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
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SUPPLEMENTARY DATA
Title: The secreted protein Rv1860 of Mycobacterium tuberculosis stimulates human
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.
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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