Transgenic Expression of RasGRP1 Induces the Maturation of Double-Negative Thymocytes and Enhances the Production of CD8 Single-Positive Thymocytes

Abstract

RasGRP1 is a guanine nucleotide exchange factor for Ras that is required for the efficient production of both CD4 and CD8 single-positive thymocytes. We found that RasGRP1 expression is rapidly up-regulated in double-negative thymocytes following pre-TCR ligation. Transgenic overexpression of RasGRP1 compensated for deficient pre-TCR signaling in vivo, enabling recombinase-activating gene 2−/− double-negative thymocytes to mature to the double-positive stage. RasGRP1 transgenic mice had a 4-fold increase in CD8 single-positive thymocytes, most of which had atypically low levels of CD3. The RasGRP1 transgene lowered the threshold of TCR signaling needed to initiate proliferation of single-positive thymocytes, with this effect being particularly evident among CD8 single-positive cells. In 3-day cultures, TCR stimulation via anti-CD3 caused a 10-fold increase in the ratio of CD8 to CD4 thymocytes among RasGRP1 transgenic vs nontransgenic thymocytes. These results demonstrate that in addition to driving the double-negative to double-positive transition, increased expression of RasGRP1 selectively increases CD8 single-positive thymocyte numbers and enhances their responsiveness to TCR signaling.

Deregulated expression of RasGRP1 initiates thymic lymphomagenesis independently of T-cell receptors

“…RasGRP1 is activated in response to TCR ligation Bivona et al, 2003), and transduces signals that enable TCR-mediated positive selection at the DP stage (Dower et al, 2000;Priatel et al, 2002;Layer et al, 2003) as well as enhanced survival and TCR-induced proliferation at the SP stage (Norment et al, 2003). This raised the question of whether the lymphomas in RasGRP1 transgenic mice were caused by amplified signaling downstream of TCR, leading to an exaggeration of the survival and proliferative responses which confer positive selection.…”

“…In RasGRP1 Tg þ /Rag2 À/À mice, the developmental block due to pre-TCR deficiency was over-ridden, resulting in a 10-fold expansion of the DN population and the generation of ISP and DP thymocytes (Figure 7a, middle panel). This reflects the ability of RasGRP1 to provide proliferative and developmental progression signals which partially compensate for pre-TCR deficiency (Norment et al, 2003). The RasGRP1 Tg þ / Rag2 À/À mice had a high death rate (Figure 7b) due to thymic lymphomas comprised of transformed DN, ISP and DP thymocytes (example shown in Figure 7a, right panel).…”

“…The efficiency of TCR-directed positive selection of DP thymocytes is severely compromised in RasGRP1-deficient mice (Dower et al, 2000;Priatel et al, 2002;Layer et al, 2003). Conversely, RasGRP1 transgenic mice have increased numbers of CD8 SP thymocytes that have enhanced proliferative and survival responses to TCR ligation (Norment et al, 2003). Thus, RasGRP1 serves to transduce signals from pre-TCR and TCR which allow thymocytes to pass through the two critical checkpoints during T-cell development, b selection and positive selection.…”

“…The transgene-encoded RasGRP1 protein is expressed at moderate to low levels in all thymocyte subsets of normal (lymphoma-free) mice of the AM1268 line (Figure 3), resulting in a net doubling of RasGRP1 protein levels relative to nontransgenic thymocytes (Norment et al, 2003). Transgenic RasGRP1 expression was similar in the 293H line except for minimal expression in DP thymocytes (Figure 3, mouse S), whereas in the 13H line expression of transgenic RasGRP1 protein was detected only in a minor portion In contrast, transgenic RasGRP1 expression was high in all transformed thymocytes within lymphomas arising in RasGRP1 transgenic mice (e.g.…”

“…Pre-TCR ligation or TCR-MHC interactions result in transcriptional induction of the RasGRP1 gene (Norment et al, 2003;Tiong Ong et al, 2003), and biochemical activation of RasGRP1 is dependent on signal transduction from TCR via phospholipase Cg1 Bivona et al, 2003). Although RasGRP1 is not required for pre-TCR-directed b selection in normal mice (Dower et al, 2000), constitutive transcription of RasGRP1 from a transgene confers partial developmental progression up to the DP stage in the absence of pre-TCR (Norment et al, 2003). The efficiency of TCR-directed positive selection of DP thymocytes is severely compromised in RasGRP1-deficient mice (Dower et al, 2000;Priatel et al, 2002;Layer et al, 2003).…”