MAIT CELL RESPONSES IN TOXIC SHOCK SYNDROME

MAIT cells have been implicated in multiple inflammatory and infectious diseases. Their responsiveness to bacterial riboflavin metabolites is well established but their ability to respond to bacteria lacking riboflavin metabolism, such as GAS had been largely overlooked (172, 173, 175). GAS produce superantigens which activate T cells by binding to MHC class II and the Vb region of the TCR, thereby bypassing conventional antigen processing and presentation (235, 236). This results in massive cytokine release which underlies the vascular leakage,

shock, and multiorgan failure of STSS. The MAIT cell TCR repertoire includes high frequencies of some of the Vb-segments commonly recognized by superantigens, in particular Vb2 (179, 237). In addition, it had been shown that MAIT cells respond to the staphylococcal superantigen SEB and are significant contributors to the cytokine response to this toxin (85).

In paper II, we therefore hypothesized that MAIT cells can respond to GAS and may play a role in the pathogenesis and cytokine storm of STSS.

Figure 8. MAIT cells are major contributors to the cytokine production in response to GAS. Pie charts depict the frequency of MAIT cells among total CD3+ T cells and among IFNg and TNF-producing T cells after 24 hours of stimulation with fixed GAS or GAS supernatant. Assessed by flow cytometry.

In contrast to previous studies (175, 221), paper II revealed a strong MAIT cell response to both whole mildly fixed GAS and to GAS supernatants containing secreted superantigens. Up to 40% of all IFNg-producing T cells in response to fixed GAS, and 20% of TNF-producing T cells in response to GAS supernatants were MAIT cells, which was striking given that the MAIT cell frequency in circulation ranged from 0.1% to 10% (Figure 8). The MAIT cell response to GAS supernatants occurred through two different mechanisms. The early response was largely mediated by superantigen engagement of MAIT cell TCRs carrying Vb2 and functionally dominated by TNF production. After 8 hours of stimulation, only Vb2+ MAIT cells were activated. At later timepoints (after 12-24 hours of stimulation) both Vb2+ and Vb2- MAIT cells started to produce IFNg as well. In addition, we saw an amplified TNF response and increased expression of activation markers on the MAIT cell surface. This second wave of MAIT cell activation was almost completely inhibited by addition of blocking antibodies against IL-12 and IL-18, indicating that the activation was mediated by innate cytokines produced by other immune cells in response to factors secreted by GAS. Similar response kinetics occurred after stimulation of MAIT cells with the recombinant Vb2-specific superantigens SpeJ and SpeC. The MAIT cell response to fixed GAS was largely driven by IL-12 and IL-18 and occurred later than the superantigen-driven response. Blocking of MR1 using antibodies had no effect on the MAIT cell response to GAS, consistent with the lack of riboflavin metabolism in GAS. Instead, fixed GAS initiate IL-12 and IL-18 production by myeloid cells, likely by binding to TLRs. We also investigated stimulation of MAIT cells with group G streptococci (GGS, Streptococcus dysgalactiae) which show no superantigenic activity. Whole fixed GGS induced a strong IL-12 and IL-18-dependent IFNg response, similar

to GAS. However, MAIT cells did not respond to GGS supernatants, underscoring the importance of superantigens in GAS-mediated MAIT cell activation. The MAIT cell contribution to the overall cytokine response to GAS was further evaluated by measuring cytokine concentrations in GAS-stimulated cultures of PBMC where MAIT cells had been depleted. Removal of MAIT cells resulted in a significant reduction in the concentration of cytokines known to be produced by MAIT cells, such as IFNg and IL-2, but also in the concentration of cytokines not produced by MAIT cells such as IL-1b and TNFb. This suggests that MAIT cells contribute to the overall cytokine response both directly and indirectly, by influencing the cytokine production by other immune cells.

The MAIT cell activation observed in vitro was further supported by analyses of PBMC from STSS patients (Paper II). Investigation of cytokine responses in these patients was not possible in the samples we had access to, but we found high levels of activation markers CD69, CD38, CD25, PD-1, and HLA-DR as well as the proliferation marker Ki67 (Figure 9A). In three out of 10 patients the observed expression of these markers was restricted to Vb2-expressing MAIT cells, suggesting that the activation was mediated by Vb2-binding superantigens (Figure 9B).

Taken together, paper II identifies MAIT cell as rapid responders to GAS infections and significant contributors to the cytokine storm, thereby implicating MAIT cells as potentially detrimental to the immunopathogenesis of STSS.

Figure 9. MAIT cells are activated in STSS patients. (A) Expression of activation markers on MAIT cells in STSS patients at day 1, 28 and 180. (B) Vb2-specific activation in one patient. Assessed by flow cytometry.

In paper III, we continued to explore the mechanisms behind the robust cytokine response in MAIT cells during GAS infections. We extended the study by also including another superantigen-producing bacterium capable of causing TSS, S. aureus. GAS infections and superantigens have been associated with increased production of ROS in leukocytes (238-242).

As ROS have been linked to production of IFNg, TNF, IL-12, and IL-18, as well as to IL-12R and IL-18R signaling (104, 106, 107, 112), we hypothesized that ROS signaling is important in controlling the MAIT cell response in streptococcal and staphylococcal TSS and that antioxidants can be used to modulate MAIT cell responses.

In paper III, we found that ROS levels in MAIT cells both before and after activation were considerably lower than ROS levels in conventional T cells. MAIT cell production of IFNg and TNF in response to GAS and S. aureus supernatants was inhibited by NAC and GSH. In contrast, the cytokine production by conventional T cell subsets was not inhibited by antioxidants, or rather even increased slightly. This points to an important role of ROS in controlling MAIT cell function. Targeting ROS using NAC or GSH inhibited both Vb2+ and Vb2- MAIT cells suggesting that the antioxidants do not specifically affect the direct activation of MAIT cells by superantigens. Instead, NAC and GSH inhibited the IL-12 and IL-18-dependent MAIT cell activation. Stimulation of purified Va7.2+ cells with recombinant IL-12 and IL-18 was inhibited by NAC and GSH, suggesting that the ROS could be involved in controlling the expression of IL-12R and IL-18R, or be involved in receptor signaling. It was recently demonstrated that ROS is required for signaling downstream of IL-12R and IL-18R in mouse CD4+ T cells (112). We found that cytokine production of conventional T cells as well as NK cells in response to recombinant IL-12 and IL-18 was also inhibited by antioxidants.

This suggests that the ROS-dependency of IL-12 and IL-18-mediated activation is not limited to MAIT cells. However, as MAIT cells are more robust responders to IL-12 and IL-18 compared to other T cells, they are more affected by antioxidant treatment. In addition, production of IL-18 by other immune cells was also inhibited by antioxidants, indirectly affecting the IL-18-mediated MAIT cell activation. ROS have been suggested to play a role in the formation of the NLRP3 inflammasome driving IL-18 activation and secretion (106, 107).

In addition, glutathionylation of caspase-1 prevents the assembly of the inflammasome (119).

This may explain the decreased IL-18 production after antioxidant treatment. The significant difference in cytokine responses between MAIT cells and other T cells observed in paper II is intriguing. In paper III, we find that the involvement of redox signaling in MAIT cell activation in response to GAS also differs from other T cells. It is possible that there is a link between these observations. Release of ROS plays a role in combating infections and the overall ROS production by the immune system is elevated in STSS, sepsis, as well as other infectious diseases. Our data indicate that the increased ROS levels also promote MAIT cell activation in these conditions. However, whether MAIT cell activation is dependent on endogenous ROS, or ROS produced by other cells remains to be investigated. Taken together, paper III reveals a novel role of ROS in MAIT cell activation and suggests that antioxidants have the potential of dampening the MAIT cell response in inflammatory diseases.