Activation of human γδ T cells and NK cells by Staphylococcal enterotoxins requires both monocytes and conventional T cells

Staphylococcal enterotoxins (SE) pose a great threat to human health due to their ability to bypass antigen presentation and activate large amounts of conventional T cells resulting in a cytokine storm potentially leading to toxic shock syndrome. Unconventional T‐ and NK cells are also activated by SE but the mechanisms remain poorly understood. In this study, the authors aimed to explore the underlying mechanism behind SE‐mediated activation of MAIT‐, γδ T‐, and NK cells in vitro. CBMC or PBMC were stimulated with the toxins SEA, SEH, and TSST‐1, and cytokine and cytotoxic responses were analyzed with ELISA and flow cytometry. All toxins induced a broad range of cytokines, perforin and granzyme B, although SEH was not as potent as SEA and TSST‐1. SE‐induced IFN‐γ expression in MAIT‐, γδ T‐, and NK cells was clearly reduced by neutralization of IL‐12, while cytotoxic compounds were not affected at all. Kinetic assays showed that unconventional T cell and NK cell‐responses are secondary to the response in conventional T cells. Furthermore, co‐cultures of isolated cell populations revealed that the ability of SEA to activate γδ T‐ and NK cells was fully dependent on the presence of both monocytes and αβ T cells. Lastly, it was found that SE provoked a reduced and delayed cytokine response in infants, particularly within the unconventional T and NK cell populations. This study provides novel insights regarding the activation of unconventional T‐ and NK cells by SE, which contribute to understanding the vulnerability of young children towards Staphylococcus aureus infections.

skin, and gut. However, S. aureus is also a major cause of nosocomial infections, food poisoning, and sepsis due to production of a wide range of staphylococcal enterotoxins (SE). 1 In addition to causing food poisoning and toxic shock syndrome, 2,3 certain SE could potentially be weaponized and used in biological warfare. 4 The virulence of S. aureus is mainly determined by its enterotoxins and 26 SE and SE-like proteins have been described and classified. 5 SE are known to induce polyclonal activation of conventional T cells. This is independent of Ag presentation and potentiated by cross-linking the T cell receptor (TCR) and the MHC class II receptor resulting in proliferation, cytotoxicity and secretion of the cytokines IL-2, IFN-γ, and TNF. 5 Two frequently studied SE, SEA and toxic shock syndrome toxin-1 (TSST-1), mediate conventional T cell activation through several TCR variable β-chains (TRBV), while the less studied SEH specifically binds the alpha chain (TRAV27). 6 Notably, although unconventional T cells and NK cells are also readily activated by S. aureus and its SE, [7][8][9] the mechanisms involved in their activation by SE still remain poorly understood. Unconventional T cells are non-MHC restricted and include, among others, mucosal associated invariant T (MAIT) cells and γδ T cells. These cell types are described as innate-like lymphocytes that recognize a limited set of microbe-derived antigens and quickly secrete cytokines and act cytotoxic upon immune challenge. 10,11 MAIT cells are restricted by the MHC class I-like receptor (MR1) and characterized by expression of a semi-variable αβ TCR with a fixed αchain (TRAV2-1) and high IL-18R and CD161 expression. 12 γδ T cells express a γand δ-chain TCR of which the majority respond to small, phosphorylated bacterial metabolites or lipid based self-ligands via the receptor butyrophilin 3A1 (BTN3A1) or CD1d, respectively. [13][14][15][16] cells are innate lymphocytes lacking a TCR, but capable of clearing infected and transformed cells through cytolytic activities and cytokine production. 17,18 Although SEA has been reported to directly activate γδ T cells, 19 the binding restrictions of most SE suggest a theoretical inability to directly activate γδ T cells and NK cells, while SEA and TSST-1, but not SEH, could potentially directly activate MAIT cells. On the other hand, these cells are also activated by the presence of inflammatory cytokines in the local environment where APC-derived cytokines are of particular importance. For example, NK cell effector responses are up-regulated by IL-12, IL-15, and IL-18 20 and IL-12 and IL-18 can induce effector responses in MAIT cells through a TCR-independent mechanism. 8,21 However, the involvement of innate cytokines in SE-mediated activation of unconventional T cells and NK cells require further investigations.
In the present study, we aimed to investigate underlying mechanisms of SE-mediated activation of MAIT cells, γδ T cells and NK cells in vitro. Further, as S. aureus is one of the leading causes of neonatal sepsis 22 and the immune system of neonates and infants have altered characteristics compared to adults, 23,24 we also wanted to compare neonatal and adult immune cell responses toward SE.

Ethics statement and isolation of cord/peripheral blood mononuclear cells
For the majority of the experiments (Figures 1-4; all Supplementary figures), peripheral blood mononuclear cells (PBMC) from adult volunteers were used, which was approved by the regional Ethics committee at the Karolinska Institute, Stockholm, Sweden. All adult study subjects gave their informed written consent.
In Figure 4, cord blood mononuclear cells (CBMC) were collected from healthy neonates at term deliveries, which was approved by the regional Ethics committee at the Karolinska Institute, Stockholm, Sweden. All mothers gave their informed written consent.
In Figure 5, CBMC/PBMC from healthy children at birth, 2, and 7 years of age, and adults, participating in 2 different cohorts described elsewhere 25,26 were included. These studies were approved by the

2.2
Production of SEA, SEAD227A, and SEH The superantigens (SEA, SEH, and SEA-D227A) were produced as earlier described with minor changes. 27

Transwell experiments
Monocytes, total CD3 + T cells, γδ T cells, and NK cells were isolated using STEMCELL Technology kits as described above. A total of 1 × 10 5 γδ T cells or 1 × 10 5 NK cells were added to the inserts and 1 × 10 5 monocytes and 4 × 10 5 total CD3 + T cells were added to the bottom wells of a Costar ® 6.5 mm Transwell ® 0.

Blocking experiments
PBMCs were stimulated in the presence of 2 μg/mL anti-human IL-12

Degranulation assay
Anti-human CD107a V450 antibody (clone H4A3; BD Biosciences) was added to the cells at 0.5 μg/mL immediately prior to stimulation with SEA and TSST-1. Cells were incubated for 48 h and analyzed by flow cytometry.

2.12
Online supplemental material Figure S1 shows IL-6 secretion upon stimulation with in-house produced SE. Figure S2 shows a complete gating strategy used for flow cytometric analyses. Figure S3 shows the proportion of CD130 expressing cells within unconventional T-and NK cells at baseline and after stimulation. Figure S4 shows a representative FACS plot for the activation kinetics of T-and NK cells. Figure S5 shows a representative FACS plot for the degranulation staining after SEA stimulation.

SE-mediated activation of γδ T-and NK cells is dependent on physical contact with monocytes and conventional T cells
Although structurally and functionally similar, SEA, SEH, and TSST-1 all differ in their binding orientation and specificities toward the MHC class II and TCR. 28 If and how this affects the subsequent immunological response is largely unknown. Following PBMC-stimulation with these three toxins, all cell types investigated -conventional T cells, γδ T cells, MAIT cells, and NK cells -were induced to express IFNγ, although SEH was comparably less potent (Fig. 1A). Conventional T cells were clearly activated at lower toxin concentrations compared to the other cell types investigated. In addition, following stimulation with a SEA-D227A mutant toxin, which is unable to bind MHC class II, the IFN-γ production was significantly reduced in all investigated cell types at comparable concentrations (Fig. 1B).
γδ T-and NK cells lack the canonical receptors for superantigen-TCR interaction. Still, SEA also interacts with, and promote signaling through, the IL-6 co-receptor CD130 on adipocytes. 29  were added to SE-primed autologous monocytes followed by analyses of intracellular cytokine responses using flow cytometry. Interestingly, none of the SE were able to induce IFN-γ in pure γδ T-or NK cell cultures, nor were SE-primed monocytes able to induce IFN-γ expression in autologous γδ T-or NK cells (Fig. 1C). However, addition of total CD3 + T cells to either co-culture systems did result in activation of both γδ T-and NK cells (Fig. 1C), suggesting that SE-induced activation of these cells requires the presence of both αβ T cells and monocytes. In order to determine whether cell-to-cell contact was required for activation, γδ T-and NK cells were seeded into inserts of 0.4 μm pore membrane transwell plates with SEA-stimulated monocytes and total CD3 + T cells in the bottom well. In the absence of physical contact, γδ T-and NK cell derived IFN-γ was lost (Fig. 1D), suggesting an important role for surface-expressed co-stimulatory receptors. In addition, conventional T cells expressed IL-2 and IFN-γ already after 4 hours of stimulation whereas NK-, γδ T-, and MAIT cell-activation required longer durations of stimulation ( Fig. 1E; Supplementary Fig. S4). Collectively, this indicates that SE-mediated activation of γδ T-and NK cells occur through an indirect mechanism dependent on the presence of monocytes and conventional T cells.

SE-induced IFN-γ expression in T-and NK cells is IL-12 dependent
To study the involvement of APC-derived cytokines in SE-mediated activation, we isolated RNA from stimulated PBMC and analyzed the expression of IL-12, IL-15, and IL-18 using RT-qPCR. We observed an up-regulation of IL-12 transcription by all SE while IL-15 and IL-18 levels remained unchanged at both timepoints investigated ( Fig. 2A).  (Fig. 2B, top row), however the frequency of CD107a + cells remained unaffected (Fig. 2B, bottom row and supporting information Fig. S5). Furthermore, quantification of secreted perforin and granzyme B in PBMC culture supernatants revealed no differences after neutralization of IL-12 (Fig. 2C). Collectively, our results indicate that SE-induced IFN-γ-responses but not cytotoxic granule release, are dependent on IL-12 production.

δ-Chain usage and phenotypic markers associate with SE-induced IFN-γ expression in γδ T cells
γδ T cells are commonly subdivided according to Vδ-chain usage, which also correlates with tissue distribution and function where TRDV2 + cells are more frequently found in circulation and TRDV1/3 + at mucosal sites. 11, 30 We first investigated IFN-γ expression in TRDV1 + , TRDV2 + , and TRDV1 -TRDV2γδ T cells upon SE stimulation of PBMC using flow cytometry (Supporting information Fig. S2B). All three γδ T cell subsets expressed IFN-γ upon stimulation and we observed that SEA and TSST-1 induced a stronger response compared to SEH within each subset (Fig. 3A).
In the circulation, the TRDV2 + subpopulation is the most prevalent among γδ T cells. We therefore investigated whether SEinduced responses in TRDV2 + T cells associated with a particular phenotype. We specifically investigated the memory-associated markers CD27, CD28, and CD161, commonly associated with pro-inflammatory responses in memory T cells. 31 Both CD27and CD28 expression correlated with increased IFN-γ expression upon SEA stimulation (Fig. 3B). Also, we have previously shown that SEA induces up-regulation of CD161 followed by increased production of pro-inflammatory cytokine expression in CD4 + FOXP3 + T cells. 32 However, we observed no significant association between IFN-γ and CD161 expression within the TRDV2 + T cell population (Fig. 3B).

CD57 and NKG2C mark NK cells of a more differentiated state
where NKG2C + NK cells associate with increased IFN-γ production during human cytomegalovirus infection. 33,34 However, we observed no correlation between IFN-γ expression and surface expression of the markers CD57 or NKG2C among NK cells in SEA-stimulated PBMC cultures (Fig. 3C). The relationship between CD161 expression and NK cell activity has been debated. In our current setting, there was no association between IFN-γ-and CD161 expression among NK cells (Fig. 3C).

The response to SE is delayed and weaker in newborns compared to adults
In order to investigate the response to SE at different stages of immune maturation, we analyzed a set of cytokines (IL-2, IL-10, IL-17A, IL-21, IFN-γ, and TNF) and cytotoxic compounds (granzyme B and perforin) released by CBMC and adult PBMC upon stimulation using ELISA.
Twenty-four hours after stimulation, only IL-2 was detected in CBMC supernatants, whereas all the other factors were detected in adult PBMC supernatants (Fig. 4A). After 48 h, CBMC secreted detectable levels of IL-2, IFN-γ, TNF, granzyme B, and perforin, but still in lower amounts compared to adult PBMC (Fig. 4B). IL-10, IL-17A, and IL-21 were not detected or barely detected in cord blood after 48 h of stimulation, although readily secreted by adult PBMC (Figs. 4A, B)  First, we assessed the frequency of each cell type in absence of stimulation (Fig. 5A). The highest frequency of MAIT cells was found in adults but was barely detectable in cord blood, which is in accordance with previous studies. 35,36 γδ T cells were detected in all age groups, and at the highest frequency in 2-year-olds, while NK cells were found in highest frequency in cord blood (Fig. 5A). In terms of the response to SE, conventional T cells from all age groups produced IFN-γ upon stimulation with SEA and TSST-1, with the highest expression noted in adult cells (Fig. 5B) Since we observed a significant role of IL-12 production for efficient IFN-γ responses in adult cells, we stimulated PBMC of 2-and 7-

SE do not induce IFN-γ in unconventional T cells and NK cells during the neonatal and infant period
year-olds with SEA for 8 h and analyzed IL-12 induction by RT-qPCR.
Interestingly, SEA-induced IL-12 transcription was clearly hampered in both 2-and 7-year-olds, (Fig. 5C) compared to adults ( Fig. 2A). Altogether, we have shown that unconventional T-and NK cells from cord blood and 2-year-old children failed to produce IFN-γ in response to SE, and that SEA-induced IL-12 transcription was lower in early life.

Several reports have shown that unconventional T cells and NK
cells can be activated by bacterial toxins, although the mechanisms and physiological consequences are only just beginning to be revealed. 7,8,37,38 We have previously shown that SEA induces IFN-γ production in MAIT-, γδ T-, and NK cells in a partly IL-12 dependent manner. 7 Here, we have used the toxins TSST-1 and SEH, as well as and is also the only included toxin containing two separate binding sites for the MHC class II receptor. 5 This results in the ability to cross-link multiple MHC class II molecules on the monocyte. 39 We also report that higher concentrations of the mutant SEA-D227A are required in order to induce the same level of activation as wild-type SEA. The fact that SEA-D227A binds to MHC class II with a much-reduced affinity, highlights the importance of this interaction to activate also unconventional T cells and NK cells. Indeed, SEA tended to induce the highest frequency of IFN-γ + cells and the highest levels of secreted IFN-γ compared with the other toxins used.
Despite the fact that γδ T-and NK cells are unlikely to be direct targets for SE, reports suggest alternative modes of activation that do not rely on crosslinking between the αβ TCR and MHC class II. SEA has been shown to activate T cells in a TCR-independent manner by binding to the laminin receptor-subunit LAMA2. 40 Furthermore, SEA binds to the IL-6 co-receptor CD130 and activates STAT3 signaling in human adipocytes 29 and a binding site for SEA on the γδ TCR has been reported. 19 However, the proportion of unconventional T cells and NK cells expressing CD130 was negligible both at baseline and upon stimulation. S. aureus infected monocyte-derived dendritic cells specifically activate TRDV2 + γδ T cells in co-culture models, which is dependent on both IL-12 production and surface expressed receptors including the γδ TCR. 41 However, by performing co-culture experiments with isolated cell populations, we here demonstrate that activation of γδ T-and NK cells by SEA was dependent on both monocytes and total CD3 + T cells in a contact-dependent manner, but also that IL-12 was necessary for the activation of these cells. We also observed a marked IFN-γ response in TRDV1 + and TRDV1  Here, we observed no difference in IFN-γ expression between CD161negative and -positive NK cells or TRDV2 + γδ T cells. CD161 expression correlates with increased expression of the IL-18Rα on both conventional and γδ T cells. Moreover, stimulation of these cells with IL-12 + IL-18 results in production of IFN-γ mainly within the CD161 + γδ T cells. 35 SE failed to induce IL-18 transcription, and IL-18 neutralization had no effect on IFN-γ expression (data not shown), which could explain the observed lack of correlation between CD161 and IFN-γ expression among NK and γδ T cells in our study.
Throughout this study, we have demonstrated that SE activate adult unconventional T cells and NK cells, and that these seem to contribute to the bulk of IFN-γ production. To determine if these responses were influenced by age and level of immune maturation, we investigated the cytokine profile and cellular responses upon stimulation with SE at several time-points during early childhood. NK cells have been reported to be more abundant in cord blood than in adults, 57 which is in agreement with our results. In addition to being more abundant, cord blood NK cells seem to mount a strong IFN-γ response when stimulated with IL-12 and IL-18. 58 Also γδ T cells have been shown to be potent in early life and possess a mature phenotype and a level of response similar to adults already at 2 years of age. 59  mRNA are not due to decreased transcription but to a lower stability of the transcript. 60 Here, we assessed IL-12 transcription in 2-and 7- year-olds, which was clearly reduced in response to SEA compared to adults. Altogether, this suggests that the relative unresponsiveness of unconventional T cells and NK cells during infancy could be a consequence of reduced conventional T cell activation due to a decreased APC response.
In this study, we conclude that several SE, with different TCR Vchain specificities, are able to induce IFN-γ expression in MAIT cells, γδ T cells and NK cells and suggest that their activation is secondary to and requires the presence of αβ TCR-expressing T cells, APC and IL-12.
Importantly, we confirm that neonatal T-and NK cells have a reduced capacity to mount a response towards SE in vitro compared with adults.
S. aureus is a major contributor to hospital and community acquired infections frequently causing severe conditions such as toxic shock and sepsis. Unconventional T cells have a clear role in these life-threatening conditions and elucidating the mechanism behind their involvement is important. 37,63,64 Our study expands the understanding of the mechanisms involved in SE-induced activation of unconventional T cells and NK cells and suggests a possible contributor to the vulnerability to infections observed during the neonatal and infant period.