Unexpected role of clathrin adaptor AP-1 in MHC-dependent positive selection of T cells

Abstract

Trafficking of transmembrane receptors to a specific intracellular compartment is conducted by adaptor molecules that bind to target motifs within the cytoplasmic domains of cargo proteins. We generated mice containing a lymphoid-specific deficiency of AP-1 using RNAi knockdown technology. Inhibition of AP-1 expression in thymocytes blocks progression from double-positive immature thymocytes, resulting in complete absence of CD4(+) single-positive thymocytes and severe reduction of CD3(+)CD8(+) single-positive thymocytes. Analysis of the contribution of AP-1 deficiency on the interaction between mature CD4(+) T cells and antigen-presenting cells revealed that AP-1 is essential to efficient immune synapse formation and associated T cell activation, suggesting a possible mechanism of AP-1 function in thymocyte development.

Fig. 1.

AP-1 is up-regulated in DP thymocytes. (A) Quantitative real-time PCR analysis of AP-1γ subunit. Cells isolated from 4- to 5-week-old C57BL/6 mice were sorted to >98% purity. AP-1 expression in different subsets was normalized to β-actin, and AP-1 expression was determined relative to peripheral CD4 (pCD4) cells, which was set as 1. Data shown represent mean ± SD of three independent experiments. BM, bone marrow; DN1, CD44⁺CD25⁻; DN2, CD44⁺CD25⁺; DN3, CD44⁻CD25⁺; DN4, CD44⁻CD25⁻; DN, CD4⁻CD8⁻; DP, CD4⁺CD8⁺; thSP, thymic single-positive cells; pSP, peripheral single-positive cells. (B) AP-1γ protein expression was analyzed by immunoblotting with anti-γ1-adaptin. Membrane was stripped and reprobed with anti-β-actin mAb. (C) Quantitative real-time PCR of AP-1 σ, μ and β subunits, as performed as in A.

Fig. 2.

Analysis of lymphocyte subsets in AP-1 KD mice. (A) Comparison of cell numbers between AP-1 KD and control mice in different organs. TH, thymus; LN, lymph nodes. (B) Flow cytometry of thymic subpopulations in AP-1–deficient mice (AP-1 KD) vs. GFP control mice (plasmid only). The percentage of different subpopulations is shown. (C) Comparison of cell numbers between AP-1 KD and GFP control mice in different thymocyte subsets: 1, DN cells; 2, CD4^loCD8^lo (DP low); 3, CD4^hiCD8^hi (DP high); 4, CD4⁺CD8^lo; 5, mature CD4 SP; 6, CD4^lo CD8^hi; 7, mature CD8 SP. Only GFP⁺ cells carrying lentivirus construct with GFP reporter were included in analysis.

Fig. 3.

AP-1 KD impairs preselection thymocyte maturation. Flow cytometric analysis of positive selection markers expressed on thymocyte subsets from AP-1 KD (gray) or GFP control mice (solid line). Thymocyte populations were gated as in Fig. 2B. (A) Histograms of CD69, CD5, and CD24 surface expression. (B) Histograms of TCRβ and CD3ε surface expression.

Fig. 4.

Intrathymic transfer of cells from GFP control or AP-1 KD mice. Approximately 10⁴ thymocytes at the DN stage were isolated from both strains of mice. Cells were transferred to (A) irradiated C57BL/6 mice or (B) MHC^−/− hosts. The number of GFP⁺TCRβ⁺ cells was calculated at the indicated time points.

Fig. 5.

AP-1 KD impairs peripheral CD4⁺ T cell function. (A) Representative AP-1 KD in peripheral CD4⁺ T cells. Cells were isolated from 5- to 7-week-old OT-II mice and lentivirally infected with vector only (GFP) or shRNA against AP-1β subunit for 4 to 5 h. Cells were cultured for 48 h in the presence of APC and 1 μg/mL anti-CD3ε. AP-1 complex expression was monitored by immunoblotting with anti-γ1-adaptin. Membrane was stripped and reprobed with anti-β-actin mAb. (B). Purified OT-II CD4⁺ T cells were infected as in A. T cells (1 × 10⁵) were cocultured with irradiated splenocytes (4 × 10⁵) from C57BL/6 in the presence of incremental concentrations of OVA peptide for 72 h. [³H] thymidine was included in cultures during the last 16–18 h. (C) Mouse CD4⁺ T cells were enriched by negative selection and infected with either GFP control or shRNA against AP-1γ. Cells (10⁵) were cultured for 72 h with 2 μg/mL anti-CD28 and increasing concentrations of anti-CD3ε Ab in the presence of [³H] thymidine during the last 18 h of culture. Data represent triplicates of three independent experiments.

Fig. 6.

AP-1 regulates immune synapse formation. (A) Primary mouse OT-II cells were lentivirally infected with vector only (GFP) or shRNA against AP-1γ subunit. Cells were cultured for 72 h and conjugated with OVA-pulsed B cells for 20–30 min at 37 °C on 0.01% poly-L-lysine–coated slides. Conjugates were fixed/permeabilized with methanol and stained with anti-CD3ε and anti-I-A^b. (B) Percentage of T cells that formed conjugates are mean ± SD from three independent experiments. N conjugates were analyzed for each group. Percentage synapse was calculated as TCR enrichment at the cell interface relative to the T cell periphery. Percentage IC polarization is percentage of T cells in conjugates that reposition TCR in intracellular compartment toward synapse.