Staphylococcal Superantigens Use LAMA2 as a Coreceptor To Activate T Cells

The Gram-positive bacterium Staphylococcus aureus is a common commensal in the upper respiratory tract of 30–50% of healthy adults at any given time (1–3). S. aureus is also a frequent pathogen, causing various infections including dermatitis, cellulitis, pneumonia, and endocarditis. In addition, most S. aureus isolates produce superantigens, which are pyrogenic exotoxins that cause toxic shock syndrome (TSS) (4).

TSS is a serious condition (5) and the pathogenesis of this disease is explained by the ability of superantigens to trigger massive proinflammatory cytokine release following intense T cell activation (5, 6). In contrast to the very small (<0.01%) percentage of T cells activated by a nominal Ag, even picomolar concentrations of superantigens can activate up to 30% of the body’s T cells (7, 8). The massive T cell activation induced by superantigens is secondary to its capacity to bind directly to the V region of selective β-chains of the TCR and to MHC class II molecules on APCs (9, 10). The result is an overwhelming hyperinflammatory response that causes tremendous collateral tissue damage, cardiovascular dysfunction, and hemodynamic collapse, and directly threatens the viability of the host. There are no effective treatments for superantigen-driven staphylococcal TSS. Current therapy is aimed at hemodynamic stabilization, corticosteroid treatment, and other types of supportive care (5).

The differences between nominal Ags and superantigens are not only apparent in terms of recognition by the TCR but are also evident in the early signaling mechanisms to activate T cells. Activation of TCR signal transduction by nominal Ags requires the function of LCK, a tyrosine kinase associated with CD4 and CD8 in T cells. However, previous studies have demonstrated that superantigens can also activate human T cells through an LCK-independent pathway that requires a Gα11-containing G protein–coupled receptor (GPCR) and PLCβ (11, 12). Yet, the molecular players involved in the activation of this pathway have thus far remained a mystery. Here, we report that LAMA2, the α2 subunit of laminin, links superantigen recognition with GPCR signaling. Furthermore, our data suggest that a member of the GPCR class C family of the calcium-sensing receptor (CaSR) type is likely involved because superantigen-induced effects were partially or completely blocked in the presence of the CaSR antagonist NPS 2143 in vitro and in vivo. Based on our findings, blocking the interaction between superantigens and LAMA2 may represent a new therapeutic strategy to slow the development of full-blown TSS during S. aureus infections.

The Jurkat E6.1 T cell line and the LCK-deficient Jurkat subline JCaM1.6 were purchased from American Tissue Culture Collection. The lymphoblastoid B cell line LG2 was provided by Dr. E. Long (National Institutes of Health). All cells were cultured in RPMI 1640 medium (Thermo Scientific) supplemented with l-glutamine, nonessential amino acids, sodium pyruvate, penicillin-streptomycin, and 10% FBS.

Laminin 211 (LN211) and laminin 221 (LN221) were from BioLamina. Staphylococcal enterotoxin E (SEE; cat # ET404) and biotinylated SEE (cat # ET404-B) were from Toxin Technology. Streptococcal mitogenic exotoxin (SME-Z), biotinylated staphylococcal enterotoxin B (SEB) and biotinylated TSS toxin-1 (TSST-1) were generated as previously described (11). Biotinylation tags were engineered onto the C terminus of each protein.

Peptide MG73 (sequence: KNRLTIELEVRT) derived from human LAMA2 (aa 2784–2795), and scrambled peptide MG73s (sequence: RTLEVINTKLER) were synthesized by GenScript. For the pull-down assays, N-terminal biotin-modified MG73 and MG73s were used.

Ligand-receptor capture technology LRC-TriCEPS (Dualsystems Biotech) was performed as previously described (13, 14). Briefly, SEE (Toxin Technology) was coupled with a trifunctional cross-linker TriCEPS, which has an N-hydroxysuccinimide ester for ligand coupling, a hydrazine cross-linker for receptor capture, and biotin for purification. Insulin was used as a control ligand. SEE-TriCEPS was incubated with oxidized sodium metaperiodate and LCK-deficient Jurkat T cells (JCaM1.6) and B cell APCs (LG2 cells) in PBS pH 6.5 at 4°C for 90 min. Cells were collected and lysed, and the lysate subjected to trypsinization and biotin-mediated affinity purification. Released receptor glycopeptides were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). SEE and insulin datasets were quantitatively compared and presented as a volcano plot.

Immunoplate 96-well plates were coated (4°C overnight) with LN211 and LN221 (both from BioLamina), as well as BSA (negative control), at 5 μg/ml in PBS pH 7.4 containing 0.9 mM CaCl₂ and 0.4 mM MgCl₂ (PBS-CM). After washing with PBS-CM containing 0.02% (v/v) Tween 20, the wells were blocked with diluent buffer (PBS-CM containing 1% (w/v) BSA) for 1 h at room temperature (RT). Washed again, the wells were then incubated with biotinylated SEE, biotinylated SEB, or biotinylated TSST-1 in diluent buffer (5 h at RT). Washed again, the wells were then incubated with avidin-HRP (eBioscience) in diluent buffer (1 h at RT) before adding the 3,3′,5,5′-tetramethylbenzidine substrate (eBioscience). The colored reaction was terminated with stop solution (2 M sulfuric acid) and the absorbance in each well was measured using a microplate reader.

Streptavidin beads (Invitrogen) coated with the same amount of biotin-MG73 or biotin-MG73s peptides were blocked with free D-biotin (Santa Cruz Biotechnology) and BSA for 1 h. Beads were then washed and coincubated with 5 μg of nonbiotinylated SEE for 2 h. After extensive washes, beads were heated at 95°C for 7 min in 1× SDS sample buffer. Eluted samples were then subjected to SDS-PAGE and immunoblotted using anti-SEE Ab (Toxin Technology).

Based upon the method of Casu et al. (15), surface plasmon resonance (SPR) was performed at 25°C using a BIACORE 3000 system with PBS-CM running buffer containing 0.05% (v/v) Tween 20. LN211, LN221, and reference (no protein) surfaces were amine-coupled to CM5 sensors at neutral pH as recommended by the manufacturer (20 μg/ml laminin in PBS-CM pH 7.4 containing 1.4 mM cetyltrimethylammonium bromide detergent [Calbiochem]; 1500–3000 resonance units final density). Free biotin (0–100 μM), BSA (0–5 μM), and biotin-SEE (0–5 μM) were titrated in tandem over reference and active laminin surfaces in single-cycle (50 μl/min × 60 s association + 30–300 s dissociation) and/or multicycle mode (15 μl/min × 10 min association + 20 min dissociation). The surfaces were regenerated at 50 μl/min using two 30 s pulses of PBS-CM containing 1 M NaCl and 0.1% (v/v) Empigen detergent (Anatrace). Because the acquired titration data deviated from a simple “1:1 kinetic” binding model (i.e., likely because of sample heterogeneity in the purified LN211/221 and/or biotin-SEE preparations), the “steady-state affinity” model (BIAevaluation software) was used to predict the apparent equilibrium dissociation constant (K_D).

Lentiviral particles (# HS0000015349; Sigma-Aldrich) containing sequences of guide RNA (5′-TCACGACCAATGCAACATGTGG-3′) and Cas9 in the construct of U6-gRNA/EF1a-puro-2A-Cas9-2A-GFP were used to infect JCaM1.6 T cells. Transfectants were selected by puromycin and single cell sorted. Expression of LAMA2 in single cell colonies was assessed by Western blot.

JCaM1.6 T cells (5 × 10⁴ per group) and LG2 cells (2.5 × 10⁴ per group), or PBMCs (2 × 10⁵ per group) were preincubated with or without the indicated concentration of inhibitors (chemical inhibitors were purchased from Tocris Bioscience; CD97 neutralizing Ab and control monoclonal mouse IgG2A Clone # 380903 were purchased from R&D Systems) or peptides for 1 h, followed by stimulation with the indicated concentration of superantigen for 8, 18, or 24 h. For splenocytes (5 × 10⁵ per group) isolated from HLA-DR4 mice, SEB was used. For T cell activation by canonical TCR signaling, Jurkat E6.1 T cells (1 × 10⁵ per group) were preincubated with indicated concentration of NPS 2143 for 1 h, and then incubated with 0.25 μg/ml anti-CD3 and anti-CD28 Abs for 18 h. Accumulation of IL-2 in culture supernatants was determined by ELISA (BioLegend and eBioscience). To determine the capacity of IL-2 production by different T cell clones, the same number of T cells were stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin for 18 h.

JCaM1.6 T cells (2 × 10⁶ per group) and LG2 cells (1 × 10⁶ per group), preincubated with or without 10 μM NPS 2143 for 1 h, were stimulated with 10 ng/ml SEE or vehicle control for 15 min. Cell lysates were prepared in 1% Triton X-100 lysis buffer. Western blot was performed as previously described (16). Anti-phosphorylated ERK1/2 Ab, anti-CD29 Ab, and anti–β-actin Ab were purchased from Cell Signaling Technology. Anti-total ERK1/2 Ab was purchased from StressGen (Victoria, BC, Canada). Anti-LAMA2 Ab (B-4) was purchased from Santa Cruz Biotechnology.

Jurkat E6.1, JCaM1.6, LG2, HEK293, and human CaSR-transfected HEK293 cells were subjected to RNA extraction using the RNeasy Plus Kit (Qiagen). cDNA was synthesized using the cDNA Reverse Transcription Kit (Applied Biosystems). PCR was performed using Taq DNA polymerase (FroggaBio) and the primers listed in Table I, with a program of 94°C for 3 min, followed by 40 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 45 min.

JCaM1.6 T cells were stimulated with LG2 cells and SEE in the presence or absence of 10 μM NPS 2143 for 18 h. Cells were harvested and stained with the following fluorochrome-conjugated Abs: anti-human CD3 APC-eFluor 780 (clone UCHT1; eBioscience), anti-human CD28 FITC (clone CD28.2; eBioscience), and anti-human CD69 Alexa Fluor 647 (clone FN50; BioLegend) Abs. The Zombie Aqua Fixable Viability dye (BioLegend) was used to exclude dead cells. Flow cytometry was performed with LSRII Fortessa (BD Biosciences) and Diva software (BD Biosciences) and data analyzed with FlowJo version 10.x (Tree Star, Ashland, OR).

C57BL/6 HLA-DR4 mice (17) were purchased from Taconic Biosciences and maintained in the animal facility at McGill University with the approval of the Comparative Medicine and Animal Resource Centre in accordance with Canadian Council on Animal Care guidelines. To induce TSS, 6–10-wk-old mice were i.p. injected with 30 mg of D-(+)-galactosamine hydrochloride (Sigma) followed, 1 h later, by i.p. injection of SEB (5 μg per mouse) (18). To test the effect of CaSR antagonism, NPS 2143 was dissolved in vehicle (20% aqueous solution of 2-hydroxypropyl-cyclodextrin; Sigma) or vehicle control and administrated (30 mg/kg mouse body weight per injection) 2 h before and 8 h after SEB injection. Mice were monitored every 10–15 min during the initial phase postinjection and monitored every hour from 12 to 48 h postinjection. To determine serum cytokine levels, mice were sacrificed 2 h post–SEB injection and serum collected for ELISA.

Statistical analysis was performed using ANOVA and Student t test or Kaplan–Meier analysis with GraphPad Prism. A p value <0.05 was deemed significant.

Previous findings have demonstrated that bacterial superantigens trigger T cell activation not only through the canonical LCK-dependent signaling pathway, but also through a G protein–dependent alternative pathway (11, 12). To identify the molecular partner(s) linking the superantigen with GPCR signaling, we used the ligand-receptor capture technology LRC-TriCEPS (13, 14). LCK-deficient T cells were stimulated with LG2 cells as APCs in the presence of TriCEPS-conjugated superantigen SEE. Glycosylated receptor proteins that interacted with SEE were captured by TriCEPS molecules and identified by LC-MS/MS. Insulin was used as a control ligand. As expected, the insulin receptor INSR was captured by insulin and the TCR β-chain was captured by SEE (9, 10). Notably, quantitative comparisons between the interactomes of SEE and insulin revealed LAMA2 as a highly significant hit for interacting partners of SEE (Fig. 1A).

LAMA2 is the α2 subunit of heterotrimeric laminins, extracellular proteins that interact with cell surface adhesion molecules and GPCRs (19, 20). LAMA2-containing laminins are expressed in skeletal muscle tissues, thymus, and blood vessels, as well as lymphoblastoid cell lines (21–25). To validate our TriCEPS finding in cells, we first confirmed the expression of LAMA2 in LCK-competent and LCK-deficient Jurkat T cells, as well as LG2 cells. In both Jurkat T cell lines, LAMA2 was highly expressed in terms of both mRNA and protein levels (Fig. 1B, 1C, respectively). In contrast, LAMA2 protein was detected at much lower levels in LG2 APCs although LAMA2 mRNA was highly expressed.

To demonstrate that SEE can directly bind to LAMA2, we assessed the interaction of SEE using two heterotrimeric proteins that contain LAMA2, namely LN211 or LN221. By ELISA, the binding of SEE (biotinylated for avidin-HRP detection) to the coated laminins yielded similar responses, which were significant compared with “sticky” BSA as a negative control (Fig. 2A). Two additional staphylococcal superantigens, SEB and TSST-1, also interacted with the laminins, but they exhibited lower binding responses compared with SEE under similar ELISA conditions (Supplemental Fig. 1). Representative of both laminins, an additional titration showed that the dose-dependent binding of biotin-SEE to coated LN221 approached saturation around 50 μg/ml (equivalent to 2.5 μM biotin-SEE) (Supplemental Fig. 2A).

For cross-validation, nonbiotinylated SEE was used to perform classical pull-down assays (Fig. 2B). SEE specifically bound to biotinylated MG73, a 12-aa peptide derived from the G4 domain of LAMA2 which has been reported to mediate cell adhesion (26, 27), whereas there was little or no binding to the corresponding biotinylated scrambled peptide MG73s.

Consistent with the ELISA data and the pull-down data, single-cycle SPR experiments (Supplemental Fig. 2B) showed that biotin-SEE specifically bound to amine-coupled laminins in a dose-dependent manner (saturable response approaching 5 μM). Complementary multicycle SPR experiments (Fig. 2C, 2D) showed that biotin-SEE possessed similar equilibrium dissociation constants for LN211 (K_D = 1.5 μM) and LN221 (K_D = 1.8 μM). Collectively, our ELISA, pull-down, and SPR data demonstrate that SEE can directly interact with the G4 domain of LAMA2 and LAMA2-containing laminins on T cells.

Next, we examined the effect of targeting LAMA2 on SEE-induced T cell activation using the LAMA2-derived MG73 peptide and a scrambled (MG73s) control peptide. Neither peptide was able to induce IL-2 production by LCK-deficient T cells in the absence of SEE (Fig. 3A). However, in the presence of SEE, addition of MG73 significantly enhanced T cell activation whereas the control MG73s did not (Fig. 3A).

To further demonstrate the involvement of LAMA2 in T cell activation, we generated LAMA2-deficient, LCK-deficient JCaM1.6 T cells using CRISPR/Cas9 technology targeting the second exon of the LAMA2 gene. Deficiency of LAMA2 in these T cells was confirmed by Western blotting (Fig. 3B). We found that superantigen-induced activation of LAMA2-deficient T cells was dramatically diminished as illustrated by reduced IL-2 production (Fig. 3C) and CD69 expression (Fig. 3E) compared with parental JCaM1.6 T cells. Similar effects were also observed in T cells responding to SME-Z (Supplemental Fig. 3), another TCR Vβ8-binding superantigen. The minimal IL-2 response remaining in LAMA2-deficient T cells is likely secondary to the low levels of LAMA2 expression by the APCs, or to other coreceptors. Moreover, the significant reduction in the IL-2 response to superantigens was not due to an intrinsic defect in IL-2 production by these cells because PMA and ionomycin induced a strong IL-2 response, even higher than that observed in parental cells (Fig. 3D). Taken together, these data demonstrate an important role of LAMA2 in superantigen-triggered T cell activation.

As superantigens activate GPCR signaling, we next investigated GPCR candidates that might be activated by SEE and LAMA2. Integrins act as primary laminin receptors (28) and physically interact with and activate the CaSR to regulate cell adhesion and migration (29). Similar to superantigens, CaSR ligands can trigger Gαq/11 signaling (30). Thus, we first tested the involvement of the CaSR using its antagonist NPS 2143. In the presence of NPS 2143, activation of LCK-deficient T cells in response to SEE was significantly inhibited in a dose-dependent manner as illustrated by the diminished IL-2 response (Fig. 4A), lack of upregulation of the T cell activation marker CD69 (Fig. 4B), and minimal ERK activation (Fig. 4C). This effect was specific for the alternative T cell signaling as activation of LCK-competent Jurkat T cells by anti-CD3/anti-CD28 Abs was not affected by NPS 2143 (Fig. 4D). Furthermore, these results were not due to toxicity of the compound as cell viability was not affected (data not shown). The effect was not unique to Jurkat cells as the inhibitory effect of NPS 2143 was also observed in human and mouse primary T cells (Fig. 5).

We next tested whether NPS 2143 could downregulate T cell activation in vivo and prevent TSS. In HLA-DR4 transgenic mice, administration of SEB resulted in TSS and mortality with a mean survival of 7.75 h (Fig. 6A). Concomitant administration of NPS 2143 and SEB resulted in a significant doubling of mean survival (16 h) although mortality at 48 h was not affected. Prolonged survival correlated with downregulation of IFN-γ and TNF-α production, although IL-2 production did not change significantly (Fig. 6B). The lack of effect on IL-2 and the inability to completely prevent TSS may be secondary to the activation of the canonical LCK-dependent pathway which is still active in vivo.

The mechanism of action of NPS 2143 has been linked to selective antagonism of the CaSR or its secondary target GPRC6A (31, 32). Thus, we next examined whether these two receptors were involved in SEE-induced LCK-independent T cell activation. To our surprise, we did not detect expression of the CaSR or GPRC6A (data not shown) in Jurkat T cells by RT-PCR or Western blotting. These findings implied that there are other NPS 2143–sensitive receptors. Additional screening of Jurkat T cells for expression of class C GPCRs (Table I), to which the CaSR belongs, revealed expression of GABBR1, GRM2, GRM4, GRM8, and GPRC5B, as well as expression of the adhesion GPCRs LPHN1, GPR125, BAI1, BAI2, CELSR1, CELSR2, CELSR3, and CD97, which interact with laminin and extracellular matrix (ECM) proteins, and of Gαq/11-coupled GPCRs GPR180, AVPR2, PAR1, PAR2, S1PR2, and S1PR4 (Fig. 7). Taken together, these data suggest that not the CaSR itself but CaSR-type GPCRs are associated with laminin signaling in superantigen-induced T cell activation.

Full T cell activation, in response to TCR binding to peptide-MHC complexes or Ab-mediated TCR cross-linking, requires the expression of functional LCK and ZAP-70. In contrast, staphylococcal superantigens are able to fully activate LCK-deficient and ZAP-70–deficient T cells (11, 33). They do so by turning on an alternative signaling pathway utilizing a Gα11-coupled receptor and PLCβ (11). Our results here identify laminin and its LAMA2 subunit as an important player in this process. By directly interacting with the staphylococcal superantigen and bridging it with GPCR signaling, this leads to T cell activation and IL-2 production. Because activation of this alternative pathway still requires TCR engagement by the superantigen (11), we propose that the role of LAMA2 fulfills the operational definition of coreceptor.

Components of the ECM have been reported to play important roles in T cell signaling (34, 35). Fibronectin, fibrinogen, and vitronectin not only act synergistically on anti-CD3 Ab-induced T cell activation, but can also trigger responses by themselves (34). In addition, it has been reported that laminin 5 promotes T cell activation and apoptosis (35). In these studies, it is important to note that the ECM components were interacting with its receptors on T cells independently of TCR engagement, and their effect was an additive effect on canonical TCR signaling. In contrast, under conditions of superantigen-induced T cell activation, our data show that the role of laminin involves activation on an alternative signaling pathway.

Integrin β1 is the major receptor for ECM proteins in leukocytes and acts as a costimulator for TCR:CD3-driven T cell activation (36). In this context, it was expected that LAMA2 played its role by using integrin β1 signaling. However, we were not able to confirm this possibility given that the integrin β1–deficient Jurkat T cell line has an intrinsic defect in IL-2 production. In addition, a neutralizing Ab against integrin β1 only partially inhibited IL-2 production in response to superantigen-stimulated, LCK-deficient T cells (data not shown). Based on these results, we cannot exclude that the activation of the alternative signaling pathway may in part involve LAMA2 interacting with integrin β1.

How does LAMA2 connect TCR-engaged SEE with G protein signaling then? The results presented here point to the interaction of LAMA2 with multiple CaSRs. The initial candidate was the CaSR, a prototypic receptor activated by extracellular calcium, as well as gadolinium, magnesium, L-α-amino acids and some antibiotics, resulting in the activation of a Gαq/11 signaling cascade (37). The CaSR plays important roles in immune responses including activation of the NLRP3 inflammasome (38) and of the MAPKs and NF-κB pathways resulting in production of proinflammatory cytokines IL-6 and TNF-β (39). In addition, the CaSR physically interacts with the laminin receptor integrin β1 (29). Our observation that the CaSR antagonist NPS 2143 inhibited T cell activation in vitro and in vivo in response to superantigens suggested that the CaSR was mediating the activation of the alternative TCR signaling pathway. However, we were not able to detect the expression of this GPCR in JCaM1.6 T cells. This observation is similar to what has been reported in B cells, in which extracellular calcium promoted B cell activation and function but the CaSR itself was not detectable (40). We have also excluded GPRC6A because it is not expressed by JCaM1.6 T cells. In addition, the metabotropic glutamate receptors mGluR1 and mGluR5, although highly expressed in Jurkat T cells (41), did not seem to play a role in the IL-2 response of JCaM1.6 T cells to superantigens based on the lack of effect of antagonists for these receptors. Overall, these findings lead us to suggest that there are other CaSR–related receptors sensitive to NPS 2143 and that may mediate such an effect.

It is plausible that the effect of LAMA2 in activating the alternative pathway of T cell activation by superantigens involves a coordinated response of multiple CaSR-type GPCRs that interact with laminin. This model is in line with the array of receptor interactions with ECM proteins (28, 42, 43). Several classes of receptors may be involved in signal transduction by LAMA2. One group is the target voltage-gated calcium channels (VGCC), which have been associated with ECM laminin 9 signaling (44). Of interest, NPS 2143 inhibits CaSR-independent, VGCC-mediated vascular reactivity (45). VGCC, specifically l-type VGCC is highly expressed in Jurkat T cells (46, 47). Furthermore, isradipine, a selective l-type VGCC blocker, downregulated SEE-induced IL-2 response in JCaM1.6 T cells. However, the required dose of isradipine to inhibit IL-2 production in response to SEE was very high (EC₅₀ of 50 μM, Supplemental Fig. 4A) casting the uncertainty of its role in mediating LAMA2-dependent alternative T cell signaling.

Adhesion GPCRs may also be candidates for LAMA2 signaling to T cells. These are a class of protein receptors that interact with ECM and facilitate cell adhesion and migration (48). LAMA2 has been reported to physically interact with the adhesion GPCR GPR126 (49). However, GPR126 is not involved in the superantigen-induced alternative signaling because it was not expressed in JCaM1.6 T cells (data not shown); also, GPR126 is associated with Gαs/i instead of Gαq/11 signaling (50). We have ruled out CD97 because the IL-2 response to superantigen was not affected in the presence of an anti-CD97 neutralizing Ab. However, there are other receptors such as the Gq/11-coupled GPCRs GPR180 and PAR1, the Gαq/11 protein–coupled GPCR S1PR2, and the Gαi/Gα12/13-coupled S1PR4, which are expressed in JCaM1.6 T cells. Of interest, the S1PR4 antagonist CYM 50358 inhibited the alternative T cell signaling in a dose-dependent manner (Supplemental Fig. 4B) suggesting that it may also be involved in superantigen-induced LAMA2 signaling. Further experiments are needed to investigate its role in T cell activation and the specificity of this antagonist. However, the observation of partial effects upon antagonism on several of these GPCRs is consistent with the idea that LAMA2 interacts with multiple receptors and its effect on activating alternative signaling in T cells is a cumulative effect.

Superantigens directly bind to MHC class II and TCR V β-chain. It has also been reported that superantigens interact with CD28 (51). However, the contribution of CD28 to the alternative signaling by superantigens is unknown. In our experience, the T cell response to superantigens does not require CD28 expression on T cells although such a response is enhanced if CD28 is available (G. Criado and J. Madrenas, unpublished observations). In addition, the potential cross-talk between canonical and alternative signaling pathways and the specific involvement that CD28 has in bridging them in light of the role reported here for LAMA2 remain to be elucidated. Because LCK is required for the negative regulation of TCR signaling by activating SHP-1 and other downstream regulators (52), it is therefore plausible that, in the presence of active LCK, the contribution of alternative TCR signaling by superantigens may be underappreciated.

In summary, we have demonstrated that the laminin subunit LAMA2 directly binds superantigens and bridges the superantigens with NPS 2143–sensitive activation of a CaSR-type GPCR(s). This mechanism turns on an LCK-independent, alternative pathway that potentiates the activation of T cells by staphylococcal superantigens. Our findings provide new insights for the development of novel therapeutic strategies for bacterial superantigen-induced diseases that target the interaction of the superantigen with LAMA2.

We thank Camille Stegen (McGill’s Department of Microbiology and Immunology Flow Cytometry and Cell Sorting Core Facility) for assistance with flow cytometry experiments. We thank the members of the Madrenas Laboratory for helpful comments and criticisms.

The authors have no financial conflicts of interest.