No difference in TCR-V usage was observed between the two populations (Fig. massive accumulation of Th2 cells in the lung and intestine.2 Similarly, high frequencies of Th2 cells are found in several tissues after contamination with have been shown to contain large glycoproteins that promote Th2 cell differentiation by polarized activation of dendritic cells.4,5 A Th2-inducing component of egg antigen was recently identified as Omega-1, a T2 ribonuclease that reduces the contact time between dendritic cells and T cells and L-655708 stimulates dendritic cells for Th2 cell activation.6,7 Other Th2-inducing factors from helminths include complex glycans and proteases.8,9 However, receptors on antigen-presenting cells that recognize these Th2-inducing factors remain largely unknown. The Th2 cells might also be induced by helminth-associated superantigens that could induce massive oligoclonal growth of T cells by activation of T-cell receptors (TCR) of a certain V family, as has been exhibited for viral and bacterial superantigens.10 Evidence for helminth-associated superantigens comes from studies with or result in high levels of IgE or IgG1 that appear to be unspecific for these parasites.22 One might speculate Mouse Monoclonal to 14-3-3 that this unspecific B-cell response results from an unspecific activation of T cells. Furthermore, it remains unclear whether a polyclonal TCR repertoire is required for a protective T-cell response against helminths. The correlation between TCR diversity and efficiency of worm expulsion can be determined by contamination of TCR-transgenic mice. The majority of T cells in these mice express a transgenic TCR that does not react with helminth antigens. However, allelic exclusion by the transgenic TCR can be leaky so that a second, endogenous TCR -chain is expressed, resulting in a peripheral T-cell pool with oligoclonal TCR specificities. Here, we demonstrate that contamination of mice with the helminth induces a polyclonal T-cell response that is reflected by unbiased distribution of TCR-V families among naive and activated CD4 T cells. The broad diversity of the TCR repertoire is required for protective immunity. Superantigens or cytokine-driven bystander activation do not contribute to the Th2 response against this pathogen. Materials and methods Mice Interleukin-4 reporter mice (4get mice; C.129-gene. DO11.10 TCR-transgenic (tg) mice23 were originally obtained from The Jackson Laboratory (Bar Harbor, ME). Smarta TCR-tg mice24 were kindly provided by A. Oxenius. Both TCR-tg strains were crossed to 4get mice to generate DO11/4get and Smarta/4get mice. Rag2?/? mice on a BALB/c background were purchased from Taconic Farms (Germantown, NY). They were bred to DO11/4get mice to generate DO11/4get/Rag?/? mice. Rag1?/? mice on a C57BL/6 background were originally obtained from The Jackson Laboratory. Mice were housed according to institutional guidelines and used at 6C8 weeks of age. All animal experiments were approved by the local federal government. N. brasiliensis were washed extensively in sterile 09% saline (37) and injected subcutaneously (500 organisms) into mice. Mice were given antibiotics contained in water (2 g/l neomycin sulphate, 100 mg/l polymyxin B sulphate, Sigma-Aldrich, St Louis, MO) for the first 5 days after contamination. Worm expulsion was determined by counting adult worms in the small intestine on day 9 after contamination. Eggs in faecal pellets were counted using McMaster counting chambers. Flow cytometry Single-cell suspensions were generated from lymph nodes, spleen or PBS-perfused lung samples that had been cut into small pieces and mechanically dispersed using a 70-m nylon strainer (BD Falcon, Bedford, MA). Samples were L-655708 washed once in FACS buffer (PBS / 2% fetal bovine serum /1 mg/ml sodium azide), incubated with anti-CD16/CD32 blocking monoclonal antibody (mAb; 2.4G2) for 5 min at room L-655708 heat, and stained with diluted mAb mixtures. The following mAbs were used: phycoerythrin (PE)-Cy5.5-labelled anti-CD4 (clone RM4-5), biotinylated anti-CD11a (M17/4), PE-labelled anti-CD25 (PC61.5), allophycocyanin (APC)-labelled anti-CD29 (eBioHMb1-1), PE-labelled anti-CD44 (IM7), PE- or APC-labelled anti-DO11.10 TCR (KJ1-26), APC-labelled anti-V2 (B20.1) and PE-labelled anti-TCR-V8.3 (B21.14) were all purchased from eBioscience (San Diego, CA). Biotinylated anti-CD62 ligand (CD62L; MEL-14) and PE-labelled anti-CD69 were purchased from Invitrogen-Caltag (Carlsbad, CA). Biotinylated anti-TCR-V3.2 (RR3-16), anti-TCR-V11.1/11.2 (RR8-1), anti-TCR-V3 (KJ25), anti-TCR-V4 (KT4), anti-TCR-V5.1/5.2 (MR9-4), anti-TCR-V6 (RR4-7), anti-TCR-V8.1/8.2 (MR5-2), anti-TCR-V14 (14-2), the FITC-labelled mouse V TCR screening panel and PE-labelled anti-Siglec-F (E50-2440) were purchased from BD Biosciences (San Jose, CA). Biotinylated anti-IgE (23G3) was purchased from Southern Biotechnology Associates (Birmingham, AL). An APC-labelled streptavidin (Southern Biotechnology Associates) was used to visualize biotinylated mAbs. Samples were acquired on a FACSCalibur or FACS Canto II instrument (BD Immunocytometry Systems, San Jose, CA) and analysed using FlowJo software (Tree Star, Ashland, OR). IL-4 cytokine capture assay T cells from mediastinal lymph nodes of alone (Nb) or with a mixture of and 100 g of ovalbumin (Nb-OVA). The Nb-infected mice were analysed.