CD8αα and -αβ isotypes are equally recruited to the immunological synapse through their ability to bind to MHC class I

Abstract

Bimolecular fluorescence complementation was used to engineer CD8 molecules so that CD8αα and CD8αβ dimers can be independently visualized on the surface of a T cell during antigen recognition. Using this approach, we show that CD8αα is recruited to the immunological synapse almost as well as CD8αβ, but because the kinase Lck associates preferentially with CD8αβ in lipid rafts, CD8αα is the weaker co-receptor. During recognition of the strong CD8αα ligand H2-TL, CD8αα is preferentially recruited. Thus, recruitment of the two CD8 species correlates with their relative binding to the available ligands, rather than with the co-receptor functions of the CD8 species.

Figure 1

Principle of the bimolecular fluorescence complementation (BiFC) assay and specific CD8α1 and CD8β detection. (A) CD8α2–CC155 forms CD8αα when expressed with CD8α1–CN173, or CD8αβ when expressed with CD8β–CN173. Note that non-fluorescent CD8αα or CD8αβ can also be formed from CD8α1 or CD8α2 homodimers, or from CD8α1–CD8β heterodimers. (B,C) Cells expressing only the fluorescent molecules CD8α2α1 (Cerulean: shown as green) or CD8α2β (Venus, shown as red) were mixed at a 1:1 ratio and stained with allophycocyanin (APC)–anti-CD8α1 (B) or APC–anti-CD8β (C). The antibody staining is shown in blue.

Figure 2

Separate capping of CD8αα and CD8αβ. (A,B) Representative images of co-capping with anti-CD8α1 (anti-Ly2.1) (A) or anti-CD8β (B) monoclonal antibodies on cells expressing all three bimolecular fluorescence complementation (BiFC) constructs. On panels labelled Venus/Cerulean, the intensity ratio of Venus/Cerulean is represented using a high/low scale in which blue indicates a lower ratio value (more CD8α2α1) and red a higher ratio value (more CD8α2β). (C) Shows quantification of the differential capping of CD8α2α1 and CD8α2β as percentage of increase. Negative values reflect preferential recruitment of CD8α2α1 and positive values the preferential recruitment of CD8α2β. Error bars show s.e.m., n=16 for anti-α1 and n=30 for anti-β. APC, allophycocyanin.

Figure 3

CD8αα or αβ recruitment to the IS depends on the ability to bind to MHC-I. (A) T cell expressing CD8αα-Cerulean and CD8αβ-Venus interacting with a Cy5-labelled antigen-presenting cell (APC). For clarity, Cerulean and Venus channels merged with the Cy5 channel are presented separately. (B) Time course of differential recruitment of CD8αα-Cerulean and CD8αβ-Venus in cells responding to RMA-S cells presenting K^b-OVA. Graph shows the percentage of increase ±s.e.m., n⩾20. (C) T cells expressing CD8αα+CD8αβ, CD8αα+CD8αβ′ or CD8αα′+CD8αβ fluorescent proteins during interaction with ovalbumin (OVA) peptide-loaded EL4 cells. Cerulean and Venus channels are presented separately (left and centre) and merged with Cy5 (APC) channel in right panels. T cells and APCs are labelled on the merged image (T and A, respectively). (D) Preferential recruitment of wild-type co-receptor species compared with non-major histocompatibility complex-I-binding mutants to the immunological synapse (IS). Negative values reflect preferential recruitment of CD8αα and positive values the preferential recruitment of CD8αβ. Graph shows the percentage of increase ±s.e.m. n=17, 18 and 15.

Figure 4

Ligand dependence of the differential recruitment of CD8αα and CD8αβ. Data are presented for T cells expressing CD8αα-Cerulean and CD8αβ-Venus responding to RMA-S cells presenting K^b-OVA (OVA), RMA-S cells expressing thymus leukaemia antigen (H2-TL or TL) and also presenting K^b-OVA (TL+OVA) and RMA-S cells expressing TL alone (TL). Error bars represent s.d. n=25 for each group.