VEGF Requires the Receptor NRP-1 To Inhibit Lipopolysaccharide-Dependent Dendritic Cell Maturation.

To stimulate a productive T cell response, dendritic cells (DC) must undergo maturation characterized by heightened cell surface expression of MHC and costimulatory molecules as well as cytokine production. Conversely, the inhibition of DC maturation is a central mechanism of immune tolerance. The control of the DC maturation process relies on the integration of several cellular stimulatory or inhibitory signals. The soluble factors and their receptors controlling this central aspect of DC biology are incompletely characterized. We show that murine bone marrow-derived DC (BMDC) maturation induced by LPS, as opposed to polyinosinic:polycytidylic acid or cytosine-phosphate-guanine, is robustly inhibited by vascular endothelial growth factor (VEGF), a previously identified immunosuppressive cytokine. Using BMDC from wild type and conditional knockout mice, we show that neuropilin-1 (NRP-1), a known receptor of VEGF, is necessary to suppress LPS-dependent BMDC maturation. The absence of NRP-1 had no ostensible effects on the biology of BMDC in the absence of VEGF. However, NRP-1-deficient BMDC remained completely insensitive to the VEGF-dependent inhibition of BMDC maturation in culture. In the presence of VEGF, NRP-1 directly interacted with the LPS receptor TLR4 and suppressed downstream signaling through ERK and NF-κß, resulting in a sharp inhibition of MHC class II and costimulatory molecules (CD40, CD86) expression as well as proinflammatory cytokine production. Consequently, we identify NRP-1 as a target to optimize DC maturation within environments that are rich in VEGF, such as tumors.

Insufficient Body Weight of Adults Born With Esophageal Atresia.

OBJECTIVES: Impaired growth has been reported in children born with esophageal atresia (EA). Their nutritional fate at adulthood remains uncertain though. Our objectives were to determine the body mass index (BMI) of adult patients with EA followed up from 2009 to 2011 in the EA clinic of a university-affiliated hospital in Quebec (Canada), and investigate characteristics associated with underweight. METHODS: The 40 adult patients with EA attending the clinic were invited to participate. Height and weight were measured and BMI calculated. Patients with BMI < 18.5 kg/m were deemed underweight. Patients' characteristics were obtained, including digestive symptoms and compensatory eating behaviors. Nonparametric tests were used to compare the proportion of underweight among patients with EA with that found in the Quebec population and the characteristics between patients with EA deemed underweight and those with BMI ≥ 18.5 kg/m. RESULTS: The final sample included 16 women and 21 men, ages 18 to 44 years. Mean BMI was 21.3 ±â€Š4.9 kg/m and 24.3% had BMI < 18.5 kg/m, which is higher than in the Quebec adult population (2.5%, P < 0.001). Compared with patients with EA having BMI ≥ 18.5 kg/m, underweight patients had more often experienced failure to thrive during their adolescence (55.6% vs 7.4%, P = 0.006), and reported severe postprandial fullness (62.5% vs 21.4%, P = 0.040), the need to eat slowly (87.5% vs 46.4%; P = 0.045), and severe difficulties to swallow dry solid foods (50.0% vs 14.3%, P = 0.054). CONCLUSIONS: Insufficient body weight is prevalent in this sample of adult patients with EA and could result from digestive symptoms. Follow-up with a gastroenterologist and nutritional counseling should be considered for adult patients with EA.

Defining novel parameters for the optimal priming and expansion of minor histocompatibility antigen-specific T cells in culture.

BACKGROUND: Adoptive transfer of minor histocompatibility antigen (MiHA)-specific T cells is a promising therapy for patients with hematological cancers. However, the efficacy of the transferred cells is hampered by the acquisition of terminal effector differentiation and exhaustion features during expansion in vitro thus preventing their function and persistence in vivo. Yet, the factors that induce T-cell differentiation and functional impairment in culture remain poorly defined and are likely to vary depending on the method used for expansion. METHODS: Using the clinically relevant HLA-A0201-restricted MiHA HA-1 as well as reagents and procedures that are readily transferable to a clinical environment, we designed a novel culture protocol and defined how exhaustion features appeared in function of time. The optimal time points for the expansion of "fit" MiHA-specific T cells were delineated using phenotypic and functional assessments including KLRG-1 and PD-1 surface markers as well as Ki67 staining and cytokine secretion assays. RESULTS: Following a priming phase, an enrichment step and a rapid expansion stage, our method generates MiHA-specific T-cell lines. Evidence of phenotypic and functional dysfunction appear in function of culture duration, but display different characteristics following the extension of the priming or rapid expansion phases. While repeated antigen exposure during the priming phase induced the decline of the antigen-specific population and the expression of PD-1 and KLRG-1 on antigen-specific CD8+ T cells, the prolongation of an antigen-free expansion phase induced proliferation arrest and the relative loss of antigen-specific cells without impairing polyfunctional cytokine secretion or inducing PD-1 and KLRG-1 expression. A similar pattern was also observed after stimulating a virus-specific memory repertoire, except for the more rapid acquisition of exhaustion features upon repeated antigen exposure. CONCLUSION: Our results offer novel insights on the impact of culture duration on the acquisition of T-cell exhaustion features. Using a new clinical-compliant protocol, we define critical parameters to monitor in order to optimally differentiate and expand MiHA-specific T cells in culture prior to adoptive transfer.

Early exposure to interleukin-21 limits rapidly generated anti-Epstein-Barr virus T-cell line differentiation.

BACKGROUND AIMS: The adoptive transfer of ex vivo-expanded Epstein-Barr virus (EBV)-specific T-cell lines is an attractive strategy to treat EBV-related neoplasms. Current evidence suggests that for adoptive immunotherapy in general, clinical responses are superior if the transferred cells have not reached a late or terminal effector differentiation phenotype before infusion. The cytokine interleukin (IL)-21 has shown great promise at limiting late T-cell differentiation in vitro, but this remains to be demonstrated in anti-viral T-cell lines. METHODS: We adapted a clinically validated protocol to rapidly generate EBV-specific T-cell lines in 12 to 14 days and tested whether the addition of IL-21 at the initiation of the culture would affect T-cell expansion and differentiation. RESULTS: We generated clinical-scale EBV-restricted T-cell line expansion with balanced T-cell subset ratios. The addition of IL-21 at the beginning of the culture decreased both T-cell expansion and effector memory T-cell accumulation, with a relative increase in less-differentiated T cells. Within CD4 T-cell subsets, exogenous IL-21 was notably associated with the cell surface expression of CD27 and high KLF2 transcript levels, further arguing for a role of IL-21 in the control of late T-cell differentiation. CONCLUSIONS: Our results show that IL-21 has profound effects on T-cell differentiation in a rapid T-cell line generation protocol and as such should be further explored as a novel approach to program anti-viral T cells with features associated with early differentiation and optimal therapeutic efficacy.