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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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