SUPPLEMENTARY DATA

Title: The secreted protein Rv1860 of Mycobacterium tuberculosis stimulates human

polyfunctional CD8+ T cells.

Vijaya Satchidanandam, Naveen Kumar, Sunetra Biswas, Rajiv S. Jumani, Chandni Jain, Rajni

Rani, Bharti Aggarwal, Jaya Singh, Mohan Rao Kotnur, and Anand Sridharan.

The supplementary information file contains supplementary Methods, supplementary tables of

volunteer information, sequences of peptides used, sequence of full length Rv1860 protein, HLA

allele data and supplementary figures showing human polyfunctional T cell response to Rv1860

and well-studied secreted proteins of Mycobacterium tuberculosis.

Supplementary Methods

Intracellular cytokine detection. Whole, heparinized (sodium heparin) blood was diluted 1:1

with RPMI 1640 and 1 ml aliquots were stimulated with peptides, each at a concentration of

5µg/ml in 13-ml tubes for 18 hours as described (1). This peptide concentration was arrived at

after testing concentrations ranging from 0.5 to 10 µg/ml. 2 hrs after peptide addition, brefeldin

A (Sigma, 10 µg/ml) was added to the samples. Monensin was added 6 hrs after peptide addition

at a concentration of 0.75 µM, a concentration that we determined to effectively block secretion

of cytokines without adversely affecting cell viability following 12 hrs exposure. We chose to

use both these secretion inhibitors as they have been reported to differentially block surface

expression/secretion of several markers studied (2, 3). Samples were removed from the 37oC

incubator 12 hrs after monensin addition, and erythrocytes lysed by addition of 10 volumes of

ammonium chloride lysis solution followed by vigorous vortexing for 1 min. Leukocytes

collected by centrifugation were fixed using 2% paraformaldehyde on ice for 10 min. followed

by washing with PBS-0.1 % sodium azide solution.

Following permeabilization of stimulated cells with saponin (0.1% saponin and 0.1% bovine

serum albumin, in PBS) for 15 min. on ice, intracellular cytokines were detected using an

antibody cocktail (made up in saponin-BSA-PBS solution) consisting of titrated amounts of anti-

CD3-APC-H7 (clone SK7), anti-CD8-PerCP (clone SK1), anti-IFN--PECy7 (clone B27), anti-

IL-2-FITC (MQ1-17H12), anti-TNFα-APC (6401-1111) and anti-MIP-1β-PE (11A3), all from

BD Pharmingen, San Diego, CA. Data were acquired on a BD-FACS Canto II flow cytometer

(Becton Dickinson, San Jose, CA). Singlet small lymphocytes were collected by gating on

forward versus side scatter and then gated on singlet cells, followed by CD3-high T lymphocytes

(Supplementary Figure S1A). We excluded dead cells (verified by separate live/dead staining to

be less than 0.01% of CD3+ T cells) by avoiding cells high on SSC and those close to the Y axis,

which was feasible as all staining was done on fresh PBMC. For each analysis, a minimum of

100,000 CD4+/CD8+ T cell subsets were acquired and data analyzed using FlowJo, (Treestar)

PESTLE and SPICE (Mario Roederer, NIH, USA) software. Positive staining was affirmed by

comparing the dot-plots of antibody-stained unstimulated and antigen-stimulated cells; a

minimum number of 50 events was used as cut-off for positive response. Gates were positioned

to ensure that the response of unstimulated cells were ≤0.01 % of total CD4+ or CD8+ T cells

for IFN-γ, TNF-α and IL-2 secreting T cells, while it was ≤0.05 for MIP-1β single cytokine

secreting T cells.

Supplementary Table S1. Sequence of 20 mer overlapping peptides covering the 325 amino acidsof Rv1860,

Peptide No. Amino acid Position Sequence

1801. 1-20 MHQVDPNLTR RKGRLAALAI

1802. 11-30 RKGRLAALAI AAMASASLVT

1803. 21-40 AAMASASLVT VAVPATANAD

1804. 31-50 VAVPATANAD PEPAPPVPTT

1805. 41-60 PEPAPPVPTT AASPPSTAAA

1806. 51-70 AASPPSTAAA PPAPATPVAP

1807. 61-80 PPAPATPVAP PPPAAANTPN

1808. 71-90 PPPAAANTPN AQPGDPNAAP

1809. 81-100 AQPGDPNAAP PPADPNAPPP

1810. 91-110 PPADPNAPPP PVIAPNAPQP

1811. 101-120 PVIAPNAPQP VRIDNPVGGF

1812. 111-130 VRIDNPVGGF SFALPAGWVE

1813. 121-140 SFALPAGWVE SDAAHFDYGS

1814. 131-150 SDAAHFDYGS ALLSKTTGDP

1815. 141-160 ALLSKTTGDP PFPGQPPPVA

1816. 151-170 PFPGQPPPVA NDTRIVLGRL

1817. 161-180 NDTRIVLGRL DQKLYASAEA

1818. 171-190 DQKLYASAEA TDSKAAARLG

1819. 181-200 TDSKAAARLG SDMGEFYMPY

1820. 191-210 SDMGEFYMPY PGTRINQETV

1821. 201-220 PGTRINQETV SLDANGVSGS

1822. 211-230 SLDANGVSGS ASYYEVKFSD

1823. 221-240 ASYYEVKFSD PSKPNGQIWT

1824. 231-250 PSKPNGQIWT GVIGSPAANA

1825. 241-260 GVIGSPAANA PDAGPPQRWF

1826. 251-270 PDAGPPQRWF VVWLGTANNP

1827. 261-280 VVWLGTANNP VDKGAAKALA

1828. 271-290 VDKGAAKALAESIRPLVAPP

1829. 281-300 ESIRPLVAPP PAPAPAPAEP

1830. 291-310 PAPAPAPAEP APAPAPAGEV

1831. 301-320 APAPAPAGEV APTPTTPTPQ

1832. 311-325 APTPTTPTPQ RTLPA

Complete amino acid sequence of Rv1860. The signal sequence is underlined.

MHQVDPNLTRRKGRLAALAIAAMASASLVTVAVPATANADPEPAPPVPTTAASPPSTAAAPPAPATPVAPPPPAAANTPNAQPGDPNAAPPPADPNAPPPPVIAPNAPQPVRIDNPVGGFSFALPAGWVESDAAHFDYGSALLSKTTGDPPFPGQPPPVANDTRIVLGRLDQKLYASAEATDSKAAARLGSDMGEFYMPYPGTRINQETVSLDANGVSGSASYYEVKFSDPSKPNGQIWTGVIGSPAANAPDAGPPQRWFVVWLGTANNPVDKGAAKALAESIRPLVAPPPAPAPAPAEPAPAPAPAGEVAPTPTTPTPQRTLPA

Supplementary Table S2: Volunteer Information

Healthy volunteers

(HV)

TB patients

(PAT) p valueN 28 20

Male

Female

19 (68%)

9 (32%)

17 (85%)

3 (15%)

0.16a

0.16

Age: Male

Female

41 ± 7 years

39 ± 6 years

47 ± 8 years

37 ± 6 years

0.007b

0.24b

Weight: Male

Female

57 ± 7 Kg

47 ± 4 Kg

54 ± 6 Kg

45 ± 5 Kg

0.11b

0.14b

a Both the Z test as well as Fisher's exact test indicated that the differences between theproportions of males and females were not statistically significant ( p > 0.16 ). bAge and weightof males and females were compared using the 2 tailed Student’s t test. Age, weight and sextrends for SI and cytokine responses were analyzed using logistic regression and were found notto significantly influence the outcome.

Supplementary Table S3A. HLA alleles of PPD-positive healthy volunteers.

SampleID

HLA-A HLA-B HLA-DRB1allele1 allele2 allele1 allele2 allele1 allele2

LAT1 01:01 30:01 13:02 15:18 04:03 07:01LAT2 02:06 24:02 15:02 51:01 11:01 15:01LAT3 02:11 68:01 40:04 51:01 15:01 15:02LAT4 01:01 11:01 15:17 52:01 04:03 13:02LAT5 11:01 23:01 08:01 40:01 03:07 13:01LAT6 01:01 11:01 40:04 52:01 08:03 13:01LAT7 02:11 30:01 13:01 40:06 07:01 14:04LAT8 02:03 24:02 38:02 52:01 15:01 15:02LAT9 02:01 24:07 35:03 44:03 07:01 15:02LAT10 01:01 11:01 40:06 51:06 15:01 15:02LAT11 01:01 68:01 35:01 44:03 07:01 15:02LAT12 01:01 02:11 07:05 15:12 12:15 15:01LAT13 03:01 29:01 07:02 07:05 10:01 15:01LAT14 01:01 33:01 37:01 44:03 07:01 10:01LAT15 01:01 33:01 15:17 35:01 01:12 13:01LAT16 01:01 01:01 58:01 58:01 03:01 03:06LAT17 02:01 33:01 35:04 44:03 11:01 14:04LAT18 0206 1101 1501 4403 0701 1506LAT19 02:01 24:07 15:35 58:01 03:01 14:01LAT20 02:05 11:01 50:01 51:04 11:01 15:02LAT21 02:11 26:01 07:05 08:01 03:01 07:01LAT22 01:01 01:01 40:06 57:01 07:01 07:01LAT23 03:01 68:01 15:18 50:01 07:01 07:01LAT24 01:01 24:02 44:03 57:01 07:01 07:01LAT25 02:01 03:01 07:18 51:04 10:01 15:02LAT26 01:01 29:01 07:18 15:25 15:01 15:01LAT27 24:02 29:01 40:50 51:06 07:01 15:02LAT28 02:01 31:01 35:03 51:01 04:01 15:02LAT29 33:01 33:01 44:03 51:01 03:01 15:01LAT30 2402 6801 3501 4006 1101 1501

Supplementary Table S3B. HLA alleles of TB patients.

SampleID

HLA-A HLA-B HLA-DR

allele1 allele2 allele1 allele2 allele1 allele2TB5 02:01 11:01 07:02 40:09 08:03 14:39TB6 02:01 02:06 08:33 13:01 04:01 14:04TB7 01:01 32:01 07:18 57:01 07:01 15:28TB8 24:02 33:01 40:04 44:03 07:01 15:04TB10 01:01 11:01 52:01 57:01 07:01 15:01TB11 24:02 33:01 07:18 07:18 07:01 10:01TB12 02:01 02:11 07:05 40:02 07:01 12:02TB13 01:01 24:02 40:04 51:01 01:01 04:05TB14 02:01 31:01 51:01 52:01 01:01 04:05TB15 11:01 26:01 18:02 51:04 14:04 14:04TB21 02:01 33:01 44:03 44:03 07:01 07:01TB22 33:01 33:01 27:03 27:04 09:01 14:04TB23 11:01 24:02 27:03 51:01 13:23 15:01TB24 01:01 11:01 15:02 57:01 07:01 12:02TB25 01:01 11:01 18:01 57:01 07:01 14:04TB26 02:11 68:01 40:04 52:01 13:01 14:01TB27 02:06 68:01 48:01 52:01 03:01 08:03TB28 33:01 68:01 52:01 58:01 03:01 15:02TB29 02:01 33:07 35:03 35:03 04:03 14:04TB30 11:09 33:07 44:03 51:01 04:04 07:01

Supplementary Figure S1(A). Gating strategy for analysis of flow cytometry data. FlowJo

software (Treestar) was used. Small lymphocytes were gated on the forward versus side scatter

plot followed by sequential gating on singlet cells and CD3-high cells. These were resolved into

CD8-high and CD8-low (CD4+). Production of IFN-γ (PE-Cy7), IL-2 (FITC), TNF-α (APC) and

MIP-1β (PE) were then determined for each subset as shown. Gates were positioned to ensure

that the percentages of fully stained unstimulated cells were ≤0.01 % of total CD4+ or CD8+ T

cells for IFN-γ, TNF-α and IL-2 secreting T cells, while it was ≤0.05 for MIP-1β secreting T

cells and these values were deducted from the percentage values for peptide stimulated cells.

Supplementary Figure S1 (B). Whole blood from a representative HV and PAT, stimulated

with the peptides 1803 and 1820, respectively, was processed as described in Methods.

Unstimulated and peptide-stimulated cells were stained with the panel of antibodies described in

the Methods section and data acquired using BD FACS-DIVA software for detecting

intracellular cytokines IFN-γ (PE-Cy7), IL-2 (FITC), TNF-α (APC) and MIP-1β (PE) from

CD8+ (PerCP-high) and CD4+ (PerCP-low) T cells are shown.

Supplementary Figure S2. Polychoromatic flow cytometry analysis of cytokine production

profiles of peptide-specific T cells. Whole blood ICC samples stimulated with a mixture of

peptides 1803, 1821 and 1826 were stained and analyzed as described in methods. Percentage of

CD4+ T cell subsets secreting the different combinations of cytokines IFN-γ, IL-2, TNF-α and

MIP-1β indicated below the pairs of bars as +/-, by healthy volunteers (HV, blue dots, left bar in

each pair) were compared with TB patients (PAT, red dots, right bar in each pair). Boxes

represent the 25th and 75th percentile values while bars denote median. Dots represent individual

values. Following Bonferroni’s correction for multiple comparisons, none of the p values

computed by the non-parametric Wilcoxon test available within SPICE on log-transformed data

for difference between PAT and HV (#) were significant (p<0.0033).

Supplementary Figure S3. Polychoromatic flow cytometry analysis of cytokine production

profiles of peptide-specific T cells. Whole blood ICC samples stimulated with a mixture of

peptides 1803, 1821 and 1826 were stained and analyzed as described in methods. Percentage of

CD8+ T cells secreting the different combinations of cytokines IFN-γ, IL-2, TNF-α and MIP-1β

indicated below the pairs of bars as +/-, by healthy volunteers (HV, blue dots, left bar in each

pair) were compared with TB patients (PAT, red dots, right bar in each pair). Boxes represent the

25th and 75th percentile values while bars denote median. Dots represent individual values. P

values computed by the non-parametric Wilcoxon test available within SPICE on log-

transformed data for significant difference between PAT and HV (#) that remained significant

after Bonferroni’s correction was applied (p<0.0033) are given above the bars (*).

Supplementary Figure S4. Polyfunctional T cell response to secreted antigen CFP-10. The plots

show comparison of frequency of CD4+ (blue dots, left bar in each pair) with CD8+ (red dots,

right bar in each pair) T cell subsets secreting the different combinations of cytokines IFN-γ, IL-

2, TNF-α and MIP-1β indicated below the pairs of bars as +/-, by HV (upper panel) and PAT

(lower panel). P values computed by the non-parametric Wilcoxon test available within SPICE

on log-transformed data for significant difference between PAT and HV (#) that remained

significant after Bonferroni’s correction was applied (p<0.0033) are given above the bars (*).

Supplementary Figure S5. Polyfunctional T cell response to secreted antigens ESAT-6, CFP-

10, Ag85A and Ag85B of MTB. The average response to the four antigens ESAT-6, CFP-10,

Ag85A and Ag85B was computed in HV (upper panel) and PAT (lower panel) and compared

between CD4+ (blue dots, left bar in each pair) and CD8+ (red dots, right bar in each pair) T cell

cell subsets secreting the different combinations of cytokines IFN-γ, IL-2, TNF-α and MIP-1β

indicated below the pairs of bars as +/-. P values computed by the non-parametric Wilcoxon test

available within SPICE on log-transformed data for significant difference between PAT and HV

(#) that remained significant after Bonferroni’s correction was applied (p<0.0033) are indicated

above the bars (*).

Supplementary Figure S6. Polyfunctional T cell response to secreted antigens ESAT-6, CFP-

10, Ag85A and Ag85B of MTB. The average response to the four antigens was computed for

CD4+ (upper panel) and CD8+ (lower panel) T cell cell subsets secreting the different

combinations of cytokines IFN-γ, IL-2, TNF-α and MIP-1β indicated below the pairs of bars as

+/-, and compared between HV (blue dots, left bar in each pair) and PAT (red dots, right bar in

each pair). P values computed by the non-parametric Wilcoxon test available within SPICE on

log-transformed data for significant difference between PAT and HV (#) that remained

significant after Bonferroni’s correction was applied (p<0.0033) are indicated above the bars (*).

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