Regulatory T cells (Treg) that prevent autoimmune diseases by suppression of self-reactive T cells may also suppress the immune response against cancer. In mice, depletion of Treg by Ab therapy leads to more efficient tumor rejection. Treg-mediated suppression of antitumor immune responses may partly explain the poor clinical response to vaccine-based immunotherapy for human cancer. In this study, we measured the prevalence of Treg that coexpress CD4 and CD25 in the PBLs, tumor-infiltrating lymphocytes, and regional lymph node lymphocytes from 65 patients with either pancreas or breast cancer. In breast cancer patients (n = 35), pancreas cancer patients (n = 30), and normal donors (n = 35), the prevalence of Treg were 16.6% (SE 1.22), 13.2% (SE 1.13), and 8.6% (SE 0.71) of the total CD4+ cells, respectively. The prevalence of Treg were significantly higher in breast cancer patients (p < 0.01) and pancreas cancer patients (p < 0.01) when compared with normal donors. In tumor-infiltrating lymphocytes and lymph node lymphocytes, the Treg prevalence were 20.2% (SE 3.93) and 20.1% (SE 4.3), respectively. Treg constitutively coexpressed CTLA-4 and CD45RO markers, and secreted TGF-β and IL-10 but did not secrete IFN-γ. When cocultured with activated CD8+ cells or CD4+25− cells, Treg potently suppressed their proliferation and secretion of IFN-γ. We conclude that the prevalence of Treg is increased in the peripheral blood as well as in the tumor microenvironment of patients with invasive breast or pancreas cancers. These Treg may mitigate the immune response against cancer, and may partly explain the poor immune response against tumor Ags.

We examined the function and intracellular localization of the product of the Na(+)/H(+) antiporter gene (OsNHX1) cloned from rice (Oryza sativa). OsNHX1 has the ability to suppress Na(+), Li(+) and hygromycin sensitivity of yeast nhx1 mutants and sensitivity to a high K(+) concentration, a novel phenotype of the nhx1 mutants. Analysis using rice cells expressing a fusion protein of OsNHX1 and green fluorescent protein and Western blot analysis using antibodies specific for OsNHX1 confirmed the localization of OsNHX1 on the tonoplasts. These results indicate that the OsNHX1 gene encodes a vacuolar (Na(+), K(+))/H(+) antiporter. Treatment with high concentrations of NaCl and KCl increased the transcript levels of OsNHX1 in rice roots and shoots. In addition, overexpression of OsNHX1 improved the salt tolerance of transgenic rice cells and plants. These results suggest that OsNHX1 on the tonoplasts plays important roles in the compartmentation of Na(+) and K(+) highly accumulated in the cytoplasm into the vacuoles, and the amount of the antiporter is one of the most important factors determining salt tolerance in rice.

We previously cloned a vacuolar Na+/H+ antiporter gene (OsNHX1) from rice (Oryza sativa). Here we identified four additional NHX-type antiporter genes in rice (OsNHX2 through OsNHX5) and performed molecular and functional analyses of those genes. The exon-intron structure of the OsNHX genes and the phylogenetic tree of the OsNHX proteins suggest that the OsNHX proteins are categorized into two subgroups (OsNHX1 through OsNHX4 and OsNHX5). OsNHX1, OsNHX2, OsNHX3, and OsNHX5 can suppress the Na+, Li+, and hygromycin sensitivity of yeast nhx1 mutants and their sensitivity to a high K+ concentration. The expression of OsNHX1, OsNHX2, OsNHX3, and OsNHX5 is regulated differently in rice tissues and is increased by salt stress, hyperosmotic stress, and ABA. When we studied the expression of β-glucuronidase (GUS) driven by either the OsNHX1 or the OsNHX5 promoter, we observed activity in the stele, the emerging part of lateral roots, the vascular bundle, the water pore, and the basal part of seedling shoots with both promoters. In addition, each promoter had a unique expression pattern. OsNHX1 promoter-GUS activity only was localized to the guard cells and trichome, whereas OsNHX5 promoter-GUS activity only was localized to the root tip and pollen grains. Our results suggest that the members of this gene family play important roles in the compartmentalization into vacuoles of the Na+ and K+ that accumulate in the cytoplasm and that the differential regulation of antiporter gene expression in different rice tissues may be an important factor determining salt tolerance in rice.

Summary Capsaicinoids are responsible for the spicy flavor of pungent peppers (Capsicum). The cultivar CH‐19 Sweet is a non‐pungent pepper mutant derived from a pungent pepper strain, Capsicum annuum CH‐19. CH‐19 Sweet biosynthesizes capsaicinoid analogs, capsinoids. We determined the genetic and metabolic mechanisms of capsinoid biosynthesis in this cultivar. We analyzed the putative aminotransferase (pAMT) that is thought to catalyze the formation of vanillylamine from vanillin in the capsaicinoid biosynthetic pathway. Enzyme assays revealed that pAMT activity catalyzing vanillylamine formation was completely lost in CH‐19 Sweet placenta tissue. RT‐PCR analysis showed normal mRNA transcription of the pAMT gene; however, SNP analysis of the cDNA sequence showed a T nucleotide insertion at 1291 bp in the pAMT gene of CH‐19 Sweet. This insertion formed a new stop codon, TGA, that prevented normal translation of the gene, and the pAMT protein did not accumulate in CH‐19 Sweet as determined using Western blot analysis. We developed a dCAPS marker based on the T insertion in the pAMT gene of CH‐19 Sweet, and showed that the pAMT genotype co‐segregated with the capsinoid or capsaicinoid fruit phenotype in the F2 population. The T insertion was not found in other pungent and non‐pungent Capsicum lines, suggesting that it is specific to CH‐19 Sweet. CH‐19 Sweet’s pAMT gene mutation is an example of a nonsense mutation in a single gene that alters a secondary metabolite biosynthetic pathway, resulting in the biosynthesis of analogs. The dCAPS marker will be useful in selecting lines with capsinoid‐containing fruits in pepper‐breeding programs.

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