Antimicrobial activity of mucosal-associated invariant T cells
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1 correction notice Nat. Immunol.; doi:1.138/ni.189; corrected online 4 July 21 Antimicroial activity of mucosal-associated invariant T cells Lionel Le Bourhis, Martin, Isaelle Péguillet, Amélie Guihot, Nathalie Froux, Maxime Coré, Eva Lévy, Mathilde Dusseaux, Vanina Meyssonnier, Virginie Premel, Charlotte Ngo, Béatrice Riteau, Livine Duan, Delphine Roert, Martin Rottman, Claire Soudais & Olivier Lantz In the version of this supplementary file originally posted online, some lines and symols were missing. The errors have een corrected in this file as of 4 July 21.
2 Sorted V α Gated on CD3 + Sorted V α 7.2 Gated on CD3 + CD161 CD4 CD161 CD4 V α 7.2 V α 7.2 CD4 CD4 CD69 CD69 Sup Figure 1: Mainstream CD4 and T cells are not early responders to acterial infection. (a) Activation of human sorted V α cells (as in Figure 1) were co-cultured with infected (lack) or non-infected (grey) autologous monocytes, gating on CD161 - mainstream CD4 or T cells. () Human sorted V α cells were analyzed as in (a).
3 1 *** *** * 1 ns ns ns MAIT / µl γδ / µl c Pneumopathy group Tuerculosis (TB) group Cancer group Patient (#) Sex (male #/ female #) 15 / 7 8 / 9 / 24 Age (mean ±SD) 62 ± ± ± 14.4 Diagnosis of acteriological pneumopathy ased upon : - clinical findings alone (8) - clinical and X-ray findings (11) - clinical findings and acteriology (3) Healthy donor group : anonymous lood donors matching EFS guidelines Site of tuerculosis : - pulmonary (8) - disseminated/miliary (4) - lymph nodes TB (2) - ones and joints (3) Cancer type : - reast cancer (17) - uterine cervix cancer (7) Sup Figure 2: γδ T cell percentage in human lood is stale during acterial infection. (a) Gated on CD3 + γδ, asolute MAIT cell numers are indicated for each patient. () Gated on CD3 +, γδ T cell asolute numer was calculated for each patients and plotted according to their pathologies. (c) Human sample information.
4 iv α 19 Tg iv α 19-V β 6 Tg V β 6 Tg Tap -/- Ii -/- Gated on TCRβ +, V β 6 + or V β 8 +, + or DN Gated on TCRβ +, V β 6 +, + or DN Gated on TCRβ + T cells T cells T cells c DN DC MR1 + DC MR1 + Sup Figure 3: Study of the acterial reactive MAIT repertoire using various transgenic T cells as responders. MAIT cells were stimulated as in Figure 2. MAIT cells were prepared from iv α 19 (a) iv α 19-V β 6 () or V β 6 Tap -/- Ii -/- (c) mice. For each transgenic mouse type, responder T cells were isolated from the indicated mouse (, MR1 -/- or Rag -/- mice). Each T cell type was stimulated on or MR1 -/- BMDC. For iv α 19-V β 6 Tg, T cells were gated on TCRβ + and + or DN cells. For iv α 19 Tg, T cells were gated on TCRβ +, + or DN, V β 6 + or V β 8 + events. For V β 6 Tg, T cells were gated on TCRβ + and sudivided y expression or asence of CD4 and staining as indicated. DC DN
5 % CD69 + cells EMCV E. coli Para3 Sendai V HSV NDV ctr α-mr Sup Figure 4: Virus infected APCs do not activate MAIT cells iv α 19-V β 6 MACS sorted Tg T cells were co-cultured with BMDC previously infected with viruses or E. coli as control. Activation of the MAIT cells was assessed y CD69 up-regulation.
6 9 E. coli 1 E. coli + (%) CpG Pam CpG Pam (%) polyic 1 LPS 1 1 polyic 1 LPS (%) 6 3 µg/ml (log2) 1 1 µg/ml (log2) 5 25 µg/ml (log2) 1 µg/ml (log2) 1 Sup Figure 5: Innate immune ligands are not sufficient for MAIT cell activation. BMDC from ( ) or ( ) animals were stimulated with TLR specific ligands (Pam3Cys, Poly- IC, LPS, CpG) or infected with E. coli and co-cultured with iv α 19-V β 6 Tg T cells. (a) MAIT cells or () DCs activation is assessed y up-regulation.
7 Myd88 -/- Trif -/- Myd88 -/- Trif -/- Nod1 -/- Nod2 -/- Nod1 -/- Nod2 -/- Nlrp3 -/- Asc -/- Ips1 -/- CD Myd88 -/- Trif -/- Myd88 -/- Trif -/ Nod1 -/- Nod2 -/- Nod1 -/- Nod2 -/ Nlrp3 -/- Asc -/- Ips1 -/- CD4 MFI (x1 3 ) Myd88 -/ Nod1 -/- Nod2 -/- Nod1 -/- Nod2 -/- Ips1 -/ Mr1+ Asc -/- Nlrp3 -/- Sup Figure 6: Innate immune receptors play a marginal role in MAIT cell activation. iv α 19-V β 6 MACS sorted Tg T cells were co-cultured with the BMDC from or indicated knockout animals that were infected with E. coli at the indicated. (a) MAIT cells (% CD69) or () DCs (CD4 MFI) activation is plotted.
8 iv α 19-V β 6 Tg Human MAIT a c CD69 + CD25 + (%) 5 25 Live cells * α-mr1 WT3 * WT3m α-mr1 6 3 Fixed cells * CD69 MFI (x1 3 ) Live cells HeLa Fixed cells HeLa-hMR1 Sup Figure 7: activation of human MAIT cells and iv α 19-V β 6 Tg T cells y epithelial and firolastic cell lines expressing MR1 co-cultured with E. coli. (a) Mouse firolastic cell line WT3 and the staly transduced over-expressing mouse MR1, WT3m, were co-cultured with E. coli at the indicated. These cells were left untreated (live), or () fixed prior to infection (fixed) efore adding iv α 19-V β 6 Tg cells alone or in the presence of locking anti- MR1 (α-mr1). Activation of MAIT cells was assessed after overnight culture y up-regulation of CD69 and CD25. (c) Similar experiment as in a. using the human cell line HeLa and the staly transduced HeLa-hMR1 over-expressing the human MR1 molecule. Cells were infected and sorted human MAIT cells seeded on this cell layer.
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