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Supplementary appendixThis appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors.

Supplement to: Skrahin A, Ahmed RK, Ferrara G, et al. Autologous mesenchymal stromal cell infusion as adjunct treatment in patients with multidrug and extensively drug-resistant tuberculosis: an open-label phase 1 safety trial. Lancet Respir Med 2014; published online Jan 9. http://dx.doi.org/10.1016/S2213-2600(13)70234-0.

Open-label Phase I safety trial of autologous mesenchymal stromal cell (MSC) infusion as adjunct treatment in patients with multi- and extensively-drug resistant TB

Supplementary Data Sets

Page Number Item Topic

1-2 Text S1 Immune cell phenotyping, functional Assays

3-7 Table S1A List of antibodies and molecular probes

8-11 Table S1B List of M.tb target antigens for functional assays

12 Table S2 Xray Score analysis

13 Table S3 Clinical characteristics TB+ patients, non-MSC treated

14 Figure S1 MSC-treated patients: microscopy and TB culture

15-24 Figure S2 MSC and non-MSC treated patients:

Hematology and Biochemistry

25-35 Figure S3 Laboratory values in MSC-treated subgroups

36 Figure S4 TB treatment outcome: MSC treated patients

37 Figure S5 TB treatment outcome: non-MSC treated patients

38-40 Figure S6 Intracellular cytokine staining

41-43 Figure S7 T-cell population analysis

44 Figure S8 T-cell receptor zeta chain analysis

45-47 Figure S9 IFNγ production in the T-cell expansion assay

1

Suppl.data p. 1 Skrahin et al., online Material Text S1:

Immune cell phenotyping, Functional Immune Assays

Intracellular cytokine staining. Cytokine production was analyzed in frozen PBMCs. Cells were thawed,

rested overnight, and stimulated for 6 hours in the presence of Brefeldin A (10 mg/mL) from Sigma-

Aldrich (Sweden AB, Stockholm, Sweden) either with medium, i.e. RPMI 1640 containing L-glutamine (2

mM), penicillin (100 IU/mL) and streptomycin (10 mg/mL), 10% heat-inactivated FBS (Gibco,

Invitrogen), or medium and phorbol 12-myristate 13-acetate (PMA)-ionomycin (respectively 25 ng/mL and

1 mg/mL; Sigma-Aldrich) as a positive control. M.tb specific responses were tested using a TB10.4 peptide

pool, covering the entire TB10.4 protein. T-cells were stained with cell surface marker mAbs: CD3, CD4,

CD8 in the presence of the live/dead cell marker (Aqua LIVE/DEAD; Invitrogen) for 30 min at 4oC. After

washing with PBS, cells were fixed and permeabilized using the IntraPrep Fix/Perm Kit (Beckman Coulter)

and incubated with Abs specific for intracellular cytokines for 30 min at 4oC: anti-IL-2, -IFN-γ, -TNF-α

and anti-IL-17. Cells were analyzed using a BD FACSCAria flow cytometer (BD Biosciences) and data

analysis was performed using FlowJo software. All antibodies are listed below. PBMCs from healthy blood

donors were used as positive controls and provided in the supplementary material, Table S1.

Cytokine induced STAT-5 phosphorylation assay. Constitutive and cytokine-induced phosphorylated

STAT5 (P-STAT5) expression was evaluated in frozen PBMCs as described. Overnight starved, thawed

PBMCs were incubated with recombinant human IL-7 (rhIL-7-100ng for 105 cells, provided by Dr. Michel

Morre, Cytheris, Issy-les-Moulineaux, France) or IL-2 (100 IU) for 15 min at 37oC. Cells were then

incubated for 15 min at 4oC stained with cell surface markers, fixed with 2% paraformaldehyde,

permeabilized with 90% methanol for 30 min on ice, followed by two washes and staining for P-STAT5a

mAb for 1 hr at room temperature and analyzed immediately using a FACSAria flow cytometer, the

detailed antibody specifications are listed in the supplementary Table S1.

TCR zeta chain testing and tetramer analysis. TCR zeta-chain expression was analyzed in CD3+ T-

cells, which were fixed and lysed according to the protocol listed above (STAT5-phosphorylation assay)

followed by TCR-zeta-chain determination. Analysis was performed using a FACSAria flow cytometer and

data analyzed by using FlowJo software.

2

Gene expression in MSCs Suppl.data p. 2

Total RNA from MSC cultures was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and

transcribed into cDNA by Superscript III first strand cDNA synthesis kit (Invitrogen). cDNA was used to

analyze gene expression using a TaqMan gene expression assays on a ABI7900 real time instrument

according to the suppliers’ instruction. Data were analyzed by relative quantitation of immune and stem

cell markers within the sample using CD90 expression in each sample as a baseline. The gene probe

designation numbers are listed in the online material Table S1, MSC expressed the typical gene patterns,

i.e. CD90, CD73 and CD105.

Whole blood assay. The whole blood assay (WBA) was used to determine IFN-γ production in response to

M.tb antigens. Venous whole blood was obtained using heparinized blood collection tubes and diluted 1:2.5

in RPMI 1640-medium supplemented with 1% Hepes, 0.5% Penicillin/ (100 IU/ml) and streptomycin (10

mg/ml), (Gibco Invitrogen). 100 μl of diluted blood was added into 96-well round bottom plates (Nunc,

Roskilde, Denmark) in duplicate wells pre-coated with the specific antigen diluted in 100 μl medium.

Cultures were incubated at 37°C, 5% CO2. After 7 days, 75μl of cell culture supernatant was removed from

each duplicate well, pooled, and stored at -80°C until IFN-γ was determined by ELISA. The antigens tested

in the WBA are listed in the supplementary Table S1. IFN-γ in cell culture supernatants was measured by

ELISA (Eli-pair DIACLONE, Biosite, Stockholm, Sweden). The assays were performed according to the

manufacturer’s instruction. In brief, Nunc-Immuno™ Maxisorp 96-well plates (Nunc, GTF, Stockholm,

Sweden) were coated with the specific capture antibody overnight at 4°C. The plates were then washed

with PBS containing 0.05% tween-20 and then blocked with PBS containing 5% bovine serum albumin

(BSA, Karolinska Hospital, Stockholm, Sweden). Supernatants collected from WBA (150µl) were thawed

and diluted (1:1.47 for IFN-γ [75µl]) with PBS containing 1% BSA. Values were multiplied by their

corresponding dilution factor, background from un-stimulated medium control were subtracted from each

antigen response; the cytokine concentration was expressed in pg/ml. The detection range for IFN-γ was

400-7 pg/ml. An additional standard IFN-γ recombination protein (purchased from R&D, Minneapolis,

MN, USA) was used as an internal control to gauge for differences between ELISA assay performances.

The WBA assay measures effector and memory T-cell expansion in CD4+ and CD8+ T-cells during the 7

day expansion period and not NK-cell function in response to antigenic stimulation.

Skrahin et al Supplementary Table S1 A Suppl.data p. 3

Monoclonal antibodies used for immune cell, MSC phenotyping and intracellular staining.

Immune cell phenotyping

Antibody Clone Company Isotype

CD19-FITC SJ25C1 BD Biosciences, San Jose, USA Mouse IgG1 k

CD45RA-ECD 2H4 Beckman Coulter , Miami, USA Mouse IgG1

TCRalpha/beta-PerCP WT31 BD Biosciences, (Custom), San Jose, USA Mouse IgG1

TCRgamma-delta PE Cy5.5 IMMU510 Beckman Coulter , (Custom), Miami, USA Mouse IgG1

CCR7-PECy7 3D12 BD Pharmingen, San Jose, USA Rat IgG2a

CD127- APC R34.34 Beckman Coulter , (Custom), Miami, USA Mouse IgG1

CD107a- APCAlexa Fluor700 H4A3 Beckman Coulter , (Custom), Miami, USA Mouse IgG1

CD8 -APC Alexa750 T8 Beckman Coulter , (Custom), Miami, USA Mouse IgG1

CD4- Pacific Blue SFC1 12T4D11 Beckman Coulter , (Custom), Miami, USA Mouse IgG1

ICS (Intracellular cytokine staining) Suppl.data p. 4


CD3-Pacific Blue SP34-2 BD Biosciences, San Jose, USA Mouse IgG1

CD4-PerCPCy5.5 L200 BD Biosciences, San Jose, USA Mouse IgG1 k

CD8a-APCCy7 SK1 Beckman Coulter , (Custom), Miami, USA Mouse IgG1

IL-17A-FITC BL168 Biolegend Mouse IgG1

IL2-PE MQ1-17H12 BD Biosciences, San Jose, USA Rat IgG2a k

INFgamma PECy7 B27 BD Biosciences, San Jose, USA Mouse IgG1 k

TNFalpha-APC Mab11 BD Biosciences, San Jose, USA Mouse IgG1 k

IL10-Alexa Fluor 647 JES3-9D7 eBioscience Rat IgG1 k

CD279 (PD1)-PE EH12.1 BD Pharmingen, San Jose, USA Mouse IgG1 k

STAT5 Panel - STAT5 phosphorylation assay

Antibody Clone Company Isotype control

CD3-ECD UCHT1 Beckman Coulter , Miami, USA Mouse IgG1

CD4-PCy5 13B8.2 Beckman Coulter, Marseille, France Mouse IgG1

CD8a-APCCy7 T8 Beckman Coulter , Miami, USA Mouse IgG1

CD19-PE SJ25C1 BD Biosciences, San Jose, USA Mouse IgG1 k

CD25-PECy7 2A3 BD Biosciences, San Jose, USA Mouse IgG1

pSTAT5-Alexa Fluor 488 47 BD Biosciences, San Jose, USA Mouse IgG1 k

Treg and TCR-zeta-chain analysis Suppl.data p. 5



CD8-PerCP SK1 BD Biosciences, San Jose, USA Mouse IgG1 k

CD25-PECy7 2A3 BD Biosciences, San Jose, USA Mouse IgG1

CD4-Pacific Blue SFC1 12T4D11 Beckman Coulter , (Custom), Miami, USA Mouse IgG1

CD127-APC R34.34 Beckman Coulter , (Custom), Miami, USA Mouse IgG1

Foxp3-Alexa Fluor 488 259D Biolegend, San Jose, USA IgG1 k

TCR zeta-chain PE 6B10.2 Santa-Cruz Biotechnology, USA Mouse IgG1

MSC panel 1

Antibody Clone Company Isotype control

CD105-FITC 43A3 Biolegend, San Jose, USA Mouse IgG1 k

CD90-APC 5E10 BD Biosciences, San Jose, USA Mouse IgG1 k

CD73-PE AD2 BD Biosciences, San Jose, USA Mouse IgG1 k

CD25-PECy7 2A3 BD Biosciences, San Jose, USA Mouse IgG1 k

CD8-PerCP SK1 BD Biosciences, San Jose, USA Mouse IgG1 k

HLA-DR-Pacific Blue L243 Beckman Coulter , (Custom), Miami, USA Mouse IgG2a, κ

CD127-Alexa Fluor 700 R34.34 Beckman Coulter, (Custom), Miami, USA Mouse IgG1 k

CD117-Pacific Orange YB5.B8 BD Biosciences, San Jose, USA Mouse IgG1 k

MSC panel 2 Suppl.data p. 6



CD45-PerCP 2D1 BD Biosciences, San Jose, USA Mouse IgG1 k

CD34-PeCy7 8G12 BD Biosciences, San Jose, USA Mouse IgG1 k

HLA-DR-APCCy7 L243 (G46-6) BD Biosciences, San Jose, USA Mouse IgG2a, κ

CD19-FITC SJ25C1 BD Biosciences, San Jose, USA Mouse IgG1 k

CD14-PE MφP9 BD Biosciences, San Jose, USA Mouse IgG1 k

MSC panel 3

CD166-PE 3A6 BD Pharmingen, San Jose, USA Mouse IgG1 k

CD44-FITC G44-26 BD Pharmingen, San Jose, USA Mouse IgG2b, κ

Target gene designation implemented for Q-PCR Gene Expression Analysis, typical MSC markers are marked in red. Suppl.data p. 7

No Target Assay ID Provider

1 18S RNA Hs99999901_s1 Applied Biosystems

2 Beta-actin Hs00357333_g1 Applied Biosystems

3 BMPR2 Hs00176148_m1 Applied Biosystems

4 Endoglin (CD105) Hs00923997_g1 Applied Biosystems

5 IL7 Hs00174202_m1 Applied Biosystems

6 IFNA1 Hs00855471_g1 Applied Biosystems

7 IFNA2 Hs00265051_s1 Applied Biosystems

8 IL7R Hs00902334_m1 Applied Biosystems

9 KITLG Hs00295067_s1 Applied Biosystems

10 NT5E (CD73) Hs01573922_m1 Applied Biosystems

11 TGFB1 Hs00171257_m1 Applied Biosystems

12 THY1 (CD90) Hs00174816_m1 Applied Biosystems

13 GAPDH Hs99999905_m1 Applied Biosystems

Supplementary Table S1B M.tb targets for the T-cell expansion assay (Whole Blood Assay, WBA) Suppl.data p. 8

Gene locus RefSeq M.tb Antigens aa Comment

Targ

ets

Rv0447c NP854118 Probable cyclopropane fatty acyl phospholipid synthase.

427 Methyl transferase activity. Cyclopropane fatty acyl phospholipid synthase activity. Lipid biosynthetic process [1, 2]

Rv2940c YP_976584 Mycocerosic acid synthase 2111 Lipid biosynthetic process. Oxido-reduction and transferase activity, Cofactor binding. Location at the cell wall. [1, 3-7]

Rv3347c YP_177963 PPE family protein 3157 Function unknown. Gly-Ala-Asn rich protein, interacts with the host system by inhibition of antigen processing. [2, 7-10]

Rv2453c CAA16030 Probable molybdopterin-guanine dinucleotide biosynthesis Protein A

201 Molybdenum cofactor biosynthesis. Molecular function as GTP binding. Located at the cytoplasm membrane. [2, 7, 8, 11]

Rv1886 CAB10044 Antigen 85B 325 Fibronectin binding protein. Acyltransferase activity. Secreted protein, also located at cell wall, plasma membrane. [2, 12-15], strongly immunogenic

Rv1690 CAB10947 Probable lipoprotein 127 Putative uncharacterized protein. Protein binding, cellular component plasma membrane.[2, 8, 16]

Rv3019c CAA16104 ESAT-6 like protein 96 Belongs to ESAT-6 (esx) family, Protein-protein interaction [2, 8] Rv2957 CAB05419 PGL/p-HBAD biosynthesis glycosyltransferase

MT3031 256 Glycosyl transferase activity, transferring hexosyl groups. Glycolipid biosynthetic

function. Identified as a drug target. [2, 8, 14, 17, 18] Rv1085c CAA17201 UPF0073 membrane protein 242 Belongs to the UPF0073 (HIy-III) family. Cytolysis. Sub cellular location in cell membrane

[2, 8] Rv0066c CAA16247 Isocitrate dehydrogenase, NADP-dependent- icd2. 745 Oxidoreductase function. NAD or NADH binding. Isocitrate dehidrogenase (NADP+)

activity. Magnesium ion binding. Protein homodimerization. [2, 6-8, 19, 20] Rv2958c CAB05418 PGL/p-HBAD biosynthesis glycosyltransferase 428 Glycolipid biosynthetic process, pathogenesis, Glycosyl transferase activity. Evasion or

tolerance concerning to the host immune response. [2, 6, 8, 17, 21] Rv2962c CAB05415 PGL/p-HBAD biosynthesis rhamnosyl-transferase 449 Glycolipid biosynthetic process, pathogenesis. Glycosyl transferase activity. Evasion or

tolerance concerning to the host immune response. [2, 6, 8, 14, 17, 21] Rv0288 CAA17363 TB10.4. Low molecular weight protein. (M.tb) 96 Belongs to theESAT-6 (esx) family. Molecular function protein binding. Involved in

virulence. Immunogenic. [2, 8, 22-25]

References related to M.tb targets (see table above) Suppl.data p. 9

1. Garnier, T., et al., The complete genome sequence of Mycobacterium bovis. Proc Natl Acad Sci U S A, 2003. 100(13): p. 7877-82.

2. Cole, S.T., et al., Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature, 1998. 393(6685): p. 537-44.

3. Mathur, M. and P.E. Kolattukudy, Molecular cloning and sequencing of the gene for mycocerosic acid synthase, a novel fatty acid elongating multifunctional enzyme, from Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guerin. J Biol Chem, 1992. 267(27): p. 19388-95.

4. Rainwater, D.L. and P.E. Kolattukudy, Fatty acid biosynthesis in Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guerin. Purification and characterization of a novel fatty acid synthase, mycocerosic acid synthase, which elongates n-fatty acyl-CoA with methylmalonyl-CoA. J Biol Chem, 1985. 260(1): p. 616-23.

5. Camus, J.C., et al., Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv. Microbiology, 2002. 148(Pt 10): p. 2967-73.

6. Mawuenyega, K.G., et al., Mycobacterium tuberculosis functional network analysis by global subcellular protein profiling. Mol Biol Cell, 2005. 16(1): p. 396-404.

7. Gu, S., et al., Comprehensive proteomic profiling of the membrane constituents of a Mycobacterium tuberculosis strain. Mol Cell Proteomics, 2003. 2(12): p. 1284-96.

8. Fleischmann, R.D., et al., Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J Bacteriol, 2002. 184(19): p. 5479-90.

Suppl.data p. 10

9. Gey van Pittius, N.C., et al., Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions. BMC Evol Biol, 2006. 6: p. 95.

10. Singh, K.K., et al., Immunogenicity of the Mycobacterium tuberculosis PPE55 (Rv3347c) protein during incipient and clinical tuberculosis. Infect Immun, 2005. 73(8): p. 5004-14.

11. Guse, A., et al., Biochemical and structural analysis of the molybdenum cofactor biosynthesis protein MobA. J Biol Chem, 2003. 278(28): p. 25302-7.

12. De Wit, L., M. Palou, and J. Content, Nucleotide sequence of the 85B-protein gene of Mycobacterium bovis BCG and Mycobacterium tuberculosis. DNA Seq, 1994. 4(4): p. 267-70.

13. Harth, G., et al., Novel insights into the genetics, biochemistry, and immunocytochemistry of the 30-kilodalton major extracellular protein of Mycobacterium tuberculosis. Infect Immun, 1996. 64(8): p. 3038-47.

14. Raman, K., K. Yeturu, and N. Chandra, targetTB: a target identification pipeline for Mycobacterium tuberculosis through an interactome, reactome and genome-scale structural analysis. BMC Syst Biol, 2008. 2: p. 109.

15. Anderson, D.H., et al., An interfacial mechanism and a class of inhibitors inferred from two crystal structures of the Mycobacterium tuberculosis 30 kDa major secretory protein (Antigen 85B), a mycolyl transferase. J Mol Biol, 2001. 307(2): p. 671-81.

16. Steyn, A.J., J. Joseph, and B.R. Bloom, Interaction of the sensor module of Mycobacterium tuberculosis H37Rv KdpD with members of the Lpr family. Mol Microbiol, 2003. 47(4): p. 1075-89.

17. Perez, E., et al., Characterization of three glycosyltransferases involved in the biosynthesis of the phenolic glycolipid antigens from the Mycobacterium tuberculosis complex. J Biol Chem, 2004. 279(41): p. 42574-83.

Suppl.data p. 11

18. Berg, S., et al., The glycosyltransferases of Mycobacterium tuberculosis - roles in the synthesis of arabinogalactan, lipoarabinomannan, and other glycoconjugates. Glycobiology, 2007. 17(6): p. 35-56R.

19. Banerjee, S., et al., Comparison of Mycobacterium tuberculosis isocitrate dehydrogenases (ICD-1 and ICD-2) reveals differences in coenzyme affinity, oligomeric state, pH tolerance and phylogenetic affiliation. BMC Biochem, 2005. 6: p. 20.

20. Malen, H., et al., Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv. Proteomics, 2007. 7(10): p. 1702-18.

21. Miller, B.H. and T.M. Shinnick, Evaluation of Mycobacterium tuberculosis genes involved in resistance to killing by human macrophages. Infect Immun, 2000. 68(1): p. 387-90.

22. Skjot, R.L., et al., Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens. Infect Immun, 2000. 68(1): p. 214-20.

23. Majlessi, L., et al., CD8+-T-cell responses of Mycobacterium-infected mice to a newly identified major histocompatibility complex class I-restricted epitope shared by proteins of the ESAT-6 family. Infect Immun, 2003. 71(12): p. 7173-7.

24. Okkels, L.M. and P. Andersen, Protein-protein interactions of proteins from the ESAT-6 family of Mycobacterium tuberculosis. J Bacteriol, 2004. 186(8): p. 2487-91.

25. Lightbody, K.L., et al., Characterisation of complex formation between members of the Mycobacterium tuberculosis complex CFP-10/ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility. FEMS Microbiol Lett, 2004. 238(1): p. 255-62.

Supplementary Table 2 XRay score analysis Suppl.data p. 12

The Xray score was obtained at 3 time-points according to Ralph et al. Thorax 2010; 65: 863-869: CXR score = proportion of total lung affected (%) + 40 if cavitation present. The difference between the scores at time 0 and after 6 months was calculated according to: Difference (%) = (Score at time 0 – Score after 6 months)/Score at timepoint 0 x 100

A difference of at least -5% (indicating an increase in the score after 6 months) was considered as a worsening; a decrease of 10% or more was considered as an improvement (indicating a decrease in the score after 6 months). + Note that the difference in the Xray score from patient 28 was obtained using the 2month score and the Xray score at 6 month.

ID Score Timepoint 0 Score after 2 months Score after 6 months Difference beetween scores at timepoints 0 and

6 months (%)

Xray score after 6 months

1 65 5 10 85 Improved 2 65 55 55 15 Improved 3 100 115 105 -5 Worsened 4 105 110 105 0 Stable 5 70 65 80 -14 Worsened 6 10 5 5 50 Improved 7 90 80 80 11 Improved 8 100 100 100 0 Stable 9 50 45 5 90 Improved

10 60 55 50 17 Improved 11 110 95 80 27 Improved 12 55 50 10 82 Improved 13 65 50 50 23 Improved 14 20 15 10 50 Improved 15 55 50 5 90 Improved 16 45 45 5 88 Improved 17 50 5 5 90 Improved 18 55 50 50 10 Improved 19 10 5 5 50 Improved 20 10 10 10 0 Stable 21 10 5 <5 50 Improved 22 55 10 10 82 Improved 23 10 10 5 50 Improved 24 60 55 65 -8 Worsened 25 25 20 15 40 Improved 26 10 5 5 50 Improved 27 65 75 95 -46 Worsened 28 5 45 <5 88+ Improved 29 10 10 10 0 Stable 30 10 10 10 0 Stable

Supplementary Table S3 Suppl.data p. 13 Overview of clinical characteristics of TB patients who received standard treatment and not adjunct MSC treatment

ID Sex Age Case definition DR Status

DST

N0. of previous SLT SLT regimen at the time

of inclusion in the study Microscopy/culture

at SLT’s start Radiology

after 6-8 months

1 M 46 New Case MDR H,S,R,E 0 Z, Km,Ofx,Pto,Cs +/+ Improved 2 M 62 Previously treated MDR H,S,R,E 1 Z,Km,Lfx, ,Pto,Cs +/+ Stable 3 M 39 Previously treated MDR H,S,R,Z,Km 1 Z,Cm,Lfx,Pas,Pto, Cs -/+ Improved 4 M 29 New Case MDR H,S,R,Е,Z,Km 0 Z,Сm,Lfx,Pas,Pto, Cs -/+ Worsened 5 M 39 New Case MDR H,S,R 0 Z,E,Km,Ofx,Pto +/+ Improved 6 F 32 New Case MDR H,S,R,E 0 Z, ,Km,Ofx,Pto, Pas -/+ Improved 7 M 39 New Case MDR H,S,R,E 1 Z,Km,Ofx,Pas,Pto -/+ Improved 8 M 27 New Case MDR H,S,R 0 Z,E,Km,Ofx,Pto -/+ Improved 9 M 37 New Case MDR H,S,R 0 Е,Z,Km,Ofx,Pas,Pto +/+ Stable 10 F 20 New Case MDR H,S,R 0 Е,Km,Ofx,Pas,Еto,Cs -/+ Improved 11 M 46 New Case MDR H,S,R,E 0 Ofx,Km,Cs,Pto,Pas,Z -/+ Improved 12 M 25 Previously treated MDR H,S,R,E,Eto,Ofx 0 Lvx,Cm,Cs,Pto,Pas -/+ Improved 13 M 62 Previously treated MDR H,S,R,E,Eto,Km 0 Lfx,Cm,Cs,Pto,Pas -/+ Improved 14 M 30 Previously treated MDR H,S,R,E,Km,Cm 0 Ofx, Am,Cs,Pto,Pas -/+ Improved 15 F 26 Previously treated XDR H,R,E,S,Ofx,PAS,Km 1 Amx/clvMfxPtoCsCmPAS +/+ Improved 16 M 28 Previously treated MDR H,R,E,S,Ofx,Eto 1 ,Am,Amx/clv,Lfx,Pto,Cs,PAS +/+ Stable 17 M 43 New Case MDR H,R,E,S,Z 0 Cm,Ofx,Pro,Cs,PAS +/+ Improved 18 M 59 Previously treated MDR H,R,E,S,Ofx,Eto 1 Am,Amx/clv,Lfx,Pto,Cs,PAS +/+ Improved

19 M 54 New Case XDR H,R,E,Z,S,Am,Km,Ofx,Eto,Cm,Cs 0 Lfx,PAS,Pto, Cm,Cs,Amx/clv,Clr +/+ Improved

20 M 54 New Case MDR H,R,Z,S 0 E,Ofx,PAS,Pto,Km +/+ Improved 21 M 57 New Case MDR H,R,E,S,Z,Km, 0 Am,Ofx,PAS,Pto,Cs -/+ Improved 22 F 21 Chronic MDR H,R,S,Ofx 0 Lfx,Pto,PAS,Am,Cs § -/+ Improved 23 F 25 New Case MDR H,R,E,Z,S 0 Ofx,Eto,PAS,Km,Cs -/+ Improved 24 M 30 Chronic XDR H,R,E,Z,S,Km,Ofx 0 Pto,Am,Lfx,Cs,PAS, Amx/clv +/+ Worsened 25 F 55 New Case MDR H,R,E,S,Km,Am 0 Z,Cm,Lfx,Pto,Cs,PAS -/- Improved

26 F 30 New Case MDR H,R,S,E 0 Z,Km,Lfx,Pto,Cs -/+ Improved

27 M 61 New MDR H,R,E,S,Eto,Cs,Km 0 Am, ,Lfx,Pto,Cs,PAS,Amx/Clv +/+ Worsened

28 F 46 Previously treated MDR H,R,E,S,Z,Km,Cs 0 Am,Lfx,Pto,PAS,Amx/Clv +/+ Improved

29 M 32 Chronic XDR H,R,S,Z,Ofx,Eto,Km 2 Lfx,PAS,Pto,Cs,Cm,Amx/Clv -/+ Worsened

30 M 29 New case MDR H,R,E,S,Z,Km,Am,Cm,Pto,PAS,Cs 0 Z,Cm,Lfx,Pto,Cs,Pas, Amx/Clv -/- Improved Abbreviations: DR: drug resistance; DST: drug susceptibility testing; SLT: second-line treatment; M: male, F: female; MDR: multidrug-resistant; XDR: extensively drug resistant; TB: tuberculosis; H: isoniazid; R: rifampicin; S: streptomycin; E: ethambutol; Z: pyrazinamide; Am: amikacin; Eto: ethionamide; Cm: capreomycin; Cs: cycloserine; Km: kanamycin; Ofx: ofloxacin; PAS: para-amino-salicylic acid; Mfx: moxifloxacin; Pto: prothionamide; Cfz: clofazimine; Lfx: levofloxacine; Amx-Clv: amoxicillin-clavulanate; Clr: claritromycin. § lobectomy right upper lobe during therapy. The patients were consecutively recruited into the study and fulfilled the inclusion criteria, yet they did not chose to obtain MSC adjunct therapy.

L-J diagnostic : 0 vs 1 p < 0.0001 0 vs 2 p < 0.0001 0 vs 3 p < 0.0001 0 vs 4 p < 0.0001 0 vs 5 p < 0.0001 0 vs 6 p < 0.0001 0 vs 8 p < 0.0001 1 vs 2 (p=0.06) 1 vs 3 p=0.0149 1 vs 4 p=0.0227 1 vs 5 p=0.0024 1 vs 6 p=0.0097 1 vs 8 p=0.0033

Suppl. Figure S1

Clinical outcome of 30 individuals with TB (see Table 1) after MSC infusion. Acid fast stain results and culture in months after MSC infusion . The Fisher test was used to examined differences between different timepoints (months) after MSC infusion using the GraphPad program.

Suppl.data p. 14

Safety / adverse events in TB + patients after MSC infusion: Followup of M.tb microscropy and culture

TB + patients, no MSC MSC infused patients

0vs2 p=0,0475 2vs6 p=0,0170

0vs2 p=0,0409 0vs3 p=0,0126

3vs6 p=0,042

Supplementary data Figure S 2A

Suppl.data p. 15 Safety data: hematological analysis and biochemistry

TB + patients, no MSC

MSC infused patients

0vs6 p=0,0344 1vs6 p=0,0054 1vs5 p=0,0123

1vs4 p=0,0205 1vs3 p=0,0433

Suppl.data p. 16 Supplementary data Figure S2 B



0vs6 p=0,0269 0vs5 p=0,0103 2vs5 p=0,0369 2vs6 p=0,0210 3vs5 p=0,0067 3vs6 p=0,0488

3vs4 P value 0,0068

Suppl.data p. 17 Supplementary data Figure S2 C



0vs2 p=0,0256 2vs3 p=0,0479 2vs6 p=0,0215

1vs4 p=0,0182

2vs4 p=0,0366

Neutrophils

Neutrophils

Neutrophils

Neutrophils

Suppl.data p. 18 Supplementary data Figure S2 D


0vs1 p=0,0156

Suppl.data p. 19 Supplementary data Figure S2 E




Globulin Globulin

Suppl.data p. 20 Supplementary data Figure S2F



4vs6 p=0,0258 2vs6 p=0,0125 1vs6 p=0,0095

Suppl.data p. 21 Supplementary data Figure S2 G



0vs1 p=0,0269 0vs2 p=0,0005 0vs3 p<0.0001 0vs4 p=0,0078

Suppl.data p. 22 Supplementary data Figure S2 H



LDH LDH

IU/L

Suppl.data p. 23 Supplementary data Figure S2 I



IU/L

Suppl.data p. 24 Supplementary data Figure S2 J

Supplementary Data Set Figure S3 Hematological analysis and biochemistry in TB+ patient subsets who received MSCs 30 TB patients received adjunct MSC treatment (see Table 1 in the report); no major alterations concerning hematological or biochemical values were observed (see Figure 2 in the report). The group of 30 individuals were further analyzed based on i) Age (˃35 and ≤ 35 years) ii) Case definition (new or previously treated case) iii) Resistance pattern (XDR / MDR TB) iv) Second line treatment (˃6 and ≤ 6 drugs) v) Radiology (improved / non-improved) according to the patient information in Table 1. The groups were analyzed in detail concerning ASAT, ALAT, CRP, ESR, Glucose levels as well as for absolute leucocyte and lymphocyte count) to detect differences in the subgroups. No gross differences were observed. Larger patient cohort would need to be analyzed in order to allow for a multivariate analysis.

Suppl.data p. 25

Age

ESR

mm

/hou

r

CRP

mg/

L

ASAT

ALAT

Leukocytes

Lymphocytes

109 /

L 10

9 /L

before 1 2 3 4 5 6 before 1 2 3 4 5 6 before 1 2 3 4 5 6


m p. MSC infusion

0m p= 0,0251 1m p= 0,0182

Age ˃35 ≤ 35 years

Suppl.data p. 26

Age

Glucose

before 1 2 3 4 5 6

Mm

ol/L

m p. MSC infusion

Age ˃35 ≤ 35

0m p= 0,0392 4m p= 0,0083 6m p= 0,0229

years

Suppl.data p. 27

Case definition

ESR ASAT Leukocytes

CRP ALAT Lymphocytes

mm

/hou

r m

g/L

109 /

L 10

9 /L



m p. MSC infusion

Case definition new case already treated

1m p= 0,0168

0m p= 0,0403 1m p= 0,0162 6m p= 0,0078

IU

IU

Suppl.data p. 28

Case definition

m p. MSC infusion

Case definition new case already treated

Glucose

before 1 2 3 4 5 6

Mm

ol/L

Suppl.data p. 29

Resistance ESR ASAT


mm

/hou

r m

g/L

109 /

L 10

9 /L

Leukocytes

m p. MSC infusion


before 1 2 3 4 5 6 Before 1 2 3 4 5 6 before 1 2 3 4 5 6

Resistance XDR MDR

0m p= 0,0423 0m p= 0,0149

IU

IU

Suppl.data p. 30

Resistance

m p. MSC infusion

Resistance XDR MDR

Glucose

before 1 2 3 4 5 6

mm

ol/L

3m p= 0,0465

Suppl.data p. 31

Second Line Treatment (SLT)

ESR ASAT Leukocytes


mm

/hou

r m

g/L

109 /

L 10

9 /L

m p. MSC infusion


before 1 2 3 4 5 6 Before 1 2 3 4 5 6 before 1 2 3 4 5 6

Second Line treatment >6 ≤6

5m p= 0,0431

0m p= 0,0358 5m p= 0,0134

3m p= 0,0275 6m p= 0,0182

IU

IU

Suppl.data p. 32

SLT

m p. MSC infusion Second Line treatment >6 ≤6

before 1 2 3 4 5 6

mm

ol/L

Glucose 3m p= 0,0034

Suppl.data p. 33

Radiology ESR ASAT Leukocytes


mm

/hou

r m

g/L

109 /

L 10

9 /L



m p. MSC infusion

Radiology improved not improved

0m p= 0,018

IU

IU

Suppl.data p. 34

Radiology

m p. MSC infusion Radiology improved not improved

before 1 2 3 4 5 6

mm

ol/L

Glucose 0m p= 0,0078

Suppl.data p. 35

TB+ patients, MSC treatment

Cured

Treatment completed

Default

Death

Treatment failure

Still on treatment

Suppl. Figure S4

Outcome # patients Cured 16 Treatment completed 2 Default 1 Death 0 Treatment failure 3 Still on treatment 8

Clinical outcome of 30 individuals with TB (see Table 1) after MSC infusion. Cure: defined as 18 month negative M.tb cultures.

Suppl.data p. 36

TB+ patients, no MSC treatment

Cured

Treatment completed

Default

Death

Treatment failure

Still on treatment

Suppl. Figure S5

Outcome # of patients Cured 5 Treatment completed 5 Default 5 Death 5 Treatment failure 6 Still on treatment 4

Clinical outcome of 30 individuals with TB (see Table 1) without MSC infusion. Cure: defined as 18 month negative M.tb cultures. Clinical characteristics are provided in the online Table S2.

Suppl.data p. 37

Suppl. Figure S6A

Intracellular cytokine staining in CD4+ T-cells responding to positive control (PMA), TB10.4 peptides and medium (unstimulated). T-cells from healthy donors (HD) served as positive controls.

Suppl.data p. 38 Intracellular cytokine production

Suppl. Figure S6B

Intracellular cytokine staining in CD8+ T-cells responding to positive control (PMA), TB10.4 peptides and medium (unstimulated). T-cells from healthy donors (HD) served as positive controls.

Suppl.data p. 39

Suppl. Figure S6C

Intracellular cytokine staining in CD8+ T-cells from TB patients responding to positive control (PMA), TB10.4 peptides and medium (unstimulated) at different time points after MSC infusion. Some T-cells from individual patients recovered and showed cytokine production, yet this was not significant.

Suppl.data p. 40

Suppl. Figure S7A

Figure 8 A-C: Left: CD4+ T-cells, CD8+ (middle panel); left: TCRalpha/beta+ T-cells negative for CD4+ and CD8+. This T-cell population is often increased in chronic inflammations. Frequencies within TCRalpha/beta + T-cells. No significant differences at different time points after MSC infusion.

Suppl.data p. 41

T-cell populations in PBMCs from TB patients at different timepoints after MSC infusion

Precursor Central memory Effector memory Terminally differentiated

Suppl. Figure S7B

T-cell populations after MSC infusion, frequencies in the parental (CD4+, CD8+ or CD4-CD8-) T-cell populations. Most T-cells reside in the central memory T-cell subset (CD45RA-CCR7+).

CD4+

CD8+

CD4-CD8-

Suppl.data p. 42

CD45RA+ CCR7+ CD45RA- CCR7+ CD45RA- CCR7- CD45RA+ CCR7-

Suppl. Figure S7C

T-cell populations after MSC infusion, frequencies in the parental (CD4+, CD8+ or CD4-CD8-) T-cell populations. CD107a is a marker of recent degranulation. CD127 is the alpha chain of the IL-7 receptor and mediates survival signals.

CD4+

CD8+

CD4-CD8-

Suppl.data p. 43

fluor

esce

nce

inte

nsity

Supplementary Figure S8

timepoint (d after MSC)

control

0

7

21

28

60

90

180

CD3zeta chain in CD3+ T-cells

healthy P 1 cured P2 failed

P4 failed P 7 cured P9 cured

100% 82% 100% 99.5%

0.1% 0.09%

90% 99% 93%

100% 98%

99.5% 99.7%

Differential TCR zeta chain expression in PBMCs from patients with TB. Left, upper right panel: Representative flow cytometric staining of PBMCs from a healthy individual. PBMCs are first gated on CD3+ T-cells, followed by TCR zeta chain staining. 100 % of CD3+ T-cells stain positive for the TCR zeta chain. This was not true for patients with TB who showed variable TCR zeta chain expression in CD3+ T-cells after MSC infusion. Grey: unstained cells, the time points after MSC infusion are marked with different colors.

Suppl.data p. 44 T-cell receptor zeta chain analysis

Suppl. Figure S9A

Figure 10A-C: Target M.tb antigens are listed in the supplementary Table S1. Improved or decreased IFN responses in blood from some patients to M.tb targets. The tests compared the IFN responses prior to MSC infusion and after 6 month. Test data from 10 individuals. No significant differences concerning IFN responses. Significant differences are provided in the Figure 3. A high number of M.tb target antigens was chosen to cover potential different immune response patterns associated with M.tb exposure and genetic background (e.g. MHC) of the host.

Suppl.data p. 45

Additional data points for IFNgamma responses in the T-cell memory assay (WBA).

Suppl. Figure S9B Suppl.data p. 46

Suppl. Figure S9C Suppl.data p. 47

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