As shown in Fig.4, splenocytes from all infected mice produced high levels of IL-17 upon restimulation with the dominant P.69 Prn7-24epitope. cross-react with natural variants of the epitope as present inBordetella parapertussisandBordetella bronchiseptica. Finally, it was found that the immunodominant P.69 Prn epitope is broadly recognized in the human population by CD4+T cells in an HLA-DQ-restricted manner. DuringB. pertussisinfection, the epitope was associated with a Th1-type CD4+T-cell Rabbit Polyclonal to DARPP-32 response. Hence, this novel P.69 Prn epitope is involved in CD4+T-cell immunity afterB. pertussisvaccination and illness in mice and, more importantly, in humans. Therefore, it may provide a useful tool for the evaluation of the type, magnitude, and maintenance ofB. pertussis-specific CD4+T-cell mechanisms in preclinical and medical vaccine studies. Whooping cough, or pertussis, is an acute infection of the upper respiratory tract that is most severe in young children (8). The disease is definitely caused primarily Peficitinib (ASP015K, JNJ-54781532) from the gram-negative bacteriumBordetella pertussis. Although vaccination against pertussis has been very successful in reducing morbidity and mortality among children worldwide, during the past 2 decades the disease offers reemerged in countries with well-implemented infant vaccination programs (2,5,9,11,15,17). In addition to changes in transmission patterns and the emergence of new variants ofB. pertussis(18,35,36,48), waning immunity has been proposed as a major factor in the increasing incidence of whooping cough (19,50). Knowledge of the mechanisms of immunity to pertussis is still incomplete (3,30,40). Antibody reactions against virulence factors have been associated with protection in various clinical studies (7,45), while, on the other hand, immunogenicity studies after pertussis vaccination failed to demonstrate an unequivocal correlation between titers of antibodies to vaccine antigens and vaccine effectiveness (1,13). Studies with mice have indicated that protecting immunity toB. pertussisinfection not only depends on humoral immunity but also requires a CD4+T-cell response (26,27,30,38). CD4+T cells can add to pertussis resistance by regulating specific B-cell reactions and by generating protecting Th1- and Th17-type cytokines, such as gamma interferon (IFN-) (31,39) and interleukin 17 (IL-17) (20), respectively. To further substantiate the part and maintenance of CD4+T cells in protecting pertussis immunity, however, more knowledge about specific targeted antigens and CD4+T-cell epitopes is required. P.69 pertactin (P.69 Prn), the focus of this study, is regarded as an important antigen in pertussis vaccines. P.69 Prn is polymorphic, and among the 12 variants described to date (14), variation in P.69 Prn is essentially found only in two regions (region 1 and 2) composed of sequence repeats (34). Several studies have shown that P.69 Prn is important for immune protection againstB. pertussisinfection (7,10,45). Furthermore, acellular vaccines comprising only pertussis toxin and filamentous hemagglutinin appear considerably less effective than vaccines comprising P.69 Prn as well (16,29,37). In mice, passive and active vaccination showed that P.69 Prn confers protective immunity (24,25). Taken together, these results strongly support a role for P.69 Prn in protection against whooping cough. To gain insight into the part of P.69 Prn like a CD4+T-cell target, we founded specific T-cell hybridomas (TCH) from primed BALB/c mouse lymph node cells. This approach led to the identification of an immunodominantB. pertussisconserved I-Ad-restricted CD4+T-cell epitope in P.69 Prn that evokes strong proliferative and cytokine responses after infection or vaccination of BALB/c mice. Moreover, the P.69 Prn epitope is also associated with HLA-DQ-restricted CD4+T-cell immunity in humans. == MATERIALS AND METHODS == == Mice and cell suspensions. == Female specific-pathogen-free BALB/c mice were purchased from Harlan and kept in-house under standard conditions. All experiments were authorized by the Animal Ethics Committee of the Netherlands Vaccine Institute (NVI). After section, single-cell suspensions of splenocytes and lymph node cells were produced by mechanical dissociation of organs through 70-m-pore-size nylon filters. Red blood cells in splenocyte suspensions were lysed with 10 mM KHCO3-0.1 mM EDTA for 2 min at 4C. Splenocytes were resuspended in total IMDM-10 (Iscove’s altered Dulbecco’s medium [Gibco-BRL] supplemented with 10% fetal bovine serum [HyClone] and penicillin, streptomycin, andl-glutamine [Pen/Strep/Glu; Gibco-BRL]). Lymph node cells were resuspended in total IMDM-5 (Iscove’s altered Dulbecco’s medium supplemented with 5% normal mouse serum [Harlan] and Pen/Strep/Glu). == Isolation of PBMC. == Peripheral blood was acquired after educated consent from HLA-oligotyped blood standard bank donors from a birth cohort associated with pertussis vaccination (S03.0015-x; Sanquin) and from two pertussis individuals within 4 Peficitinib (ASP015K, JNJ-54781532) weeks after laboratory-confirmed analysis ofB. pertussisinfection (NVI-243). Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation of buffy coating cells on Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) and were used directly or after cryopreservation. == Cell lines. == The BW1100 cell collection, a TCR/variant of BW5147 (51), was used like a fusion partner for the production of TCH and was kindly provided by D. Canaday (Case Western Reserve Peficitinib (ASP015K, JNJ-54781532) University or college and University Private hospitals, Cleveland, OH). The A20 BALB/c B-cell lymphoma cell collection was from the ATCC. The BW1100 and A20 cell lines were grown in total DMEM-10 (Dulbecco’s minimal essential medium supplemented with 10% fetal bovine.