Requirements for CD1d recognition by human invariant Valpha24+ CD4-CD8- T cells

Abstract

A subset of human CD4-CD8- T cells that expresses an invariant Valpha24-JalphaQ T cell receptor (TCR)-alpha chain, paired predominantly with Vbeta11, has been identified. A series of these Valpha24 Vbeta11 clones were shown to have TCR-beta CDR3 diversity and express the natural killer (NK) locus-encoded C-type lectins NKR-P1A, CD94, and CD69. However, in contrast to NK cells, they did not express killer inhibitory receptors, CD16, CD56, or CD57. All invariant Valpha24(+) clones recognized the MHC class I-like CD16 molecule and discriminated between CD1d and other closely related human CD1 proteins, indicating that recognition was TCR-mediated. Recognition was not dependent upon an endosomal targeting motif in the cytoplasmic tail of CD1d. Upon activation by anti-CD3 or CD1d, the clones produced both Th1 and Th2 cytokines. These results demonstrate that human invariant Valpha24+ CD4-CD8- T cells, and presumably the homologous murine NK1+ T cell population, are CD1d reactive and functionally distinct from NK cells. The conservation of this cell population and of the CD1d ligand across species indicates an important immunological function.

Figure 1

Invariant Vα24⁺ TCR-α nucleotide and derived amino acid sequences. DN invariant Vα24⁺ T cell clone cDNA was sequenced. One clone (DN2.C7) had a distinct nucleotide sequence, which resulted in an identical amino acid sequence as shown.

Figure 2

Expression of NK-associated proteins by invariant Vα24⁺ T cells. FACS^® profiles of a representative DN invariant Vα24⁺ T cell clone (DN2.C9) 4 wk after PHA stimulation. T cells were stained with mAbs against the antigens shown or isotype-matched control mAb at 10 μg/ml and with anti-IgG FITC conjugate for 30 min each before FACS^® analysis with propidium iodide gating on viable cells. (Top, left to right) P3 isotype control (open histogram) and Vα24 (solid histogram), NKR-P1A, CD69, CD94. (Bottom, left to right) p58 KIR (GL183 shown), CD16, CD56, CD57.

Figure 3

Invariant Vα24⁺ T cells responded to CD1d CHO transfectants specifically. DN invariant Vα24⁺ T cell clones (DN2.B9, C6, C7, C9, and D6) and control CD4⁺ Vα24⁺ invariant TCR-negative T cell clones (SP3.4G9 and 5B2), all at 2 × 10⁵/well were stimulated with 0.05% glutaraldehyde-fixed CD1d⁺ CHO transfectants or control CHO cells (2 × 10⁵/ well). PMA (1 ng/ml) and IL-2 (1 nM) were included, and secreted IL-4 and IFN-γ measured at 48 h by ELISA in triplicate (standard deviations shown). Similar results were obtained without IL-2. (a) IL-4; (b) IFN-γ.

Figure 3

Invariant Vα24⁺ T cells responded to CD1d CHO transfectants specifically. DN invariant Vα24⁺ T cell clones (DN2.B9, C6, C7, C9, and D6) and control CD4⁺ Vα24⁺ invariant TCR-negative T cell clones (SP3.4G9 and 5B2), all at 2 × 10⁵/well were stimulated with 0.05% glutaraldehyde-fixed CD1d⁺ CHO transfectants or control CHO cells (2 × 10⁵/ well). PMA (1 ng/ml) and IL-2 (1 nM) were included, and secreted IL-4 and IFN-γ measured at 48 h by ELISA in triplicate (standard deviations shown). Similar results were obtained without IL-2. (a) IL-4; (b) IFN-γ.

Figure 4

CD1d antibodies inhibited invariant Vα24⁺ T cell recognition of targets. DN invariant Vα24⁺ T cell clone DN2.D5 (10⁵/well) was stimulated with fixed CD1d⁺ CHO cell transfectants (10⁵/well) as in Fig. 3. Control and CD1d-specific mAb were included at 0.67 μg/ml, and secreted IFN-γ measured by ELISA. Higher concentrations of mAb gave similar results except for 68.2, where inhibition was more complete.

Figure 5

Invariant Vα24⁺ T cells responded to CD1d B cell transfectants. T cell clones (10⁵/ well) were incubated for 48 h with either fixed (0.025 or 0.05% glutaraldehyde, latter shown) or unfixed C1R human B cells (10⁵/well) transfected with CD1a, b, c, d, or plasmid alone. Results with the two fixations were indistinguishable. PMA (1 ng/ml) was included and PHA as positive control is shown for comparison. Representative IFN-γ cytokine ELISA results of multiple experiments are shown. (a) Production of IFN-γ by DN2.C9 incubated for 48 h with C1R ± fixation. (b) IFN-γ cytokine responses of three representative DN2 T cell clones incubated for 48 h with unfixed C1R transfected with CD1a, b, c, d, mock, or mock with PHA.

Figure 5

Invariant Vα24⁺ T cells responded to CD1d B cell transfectants. T cell clones (10⁵/ well) were incubated for 48 h with either fixed (0.025 or 0.05% glutaraldehyde, latter shown) or unfixed C1R human B cells (10⁵/well) transfected with CD1a, b, c, d, or plasmid alone. Results with the two fixations were indistinguishable. PMA (1 ng/ml) was included and PHA as positive control is shown for comparison. Representative IFN-γ cytokine ELISA results of multiple experiments are shown. (a) Production of IFN-γ by DN2.C9 incubated for 48 h with C1R ± fixation. (b) IFN-γ cytokine responses of three representative DN2 T cell clones incubated for 48 h with unfixed C1R transfected with CD1a, b, c, d, mock, or mock with PHA.

Figure 6

Invariant Vα24⁺ T cells that did not express Vβ11 responded to CD1d. (a) Representative FACS^® profiles of two DN invariant TCR⁺ T cell lines (DN2.Vβ11+, left; and DN2.Vβ11−, right). T cells were stained with 10 μg/ml of mAbs against Vα24, Vβ11, or isotype control mAb and with anti-IgG FITC conjugate for 30 min each before FACS^® analysis of viable cells. (b) T cell lines (10⁵/well) were incubated with unfixed C1R human B cells (10⁵/well) transfected with CD1a, b, c, d, mock, or mock with PHA as in Fig. 5 and IFN-γ production results are shown.

Figure 6

Invariant Vα24⁺ T cells that did not express Vβ11 responded to CD1d. (a) Representative FACS^® profiles of two DN invariant TCR⁺ T cell lines (DN2.Vβ11+, left; and DN2.Vβ11−, right). T cells were stained with 10 μg/ml of mAbs against Vα24, Vβ11, or isotype control mAb and with anti-IgG FITC conjugate for 30 min each before FACS^® analysis of viable cells. (b) T cell lines (10⁵/well) were incubated with unfixed C1R human B cells (10⁵/well) transfected with CD1a, b, c, d, mock, or mock with PHA as in Fig. 5 and IFN-γ production results are shown.

Figure 7

Invariant Vα24⁺ T cells responded to chimeric CD1d with intracellular CD1a. (a) FACS^® profiles of C1R CD1d (left) and CD1d/a chimera (right) transfectants stained with normal mouse serum (open histogram) or 42.1 CD1d-specific mAb (solid histogram). (b) DN2.D6 T cell clone (10⁵/well) was incubated with unfixed C1R human B cell CD1d/a chimera or mock transfectants (10⁵/well) and IFN-γ cytokine ELISA results obtained.

Figure 7

Invariant Vα24⁺ T cells responded to chimeric CD1d with intracellular CD1a. (a) FACS^® profiles of C1R CD1d (left) and CD1d/a chimera (right) transfectants stained with normal mouse serum (open histogram) or 42.1 CD1d-specific mAb (solid histogram). (b) DN2.D6 T cell clone (10⁵/well) was incubated with unfixed C1R human B cell CD1d/a chimera or mock transfectants (10⁵/well) and IFN-γ cytokine ELISA results obtained.