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CD161 (NKR-P1A) costimulation of CD1d-dependent activation of human T cells expressing invariant V alpha 24 J alpha Q T cell receptor alpha chains - PubMed

  • ️Thu Jan 01 1998

CD161 (NKR-P1A) costimulation of CD1d-dependent activation of human T cells expressing invariant V alpha 24 J alpha Q T cell receptor alpha chains

M Exley et al. J Exp Med. 1998.

Abstract

A population of human T cells expressing an invariant V alpha 24 J alpha Q T cell antigen receptor (TCR) alpha chain and high levels of CD161 (NKR-P1A) appears to play an immunoregulatory role through production of both T helper (Th) type 1 and Th2 cytokines. Unlike other CD161(+) T cells, the major histocompatibility complex-like nonpolymorphic CD1d molecule is the target for the TCR expressed by these T cells (V alpha 24(invt) T cells) and by the homologous murine NK1 (NKR-P1C)+ T cell population. In this report, CD161 was shown to act as a specific costimulatory molecule for TCR-mediated proliferation and cytokine secretion by V alpha 24(invt) T cells. However, in contrast to results in the mouse, ligation of CD161 in the absence of TCR stimulation did not result in V alpha 24(invt) T cell activation, and costimulation through CD161 did not cause polarization of the cytokine secretion pattern. CD161 monoclonal antibodies specifically inhibited V alpha 24(invt) T cell proliferation and cytokine secretion in response to CD1d+ target cells, demonstrating a physiological accessory molecule function for CD161. However, CD1d-restricted target cell lysis by activated V alpha 24(invt) T cells, which involved a granule-mediated exocytotic mechanism, was CD161-independent. In further contrast to the mouse, the signaling pathway involved in V alpha 24(invt) T cell costimulation through CD161 did not appear to involve stable association with tyrosine kinase p56(Lck). These results demonstrate a role for CD161 as a novel costimulatory molecule for TCR-mediated recognition of CD1d by human V alpha 24(invt) T cells.

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Figures

Figure 1
Figure 1

Expression of NK cell–associated proteins by Vα24invt T cells. Representative FACS® analysis of DN Vα24invt T cell clones DN2.D5 (top) and DN1.10B3 (bottom) ∼3 wk after stimulation with PHA and irradiated feeders. T cells were stained with mAb against the antigens shown and with anti-IgG FITC conjugate before gating on live cells. Left to right, Normal mouse serum (outline) and CD28 (solid line); CD69; CD94; CD161.

Figure 2
Figure 2

Costimulation of Vα24invt T cells by NK locus–encoded C-type lectins. CD161+CD94+ Vα24invt T cell clone DN2.D6 (105/well, 2–4 wk after restimulation, except where shown) was stimulated with limiting quantities of plate-bound CD3 mAb (OKT3 at 1 μg/ ml without PMA, or 0.1 μg/ml with PMA at 1 ng/ml as shown) and/or plate-bound or soluble accessory mAb (10 μg/ml, except as shown). For soluble mAb, cross-linking anti-IgG was also added at molar equivalence. T cell proliferation measured by [3H]thymidine incorporation (cpm) was determined in triplicate at 72 h (SEM shown). Similar results were obtained by IL-4 and IFN-γ cytokine ELISA at 48 h (not shown). (A and B) Titration of anti-CD3 mAb in the presence of 10 μg/ml potential accessory molecule antibodies without (A) or with (B) PMA. (C) Titration of soluble and plate-bound CD161 costimulatory mAb 191.B8 with 0.1 μg/ml anti-CD3 mAb and PMA. (D) 1.0 μg/ml anti-CD3 mAb and/or 10 μg/ml CD161 and/or CD94 costimulation; no PMA. (E) Partially rested T cells at only 9 d after restimulation, with 1.0 μg/ml anti-CD3 mAb, 10 μg/ml CD161 and/or CD94 costimulation, and no PMA, or with PHA only.

Figure 2
Figure 2

Costimulation of Vα24invt T cells by NK locus–encoded C-type lectins. CD161+CD94+ Vα24invt T cell clone DN2.D6 (105/well, 2–4 wk after restimulation, except where shown) was stimulated with limiting quantities of plate-bound CD3 mAb (OKT3 at 1 μg/ ml without PMA, or 0.1 μg/ml with PMA at 1 ng/ml as shown) and/or plate-bound or soluble accessory mAb (10 μg/ml, except as shown). For soluble mAb, cross-linking anti-IgG was also added at molar equivalence. T cell proliferation measured by [3H]thymidine incorporation (cpm) was determined in triplicate at 72 h (SEM shown). Similar results were obtained by IL-4 and IFN-γ cytokine ELISA at 48 h (not shown). (A and B) Titration of anti-CD3 mAb in the presence of 10 μg/ml potential accessory molecule antibodies without (A) or with (B) PMA. (C) Titration of soluble and plate-bound CD161 costimulatory mAb 191.B8 with 0.1 μg/ml anti-CD3 mAb and PMA. (D) 1.0 μg/ml anti-CD3 mAb and/or 10 μg/ml CD161 and/or CD94 costimulation; no PMA. (E) Partially rested T cells at only 9 d after restimulation, with 1.0 μg/ml anti-CD3 mAb, 10 μg/ml CD161 and/or CD94 costimulation, and no PMA, or with PHA only.

Figure 2
Figure 2

Costimulation of Vα24invt T cells by NK locus–encoded C-type lectins. CD161+CD94+ Vα24invt T cell clone DN2.D6 (105/well, 2–4 wk after restimulation, except where shown) was stimulated with limiting quantities of plate-bound CD3 mAb (OKT3 at 1 μg/ ml without PMA, or 0.1 μg/ml with PMA at 1 ng/ml as shown) and/or plate-bound or soluble accessory mAb (10 μg/ml, except as shown). For soluble mAb, cross-linking anti-IgG was also added at molar equivalence. T cell proliferation measured by [3H]thymidine incorporation (cpm) was determined in triplicate at 72 h (SEM shown). Similar results were obtained by IL-4 and IFN-γ cytokine ELISA at 48 h (not shown). (A and B) Titration of anti-CD3 mAb in the presence of 10 μg/ml potential accessory molecule antibodies without (A) or with (B) PMA. (C) Titration of soluble and plate-bound CD161 costimulatory mAb 191.B8 with 0.1 μg/ml anti-CD3 mAb and PMA. (D) 1.0 μg/ml anti-CD3 mAb and/or 10 μg/ml CD161 and/or CD94 costimulation; no PMA. (E) Partially rested T cells at only 9 d after restimulation, with 1.0 μg/ml anti-CD3 mAb, 10 μg/ml CD161 and/or CD94 costimulation, and no PMA, or with PHA only.

Figure 2
Figure 2

Costimulation of Vα24invt T cells by NK locus–encoded C-type lectins. CD161+CD94+ Vα24invt T cell clone DN2.D6 (105/well, 2–4 wk after restimulation, except where shown) was stimulated with limiting quantities of plate-bound CD3 mAb (OKT3 at 1 μg/ ml without PMA, or 0.1 μg/ml with PMA at 1 ng/ml as shown) and/or plate-bound or soluble accessory mAb (10 μg/ml, except as shown). For soluble mAb, cross-linking anti-IgG was also added at molar equivalence. T cell proliferation measured by [3H]thymidine incorporation (cpm) was determined in triplicate at 72 h (SEM shown). Similar results were obtained by IL-4 and IFN-γ cytokine ELISA at 48 h (not shown). (A and B) Titration of anti-CD3 mAb in the presence of 10 μg/ml potential accessory molecule antibodies without (A) or with (B) PMA. (C) Titration of soluble and plate-bound CD161 costimulatory mAb 191.B8 with 0.1 μg/ml anti-CD3 mAb and PMA. (D) 1.0 μg/ml anti-CD3 mAb and/or 10 μg/ml CD161 and/or CD94 costimulation; no PMA. (E) Partially rested T cells at only 9 d after restimulation, with 1.0 μg/ml anti-CD3 mAb, 10 μg/ml CD161 and/or CD94 costimulation, and no PMA, or with PHA only.

Figure 2
Figure 2

Costimulation of Vα24invt T cells by NK locus–encoded C-type lectins. CD161+CD94+ Vα24invt T cell clone DN2.D6 (105/well, 2–4 wk after restimulation, except where shown) was stimulated with limiting quantities of plate-bound CD3 mAb (OKT3 at 1 μg/ ml without PMA, or 0.1 μg/ml with PMA at 1 ng/ml as shown) and/or plate-bound or soluble accessory mAb (10 μg/ml, except as shown). For soluble mAb, cross-linking anti-IgG was also added at molar equivalence. T cell proliferation measured by [3H]thymidine incorporation (cpm) was determined in triplicate at 72 h (SEM shown). Similar results were obtained by IL-4 and IFN-γ cytokine ELISA at 48 h (not shown). (A and B) Titration of anti-CD3 mAb in the presence of 10 μg/ml potential accessory molecule antibodies without (A) or with (B) PMA. (C) Titration of soluble and plate-bound CD161 costimulatory mAb 191.B8 with 0.1 μg/ml anti-CD3 mAb and PMA. (D) 1.0 μg/ml anti-CD3 mAb and/or 10 μg/ml CD161 and/or CD94 costimulation; no PMA. (E) Partially rested T cells at only 9 d after restimulation, with 1.0 μg/ml anti-CD3 mAb, 10 μg/ml CD161 and/or CD94 costimulation, and no PMA, or with PHA only.

Figure 3
Figure 3

CD1d and CD161 mAbs specifically inhibit Vα24invt T cell response to CD1d. Vα24invt T cells (105/well) were stimulated with mitomycin C–treated CD1d+ C1R cell transfectants (105/well) and 1 ng/ml PMA. Control, CD1d-specific, and other mAbs were included in incubations at 10 μg/ ml. Representative results from five independent experiments are shown. (A) DN2.D6 T cell proliferation (cpm) was determined in triplicate at 72 h. (B) IFN-γ cytokine ELISA was determined at 48 h from the same experiment as in A. (C) IL-4 determined as for IFN-γ. Control mock C1R-containing wells had background proliferation of 6,000 cpm, 6.4 ng/ml IFN-γ, and 2.4 ng/ml IL-4. In a further experiment with two Vα24invt T cell clones, three different CD161 mAbs were used. (D) DN2.D6. (E) DN2.D5.

Figure 3
Figure 3

CD1d and CD161 mAbs specifically inhibit Vα24invt T cell response to CD1d. Vα24invt T cells (105/well) were stimulated with mitomycin C–treated CD1d+ C1R cell transfectants (105/well) and 1 ng/ml PMA. Control, CD1d-specific, and other mAbs were included in incubations at 10 μg/ ml. Representative results from five independent experiments are shown. (A) DN2.D6 T cell proliferation (cpm) was determined in triplicate at 72 h. (B) IFN-γ cytokine ELISA was determined at 48 h from the same experiment as in A. (C) IL-4 determined as for IFN-γ. Control mock C1R-containing wells had background proliferation of 6,000 cpm, 6.4 ng/ml IFN-γ, and 2.4 ng/ml IL-4. In a further experiment with two Vα24invt T cell clones, three different CD161 mAbs were used. (D) DN2.D6. (E) DN2.D5.

Figure 3
Figure 3

CD1d and CD161 mAbs specifically inhibit Vα24invt T cell response to CD1d. Vα24invt T cells (105/well) were stimulated with mitomycin C–treated CD1d+ C1R cell transfectants (105/well) and 1 ng/ml PMA. Control, CD1d-specific, and other mAbs were included in incubations at 10 μg/ ml. Representative results from five independent experiments are shown. (A) DN2.D6 T cell proliferation (cpm) was determined in triplicate at 72 h. (B) IFN-γ cytokine ELISA was determined at 48 h from the same experiment as in A. (C) IL-4 determined as for IFN-γ. Control mock C1R-containing wells had background proliferation of 6,000 cpm, 6.4 ng/ml IFN-γ, and 2.4 ng/ml IL-4. In a further experiment with two Vα24invt T cell clones, three different CD161 mAbs were used. (D) DN2.D6. (E) DN2.D5.

Figure 3
Figure 3

CD1d and CD161 mAbs specifically inhibit Vα24invt T cell response to CD1d. Vα24invt T cells (105/well) were stimulated with mitomycin C–treated CD1d+ C1R cell transfectants (105/well) and 1 ng/ml PMA. Control, CD1d-specific, and other mAbs were included in incubations at 10 μg/ ml. Representative results from five independent experiments are shown. (A) DN2.D6 T cell proliferation (cpm) was determined in triplicate at 72 h. (B) IFN-γ cytokine ELISA was determined at 48 h from the same experiment as in A. (C) IL-4 determined as for IFN-γ. Control mock C1R-containing wells had background proliferation of 6,000 cpm, 6.4 ng/ml IFN-γ, and 2.4 ng/ml IL-4. In a further experiment with two Vα24invt T cell clones, three different CD161 mAbs were used. (D) DN2.D6. (E) DN2.D5.

Figure 3
Figure 3

CD1d and CD161 mAbs specifically inhibit Vα24invt T cell response to CD1d. Vα24invt T cells (105/well) were stimulated with mitomycin C–treated CD1d+ C1R cell transfectants (105/well) and 1 ng/ml PMA. Control, CD1d-specific, and other mAbs were included in incubations at 10 μg/ ml. Representative results from five independent experiments are shown. (A) DN2.D6 T cell proliferation (cpm) was determined in triplicate at 72 h. (B) IFN-γ cytokine ELISA was determined at 48 h from the same experiment as in A. (C) IL-4 determined as for IFN-γ. Control mock C1R-containing wells had background proliferation of 6,000 cpm, 6.4 ng/ml IFN-γ, and 2.4 ng/ml IL-4. In a further experiment with two Vα24invt T cell clones, three different CD161 mAbs were used. (D) DN2.D6. (E) DN2.D5.

Figure 4
Figure 4

Cytolytic responses of Vα24invt T cells to CD1d+ target cells. Vα24invt DN2.D6 T cells were stimulated with 51Cr-loaded CD1d+ or mock C1R cell transfectants. (A). E/T ratio titration. CD1d (51.1) antibody inhibition of target cell lysis (1 and 10 μg/ml). (B) Antibody inhibition of target cell lysis. CD1d (51.1) at 10 μg/ml or CD161-specific mAb (DX-1) with 51.1 at 0.08 μg/ml were included in incubations.

Figure 4
Figure 4

Cytolytic responses of Vα24invt T cells to CD1d+ target cells. Vα24invt DN2.D6 T cells were stimulated with 51Cr-loaded CD1d+ or mock C1R cell transfectants. (A). E/T ratio titration. CD1d (51.1) antibody inhibition of target cell lysis (1 and 10 μg/ml). (B) Antibody inhibition of target cell lysis. CD1d (51.1) at 10 μg/ml or CD161-specific mAb (DX-1) with 51.1 at 0.08 μg/ml were included in incubations.

Figure 5
Figure 5

Association of p56Lck with murine NK1, but not human Vα24invt T cell CD161. (A) Comparison of human (reference 36) and murine NKR-P1 (references and 2) amino acid sequences around the functional p56Lck binding motif (reference 47) found in murine NKR-P1C. (B) p56Lck immunoblot of nonreduced murine p56Lck, NK1.1, and control immunoprecipitations from Vα14invt T-T hybridoma DN32.D3. (C) p56Lck immunoblot of CD3, CD161, and Con A precipitations from Vα24invt T cell clone DN2.B9. (D) CD161 (HP-3G10) immunoblot of nonreduced p56Lck, CD161 (DX-1), or control mAb (Cont.) immunoprecipitations from Vα24invt T cell clone DN2.B9.

Figure 5
Figure 5

Association of p56Lck with murine NK1, but not human Vα24invt T cell CD161. (A) Comparison of human (reference 36) and murine NKR-P1 (references and 2) amino acid sequences around the functional p56Lck binding motif (reference 47) found in murine NKR-P1C. (B) p56Lck immunoblot of nonreduced murine p56Lck, NK1.1, and control immunoprecipitations from Vα14invt T-T hybridoma DN32.D3. (C) p56Lck immunoblot of CD3, CD161, and Con A precipitations from Vα24invt T cell clone DN2.B9. (D) CD161 (HP-3G10) immunoblot of nonreduced p56Lck, CD161 (DX-1), or control mAb (Cont.) immunoprecipitations from Vα24invt T cell clone DN2.B9.

Figure 5
Figure 5

Association of p56Lck with murine NK1, but not human Vα24invt T cell CD161. (A) Comparison of human (reference 36) and murine NKR-P1 (references and 2) amino acid sequences around the functional p56Lck binding motif (reference 47) found in murine NKR-P1C. (B) p56Lck immunoblot of nonreduced murine p56Lck, NK1.1, and control immunoprecipitations from Vα14invt T-T hybridoma DN32.D3. (C) p56Lck immunoblot of CD3, CD161, and Con A precipitations from Vα24invt T cell clone DN2.B9. (D) CD161 (HP-3G10) immunoblot of nonreduced p56Lck, CD161 (DX-1), or control mAb (Cont.) immunoprecipitations from Vα24invt T cell clone DN2.B9.

Figure 5
Figure 5

Association of p56Lck with murine NK1, but not human Vα24invt T cell CD161. (A) Comparison of human (reference 36) and murine NKR-P1 (references and 2) amino acid sequences around the functional p56Lck binding motif (reference 47) found in murine NKR-P1C. (B) p56Lck immunoblot of nonreduced murine p56Lck, NK1.1, and control immunoprecipitations from Vα14invt T-T hybridoma DN32.D3. (C) p56Lck immunoblot of CD3, CD161, and Con A precipitations from Vα24invt T cell clone DN2.B9. (D) CD161 (HP-3G10) immunoblot of nonreduced p56Lck, CD161 (DX-1), or control mAb (Cont.) immunoprecipitations from Vα24invt T cell clone DN2.B9.

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