Dynamics of Proximal Signaling Events after TCR/CD8-Mediated Induction of Proliferation or Apoptosis in Mature CD8+ T Cells

Abstract

Engagement of the TCR can induce different functional outcomes such as activation, proliferation, survival, or apoptosis. How the TCR-mediated signaling cascades generating these distinct cellular responses are organized on the molecular level is so far not completely understood. To obtain insight into this question, we analyzed TCR/CD8-mediated signaling events in mature OT-I TCR transgenic T cells under conditions of stimulation that lead to either proliferation or apoptosis. These experiments revealed major differences in the phosphorylation dynamics of LAT, ZAP70, protein kinase B, phospholipase C-γ1, protein kinase D1, and ERK1/2. Moreover, input signals leading to apoptosis induced a strong, but transient activation of ERK1/2 mainly at sites of TCR-engagement. In contrast, stimuli promoting survival/proliferation generated a low and sustained activation of ERK1/2, which colocalizes with Ras in recycling endosomal vesicles. The transient activation of ERK1/2 under pro-apoptotic conditions of stimulation is at least partially due to the rapid polyubiquitination and subsequent degradation of ZAP70, whereas the sustained activation of ERK1/2 under survival promoting conditions is paralleled by the induction/phosphorylation of anti-apoptotic molecules such as protein kinase B and Bcl-xL. Collectively, our data provide signaling signatures that are associated with proliferation or apoptosis of T cells.

Analysis of TCR activation kinetics in primary human T cells upon focal or soluble stimulation

“…Indeed, we and others have demonstrated that OT-I transgenic T cells and cytotoxic T-lymphocyte clones are not activated and do not differentiate upon stimulation with antibodies cross-linked in suspension (Berg et al, 1998;Wang et al, 2008). Here, we have stimulated primary human T cells with either sAbs or iAbs and analyzed their functional responses.…”

“…For more than 30 years, this method has been extensively employed and indeed, has provided significant insight into the molecular events occurring during T-cell activation. However, stimulations with soluble ligands have clear limitations, as they fail to reproduce the extra dimension of a cell-cell contact and, more importantly, they do not induce T-cell responses (Berg et al, 1998;Wang et al, 2008). Thus, it is questionable whether TCR signaling induced upon stimulation with soluble Abs mirrors the physiologic signaling cascade leading to productive T-cell responses.…”

RasGRP1 Regulates Antigen-Induced Developmental Programming by Naive CD8 T Cells

“…Using OT-1 TCR Tg preselection CD4 + CD8 + thymocytes and specific peptides spanning the boundary of positive and negative selection, it was found that negatively selecting peptides recruited Ras, Raf-1, and RasGRP1 to the PM, whereas these molecules colocalized to internal cellular sites, perhaps the Golgi and/or other endomembranes, when thymocytes were stimulated with a peptide mediating positive selection (56). In addition, studies on peripheral OT-1 CD8 T cells correlated active ERK being localized to the PM when cells were induced to undergo apoptosis, whereas a condition promoting survival and proliferation was associated with ERK activation present at endosomal compartments (57). The rationale for alternate cellular sites of Ras activity may be to subject it to differential regulation and/or serve to pair it with a unique subset of effectors.…”

Duality of the murine CD8 compartment

“…Although CD8αα and CD8αβ bind similarly to MHC class I molecules (8), CD8β endows CD8 with coreceptor functions. Indeed, CD8αβ, but not CD8αα, associates with TCR/ CD3, strengthens pMHC binding (3-5, 9, 10), and promotes CD8 association with lipid rafts and p56 lck (lymphocyte-specific tyrosine kinase, lck) and hence TCR signaling via lck-mediated phosphorylation of CD3 ITAMs, followed by recruitment and activation of Zeta-chain-associated protein kinase 70 (ZAP-70), phosphorylation of Linker for activation of T cells (LAT), and diverse downstream signaling cascades, including activation of phospholipase C-γ (PLCγ), mobilization of intracellular Ca 2+ , and translocation of the transcription factor, Nuclear factor of activated T cells (NFAT) (3)(4)(5)(9)(10)(11). CD8β-KO mice have two-to-threefold lower numbers of CD8 + T cells, showing that CD8β plays an important but not essential role in the thymic selection of CD8 + T cells (5,12,13).…”