This leads us to speculate that with tools of the appropriate sensitivity,
one should be able to find a large number of autoreactive T cells, even in a normal repertoire, maintained in a tolerant state by nondeletional mechanisms. Mice from the NIAID contract facility (Taconic Farms, Germantown, NY, USA) were housed pathogen free. B10.A CD45.2 mice were also crossed to B6,CD45.1 mice to generate a B10.A,CD45.1 strain . To generate B10.A, mPCC(tg),CD45.1 mice, B10.A mPCC-transgenic, CD45.2 mice  were bred to B10.A,CD45.1. The IEk restricted MCC (Moth Cytochrome C)/PCC specific TCR transgenic 5C.C7 mice on Rag2−/−, CD45.1+/+, and CD45.2+/+ backgrounds have been previously described . A1(M) mice originally from Steve Cobbold selleck  on a CBA/Ca background were backcrossed 11 times onto a B10.A,Rag2−/− background  and maintained by homozygous breeding. All animal protocols were as approved by the NIAID animal care and use committee. For adoptive cell transfers, cell suspensions from pooled lymph nodes of donor TCR-Tg Rag2−/− mice (>90% CD4+ T cells) were used without further enrichment and injected by the suborbital route. Acute antigen challenges were performed by intraperitoneal
injections of 30 μg of antigenic peptide (DbY or PCC; Anaspec or Bachem, USA) mixed with 5 μg of LPS (Sigma, MI, USA). T cells in transfer recipients were enumerated by isolating all lymph nodes and spleen, chopping them to approximately 1 mm cubes and digesting buy RG-7388 with 2 mg/mL collagenase-D (Roche, USA) solution containing 3 mM CaCl2 in 1× PBS, at 37°C for 45 min. Digested tissue was dissociated using gentleMACS dissociator and gentleMACS dissociator C tubes (Miltenyi biotec, Germany) with manufacturer’s programmed settings m_Spleen 2.01 followed by m_Spleen 3.02 run serially on each sample. A total of Dynein 500 μL aliquots of the single cell suspensions were stained to obtain the percentage of CD4+ T cells and used to calculate the number of CD4+ T cells in each animal without any further manipulation. However, in order to track exceedingly low numbers
of transferred T cells, further enrichment was necessary. Following absolute counts, as stated above, as remaining cells were washed and centrifuged over Ficoll-Paque PLUS (GE Healthcare Bioscience) followed by enrichment for T cells by negative selection. Briefly, a cocktail of mouse and rat antibodies to B220 (RA3-6B2), CD11b (M1/770), I-EK (14.4.4s), CD8 (53-6.7), and MHC II (M5.114) (BD Bioscience) were used to label the cells and the bound fraction, pulled out using anti-mouse IgG and anti-rat IgG coated Dynabeads (Dynal Invitrogen). T cells were analyzed on a FACS Canto II cytometer (BD Immunocytometry) after staining with appropriate fluorophore coupled antibodies (Biolegend, Ebioscience or BD). We thank Eleanore Chuang for assistance with experiments, and Pascal Chappert for discussions. This research was supported by the Intramural Research Program of the NIH, NIAID.