Bacterial toxins, called superantigens, are produced by Staphylococcus aureus and are known to activate γδ T cells. γδ T cells contribute to long-lasting immunity against bacterial skin infections caused by S. aureus. γδ T cells are a distinct subgroup of T cells containing the T cell receptor (TCR) γ and δ chains. The γδ TCR harbouring the variable chains TRGV9/TRDV2 is the most common TCR in human peripheral blood and is known to be activated by superantigens and are also promising candidates for tumor immunotherapy. However, detailed analyses of antigen binding to γδ TCR have been severely hampered by difficulties in producing large amounts of γδ TCR. In this study, we report a protocol to produce recombinant γδ TCR (TRGV9/TRDV2) by fusing the variable domains γδ TCR with the constant domains of αβ TCR. Subsequently, binding analyses were executed applying microscale thermophoresis showing a clear binding between superantigen and the γδ TCR in the micro molar range. In addition, the superantigen SEA was shown to induce cytokine expression in γδ T cells with moderate MHC dependence, suggesting that other receptors can act as antigen presenting molecules upon γδ T cell activation. These results pave the way towards a better understanding of superantigen recognition by the γδ T cells and facilitates the future use of γδ TCR in cellular tumor immunotherapy.

Staphylococcus (S.) aureus is a common commensal and opportunistic pathogen that can produce several potent virulence factors, among which are the staphylococcal enterotoxins (SE). SE act as superantigens by crosslinking the T cell receptor (TCR) with the MHC-II on antigen presenting cells (APCs), subsequently leading to polyclonal T cell activation that can result in toxic shock syndrome. In this thesis, we have investigated different aspects of the response to SEA, one of the most produced SE by S. aureus. 

In paper I we focused on the activation of monocytes, and showed increased transcriptional responses related to inflammation, infection and dermatitis upon SEA stimulation. We demonstrated that posterior differentiation of these monocytes into monocyte-derived macrophages (MDMs) led to an altered phenotype, including reduced expression of antigen presentation and costimulatory molecules, but paradoxically increased induction of T cell proliferation and IFN-γ production. 

In paper II, we investigated how SE are able to activate MAIT cells, γδ T cells and NK cells, which lack the appropriate receptors to directly interact with SE. We demonstrated that their activation is secondary to that of APCs and conventional T cells. Upon SE encounter, APCs will produce high levels of IL-12, which induces the prompt activation and secretion of IL-2 and IFN-γ by conventional T cells. Cytokine signaling together with direct cell-to-cell contact with APCs and conventional T cells then lead to extensive IFN-γ production by MAIT, γδ T and NK cells.

Last, we investigated the effects of SEA across different immune landscapes, namely early life, pregnancy and allergy. In paper II, we observed an early life diminished immune response to SEA, characterized by delayed and lower cytokine secretion and impaired activation of γδ T cells and NK cells in cord blood and blood samples from 2-year-olds. In paper III we demonstrated that SEA stimulation leads to diminished IFN-γ and TNF responses in allergic individuals, most prominently in MAIT, γδ T and NK cells. However, these differences are overridden in the context of pregnancy. We also assessed APC markers, finding pregnancy-related and allergy-driven alterations in CD14, CD16, CD163, and HLA-DR expression, highlighting divergent immune regulation in these contexts.

Overall, we provide evidence for the crucial role of SEA in shaping immune function and regulation. We show that conventional T cells and APCs are essential in activating other lymphocytes, which subsequently contribute to the bulk of the excessive immune response caused by SEA. By examining the influence of different immune landscapes in the response to SEA, we not only enhance our understanding of superantigen biology but also expand our knowledge of factors that contribute to the immune responses in S. aureus-related diseases.

To the Editor,

Staphylococcus (S.) aureus is an intermittent or permanent skin colonizer in 90% of patients with airway diseases, and staphylococcal enterotoxin-IgE serum levels have been linked to both allergy and severe asthma.1, 2 During pregnancy, immune adaptation is required to ensure fetal growth,3 and type 2 responses are enhanced. These changes potentially worsen allergic conditions and increase the susceptibility to certain infections.4

Here we investigate the immune response to Staphylococcal enterotoxin A (SEA), a strong inducer of type 1 responses, in individuals with Th2-skewing,5 using peripheral blood mononuclear cells (PBMC) from allergic and non-allergic, pregnant and non-pregnant women6 (Figure 1A). Staphylococcal enterotoxins cause polyclonal T cell activation crosslinking the MHC-II on antigen-presenting cells (APCs) to the T-cell receptor (TCR) on T-cells (Figure 1B), leading to a strong proinflammatory response, potentially increasing IgE-production or disrupting the maternal-fetal tolerance.

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.

Memory-like responses in innate immune cells confer nonspecific protection against secondary exposures. A number of microbial agents have been found to induce enhanced or diminished recall responses in innate cells, however, studies investigating the ability of probiotic bacteria to trigger such effects are lacking. Here, we show that priming of human monocytes with a secretome from the gut probiotic bacterium Limosilactobacillus (L.) reuteri induces a mixed secondary response phenotype in monocyte-derived dendritic cells (mo-DCs), with a strong IL-6 and IL-1β response but low TNFα, IL-23 and IL-27 secretion. Instead, blood DC priming with L. reuteri-secretome resembles a tolerant state upon secondary exposure. A similar pattern was found in conventional and gut-like (retinoic acid exposed) DCs, although retinoic acid hampered TNFα and IL-6 production and enrichment of histone modifications in L. reuteri-secretome primed mo-DC cultures. Further, we show that the memory-like phenotype of mo-DCs, induced by priming stimuli, is important for subsequent T helper (Th) cell differentiation pathways and might determine the inflammatory nature of Th cells. We also show enhanced recall responses characterized by robust inflammatory cytokines and lactate production in the gut-like mo-DCs derived from β-glucan primed monocytes. Such responses were accompanied with enriched histone modifications at the promoter of genes associated with a trained phenotype in myeloid cells. Altogether, we demonstrate that a gut commensal-derived secretome prompts recall responses in human DCs which differ from that induced by classical training agents such as β-glucan. Our results could be beneficial for future therapeutic interventions where T cell responses are needed to be modulated.

The initiation of an immune response is dependent on the activation and maturation of dendritic cells after sensing pathogen associated molecular patterns by pattern recognition receptors. However, the response needs to be balanced as excessive pro-inflammatory cytokine production in response to viral or stress-induced pattern recognition receptor signaling has been associated with severe influenza A virus (IAV) infection. Here, we use an inhibitor of Toll-like receptor (TLR)3, a single-stranded oligonucleotide (ssON) with the capacity to inhibit certain endocytic routes, or a TLR3 agonist (synthetic double-stranded RNA Polyl:C), to evaluate modulation of innate responses during H1N1 IAV infection. Since IAV utilizes cellular endocytic machinery for viral entry, we also assessed ssON's capacity to affect IAV infection. We first show that IAV infected human monocyte-derived dendritic cells (MoDC) were unable to up-regulate the co-stimulatory molecules CD80 and CD86 required for T cell activation. Exogenous TLR3 stimulation did not overcome the IAV-mediated inhibition of co-stimulatory molecule expression in MoDC. However, TLR3 stimulation using Polyl:C led to an augmented pro-inflammatory cytokine response. We reveal that ssON effectively inhibited Polyl:C-mediated pro-inflammatory cytokine production in MoDC, notably, ssON treatment maintained an interferon response induced by IAV infection. Accordingly, RNAseq analyses revealed robust up-regulation of interferon-stimulated genes in IAV cultures treated with ssON. We next measured reduced IAV production in MoDC treated with ssON and found a length requirement for its anti-viral activity, which overlapped with its capacity to inhibit uptake of Polyl:C. Hence, in cases wherein an overreacting TLR3 activation contributes to IAV pathogenesis, ssON can reduce this signaling pathway. Furthermore, concomitant treatment with ssON and IAV infection in mice resulted in maintained weight and reduced viral load in the lungs. Therefore, extracellular ssON provides a mechanism for immune regulation of TLR3-mediated responses and suppression of IAV infection in vitro and in vivo in mice.

Staphylococcal enterotoxins (SE) crosslink the MHC-II on antigen-presenting cells (APC) with the T-cell receptor, inducing a polyclonal T-cell response. Although APCs are the initial targets of SE and are critical in shaping subsequent T-cell activation, the effects of SE on APC function remain poorly understood. This study investigates the immunomodulatory effects of staphylococcal enterotoxin A (SEA) on monocytes and their differentiation into monocyte-derived dendritic cells (moDC) or macrophages (MDM). Transcriptomic analyses of human monocytes via RNA sequencing revealed SEA-induced enrichment of gene pathways associated with inflammation, infection and dermatitis, with these effects amplified in the presence of T-cells. Phenotypic and functional characterization showed that SEA-primed monocytes differentiated into MDM with an altered polarization, deviating from classical M1/M2 pathways. SEA-primed MDM exhibited downregulayion of key markers including HLA-DR, CD80. CD86 and PD-L1. Functional assays demonstrated that SEA-primed MDM drived hyperinflammatory T-cell responses, with significantly enhanced proliferation and IFN-γ secretion. In contrast. moDC retained robust antigen-presenting capabilities even upon SEA-priming. These findings provide mechanistic insights into SEA-mediated immune modulation, illustrating how SEA reprograms MDM functions and amplify proinflammatory T-cell responses. This advances our understanding of superantigen-driven immune interactions, offering a foundation for developing therapeutic strategies to mitigate superantigen-mediated immune conditions.