CD8 beta increases CD8 coreceptor function and participation in TCR-ligand binding

Abstract

To study the role of CD8 beta in T cell function, we derived a CD8 alpha/beta-(CD8-/-) T cell hybridoma of the H-2Kd-restricted N9 cytotoxic T lymphocyte clone specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260. This hybridoma was transfected either with CD8 alpha alone or together with CD8 beta. All three hybridomas released interleukin 2 upon incubation with L cells expressing Kd-peptide derivative complexes, though CD8 alpha/beta cells did so more efficiently than CD8 alpha/alpha and especially CD8-/- cells. More strikingly, only CD8 alpha/beta cells were able to recognize a weak agonist peptide derivative variant. This recognition was abolished by Fab' fragments of the anti-Kd alpha 3 monoclonal antibody SF1-1.1.1 or substitution of Kd D-227 with K, both conditions known to impair CD8 coreceptor function. T cell receptor (TCR) photoaffinity labeling indicated that TCR-ligand binding on CD8 alpha/beta cells was approximately 5- and 20-fold more avid than on CD8 alpha/a and CD8-/- cells, respectively. SF1-1.1.1 Fab' or Kd mutation D227K reduced the TCR photoaffinity labeling on CD8 alpha/beta cells to approximately the same low levels observed on CD8-/- cells. These results indicate that CD8 alpha/beta is a more efficient coreceptor than CD8alpha/alpha, because it more avidly strengthens TCR-ligand binding.

Figure 1

CD8α/β cells more efficiently recognize IASA-YIPSAEK (ABA)I and IASA-YIPSAEK(BA)I than CD8α/α or CD8^−/−cells. The IL-2 released by CD8^−/− (A), CD8α/α (B), and CD8α/β (C) cells was measured as [³H]thymidine uptake by CTLL indicator cells after incubation with L-K^d cells sensitized with IASA-YIPSAEK(ABA)I (○) or IASA-YIPSAEK(BA)I (•). In insets, the IL-2 responses are shown as observed after incubation with anti-CD3 mAb or PMA and ionomycin.

Figure 3

TCR photoaffinity labeling with soluble ligand K^d–peptide derivative complexes. CD8α/β, CD8α/α or CD8^−/− cells were incubated with K^d-¹²⁵ IASA-YIPSAEK(ABA)I in absence (black bars) or presence of SF1-1.1.1 Fab′ (20 μg/ml) (white bars) or 20-8-4S mAb (10 μg/ml) (hatched bars), and TCR photoaffinity labeling was evaluated by SDSPAGE and autoradiography (A) and densitometry (B). Alternatively, CD8α/β and CD8^−/− cells, respectively, were tested likewise, either with K^d-IASA-YIPSAEK(ABA)I (black bars) or mutant K^dD227K-IASA-YIPSAEK(BA)I (white bars) (C). In both experiments, 100% refers to the TCR labeling observed on CD8α/β cells with the wild type ligand. The TCR binding of K^d-IASA-YIPSAEK(BA)I was assessed by its ability to inhibit the TCR photoaffinity labeling by K^d-¹²⁵ IASA-YIPSAEK (ABA)I on N9 CTL (D). Mean values and standard deviations were calculated from at least three independent experiments.

Figure 3

TCR photoaffinity labeling with soluble ligand K^d–peptide derivative complexes. CD8α/β, CD8α/α or CD8^−/− cells were incubated with K^d-¹²⁵ IASA-YIPSAEK(ABA)I in absence (black bars) or presence of SF1-1.1.1 Fab′ (20 μg/ml) (white bars) or 20-8-4S mAb (10 μg/ml) (hatched bars), and TCR photoaffinity labeling was evaluated by SDSPAGE and autoradiography (A) and densitometry (B). Alternatively, CD8α/β and CD8^−/− cells, respectively, were tested likewise, either with K^d-IASA-YIPSAEK(ABA)I (black bars) or mutant K^dD227K-IASA-YIPSAEK(BA)I (white bars) (C). In both experiments, 100% refers to the TCR labeling observed on CD8α/β cells with the wild type ligand. The TCR binding of K^d-IASA-YIPSAEK(BA)I was assessed by its ability to inhibit the TCR photoaffinity labeling by K^d-¹²⁵ IASA-YIPSAEK (ABA)I on N9 CTL (D). Mean values and standard deviations were calculated from at least three independent experiments.

Figure 3

TCR photoaffinity labeling with soluble ligand K^d–peptide derivative complexes. CD8α/β, CD8α/α or CD8^−/− cells were incubated with K^d-¹²⁵ IASA-YIPSAEK(ABA)I in absence (black bars) or presence of SF1-1.1.1 Fab′ (20 μg/ml) (white bars) or 20-8-4S mAb (10 μg/ml) (hatched bars), and TCR photoaffinity labeling was evaluated by SDSPAGE and autoradiography (A) and densitometry (B). Alternatively, CD8α/β and CD8^−/− cells, respectively, were tested likewise, either with K^d-IASA-YIPSAEK(ABA)I (black bars) or mutant K^dD227K-IASA-YIPSAEK(BA)I (white bars) (C). In both experiments, 100% refers to the TCR labeling observed on CD8α/β cells with the wild type ligand. The TCR binding of K^d-IASA-YIPSAEK(BA)I was assessed by its ability to inhibit the TCR photoaffinity labeling by K^d-¹²⁵ IASA-YIPSAEK (ABA)I on N9 CTL (D). Mean values and standard deviations were calculated from at least three independent experiments.

Figure 2

The recognition of IASA-YIPSAEK(BA)I by CD8α/β cells is abrogated by SF1-1.1.1 Fab′ and K^d mutation D227K. The IL-2 release by CD8α/β cells was measured after incubation with L-K^d sensitized with IASA-YIPSAEK(BA)I in the absence (○) or presence (•) of SF11.1.1 Fab′ (A), as described for Fig. 1. Alternatively, L-K^d cells expressing K^d D227K were used as presenting cells, which were sensitized either with IASA-YIPSAEK(ABA)I (○) or IASA-YIPSAEK(BA)I (•) (B).