The expansion of thymopoiesis in neonatal mice is dependent on expression of high mobility group a 2 protein (Hmga2).

Cell number in the mouse thymus increases steadily during the first two weeks after birth. It then plateaus and begins to decline by seven weeks after birth. The factors governing these dramatic changes in cell production are not well understood. The data herein correlate levels of High mobility group A 2 protein (Hmga2) expression with these temporal changes in thymopoiesis. Hmga2 is expressed at high levels in murine fetal and neonatal early T cell progenitors (ETP), which are the most immature intrathymic precursors, and becomes almost undetectable in these progenitors after 5 weeks of age. Hmga2 expression is critical for the initial, exponential expansion of thymopoiesis, as Hmga2 deficient mice have a deficit of ETPs within days after birth, and total thymocyte number is repressed compared to wild type littermates. Finally, our data raise the possibility that similar events occur in humans, because Hmga2 expression is high in human fetal thymic progenitors and falls in these cells during early infancy

IND-Enabling Studies for a Clinical Trial to Genetically Program a Persistent Cancer-Targeted Immune System

PURPOSE: To improve persistence of adoptively transferred T-cell receptor (TCR)-engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. EXPERIMENTAL DESIGN: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)-compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)-compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. RESULTS: TCR genetically modified and ex vivo-cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. CONCLUSIONS: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471

Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma

BACKGROUND: Approximately 75% of objective responses to anti–programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown. METHODS: We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti–PD-1 therapy (pembrolizumab) followed by disease progression months to years later. RESULTS: Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated loss-of-function mutations in the genes encoding interferon-receptor–associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I. CONCLUSIONS: In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.

Human mast cells express intracellular TRAIL

Recently we demonstrated that human mast cells (MC) express functional TRAIL death receptors. Here we assessed the expression of TRAIL on both mRNA and protein level in cord blood derived MC (CBMC) and HMC-1. The TRAIL release either spontaneous or induced by LPS, IFN-gamma and IgE-dependent activation, was evaluated as well. The protein location was restricted to the intracellular compartment in CBMC, but not in HMC-1. The intracellular TRAIL was not localized inside the granules. The treatment with IFN-gamma and LPS up-regulated intracellular TRAIL expression in CBMC, but did not induce its release. These in vitro data show that human MC can produce and express intracellular TRAIL whose location could not be altered by different stimuli

Human mast cells undergo TRAIL-induced apoptosis

Mast cells (MC), supposedly long-lived cells, play a key role in allergy and are important contributors to other inflammatory conditions in which they undergo hyperplasia. In humans, stem cell factor (SCF) is the main regulator of MC growth, differentiation, and survival. Although human MC numbers may also be regulated by apoptotic cell death, there have been no reports concerning the role of the extrinsic apoptotic pathway mediated by death receptors in these cells. We examined expression and function of death receptors for Fas ligand and TRAIL in human MC. Although the MC leukemia cell line HMC-1 and human lung-derived MC expressed both Fas and TRAIL-R, MC lines derived from cord blood (CBMC) expressed only TRAIL-R. Activation of TRAIL-R resulted in caspase 3-dependent apoptosis of CBMC and HMC-1. IgE-dependent activation of CBMC increased their susceptibility to TRAIL-mediated apoptosis. Results suggest that TRAIL-mediated apoptosis may be a mechanism of regulating MC survival in vivo and, potentially, for down-regulating MC hyperplasia in pathologic conditions

Distinct Genetic Networks Orchestrate the Emergence of Specific Waves of Fetal and Adult B-1 and B-2 Development

B cell development is often depicted as a linear process initiating in the fetus and continuing postnatally. Using a PU.1 hypomorphic mouse model, we found that B-1 and B-2 lymphopoiesis occurred in distinct fetal and adult waves differentially dependent on the Sfpi1 14 kB upstream regulatory element. The initial wave of fetal B-1 development was absent in PU.1 hypomorphic mice, while subsequent fetal and adult waves emerged. In contrast, B-2 lymphopoiesis occurred in distinct fetal and adult waves. Whole-transcriptome profiling of fetal and adult B cell progenitors supported the existence of three waves of B-1 and two waves of B-2 development and revealed that the network of transcription factors governing B lineage specification and commitment was highly divergent between B-1 and B-2 progenitors. These findings support the view that the B-1 and B-2 lineages are distinct and provide a genetic basis for layering of immune system development

Human Mast Cell Activation by Staphylococcus aureus: Interleukin-8 and Tumor Necrosis Factor Alpha Release and the Role of Toll-Like Receptor 2 and CD48 Molecules▿

The ability of Staphylococcus aureus to invade and survive within host cells is believed to contribute to its propensity to cause persistent and metastatic infections. In addition, S. aureus infections often are associated with atopic diseases such as dermatitis, rhinitis, and asthma. Mast cells, the key cells of allergic diseases, have a pivotal role in innate immunity and have the capacity of phagocytosis, and they can destroy some pathogenic bacteria. However, little is known about the ability of some other bacteria to survive and overcome mast cell phagocytosis. Therefore, we were interested in evaluating the interplay between mast cells and S. aureus. In this study, we show that human cord blood-derived mast cells (CBMC) can be infected by pathogenic S. aureus. S. aureus displayed a high adherence to mast cells as well as invasive and survival abilities within them. However, when infections were performed in the presence of cytochalasin D or when CBMC were preincubated with anti-Toll-like receptor 2 (TLR2) or anti-CD48 antibodies, the invasiveness and the inflammatory response were abrogated, respectively. Furthermore, we observed an increase of TLR2 and CD48 molecules on CBMC after S. aureus infection. The infection of CBMC with S. aureus also caused the release of tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8). Both live and killed S. aureus organisms were found to trigger TNF-α and IL-8 release by CBMC in a time-dependent manner. Cumulatively, these findings suggest that S. aureus internalizes and survives in mast cells. This may play an important role in infections and in atopic diseases associated with S. aureus

The expansion of thymopoiesis in neonatal mice is dependent on expression of high mobility group a 2 protein (Hmga2).

Cell number in the mouse thymus increases steadily during the first two weeks after birth. It then plateaus and begins to decline by seven weeks after birth. The factors governing these dramatic changes in cell production are not well understood. The data herein correlate levels of High mobility group A 2 protein (Hmga2) expression with these temporal changes in thymopoiesis. Hmga2 is expressed at high levels in murine fetal and neonatal early T cell progenitors (ETP), which are the most immature intrathymic precursors, and becomes almost undetectable in these progenitors after 5 weeks of age. Hmga2 expression is critical for the initial, exponential expansion of thymopoiesis, as Hmga2 deficient mice have a deficit of ETPs within days after birth, and total thymocyte number is repressed compared to wild type littermates. Finally, our data raise the possibility that similar events occur in humans, because Hmga2 expression is high in human fetal thymic progenitors and falls in these cells during early infancy