Lung-residing myeloid-derived suppressors display dual functionality in murine. pulmonary tuberculosis

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1 Lung-residing myeloid-derived suppressors display dual functionality in murine pulmonary tuberculosis Julia K. Knaul, Sabine Jörg, Dagmar Oberbeck-Mueller, Ellen Heinemann, Lisa Scheuermann, Volker Brinkmann, Hans-Joachim Mollenkopf, Vladimir Yeremeev, Stefan H.E. Kaufmann, nca Dorhoi This Online Data Supplement includes: Supplementary Methods Supplementary Figure legends E1 E8

2 Supplementary Methods Reagents used in flow cytometry studies For phenotypic characterizations the following antibodies were used: anti-cd16/cd3 (4G, TCC) anti-cd19 (1D3, TCC), anti-cd3 (145-C11, TCC), anti-nk1.1 (PK136, TCC), anti-nkp46 (91.4, ebioscience, Frankfurt, Germany), anti-ly6g (18, BD), anti-ly6c (L-1, BD), anti-cd14 (mil4r-m1, BD), anti-cd11b (M1/7, BD), anti-cd11c (N418, TCC or BD), anti-gr1 (RB6-8C5, BD), CD86 (GL-1, BioLegend), anti-cd31 (MEC 13.3, BD), anti-mhcii (Tib1, TCC), anti-mhci (B.49, TCC) anti-cd49d (R1-, ebioscience), anti-cd115 (FS98, ebioscience) and anti-f4/8 (BM8, ebioscience), anti-cd3 (UCHT1, BD), anti-cd4 (191, TCC), anti- CD8 (53-6.7, BD). For intracellular cytokine staining the following antibodies were used: anti-tnfα(xt-,tcc) anti-ifnγ (XMG1., BD) and anti-il17(n49-653, BD). To guarantee consistency between experiments, gates were set using fluorescence minus one controls on whole lung (i.e. not fractionated) before being applied to fractionated samples. t least cells were acquired on a FCS-CantoII or LSRII (BD) and analyzed by FlowJo software (Tree Star Inc., shland, OR, US). Cytological analysis Cytospins of BM-MDSCs were generated using a CytoSpin III centrifuge (Thermo Scientific Shandon, Hudson, NH, US). Samples were stained by modified Giemsa (Differential Quick Stain Kit, Polyscience Inc., Warrington, P, US). t least cells from five microscopic fields were quantified to assess cell morphology. E

3 nalysis of co-culture supernatants Chemokine and cytokine concentrations were analyzed using immunoassay (Bio-Rad, Munich, Germany), commercial TNFα or IL-1 ELIS (R&D Systems, Minneapolis, MN, US) or in-house IL-6 ELIS. Cell death was assessed by detecting lactate dehydrogenase levels in co-culture supernatants (Roche, Basel, Switzerland). E3

4 Supplementary Figures E1-E4 Figure E1. Gene expression and cytokine abundance in lung homogenates/sera from TB-resistant and -susceptible inbred mouse strains Expression of MDSC-associated genes in lungs of infected C57BL/6 and 19S mice at day 7 p.i. () and day 14 p.i. (B). Fold expression + indicates upregulation in 19S mice; expression denotes elevated expression in C57BL/6 mice. Fold change calculated as: fold change in C57BL/6 over fold change in 19S lungs. (C) Cytokine levels in lung and serum at day 5 p.i. in C57BL/6 and 19S mice. (D) Gene expression of selected molecules allowing distinction between MDSCs and tumorassociated neutrophils in Mtb-infected C57BL/6 and 19S animals (MDSC profile: fold expression < to ; tumor-associated neutrophil profile: fold change < or >+). ( D) Values from three independent experiments, each with five specimens per mouse strain, median±iqr, Mann Whitney test. **** P.1 Figure E. Gating strategy applied for flow cytometry analysis Single cell suspensions from whole lung or MCS-purified cells were analyzed by 8- color flow cytometry. Leukocytes were selected before all dump + B cells (CD19 + ), T cells (CD3 + ), NK/NKT cells (NK1.1 + and/or Nkp46 + ) were excluded from the analysis. Subsequently, dendritic cells and alveolar macrophages (CD11c + ) were eliminated from the analysis. CD11b + Ly6G + cells and CD11b + Ly6G cell subsets were selected and the latter was then further gated on Ly6C expression. Finally, both CD11b + Ly6G + and CD11b + Ly6G Ly6C + cells were analyzed for marker expression. E4

5 (B) Representative plots for CD11b and Gr1 expression on unfractionated lung cells after exclusion of Dump + and CD11c + cells. (C) Representative plots for CD14 expression throughout the course of Mtb infection. Due to the heightened TBsusceptibilty of the 19S strain, analysis for days 8 and 6 p.i. could not be performed. ( C) Data from one representative of two independent experiments; n=1. Figure E3. Gr1 + cell analysis of unsorted lung cells during TB Single cell suspensions from lungs were analyzed by flow cytometry without magnetic cell isolation at day 7, 14 or 1 p.i. with Mtb. () Ly6G + Gr1 int cell number and CD14- expression. (B) nalysis of Ly6G Gr1 high cell number and marker CD14 expression. Data from two independent experiments (n=1, mean±sem, two-way NOV with Bonferroni s post-test) **** P.1, ** P.1. Figure E4. Lung Gr1 + cells do not supress TNFα and IL-17 responses ex vivo. () TNFα production of splenocytes in the presence of Ly6G + Gr1 int (left) and Ly6G Gr1 high (right) cells isolated from lungs at day 14 p.i. (top) and day 1 p.i. (bottom). (B) IL17F (top) and IL-17 (bottom) production of splenocytes in the presence or absence of Ly6G + Gr1 int (left) and Ly6G Gr1 high (right) cells isolated from lungs at day 1 p.i.. (, B) Results are from two independent experiments (n=1, mean±sem), one-way NOV with Bonferroni s post-test. (C) TNFα production (left) and cell death (right) of splenocytes in the presence or absence of either MDSCs subset or PMNs isolated from lungs of 19S animals at day 1 p.i. Results from two independent experiments (n=4 7, median±iqr Mann-Whitney t-test). (D) TNFα production of splenocytes in the E5

6 presence or absence of BM-MDSCs or BM-PMNs from C57BL/6 mice infected with Mtb. Results from three independent experiments, which three replicas each (median±iqr Mann-Whitney t-test). ** P.1, ** P.1, * P.5. Figure E5. BCG-induced response in MDSCs is comparable to Mtb-induced effects. () Frequency of BCG + macrophages and Gr1 + cells subsets. (B) Concentration of cytokines produced by BCG-infected BM-MDSCs or BM-macrophages 4 h p.i. (, B) Data from two independent experiments, with three replicates each (median±iqr, Mann-Whitney t-test). ** P.1, * P.5. Figure E6. Cytologic and phenotypic characterization of in vitro-generated MDSCs BM-derived MDSCs were generated as previously described (3). () Giemsa stain of BM-derived MDSCs (n=5 mean±sem). (B D) Phenotypic analysis of BM-derived MDSC subsets. Data from four to seven independent experiments. Figure E7. BM-MDSCs do not supress TNFα responses. () TNFα production of polyclonally stimulated splenocytes in the absence or presence of naïve or infected BM-derived MDSCs. Data from one representative of three to five independent experiments with three replicates each; mean±sem, one-way NOV with Bonferroni s post-test. (B) TNFα production (top) and cell death (bottom) of polyclonally stimulated splenocytes in the absence or presence of infected BM-derived MDSCs from WT, Nos /, rg1 / and Nos / rg1 / animals. Data from three independent E6

7 experiments with duplicates each (mean±sem). **** P.1, *** P.1, ** P.1, ** P.5. Figure E8. blation of MDSCs ameliorates pathology in TB 19S mice received TR or Placebo during Mtb-infection and were analyzed at day 1 p.i. () Frequencies and numbers of lung CD11c + cells (n=6, mean±sem,). (B) Cell numbers of lung CD49d +, CD86 +, MHCII +, CD115 + and CD14 + CD11b + Gr1 + (n=6, mean±sem). (C) Immunostaining for MPO (red) and nuclei (blue) and (D) for NOS (green), rg1 (red) and nuclei (blue) of lung parenchyma (n=1). (C) Scale bar 1 µm (top) and 1 µm (bottom), n=1. (D) Scale bar 1 µm (top/bottom), n=1. (E) IFNγ (left) and TNFα (right) production and (F) cell death of polyclonally stimulated splenocytes in co-cultures with mycobacteria-infected BM-derived MDSCs treated with TR/DMSO for 5 days. (G) Immunostaining for CD3 (red) and Ki67 (green) and nuclei (blue) (n=6, scale bar 1 µm). (-D, G) Data from two independent experiments. (E-G) Data from two independent experiments with duplicates each (mean±sem). **** P.1, *** P.1, ** P.1, * P.5. E7

8 fo ld c h a n g e C 5 7 B L /6 v s. 1 9 S lu n g c o n c e n tra tio n (p g /m l) s e ru m c o n c e n tra tio n (p g /m l) fo ld c h a n g e B 6 v s. 1 9 S fo ld c h a n g e B 6 v s. 1 9 S S u p d a y 7 lu n g m ic ro a rra y B C C 5 7 B L /6 u p S ta t3 S ta t5 b S ta t5 a Irf8 P u S u p C 5 7 B L /6 u p S ta t3 S ta t5 b S ta t5 a C 5 7 B L /6 1 9 S I L -6 P lc y S ta t6 C /e b p C /e b p Il-6 Il-6 ra G -c s f d a y 1 4 lu n g m ic ro a rra y Irf8 P u.1 P lc y S ta t6 C /e b p C /e b p Il-6 Il-6 ra G -c s f G -C S F G m -c s f M -c s f P ro k C /e b p S 1 a 8 S 1 a 9 IL -1 Id o G m -c s f M -c s f P ro k C /e b p S 1 a 8 S 1 a 9 Il-1 Id o Il-4 Il-4 ra P ro k 1 P ro k r1 P ro k r T d o C d 4 4 M m p 9 C 5 7 B L /6 1 9 S I L -6 Il-4 Il-4 ra P ro k 1 P ro k r1 P ro k r T d o C d 4 4 M m p 9 G -C S F S u p d a y 7 d a y 1 4 d a y 5 D C 5 7 B L /6 u p L R ik C c l8 C c l1 rg 1 C c l7 D a b C h 5 h M m p 1 3 S p p 1 C x c l4 E a r C 8 R ik C x c l1 6 Figure E1

9 19S B6 Gr1 19S 19S C57BL/6 Leukocytes Non-dump CD11c- Ly6G+ Ly6C+ nalysis of marker expression B Day 14 Day 1 14% 37% 13% 16% CD11b C FMO control Day Day 7 Day 14 Day 1 Day 8 Day 6,%,7% 7% 13% 47,5% ND ND C57BL/6 Ly6G,4% 1,3% 4,8% 9,5% 14% 6,5% 14% CD14 Figure E

10 L y 6 G - G r1 h ig h c e lls (x 1 5 ) L y 6 G - G r1 h ig h C D c e lls (% ) L y 6 G + G r1 in t c e lls (x 1 6 ) L y 6 G + G r1 in t C D c e lls (% ) L y 6 G + G r1 in t % C D C 5 7 B L /6 1 9 S 1 8 C 5 7 B L /6 1 9 S * * D a y s p o s t in fe c tio n D a y s p o s t in fe c tio n B L y 6 G - G r1 h ig h % C D C 5 7 B L /6 1 9 S 1 C 5 7 B L /6 1 9 S D a y s p o s t in fe c tio n D a y s p o s t in fe c tio n Figure E3

11 T N F (% re le a s e ) L a c ta te d e h y d ro g e n a s e (O D 4 9 ) T N F (% re le a s e ) IL -1 7 (% re le a s e ) IL -1 7 (% re le a s e ) IL -1 7 F (% re le a s e ) IL -1 7 F (% re le a s e ) T N F (% re le a s e ) T N F (% re le a s e ) T N F (% re le a s e ) T N F (% re le a s e ) Ly6G + Gr1 int cells Ly6G - Gr1 high cells C 5 7 B L /6 1 9 S C 5 7 B L /6 1 9 S : 1 : 1 :4 1 :8 :1 1 : 1 : 1 :4 1 :8 :1 L y 6 G + G r1 in t : S p le n o c y te s 1 : 1 : :1 1 : 1 : :1 L y 6 G - G r1 h ig h : S p le n o c y te s C 5 7 B L /6 1 9 S C 5 7 B L /6 1 9 S : 1 : 1 :4 1 :8 :1 1 : 1 : 1 :4 1 :8 :1 L y 6 G + G r1 in t : S p le n o c y te s 1 : 1 : 1 :4 1 :8 :1 1 : 1 : 1 :4 1 :8 :1 L y 6 G - G r1 h ig h :S p le n o c y te s B Ly6G + Gr1 int cells Ly6G - Gr1 high cells 6 C 5 7 B L /6 1 9 S 1 5 C 5 7 B L /6 1 9 S * 4 1 * * 5 1 : 1 : 1 :4 1 :8 :1 1 : 1 : 1 :4 1 :8 :1 L y 6 G + G r1 in t : S p le n o c y te s 1 : 1 : 1 :4 1 :8 :1 1 : 1 : 1 :4 1 :8 :1 L y 6 G - G r1 h ig h : S p le n o c y te s 6 4 C 5 7 B L /6 1 9 S * * C 5 7 B L /6 1 9 S * 6 4 * * C * * 1 : 1 : 1 :4 1 :8 :1 1 : 1 : 1 :4 1 :8 :1 L y 6 G + G r1 in t : S p le n o c y te s M D S C s P M N s S p le n o c y te s L y 6 G + G r1 in t M D S C L y 6 G - G r1 h ig h M D S C L y 6 G + P M N S p le n o c y te s 1 : 1 : 1 :4 1 :8 :1 1 : 1 : 1 :4 1 :8 :1 L y 6 G - G r1 h ig h : S p le n o c y te s D 3 1 L y 6 G + G r1 in t Figure E4 L y 6 G - G r1 h ig h L y 6 G + S p le n o c y te s. 1 : 1 :1 1 : 1 :1 1 : 1 :1 :1 C e ll s u b s e t : S p le n o c y te M D S C s P M N s S plenocytes

12 CD31 CD14 CD49d % o f c o u n te d c e lls CD115 MHCII MHCI F4/8 CD11c Day Day 4 B ll cells C Gr1 high CD11b high Gr1 low CD11b high Gr1 dim CD11b int ll singlets M o n o c y tic P o ly m o rp h o n u c le a r CD11b+ Gr1+ Subsets 1- Gr1 high CD11b high - Gr1 low CD11b high 3- Gr1 int CD11b int D a y D a y 4 D G r 1 h i g h C D 1 1 b h i g h G r 1 lo w C D 1 1 b h i g h G r 1 in t C D 1 1 b lo w C D 1 1 c F 4 /8 M H C I M H C II C D 4 9 d C D 1 4 C D 3 1 C D Figure E5

13 G r1 h ig h C D 1 1 b h ig h G r1 d im C D 1 1 b h ig h G r1 lo w C D 1 1 b in t M a c ro p h a g e s B C G + c e lls (% ) S e c re te d C y to k in e (n g /m l) B M D S C s, B C G M a c ro p h a g e s, B C G * * * * * IL -1 (O D 4 5 ) T N F IL - 6 IL - 1. Figure E6

14 L a c ta te d e h y d ro g e n a s e (O D 4 9 ) T N F (% re le a s e ) T N F (p g /m l) 4 S p le n o c y te s o n ly C o -c u ltu re -M tb C o -c u ltu re + M tb M D S C o n ly 3 * * * 1 * * B 6 4 :1 1 : 1 :4 1 :8 1 :1 6 1 : 1 :4 1 :8 1 :1 6 1 : 1 : - M tb + M tb G r1 + C D 1 1 b + c e lls : S p le n o c y te s S p le n o c y te s o n ly W T M D S C s N o s - /- M D S C s rg 1 - /- M D S C s N o s - /- rg 1 - /- M D S C s :1 1 : 1 :1 1 : 1 :4 1 : 1 :1 1 : 1 :4 1 : 1 :1 1 : 1 :4 1 : 1 :1 1 : 1 :4 G r1 + C D 1 1 b + c e lls : S p le n o c y te s 3 S p le n o c y te s o n ly W T M D S C s N o s - /- M D S C s rg 1 - /- M D S C s N o s - /- rg 1 - /- M D S C s 1 :1 1 : 1 :1 1 : 1 :4 1 : 1 :1 1 : 1 :4 1 : 1 :1 1 : 1 :4 1 : 1 :1 1 : 1 :4 G r1 + C D 1 1 b + c e lls : S p le n o c y te s Figure E7

15 L a c ta te d e h y d ro g e n a s e (O D 4 9 ) Placebo T N F (% re le a s e ) TR IF N (% re le a s e ) C D 1 1 c + L e u k o c y te s (% ) C D 1 1 b + G r1 + ce lls (x 1 6 ) T R P la c e b o T R P la c e b o C D 1 1 c + c e lls (x1 6 ) B 4 T R P la c e b o 3 1 C D TR Placebo TR Placebo C D 4 9 d C D 8 6 M H C II C D C D 1 4 E D M S O M T R S p le n o c y te s G * * * 5 n.d. n.d. 1 : 1 : 1 :4 1 : 1 : 1 :4 :1 M D S C : S p le n o c y te 6 D M S O M T R S p le n o c y te s 4 * * * * * 1 : 1 : 1 :4 1 : 1 : 1 :4 :1 F 1. M D S C : S p le n o c y te D M S O S p le n o c y te s M T R : 1 : 1 :4 1 : 1 : 1 :4 :1 Figure E8 M D S C : S p le n o c y te