CD2 sets quantitative thresholds in T cell activation

Abstract

It has been proposed that CD2, which is highly expressed on T cells, serves to enhance T cell-antigen presenting cell (APC) adhesion and costimulate T cell activation. Here we analyzed the role of CD2 using CD2-deficient mice crossed with transgenic mice expressing a T cell receptor specific for lymphocytic choriomeningitis virus (LCMV)-derived peptide p33. We found that absence of CD2 on T cells shifted the p33-specific dose-response curve in vitro by a factor of 3-10. In comparison, stimulation of T cells in the absence of lymphocyte function-associated antigen (LFA)-1-intercellular adhesion molecule (ICAM)-1 interaction shifted the dose-response curve by a factor of 10, whereas absence of both CD2-CD48 and LFA-1-ICAM-1 interactions shifted the response by a factor of approximately 100. This indicates that CD2 and LFA-1 facilitate T cell activation additively. T cell activation at low antigen density was blocked at its very first steps, as T cell APC conjugate formation, TCR triggering, and Ca(2+) fluxes were affected by the absence of CD2. In vivo, LCMV-specific, CD2-deficient T cells proliferated normally upon infection with live virus but responded in a reduced fashion upon cross-priming. Thus, CD2 sets quantitative thresholds and fine-tunes T cell activation both in vitro and in vivo.

Figure 1

CD2–CD48 and LFA-1–ICAM-1 interactions enhance T cell proliferation at low peptide densities. Thioglycollate-elicited macrophages derived from control (filled symbols) or ICAM-1–deficient (open symbols) mice were pulsed with various doses of peptide p33 or the low-affinity ligand A4Y and used to stimulate T cells derived from TCR-transgenic control (squares) or CD2-deficient (circles) mice. Proliferation was assessed 36 h later by means of [³H]thymidine incorporation. Two independent experiments are shown. ▪, CD2⁺ICAM⁺; •, CD2⁻ICAM⁺; □, CD2⁺ICAM⁻; ○, CD2⁻ICAM⁻.

Figure 3

CD2–CD48 and LFA-1–ICAM-1 enhance T cell activation by altering signal 1. Thioglycollate-elicited macrophages derived from control or ICAM-1–deficient mice were pulsed with various doses of peptide p33, mixed with T cells derived from TCR-transgenic control or CD2-deficient mice, and centrifuged together. Expression of TCR (Vα2) was assessed 4 h later on CD8⁺ T cells. (A) TCR expression is shown for various combinations after stimulation with 10⁻¹⁰ M p33-pulsed macrophages. (B) Mean fluorescence of TCR expression is shown as a function of the peptide concentration for the various combinations. One representative experiment of three is shown. ▪, CD2⁺ICAM⁺; •, CD2⁻ICAM⁺; □, CD2⁺ICAM⁻; ○, CD2⁻ICAM⁻.

Figure 3

CD2–CD48 and LFA-1–ICAM-1 enhance T cell activation by altering signal 1. Thioglycollate-elicited macrophages derived from control or ICAM-1–deficient mice were pulsed with various doses of peptide p33, mixed with T cells derived from TCR-transgenic control or CD2-deficient mice, and centrifuged together. Expression of TCR (Vα2) was assessed 4 h later on CD8⁺ T cells. (A) TCR expression is shown for various combinations after stimulation with 10⁻¹⁰ M p33-pulsed macrophages. (B) Mean fluorescence of TCR expression is shown as a function of the peptide concentration for the various combinations. One representative experiment of three is shown. ▪, CD2⁺ICAM⁺; •, CD2⁻ICAM⁺; □, CD2⁺ICAM⁻; ○, CD2⁻ICAM⁻.

Figure 2

CD2–CD48 and LFA-1–ICAM-1 interaction enhances IFN-γ production at low peptide densities. Thioglycollate-elicited macrophages derived from control (filled symbols) or ICAM-1–deficient (open symbols) mice were pulsed with various doses of peptide p33 or the low-affinity ligand A4Y and used to stimulate T cells derived from TCR-transgenic control (squares) or CD2-deficient (circles) mice. Production of IFN-γ was assessed by ELISA 3 d later from culture supernatants. Two independent representative experiments are shown. ▪, CD2⁺ICAM⁺; •, CD2⁻ICAM⁺; □, CD2⁺ICAM⁻; ○, CD2⁻ICAM⁻.

Figure 4

CD2–CD48 and LFA-1–ICAM-1 enhance Ca²⁺ fluxes at low antigen concentration. Thioglycollate-elicited macrophages derived from control or ICAM-1–deficient mice were pulsed with various doses of peptide p33, mixed with INDO-1–pulsed, purified CD8⁺ T cells derived from TCR-transgenic control or CD2-deficient mice, and centrifuged together. Elevation of [Ca²⁺]_i was assessed by measuring the FL5/FL4 ratio. (A) FL5/FL4 ratio is shown after stimulation with 10⁻¹¹ M p33-pulsed macrophages. (B) Mean FL5/FL4 ratios are shown as a function of the peptide concentration for the various combinations. Baseline FL5/FL4 values were subtracted for the calculation. One representative experiment of two is shown. ▪, CD2⁺ ICAM⁺; •, CD2⁻ICAM⁺; □, CD2⁺ICAM⁻; ○, CD2⁻ICAM⁻.

Figure 4

CD2–CD48 and LFA-1–ICAM-1 enhance Ca²⁺ fluxes at low antigen concentration. Thioglycollate-elicited macrophages derived from control or ICAM-1–deficient mice were pulsed with various doses of peptide p33, mixed with INDO-1–pulsed, purified CD8⁺ T cells derived from TCR-transgenic control or CD2-deficient mice, and centrifuged together. Elevation of [Ca²⁺]_i was assessed by measuring the FL5/FL4 ratio. (A) FL5/FL4 ratio is shown after stimulation with 10⁻¹¹ M p33-pulsed macrophages. (B) Mean FL5/FL4 ratios are shown as a function of the peptide concentration for the various combinations. Baseline FL5/FL4 values were subtracted for the calculation. One representative experiment of two is shown. ▪, CD2⁺ ICAM⁺; •, CD2⁻ICAM⁺; □, CD2⁺ICAM⁻; ○, CD2⁻ICAM⁻.

Figure 5

CD2–CD48 interaction enhances T cell–APC conjugate formation. Thioglycollate-elicited macrophages were pulsed with various doses of peptide p33, mixed with INDO-1–pulsed, purified CD8⁺ T cells derived from TCR-transgenic control (▪) or CD2-deficient (•) mice, and centrifuged together, and conjugate formation was assessed. Data from two independent experiments were pooled, and the average and SD is shown.

Figure 7

Normal in vivo expansion of LCMV-GP–specific, CD2-deficient, TCR-transgenic T cells upon infection with live virus. CD45.1⁺ TCR-transgenic control spleen cells were mixed 1:1 with CD45.1⁺ CD2-deficient spleen cells and adoptively transferred into C57BL/6 recipient mice. (A) Left panel: 1:1 distribution of CD2⁺ and CD2⁻ TCR⁺ T cells was confirmed before transfer. Center and right panels: splenocytes from mice that received 10⁶ cells of the mixture shown in the left panel 6 d earlier in the absence of an infection were stained for Vα2, CD8, and CD45.1 expression; <2% of CD8⁺Vα2⁺ T cells were derived from the adoptively transferred T cells. (B and C) Recipient mice were infected with LCMV (200 PFU; B) or recombinant vaccinia virus expressing LCMV-GP (2 × 10⁶ PFU; C). CD45.1 expression was assessed for CD8⁺Vα2⁺ T cells, revealing expansion of CD2⁺ control T cells (upper right panels). CD2 expression was assessed similarly for CD8⁺Vα2⁺ T cells, revealing expansion of CD2-deficient T cells (lower right panels). Similar results were obtained 8 d after infection. One representative experiment of three is shown.

Figure 6

CD2 reduces the amount of peptide required for in vivo induction of CD44 expression. TCR-transgenic control (▪) and CD2-deficient (•) mice were injected with various amounts of peptide p33, and expression of CD44 was assessed 24 h later on CD8⁺Vα2⁺ T cells. The average of two mice is shown per dose. One representative experiment of three is shown.

Figure 8

Impaired in vivo expansion of LCMV-GP–specific, TCR-transgenic, CD2-deficient T cells upon cross-priming. CD45.1⁺ TCR-transgenic control spleen cells were mixed 1:1 with CD45.1⁺ CD2-deficient spleen cells and adoptively transferred into C57BL/6 recipient mice. (A) Recipient mice were immunized with UV light–inactivated recombinant vaccinia virus expressing LCMV-GP, and spleen cells were analyzed 6 d later. CD45.1 expression was assessed for CD8⁺Vα2⁺ T cells, revealing expansion of CD2⁺ control T cells (upper right panels). CD2 expression was assessed similarly for CD8⁺ Vα2⁺ T cells, revealing expansion of CD2-deficient T cells (lower right panels). (B) Recipient mice were immunized with LCMV-GP associated with cellular debris, and spleen cells were analyzed 6 d later. CD45.1 expression was assessed for CD8⁺Vα2⁺ T cells, revealing expansion of CD2⁺ control T cells (upper right panels). CD2 expression was assessed similarly for CD8⁺Vα2⁺ T cells, revealing expansion of CD2-deficient T cells (lower right panels). Percentage of CD45.1⁺ control T cells versus CD2-deficient T cells was <3% in the absence of immunization. One representative experiment of two is shown.