Proc. Nati. Acad. Sci. USA Vol. 88, pp. 8705-8709, October 1991 Immunology
Conventional antigen and superantigen may be coupled to distinct and cooperative T-cell activation pathways (T-cell antigen receptor/bacterial enterotoxm/signal transduction)
Hsi LIU*t, MARILYN A. LAMPE*t, MARIA VICTORIA IREGUIt, AND HARVEY CANTOR*t* *Department of Pathology, Harvard Medical School, and tLaboratory of Immunopathology, Dana-Farber Cancer Institute, 44 Binney Street, Boston,
MA 02115
Communicated by Edward A. Boyse, July 8, 1991 (received for review May 1, 1991)
CD4' T cells are equipped to detect two ABSTRACT major classes of ligands. Infectious microbial agents, including bacteria and retroviruses, carry a class of proteins termed superantigens that are recognized by the T-cell receptor in association with class II products of the major histocompatibility complex. Proteins expressed by other cells and organisms are processed by macrophages into peptides that are presented to CD4' T cells by class II molecules. We have examined CD4' T-cell clones that proliferate vigorously in response both to conventional peptide antigens and to bacterial or retroviral superantigens. The response to peptide antigen is characterized by a rapid and sustained increase in the levels of intracellular free Ca2" and a vigorous cytokine response. In contrast, the proliferative response of these clones to bacterial or retroviral superantigen is not accompanied by detectable increases in intracellular Ca2+ or by signifcant cytokine production. Further analysis of T-cell activation indicates that interaction of a single T-cell receptor with the two types of ligand may be coupled to functionally distinct signaling pathways that interact in a synergistic fashion to achieve T-cell activation. CD4+8- T cells that mediate helper function recognize two general classes of ligands in association with class II products of the major histocompatibility complex (MHC) (1-3). Conventional ligands consist of peptides embedded in the antigen-binding groove of MHC class II molecules (4, 5). A second class of ligand consists of cell-surface Mls products and some bacterial enterotoxins. These have been collectively termed superantigens because they are recognized by a high frequency of CD4' T cells in association with class II MHC products (6, 7). Recent studies have suggested that superantigens interact with MHC class II molecules outside the antigen-binding groove (8) and may bind to the T-cell receptor (TCR) away from the predicted combining site for conventional ligands (9). These findings have raised the possibility that the interaction of the TCR with conventional peptide antigen and with superantigen may have distinct functional consequences (10). We have addressed this question using murine T helper (TH) clones that bear receptors that allow clonal proliferation in response to both classes of ligand. We find that interaction of a single TCR with the two types of ligand may be coupled to functionally distinct intracellular activation pathways that lead to different patterns of cytokine gene expression.
EXPERIMENTAL PROCEDURES Mice. BALB/c and DBA/2J mice were obtained from The Jackson Laboratory and were maintained at the Redstone Animal Facility (Dana-Farber Cancer Institute, Boston). T-Cell Clones and Cell Proliferation Assay. The Ar5 THO clone, from a (BALB/c x A/J)F1 donor, was selected for its
proliferative response to arsonylated proteins [including arsonylated ovalbumin (Ar-Ova)] in association with I-Ad antigen-presenting cells (APCs) (11). 03 is a CD4' THO clone derived from BALB/c mice after in vitro selection for proliferation in response to Ova in association with BALB/c APCs (12). To assay cell proliferative responses, 03 cells (2 x 104) were added to wells containing either mitomycintreated BALB/c spleen cells (5 x 105) and Ova (10 ,ug/ml) or mitomycin-treated DBA/2 spleen cells (5 x 105). In some experiments, splenic adherent cells were prepared by incubating 106 spleen cells after treatment with mitomycin C in Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum for 2 hr before washing with balanced salt solution containing 2% fetal bovine serum to remove nonadherent cells (>90%o of the total cell population). After 24 hr, each well received 1 jCi (37 kBq) of [3H]thymidine and was incubated for an additional 16 hr. Cultures were harvested onto fiberglass filters (Whatman) with a PHD cell harvester (Cambridge Technology, Cambridge, MA) and [3H]thymidine incorporation was measured by liquid scintillation counting. The results are expressed as the mean of triplicate cultures. Assay of Intracellular Free Ca2+ Concentration ([Ca2+]1). [Ca2+], was estimated using 2 x 107 cloned T cells (03 or Ar5) that had been loaded with 8 gM indo-1 acetoxymethyl ester as described (13), washed and resuspended to 2 x 107 cells per ml in DMEM, and kept at room temperature. One hundred microliters of indo-1-loaded cells was added to an equal volume containing 2 x 107 BALB/c or DBA/2 spleen cells alone or 2 x 107 BALB/c spleen cells that had been incubated at 370C with Ova (100 pkg/ml), Ar-Ova (100 ,ug/ml), or staphylococcal enterotoxin B (SEB, 40 gg/ml) for 4 hr. Anti-CD3 (145.2C11) at 1:5 final dilution was added to BALB/c spleen cells just before addition of T cells. In some experiments, additional SEB (100 Ig/ml) was added to spleen cell cultures immediately before mixing with T cells, to rule out the possibility that this antigen had lost its activity during the 4-hr incubation period. This did not affect the results shown here. After addition of the T-cell clone to spleen cells, the mixture was immediately centrifuged for 20 sec at 200 x g, and the cells were resuspended gently by four strokes of a Gilson pipette, and placed into a Nunc freezing vial for analysis in an EPICS C flow cytometer (Coulter) equipped with an argon laser. The ratio of 485-nm to 404-nm fluorescence (linear scale) was calculated by analogue ratio circuitry on the Coulter EPICS C and the indo-1 fluorescence signal was calibrated for [Ca2+], as described (13). Cytokine Assays. Fifty microliters of culture supernatant fluids was added to 96-well tissue culture plates in 2-fold Abbreviations: APC, antigen-presenting cell; IL-n, interleukin n; MHC, major histocompatibility complex; Ova, ovalbumin; Ar-Ova, arsonylated Ova; SEB, staphylococcal enterotoxin B; TCR, T-cell receptor; TH, T helper. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
8705
8706
Proc. Natil. Acad. Sci. USA 88 (1991)
Immunology: Liu et al.
dilutions. HT-2 or MC/9 cells (5 x 103) were added and incubated for 24 hr at 370C in a 5% CO2 atmosphere. [3H]Thymidine (1 puCi per well) was added during the last 18 hr of culture. Cells were harvested onto fiberglass filters with a PHD harvester and [3H]thymidine incorporation was determined by a scintillation counter (Beckman LS1800). Results (cpm) were converted to units/ml based on the halfmaximum proliferative response of these indicator cells in control cultures containing recombinant interleukin 2 (IL-2; Boehringer Mannfeimq) or recombinant IL-3 (Genzyme) (14).
The T-cell clones used do not produce IL4 (determined by assay with CT.4S cell line, kindly provided by William Paul, National Institutes of Health). In addition, monoclonal antibodies against the IL-2 receptor (7D4 and TIB222; American Type Culture'Collection) and against IL-4 (l1B11) were used to confirm the specificity of these cytokine assays.
RESULTS We studied the response of two independently derived murine CD4' THi clones to conventional protein antigens and to
A 03 -
120
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Er a
-5.& Ce
60 30
0
: S~~~~~~~~~ 1
100
10
0
0.
Antigen, ,ug/ml
B 03
0 400 .
Ar5 2a
Anti-CD3
Ar-Ova
. -I
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. .U I I
v
w
No antigen
SEB
300 ..
F
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4 Fin a
1 VW 00.
a
I
200
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200
400
0
Time,
.
.
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I
200
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-a
400
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0
200
400
600
sec
FIG. 1. (A) Proliferative response of clones 03 and Ar5 to antigen. Cells (2 x 104) from clone 03 (Left) or ArS (Right) were incubated with 105 irradiated (2900 rads; 1 rad = 0.01 Gy) BALB/c spleen cells and the indicated concentrations of conventional antigen [Ova (03) or Ars-Ova (Ar5)] (o) or' SEB '(). After 24 hr, [3H]thymidine (1 pCi per well) was added and the cells were harvested 18 hr later. The responses represent the mean of triplicate cultures; the standard error of all groups shown was
Conventional antigen and superantigen may be coupled to distinct and cooperative T-cell activation pathways (T-cell antigen receptor/bacterial enterotoxm/signal transduction)
Hsi LIU*t, MARILYN A. LAMPE*t, MARIA VICTORIA IREGUIt, AND HARVEY CANTOR*t* *Department of Pathology, Harvard Medical School, and tLaboratory of Immunopathology, Dana-Farber Cancer Institute, 44 Binney Street, Boston,
MA 02115
Communicated by Edward A. Boyse, July 8, 1991 (received for review May 1, 1991)
CD4' T cells are equipped to detect two ABSTRACT major classes of ligands. Infectious microbial agents, including bacteria and retroviruses, carry a class of proteins termed superantigens that are recognized by the T-cell receptor in association with class II products of the major histocompatibility complex. Proteins expressed by other cells and organisms are processed by macrophages into peptides that are presented to CD4' T cells by class II molecules. We have examined CD4' T-cell clones that proliferate vigorously in response both to conventional peptide antigens and to bacterial or retroviral superantigens. The response to peptide antigen is characterized by a rapid and sustained increase in the levels of intracellular free Ca2" and a vigorous cytokine response. In contrast, the proliferative response of these clones to bacterial or retroviral superantigen is not accompanied by detectable increases in intracellular Ca2+ or by signifcant cytokine production. Further analysis of T-cell activation indicates that interaction of a single T-cell receptor with the two types of ligand may be coupled to functionally distinct signaling pathways that interact in a synergistic fashion to achieve T-cell activation. CD4+8- T cells that mediate helper function recognize two general classes of ligands in association with class II products of the major histocompatibility complex (MHC) (1-3). Conventional ligands consist of peptides embedded in the antigen-binding groove of MHC class II molecules (4, 5). A second class of ligand consists of cell-surface Mls products and some bacterial enterotoxins. These have been collectively termed superantigens because they are recognized by a high frequency of CD4' T cells in association with class II MHC products (6, 7). Recent studies have suggested that superantigens interact with MHC class II molecules outside the antigen-binding groove (8) and may bind to the T-cell receptor (TCR) away from the predicted combining site for conventional ligands (9). These findings have raised the possibility that the interaction of the TCR with conventional peptide antigen and with superantigen may have distinct functional consequences (10). We have addressed this question using murine T helper (TH) clones that bear receptors that allow clonal proliferation in response to both classes of ligand. We find that interaction of a single TCR with the two types of ligand may be coupled to functionally distinct intracellular activation pathways that lead to different patterns of cytokine gene expression.
EXPERIMENTAL PROCEDURES Mice. BALB/c and DBA/2J mice were obtained from The Jackson Laboratory and were maintained at the Redstone Animal Facility (Dana-Farber Cancer Institute, Boston). T-Cell Clones and Cell Proliferation Assay. The Ar5 THO clone, from a (BALB/c x A/J)F1 donor, was selected for its
proliferative response to arsonylated proteins [including arsonylated ovalbumin (Ar-Ova)] in association with I-Ad antigen-presenting cells (APCs) (11). 03 is a CD4' THO clone derived from BALB/c mice after in vitro selection for proliferation in response to Ova in association with BALB/c APCs (12). To assay cell proliferative responses, 03 cells (2 x 104) were added to wells containing either mitomycintreated BALB/c spleen cells (5 x 105) and Ova (10 ,ug/ml) or mitomycin-treated DBA/2 spleen cells (5 x 105). In some experiments, splenic adherent cells were prepared by incubating 106 spleen cells after treatment with mitomycin C in Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum for 2 hr before washing with balanced salt solution containing 2% fetal bovine serum to remove nonadherent cells (>90%o of the total cell population). After 24 hr, each well received 1 jCi (37 kBq) of [3H]thymidine and was incubated for an additional 16 hr. Cultures were harvested onto fiberglass filters (Whatman) with a PHD cell harvester (Cambridge Technology, Cambridge, MA) and [3H]thymidine incorporation was measured by liquid scintillation counting. The results are expressed as the mean of triplicate cultures. Assay of Intracellular Free Ca2+ Concentration ([Ca2+]1). [Ca2+], was estimated using 2 x 107 cloned T cells (03 or Ar5) that had been loaded with 8 gM indo-1 acetoxymethyl ester as described (13), washed and resuspended to 2 x 107 cells per ml in DMEM, and kept at room temperature. One hundred microliters of indo-1-loaded cells was added to an equal volume containing 2 x 107 BALB/c or DBA/2 spleen cells alone or 2 x 107 BALB/c spleen cells that had been incubated at 370C with Ova (100 pkg/ml), Ar-Ova (100 ,ug/ml), or staphylococcal enterotoxin B (SEB, 40 gg/ml) for 4 hr. Anti-CD3 (145.2C11) at 1:5 final dilution was added to BALB/c spleen cells just before addition of T cells. In some experiments, additional SEB (100 Ig/ml) was added to spleen cell cultures immediately before mixing with T cells, to rule out the possibility that this antigen had lost its activity during the 4-hr incubation period. This did not affect the results shown here. After addition of the T-cell clone to spleen cells, the mixture was immediately centrifuged for 20 sec at 200 x g, and the cells were resuspended gently by four strokes of a Gilson pipette, and placed into a Nunc freezing vial for analysis in an EPICS C flow cytometer (Coulter) equipped with an argon laser. The ratio of 485-nm to 404-nm fluorescence (linear scale) was calculated by analogue ratio circuitry on the Coulter EPICS C and the indo-1 fluorescence signal was calibrated for [Ca2+], as described (13). Cytokine Assays. Fifty microliters of culture supernatant fluids was added to 96-well tissue culture plates in 2-fold Abbreviations: APC, antigen-presenting cell; IL-n, interleukin n; MHC, major histocompatibility complex; Ova, ovalbumin; Ar-Ova, arsonylated Ova; SEB, staphylococcal enterotoxin B; TCR, T-cell receptor; TH, T helper. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
8705
8706
Proc. Natil. Acad. Sci. USA 88 (1991)
Immunology: Liu et al.
dilutions. HT-2 or MC/9 cells (5 x 103) were added and incubated for 24 hr at 370C in a 5% CO2 atmosphere. [3H]Thymidine (1 puCi per well) was added during the last 18 hr of culture. Cells were harvested onto fiberglass filters with a PHD harvester and [3H]thymidine incorporation was determined by a scintillation counter (Beckman LS1800). Results (cpm) were converted to units/ml based on the halfmaximum proliferative response of these indicator cells in control cultures containing recombinant interleukin 2 (IL-2; Boehringer Mannfeimq) or recombinant IL-3 (Genzyme) (14).
The T-cell clones used do not produce IL4 (determined by assay with CT.4S cell line, kindly provided by William Paul, National Institutes of Health). In addition, monoclonal antibodies against the IL-2 receptor (7D4 and TIB222; American Type Culture'Collection) and against IL-4 (l1B11) were used to confirm the specificity of these cytokine assays.
RESULTS We studied the response of two independently derived murine CD4' THi clones to conventional protein antigens and to
A 03 -
120
(DX CE
;5E90
Er a
-5.& Ce
60 30
0
: S~~~~~~~~~ 1
100
10
0
0.
Antigen, ,ug/ml
B 03
0 400 .
Ar5 2a
Anti-CD3
Ar-Ova
. -I
--v--
. .U I I
v
w
No antigen
SEB
300 ..
F
co 200
4 Fin a
1 VW 00.
a
I
200
400
.JL 0
t
a
200
400
0
Time,
.
.
R
I
200
Il
-a
400
JAA
0
200
400
600
sec
FIG. 1. (A) Proliferative response of clones 03 and Ar5 to antigen. Cells (2 x 104) from clone 03 (Left) or ArS (Right) were incubated with 105 irradiated (2900 rads; 1 rad = 0.01 Gy) BALB/c spleen cells and the indicated concentrations of conventional antigen [Ova (03) or Ars-Ova (Ar5)] (o) or' SEB '(). After 24 hr, [3H]thymidine (1 pCi per well) was added and the cells were harvested 18 hr later. The responses represent the mean of triplicate cultures; the standard error of all groups shown was
