Tricia Bhatti

Foxp3 Reprograms T Cell Metabolism to Function in Low-Glucose, High-Lactate Environments

Immune cells function in diverse metabolic environments. Tissues with low glucose and high lactate concentrations, such as the intestinal tract or ischemic tissues, frequently require immune responses to bexa0more pro-tolerant, avoiding unwanted reactions against self-antigens or commensal bacteria. T-regulatory cells (Tregs) maintain peripheral tolerance, but how Tregs function in low-glucose, lactate-rich environments is unknown. We report that the Treg transcription factor Foxp3 reprograms Txa0cell metabolism by suppressing Myc and glycolysis, enhancing oxidative phosphorylation, and increasing nicotinamide adenine dinucleotide oxidation. These adaptations allow Tregs a metabolic advantage in low-glucose, lactate-rich environments; they resist lactate-mediated suppression of Txa0cell function and proliferation. This metabolic phenotype may explain how Tregs promote peripheral immune tolerance during tissue injury but also how cancer cells evade immune destruction in the tumor microenvironment. Understanding Treg metabolism may therefore lead to novel approaches for selective immune modulation in cancer and autoimmune diseases.

Identification of GPC2 as an Oncoprotein and Candidate Immunotherapeutic Target in High-Risk Neuroblastoma

We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target.

Phosphorylation of pRb: mechanism for RB pathway inactivation in MYCN-amplified retinoblastoma

A small, but unique subgroup of retinoblastoma has been identified with no detectable mutation in the retinoblastoma gene (RB1) and with high levels of MYCN gene amplification. This manuscript investigated alternate pathways of inactivating pRb, the encoded protein in these tumors. We analyzed the mutation status of the RB1 gene and MYCN copy number in a series of 245 unilateral retinoblastomas, and the phosphorylation status of pRb in a subset of five tumors using immunohistochemistry. There were 203 tumors with two mutations in RB1 (RB1−/−, 83%), 29 with one (RB1+/−, 12%) and 13 with no detectable mutations (RB1+/+, 5%). Eighteen tumors carried MYCN amplification between 29 and 110 copies: 12 had two (RB1−/−) or one RB1 (RB1+/−) mutations, while six had no mutations (RB1+/+). Immunohistochemical staining of tumor sections with antibodies against pRb and phosphorylated Rb (ppRb) displayed high levels of pRb and ppRb in both RB1+/+ and RB1+/− tumors with MYCN amplification compared to no expression of these proteins in a classic RB1−/−, MYCN‐low tumor. These results establish that high MYCN amplification can be present in retinoblastoma with or without coding sequence mutations in the RB1 gene. The functional state of pRb is inferred to be inactive due to phosphorylation of pRb in the MYCN‐amplified retinoblastoma without coding sequence mutations. This makes inactivation of RB1 by gene mutation or its protein product, pRb, by protein phosphorylation, a necessary condition for initiating retinoblastoma tumorigenesis, independent of MYCN amplification.

Functional and Metabolomic Consequences of KATP Channel Inactivation in Human Islets

Loss-of-function mutations of β-cell KATP channels cause the most severe form of congenital hyperinsulinism (KATPHI). KATPHI is characterized by fasting and protein-induced hypoglycemia that is unresponsive to medical therapy. For a better understanding of the pathophysiology of KATPHI, we examined cytosolic calcium ([Ca2+]i), insulin secretion, oxygen consumption, and [U-13C]glucose metabolism in islets isolated from the pancreases of children with KATPHI who required pancreatectomy. Basal [Ca2+]i and insulin secretion were higher in KATPHI islets compared with controls. Unlike controls, insulin secretion in KATPHI islets increased in response to amino acids but not to glucose. KATPHI islets have an increased basal rate of oxygen consumption and mitochondrial mass. [U-13C]glucose metabolism showed a twofold increase in alanine levels and sixfold increase in 13C enrichment of alanine in KATPHI islets, suggesting increased rates of glycolysis. KATPHI islets also exhibited increased serine/glycine and glutamine biosynthesis. In contrast, KATPHI islets had low γ-aminobutyric acid (GABA) levels and lacked 13C incorporation into GABA in response to glucose stimulation. The expression of key genes involved in these metabolic pathways was significantly different in KATPHI β-cells compared with control, providing a mechanism for the observed changes. These findings demonstrate that the pathophysiology of KATPHI is complex, and they provide a framework for the identification of new potential therapeutic targets for this devastating condition.

Characteristics of Follicular Variant Papillary Thyroid Carcinoma in a Pediatric Cohort

ContextnIn adults, noninvasive follicular variant of papillary thyroid carcinoma (FVPTC) is considered a low risk for metastasis and persistent/recurrent disease.nnnObjectivenThe goal of this study was to assess the clinical, sonographic, and histopathologic features of FVPTC in a pediatric cohort.nnnDesignnA retrospective review of subjects <19 years of age with papillary thyroid carcinoma (PTC) who underwent thyroidectomy between January 2010 and July 2015.nnnSettingnMultidisciplinary academic referral center.nnnPatientsnPatients with FVPTC, defined as a tumor ≥1 cm in the largest dimension with predominant follicular growth, complete lack of well-formed papillae, and nuclear features of PTC.nnnMain Outcome MeasuresnTumor size and location, presence of a tumor capsule, capsule and vascular invasion, lymph node invasion, and distant metastasis.nnnResultsnEighteen patients with FVPTC were identified from a case cohort of 110 patients with PTC. On histopathology, 13 (72%) had unifocal nodules and 14 (78%) had completely encapsulated FVPTC. Capsule invasion was frequent (nine of 14; 64%), and vascular invasion was found in one-third of patients (six of 18; 33%). No lymph node metastases were found in the 13 patients (72%) who had a central neck lymph node dissection. One patient with vascular invasion had distant metastases.nnnConclusionnWhen strictly defined, FVPTC in pediatric patients has a low risk for bilateral disease and metastasis. Prospective studies are needed to confirm whether lobectomy with surveillance is sufficient to achieve remission in pediatric patients with low-risk FVPTC.

Congenital syphilis as a clinical and histopathologic mimic of neonatal lupus

Syphilis can be difficult to diagnose clinically and histologically because both its clinical and histologic patterns can mimic other dermatoses. In particular, we would like to highlight the potential to misdiagnose congenital syphilis as neonatal lupus erythematosus given their overlapping clinical, histologic, and serologic features. Despite the screening of mothers for syphilis as part of routine care early in pregnancy, this infection may still be acquired during the course of pregnancy and dermatopathologists and dermatologists should remember to consider it when evaluating neonates with suspected neonatal lupus. Estimation of the timing of infection can be made from a review of the mothers prenatal medical records and the constellation of clinical features present in the neonate. This article is protected by copyright. All rights reserved.

Cytotoxic T-Lymphocyte-Associated Protein 4 Haploinsufficiency-Associated Inflammation Can Occur Independently of T-Cell Hyperproliferation

Located contiguously on the long arm of the second chromosome are gene paralogs encoding the immunoglobulin-family co-activation receptors CD28 and cytotoxic T-lymphocyte-associated protein 4 (CTLA4). CD28 and CTLA4 share the same B7 ligands yet each provides opposing proliferative signals to T cells. Herein, we describe for the first time two unrelated subjects with coexisting CD28 and CTLA4 haploinsufficiency due to heterozygous microdeletions of chromosome 2q. Although their clinical phenotype, multi-organ inflammatory disease, is superficially similar to that of CTLA4 haploinsufficient autoimmune lymphoproliferative syndrome type V (ALPS5) patients, we demonstrate our subjects’ underlying immunopathology to be distinct. Unlike ALPS5 T cells which hyperproliferate to T-cell receptor-mediated activation and infiltrate organs, T cells from our subjects are hypoproliferative and do not. Instead of T cell infiltrates, biopsies of affected subject tissues demonstrated infiltrates of lineage negative lymphoid cells. This histologic feature correlated with significant increases in circulating type 3 innate lymphoid cells (ILC3s) and ILC3 cytokines, interleukin 22, and interleukin-17A. CTLA4-Ig monotherapy, which we trialed in one subject, was remarkably effective in controlling inflammatory diseases, normalizing ILC3 frequencies, and reducing ILC3 cytokine concentrations.

Tender Nodules on the Lower Legs

A man in his 20s with panhypopituitarism, septo-optic dysplasia, developmental delay, duodenalstricture,andahistoryofpancreatitispresentedwithapainfuleruptiononhisbilateral lower legs. The patient was well until 2 days prior to hospitalization when he developed bilateral lower extremity edema. The day prior to admission, he developed painful pink lesions on both legs. He had not applied any topical medications to his legs or had recent exposure to hot tubs or fish tanks at the home. He had a history of keratosis pilaris on the lower legs and was otherwise well with no fevers, recent upper respiratory infections, abdominal pain, vomiting, or diarrhea. Physical examination showed a man in no apparent distress. On the bilateral anterior legs were scattered tender, pink to purple nodules, and plaques (Figure, A). There was also a background of pinpoint folliculocentric papules on leg and thighs consistent with keratosis pilaris. There was 2+ pitting edema of the lower extremities. A punch biopsy specimen was obtained for histopathology and microbiology cultures (Figure, B-D). Routine complete blood cell count and complete metabolic panel had results within normal limits.