Formation of a central supramolecular activation cluster is not required for activation of naive CD8+ T cells

Abstract

Although both naive and effector T lymphocytes interact with antigen-expressing cells, the functional outcome of these interactions is distinct. Naive CD8(+) T cells are activated to proliferate and differentiate into effector cytolytic T lymphocytes (CTL), whereas CTL interact with specific targets, such as tumor cells, to induce apoptotic death. We recently observed that several molecules linked to actin cytoskeleton dynamics were up-regulated in effector vs. naive CD8(+) T cells, leading us to investigate whether T cell differentiation is accompanied by changes in actin-dependent processes. We observed that both naive and effector CD8(+) T cells underwent T cell receptor capping and formed stable conjugates with antigen-specific antigen-presenting cells. However, the characteristics of the immunological synapse were distinct. Whereas accumulation of signaling molecules at the T cell/antigen-presenting cell contact site was detectable in both naive and effector CD8(+) T cells, only effector cells developed a central supramolecular activation cluster as defined by punctate focusing of PKC theta, phospho-PKC theta, and phospho-ZAP70. Extended kinetics, CD28 costimulation, and high-affinity antigenic peptide did not promote PKC theta focusing in naive cells. Nonetheless, naive CD8(+) T cells polarized the microtubule organizing center, produced IL-2, proliferated, and differentiated into effector cells. Our results suggest that the formation of a central supramolecular activation cluster is not required for activation of naive CD8(+) T cells and support the notion that one role of an organized immune synapse is directed delivery of effector function.

Fig. 1.

TCR capping and conjugate formation are comparable in naive and effector CD8⁺ T cells. (A) Naive or effector T cells were preincubated with anti-CD3ε-FITC. Prewarmed goat anti-hamster Ab was added, and cells were incubated at 37°C for indicated durations of time, fixed, and analyzed by confocal microscopy. (B) Naive and effector CD8⁺ T cells and either EL4 cells or P815 cells were conjugated for the indicated times. Two-color flow cytometric analysis was performed, and percent conjugates were determined by calculating the ratio of double-positive cells to the total T cell population. Data are representative of three independent experiments.

Fig. 2.

Effector, but not naive, CD8⁺ T cells detectably focus PKCθ. (A) Naive or effector CD8⁺ T cells were cocultured for 8 min with CMAC-labeled P815 (blue), fixed, stained for talin (red) and PKCθ (green), and imaged by immunofluorescence microscopy, and z axis image reconstructions of the interface were performed. Representative images of conjugates displaying only localized PKCθ (Upper) or focused PKCθ (Lower) are shown. (B) CD8⁺ T cells in contact with P815 cells with talin enrichment at the interface were scored as conjugates. The percentage of conjugates with localized PKCθ was calculated. (C) Conjugates that localized PKCθ were rescored for punctuate focusing of PKCθ, and this percentage was calculated. Results are representative of five similar experiments.

Fig. 3.

CD28 costimulation, extended kinetics, and high-affinity peptide fail to promote PKCθ focusing in naive CD8⁺ T cells. (A and B) Effector CD8⁺ T cells were cocultured with either CMAC-labeled P815 or P815-B7.1 cells for 8 min in the presence of medium (None), isotype control Ab (Cont), or CTLA4Ig and scored for PKCθ localization and focusing. (C–F) Either naive or effector CD8⁺ T cells were cocultured for the indicated time with CMAC-labeled P815.B71 or T2-L^d either in the presence or absence of the QL9 peptide. The percentage of conjugates with localized PKCθ (C and E), and of these conjugates the percentage of those displaying focused PKCθ (D and F) was calculated. Data are representative of two independent experiments.

Fig. 4.

Neither phospho-PKCθ nor phospho-Zap70 display cSMAC focusing in naive CD8⁺ T cells. Naive or effector CD8⁺ T cells were cocultured for 8 min with CMAC-labeled P815 (blue), fixed, stained for talin (red) and either phospho-PKCθ (A–C) or phospho-Zap70 (D–F) (green), and imaged by immunofluorescence microscopy. (A) Representative images of conjugates displaying localized (Upper) or focused (Lower) phospho-PKCθ are shown. The percentages of conjugates displaying localized and focused phospho-PKCθ are shown in B and C, respectively. (D) Representative images of naive and effector conjugates showing the predominant staining patterns observed. The percentages of conjugates displaying localized and focused phospho-Zap70 among naive and effector cells are shown in E and F. Data are representative of three independent experiments.

Fig. 5.

MTOC polarization is comparable in naive and effector CD8⁺ T cells. Naive or effector CD8⁺ T cells were cocultured for 8 min with CMAC-labeled P815-B7.1 (blue), fixed, stained for tubulin, and imaged by immunofluorescence microscopy. (A) Representative images of conjugates displaying polarized MTOC are shown. (B) The percentages of conjugates displaying MTOC localized to the one-third of the T cells proximal to the interface were scored as polarized. Data are representative of two independent experiments.

Fig. 6.

Naive CD8⁺ T cells produce IL-2 despite lack of detectable cSMAC formation. (A) Either naive or effector CD8⁺ T cells were cultured in either the presence or absence of P815-B7.1 cells, and IL-2 protein secretion was assessed at 16 h. (B) Naive CD8⁺ T cells and P815-B7.1 were either cultured separately (Left) or cocultured (Right) for 4 h, and serial dilutions of cDNA were assayed for IL-2 mRNA (Upper) and β-action mRNA (Lower). C and D show, at 4 h, the percentage of conjugates with localized PKCθ and, of these conjugates, the percentage displaying focused PKCθ. Data are representative of three independent experiments.