CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms

Abstract

CTLA-4 and PD-1 are receptors that negatively regulate T-cell activation. Ligation of both CTLA-4 and PD-1 blocked CD3/CD28-mediated upregulation of glucose metabolism and Akt activity, but each accomplished this regulation using separate mechanisms. CTLA-4-mediated inhibition of Akt phosphorylation is sensitive to okadaic acid, providing direct evidence that PP2A plays a prominent role in mediating CTLA-4 suppression of T-cell activation. In contrast, PD-1 signaling inhibits Akt phosphorylation by preventing CD28-mediated activation of phosphatidylinositol 3-kinase (PI3K). The ability of PD-1 to suppress PI3K/AKT activation was dependent upon the immunoreceptor tyrosine-based switch motif located in its cytoplasmic tail, adding further importance to this domain in mediating PD-1 signal transduction. Lastly, PD-1 ligation is more effective in suppressing CD3/CD28-induced changes in the T-cell transcriptional profile, suggesting that differential regulation of PI3K activation by PD-1 and CTLA-4 ligation results in distinct cellular phenotypes. Together, these data suggest that CTLA-4 and PD-1 inhibit T-cell activation through distinct and potentially synergistic mechanisms.

FIG. 1.

CTLA-4 and PD-1 engagements inhibit T-cell activation and glucose metabolism. CD4 T cells were stimulated with immunobeads coated with the antibodies indicated, and (A) T-cell expansion, (B) IL-2 expression, (C) uptake of [³H]2-deoxyglucose, and (D) glycolysis were measured. IL-2 expression, uptake of [³H]2-deoxyglucose, and glycolysis were measured 18 h after activation. All data are representative of three independent experiments. CPM, counts per million.

FIG. 2.

CTLA-4 ligation blocks Akt but not PI3K activation. (A) CD4 T cells were preactivated with 5 mg/ml PHA and 100 U/ml IL-2 for 3 days and stimulated with immunobeads coated with the antibodies indicated for 30 min. Cells were lysed and proteins were resolved by SDS-PAGE. Akt and GSK-3 phosphorylation was assessed by immunoblotting using phosphospecific Abs (upper panel). The blot was then stripped and reprobed with anti-Akt antibody (lower panel) to demonstrate equal loading. (B) The procedure described for panel A was used, except cells were harvested 30 min (30′), 4 h, and 24 h after activation. (C) PHA blasts were stimulated with immunobeads coated with antibodies as indicated for either 30 min or 60 min. Cells were lysed, and stimulatory beads were used to precipitate the appropriate receptors. Lipid kinase activity associated with receptors was assessed by in vitro kinase assays. Reaction products were resolved by thin-layer chromatography and were visualized using a PhosphorImager. Whole-cell lysate (5 μl) was used as a positive control for the assay. PtdIns, phosphatidylinositol.

FIG. 3.

PD-1's ITSM motif is required to mediate PD-1 suppression of the PI3K/Akt pathway. CD4 T cells were activated with CD3/CD28-coated beads and transduced with lentiviral vectors expressing mCD28-PD-1 wild type (WT), mCD28-hPD-1 Y223F, mCD28-PD-1 Y248F, and the double mutant mCD28-hPD-1 Y223F, Y248F. (A) Expression of each construct was examined by staining the cells with an Ab specific for mCD28 3 days after transduction. (B) Transduced cells were left unstimulated or were restimulated with the indicated aAPCs for 30 min. Cells were lysed, and proteins were resolved by SDS-PAGE. Akt activity was assessed by immunoblotting for serine-phosphorylated Akt (upper panel). The blot was then stripped and reprobed with anti-Akt antibody (lower panel) to demonstrate equal loading. Data are representative of three independent experiments.

FIG. 4.

CTLA-4-mediated suppression, but not PD-1-mediated suppression, of AKT is blocked by the presence of okadaic acid. PHA-IL-2-cultured CD4 T cells were stimulated with immunobeads coated with the indicated antibodies in either the presence or absence of okadaic acid. Akt activity was assessed by immunoblotting for serine-phosphorylated Akt [(p)Akt] (upper panel). The blot was then stripped and reprobed with anti-Akt antibody (lower panel) to demonstrate equal loading. The relative ratio of pAKT to total AKT is shown below each lane. Data are representative of three independent experiments. Unstim, unstimulated.

FIG. 5.

(A) PI3K is necessary to mediate CTLA-4-resistant Bcl-xL induction. CD4 T lymphocytes were stimulated with immunobeads coated with the antibodies indicated in either the absence or presence of LY294002 (3 μM or 10 μM) for 24 h. Bcl-xL expression was quantified by RT-PCR and expressed as a comparison to Bcl-xL expression in unstimulated cells. The error bars indicate the standard deviations of three replicates, and data are representative of three independent experiments. (B) An intact ITSM is required to mediate PD-1 suppression of Bcl-xL induction. The same set of transduced cells described in the legend to Fig. 3 was left either unstimulated or was restimulated with either CD3/CD28/MHC I- or CD3/CD28/mCD28-coated beads for 24 h. Quantitative RT-PCR to measure Bcl-xL expression was performed as described for panel A. WT, wild type.

FIG. 6.

PD-1 and CTLA-4 engagement each uniquely affect the T-cell transcriptional profile. Primary human CD4 T cells were cultured for 24 h with either CD3/CD28/CTLA-4- or CD3/CD28/PD-1-coated beads. Total RNA was isolated, amplified, and analyzed by DNA microarray hybridization. Shown are comparisons (correlations) of genes significantly regulated (P < 0.01; intensity, >−1) in CD3/CD28/CTLA-4-stimulated T cells (x axis) versus that in CD3/CD28/PD-1-stimulated T cells (y axis). The color scheme is as follows: red genes represent genes significantly regulated under both conditions; green genes represent genes significantly regulated in the x dimension; blue genes represent genes significantly regulated in the y dimension; brown genes represent genes showing opposite regulation in the two conditions; gray genes are transcripts not significantly regulated in either dimension. This experiment was repeated twice with equivalent results. The gene lists for each of these distinct regulations are found in Table S1 in the supplemental material.

FIG. 7.

Model of CTLA-4- and PD-1-mediated T-lymphocyte inhibition. Signaling by CTLA-4 preserves PI3K activity, allowing expression of certain genes such as Bcl-xL, but inhibits Akt directly by activation of the phosphatase PP2A. In contrast, PD-1 antagonizes PI3K activity directly, perhaps by effecting a more global inhibition of T lymphocyte function. Since CTLA-4 and PD-1 target Akt by different mechanisms, their effect in concert may be additive or synergistic.