1 Functionally diverse human T cells recognize non-microbial antigens

2 presented by MR1

3 Marco Lepore1, Artem Kalinichenko1, Salvatore Calogero1, Pavanish Kumar2, Bhairav Paleja2,

4 Mathias Schmaler1, Vipin Narang2, Francesca Zolezzi2, Michael Poidinger2,3, Lucia Mori1,2,

5 Gennaro De Libero1,2*

6

7 1Department of Biomedicine, University Hospital and University of Basel, 4031 Basel,

8 Switzerland; 2Singapore Immunology Network, A*STAR, 138648 Singapore; 3Singapore

9 Institute for Clinical Sciences, A*STAR, 117609 Singapore

10 *For correspondence: [email protected]

11

12 Subject area: Immunology

13 Senior editor: Tadatsugu Taniguchi

14

15 16 Abstract

17 MHC class I-related molecule MR1 presents riboflavin- and folate-related metabolites to

18 mucosal-associated invariant T cells, but it is unknown whether MR1 can present alternative

19 antigens to other T cell lineages. In healthy individuals we identified MR1-restricted T cells

20 (named MR1T cells) displaying diverse TCRs and reacting to MR1-expressing cells in the

21 absence of microbial ligands. Analysis of MR1T cell clones revealed specificity for distinct cell-

22 derived antigens and alternative transcriptional strategies for metabolic programming, cell cycle

23 control and functional polarization following antigen stimulation. Phenotypic and functional

24 characterization of MR1T cell clones showed multiple chemokine receptor expression profiles

25 and secretion of diverse effector molecules, suggesting functional heterogeneity. Accordingly,

26 MR1T cells exhibited distinct T helper-like capacities upon MR1-dependent recognition of target

27 cells expressing physiological levels of surface MR1. These data extend the role of MR1 beyond

28 microbial antigen presentation and indicate MR1T cells are a normal part of the human T cell

29 repertoire.

30

31 Impact statement

32 MR1T cells are human polyclonal T cells endowed with diverse effector functions in response to

33 endogenous antigens presented by MHC-class 1-related molecule, MR1.

34

2 35 Introduction

36 T lymphocytes can detect an incredibly diverse range of microbial and self-antigens,

37 including lipids presented by non-polymorphic CD1 molecules (Zajonc, 2007), and

38 phosphorylated isoprenoids bound to Butyrophilin 3A1 (Sandstrom, 2014, Vavassori, 2013),

39 which stimulate TCR Vγ9-Vδ2 cells, the major population of TCR γδ cells in human blood. The

40 heterogeneous phenotypic and functional properties of these T cells support specialized roles in

41 host protection against infections, cancers, and autoimmunity (Cohen, 2009, Mori, 2016, Tyler,

42 2015). The repertoire of T cells specific for non-peptide antigens was expanded to include

43 mucosal associated invariant T (MAIT) cells, which are restricted to MHC I-related protein MR1

44 and were originally described within mouse gut (Treiner, 2003). Elegant functional studies

45 showed that MAIT cells react to different bacteria by recognizing non-proteinaceous antigens

46 (Gold, 2010, Le Bourhis, 2010). Finally, the stimulatory antigens were identified as small

47 riboflavin precursors produced by a wide range of yeasts and bacteria (Corbett, 2014, Kjer-

48 Nielsen, 2012, Soudais, 2015). MAIT cells display one of three alternative semi-invariant TCRs

49 in which the TRAV1-2 gene is rearranged with genes encoding TRAJ33, J12 or J20 (Gold, 2014,

50 Lepore, 2014, Reantragoon, 2012), and is combined with an oligoclonal TCR-β repertoire

51 (Lepore, 2014). Cells expressing these evolutionary-conserved TCRs are frequent in human

52 blood, kidney and intestine, and comprise a major fraction of T cells resident in the liver

53 (Dusseaux, 2011, Lepore, 2014, Tang, 2013). Following activation, MAIT cells release an array

54 of pro-inflammatory and immunomodulatory cytokines, and can mediate direct killing of

55 microbe-infected cells (Dusseaux, 2011, Kurioka, 2015, Le Bourhis, 2013, Lepore, 2014, Tang,

56 2013). MAIT cells react to a broad range of microbes, but it remains unknown whether the role

57 of MR1 extends beyond presentation of microbial metabolites to MAIT cells.

3 58 MR1 is a non-polymorphic MHC I-like protein that is expressed at low to undetectable

59 levels on the surface of many cell types (Huang, 2008, Miley, 2003). Following bacterial

60 infection, antigen-presenting cells (APC) up-regulate surface expression of MR1 due to protein

61 stabilization upon antigen binding (Huang, 2008, McWilliam, 2016, Miley, 2003). MR1 is

62 highly conserved across multiple species, with human and mouse MR1 sharing >90% sequence

63 homology at the protein level (Riegert, 1998). To date, the MR1-bound ligands reported to

64 simulate MAIT cells include three ribityl lumazines (RLs), microbial pterin-like compounds

65 displaying a ribitol at carbon 8 (Kjer-Nielsen, 2012), and two unstable adducts formed by non-

66 enzymatic reaction of microbial 5-ribityl amino uracil (5-RAU) with either host-derived or

67 bacterial carbonyls such as glyoxal or methyl-glyoxal (Corbett, 2014). RLs bind to MR1 via

68 hydrogen bonds and hydrophobic interactions with amino acids in the antigen binding cleft

69 (Kjer-Nielsen, 2012, Patel, 2013), whereas binding of unstable 5-RAU-derivatives requires

70 covalent trapping within the MR1 pocket via formation of a Schiff base with Lysine 43 (Corbett,

71 2014, Patel, 2013). While three additional molecules have also been demonstrated to bind MR1

72 via the same mechanism, including 6-formyl-pterin (6-FP), acetyl-6-formyl-pterin (Ac-6-FP),

73 and acetylamino-4-hydoxy-6-formylpteridine dimethyl acetal (Kjer-Nielsen, 2012, Patel, 2013,

74 Soudais, 2015), these compounds lack the ribityl moiety necessary for MAIT cell activation and

75 instead inhibit stimulation by microbial ligands (Corbett, 2014, Kjer-Nielsen, 2012, Soudais,

76 2015). A recent study reported that 6-FP and Ac-6-FP can also stimulate a second population of

77 ‘atypical’ MR1-restricted T cells of unknown function in the blood of healthy human donors

78 (Gherardin, 2016), but this population, did not express the canonical TRAV1-2 gene which

79 defines MAIT cells. Together, these findings indicate a degree of MR1 plasticity in

4 80 accommodating diverse chemical structures and suggest that other MR1-restricted T cell

81 populations may exist in vivo.

82 Here we report a novel population of MR1-restricted T cells (hereafter termed MR1T

83 cells) that is readily detectable in blood from healthy individuals. MR1T cells express diverse

84 TCRα and β genes and were not able to recognize previously identified microbial or folate-

85 derived ligands of MR1. Instead, MR1T cells recognized self-antigens presented by MR1 and

86 expressed a broad selection of cytokines and chemokine receptors. Functionally, MR1T cells

87 were capable of inducing dendritic cell (DC) maturation and promoted innate defense in

88 intestinal epithelial cells. These findings demonstrate that MR1 can present non-microbial

89 antigens to a novel population of functionally diverse human T cells with potentially wide-

90 ranging roles in human immunity.

91

92 Results

93 MR1-reactive T cells are readily detectable in healthy individuals

94 During our previous study on the repertoire of human MAIT cells (Lepore, 2014), we

95 detected an atypical MR1-restricted T cell clone that did not react to microbial ligands. This T

96 cell clone (DGB129) recognized cell lines constitutively displaying surface MR1 (CCRF-SB

97 lymphoblastic leukemia cells, or THP-1 monocytic leukemia cells; Figure 1A) or A375

98 melanoma cells (A375-MR1) transfected with an MR1-β2m fusion gene construct (Lepore,

99 2014) (Figure 1A) in the absence of any exogenously added antigens (Figure 1B). Sterile

100 recognition of MR1+ target cells was fully inhibited by blocking with anti-MR1 monoclonal

101 antibodies (mAbs) (Figure 1B), and thus resembled the MAIT cell response to E. coli-derived

102 antigens assessed in parallel (Figure 1C). Importantly, DGB129 T cells also failed to recognize

5 103 the synthetic MAIT cell agonist 6,7-dimethyl-8-D-ribityllumazine (RL-6,7-diMe; Figure 1D)

104 (Kjer-Nielsen, 2012), differently from a control MAIT cell clone, which instead was stimulated

105 in MR1-dependent manner by this compound (Figure 1E). Unlike the canonical semi-invariant

106 TCR typical of MAIT cells, DGB129 cells displayed a TCRαβ heterodimer comprising

107 TRAV29/DV5 rearranged to TRAJ23 and TRBV12-4 rearranged to TRBJ1-1. Expression of

108 these TCRα and β genes in a TCR-deficient cell line (SKW-3 T lymphoblastic leukemia)

109 conferred MR1 reactivity in the absence of exogenous antigens comparable to that displayed by

110 DGB129 cells (Figure 1F), while in control experiments, transduction of TCRα and β genes of a

111 representative MAIT cell clone conferred the ability to recognize the same target cells in MR1-

112 dependent manner only in the presence of E. coli antigens (Figure 1G). Collectively, these data

113 highlight a critical role of the TCR in mediating DGB129 cell recognition of MR1-expressing

114 APCs and suggest that MR1 can present non-microbial antigens to T cells other than MAIT cells.

115 To investigate the presence of these unpredicted MR1-restricted T cells in different

116 individuals, we performed combined in vitro stimulation and single cell cloning experiments

117 using total blood T cells. Purified T cells from two healthy donors were labeled with the

118 proliferation marker CellTrace violet (CTV) and stimulated with irradiated A375-MR1 cells in

119 the absence of exogenous antigens. The choice of A375-MR1 cells relied on their potent capacity

120 of inducing sterile DGB129 T cell activation (Figure 1B) and their high expression of surface

121 MR1 molecules (Figure 1A), which we reasoned could maximize the chance of stimulating and

122 thus expanding DGB129-like MR1-restricted T cells present in the blood. Amongst the T cells of

123 both donors, we observed a significant fraction of proliferating cells that expressed high levels of

124 the activation marker CD137 following re-challenge with A375-MR1 cells. These activated T

125 cells were sorted and cloned by limiting dilution (Figure 2A). Individual T cell clones were then

6 126 interrogated for their capacity to recognize A375-MR1 and A375 cells lacking MR1 (A375-WT).

127 In both donors we found that a major fraction of T cell clones (126/195 and 37/57, respectively)

128 displayed specific recognition of A375-MR1 cells (Figure 2B,D), which was potently inhibited

129 by anti-MR1 blocking mAbs (Figure 2C,E). Staining with TCR Vβ-specific mAbs of 12 MR1-

130 reactive T cell clones revealed that they expressed 7 different TRBV chains (TRBV4-3, 6-5/6-

131 6/6-9, 9, 18, 25-1, 28, 29-1) with some of the clones sharing the same TRBV gene. Furthermore,

132 none expressed the TRAV1-2 chain, canonical for MAIT cells. These data suggested that MR1-

133 restricted T cells other than MAIT cells exist in the blood of healthy donors, can express diverse

134 TCRs and are able of clonal expansion following in vitro stimulation in the absence of microbial

135 ligands.

136 Lack of specific markers did not allow univocal identification of these novel T cells ex

137 vivo by standard flow cytometry. Therefore their frequency was estimated by combining flow

138 cytometry analysis after very short-time in vitro stimulation and single T cell cloning

139 experiments. Purified blood T cells from five healthy donors were co-cultured overnight with

140 A375 cells either lacking or over-expressing MR1 and analyzed for the expression of the

141 activation markers CD69 and CD137 (Figure 3A). In all of the five donors screened, the

142 percentage of CD69highCD137+ T cells detected was consistently higher after stimulation with

143 A375-MR1 cells (range 0.034-0.072% of T cells) than after co-culture with A375-WT cells

144 (range 0.015-0.032%) (Figure 3A,B). As the two types of APCs differed for MR1 expression,

145 we assumed that MR1-reactive T cells might account for the increased numbers of activated T

146 cells after stimulation with MR1-positive APCs. Using this approach, we estimated that the

147 circulating T cell pool of the analyzed individuals contained A375-MR1-reactive T cells at

148 frequency ranging between 1:2,500 (0.072-0.032=0.04%) and 1:5,000 (0.034-0.015=0.019%).

7 149 This estimated frequency, although representing an approximation, is within the range of the

150 frequency of peptide-specific CD4+ T cells after antigen exposure (Lucas, 2004, Su, 2013). We

151 also performed parallel experiments in which overnight-activated CD69highCD137+ T cells

152 (0.065%) were sorted from one of these donors (Donor C, Figure 3A, right panel) and were

153 cloned. Indeed, 31 out of 96 screened T cell clones (32%) displayed specific reactivity to A375-

154 MR1 cells (Figure 3C), which was inhibited by anti-MR1 mAbs (Figure 3D). Accordingly, we

155 calculated that the frequency of A375-MR1-responsive T cells among blood T cells of this donor

156 was 1:5,000 (0.065x0.32= 0.02%), a value consistent with the previously estimated range.

157 Detailed analysis of representative T cell clones derived from three donors confirmed that they

158 displayed diverse TCRα and β chains and indicated differential expression of CD4, CD8 and

159 CD161 (Table 1).

160 Collectively, these findings suggested that identified MR1-responsive T cells are a novel

161 yet readily detectable polyclonal population of lymphocytes in the blood of healthy human

162 individuals (hereafter termed MR1T cells).

163

164 MR1T cells recognize antigens bound to MR1

165 We next studied the basis of MR1T cell reactivity. Firstly, we tested whether the MR1T

166 cell clones could recognize microbial antigens, in analogy to MAIT cells. While a control MAIT

167 cell clone reacted to A375-MR1 cells only in the presence of E. coli lysate, activation of different

168 MR1T cell clones was not enhanced by the E. coli lysate (Figure 4A). Consistent with these

169 data, MR1-negative A375-WT cells failed to stimulate either type of T cell, irrespective of

170 whether E. coli lysate was added (Figure 4A) and importantly, anti-MR1 mAbs efficiently

171 blocked both MR1T and MAIT cell responses (Figure 4A). These findings confirmed that

8 172 microbial ligands present in E. coli and stimulating MAIT cells do not stimulate the tested MR1T

173 cells.

174 We then tested the response of MR1T cells to the known MR1 ligands 6-FP and Ac-6-FP,

175 which have previously been reported to stimulate a rare subset of TRAV1-2-negative T cells

176 (Gherardin, 2016) and inhibit MAIT cell activation by microbial antigens (Corbett, 2014, Kjer-

177 Nielsen, 2012, Soudais, 2015). MR1T cell stimulation was impaired in the presence of 6-FP or

178 Ac-6-FP ligands, which also impaired E. coli stimulation of control MAIT cells, but did not

179 disrupt TCR γδ cell responses to cognate antigen presented by the same APCs (Figure 4B,C and

180 Figure 4–figure supplement 1A-C). Notably, 6-FP or Ac-6-FP failed to inhibit the activation of

181 MR1T cells or MAIT cells when the target A375 cells were transduced to express mutant MR1

182 molecules with defective ligand binding capacity (blockade of Schiff base formation with ligands

183 by mutation of Lysine 43 into Alanine, A375-MR1 K34A; Figure 4B,C and Figure 4–figure

184 supplement 1D,E). The specific inhibition observed with 6-FP or Ac-6-FP indicated that MR1T

185 cells i) do not recognize 6-FP and Ac-6-FP; ii) react to MR1-bound cellular antigens; iii) are

186 stimulated by ligands that do not require the formation of a Schiff base with MR1.

187 To gain information on the origin of the recognized antigens we firstly asked whether the

188 stimulatory capacity of target cells was dependent on culture medium constituents, as some MR1

189 ligands, e.g. 6-FP, may derive from folate present in RPMI 1640 medium used for cell culture

190 (Kjer-Nielsen, 2012). Both THP-1 and A375-MR1 cells were extensively washed and cultivated

191 4 days in phosphate buffered saline solution (PBS) supplemented exclusively with 5% human

192 serum. Cells were washed daily before being used to stimulate DGB129 MR1T cells and the T

193 cell activation assays were performed in PBS. Both THP-1 and A375-MR1 cells grown in RPMI

194 1640 or in PBS showed the same stimulatory capacity (Figure 5A,B), thus indicating that

9 195 medium constituents are not responsible for MR1T cell activation. To directly investigate

196 whether the stimulatory antigens were present in target cells, we performed T cell activation

197 assays using as source of antigen two types of lysates. The first lysate was obtained from in vitro

198 cultured THP-1 cells, while the second one was prepared from mouse breast tumors immediately

199 after resection. Two hydrophobic and four hydrophilic fractions were obtained and tested using

200 as APCs THP-1 cells that constitutively express low levels of MR1. The DGB129 clone reacted

201 only to fraction N4, containing highly hydrophilic compounds isolated from both freshly

202 explanted mouse tumor and in vitro cultured THP-1 cells (Figure 5C,D). These results ruled out

203 the possibility that stimulatory antigens were derived from RPMI 1640 components and

204 indicated their cellular origin. We also tested the fractions generated from THP-1 lysates with

205 DGB70, another representative MR1T cell clone. DGB70 cells recognized fraction N3 and not

206 N4, (Figure 5E), suggesting that at least two distinct compounds differentially stimulated the

207 two MR1T clones. The same fractions were also loaded onto plastic-bound MR1 molecules and

208 showed alternative and specific stimulatory capacity, i.e. N3 stimulated only DGB70 cells, while

209 N4 stimulated only DGB129 cells (Figure 5F). In the absence of N3 and N4 fractions, the two

210 clones did not react to MR1, further indicating the requirement of specific antigens.

211 In conclusion, these data indicated that MR1T cells recognize MR1 complexed with

212 ligands not derived from culture medium and present also in tumor cells grown in vivo. At least

213 two diverse molecules were stimulatory, whose nature and structure will be the subject of future

214 studies. As DGB129 and DGB70 clones were isolated from the same donor, these findings also

215 suggested that MR1T cells with different antigen specificities are present in the same individual.

216

217

10 218 Differential transcriptomes of antigen-stimulated MR1T cells

219 We next examined the diversity of MR1T cells by comparing the transcriptional response

220 to antigen stimulation of the two representative MR1T cell clones responding to diverse THP-1

221 lysate fractions (DGB129 and DGB70). MR1T cells were first rested for 3 weeks before being

222 stimulated with A375-MR1 cells for 20 hours. The transcriptional profiles of the sorted activated

223 cells (expressing similar levels of both CD25 and CD137 activation markers) were subsequently

224 compared with their unstimulated control counterparts (negative for the two markers) by RNA-

225 sequencing (Figure 6–figure supplement 1). Biological replicates were analyzed using stringent

226 cut-off criteria (FDR<0.05, minimum log2 fold change >2). The RNA-seq datasets revealed that

227 MR1+ APC stimulated DGB129 cells upregulated 403 genes and downregulated 413 genes,

228 whereas DGB70 cells up-regulated 432 genes and down-regulated 285 genes (Supplementary

229 file 1).

230 We then identified key transcription factors in each clone using global transcription factor

231 gene regulatory network analysis (Narang, 2015). A subnetwork was created from the identified

232 genes whose expression was modulated upon antigen stimulation, and the key nodes were

233 defined using centrality measures. This approach showed that some master transcription factors

234 were shared between MR1T cell clones, whereas others appeared to be clone-specific (Figure

235 6A,B). Whether assessed for betweenness centrality or tested by the PageRank algorithm, both T

236 cell clone responses were identified as being regulated by genes TBX21, FOXP3, FOS, RXRA,

237 FOSL2, IRF4, BATF and TRIB1. However, while DGB129 cells were further influenced by

238 expression of MYC, HSP90AB1 and CREM, DGB70 cells were instead regulated by EGR1,

239 JUNB, and SREBF1 (Figure 6C).

240

11 241

242 MR1T cells are functionally heterogeneous

243 We next analyzed the cytokine secretion profile of representative MR1T cell clones upon

244 stimulation by A375-MR1 APCs. All clones tested released IFN-γ (Figure 7–figure supplement

245 1A), probably as a consequence of in vitro expansion (Becattini, 2015). However, we also

246 observed diverse expression profiles of Th1 (IL-2, TNF-α and TNF-β), Th2 (IL-3, IL-4, IL-5, IL-

247 6, IL-10, IL-13) and Th17 cytokines (IL-17A, G-CSF, GM-CSF), and other soluble factors

248 (MIP-1β, soluble CD40L, PDGF-AA and VEGF; Figure 7A and Figure 7–figure supplement

249 1B). The variable combinations and quantities of cytokines expressed by MR1T cells suggested

250 considerable functional plasticity within this population. For example, clone DGA4 secreted

251 large quantities of IL-17A, IL-6, TNF-α and GM-CSF, but failed to secrete the prototypic Th2

252 cytokines IL-4, IL-5, IL-10 or IL-13, and thus displayed an ‘atypical’ Th17-like phenotype. In

253 contrast, clone TC5A87 released substantial amounts of VEGF and PGDF-AA, but only little

254 Th1 or Th2 cytokines, and no IL-17A. Notably, four of the seven clones studied (DGB129,

255 CH9A3, DGB70, JMA) displayed a Th2-skewed profile of cytokine release. In the same

256 experiments, both control MAIT cell clones, although derived from two different donors, showed

257 uniform Th1-biased responses following activation by A375-MR1 cells pulsed with E. coli

258 lysate. Given that the culture conditions and APCs used in these experiments were identical for

259 all clones, these data indicate that MR1T cells exhibit intrinsic functional heterogeneity.

260 To further support these findings, we next investigated the expression of three selected

261 chemokine receptors known to be differentially expressed by T cell subsets with distinct

262 functions (Becattini, 2015) and whose alternative combined expression regulates T cell

263 recirculation and migration to diverse homing sites (Al-Banna, 2014, Bromley, 2008, Thomas,

12 264 2007). Both resting MR1T and MAIT cell clones displayed high levels of CXCR3 (with the

265 exception of the DGA4 clone only; Figure 7B), However, we observed divergent expression

266 patterns of CCR4 and CCR6 (Figure 7B), which further suggested that MR1T cells are

267 heterogeneous and different from MAIT cells.

268 Taken together, these data indicated that the MR1-reactive clones tested here are

269 phenotypically and functionally heterogeneous, thus suggesting that MR1T cells include multiple

270 subsets with diverse recirculation patterns and tissue homing capacity and likely different

271 specialized roles in human immunity.

272

273 MR1T cells recognize cells constitutively expressing low surface MR1 and show

274 diverse T helper-like functions.

275 The MR1 expression level on the surface of APCs is physiologically regulated to be low

276 to undetectable in the absence of microbial ligands (Huang, 2008, McWilliam, 2016). We

277 therefore investigated whether monocyte-derived DCs (Mo-DCs) and other types of cells not-

278 overexpressing MR1, but constitutively displaying low surface MR1 levels (Figure 1A, Figure

279 8–figure supplement 1A,C and data not shown), were able to stimulate MAIT and MR1T cells.

280 All these cell types, supported MAIT cell activation in the presence of microbial antigens and in

281 an MR1-dependent manner (Figure 8A). The same cells also induced sterile MR1T cell

282 activation to various extents. Mo-DC and THP-1 cells were recognized by the majority of the

283 tested MR1T cell clones, followed by the Huh7 hepatoma cells, the LS 174T goblet-like cells

284 and the HCT116 colon carcinoma cells (Figure 8B). Importantly, all responses were blocked by

285 anti-MR1 mAbs.

13 286 Having found that MR1 T cells can recognize cells constitutively expressing

287 physiological surface levels of MR1, we next investigated whether they can also modulate the

288 functions of these target cells. First we focused on Mo-DCs, which stimulated several tested

289 MR1T cell clones. The representative DGB129 MR1T cell clone reacted to Mo-DC from

290 different donors (Figure 8–figure supplement 1B) and induced up-regulation of the maturation

291 markers CD83 and CD86 on these target cells (Figure 8C). Remarkably, Mo-DC maturation

292 promoted by DGB129 cells was fully inhibited by anti-MR1 mAbs, thus confirming that

293 recognition of MR1 was required for this T helper-like function.

294 As we observed that three MR1T cell clones reacted also to LS 174T goblet-like cells, we

295 next investigated the outcome of this interaction. LS 174T cells are used as a model to study

296 mucin gene expression regulation in intestinal epithelial cells (van Klinken, 1996) and express

297 low surface levels of MR1 (Figure 8–figure supplement 1C). The selected JMA MR1T cell

298 clone reacted to LS 174T by secreting in MR1-dependent fashion IL-8 and IL-13 (Figure 8D,E),

299 two cytokines involved in the modulation of human intestinal epithelial cell functions (Iwashita,

300 2003, Sturm, 2005). Interestingly, JMA recognition of LS 174T cells also promoted their

301 reciprocal activation resulting in increased transcription of mucin 2 gene (MUC2), which was

302 prevented by anti-MR1 blocking antibodies (Figure 8F). In control experiments, similar MUC2

303 expression upregulation was observed in LS 174T cells treated with TNF-α (Iwashita, 2003) in

304 the absence of MR1T cells (Figure 8F). These results unraveled the capacity of some MR1T

305 cells to modulate MUC2 expression in intestinal epithelial cells, and therefore suggested their

306 possible contribution to intestinal epithelial barrier homeostasis.

307 Taken together, these data identify MR1T cells as a novel population of human MR1-

308 restricted T lymphocytes that may mediate diverse immunological functions. These findings

14 309 indicate that the repertoire and reactivity of MR1-restricted T cells in healthy individuals is far

310 broader than was previously appreciated.

311

312 Discussion

313 Here we report that functionally diverse human T cells respond to MR1 in the absence of

314 microbial antigens, thus extending the role of MR1 beyond the presentation of microbial

315 riboflavin precursors to MAIT cells. While the structure of MR1 resembles that of MHC class I

316 and related proteins, this molecule also displays various unique properties. A key distinguishing

317 feature is the presence of a bulky antigen-binding pocket that binds small molecules via either

318 hydrophilic or hydrophobic interactions. The total volume of the MR1 antigen-binding pocket is

319 far larger than any of the MR1 ligands reported to date, suggesting capacity to bind a wider

320 repertoire of antigens than that known so far. A direct consequence of this is the likely existence

321 of other T cell populations that recognize MR1-presented antigens distinct from the known

322 riboflavin precursors or folate-derivatives. Our study indicates the existence of an unpredicted

323 population of MR1-restricted T cells (designated MR1T cells) outside of the MAIT cell

324 compartment. Human MR1T cell clones displayed differential recognition of a variety of target

325 cells expressing constitutive levels of MR1. The different recognition of target cells might reflect

326 different antigen presentation capacity, although they equally presented microbial antigens to

327 MAIT cells, or the presence of different antigens. Indeed, preliminary antigen purification from a

328 representative target cell line identified two diverse fractions that each stimulated only one of

329 two MR1T cell clones, both derived from the same donor. Although the nature of these

330 molecules remains to be determined, they do not derive from RPMI 1640 culture medium and

331 are present in tumor cells grown in vivo. Furthermore, initial characterization of the molecules

15 332 showed that they formed stable complexes with plastic-bound MR1 without forming a Schiff

333 base and activated specific MR1T cells without need of APC processing.

334 Using as APCs A375 cells expressing high levels of MR1, we estimated that MR1T cells

335 represent between 1:2,500 and 1:5,000 of total circulating T cells. These frequency estimates

336 could not be determined by direct ex vivo identification of MR1T cells due to lack of specific

337 markers, and were obtained using two approaches. In the first, frequencies were calculated after

338 overnight stimulation and subtracting the frequency of T cells responding to MR1-negative APC

339 to the frequency of cells responding to MR1-positive APC. In another set of experiments the

340 overnight-stimulated T cells were immediately cloned without previous in vitro expansion and

341 individual MR1T cell clones were identified with functional assays. These observations indicated

342 that MR1T cell are readily detectable in the blood of healthy individuals with a size that

343 resembles that of MHC-restricted memory T cells recognizing pathogen-derived peptides in

344 pathogen-exposed individuals (Bacher, 2013, Lucas, 2004, Su, 2013, Yu, 2015). The calculated

345 range of MR1T cell frequency could however be underestimated as A375 cells utilized for the

346 frequency studies might lack some MR1T cell antigens and therefore fail to stimulate a fraction

347 of MR1T cells. Collectively, our data suggest that MR1T cells are not rare T lymphocytes in

348 human blood.

349 In some respects, MR1T cells resemble another population of autoreactive T cells

350 restricted to non-polymorphic antigen presenting molecules and present at high frequency in

351 human blood. These T cells react to self-lipids presented by CD1 molecules (de Jong, 2010, de

352 Lalla, 2011, Dellabona, 2015). In analogy to MR1T cells, CD1-self-reactive T cells display a

353 polyclonal TCRα and β usage, and are functionally heterogeneous (de Jong, 2010, de Lalla,

354 2011, Dellabona, 2015). The physiological role of self-reactive T cells restricted by CD1 and

16 355 MR1 molecules is not well characterized. It is tempting to speculate that these cells might be

356 activated upon metabolic changes in target cells induced by environmental signals, infection or

357 cell transformation.

358 It is currently unclear whether MR1T cells can be classified into distinct subgroups based

359 on phenotype, gene expression and function. Since MR1T cells exhibit non-invariant TCRs with

360 diverse antigen specificities, express a wide range of different surface markers and release

361 multiple soluble effector molecules, it will be challenging to identify these cells using MR1

362 tetramer staining combined with conventional phenotypic analyses. Interestingly, some activated

363 MR1T cell clones also displayed atypical functions for T cells, including the release of growth

364 factors VEGF and PDGF, which support mesenchymal cell proliferation, vasculogenesis, and

365 angiogenesis (Carmeliet, 2003), suggesting a potential role in tissue remodeling processes.

366 The functional variety of MR1T cells was also accompanied by differential expression of

367 selected chemokine receptors, suggesting that this population may exert multiple functions at

368 various sites throughout the body. By comparing the transcriptomes of two select MR1T cell

369 clones, we observed that certain transcription factors were expressed in common by both cell

370 types, whereas others were only expressed in a clone-specific fashion. The DGB70 clone was

371 distinguished by expression of the regulator of T cell polarization and proliferation factor EGR-1

372 (Shin, 2009, Thiel, 2002), the mediator of lymphocyte functional differentiation JUN-B (Li,

373 1999), and the SREBF1, a member of the SREBP gene family that regulates sterol synthesis,

374 coordinates tissue-specific gene expression and controls T cell metabolism (Kidani, 2013, Shao,

375 2012). In contrast, transcription factors unique to the DGB129 clone were the proliferation and

376 apoptosis regulator MYC (Kress, 2015), involved in metabolic programming of effector T cells

377 (Hough, 2015), and the cAMP responsive element binding factor CREM, which modulates T cell

17 378 effector functions and cytokine gene expression (Rauen, 2013). DGB129 cells also uniquely

379 upregulated the HSP90AB1 heat shock protein gene, which mediates quality control of

380 cytoplasmic proteins (Taipale, 2010) and is involved in the regulation of diverse cell surface

381 receptor expression in T and NK cells (Bae, 2013). These clear distinctions between the

382 transcriptional profiles of the two MR1T cell clones indicate the use of different strategies for

383 metabolic programming, cell cycle control and functional polarization and suggest specific roles

384 for these cells. As sorted cells expressed equivalent levels of CD25 and CD137 after Ag

385 stimulation (see Figure 6-figure supplement 1), and released comparable amounts of IFN-γ (not

386 shown) we excluded a different strength of antigen stimulation as possible explanation of the

387 diverse transcriptional profiles observed. Given that the two clones were isolated from the same

388 donor, maintained in identical culture conditions and stimulated with the same type of APC,

389 these divergent gene signatures could reflect unique priming events (Boltjes, 2014).

390 A key finding of our study is the reactivity of a large fraction of MR1T cell clones to

391 diverse types of target cells expressing constitutive low MR1 surface levels. These results

392 indicated the capacity of MR1T cells to be activated in physiologic conditions, when limiting

393 amounts of MR1-antigen complexes are available on the target cell surface. As consequence,

394 these data raised the issue of what influence MR1T cells could exert on these target cells. Several

395 MR1T clones tested recognized monocyte-derived DCs, thus suggesting that MR1-restricted

396 reactivity to these APCs is quite frequent among MR1T cells. In addition, experiments

397 performed with a representative MR1T cell clone revealed its capacity to promote monocyte-

398 derived DCs maturation. These data suggest that MR1T cells may modulate DC functions as it

399 has already been reported for other T lymphocyte populations including TCR γδ (Devilder,

400 2006) and CD1-restricted T cells (Bendelac, 2007, Vincent, 2002). Some MR1T cell clones

18 401 were also stimulated by human intestinal epithelial LS 174T cells. In co-culture experiments, we

402 observed reciprocal MR1-dependent activation of both MR1T and LS 174T cells, with the first

403 releasing IFN-γ, IL-8 and IL13, and the latters up-regulating mucin 2 gene expression. These

404 data suggested that some MR1T cells might influence epithelial cell function at mucosal sites,

405 perhaps promoting innate defense and/or inflammation.

406 In conclusion, we report that in addition to microbial metabolite-sensitive MAIT cells,

407 human blood contains a novel population of MR1-restricted T cells that can recognize different

408 antigens present in distinct target cells and exhibits a variety of effector functions. Based on the

409 diversity of effector molecules they release following antigen stimulation, MR1T cells might

410 drive inflammatory responses, support B cell function, mediate DC licensing, promote tissue

411 remodeling, and contribute to mucosal homeostasis by enhancing innate defenses at the epithelial

412 barrier. Future studies are therefore likely to uncover the roles of MR1T cells in human diseases.

413

19 414 Materials and methods

415 Cells

416 The following cell lines were obtained from American Type Culture Collection: A375 (human

417 melanoma), THP-1 (myelomonocytic leukemia), J.RT3-T3.5 (TCRβ-deficient T cell leukemia),

418 HEK 293 (human embryonic kidney), CCRF-SB (acute B cell lymphoblastic leukemia), Huh7

419 (human hepatoma), HCT116 (human colorectal carcinoma), LS 174T (goblet-like cells from

420 colon adenocarcinoma), and EMT6 (mouse breast carcinoma). SKW-3 cells (TCRα- and β-

421 deficient human T cell leukemia) were obtained from the Leibniz-Institute DSMZ-German

422 Collection of Microorganisms and Cell Cultures. All the used cells were routinely tested for

423 mycoplasma contamination and were negative. None of the cell lines used in this study is present

424 in the database of commonly misidentified cell lines. Cells lines were not authenticated. Two

425 representative MAIT clones (MRC25 and SMC3) were used in this study generated from blood

426 of two different healthy donors and maintained in culture as previously described (Lepore,

427 2014). MR1T cells were isolated from the peripheral blood of healthy individuals. Briefly, T

428 cells purified by negative selection were stimulated with irradiated (80 Gray) A375-MR1 cells

429 (ratio 2:1) once a week for three weeks. Human rIL-2 (5 U/ml; Hoffmann-La Roche) was added

430 at day +2 and +5 after each stimulation. Twelve days after the last stimulation cells were washed

431 and co-cultured overnight with A375-MR1 cells (ratio 2:1). CD3+CD69+CD137high cells were

432 then sorted and cloned by limiting dilution in the presence of PHA (1 µg/ml, Wellcome Research

433 Laboratories), human rIL-2 (100 U/ml, Hoffmann-La Roche) and irradiated PBMC (5×105 cells

434 /ml). In other experiments, MR1 T cells clones were generated using the same protocol from

435 sorted CD3+CD69+CD137high upon a single overnight stimulation with A375-MR1 cells (ratio

436 2:1). T cell clones were periodically re-stimulated following the same protocol (Lepore, 2014).

20 437 Monocytes and B cells were purified (>90% purity) from PBMCs of healthy donors using

438 EasySep Human CD14 and CD19 positive selection kits (Stemcell Technologies) according to

439 the manufacturer instructions. Mo-DCs were differentiated from purified CD14+ monocytes by

440 culture in the presence of GM-CSF and IL-4 as previously described (Lepore, 2014).

441

442 Generation of cells expressing MR1A gene covalently linked with β2m

443 A human MR1A cDNA construct linked to β2m via a flexible Gly-Ser linker was generated by

444 PCR as previously described (Lepore, 2014). The K43A substitution in the MR1A cDNA was

445 introduced into the fusion construct using the following primers: MR1K43A_f 5′-

446 CTCGGCAGGCCGAGCCACGGGC and MR1K43A_r 5′GCCCGTGGCTCGGCCTGCCGAG.

447 Resulting WT and mutant constructs were cloned into a bidirectional lentiviral vector (LV)

448 (Lepore, 2014), provided by Jürg Schwaller, Department of Biomedicine, University of Basel.

449 HEK293 cells were transfected with individual LV-MR1A-β2m constructs together with the

450 lentivirus packaging plasmids pMD2.G, pMDLg/pRRE and pRSV-REV (Addgene) using

451 Metafectene Pro (Biontex) according to the manufacturer instructions. A375, THP-1, CHO and

452 J558 cells were transduced by spin-infection with virus particle containing supernatant in

453 presence of 8 µg/ml protamine sulfate. Surface expression of MR1 was assessed by flow

454 cytometry and positive cells were FACS sorted.

455

456 Soluble recombinant β2m-MR1-Fc fusion protein

457 β2m-MR1-Fc fusion construct was obtained using human MR1A-β2m construct described above

458 as template. DNA complementary to β2m-MR1A gene was amplified by PCR using primers:

459 β2mXhoI_f 5’- CTCGAGATGTCTCGCTCCGTGGCCTTA and MR1-IgG1_r 5’-

460 GTGTGAGTTTTGTCGCTAGCCTGGGGGACCTG, thus excluding MR1 trans-membrane and

21 461 intracellular domains. The DNA complementary to the hinge region and CH2-CH3 domains of

462 human IgG1 heavy chain was generated using the following primers: NheI-hinge-f 5’-

463 CAGGTCCCCCAGGCTAGCGACAAAACTCACAC and IgG1NotI_r 5’-

464 GCGGCCGCTCATTTACCCGGAGACAGGGAGA from pFUSE-hIgG1-Fc1 (InvivoGen). The

465 β2m-MR1A and IgG1 PCR products were joined together using two-step splicing with overlap

466 extension PCR and the resulting construct subcloned into the XhoI/NotI sites of the BCMGSNeo

467 expression vector. CHO-K1 cells were transfected with the final construct using Metafectene Pro

468 (Biontex), cloned by limiting dilutions and screened by ELISA for the production of β2m-MR1-

469 Fc fusion protein. Selected clones, adapted to EX-CELL ACF CHO serum-free medium (Sigma),

470 were used for protein production and β2m-MR1-Fc was purified using Protein-A-Sepharose

471 (Thermo Fisher Scientific) according to the manufacturer instructions. Protein purity was

472 verified by SDS-PAGE and Western Blot. Protein integrity was assessed by ELISA, using anti-

473 β2M mAb (HB28) as capture and the conformation-dependent anti-MR1 mAb 25.6 as reveling

474 (Kjer-Nielsen, 2012).

475

476 Flow cytometry and antibodies

477 Cell surface labeling was performed using standard protocols. Intracellular labeling was

478 performed using the True-Nuclear™ Transcription Factor Buffer Set according to the

479 manufacturers’ instructions. The following anti-human mAbs were obtained from Biolegend:

480 CD4-APC (OKT4), CD8α-PE (TuGh4), CD161-Alexa Fluor 647 (HP-3G10), CD69-PE (FN50),

481 CD3-PE/Cy7, Brilliant Violet-711, or Alexa-700 (UCHT1), CD137-biotin (n4b4-1), CXCR3-

482 Brilliant Violet 421 (G025H7), CD83-biotin (HB15e) and TRAV1-2- PE (10C3). CD86-FITC

483 (2331), CCR4-PECy7 (1G1) and CCR6-PE (11A9) mAbs were from BD Pharmingen. All these

22 484 mAbs were used at 5 µg/ml. Biotinylated mAbs were revealed with streptavidin-PE, -Alexa

485 Fluor 488, or -Brilliant violet 421 (2 µg/ml, Biolegend). The MR1-specific mAb clone 26.5

486 (mouse IgG2a) was provided by Ted Hansen, Marina Cella and Marco Colonna, Washington

487 University School of Medicine, St. Louis (MO) (Lepore, 2014). Unlabeled MR1-specific mAbs

488 were revealed with goat anti-mouse IgG2a-PE (2 µg/ml, Southern Biotech). Samples were

489 acquired on LSR Fortessa flow cytometer (Becton Dickinson). Cell sorting experiments were

490 performed using an Influx instrument (Becton Dickinson). Dead cells and doublets were

491 excluded on the basis of forward scatter area and width, side scatter, and DAPI staining. All data

492 were analyzed using FlowJo software (TreeStar).

493

494 TCR gene analysis of MR1T cell clones

495 TCRα and β gene expression by MR1T cell clones was assessed either by RT-PCR using total

496 cDNA and specific primers, or by flow cytometry using the IOTest® Beta Mark TCR Vβ

497 Repertoire Kit (Beckman Coulter) according to the manufacturers’ instructions. For RT-PCR,

498 RNA was prepared using the NucleoSpin RNA II Kit (Macherey Nagel) and cDNA was

499 synthesized using Superscript III reverse transcriptase (Invitrogen). TCRα and β cDNAs were

500 amplified using sets of Vα and Vβ primers as directed by the manufacturer (TCR typing

501 amplimer kit, Clontech). Functional transcripts were identified by sequencing and then analyzed

502 using the ImMunoGeneTics information system (http://www.imgt.org). The cDNA-sequencing

503 data set has been deposited in the GenBank repository under accession numbers MF085360-

504 MF085372.

505

506

23 507 Fractionation of cell and whole tumor lysates

508 Total cell lysates were generated from a single pellet of 2.5×109 THP-1 cells via disruption in

509 water with mild sonication. The sonicated material was then centrifuged (15,000g for 15 min at

510 4°C) and the supernatant collected (S1). Next, the pellet was re-suspended in methanol,

511 sonicated, centrifuged (15,000g for 15 min at 4°C), and the supernatant obtained (S2) was pooled

512 with the S1 supernatant. The final concentration of methanol was 10%. The total cell extract

513 (pool of S1 and S2) was then loaded onto a C18 Sep-Pak cartridge (Waters Corporation) and the

514 unbound material was collected and dried (fraction E-FT). Bound material was eluted in batch

515 with 75% (fraction E1) and 100% methanol (fraction E2), collected and dried. The E-FT material

516 was re-suspended in acetonitrile/water (9:1 vol/vol) and loaded onto a NH2 Sep-Pak cartridge

517 (Waters Corporation). Unbound material (fraction N-FT) and 4 additional fractions (N1-N4)

518 were eluted with increasing quantities of water. Fraction N1 was eluted with 35% H2O, fraction

519 N2 with 60% H2O, fraction N3 with 100% H2O, and fraction N4 with 100% H2O and 50 mM

520 ammonium acetate (pH 7.0). Fractions N1-N4 were then dried and stored at -70°C until use. All

521 collected dry-frozen fractions were then thawed and re-suspended in H2O 20% methanol

522 (fractions E1, E2 and N-FT) or 100% H2O (all other fractions) prior to being tested in T cell

523 activation assays.

524 Mouse EMT6 breast tumors were provided by A. Zippelius and were prepared as described

525 (Zippelius, 2015). Freshly excised tumors were extensively washed in saline, weighted and 4 g

526 masses were homogenized in 7 ml of HPLC-grade water using a Dounce tissue grinder. Tumor

527 homogenate underwent two freeze-thaw cycles, centrifuged (3,250g) for 10 min at 4°C, and

528 supernatant was collected and stored at -70°C. The pellet was extracted a second time with 2 ml

529 of HPLC-grade water, centrifuged (5,100g) for 10 min at 4°C and the supernatant was collected

24 530 and stored at -70°C. The pellet was further extracted with 9 ml of HPLC-grade methanol for 5

531 min at room temperature by vortexing, centrifuged (5,100g) for 10 min at 4°C, and supernatant

532 collected. The three supernatants were pooled, dried, and resuspended in water:methanol (10:1).

533 Material was fractionated using C18 and NH2 Sep-Pak cartridges as above.

534

535 T cell activation assays

536 MR1-restricted T cells (5×104/well unless otherwise indicated) were co-cultured with indicated

537 target cells (5×104/well) in 200 µl total volume in duplicates or triplicates. T cells were cultured

538 with indicated APCs for 24 h. In some experiments, anti-MR1 mAbs (clone 26.5) or mouse

539 IgG2a isotype control mAbs (both at 30 µg/ml) were added and incubated for 30 min prior to the

540 addition of T cells. In some experiments A375-MR1 and THP1-MR1 cells were cultured 4 days

541 in PBS supplemented with 5% human AB serum. Cells were washed every day and then used to

542 stimulate T cell clones upon assessment of target cell viability by trypan blue staining. In such

543 type of experiments, T cell activation assays were also performed in PBS supplemented with 5%

544 human AB serum. E. coli lysate was prepared from the DH5α strain (Invitrogen) grown in LB

545 medium and collected during exponential growth. Bacterial cells were washed twice in PBS and

546 then lysed by sonication. After centrifugation (15,000g for 15 min), the supernatant was

547 collected, dried, and stored at -70°C. APCs were pulsed for 4 h with E. coli lysate equivalent to

548 108 CFU/ml (unless otherwise indicated) before addition of T cells. In some experiments, APCs

549 were pre-incubated with 6-FP or Ac-6-FP (Schircks Laboratories) or pulsed with 6,7-dimethyl-8-

550 D-ribityllumazine (RL-6,7-diMe; 150 µM; Otava Chemicals) for 4 h before co-culture with T

551 cells. In control experiments with TCR γδ cells, the APCs were first treated for 6 h with

552 zoledronate (10 µg/ml, Sigma) prior to T cell addition. Activation experiments with plate-bound

25 553 recombinant human β2m-MR1-Fc were performed by coating β2m-MR1-Fc onto 96 well plates

554 (4 µg/ml) and loading with cartridge-purified cell lysates for 4 h at 37°C before washing twice

555 and adding T cells. Supernatants were collected after 24 h and IFN-γ or GM-CSF were assessed

556 by ELISA. Multiple cytokines and chemokines in cell culture supernatants were analyzed using

557 the Milliplex MAP human cytokine/chemokine magnetic bead panel – Premixed 41-plex

558 (HCYTMAG-60K-PX41; Merck Millipore) according to the manufacturer’s instructions.

559 Samples were acquired on a Flexmap 3D system (Merck Millipore) and Milliplex analyst

560 software was used to determine mean fluorescence intensity and analyte concentration.

561

562 Modulation of DC and epithelial cell functions

563 Mo-DCs were co-cultured with MR1T cell clones at 1:1 ratio. When indicated Mo-DCs were

564 pre-incubated with anti-MR1 mAbs (clone 26.5, 30 µg/ml) for 30 min prior addition of T cells.

565 Supernatants were harvested after 48h and tested for IFN-γ release by ELISA to assess T cell

566 activation. Cells were collected at the same time and analyzed by FACS for CD83 and CD86

567 expression. T cells were excluded by the analysis using anti-CD3 mAbs. LS 174T cells were co-

568 cultured with MR1T cell clones at 1:1 ratio in the presence or absence of anti-MR1 (clone 26.5)

569 or mouse IgG2a isotype control mAbs (both at 30 µg/ml, incubated with APCs 30 min prior

570 addition of T cells). MR1T cell activation was evaluated by measuring IFN-γ, IL-8 (R&D) and

571 IL-13 (Pharmingen) in the supernatants by ELISA. Q-PCR for MUC2 gene expression by LS

572 174T cells was performed in a 20 µl reaction volume containing 0.5 µM of each primer and 2.5

573 µl of cDNA in Power SYBRgreen MasterMix (Applied Biosystems). β-actin was used as

574 reference gene. All the reactions were carried out in triplicate. The following method was run

575 using an ABI 7500 Fast Real-Time PCR System (Applied Biosystem): initial incubation for 20

26 576 sec at 50°C, incubation for 10 min at 95°C, 40 cycles of 15 sec at 95°C and 1 min at 60°C. The

577 PCR primers used were as follows: β-actin_f 5′-GCCACCCGCGAGAAGATGA, β-actin_r 5′-

578 CATCACGATGCCAGTGGTA; MUC2_f 5′-ACCCGCACTATGTCACCTTC, MUC2_r 5′-

579 GGGATCGCAGTGGTAGTTGT. Changes in gene expression were quantified using the ΔΔCt

580 method.

581

582 TCR gene transfer

583 TCRα and β functional cDNA from the MAIT cell clone MRC25 were cloned into the XhoI/NotI

584 sites of the BCMGSNeo expression vectors and the resulting constructs were used to co-transfect

585 J.RT3-T3.5 cells by electroporation. Transfectants expressing TRAV1-2 and CD3 were FACS

586 sorted. The TCRα and β functional cDNA from MR1T clones were cloned into the XmaI/SalI

587 sites of a modified version of the p118 lentiviral expression vector (Amendola, 2005, Lepore,

588 2014). SKW-3 cells were transduced with virus particle-containing supernatant generated as

589 described above. Cells were FACS sorted based on CD3 expression.

590

591 Next-generation sequencing, transcriptome and gene centrality analyses

592 Comparative transcriptome analysis between resting and A375-MR1 stimulated MR1T cell

593 clones was performed by RNA-sequencing. T cells were stimulated with A375-MR1 cells and

594 after 20 h DAPI-CD3+CD25+CD137+ cells were sorted, counted, and immediately frozen. Total

595 RNA was extracted using Arcturus PicoPure RNA Isolation kit (Applied Biosystems) according

596 to the manufacturer's protocol. RNA quality, assessed using the Agilent Bioanalyzer, exhibited

597 RNA integrity number (RIN) ≥6.9. Next, cDNA libraries were prepared using 200 ng total RNA

598 and a 2 µl volume of a 1:500 dilution of ERCC RNA Spike in Controls (Ambion). Samples were

27 599 subjected to cDNA library synthesis using the TruSeq Stranded mRNA Library Preparation Kit

600 (Illumina) according to the manufacturer's protocol with minor modifications; 13 PCR cycles

601 used, and 2 additional rounds of purification with Agencourt Ampure XP SPRI beads (Beckman

602 Courter) to remove double-stranded cDNA >600bp in size. The length distribution of the cDNA

603 libraries was monitored using DNA 1,000 kits with the Agilent Bioanalyzer. The samples were

604 then subjected to an indexed PE sequencing run of 2×51 cycles on an Illumina HiSeq 2,000.

605 Raw RNA-seq reads in fastq format were aligned to the hg19 genome assembly using

606 STAR aligner (Dobin, 2013). Gene annotations were derived from GENCODE version (Harrow,

607 2012) and counts of reads mapping over gene features were obtained using the featureCount

608 method of the R/bioconductor subread packages (Liao, 2014). EdgeR (Robinson, 2010) was

609 used to generate a differentially expressed gene (DEG) list. From the DEGs, a global network of

610 transcription factors and their target genes was created using the Encode chip-seq data as

611 described (Narang, 2015). Using the list of DEGs (FDR <0.05, log2 fold-change >2), a

612 subnetwork of the global transcription factors-gene network was created and centrality scores for

613 nodes were generated by both PageRank and betweeness centrality algorithms using NetworkX

614 library in Python. The network was visualized using Cytoscape software and betweeness

615 centrality score were used to define the radius of the nodes (circles in the network). The RNA-

616 sequencing data set has been deposited in the Gene Expression Omnibus repository under

617 accession code GSE81063.

618

619 Statistics

620 For cytokine secretion assays and Q-PCR data were analyzed using Unpaired Student’s t-test

621 (Prism 6, GraphPad software).

28 622

623 Acknowledgements

624 We thank L. Angman and C. Furtwängler for TCR cloning and gene-transfer experiments, E.

625 Traunecker for cell sorting, S. Sansano for MR1 purification, T. Rutishauer for help with figure

626 preparation, A. Zippelius for EMT6 mouse tumors, J. Schwaller, G. Spagnoli and V. Governa for

627 providing reagents (all from University of Basel), D. Trono for plasmid availability through

628 Addgene, T. Hansen, M. Cella, M. Colonna (University of Washington) for MR1-specific mAbs.

629 We thank O. Lantz (Institut Curie, Paris) for helpful discussions and N. McCarthy of Insight

630 Editing London for reviewing the manuscript.

631

29 632 Additional information

633 Competing interests

634 The authors declare no competing financial interests.

635 Funding

Funder Grant reference number Author

Swiss National Science Foundation 310030-149571 Gennaro De Libero DBM, University Hospital Basel Core Funds Gennaro De Libero European Union Horizon 2020 “TBVAC 2020” 643381 Gennaro De Libero BMRC-SERC, A*STAR 1121480006 Gennaro De Libero SIgN, A*STAR Core Funds Gennaro De Libero Lucia Mori A*STAR-Australian NHMRC 1201826277 Lucia Mori University of Basel “Förderbeiträge für exzellente Marco Lepore junge Forschende” The funders had no role in study design, data collection and interpretation, or decision to submit the work for publication. 636

637 Ethics

638 Human studies: Venous blood was taken from healthy donors after informed consent obtained at

639 the time of blood collection under approval of the “Ethikkommision Nordwest und

640 Zentralschweiz/EKNZ (139/13).

641

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852

37 853 Table 1. Phenotype and TCR gene usage of selected MR1-reactive T cell clones. Clone CD4 CD8α TCRα TCRβ CD161 DGB129 - + TRAV29 TRBV12-4 - DGB70 - - TRAV5 TRBV28 - DGA28 - + TRAV21 TRBV29-1 + DGA4 - - TRAV1-2 ND + JMA - + TRAV27 TRBV25-1 - TC5A87 - + TRAV13-1 TRBV25-1 - CH9A3 - + TRAV24 TRBV5-5 - 854 ND, not determined

855

38 856 Figure legends

857

858 Figure 1. Recognition of non-microbial antigens by MR1-restricted T cells. (A) Surface

859 expression of MR1 by CCRFSB, THP-1 and A375-MR1 cells. Grey histograms indicate staining

860 with isotype-matched control mAbs. Stimulation of (B) T cell clone DGB129 or (C) MAIT cell

861 clone SMC3 by the three cell lines in A in the absence (no Ag) or presence of E. coli lysate (E.

862 coli) and/or anti-MR1 blocking mAbs (α-MR1). Columns indicate IFN-γ release (mean + SD).

863 Stimulation of (D) DGB129 MR1T or (E) SMC3 MAIT cells by THP-1 cells, constitutively

864 expressing surface MR1, loaded with synthetic 6,7-dimethyl-8-D-ribityllumazine (RL-6,7-diMe)

865 with or without anti-MR1 mAbs. Columns indicate mean IFN-γ release + SD. Stimulation of (F)

866 SKW-3 cells expressing the DGB129 TCR (SKW3-DGB129) or (G) J.RT3-T3.5 cells

867 expressing the MAIT MRC25 clone TCR (J.RT3-MAIT) with A375 cells that expressed (A375-

868 MR1) or lacked (A375-WT) MR1, with or without E. coli lysate and/or anti-MR1 mAbs. CD69

869 median fluorescence intensity (MFI) ± SD of duplicate cultures of transduced T cells are shown.

870 The CD69 MFI of transduced T cells cultured in the absence of APCs is also shown. Data are

871 representative of four (A, B and C), two (D and E), and three (F and G) independent

872 experiments. * P<0.05 (Unpaired Student’s t-test).

873

874 Figure 2. Isolation of non-MAIT MR1-restricted T cell clones after stimulation with A375-MR1

875 cells in the absence of microbial antigens. (A) FACS analysis of purified T cells previously

876 expanded with irradiated A375-MR1 cells following overnight co-culture with A375-MR1 cells.

877 Left dot plot shows CD3 and CellTrace violet (CTV) staining in live cells. Upper right and

878 bottom right dot plots show CD69 and CD137 expression on CD3+CTV- and CD3+CTV+ gated

39 879 cells, respectively. Arrows indicate gating hierarchy. Numbers indicate the percentages of cells

880 within the gates. Cells from Donor A are illustrated as a representative donor. (B, D) Cumulative

881 results of T cell clones screening from Donors A and B. T cell clones were generated from

882 CD3+CTV-CD137high sorted T cells as depicted in A. Graphs show the individual clones (x axis)

883 and their IFN-γ release (y axis), expressed as ratio between the amount of cytokine secreted in

884 response to A375-MR1 cells vs. A375 WT cells. Each dot represents a single T cell clone, tested

885 at the same time in the indicated experimental conditions. The horizontal red line marks the

886 arbitrary IFN-γ ratio cut-off of 2, above which MR1-dependent T cell clone reactivity was set.

887 The intercept of the vertical red line indicates the number of MR1-restricted T cell clones in each

888 donor. Red boxes highlight T cell clones whose reactivity was MR1-dependent. Results are

889 representative of two independent experiments. (C, E) IFN-γ release by 14 representative clones

890 from Donor A and 11 clones from Donor B after stimulation with A375 WT, A375-MR1 and

891 A375-MR1 in the presence of blocking anti-MR1 mAbs (α-MR1). Dots represent the IFN-γ

892 release (mean ± SD of duplicate cultures) by each clone. Results are representative of three

893 independent experiments. * P<0.05 (Unpaired Student’s t-test).

894

895 Figure 3. Non-MAIT MR1-restricted T cells are readily detectable in the blood of healthy

896 individuals. (A) Flow cytometry analysis of purified T cells from a representative donor (Donor

897 C) after overnight co-culture with A375 WT or A375-MR1 cells. Dot plots show CD69 and

898 CD137 expression on live CD3+ cells. Numbers indicate the percentage of cells in the gates. (B)

899 Frequency of CD69+CD137+ T cells from 5 different donors after overnight co-culture with

900 A375 WT or A375-MR1 cells. (C) Cumulative results of T cell clone stimulation assays from

901 Donor C. T cell clones were generated from CD3+CD69+CD137+ sorted T cells as depicted in A,

40 902 right dot plot. The graph shows the number of tested clones (x axis) and IFN-γ release (y axis)

903 expressed as ratio between the amount of cytokine secreted in response to A375-MR1 cells vs.

904 A375-WT cells. Each dot represents a single T cell clone, tested at the same time in the indicated

905 experimental conditions. The horizontal red line marks the arbitrary IFN-γ ratio threshold of 2

906 above which MR1-dependent T cell clone reactivity was set. The intercept of the vertical red line

907 indicates the number of MR1-restricted T cell clones. Red box highlights T cell clones whose

908 reactivity was MR1-dependent. Results are representative of two independent experiments. (D)

909 Recognition of A375-MR1 but not A375 WT cells in the absence of exogenous antigens by 8

910 representative MR1-restricted T cell clones from Donor C. Inhibition of T cell clone reactivity to

911 A375-MR1 cells by blocking anti-MR1 mAbs (α-MR1). Dots represent the IFN-γ release (mean

912 ± SD of duplicate cultures) by each clone tested in the three experimental conditions. Results are

913 representative of three independent experiments. * P<0.05 (Unpaired Student’s t-test).

914

915 Figure 4. MR1T cell clones do not react to microbial ligands or to 6-FP. (A) Response of seven

916 MR1T cell clones and one control MAIT cell clone co-cultured with A375 cells expressing

917 (A375-MR1) or not (A375 WT) MR1 in the presence or absence of E. coli lysate. Blocking of T

918 cell clone reactivity by anti-MR1 mAbs (α-MR1) is also shown. (B) Response of MR1T cell

919 clones to A375 cells expressing either WT MR1 molecules (A375-MR1) or K43A-mutated MR1

920 molecules (A375-MR1 K43A) in the presence of 6-formyl pterin (6-FP). (C) Stimulation of

921 control MAIT cell clone MRC25 or control TCR Vγ9Vδ2 clone G2B9 with A375-MR1 or

922 A375-MR1 K43A cells previously incubated with or without E. coli lysate or zoledronate,

923 respectively, either in the absence or presence of 6-FP. Results are expressed as mean ± SD of

41 924 IFN-γ measured in duplicate cultures. Results are representative of three independent

925 experiments. * P<0.05 (Unpaired Student’s t-test).

926

927 Figure 4–figure supplement 1. MR1T cell clones do not recognize Ac-6-FP. (A) Stimulation of

928 three representative MR1T cell clones by A375-MR1 cells in the absence or presence of acetyl-

929 6-formyl pterin (Ac-6-FP). (B) Stimulation of two MAIT cell clones (MRC25 and SMC3) by

930 A375-MR1 cells pulsed with E. coli lysate in the absence or presence of Ac-6-FP. (C) A375-

931 MR1 cells were treated with zoledronate (Zol) in the absence or presence of Ac-6-FP (25 µg/ml)

932 and used to stimulate a TCR Vγ9-Vδ2 cell clone (G2B9). (D) A375 cells expressing K43A

933 mutant MR1 molecules (A375-MR1 K43A) were used to stimulate the three MR1T cell clones

934 shown in A, in the absence or presence of Ac-6-FP (25 µg/ml). (E) Stimulation of the two MAIT

935 cell clones used in B by A375-MR1 K43A cells pulsed with E. coli lysate in the absence or

936 presence of Ac-6-FP (25 µg/ml). Results are expressed as mean ± SD of IFN-γ release assessed

937 in duplicate cultures and are representative of three independent experiments. * P<0.05

938 (Unpaired Student’s t-test).

939

940 Figure 5. MR1T cells recognize diverse antigens not derived from RPMI 1640 medium.

941 Stimulation of the DGB129 MR1T cell clone by MR1-overexpressing (A) A375 cells (A375-

942 MR1) and (B) THP-1 cells (THP1-MR1) grown for 4 days in RPMI 1640 or in PBS both

943 supplemented with 5% human serum. Inhibition of T cell clone reactivity by anti-MR1 blocking

944 mAbs (α-MR1) is shown. DGB129 cells recognize APCs loaded with fractions isolated from (C)

945 THP-1 cell lysate or from (D) in vivo grown mouse breast tumor EMT6. Fractions E1 and E2

946 contain hydrophobic molecules; fractions N1-N4 contain hydrophilic molecules. (E) DGB70

947 MR1T cells react to N3 fraction of THP-1 lysate. (F) Stimulation of DGB129 and DGB70 T

42 948 cells by THP-1-derived fractions N3 and N4 loaded onto plastic-bound recombinant MR1.

949 Shown is T cell release of IFN-γ or GM-CSF mean ± SD of duplicate cultures (representative of

950 three independent experiments). Total cytokine release is shown in panels A, B, F; fold increase

951 over background is shown in panels C, D, E. * P<0.05 (Unpaired Student’s t-test).

952

953 Figure 6. MR1T cell clones exhibit divergent transcriptional responses to antigen stimulation.

954 Betweeness centrality analysis illustrating the key transcription factors that were differentially

955 expressed in resting (No Ag) and antigen-activated (Ag Act) MR1T cell clones (A) DGB129 and

956 (B) DGB70. Color code represents betweeness centrality score. The size of the nodes (or hubs)

957 indicates the relative importance of individual transcription factors within the whole gene

958 network. (C) Heat-map comparing key transcription factors that were differentially expressed in

959 resting (Rest.) and antigen-activated (Act.) DGB129 and DGB70 MR1T cell clones (analysis

960 performed by PageRank algorithm).

961

962 Figure 6–figure supplement 1. FACS analysis of resting and activated DGB129 and DGB70

963 MR1T cells used for transcriptome studies. Dot-plots display the expression of CD25 and CD137

964 activation markers on T cells cultured alone (Resting) or stimulated with A375-MR1 cells (Ag

965 stimulated). Purity of CD25+CD137+ cells sorted from the Ag stimulated groups (Sorted

966 Activated) is shown. Plots are gated on CD3+ DAPI- cells. Resting and Sorted Activated

967 DGB129 and DGB70 MR1T cells were utilized for next-generation sequencing experiments.

968

969 Figure 7. Functional heterogeneity of MR1T cell clones. (A) Heat-map of cytokine expression

970 by 7 different MR1T cell clones when stimulated by MR1-expressing A375 cells. Also shown

43 971 are the cytokine profiles of control MAIT cell clones MRC25 (MAIT-1) and SMC3 (MAIT-2)

972 following activation by A375-MR1 cells pulsed with E. coli lysate. Cytokines were assessed in

973 the supernatants of duplicate cultures. The mean value for each cytokine was used to generate the

974 heat-map. Cluster analysis was performed using Pearson correlation. Graphs displaying the

975 amounts of individual cytokines released by the different clones are shown in figure supplement

976 1. (B) Flow cytometry analysis of CXCR3, CCR4 and CCR6 surface expression by resting

977 MR1T cell clones and 2 MAIT control clones (MRC25, MAIT-1 and SMC3, MAIT-2). Graphs

978 show the relative fluorescence intensity calculated by dividing the median fluorescence intensity

979 (MFI) of specific mAb staining by the MFI of the corresponding isotype control. Data are

980 representative of two independent experiments.

981

982 Figure 7–figure supplement 1. Cytokines released by antigen-stimulated MR1T and MAIT cell

983 clones. (A) IFN-γ released by 7 MR1T cell clones stimulated with A375-MR1 cells and 2 MAIT

984 cell clones (MRC25 and SMC3) stimulated with A375-MR1 cells pulsed with E. coli lysate.

985 ELISA results are expressed as mean ± SD of IFN-γ release measured in duplicate cultures. (B)

986 Analysis of 16 additional cytokines by multiplex cytokine assay performed on the same

987 supernatants for which IFN-γ is shown in A. Results are representative of two independent

988 experiments.

989

990 Figure 8. MR1T cells recognize cells constitutively expressing low surface MR1 and show

991 diverse T helper-like functions. (A) Recognition of four human cells lines expressing constitutive

992 surface levels of MR1 and Mo-DCs by the representative SMC3 MAIT cell clone in the absence

993 (no Ag) or presence of E. coli lysate (E. coli) with or without anti-MR1 blocking mAbs (α-

994 MR1). (B) Recognition of the same cell types as in A by thirteen MR1T cell clones with or

44 995 without anti-MR1 mAbs (α-MR1). Graphs show IFN-γ release (mean ±SD of duplicate cultures).

996 (C) Flow cytometry analysis of co-stimulatory molecules CD83 and CD86 on Mo-DCs after co-

997 culture with DGB129 MR1T cells with or without anti-MR1 mAbs (α-MR1). A control group

998 consisting of Mo-DCs stimulated with LPS (10 ng/ml) in the absence of T cells is also shown.

999 Numbers indicate percentages of cells in each quadrant. (D, E) Stimulation of JMA MR1T cells

1000 by LS 174T intestinal epithelial cells with or without anti-MR1 mAbs (α-MR1). Columns show

1001 (D) IL-8 (ng/ml) and (E) IL-13 (optical density, O.D.) release. (F) Q-PCR analysis of MUC2

1002 gene expression in LS 174T cells cultured alone or with JMA MR1T cells in the presence or

1003 absence of anti-MR1 mAbs (α-MR1). As control, LS 174T cells were stimulated with

1004 recombinant TNF-α (rTNF-α, 10 ng/ml) in the absence of MR1T cells. All data are expressed as

1005 mean ± SD of triplicate cultures. Results are representative of at least three independent

1006 experiments. * P<0.05 (Unpaired Student’s t-test).

1007

1008 Figure 8–figure supplement 1. MR1 surface expression by monocyte-derived dendritic cells

1009 and LS 174T cells. (A) Flow cytometry detection of MR1 surface protein expression on

1010 monocyte-derived dendritic cells (Mo-DCs) generated from blood of three healthy donors. Grey

1011 histograms represent staining with isotype-matched control mAbs. (B) Recognition of Mo-DCs

1012 from the three donors in A by DGB129 MR1T cell clones in the absence or presence of anti-

1013 MR1 (α-MR1) mAbs. IFN-γ release in the supernatants is shown and expressed as mean ± SD.

1014 Results are representative of three independent experiments. * P<0.05 (Unpaired Student’s t-

1015 test). (C) MR1 surface protein expression by LS 174T intestinal epithelial cells. Grey histograms

1016 represent staining with isotype-matched control mAbs.

1017

45 DGB129 MAIT A B C ∗ 100 CCRF-SB 20 20 ∗ no Ag

) ∗ ∗ 80 15 15 no Ag α-MR1 60 10 10 E. coli 40 ∗ ∗ - γ (ng/m l 3.0 - γ (ng/m l ) 3.0 % of Max E. coli α 20 ∗ ∗ ∗ -MR1 IF N 0 1.5 IF N 1.5 100 101 102 103 104 105 MR1 0.0 0.0 B -1 B -1 S P S P - H - H 100 THP-1 T T 375-MR1 375-MR1 80 CCRF A CCRF A 60 40 D E F SKW3-DGB129 G JRT-3-MAIT

% of Max DGB129 MAIT 15,000 40,000 20 ∗ 20 ∗ 0.50 ∗ ∗ I I 0 ) ) ∗ F 100 101 102 103 104 105 15 F 30,000 M M 10,000 MR1 0.25 10 20,000 100 - γ (ng/m l - γ (ng/m l CD69 A375-MR1 CD69 5,000 80 IF N IF N 5 10,000 60 0.00 0 0 40 0 % of Max 20 THP1 + + + + + + A375 WT − + + − − − − A375 WT − + + − − − − 0 RL-6,7- diMe − + + − + + A375-MR1 − − − + + + + A375-MR1 − − − + + + + 100 101 102 103 104 105 α-MR1 − − + − − + E. coli − − + − − + + E. coli − − + − − + + MR1 α-MR1 − − − − + − + α-MR1 − − − − + − +

Figure 1 A Donor A 104 39 103 Sorting 4 10 102

29.1 CD69 103 101 102 CD3 0 104 101 4 103 0 1 2 3 4 0 10 10 10 10 102 CD69 CTV 101

0 0 101 102 103 104 CD137

Donor A Donor A B C ∗ ∗ 70 101

50 100 A375 target ratio) - γ (ng/m l ) - γ release - 30 10-1 IF N IF N

/MR1 10 + 2 10-2 A375 WT + − −

(MR1 1 126 195 A375-MR1 − + + Number of T cell clones α-MR1 − − +

D Donor B E Donor B 2 30 10 ∗ ∗ 101 20 100 A375 target ratio) - γ (ng/m l ) - γ release -

10 -1 IF N

IF N 10 /MR1 + 2 10-2 1 37 57 A375 WT + − − (MR1 Number of T cell clones A375-MR1 − + + α-MR1 − − + Figure 2 Donor C A A375 WT A375-MR1 B 105 0.08

4 cell s +

10 cells ) 7 9 + 3 103 0.04 CD 6 0.024 0.065 CD1 3 + 102

0 (% of C D

CD6 9 0.00 2 3 4 5 010 10 10 10 A375 WT A375-MR1 CD137

Donor C Donor C C 150 D 102 ∗ ∗ ) 100 101

0 A375 target ratio)

- γ release 10 - 50 - γ (ng/m l

IF N -1 IF N 10 /MR1 + 2 10-2

(MR1 1 31 96 A375 WT + − − Number of T cell clones A375-MR1 − + + α-MR1 − − + Figure 3 JMA CH9A3 5.0 JMA A375-MR1 A375-MR1+ Ag ∗ ∗ ∗ ∗ A ns ∗ B C A375-MR1 K43A + Ag 10 ∗ 24 ∗ A375-MR1 K43A 2.5 A375-MR1 no Ag A375-MR1 K43A no Ag 12 ∗ ∗ ∗ 5 0.0 ) 12 MAIT 10 Vγ9Vδ2 0 2 5 15 25 ∗ 0 0 CH9A3 DGA4 6 6 12 5 DGB70 DGB129 - γ (ng/m l ∗ ∗ ∗ ∗ ns N ∗ ∗ 8 F 0 20 ∗ 24 ∗ 3 I 0 ∗ ∗ ∗ 0 2 5 15 25 0 2 5 15 25 4 ∗ ∗ ∗ 6-FP (µg/ml) 10 12 0 0 0 2 5 15 25 0 2 5 15 25

0 0 - γ (ng/m l ) DGB70 12 DGB129 DGA4 TC5A87 2 - γ (ng/m l ) ∗ ∗ ∗ IF N ∗ ∗ 8

IF N ns 10 ∗ 20 ∗ 1 4 ∗ ∗ ∗ ∗ ∗ ∗ 5 10 0 0 0 2 5 15 25 0 2 5 1525

0 0 10 DGA28 5.0 TC5A87 DGA28 MAIT ∗ ∗ ns ∗ 5 2.5 6 ∗ 8 ∗ ∗ ∗ ∗ ∗ ∗ ∗ 0 0.0 3 4 0 2 5 15 25 0 2 5 15 25 6-FP (µg/ml) − − 0 0 A375 WT ++− −− − ++− −− − A375-MR1 −−+ ++ + −−+ ++ + E. coli −−− +− + −−− +− + Figure 4 α-MR1 −−− −+ + −−− −+ + A C Figure 4-FigureSupplement 1 Ac-6-FP γ IFN-γ (ng/ml)

IFN- (ng/ml) 12 Zol 0 6 12 0 0 6 Ac-6-FP ∗ - - DGB70 2 5 ∗ + - ∗ (µg/ml) 15 + + ∗ 25 ∗ D IFN-γ (ng/ml) 0 4 8

IFN-γ (ng/ml) 0 12 Ac-6-FP

DGB70 0 6 ∗ 2 DGA4 5 DGA4 ∗ (µg/ml) 15 DGA28 25 ∗

IFN-γ (ng/ml)12 0 6 0 Ac-6-FP No Ac-6-FP ∗ Ac-6-FP 2 DGA28 5 ∗ (µg/ml) 15 25 ∗ E B

IFN-γ (ng/ml) IFN-γ (ng/ml)10 10 0 5 0 5 0 Ac-6-FP MAIT ∗ 2 -1 MAIT 5 ∗

MAIT (µg/ml) -1 15 -2 25 ∗

IFN-γ (ng/ml) 10 Ac-6-FP No 0 5 0 Ac-6-FP Ac-6-FP ∗ MAIT 2 5 ∗ (µg/ml) -2 15 25 ∗ E. coli no Ag A A375-MR1 B THP1-MR1 20 20 RPMI PBS RPMI α-MR1 PBS α-MR1

- γ (ng/m l ) 10 - γ (ng/m l ) 10 IF N IF N

0 0 0 10 100 0 10 100 Cell number (x10-3) Cell number (x10-3)

C DGB129 DGB129 4 D 5 ∗ ∗

F 4 3

hange ) 3 M-C S 2 G

old c 2 f ( 1 1

E1 E2 N1 N2 N3 N4 E1 E2 N1 N2 N3 N4 Source: in vitro cultured THP1 in vivo grown EMT6

E DGB70 F MR1-plate bound

) DGB129 13 ∗ 3 ∗ ∗

F 10 DGB70

(ng/m l 2 hange ) 7 F M-C S G

old c 1 f 4 M-C S ( G 1 0 E1 E2 N1 N2 N3 N4 Ag N3 N4 Source: in vitro cultured THP1 no

Figure 5 GSN CD101

PFKFB4 ZFHX3 BLVRA HMGB3P32 MGLL Low High MPP7 TPI1 FLVCR2 DGB129 DGB70 DBN1 PMCH GBP6 ADARB2 CENPA GLDC SUN2 EBI3 CSF2 CECR1 TPM2 TNFRSF18 TNFRSF9 ZBP1 SLC30A3 BATF PPFIA4 SIGIRR SCARB1 RNF44 RNASE6 B A LY9 PLXND1 CAMK2G GPI GALNT8 IFIT2 NINJ1 CLU BATF PSAT1 RIMKLA DISC1 APOBEC3H S100A4 P4HA1 NEIL3 FAM65B TCEANC AHNAK HMGCS1 SNX9 SLCO4A1 ABLIM1 PLAUR ICAM1 CAMK1 Betweenness centrality score GPR183 SERPINB8 SLC22A18 PHLDB1 ABTB2 CCR7 CLIC4 SH2D2A CSF2 ZBTB32 AHI1 AQP3 SLC2A14 ZC3H12C STRIP2 CD68 CD40LG NR4A1 ERBB3 IL21R EVI5 MAML2 NME1 BNIP3 PSMD14 PKM IRF4 TRIB1 SHF LINC00528 GPR153 KIF14 GPR56 FOSL2FRAT1 MROH6 CREM MFSD2A WARS CYB561 MTHFD2 HSP90AB1 NAMPT TRABD2A CCNB2 CDC6 NCF4 HSPA6 TNFRSF10A ALDOC FOSL2 DDIT4 SYP ASPM CCR1 ZDHHC9 TROAP DUSP3 CHI3L2 VLDLR IFI6 DHRS12 ABTB1 GAS6 IL4R YPEL1 UBE2F PLXDC1 MYOF HILPDA DLGAP5 ENO1 IRF4 CDCA8 P4HA2 GALE DAPK2 STARD4 N4BP3 HSPE1 GPT EAF2 KCNQ1 NR1D2 CCDC10H9ISTB 1H2AC HJURP FOS TFRC NR4A3 MIIP CISH PER3 JMJD7-PLA2G4B PMAIP1 NEURL1B CDCP1 IL7R PRKCE TCTN3 IL15 EGR3 FGD3 ARG2 SOCS1 CDKN2D ABCG1 BOLA3 AIM2 CYB561 ANLN E2F8 TMEM116 CIT EGR2 BCL2L1 PRR11 RNF214 BCAT1 SELPLG AGRN ZNF395 ADAM23 GAPDH IMPDH2 ZNF33B ACTN1 ZMYND10 ABCA7 LDHA METTL1 MTHFD1L STMN1 F2R RCBTB2 ZDHHC9 GPT2 SMS ANKRD33B TPI1P2 PLXNA4 SLC16A1 RSAD2 MX1 WARS SNHG15 SBF2 PBK VEGFA ANKRD24 SDC4 ZNF282 AKAP7 C3 ELL3 SKA3 NUAK2 FOS SCD HSPD1 MTHFD2 DUSP6 SPRY1 VTI1B TXK HK2 HK2 NPM3 TTC21A TRAM2 TCF7 TSKU BCL2L11 LDLR GBE1 ATP8A1 LINC00271 TNFRSF4 CMSS1 MGAT3 IL5 MYO1B WHAMMP3 PCSK4 BNIP3L PAICS SORL1 PTGIS NKD1 PFKFB3 GPAR1Q4R68 ICOSLG PIF1 IRS1 CHAC2 CORO1C BBC3 TROAP HIP1 KIF14 ZNF629 GPR160 SIK1 PPA1 PPAN MCM2 MORN2 PTGS2 NR1D2 ICAM5 AKTIP SCML4 KIF20A NME1-NME2 TCF7 HIVEP1 FBXO16 TRAF4 P2RX5 SOCS3 GATA6 ZNF449 MCM6 ENTPD1 HSPA6 PHKA1 IZUMO4 SMS OSM NCR3LG1 FOSB WHAMMP3 NFIL3 FOXD1 KIF18B MVB12B RXRA NMT2 MB21D2 GLIS2 VEGFA PGAM1 IFI30 MCM10 KIF18B CD40LG MICAL2 SETBP1 SLC41A2 RASSF1 SETBP1 PSD PSAT1 JAM3 TTYH2 CISH TRIP13 FSCN1 NUAK2 VTI1B KLHDC1 PER3 DUSP5 CEBPD RXRA ADAM23 NEURL1B AGRN RHOU TPMT CTTN GNAO1 LPAR6 RRM2 IFNLR1 COLGALT2 ARHGEF40 RTKN2 IQGAP3 DUSP2 ZMYND10 PIM3 ABCA7 FAM43A ASPM SQLE EFHC1 IL13 GPT2 CSF1 FADS2 CORO1C CAPG TNFRSF8 SCML4 RXRA PPFIA4 IL5 SLFN14 PLCD1 TRAM2 ANKRD32 NR4A2 FAM57A CDC20 SCD SREBF1 PLAUR ADAMTS17 RAB27B SYTL3 DOK4 FAM111B SOCS1 TLE2 MKI67 HAVCR1 NLRP1 MYC SDC4 NBEAL2 ATXN7L1 LINC00086 CDCP1 FSCN1 PLEC TRAF4 ZBTB32 NKD1 PFKFB4 TSPAN32 DTL MYC SLC39A10 RGS16 ABLIMA1RHGAP31 IL18RAP PTPN3 SPINT2 CDCA7L AKR1C3 ZNF449 FABP5 STOM NMUR1 VDR SLC2A14 SLC16A3 TSKU PHKA1 KCNH4 RAVER2 E2F7 FOS TPX2 SPRY1 KANK1 LMNA LEF1 EGR1 NCR3LG1 ZNF215 SORL1 ANKRD30BL FOXP3 ERMP1 DUSP5 BCL2L14 TPI1P2 PTGDR2 SLCO4A1 FZD3 MTHFD1L CCR9 LAG3 RIMKLA EGR3 MB21D2 LIMA1 EGR1 DUSP4 CDCA3 AIM1L ADAMTS10 ADM ACACB SYNE2 GPC1 SLC41A2 TNFRSF18 CCL5 DUSP6 DUSP4 LIMD2 CYP51A1 FABP5P7 SPOCKZ1WINT NSREBF1EK2 SGK223 ANLN SLC7A5 TLE1 IER3 EGR2 CTTN LINC00271 PGAM1P8 RASGRP2 FOXP3PLA2G4B SLC9A7 EVI5 LINC00086 TFRC FAM47E-STBD1 MYBL1 AK4 ZBTB16 DUSP1 MYO1B TNIP3 TTC21A SHCBP1 ST7 RAB37 LAIR2 FOS LDHA GAS6 HBEGF FADS1 PLB1 PRKCE KIF20A FRY IL10 VWCE RFPL2 BATF3 FOXD1 BATF3 PPME1 CIT CUBN CCDC109B CLCF1 DEPDC7 IER3 CTSF ZNF215 TRPS1 IL13 CTLA4 FOSL2 PLXDC1 FOXP3 HAVCR2 ADC BCL2 BCL2L1 TTK LINGO3 CCL3L3 CSGALNACT1 ENO1 IRGM IMMP2L UST ST8SIA6 TCTN3 BBS5 CD83 GINS2 TTC39B UST CCL3 ANKRD20A5P TMOD2 DACT3 TNFRSF9 RGS16 RASGEF1B S100A4 AIM2 IL24 NAMPALDT OC FOSL2 MTFP1 IL24 PIP5K1B LAIR1 PLA2G4A NDFIP2 COPG2 GBE1 DDX43 RASGRP2 CD83 P4HA2 RAMP1 SLAMF8 SYTL3 TPRG1 INADL TRIB1 GTSE1 ZCCHC18 FAIM3 NTRK2 FOXP3 GNG4 PITPNC1 TMEM204 NFKBIZ ZBED2 NR4A3 TRANK1 NFIL3 KIAA08D2T5HD1 LINC00528 NINJ1 TPI1 KCNN4 TNFRSF8 PTGIS IRF5 PARPBP MEGF6 C1QTNF6 IL7R SLC43A3 PKM SCARB1 DNAH11 SLC9A9 ENPEP BCL2 CREM JUNB RNF144A DEPDC7 KIAA0825 CPNE7 GLB1L2 CHI3L2 ESCO2 GRB10 FAM162A P2RX5 CD274 ICAM2 GPR56 SHF IQGAP3 LLGL2 ZMAT1 HLF CTLA4 MAP7 NCAM1 CCDC141 LRP5 TNFRSF10A AK4 GATA6 FGFBP2 SLC2A3 CENPF ARG2 CALHM2 IL10 LTA KCNA2 DFNB31 TMOD2 IRF8 IKZF2 MCTP1 DTHD1 MTFP1 GFOD1 HOMER2 HSPD1P1HIF1A-AS2 PRRC2B SLC29A1 KCNA2 GSTM4 PPME1 NDFIP2 TNIP3 IL4R GRB10 STK38 MYB ANK3 PLCD1 RMI2 CAMKK1 SUSD3 LAIR2 IFNG AQP3 ITGADHAF6E1 MPZL2 SIX4 AUTS2 NAB2 ZBED2 RASGRF2 DFNB31 TGM1 SYNE1 SLFN14 ST8SIA6 GRAMD3 RSPH3JUNB VCATEMS1C CRYBG3 PHLDA1 MYB NTRK2 PUS7 SLC39A14 LIMA1 VDR DENND5A MAP2K6 IPMK GAB3 PSRC1 AURKB KIF15 PARPBP ICAM1 CDCA2 CHN2 IRF4 HMMR PGK1P4HA1 YPEL3 LTB TPM2 IL12RB2 EBI3 RASGEF1A SLC17A9 ADHFE1 ANO7 H2AFY2 TOX IRF4LAIR1 PITPNM2 CLU MICAL2 BCAT1 IRGM SYP MORN3 ADRB2 BEND5 SNX9 SORD MYO1F LTB4R IL1RAP CCL1 SIK1 CCL3L3 PMCH NT5DC2 DDX4 IFIT3 BATF SPTB KLRD1 DACT3 PANX1 AGBL3 MFSD2A EPB41L4B SLC39A14 SIGIRR ENPEP KIF2C CCR7 XYLT1 RNASE6 ZNF33B IFI27 POLQ CRTAM CD38 FAM47E CECR1 PHLDA1 ANK3 ZBTB10

ATP8A1 OASL SLC35F3 MYOF OAS1 RPS6KA2 MPP7 CFH BATF MAP2K6 ZC3H12C EPSTI1 MX2 HMGB3P32 GBP6 STX11 BEND5 MAL GNG4 CD101 TC2N ADARB2 IFNG LY9 CD86 TC2N PDE4D RPS6KA2 IL2RA IL2RA P4HA3 LTA VLDLR RASGRP3 MAL

SH3GL1P3 PLB1 TIMD4 SLC2A3 TIMD4 CRTAM

FKBP6 C DGB129 DGB70 Color Key Rest. Act. Rest. Act. EGR1 -2 -1 0 1 2 JUNB Raw Z-score SREBF1 TBX21 FOXP3 FOS RXRA FOSL2 IRF4 BATF TRIB1 MYC Figure 6 HSP90AB1 CREM Resting Ag stimulated Sorted Activated

104

1,98 53,1 90,1 103 DGB129 102

101 0

0 101 102 103 104

104

3 0,45 30,4 90,9 10

DGB70 102

101 0 CD137

0 101 102 103 104

CD25

Figure 6-Figure Supplement 1 Color key 3 A and hystogram B 10 CXCR3 12 6 102 0 -2 -1 0 1 2 1 Raw Z-score 10

100 IL-3 30 CCR4 IL-4 IL-5 IL-6 Th2 20 IL-10 IL-13 10 IL-2 α TNF- Th1/Th17 1 TNF-β 3 IL-17A Relative fluorescence intensity 10 CCR6 G-CSF Th17 GM-CSF 102 MIP1β sCD40L Others 101 PDGF-AA VEGF 100 JMA DGA4 JMA DGA28 CH9A3DGB70 MAIT-1 TC5A87 DGA4 MAIT-2 DGB129 MAIT-1 DGA28 CH9A3DGB70 MAIT-2TC5A87DGB129

Figure 7 A B 15 2.0 IL-3 15 IL-13 10 IL-2 25 MIP-1β

ml ) 10 1.5 8 20 10

(ng / 6 15

5

γ 1.0 - 3 4 10 5 2 0.5 IF N 1 2 5 0 0.0 0 0 0 T cell clones 15 IL-4 2.5 IL-10 4 IL-17A 0.4 sCD40L 2.0 3 0.3 10 1.5 DGB70 2 0.2 1.0 5 DGB129 1 0.1

ml ) 0.5 JMA 0 0.0 0 0.0 (ng / 10 15 15 1.5 CH9A3 IL-5 TNF-α G-CSF PDGF-AA 8 okine 10 10 1.0 DGA28 6 Cy t DGA4 4 5 5 0.5 TC5A87 2 0 0 0 0.0 MAIT-1 15 IL-6 2.5 TNF-β 60 GM-CSF 0.8 VEGF MAIT-2 2.0 0.6 10 40 1.5 0.4 1.0 5 20 0.5 0.2 0 0.0 0 0.0

T cell clones Figure 7-Figure Supplement 1 MAIT LS 174T Huh7 HCT116 THP1 Mo-DC A B ∗ 4 101 ∗ ∗ ∗ ∗ ∗ no Ag 3 ∗ E. coli 0 ∗ 10 E. coli α-MR1 ∗

2 - γ (ng/m l ) - γ (ng/m l ) ∗

IF N -1

IF N 10 1

0 0 LS 174T Huh7 HCT116 THP1 Mo-DC α-MR1 − + − + − + − + − +

Untreated LPS n 4 LS 174T

C 6 e) 5 D LS 174T F si o ∗ ∗

10 31.6 6.40 8.33 90.5 l ) ∗ 3 104 4 chan g expre s

(ng/m 2 3 10 old L- 8

2 F I

( 1 2

10 MUC 2 0 0 101 61.6 0.40 0.89 0.30 101 102 103 104 105 JMA − + + JMA − + + − α-MR1 − − + α-MR1 − − + − DGB129 α DGB129 -MR1 rTNF-α − − − + 5 24.9 69.2 27.8 9.18 1.5 LS 174T 10 E ) . ∗ 4

10 O. D

( 1.0 103 L-13

I 0.5 102 5.39 0.60 62.1 0.95

CD86 0.0 101 101 102 103 104 105 JMA − + + Figure 8 CD83 α-MR1 − − + A Donor 1 Donor 2 Donor 3 100 80 60 40

% of Max 20 0 0 102 103 104 105 MR1

B Donor 1 Donor 2 Donor 3 Mo-DCs 0.4 ∗ 1.0 ∗ 3.0 ∗ Mo-DCs α-MR1 0.2 ∗ 0.5 ∗ 1.5 - γ (ng/m l ) - γ (ng/m l ) - γ (ng/m l ) ∗ ∗ IF N IF N IF N 0.0 0.0 0.0 0 5 10 0 5 10 0 5 10 Cell number (x10-5) C 100 LS 174T 80 60 40

% of Max 20 0 100 101 102 103 104 105 MR1 Figure 8-Figure Supplement 1