5 103 naive CD4 T cells from NIP TCR-transgenic or retrogenic mice were transferred into each mouse. Infections and immunizations LCMV Armstrong stocks were prepared and quantified as previously described (9). data suggest that the Bcl6 BTB domain is a key mediator of the differentiation of Tfh cells. Introduction The transcriptional repressor B cell CLL/lymphoma 6 (Bcl6) is essential for the differentiation of T follicular helper (Tfh) cells and germinal center (GC) B cells. Tfh cells are CD4 T cells specialized in providing help for B cells (1). The absence of Tfh cells results in the loss of GCs and, consequently, abrogated memory B cell, plasma cell, and neutralizing Ab responses. Thus, Tfh cells have critical roles in protective immune responses against pathogens, as well as deleterious roles in numerous autoimmune diseases (1, 2). Bcl6 consists of a bric-a-brac, tramtrack, broad-complex (BTB/POZ) domain, a middle domain (also known as RDII), and a zinc finger ML133 hydrochloride domain consisting of six Kruppel-like zinc fingers (1). BTB domains are evolutionarily conserved protein-interaction domains that are widely present in transcription factors (3, 4). The BTB domain forms the interface of the obligate homodimer, and the corepressors BCOR, SMRT, and NCOR bind at the cleft formed by this interface (5C8). Although Bcl6 is required for Tfh cell differentiation (9C12), the contributions of its functional domains in CD4+ T cells are not well understood. In this study, we sought to examine the role of the Bcl6 BTB domain in Tfh cell differentiation and function. Materials and Methods Mice and vectors C57BL/6J (B6) and CreCD4 ML133 hydrochloride mice were purchased from The Jackson Laboratory. (13), CD45.1-congenic, and Smarta TCRCtransgenic (SM; specific for lymphocytic choriomeningitis virus [LCMV] gp66C77 on I-Ab) (14) mice were on a full B6 background and were bred at the La Jolla Institute for Allergy and Immunology. mice, manufactured to express the Bcl6 BTB website mutant (BTBmut) from your endogenous locus, were generously provided by Dr. Ari Melnick (15). They were crossed to homozygosity in the La Jolla Institute for Allergy and Immunology for use in all experiments. NIP TCR-transgenic mice were generated as explained below and in Supplemental Fig. 1. TCR hybridomas were generated (J. White and P. Marrack, unpublished observations), and TCR sequences were cloned and sequenced using cDNA isolated from LCMV-reactive clones. TCR sequences were indicated in 58?- T cell hybridomas and tested for reactivity against LCMV-infected dendritic cells. The TCR pair showing the strongest reactivity (V1-J8 and V6-D1-J2.3 rearrangements) was chosen ML133 hydrochloride and cloned into genomic TCR expression cassette vectors. Linearized DNA fragments were ML133 hydrochloride injected into fertilized C57BL/6 eggs in the University or college California, San Diego Transgenic Mouse Facility (La Jolla, CA). Pups were genotyped (Supplemental Fig. 1). A single / TCR-transgenic founder mouse (NIP) was selected and crossed to B6.SJL mice to generate CD45.1+ NIP mice. All animal experiments were carried out in accordance with approved animal protocols. The GFP-expressing retroviral manifestation vector pMIG was used. BTBmut Bcl6 retrovirus (BTBmut-RV) was generated by inducing two point mutations in the protein connection website that do not impact dimerization (16). RV particles were ML133 hydrochloride produced as previously explained (9). Cell transfers into sponsor mice were performed as explained (9) by i.v. injection via the retro-orbital sinus. Transferred cells were allowed to rest in sponsor mice for 3C5 d before illness or immunization. 5 105 transduced Smarta cells were transferred into each mouse for day time 3 analysis, and 25 103 transduced Smarta cells were transferred into each mouse for day time 7 analysis. For protein immunization, 5 105 cells were transferred into each mouse. 5 103 naive CD4 T cells from NIP TCR-transgenic or retrogenic mice were Rabbit Polyclonal to Mouse IgG (H/L) transferred into each mouse. Infections and immunizations LCMV Armstrong stocks were prepared and quantified as previously explained (9). Infections were performed by i.p. injection of 0.5C2 .