Cutting edge: Extracellular signal-related kinase is not required for negative selection of developing T cells

Abstract

Signals initiated through the TCR during development can result in either survival and differentiation or cell death. High affinity signals that induce death elicit a robust yet transient activation of signaling pathways, including Erk, whereas low affinity ligands, which promote survival, generate a gradual and weaker activation of the same pathways. It was recently demonstrated that Erk localizes to distinct cellular locations in response to high and low affinity ligands. Although a requirement for Erk in positive selection is well established, its role in negative selection is controversial and, thus, the importance of Erk relocalization during development is not understood. In this study, we examined the role of Erk in negative selection using mice that are genetically deficient in both Erk1 and Erk2 in T cells. Results from three different models reveal that thymocyte deletion remains intact in the absence of Erk.

FIGURE 1

Erk is not required for peptide-induced death of thymocytes in vitro. A, Thymocytes were harvested from OT-I MHC^−/− (WT), OT-I MHC^−/−Erk1^−/− (Erk1^−/−), OT-I MHC^−/−Erk2^fn/fn pLck-cre (Erk2^−/−), and OT-I MHC^−/−Erk1^−/−Erk2^fn/fn pLck-cre (DKO) mice and cultured with peritoneal macrophages and OVAp for 18 h. The cells were stained with annexin V, anti-CD4, and anti-CD8 Abs and analyzed with a FACSCalibur flow cytometer. The number of live DP cells remaining in each well was calculated. Each condition was performed in triplicate and the average number of remaining live DP cells is plotted for three wells. These data are representative of three separate experiments. B, WT and DKO thymocytes were stained with Abs to Erk2, CD4, and CD8. The histograms depict the level of Erk2 after electronically gating on DN or DP thymocytes. The number above the line is the percentage of WT cells within the Erk2^low gate, and the number below the line is the percentage of DKO cells in the gate.

FIGURE 2

Erk is not required for Ag-mediated deletion of DP thymocytes. A, Thymic lobes were harvested at embryonic day 16–16.5 and cultured in medium. Two days later, 1 nM OVAp or 1 μM control peptide (P815p) was added to the cultures. The lobes were harvested 24 h later and stained with Abs to CD4 and CD8. The number of DP thymocytes from each lobe was calculated. Representative FACS plots of CD4 and CD8 expression are shown. B, The average number of DP thymocytes is plotted for one WT and one DKO lobe cultured with P815p and four WT, one Erk1^−/−, two Erk2^−/−, and four DKO lobes cultured with OVAp. These data are representative of three separate experiments. C, DP thymocytes were purified from WT or Erk2^−/− fetal lobes that had been cultured in medium for 2 days. DP thymocytes were sorted on a FACSAria (96% purity), lysed, and analyzed by Western blotting for Erk1 and Erk2 (Zymed Laboratories).

FIGURE 3

Erk is not required for Ag-mediated deletion in vivo. A, Representative plots of CD4 and CD8 expression on the OT-I donor (CD45.1^−/−CD45.2⁺) thymocytes are depicted. B, The average number of OT-I⁺ CD8 SP thymocytes in three CD45.1/1 or four CD45.1/1 RIP-mOVA mice is plotted. C, The level of Erk2 expression on electronically gated DN, DP, or CD8 OT-I donor cells is shown. The number above the line is the percentage of WT cells within the Erk2^low gate, and the number below the line is the percentage of DKO cells in the gate. D, Representative plots of Vα2 and CD8 expression on the OT-I donor (CD45.1^−/−CD45.2⁺) splenocytes are shown. E, The average number of Vα2⁺CD8⁺ splenocytes in three CD45.1/1 or four CD45.1/1 RIP-mOVA mice is plotted. F, The level of Erk2 expression on electronically gated Vα2⁺CD8⁺ OT-I donor cells is shown. These data are representative of three separate experiments.