After stimulation with each antigen [pertussis toxoid (PT), pertactin (PRN) and filamentous haemagglutinin (FHA)], percentages of CD69 expressing CD4+ T cells among 20 infants were enumerated and plotted against respective unstimulated controls (a). were restricted to CD45RA-CCR7+CD27+ phenotype, consistent with early-stage differentiated pertussis-specific memory CD4+ T cells. We show for the first time that DTaP vaccination-induced CD4+ T cells in infants are functionally and phenotypically dissimilar from those of adults. have been shown to correlate with elicitation of T helper type 1 (Th1) cytokines such as IFN-[12,17,18], whereas CD4+ T cells co-producing IL-2 and IFN- are thought to be essential contributors to long-lasting pertussis-specific protective immunity [12]. Studies in the past suggested that aP vaccination can promote cell-mediated immunity; however, those observations were made based on traditional antigen-specific T cell proliferation and enzyme-linked immunosorbent assay (ELISA)-based cytokine assays, and lacked information regarding memory generation, functional and/or phenotypic properties of vaccine-induced CD4+ T cells [18C20]. Other Abiraterone Acetate (CB7630) studies demonstrated the ability of pertussis toxin and aP vaccine to elicit a mixed Th1 and Th2 CD4+ T cell response [18,21], and that aP vaccine induces predominantly a Th2 CD4+ T cell response in vaccinated children [19,20,22]. In addition, the pattern of cytokine production on a single cell basis in DTaP-vaccinated infants and adults has not been delineated. The combination of CCR7 and CD45RA has been used Abiraterone Acetate (CB7630) extensively for classifying antigen-experienced T cells (effector memory, TEM and central memory, TCM) [23]. More recently, another classification model based on expression of chemokine Abiraterone Acetate (CB7630) receptors CCR7 and CD27 has been described [24]. Based on the differences in their telomere lengths, activation has exhibited the order of CD4+ T cell differentiation as: naive to CCR7+CD27+ to CCR7-CD27+ to CCR7-CD27-. CCR7+CD27+ cells are least differentiated, whereas CCR7-CD27- are fully differentiated CD4+ T cells due to their shortest telomere lengths. Because characterization of pertussis-specific responses in infants and adults has not been performed previously, we developed a multi-parametric circulation cytometry approach and analysis method that allowed simultaneous detection of multi-functionality and phenotypes of CD4+ T cells induced as a result of DTaP ENSA vaccination in infants compared to adults. Material and methods Subjects and peripheral blood mononuclear cells (PBMC) samples Healthy infants (= 20; aged between 9 and 12 months, median age 10 months) who have received three doses of DTaP vaccine (Sanofi Pasteur, Swiftwater, PA, USA) at 2, 4 and 6 months of age were involved in the study. The infants were from a cohort recruited as a part of a prospective study of immunity to respiratory pathogens (NIDCD R0108671). Healthy adult volunteers (= 12; median age 304 years) vaccinated with the same DTaP within the preceding 5 years were given a booster dose before bleeding them at day 7 and for two subjects 3C4 months later for PBMC isolation. Written consent was obtained from parents of the children and the adults in association with a protocol approved by the Rochester General Hospital institutional review table. Heparinized venous blood was drawn and PBMCs isolated using Ficoll gradient according to the manufacturer’s instructions. Cells were washed in phosphate-buffered saline (PBS) resuspended at a concentration of 1 1 107 cells/ml in cell recovery freezing media (Gibco, Grand Island, NY, USA) and frozen in liquid nitrogen until used. Antigens and antibodies Purified pertussis toxoid vaccine protein antigen (PT), pertactin (PRN) and filamentous haemagglutinin (FHA) were utilized for T cell activation (gifts from Sanofi Pasteur, Swiftwater, PA, USA). Antibodies utilized for staining were anti-CD3 Qdot 605 or Pacific.
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