strategy.

Reply to Lissing et al. Comment on "Ramai et al. Risk of Hepatocellular Carcinoma in Patients with Porphyria: A Systematic Review. Cancers 2022, 14, 2947".

We thank Dr. Lissing and colleagues for providing us with these helpful comments [...].

Functional type 1 regulatory T cells develop regardless of FOXP3 mutations in patients with IPEX syndrome.

Mutations of forkhead box p3 (FOXP3), the master gene for naturally occurring regulatory T cells (nTregs), are responsible for the impaired function of nTregs, resulting in an autoimmune disease known as the immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. The relevance of other peripheral tolerance mechanisms, such as the presence and function of type 1 regulatory T (Tr1) cells, the major adaptive IL-10-producing Treg subset, in patients with IPEX syndrome remains to be clarified. FOXP3(mutated) Tr1-polarized cells, differentiated in vitro from CD4(+) T cells of four IPEX patients, were enriched in IL-10(+) IL-4(-) IFN-γ(+) T cells, a cytokine production profile specific for Tr1 cells, and expressed low levels of FOXP3 and high levels of Granzyme-B. IPEX Tr1 cells were hypoproliferative and suppressive, thus indicating that FOXP3 mutations did not impair their function. Furthermore, we isolated Tr1 cell clones from the peripheral blood of one FOXP3(null) patient, demonstrating that Tr1 cells are present in vivo and they can be expanded in vitro in the absence of WT FOXP3. Overall, our results (i) show that functional Tr1 cells differentiate independently of FOXP3, (ii) confirm that human Tr1 and nTregs are distinct T-cell lineages, and (iii) suggest that under favorable conditions Tr1 cells could exert regulatory functions in IPEX patients.

Risk of Hepatocellular Carcinoma in Patients with Porphyria: A Systematic Review.

Acute porphyrias are a group of metabolic disorders resulting in defective porphyrin synthesis and reduced heme production, which carries a risk of malignancy. Porphyrias are inborn defects in the heme biosynthesis pathway resulting in neurovisceral manifestations and cutaneous photosensitivity attacks with multi-systemic involvement. Its estimated prevalence nears 5 per 100,000 patients worldwide. Subclinical liver disease is common, which can progress into transaminitis, fibrosis, cirrhosis, and malignancy. However, data on the incidence of primary liver cancer are lacking. We aim to determine the risk of hepatocellular carcinoma (HCC) in patients with porphyria. A systematic review and pooled analysis were conducted through 2021 on studies assessing blood tests, imaging, cancer development, liver transplant, surgical resection, and outcomes in porphyria. In total, 19 studies, which included 7381 patients with porphyria (3476 females), were considered for the final review. In eight studies, alpha-fetoprotein levels were elevated between 200 and 1000 IU/mL. Of the total cohort of patients with porphyria, primary liver cancer was diagnosed in 351 patients (4.8%), of whom 243 (3.3% of the total) were found to have HCC. A subset of patients was found to have cholangiocarcinoma (n = 18; 0.3% of the total). Interestingly, advanced liver disease or cirrhosis was not a prerequisite for the formation of HCC in a small group of patients. Of the total cohort, 30 patients underwent liver resection, 48 patients underwent liver transplantation, and 327 patients died. Patients with porphyria are at risk of developing primary liver malignancy. Further studies should aim to develop diagnostic and prognostic models aimed at the early detection of HCC in porphyria.

Wild-type FOXP3 is selectively active in CD4+CD25(hi) regulatory T cells of healthy female carriers of different FOXP3 mutations.

Forkhead box P3 (FOXP3) is constitutively expressed by CD4(+)CD25(hi) regulatory T cells (nTregs). Mutations of FOXP3 cause a severe autoimmune syndrome known as immune dysregulation polyendocrinopathy enteropathy X-linked, in which nTregs are absent or dysfunctional. Whether FOXP3 is essential for both differentiation and function of human nTreg cells remains to be demonstrated. Because FOXP3 is an X-linked gene subject to X-chromosome inactivation (XCI), we studied 9 healthy female carriers of FOXP3 mutations to investigate the role of wild-type (WT) versus mutated FOXP3 in different cell subsets. Analysis of active WT versus mutated (mut)-FOXP3 allele distribution revealed a random pattern of XCI in peripheral blood lymphocytes and in naive and memory CD4(+)T cells, whereas nTregs expressed only the active WT-FOXP3. These data demonstrate that expression of WT-FOXP3 is indispensable for the presence of a normal nTreg compartment and suggest that FOXP3 is not necessary for effector T-cell differentiation in humans.

Progressive Liver Fibrosis in Non-Alcoholic Fatty Liver Disease.

Non-alcoholic steatohepatitis (NASH) is a chronic and progressive form of non-alcoholic fatty liver disease. Its global incidence is increasing and makes NASH an epidemic and a public health threat. Non-alcoholic fatty liver disease is associated with major morbidity and mortality, with a heavy burden on quality of life and liver transplant requirements. Due to repeated insults to the liver, patients are at risk for developing hepatocellular carcinoma. The progression of NASH was initially defined according to a two-hit model involving an initial development of steatosis, followed by a process of lipid peroxidation and inflammation. In contrast, current evidence proposes a "multi-hit" or "multi-parallel hit" model that includes multiple pathways promoting progressive fibrosis and oncogenesis. This model includes multiple cellular, genetic, immunological, metabolic, and endocrine pathways leading to hepatocellular carcinoma development, underscoring the complexity of this disease.

Hospital Frailty Risk Score Is Independently Associated with Mortality and Encephalopathy in Hospitalized Patients with Hepatocellular Carcinoma.

Frailty represents a state of vulnerability to multiple internal physiologic factors, as well as external pressures, and has been associated with clinical outcomes. We aim to understand the impact of frailty on patients admitted with hepatocellular carcinoma (HCC) by using the validated Hospital Frailty Risk Score, which is implemented in several hospitals worldwide. We conducted a nation-wide retrospective cohort study to determine the effect of frailty on the risk of in-patient mortality, hepatic encephalopathy, length of stay and cost. Frailty was associated with a 4.5-fold increased risk of mortality and a 2.3-fold increased risk of hepatic encephalopathy. Adjusted Cox regression showed that frailty was correlated with increased risk of in-patient mortality (hazard ratio: 2.3, 95% CI 1.9-2.8, p < 0.001). Frail HCC patients had longer hospital stay (median 5 days) vs. non-frail HCC patients (median 3 days). Additionally, frail patients had higher total costs of hospitalization ($40,875) compared with non-frail patients ($31,667). Frailty is an independent predictor of hepatic encephalopathy and in-patient mortality. Frailty is a surrogate marker of hospital length of stay and cost.

STAT5-signaling cytokines regulate the expression of FOXP3 in CD4+CD25+ regulatory T cells and CD4+CD25- effector T cells.

Forkhead box P3 (FOXP3) is considered a specific marker for CD4(+)CD25(+) regulatory T (Treg) cells, but increasing evidence suggests that human CD4(+)CD25(-) effector T (Teff) cells can transiently express FOXP3 upon activation. We demonstrate that the signal transducer and activator of transcription 5 (STAT5)-signaling cytokines, IL-2, IL-15 and to a lesser extent IL-7, induce FOXP3 up-regulation in vitro in activated human Teff cells. In contrast, cytokines which do not activate STAT5, such as IL-4 or transforming growth factor-beta alone, do not directly induce FOXP3 expression in activated Teff cells. Moreover, expression of a constitutively active form of STAT5a is sufficient to induce FOXP3 expression in Teff cells. Expression of FOXP3 in activated Teff cells requires both TCR-mediated activation and endogenous IL-2, but is not dependent on cell division and does not induce suppressive function. The presence of STAT5-activating cytokines is also required to maintain high FOXP3 expression and suppressive activity of Treg cells in vitro. These data indicate that activation of STAT5 sustains FOXP3 expression in both Treg and Teff cells and contribute to our understanding of how cytokines affect the expression of FOXP3.

Molecular regulation of cellular immunity by FOXP3.

The immune system is responsible for not only eliminating threats to the body, but also for protecting the body from its own immune responses that would cause harm if left unchecked. Forkhead box protein 3 (FOXP3) is a forkhead family member with an important role in the development and function of a type of CD4+ T cell called T regulatory cells that is fundamental for maintaining immune tolerance to self. This chapter reviews the structure of FOXP3 and how its role in the immune system was discovered. Studies of patients with mutations in FOXP3 who suffer from a syndrome known as IPEX (immune dysregulation, polyendocrinopathy, enteropathy, x-linked) are also discussed. Investigation into how expression of FOXP3 is regulated and how it interacts with other proteins have recently provided considerable insight into mechanisms by which the lack of this protein could cause disease. We also discuss how FOXP3 is involved in the reciprocal development ofT regulatory cells and proinflammatory T-cells that produce IL-17. A better understanding of how FOXP3 is regulated and the molecular basis for its function will ultimately contribute to the development ofT regulatory cell-based cellular therapies that could be used to restore dysregulated immune responses.

Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative, but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and reinfused the cells after a reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical scores. Vector integration analyses revealed highly polyclonal and multilineage haematopoiesis resulting from the gene-corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes, and no aberrant clonal expansion was observed after 20 to 32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS.