The mice used in the study (herein referred to as DKO) have been described previously (30). Crk fl/fl:CrkL fl/fl mice (49) (herein referred to as WT) were crossed with CD4+ Cre mice to generate mice lacking the Crk and CrkL loci in T cells, starting at the double-positive stage. Lifeact-GFP mice were described previously (104) and crossed with Crk fl/fl:CrkL fl/fl mice. The resulting Crk fl/fl:CrkL fl/fl lifeact-GFP mice were then crossed with CD4+ Cre mice to generate DKO lifeact-GFP mice. Primary mouse CD4+ T cells were purified from lymph nodes and spleens by negative selection. Briefly, after removing red blood cells by ACK lysis, cells were washed and incubated with anti-MHCII (major histocompatibility complex class II) and anti-CD8 hybridoma supernatants (M5/114 and 2.43, respectively) for 20 min at 4°C. After washing, cells were mixed with anti-rat immunoglobulin (Ig) magnetic beads (Qiagen BioMag), incubated for 15 min at 4°C, and subjected to three rounds of magnetic separation using a bench top magnet. The resulting CD4+ T cells were then immediately activated on 24-well plates coated with anti-CD3 and anti-CD28 (2C-11 and PV1, 1 μg/ml each) at 1 × 106 cells per well. Activation was done in T cell complete medium composed of Dulbecco’s modified Eagle’s medium (DMEM) (11885-084, Gibco) supplemented with 5% fetal bovine serum (FBS), penicillin/streptomycin, nonessential amino acids, GlutaMAX, and 2 μl of 2-mercaptoethanol. Unless otherwise indicated, all tissue culture reagents were from Gibco. After 48 hours, cells were removed from activation and mixed at a 1:1 volume ratio with complete T cell medium containing recombinant interleukin-2 (IL-2) [obtained through the National Institutes of Health (NIH) AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH from M. Gately, Hoffmann–La Roche Inc. (105)] to give a final IL-2 concentration of 25 U/ml. T cells were used on days 5 to 7 after activation.

Platinum-E (Plat-E) retroviral packaging cells were a gift from M. Marks (Children’s Hospital of Philadelphia) and were cultured in DMEM supplemented with 10% FBS, penicillin/streptomycin, and nonessential amino acids. For retroviral production, Plat-E cells were plated to 50% confluence on day 0. On day 1, cells were transfected with pMSCV encoding GRP1-PH-GFP (a gift from M. Huse, Sloan Kettering) using the calcium phosphate method. On day 2, the medium was replaced, and on day 4, the virus-containing supernatant was harvested, aliquoted, and frozen at −80°C.

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