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Intercellular Adhesion Molecule-1 and Leukocyte Function-Associated Antigen-3 Provide Costimulation for Superantigen-Induced T Lymphocyte Proliferation in the Absence of a Specific Presenting Molecule¹

James G. Lamphear^2,*, Kristin Reda Stevens^2,* and Robert R. Rich^3,*,

Departments of ^* Microbiology and Immunology and Medicine, Baylor College of Medicine, Houston, TX 77030

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial superantigens can bind TCR in the absence of MHC class II molecules and activate T lymphocytes when cocultured with certain class II-deficient accessory cells. It has not been determined, however, whether these accessory cells provide direct costimulation to the T cell or serve to present superantigens via a nonconventional ligand. We have identified a human adenocarcinoma cell line, SW480, that assists in the activation of human T cells by the staphylococcal enterotoxins B (SEB), C1 (SEC1), and D (SED), but not SEA, SEC2, SEC3, or SEE. SW480 cells did not express class II molecules, and anti-class II mAbs did not inhibit T cell proliferation, supporting the hypothesis that class II is not absolutely required for enterotoxin-mediated T cell activation. The TCR Vß profile of T cells stimulated by SEB plus SW480 cells was similar to that of T cells stimulated by SEB plus class II⁺ APC, indicating that TCR-SEB interactions were preserved in the absence of class II molecules. Binding studies failed to detect specific association of SEB with SW480 cells, suggesting that SW480 cells do not express receptors for enterotoxin. SEB coupled to beads, however, stimulated T cell proliferation, but only in the presence of SW480 cells. SW480 cells express both ICAM-1 and LFA-3 molecules, and the addition of Abs to these receptors inhibited T cell proliferation. These findings support a model in which certain enterotoxins engage the TCR independent of MHC class II or other specific presenting molecules and induce T cell proliferation with signals provided by nonconventional accessory cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Staphylococcal enterotoxins act as potent immunomodulatory agents that stimulate subsets of T lymphocytes by directly cross-linking the variable regions of certain TCR ß-chains with MHC class II molecules on the surface of APC (1, 2, 3). T cells activated in this manner may undergo robust proliferation and release cytokines, including IL-2 and IFN- (4), that further enhance enterotoxin-mediated T cell activation, in part by up-regulating MHC class II and costimulatory molecules on professional (5) and nonprofessional APC (6, 7, 8). In the absence of MHC class II⁺ APC, enterotoxins can induce clonal anergy of reactive T cells (9, 10), suggesting that MHC class II presentation itself or the provision of costimulation by an APC can override the induction of anergy. Several studies, however, have demonstrated that enterotoxins presented by MHC class II-deficient AC⁴ can activate T cells. In particular, Dohlsten et al. (11) and Herrmann et al. (12) reported that picomolar concentrations of SEB and SEC1 could trigger the lysis of MHC class II-negative human cell lines by CTL expressing an appropriate Vß TCR. Avery et al. (13) reported that SEE and the three isotypes of SEC stimulated the proliferation of T cells from MHC class II-deficient mice in an APC-dependent manner and supported CTL-mediated lysis of class II-negative targets. Beharka et al. (14) provided evidence for a low affinity interaction of SEA and SEB with macrophages isolated from class II-deficient mice. These findings were interpreted to suggest that receptors for enterotoxins other than MHC class II molecules may exist on some cells, and moreover, that subsets of enterotoxins presented by these molecules can stimulate T cell proliferation and effector functions.

Based on these previous findings, we sought to define the presenting molecule for enterotoxins on human cells and to describe the mechanism of T cell activation used in the absence of MHC class II. We chose to examine an MHC class II-negative human adenocarcinoma cell line, SW480, that enabled the potent activation of purified human T cells by several enterotoxins. Specific binding of SEB to these cells, however, was not detected under a variety of circumstances, suggesting that the enterotoxins may bind and signal through the TCR as molecules free in solution or perhaps as aggregates nonspecifically associated with the surface of SW480 cells. SEB covalently cross-linked to Sepharose beads stimulated T cell proliferation in the presence of SW480 cells, suggesting that SEB may bind and directly transduce signals through the TCR. We characterized SW480 and found that several putative costimulatory molecules were expressed at the cell surface. Blocking studies further revealed that ICAM-1/LFA-1 and LFA-3/CD2 interactions played a significant role in the activation of T cells. We interpret these findings to suggest that enterotoxins can bind and signal through the TCR in the absence of a specific presenting molecule and stimulate T cell proliferation with the addition of costimulation provided by AC.

	Materials and Methods

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Cell lines, Abs, and mitogens

SW480 and SW620 are cell lines independently derived from the same human colon adenocarcinoma (15), that were obtained from the American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640/10% FCS. The human T cell hybridoma, 289D1, was provided by J. Brawley, University of Washington School of Medicine (Seattle, WA), and maintained in RPMI 1640/10% FCS/1 mM histidinol. The HLA-DR1-transfected murine L cell line, D.5-3.1, was obtained from E. Long, National Institutes of Health (Bethesda, MD), and maintained in DMEM/10% FCS. mAbs against HLA-DR (L243), HLA-DR/DQ (L227), and CD11b (OKM1 and LM2/1.6.11) were used for the negative selection of T cell populations and were purified from the culture supernatants of B cell hybridomas obtained from American Type Culture Collection. mAbs against Vß3.1 (8F10), Vß5.2/5.3 (1C1), Vß8 (16G8), Vß12.2 (S511), Vß13.1 (BAM13), LFA-3 (TS2/9), LFA-1 (B-B15), and CD2 (TS2/18) were purchased from Endogen (Cambridge, MA), and Vß17.1–2 (BA-62) was obtained from AMAC, Inc. (Westbrook, ME). mAbs against CD3 (SK7), CD4 (SK3) CD8 (SK1), CD25 (2A3), HLA-DQ1 and -DQ3 (SK10), and HLA-DP1 through DP5 (B7/21) were purchased from Becton Dickinson (Mountain View, CA). mAbs against B7-1 (BB1), B7-2 (IT2.2), and HLA-A, -B, and -C (G46-2.6) were purchased from PharMingen (San Diego, CA), and CTLA4-Ig fusion protein was obtained from Ancell (Bayport, MN). The mAb against ICAM-1 (R6.5) was provided by M. Mariscalco, Texas Children’s Hospital (Houston, TX). Staphylococcal enterotoxins were purchased from Toxin Technology (Sarasota, FL), and PHA P, pokeweed mitogen, and Con A were obtained from Sigma Chemical Co. (St. Louis, MO).

Purification of human T lymphocytes

PBMC, obtained from buffy coats of healthy donors (Gulf Coast Blood Center, Houston, TX) by density gradient centrifugation, were stained with a mixture of anti-class II (L243, L227) and anti-monocyte (LM2/1.6.11, OKM1) mAbs, and the cells were separated on goat anti-mouse Ig-conjugated magnetic beads (Advanced Magnetics, Cambridge, MA). Two rounds of negative selection typically yielded >98% CD3⁺ cells that were judged to be naive/resting based upon light scatter properties and cell surface expression of markers of activation/maturation (CD25^-, CD45RA⁺).

Measurement of accessory cell-dependent T cell proliferation

SW480 or SW620 cells (1 x 10⁷) were treated with 100 µg/ml mitomycin C (Sigma) for 1 h at 37°C, and washed extensively with HBSS/2% FCS. Purified human T cells (1.2 x 10⁵) and either mitomycin C-treated SW480 or SW620 cells (6 x 10⁴) or autologous, irradiated (1500 rad) PBMC (2.4 x 10⁵) were cultured in 200 µl of assay medium (RPMI 1640/10% FCS/100 µg/ml gentamicin/1% antibiotic-antimycotic mixture/2 mM L-glutamine/5 mM HEPES; all components from Life Technologies (Grand Island, NY)) in a 96-well flat-bottom plate (Costar, Cambridge, MA) for 3 days. The cells were labeled for an additional 18 h with 1 µCi of [³H]thymidine (DuPont-New England Nuclear, Boston, MA), harvested, and counted by liquid scintillation spectroscopy.

Flow cytometric analysis of T lymphocyte blasts

Purified T cells (5 x 10⁶) and either mitomycin C-treated SW480 (2.5 x 10⁶) or autologous, irradiated (1500 rad) PBMC (12.5 x 10⁶) were cultured in 5 ml of assay medium in six-well plates (Costar) for 3 days. Viable cells were isolated from culture by density gradient centrifugation and recultured for an additional 24 h in the presence of 18 ng/ml IL-2 (R&D Systems, Minneapolis, MN) to expand the total number of proliferating cells and to promote the restoration of TCR expressed at the cell surface. Cells were stained with one of several TCR Vß-specific or T cell subset-specific mAbs and analyzed by flow cytometry on an Epics Profile (Coulter Corp., Hialeah, FL). Forward angle and 90° light scatter patterns were used to restrict the analysis to blast-transformed or resting T cells, as initially characterized by flow cytometric measurements of total DNA content and incorporated bromodeoxyuridine as a function of proliferation (data not shown).

Detection of SEB binding to SW480 cells

SEB (250 µg) was labeled with ¹²⁵I (2 mCi) (DuPont-New England Nuclear) in 200 µl of PBS for 30 min at 25°C using two Iodobeads (Pierce Chemical Co., Rockford, IL), and the labeled protein was separated from free ¹²⁵I by gel filtration chromatography. The sp. act. of [¹²⁵I]SEB was 2.2 x 10⁶ cpm/µg. SW480 cells or HLA-DR1-transfected fibroblasts (1 x 10⁵) were incubated with [¹²⁵I]SEB in 200 µl of HBSS/2% FCS for 2 h at 37°C, the unbound [¹²⁵I]SEB was removed by centrifugation (15 s at 3000 x g) of the reaction mixture through a 0.45-µm filter, and the filters and filtrate were subsequently analyzed using a gamma counter (Packard, Downers Grove, IL).

Conjugation of SEB to Sepharose beads

SEB (5 mg) was coupled to 1 ml of cyanogen bromide-activated Sepharose 4B (Sigma) for 2 h at room temperature and blocked with 0.2 M glycine for an additional 2 h. Beads were washed with three cycles of neutral buffer (0.1 M NaHCO₃/0.5 M NaCl, pH 8.0) followed by acidic buffer (0.1 M Na acetate/0.5 M NaCl, pH 4.0) to remove unconjugated SEB and stored in PBS/0.02% Na azide at 4°C. Before use, beads were washed with three cycles of HBSS/10% FCS and counted using a hemocytometer. Free SEB was not detected in filtered bead supernatants by Western blotting.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
SW480 adenocarcinoma cells support SEB-induced T cell proliferation

To investigate the role of nonconventional AC in the activation of T cells by bacterial superantigens, we initially screened several putatively MHC class II-negative cell lines, including SW480 and SW620, for the ability to support the proliferation of purified T cells in the presence of SEB (Fig. 1 and data not shown). The responder T cell populations used in these functional assays were obtained from human PBMC rigorously depleted of MHC class II⁺ APC. Importantly, T cell populations were judged to be free from class II⁺ APC due to their inability to respond to varying concentrations of SEB (Fig. 1) or other mitogenic lectins (data not shown). The SW480 adenocarcinoma cell line was chosen for further analysis based on its more potent ability to support T cell proliferation.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. SEB stimulates proliferation of purified T cells in association with SW480 or SW620 cells. "No AC" controls contain only T cells and the indicated concentrations of SEB. Controls containing T cells plus SW480 or SW620 in the absence of SEB were <200 cpm. Data points represent the mean counts per minute of duplicate determinations ± SD.

To address whether SW480 expressed MHC class II, cells were examined for the surface expression of HLA-DR, -DQ, and -DP molecules by flow cytometry. As shown in Table I, MHC class II was not detected on SW480, whereas class I expression was robust. Similarly, MHC class I heavy chain and ß₂m could be immunoprecipitated from surface ¹²⁵I-labeled SW480 cells, whereas class II - and ß-chains could not be detected (data not shown). Taken together, these data suggest that SW480 cells lack expression of MHC class II molecules at the cell surface.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. Fixation of SW480 cells does not inhibit T cell activation by SEB. SW480 cells were fixed for 30 min on ice with PBS/1% paraformaldehyde and washed extensively. The viability of treated and untreated cells, as assessed by trypan blue dye exclusion, was >95%. Data points represent the mean counts per minute of duplicate determinations ± SD.

The peripheral human T cells used throughout these studies, although rigorously purified to remove MHC class II⁺ APC, may nonetheless up-regulate class II molecules during the course of activation (16). Furthermore, it has been demonstrated that MHC class II-positive T cell clones can act as potent APC for the induction of Ag-dependent (17, 18) as well as superantigen-dependent (19) T cell proliferation. For these reasons we sought to investigate whether MHC class II molecules might be up-regulated on T cells and play any subsequent role during the course of the proliferative response to SEB plus SW480. As revealed by flow cytometry, the level of MHC class II expression on T cell blasts stimulated by PBMC was modest (Table II), while the level of class II was approximately fivefold lower on blasts stimulated by SW480, similar in nature to the findings of Green et al. (20). In contrast, CD25 was up-regulated on both populations of blasts, indicating robust turnover of the cells. The significance of the low level of expression of MHC class II molecules on the SW480-stimulated blasts was unclear, so we attempted to ascertain a functional role for class II by using blocking Abs during the in vitro T cell response. T cell proliferation induced by SEB plus SW480 was not inhibited by a mixture of anti-class II mAbs present throughout the 4-day culture period (Table III), whereas SEB-induced T cell proliferation in the presence of HLA-DR1⁺ AC was inhibited >50% by addition of the same mAbs. These observations suggest that class II is not required for the initiation or maintenance of T cell proliferation in response to SEB plus SW480. To further address the potential role of MHC class II on T cells that may remain unblocked by the mAbs, the class II-negative human T cell hybridoma 289D1 was tested for the ability to respond to SEB plus SW480. SEB alone at high concentrations induced the release of IL-2 from 289D1 (Fig. 3), reflecting a productive interaction between SEB and the Vß3.1 TCR in the absence of APC. SEB plus SW480, however, greatly augmented IL-2 production, suggesting that the presentation of SEB via SW480 or perhaps the provision of costimulation by SW480 was able to enhance T cell activation. Taken together, these findings suggest that MHC class II expression remains low on T cells activated by SEB plus SW480 and does not facilitate T cell proliferation via binding and presenting SEB to other T cells.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. SW480 cells augment the activation of an MHC class II-negative human T cell hybridoma. The MHC class II-negative hybridoma 289D1 (Vß3.1, V1, CD4) was cultured in the presence of SEB alone or in combination with SW480 according to the accessory cell-dependent T cell proliferation protocol described in Materials and Methods. Culture supernatants were collected after 24 h and spun through a 0.45-µm filter, and the amount of IL-2 was measured by ELISA (Biosource, Camarillo, CA). Culture supernatants from unstimulated 289D1 or 289D1 plus SW480 contained <5 pg/ml IL-2, whereas supernatants from 289D1 stimulated with PHA (1 µg/ml) plus PMA (25 ng/ml) contained 8612 pg/ml IL-2.

To assess the scope of mitogens capable of stimulating T cells in this manner, a panel of superantigens and nonspecific T cell mitogens was screened for the ability to induce SW480-dependent T cell proliferation. As shown in Figure 4, SEB, SED, and SEC1 all acted as potent T cell mitogens with an approximate ED₅₀ of 10 ng/ml, whereas SEA was modestly mitogenic, and SEC2, SEC3 (data not shown), and SEE were nonstimulatory. All toxins, however, were extremely potent when presented by PBMC, with an approximate ED₅₀ of 20 pg/ml. These findings support a mechanism of T cell activation significantly different from that of MHC class II presentation, since SEA and SEE are both potent T cell mitogens when presented by class II⁺ APC (21). Interestingly, PHA and pokeweed mitogen were also stimulatory in association with SW480, while Con A was weakly mitogenic, and soluble anti-CD3 mAbs (OKT3 and 64.1) were not mitogenic over a range of concentrations (data not shown). These results suggest that SW480 cells may either bind and present a variety of mitogens to T cells, or they might provide costimulation to T cells that are partially activated through the direct interaction of mitogen with the TCR ß heterodimer in the absence of a specific presenting molecule.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. A limited repertoire of staphylococcal enterotoxins activates T cells in association with SW480 cells. Controls containing T cells plus SW480 or PBMC in the absence of enterotoxin were <400 cpm, while controls containing T cells plus enterotoxin (1 µg/ml) or lectin (2 µg/ml) alone were <800 cpm. Data points represent the mean counts per minute of duplicate determinations ± SD.

To address the nature of T cell recognition of SEB in the context of SW480, the TCR Vß specificity of T cell blasts was examined by flow cytometry. The Vß profile of T cells activated by SEB plus SW480 was qualitatively similar to that of T cells activated by SEB plus PBMC (Fig. 5), indicating that TCR-SEB interactions in the absence of class II were preserved, similar to the findings of other investigators (22). The increased proportions of Vß3, Vß12, and Vß13 T cell blasts resulting from stimulation via SW480, compared with that via PBMC, suggest that fewer Vß subsets are activated in the absence of class II. This observation may reflect a need for enterotoxin-TCR interactions of a higher affinity that are restricted to a more limited subset of Vß to activate T cells under these conditions. In addition, the percentage of CD8⁺ T cell blasts generated in the presence of SW480 (38.4%) was twice as high as the percentage of blasts generated in the presence of PBMC (17.6%), indicating an earlier or more prominent expansion of CD8⁺ T cells.

View larger version (26K): [in this window] [in a new window]   FIGURE 5. The TCR Vß profile of T cells activated by SEB plus SW480 resembles the Vß profile of T cells activated by MHC class II+ PBMC. T cells were activated with SW480 plus SEB (200 ng/ml) or PBMC plus SEB (2 ng/ml) and stained with a panel of Vß-specific mAbs. Analysis was restricted to the resting or blasting T cell population based on flow cytometric light scatter properties. Values represent specific mean linear fluorescence (MLF) units, calculated by subtracting the MLF of isotype-matched Ab controls, from one of three representative experiments.

Since MHC class II molecules are not expressed on SW480, an undefined receptor may bind and present enterotoxins in a manner similar to MHC class II. Several previous studies have reported binding of enterotoxins to various MHC class II-negative cells. In particular, Rogers et al. (23) reported that SEB bound to COS-1, a monkey kidney fibroblast-like cell, with high affinity (K_d = 5.1 x 10^-8 M), yet was unable to activate T cells in this context. In contrast, Beharka et al. (14) reported that SEB bound to peritoneal macrophages isolated from MHC class II-deficient mice with a very low affinity (K_d = 7.3 x 10^-5 M), yet was able to stimulate T cell proliferation with the addition of exogenous cytokines (24). We thus examined the association of SEB with SW480 using both functional and physical readouts. SW480 cells pulsed with SEB and washed with medium did not induce detectable T cell proliferation (Fig. 6), whereas HLA-DR1⁺ AC treated in the same manner stimulated potent T cell proliferation. These findings suggest that SEB either fails to associate with SW480 or associates with an affinity substantially below the micromolar range estimated for the interaction of SEB with HLA-DR1 (25, 26). Studies examining the association of ¹²⁵I-labeled SEB to intact SW480 cells revealed minimal (<0.15%) and nonsaturable binding with respect to protein concentration (Fig. 7), suggestive of nonspecific trapping of labeled protein by the cells. In comparison, 4% of the [¹²⁵I]SEB remained bound to HLA-DR1-transfected fibroblasts when incubated with 100 ng of [¹²⁵I]SEB. It is possible, however, that SEB associates with SW480 via a nonspecific, low affinity interaction with components of the negatively charged glycocalyx, for instance, or may form a transient ternary complex with TCR and ligand on the surface of SW480 cells. To examine these possibilities, SEB was covalently cross-linked to Sepharose beads (average 100 µm in diameter) to prevent the free association of SEB with SW480 cells in culture, and the SEB-Sepharose conjugates were examined for the ability to activate T cells. SEB-Sepharose conjugates activated T cells in the presence of SW480 cells, while SEB-Sepharose conjugates alone or BSA-Sepharose conjugates in the presence or the absence of SW480 cells did not stimulate T cell proliferation (Fig. 8). These findings demonstrate that the aggregation of SEB on the surface of the beads is not sufficient on its own to activate peripheral T cells, but must be present in combination with SW480 cells. Due to the steric constraints imposed by the close proximity of binding of SEB to the Sepharose matrix (<2 Å), it seems unlikely that an SEB molecule is able to associate simultaneously with both an SW480 cell and a T cell while complexed to a bead. These findings suggest that SEB may directly bind the TCR and transduce signals that lead to T cell proliferation with the addition of accessory signals provided by SW480 cells in trans.

View larger version (12K): [in this window] [in a new window]   FIGURE 6. SEB does not remain functionally associated with SW480 cells. SW480 cells or HLA-DR1-transfected fibroblasts were pulsed with SEB (1 µg/ml) for 1 h at 37°C and washed. SEB-pulsed AC were combined with T cells and compared with untreated AC combined with T cells plus SEB (1 µg/ml). Controls containing T cells plus AC in the absence of SEB are labeled "No toxin." Data points represent the mean counts per minute of duplicate determinations ± SD.

View larger version (22K): [in this window] [in a new window]   FIGURE 7. The binding of 125I-labeled SEB to SW480 cells is minimal and nonsaturable. Varying amounts of [125I]SEB were incubated with SW480 cells for 2 h at 37°C, and the cells were separated from unbound [125I]SEB by filtration. Data are presented as both the total amount of bound SEB as well as the percentage of bound SEB with respect to input amounts of SEB from one of two representative experiments.

View larger version (18K): [in this window] [in a new window]   FIGURE 8. SEB-Sepharose conjugates activate T cells in the presence of SW480 cells. Sepharose beads conjugated with SEB were combined with T cells in the presence or the absence of SW480 cells according to the accessory cell-dependent T cell proliferation protocol described in Materials and Methods. Controls containing BSA-Sepharose conjugates <500 cpm. Data points represent the mean counts per minute of duplicate determinations ± SD.

Two additional mechanisms may help account for the role of SW480 cells in the initiation of T cell proliferation. These include the release of soluble mediators from SW480 cells in culture and/or costimulation through cell surface accessory molecules. Coculture of SW480 cells separated from T cells by a permeable membrane in the presence of SEB did not result in T cell proliferation or up-regulation of the T cell activation marker CD25 (data not shown). These findings suggest that soluble mediators released by SW480 are unable to promote T cell activation in combination with SEB, and imply that cell-to-cell contact between the T cell and SW480 is required for activation. These observations as well as those concerning SW480 fixation (Fig. 2) support a model in which a constitutively expressed, membrane-bound receptor or cytokine on SW480 provides costimulation to T cells. SW480 cells were thus screened with a panel of mAbs to detect the expression of several costimulatory and adhesion molecules reported to assist in T cell activation. Blocking Abs, peptides, and soluble receptors directed against several of these costimulatory and adhesion molecules were also tested in culture for the ability to inhibit T cell proliferation mediated by SEB plus SW480. From these analyses, two receptor-ligand pairs, ICAM-1 (CD54)/LFA-1 (CD18) and LFA-3 (CD58)/CD2, were identified that contributed to T cell proliferation via this mechanism. SW480 were found to express intermediate levels of both ICAM-1 and LFA-3 (Table I) as well as low levels of B7-2 (CD86) (data not shown). As shown in Figure 9, T cell proliferation was inhibited to baseline levels by anti-ICAM-1 mAb in culture, while anti-LFA-3 and anti-CD2 mAbs independently reduced the level of proliferation by approximately 40%. The anti-LFA-1 mAb, however, did not block proliferation, perhaps due to the fact that the binding of this mAb to LFA-1 was unable to block cognate ligand interactions effectively. Although small amounts of B7-2 were detected on SW480, the addition of anti-B7-1 and anti-B7-2 mAbs as well as CTLA4-Ig to cultures did not inhibit T cell proliferation induced by SEB plus SW480, whereas these blocking reagents inhibited the proliferation of PBMC to tetanus toxoid nominal Ag (data not shown). These results suggest that T cell activation via SEB plus SW480 can proceed independently of B7-CD28 costimulation, similar to the findings of Damle et al. (27).

View larger version (29K): [in this window] [in a new window]   FIGURE 9. Anti-ICAM-1, anti-LFA-3, and anti-CD2 mAbs in culture inhibit T cell proliferation induced by SEB plus SW480. T cells and SW480 cells were cultured together in the presence of a fixed concentration of SEB (0.2 µg/ml) plus the indicated concentrations of specific mAbs or isotype-matched control mAb. Data points represent the mean counts per minute of duplicate determinations ± SD.

View larger version (15K): [in this window] [in a new window]   FIGURE 10. The effects of anti-ICAM-1 and anti-LFA-3 mAbs can be localized to SW480 cells. SW480 cells were pulsed with 10 µg/ml mAb on ice, washed, and treated with PBS/1% paraformaldehyde to cross-link mAbs on the cell surface. Values represent the mean percent inhibition of proliferation induced by SEB (0.2 µg/ml) of cultures containing treated SW480 cells compared with that of SW480 cells pulsed and fixed with an isotype-matched control mAb ± SD from two independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Interestingly, only a subset of the enterotoxins tested was able to activate T cells via this MHC class II-independent mechanism. Whereas SEB, SEC1, and SED were potent mitogens in the presence of SW480 cells, SEA was relatively weak, and SEC2, SEC3, and SEE were not mitogenic. Several investigators have reported similar functional differences among the staphylococcal enterotoxins. In particular, O’Hehir (9) noted that SEB and SED at high concentrations could induce the proliferation of a human Vß3 T cell clone in the absence of AC, while Yagi et al. (29) noted that SEB could activate a murine Vß8⁺ T cell clone. Dohlsten et al. (11) and Herrmann et al. (12) both reported that SEB, SEC1, and, to a lesser extent, SEA could direct CTL-mediated killing of several class II-deficient tumor cell lines. In contrast, Avery et al. (13) reported a very different pattern of enterotoxin reactivity in the activation of T cells isolated from MHC class II-deficient mice. In this system, SEE and SEC1, -2, and -3 were mitogenic, while SEA, SEB, and SED were not stimulatory. We also observed that SEB plus SW480 were unable to activate murine splenocytes isolated from MHC class II-deficient mice as well as a Vß8⁺ murine T cell hybridoma, although HLA-DR1⁺ AC plus SEB could stimulate murine T cells (data not shown). This pattern of reactivity is intriguing, since it segregates according to species, suggesting that inherent differences in the affinity of binding of the superantigens to TCR, the precursor frequency of reactive T cell subsets, or the presence of particular AC may account for the different patterns of reactivity observed between mice and humans. Within a given system, however, enterotoxin reactivity does not necessarily segregate to a particular family of related enterotoxins or correlate with other known functions, such as MHC class II binding or Vß specificity. For example, SEC1 and SEC2 are >95% identical at the amino acid level (30), are similarly potent when presented by different MHC class II molecules (31), and possess similar Vß reactivities (32), yet differ dramatically when presented in the context of SW480 cells. These findings suggest that subtle structural differences in SEC1 mediate class II-independent T cell activation, perhaps by affecting toxin-TCR interactions or protein multimerization in solution. We examined native SEC1 and SEC2 as well as a library of hybrid SEC1/SEC2 molecules and did not detect any significant differences in the abilities of these toxins to undergo homodimerization or other protein modifications, but, rather, localized this functional difference between SEC1 and SEC2 to residues mediating direct contact with the TCR. This finding suggests that T cell activation in the absence of MHC class II depends on the affinity or topology of the enterotoxin interaction with TCR and perhaps secondary interactions between MHC class II and TCR (our manuscript in preparation).

SW480 cells express both ICAM-1 and LFA-3, and treatment with blocking Abs to these receptors can significantly inhibit the proliferative response induced by SEB plus SW480. These observations suggest that the interaction of these receptors with their cognate ligands, LFA-1 and CD2, on T cells may assist in TCR-mediated activation. Indeed, it has been reported that ICAM-1 can promote SEA-mediated T cell proliferation (33, 34), while SEB could activate a human T cell clone if cross-linked on a bead with anti-CD2 mAb (35). In addition, Haffner et al. (36) reported that the presentation of SEB by an MHC class II-negative human squamous cell carcinoma cell line was significantly inhibited by an anti-ICAM-1 mAb. Engagement of either LFA-1 (37, 38) or CD2 (39, 40) by their cognate ligands may promote activation by directly transducing costimulatory signals to the T cells. LFA-1 and CD2 also serve as prominent adhesion molecules that strengthen cell-to-cell interactions (41). Thus the interaction of T cells with SW480 cells via these receptor-ligand pairs may lead to secondary interactions that promote T cell activation by other means. For example, an increase in overall cell-to-cell avidity might strengthen interactions with other costimulatory molecules. Although we have shown that B7-1 and B7-2 as well as CD40 and CD44 (data not shown) cannot account for the costimulation provided by SW480 cells, perhaps other potential T cell costimulatory molecules not addressed here are at play, such as CD27 (42) or heat-stable Ag (43). Alternatively, costimulation might be provided by novel ligands, such as those implicated by Hagerty (44) or Nieland et al. (45). ICAM-1 and LFA-3 may also facilitate T cell activation by helping to organize critical signaling components within the T cell membrane. Shaw et al. (46) recently proposed a topographical model of T cell activation in which ICAM-1/LFA-1 and LFA-3/CD2 interactions promote the formation of contact caps between T cells and APC. These areas of contact function to concentrate TCR and costimulatory molecules in the T cell membrane while excluding phosphatases, such as CD45, thereby enabling sustained signaling through the TCR that drives IL-2 production and proliferation. Perhaps SW480 cells promote the formation of contact caps on T cells via ICAM-1 and LFA-3 interactions that enable superantigens to bind and effectively transduce signals through the TCR.

Functional studies have noted that the treatment of purified T cells with SEB alone can result in intracellular Ca²⁺ flux (47), progression from G₀ to G₁, and up-regulation of IL-2R (20), as well as the induction of anergy (10). The manner in which enterotoxins bind and signal through the TCR in the absence of MHC class II, however, is unclear. It is generally thought that TCR ligands must be presented as a multivalent complexes capable of cross-linking the TCR to achieve activation. Recent studies have demonstrated that SEB and SEC1, -2, and -3 are able to bind soluble TCR in the absence of MHC class II molecules (48, 49). These interactions are characterized by a moderate affinity (K_d = 0.9 µM) and moderate to fast off-rates (k_off = 1.1 x 10^-2 to >0.1 s^-1), suggesting that the enterotoxins free in solution not only engage the TCR, but may also support the rapid, serial triggering of multiple TCR described by Valitutti et al. (50) as a mechanism to activate T cells in the presence of limiting peptide-MHC complexes. Indeed, if TCR were consolidated in a contact cap, the relative high density of TCR and fast kinetics of SEB-TCR interactions may promote productive signaling by SEB monomers in the absence of TCR cross-linking. Alternatively, the enterotoxins may form functional homodimers, such as described for SEB (51) and SED (52), or multivalent aggregates in solution that themselves can cross-link TCR and promote T cell activation in a manner similar to soluble anti-CD3 mAbs. In either case, ICAM-1/LFA-1 or LFA-3/CD2 interactions may augment signaling and promote activation.

In conclusion, we have characterized a mechanism by which MHC class II-negative cells can support the activation of human T cells by bacterial superantigens provided they express appropriate accessory molecules. This mechanism may be employed in vivo to initiate T cell activation in the absence of MHC class II⁺ APC and thereby serve as a means to amplify or alter an immune response. It remains to be seen, however, how this class II-independent pathway may ultimately affect the activation of T cells, since it may use signaling mechanisms or lead to states of immunologic activation or tolerance that differ from conventional MHC class II-dependent pathways.

	Acknowledgments

 
We thank Mari Hagiwara and Mai Van for expert technical assistance.

	Footnotes

 
¹ This work was supported by U.S. Public Health Service Grant RO1AI30036 (to R.R.R.).

² Member of the Medical Scientist Training Program.

³ Address correspondence and reprint requests to Dr. Robert R. Rich, Department of Microbiology and Immunology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

⁴ Abbreviations used in this paper: AC, accessory cell; SEA through SEE, staphylococcal enterotoxins A through E.

Received for publication July 15, 1997. Accepted for publication September 29, 1997.

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