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MIG (CXCL9) is a more sensitive measure than IFN-gamma of vaccine induced T-cell responses in volunteers receiving investigated malaria vaccines.

Berthoud TK, Dunachie SJ, Todryk S, Hill AV, Fletcher HA - J. Immunol. Methods (2008)

Bottom Line: MIG secretion could provide a measure of bio-active IFN-gamma and a functional IFN-gamma signalling pathway.We report that detecting MIG by flow cytometry and by RT-PCR can be more sensitive than the detection of IFN-gamma using these methods.Measurement of MIG alongside IFN-gamma may provide a fuller picture of Th1 type responses post-vaccination.

View Article: PubMed Central - PubMed

Affiliation: University of Oxford, Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, OX3 7LJ, UK.

ABSTRACT
For many years the IFN-gamma ex vivo ELISPOT has been a major assay for assessing human T-cell responses generated by malaria vaccines. The ELISPOT assay is a sensitive assay, but an imperfect correlate of protection against malaria. Monokine induced by gamma (MIG), or CXCL9, is a chemokine induced by IFN-gamma and has the potential to provide amplification of the IFN-gamma signal. MIG secretion could provide a measure of bio-active IFN-gamma and a functional IFN-gamma signalling pathway. We report that detecting MIG by flow cytometry and by RT-PCR can be more sensitive than the detection of IFN-gamma using these methods. We also find that there is little inter-individual variability in MIG secretion when detected by flow cytometry and that the MIG assay may be used to estimate the amount of bio-active IFN-gamma present. Measurement of MIG alongside IFN-gamma may provide a fuller picture of Th1 type responses post-vaccination.

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(A) MIG ICS response to CEF peptides. (B) MIG ICS response to PPD. (C) IFN-γ ICS responses to CEF peptides. (D) IFN-γ ICS responses to PPD. Optimization of the incubation time with CD8+ viral epitopes (CEF) (panels A and C) and PPD (panels B and D) before addition of brefeldin A. Brefeldin A was added at 2 hourly intervals after addition the antigens. Samples were analysed after a further 18 h of incubation. The data is presented as the mean percentage of MIG+ cells ± S.E (panels A and B) (n = 5) and the mean percentage of IFN-γ+ cells ± S.E (panels C and D) (n = 9), with background media only wells deleted. A significant difference in the CEF stimulated samples (panel A) was seen between 0 and 10 h, 2 and 6 h, and 2 and 8 h (P = 0.002 for 0 and 10 h, P = 0.034 for 2 and 6 h, P = 0.049 for 2 and 8 h, Student's paired T-test). A significant difference in the PPD stimulated samples (panel B) was seen between 6 and 8 h (P = 0.02 Student's paired T-test). A significant difference in IFN-γ productions following CEF stimulation (panel C) was seen between 4 and 8 h, 6 and 8 h, and 4 and 12 h (P = 0.042 for 4 and 8 h, P = 0.042 for 6 and 8 h, P = 0.042 for 4 and 12 h, Wilcoxon signed rank test). A significant difference in IFN-γ production was seen in the PPD stimulated samples (panel D) was seen between 6 and 12 h (P = 0.042 Wilcoxon signed rank test).
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fig2: (A) MIG ICS response to CEF peptides. (B) MIG ICS response to PPD. (C) IFN-γ ICS responses to CEF peptides. (D) IFN-γ ICS responses to PPD. Optimization of the incubation time with CD8+ viral epitopes (CEF) (panels A and C) and PPD (panels B and D) before addition of brefeldin A. Brefeldin A was added at 2 hourly intervals after addition the antigens. Samples were analysed after a further 18 h of incubation. The data is presented as the mean percentage of MIG+ cells ± S.E (panels A and B) (n = 5) and the mean percentage of IFN-γ+ cells ± S.E (panels C and D) (n = 9), with background media only wells deleted. A significant difference in the CEF stimulated samples (panel A) was seen between 0 and 10 h, 2 and 6 h, and 2 and 8 h (P = 0.002 for 0 and 10 h, P = 0.034 for 2 and 6 h, P = 0.049 for 2 and 8 h, Student's paired T-test). A significant difference in the PPD stimulated samples (panel B) was seen between 6 and 8 h (P = 0.02 Student's paired T-test). A significant difference in IFN-γ productions following CEF stimulation (panel C) was seen between 4 and 8 h, 6 and 8 h, and 4 and 12 h (P = 0.042 for 4 and 8 h, P = 0.042 for 6 and 8 h, P = 0.042 for 4 and 12 h, Wilcoxon signed rank test). A significant difference in IFN-γ production was seen in the PPD stimulated samples (panel D) was seen between 6 and 12 h (P = 0.042 Wilcoxon signed rank test).

Mentions: Brefeldin A blocks proteins from being secreted. Brefeldin A may therefore prevent IFN-γ from being secreted and thus from inducing MIG. PBMC from 5 normal human donors were stimulated with either a mixture of viral CD8+ T-cell epitopes (CEF) (Fig. 2A) or PPD (Fig. 2B). The cells were incubated for 0, 2, 4, 6, 8 and 10 h before brefeldin A was added, and incubated for a further 18 h after brefeldin A was added.


MIG (CXCL9) is a more sensitive measure than IFN-gamma of vaccine induced T-cell responses in volunteers receiving investigated malaria vaccines.

Berthoud TK, Dunachie SJ, Todryk S, Hill AV, Fletcher HA - J. Immunol. Methods (2008)

(A) MIG ICS response to CEF peptides. (B) MIG ICS response to PPD. (C) IFN-γ ICS responses to CEF peptides. (D) IFN-γ ICS responses to PPD. Optimization of the incubation time with CD8+ viral epitopes (CEF) (panels A and C) and PPD (panels B and D) before addition of brefeldin A. Brefeldin A was added at 2 hourly intervals after addition the antigens. Samples were analysed after a further 18 h of incubation. The data is presented as the mean percentage of MIG+ cells ± S.E (panels A and B) (n = 5) and the mean percentage of IFN-γ+ cells ± S.E (panels C and D) (n = 9), with background media only wells deleted. A significant difference in the CEF stimulated samples (panel A) was seen between 0 and 10 h, 2 and 6 h, and 2 and 8 h (P = 0.002 for 0 and 10 h, P = 0.034 for 2 and 6 h, P = 0.049 for 2 and 8 h, Student's paired T-test). A significant difference in the PPD stimulated samples (panel B) was seen between 6 and 8 h (P = 0.02 Student's paired T-test). A significant difference in IFN-γ productions following CEF stimulation (panel C) was seen between 4 and 8 h, 6 and 8 h, and 4 and 12 h (P = 0.042 for 4 and 8 h, P = 0.042 for 6 and 8 h, P = 0.042 for 4 and 12 h, Wilcoxon signed rank test). A significant difference in IFN-γ production was seen in the PPD stimulated samples (panel D) was seen between 6 and 12 h (P = 0.042 Wilcoxon signed rank test).
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fig2: (A) MIG ICS response to CEF peptides. (B) MIG ICS response to PPD. (C) IFN-γ ICS responses to CEF peptides. (D) IFN-γ ICS responses to PPD. Optimization of the incubation time with CD8+ viral epitopes (CEF) (panels A and C) and PPD (panels B and D) before addition of brefeldin A. Brefeldin A was added at 2 hourly intervals after addition the antigens. Samples were analysed after a further 18 h of incubation. The data is presented as the mean percentage of MIG+ cells ± S.E (panels A and B) (n = 5) and the mean percentage of IFN-γ+ cells ± S.E (panels C and D) (n = 9), with background media only wells deleted. A significant difference in the CEF stimulated samples (panel A) was seen between 0 and 10 h, 2 and 6 h, and 2 and 8 h (P = 0.002 for 0 and 10 h, P = 0.034 for 2 and 6 h, P = 0.049 for 2 and 8 h, Student's paired T-test). A significant difference in the PPD stimulated samples (panel B) was seen between 6 and 8 h (P = 0.02 Student's paired T-test). A significant difference in IFN-γ productions following CEF stimulation (panel C) was seen between 4 and 8 h, 6 and 8 h, and 4 and 12 h (P = 0.042 for 4 and 8 h, P = 0.042 for 6 and 8 h, P = 0.042 for 4 and 12 h, Wilcoxon signed rank test). A significant difference in IFN-γ production was seen in the PPD stimulated samples (panel D) was seen between 6 and 12 h (P = 0.042 Wilcoxon signed rank test).
Mentions: Brefeldin A blocks proteins from being secreted. Brefeldin A may therefore prevent IFN-γ from being secreted and thus from inducing MIG. PBMC from 5 normal human donors were stimulated with either a mixture of viral CD8+ T-cell epitopes (CEF) (Fig. 2A) or PPD (Fig. 2B). The cells were incubated for 0, 2, 4, 6, 8 and 10 h before brefeldin A was added, and incubated for a further 18 h after brefeldin A was added.

Bottom Line: MIG secretion could provide a measure of bio-active IFN-gamma and a functional IFN-gamma signalling pathway.We report that detecting MIG by flow cytometry and by RT-PCR can be more sensitive than the detection of IFN-gamma using these methods.Measurement of MIG alongside IFN-gamma may provide a fuller picture of Th1 type responses post-vaccination.

View Article: PubMed Central - PubMed

Affiliation: University of Oxford, Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, OX3 7LJ, UK.

ABSTRACT
For many years the IFN-gamma ex vivo ELISPOT has been a major assay for assessing human T-cell responses generated by malaria vaccines. The ELISPOT assay is a sensitive assay, but an imperfect correlate of protection against malaria. Monokine induced by gamma (MIG), or CXCL9, is a chemokine induced by IFN-gamma and has the potential to provide amplification of the IFN-gamma signal. MIG secretion could provide a measure of bio-active IFN-gamma and a functional IFN-gamma signalling pathway. We report that detecting MIG by flow cytometry and by RT-PCR can be more sensitive than the detection of IFN-gamma using these methods. We also find that there is little inter-individual variability in MIG secretion when detected by flow cytometry and that the MIG assay may be used to estimate the amount of bio-active IFN-gamma present. Measurement of MIG alongside IFN-gamma may provide a fuller picture of Th1 type responses post-vaccination.

Show MeSH
Related in: MedlinePlus