T-cell activation is enhanced by targeting IL-10 cytokine production in toll-like receptor-stimulated macrophages

Toll-like receptor (TLR) agonists represent potentially useful cancer vaccine adjuvants in their ability to stimulate antigen-presenting cells (APCs) and subsequently amplify the cytotoxic T-cell response. The purpose of this study was to characterize APC responses to TLR activation and to determine the subsequent effect on lymphocyte activation. We exposed murine primary bone marrow-derived macrophages to increasing concentrations of agonists to TLRs 2, 3, 4, and 9. This resulted in a dose-dependent increase in production of not only tumor necrosis factor–alpha (TNF-α), a surrogate marker of the proinflammatory response, but also interleukin 10 (IL-10), a well-described inhibitory cytokine. Importantly, IL-10 secretion was not induced by low concentrations of TLR agonists that readily produced TNF-α. We subsequently stimulated lymphocytes with anti-CD3 antibody in the presence of media from macrophages activated with higher doses of TLR agonists and observed suppression of interferon gamma release. Use of both IL-10 knockout macrophages and IL-10 small-interfering RNA (siRNA) ablated this suppressive effect. Finally, IL-10 siRNA was successfully used to suppress CpG-induced IL-10 production in vivo. We conclude that TLR-mediated APC stimulation can induce a paradoxical inhibitory effect on T-cell activation mediated by IL-10

Are Immune Modulating Single Nucleotide Polymorphisms Associated with Necrotizing Enterocolitis?

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency. The purpose of this study is to determine if functional single nucleotide polymorphisms (SNPs) in immune-modulating genes pre-dispose infants to NEC. After Institutional Review Board approval and parental consent, buccal swabs were collected for DNA extraction. TaqMan allelic discrimination assays and BglII endonuclease digestion were used to genotype specific inflammatory cytokines and TRIM21. Statistical analysis was completed using logistic regression. 184 neonates were analyzed in the study. Caucasian neonates with IL-6 (rs1800795) were over 6 times more likely to have NEC (p = 0.013; OR = 6.61, 95% CI 1.48-29.39), and over 7 times more likely to have Stage III disease (p = 0.011; OR = 7.13, (95% CI 1.56-32.52). Neonates with TGFβ-1 (rs2241712) had a decreased incidence of NEC-related perforation (p = 0.044; OR = 0.28, 95% CI: 0.08-0.97) and an increased incidence of mortality (p = 0.049; OR = 2.99, 95% CI: 1.01 - 8.86). TRIM21 (rs660) was associated with NEC-related intestinal perforation (p = 0.038; OR = 4.65, 95% CI 1.09-19.78). In premature Caucasian neonates, the functional SNP IL-6 (rs1800795) is associated with both the development and increased severity of NEC. TRIM21 (rs660) and TGFβ-1 (rs2241712) were associated with NEC- related perforation in all neonates in the cohort. These findings suggest a possible genetic role in the development of NEC

Genomics in premature infants: A non-invasive strategy to obtain high-quality DNA

We used a cost-effective, non-invasive method to obtain high-quality DNA from buccal epithelial-cells (BEC) of premature infants for genomic analysis. DNAs from BEC were obtained from premature infants with gestational age ≤ 36 weeks. Short terminal repeats (STRs) were performed simultaneously on DNA obtained from the buccal swabs and blood from the same patient. The STR profiles demonstrated that the samples originated from the same individual and exclude any contamination by external DNAs. Whole exome sequencing was performed on DNAs obtained from BEC on premature infants with and without necrotizing enterocolitis, and successfully provided a total number of reads and variants corroborating with those obtained from healthy blood donors. We provide a proof of concept that BEC is a reliable and preferable source of DNA for high-throughput sequencing in premature infants

Genetics of transfusion recipient alloimmunization: Can clues from susceptibility to autoimmunity pave the way?

The search for genetic determinants of alloimmunization in sickle cell disease transfusion recipients was based on two premises: i) that polymorphisms responsible for stronger immune and/or inflammatory responses and hemoglobin β(S) mutation were co-selected by malaria; and ii) that stronger responder status contributes to development of lupus. We found a marker of alloimmunization in the gene encoding for Ro52 protein, also known as Sjögren syndrome antigen 1 (SSA1) and TRIM21. Surprisingly, the nature of the association was opposite of that with lupus; the same variant of a polymorphism (rs660) that was associated with lupus incidence was also associated with induction of tolerance to red blood cell antigens during early childhood. The dual function of Ro52 can explain this apparent contradiction. We propose that other lupus/autoimmunity susceptibility loci may reveal roles of additional molecules in various aspects of alloimmunization induced by transfusion as well as during pregnancy

Cutting Edge: Developmental Up-Regulation of IFN-gamma-Inducible Lysosomal Thiol Reductase Expression Leads to Reduced T Cell Sensitivity and Less Severe Autoimmunity

Abstract Reactivity to self-peptide/MHC complexes is required for selection of the TCR repertoire in the thymus but can also promote autoimmunity. Reduced TCR sensitivity of mature T cells is thought to help control the autoreactivity in peripheral T cells. The molecular basis for reduced sensitivity of peripheral T cells is not known. We found that peripheral T cells, but not immature thymocytes, lacking IFN-γ-inducible lysosomal thiol reductase (GILT) display increased sensitivity to TCR ligation. GILT−/− peripheral T cells express reduced levels of mitochondrial superoxide dismutase 2 and consequently display higher levels of reactive oxygen radicals and ERK1/2 phosphorylation following activation. The increased sensitivity of GILT-deficient T cells results in a more severe hyperglycemia associated with streptozotocin-induced diabetes. GILT expression levels progressively increase in T cells with maturation. These data suggest that regulation of GILT expression may be a mechanism of T cell differentiation-associated changes in sensitivity to TCR engagement.</jats:p

Physical and Functional Association of the Major Histocompatibility Complex Class I Heavy Chain �3 Domain with the Transporter Associated with Antigen Processing

CD8 � T lymphocytes recognize antigens as short, MHC class I-associated peptides derived by processing of cytoplasmic proteins. The transporter associated with antigen processing translocates peptides from the cytosol into the ER lumen, where they bind to the nascent class I molecules. To date, the precise location of the class I-TAP interaction site remains unclear. We provide evidence that this site is contained within the heavy chain �3 domain. Substitution of a 15 amino acid portion of the H-2D b �3 domain (aa 219-233) with the analogous MHC class II (H-2IA d) �2 domain region (aa 133-147) results in loss of surface expression which can be partially restored upon incubation at 26�C in the presence of excess peptide and �2-microglobulin. Mutant H-2D b (D b 219-233) associates poorly with the TAP complex, and cannot present endogenously-derived antigenic peptides requiring TAP-dependent translocation to the ER. However, this presentation defect can be overcome through use of an ER targeting sequence which bypasses TAP-dependent peptide translocation. Thus, the �3 domain serves as an important site of interaction (directly or indirectly) with the TAP complex and is necessary for TAP-dependent peptide loading and class I surface expression. The MHC class I molecule is a heterotrimeric comple

Molecular and functional analysis of anchorage independent, treatment-evasive neuroblastoma tumorspheres with enhanced malignant properties: A possible explanation for radio-therapy resistance

Despite significant advances in cancer treatment and management, more than 60% of patients with neuroblastoma present with very poor prognosis in the form of metastatic and aggressive disease. Solid tumors including neuroblastoma are thought to be heterogeneous with a sub-population of stem-like cells that are treatment-evasive with highly malignant characteristics. We previously identified a phenomenon of reversible adaptive plasticity (RAP) between anchorage dependent (AD) cells and anchorage independent (AI) tumorspheres in neuroblastoma cell cultures. To expand our molecular characterization of the AI tumorspheres, we sought to define the comprehensive proteomic profile of murine AD and AI neuroblastoma cells. The proteomic profiles of the two phenotypic cell populations were compared to each other to determine the differential protein expression and molecular pathways of interest. We report exclusive or significant up-regulation of tumorigenic pathways expressed by the AI tumorspheres compared to the AD cancer cells. These pathways govern metastatic potential, enhanced malignancy and epithelial to mesenchymal transition. Furthermore, radio-therapy induced significant up-regulation of specific tumorigenic and proliferative proteins, namely survivin, CDC2 and the enzyme Poly [ADP-ribose] polymerase 1. Bio-functional characteristics of the AI tumorspheres were resistant to sutent inhibition of receptor tyrosine kinases (RTKs) as well as to 2.5 Gy radio-therapy as assessed by cell survival, proliferation, apoptosis and migration. Interestingly, PDGF-BB stimulation of the PDGFRβ led to transactivation of EGFR and VEGFR in AI tumorspheres more potently than in AD cells. Sutent inhibition of PDGFRβ abrogated this transactivation in both cell types. In addition, 48 h sutent treatment significantly down-regulated the protein expression of PDGFRβ, MYCN, SOX2 and Survivin in the AI tumorspheres and inhibited tumorsphere self-renewal. Radio-sensitivity in AI tumorspheres was enhanced when sutent treatment was combined with survivin knock-down. We conclude that AI tumorspheres have a differential protein expression compared to AD cancer cells that contribute to their malignant phenotype and radio-resistance. Specific targeting of both cellular phenotypes is needed to improve outcomes in neuroblastoma patients