RhoH as an Adaptor for ZAP-70 Figure 4. Lck enhances the association of RhoH with ZAP-70. HEK293 cells were transfected with combinations of plasmids expressing HA- RhoH, constitutively active Lck (CA-Lck), or ZAP-70. Total lysates of transfected HEK293 cells were immunoprecipitated using anti-HA or anti-ZAP-70 antibodies. IP products (A) and total lysates (B) were analyzed by immunoblotting. Asterisks denote IgG light chains. (C) Binding activities of CA-Lck and HA-RhoH to ZAP-70 and Tyr-phosphorylation levels of HA-RhoH. Relative amounts of bound ZAP-70 or total protein levels of the Lck and HA- RhoH and Tyr-phosphorylated HA-RhoH in (A) and (B) were quantified by densitometric measurements. Binding activities were calculated by the ratio of protein amounts of CA-Lck and HA-RhoH bound in ZAP-70 IP products to total lysates. Figure shows data from one of at least three independent experiments. doi:10.1371/journal.pone.0013970.g004 Next, we analyzed the engraftment and T cell development of Myr-ZAP-70-expressing Rhoh-/- bone marrow cells transplanted into Rag2-/- mice. Rhoh-/- bone marrow (BM) cells were transduced with a bi-cistronic retroviral vector co-expressing enhanced green fluorescent protein (EGFP) and HA-RhoH, Myr-ZAP-70 or EGFP alone (as control). Transduced and EGFP+-sorted BM cells were transplanted via intravenous injection into the sub-lethally irradiated Rag2-deficient recipient mice. Development of CD8 single positive (SP) thymocytes at 8 weeks post-transplantation in Rag2-/- mice infused with Myr-ZAP-70-transduced RhoH-/- BM cells was significantly increased compared with mice transplanted with Rhoh-/- BM cells transduced with the empty-vector and reached levels equivalent to mice transplanted with cells transduced and expressing the RhoH cDNA (Fig. 7A). In addition, there was a trend toward correction of CD4 SP thymocytes in these mice, although this correction did not reach statistical significance (2.3960.88 vs 16.75614.256106; CD4 SP, mean 6 SD; EGFP vs Myr-ZAP-70; N = 3). Total thymic cellularity was also increased in mice transplanted with Myr-ZAP-70-transduced Rhoh-/- BM cells (44.663.76106/mouse vs 104.8645.66106; mean 6 SD; EGFP vs Myr-ZAP-70; N = 3; p,0.05). We next investigated whether TCR signaling was corrected in Rhoh-/- thymocytes expressing Myr-ZAP-70. As previously not- ed[3], we confirmed that Rhoh-/- thymocytes showed impaired activation of LAT, CD3f and p44/42 (Fig. 7), all molecules involved in TCR signaling. Expression of Myr-ZAP-70 partially restored the activation of these molecules after anti-CD3/28 Abs stimulation. While we could not detect the phosphorylation of CD3f and LAT after anti-CD3/28 Abs stimulation in Rhoh-/- thymocytes expressing EGFP alone, expession of Myr-ZAP-70 in Rhoh-/- thymocytes led to increased phosphorylation of CD3f and LAT (Fig. 7B). To examine whether the defective translocation of Lck to the immunologic synapse was corrected in Rhoh-/- T cells expressing Myr-ZAP-70, we used anti-CD3/CD28 conjugated microbeads to activate T cells and induce IS formation[37,38]. We transduced Rhoh-/- lymph node (LN) T cells with retroviruses co- expressing EGFP and Myr-ZAP-70 or EGFP alone. EGFP+ sorted LN T cells were incubated with anti-CD3e and CD28 Abs, and then conjugated with Dynabeads. Lck was polarized towards the microbead interface of Rhoh-/- T cells expressing Myr-ZAP-70, while Lck was widely dispersed over the membrane of Rhoh-/- T cells expressing only EGFP (Fig. 7C left panel). The association of PLoS ONE | www.plosone.org 7 November 2010 | Volume 5 | Issue 11 | e13970