Diverse Streptococcus pneumoniae strains drive a MAIT cell response through MR1-dependent and cytokine-driven pathways

Mucosal Associated Invariant T (MAIT) cells represent an innate T cell population of emerging significance. These abundant cells can recognize ligands generated by microbes utilizing the riboflavin synthesis pathway, presented via the major histocompatibility complex (MHC) class I-related molecule MR1 and binding of specific T cell receptors (TCR). They also possess an innate functional programme allowing microbial sensing in a cytokine-dependent, TCR-independent manner. Streptococcus pneumoniae is a major human pathogen that is also associated with commensal carriage, thus host control at the mucosal interface is critical. The recognition of S. pneumoniae strains by MAIT cells has not been defined, nor have the genomics and transcriptomics of the riboflavin operon (Rib genes). We examined the expression of Rib genes in S. pneumoniae at rest and in response to metabolic stress and linked this to MAIT cell activation in vitro. We observed robust recognition of S. pneumoniae strains at rest and following stress, using both TCR-dependent and TCR-independent pathways. The pathway used was highly dependent on the antigen-presenting cell, but was maintained across a wide range of clinically-relevant strains. The riboflavin operon was highly conserved across a range of 571 S. pneumoniae from 39 countries dating back to 1916, and different versions of the riboflavin operon were also identified in related Streptococcus species. These data indicate an important functional relationship between MAIT cells and S. pneumoniae,which may be tuned by local factors, including the metabolic state of the organism and the antigen-presenting cell that it encounters. Author Summary Streptococcus pneumoniae is the leading cause of bacterial pneumonia, causes invasive diseases such as meningitis and bacteraemia, and is associated with significant morbidity and mortality, particularly in children and the elderly. Here, we demonstrate that a novel T cell population called Mucosal-associated invariant T (MAIT) cells is able to respond to a diverse range of S. pneumoniae strains. We found that this response was dependent on the T cell receptor (which recognises metabolites of the bacterial riboflavin biosynthesis pathway), cytokines, and the type of antigen-presenting cell. A population genomics approach was also used to assess the prevalence and diversity of the genes encoding the riboflavin biosynthesis pathway among a large and diverse collection of S. pneumoniae. These genes were highly conserved across a range of 571 S. pneumoniae from 39 countries dating back to 1916, and was also present in other related Streptococcus species. Given the low levels of MAIT cells in neonates and MAIT cell decline in the elderly, both of whom are at the highest risk of invasive pneumococcal disease, further understanding of the functional role of MAIT cells in host defense against this major pathogen may allow novel therapeutics or vaccines to be designed.

We also tested whether using live S. pneumoniae strain PMEN34 or the supernatant 231 of S. pneumoniae growth culture, instead of fixed bacteria, would stimulate MAIT 232 cells through the MR1-pathway ( Figure 3D); however, these responses were small 233 and could not be significantly blocked by an anti-MR1 blocking antibody. We 234 enriched for CD8+ T cells and added these to the assay instead of PBMCs in case 235 other cells were interfering with MAIT cell activation through MR1, but these 236 responses were similar to those using PBMCs and not affected by  Thus, in the presence of monocytes, MAIT cells are activated mainly through innate 238 cytokines rather than MR1, regardless of temperature or riboflavin availability. We next tested whether monocyte-derived macrophages may be able to present the 244 MR1-ligand to activate MAIT cells more effectively through MR1, as alveolar 245 macrophages play an important role in the immune response to S. pneumoniae 246 (21,22). Furthermore, we investigated whether temperature or the abundance of 247 riboflavin in the media affects MR1-dependent activation of MAIT cells in the 248 presence of macrophages. For this, S. pneumoniae strain PMEN34 was grown for 249 16 hours in THB media, with or without yeast extract (THB-Y and THB, respectively) 250 at either 36˚C or 40˚C. Given that the riboflavin synthesis pathway was upregulated 251 by a short incubation at 40˚C (Figure 1), we also transferred half of the bacteria 252 grown overnight at 36˚C to 40˚C for 4 hours. The bacteria were fixed immediately 253 and added to PBMCs and monocyte-derived macrophages overnight ( Figure 4A, B). 254 255 Surprisingly, we found that when using monocyte-derived macrophages, S. 256 pneumoniae induced IFNγ expression from MAIT cells that was reduced by an anti-257 MR1 blocking antibody, suggesting that the response was MR1-dependent, in 258 contrast to the response in the presence of monocytes. The addition of the anti-MR1 259 blocking antibody significantly reduced the frequency of IFNγ-expressing MAIT cells 260 regardless of the temperature and media in which the S. pneumoniae were grown. 261 Interestingly, there was a clear increase in activation induced by bacteria grown in 262 THB media without the addition of yeast, compared to bacteria grown in the 263 presence of yeast. Degranulation was also induced by S. pneumoniae grown in THB 264 media and was blocked by the anti-MR1-blocking antibody to a varying degree 265 ( Figure 4C, D).  Table 2). The majority of these 306 riboflavin operons were located between genes involved in arginine biosynthesis 307 (argC, argJ, argB, argD) and ribonucleotide reduction (nrdF2, nrdE2, nrdH) (Figure 308  Autolysis of S. pneumoniae has been shown to specifically reduce the phagocytosis 376 of intact bacteria, even in the absence of a polysaccharide capsule, and reduce the 377 production of phagocyte-activating cytokines such as TNFa, IFNγ and IL-12 (31). 378 The uniquely high sensitivity of MAIT cells to cytokines such as IL-12 (32) allows 379 these cells to boost the immune response and provide early IFNγ production. 380

381
The biggest disease burden caused by S. pneumoniae by far is pneumonia, which is 382 the leading infectious cause of mortality in children under five years of age (1), as 383 well as accounting for a large part of community-acquired pneumonia in the elderly 384 (2). Given that we show the mechanism by which MAIT cells respond to S. Interestingly, we found that temperature and riboflavin availability for S. pneumoniae 401 have the potential to affect riboflavin synthesis and MAIT cell activation. In our 402 models using macrophages, there was a difference in MR1-dependent MAIT cell 403 activation induced by S. pneumoniae cultured in THB media compared to THB-Y 404 media, the latter of which is supplemented with riboflavin-rich yeast extract. Although 405 the THB media alone supports the growth of riboflavin-auxotrophs such as 406 Streptococcus pyogenes, this has been shown to be due to the ability of S. 407 pyogenes to utilize THB-derived components as a substitute for riboflavin, and 408 mutant strains without this ability require the addition of riboflavin or riboflavin-rich 409 yeast in order to grow in THB media (40). Thus, THB media on its own does not 410 contain riboflavin. We found that S. pneumoniae grown in THB media consistently 411 induced greater MAIT cell activation, which may be partly due to the increased 412 production of riboflavin in bacteria grown in riboflavin-deficient THB media, 413 compared to those grown in riboflavin-rich THB-Y media. We also found that 414 operon (at least one that was detectable by our screening method) and there was 441 variation among the genomes that did, either in that more than one version of 442 riboflavin operon was detected within a bacterial species, or that the presence of a 443 riboflavin operon was not ubiquitous among genomes of a particular bacterial 444 species. Hence, caution must be exercised when extrapolating findings based on a 445 small number of bacterial strains to the population as a whole since they may not be 446 representative, and ideally a population-based approach is used whenever possible.   Table 2). 34 genomes were newly 570 sequenced as above and the rest were downloaded from the ribosomal multilocus 571 sequence typing (rMLST) database (52). For each species, the number of genomes 572 included in this study dataset was capped at 50: if fewer than 50 genomes were 573 available in the rMLST database for a given species, then all available genomes 574 were included, but if more than 50 sequenced genomes were available then 575 genomes were manually selected for inclusion. In these instances, the population 576 structure of the species was depicted using PHYLOViZ and 50 genomes were 577 selected with the aim of maximising the population-level diversity of that species 578 from the available genomes (53).

RNA-sequencing experiments and analyses 586
S. pneumoniae isolate 2/2 was cultured in seven 10 ml tubes of brain-heart infusion 587 broth and incubated at 40°C + 5% CO 2 for 6 hours to mimic heat shock. The 588 experimental control was the same isolate 2/2, also cultured in seven 10 ml tubes of 589 brain-heart infusion broth but incubated at standard conditions of 37°C + 5% CO 2 . 590 Broth cultures at five time points (2, 3, 4, 5 and 6 hours of incubation) were removed 591 and 19 ml of RNAprotect Bacteria Reagent (Qiagen) was added to stabilise the RNA. 592 RNA was extracted from the samples using the Promega Maxwell® 16 Instrument 593 and LEV simplyRNA Cells purification kit, following the manufacturer's protocol. 594 Extracted RNA samples were sent to the Oxford Genomics Centre for processing. 595 Library preps were made using RNA-Seq Ribozero kits (Illumina, Inc) and 596 sequencing was performed on the MiSeq (Illumina, Inc). 597

598
The sequenced forward and reverse reads were paired and mapped to the S. 599 pneumoniae strain 2/2 genome using Bowtie2 with the highest sensitivity option (60). within the Geneious environment to calculate the percentage identity matrix.

Assessment of the relationships among Streptococcus spp. 641
A phylogenetic tree was built using concatenated sequence data from the ribosomal 642 loci using the neighbour-joining method as implemented using the BIGSdb 643 PhyloTree plugin (52,69). The tree was annotated using iTOL (70) and Inkscape (71).