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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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Investigation into the amount of rhIFN-γ needed to induce a MIG ICS response. Increasing concentrations of rhIFN-γ were added to PBMC from 6 donors. The cells were then incubated for 6 hours before and after the addition of brefeldin A. The data are presented as the mean percentage of MIG+ cells within the CD14+ population ± standard deviation (SD) (fig. 4A). A significant difference in MIG secretion was seen between the media and 0.01 ng/ml rhIFN-γ (P=0.005 -Student’s paired T-test), and all the higher concentrations of rhIFN-γ. Dot plots of one volunteer’s MIG responses to rhIFN-γ are shown in figure 4B. Firstly dead cells were excluded with 7AAD dead cell marker. Secondly the CD14+ cells were then selected against forward scatter. The CD14+ population is shown in green in plot (i). The following plots (ii-vi) show the MIG production within the CD14+ population. Responses to media (ii), 0.0001 ng/ml rhIFN-γ (iii), 0.001 ng/ml rhIFN-γ (iv), 0.01 ng/ml rhIFN-γ (v) and 0.1 ng/ml rhIFN-γ (vi) are shown.
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fig4: Investigation into the amount of rhIFN-γ needed to induce a MIG ICS response. Increasing concentrations of rhIFN-γ were added to PBMC from 6 donors. The cells were then incubated for 6 hours before and after the addition of brefeldin A. The data are presented as the mean percentage of MIG+ cells within the CD14+ population ± standard deviation (SD) (fig. 4A). A significant difference in MIG secretion was seen between the media and 0.01 ng/ml rhIFN-γ (P=0.005 -Student’s paired T-test), and all the higher concentrations of rhIFN-γ. Dot plots of one volunteer’s MIG responses to rhIFN-γ are shown in figure 4B. Firstly dead cells were excluded with 7AAD dead cell marker. Secondly the CD14+ cells were then selected against forward scatter. The CD14+ population is shown in green in plot (i). The following plots (ii-vi) show the MIG production within the CD14+ population. Responses to media (ii), 0.0001 ng/ml rhIFN-γ (iii), 0.001 ng/ml rhIFN-γ (iv), 0.01 ng/ml rhIFN-γ (v) and 0.1 ng/ml rhIFN-γ (vi) are shown.

Mentions: It has been shown previously that MIG is induced by IFN-γ (Farber, 1990, 1993; Amichay et al., 1996). PBMC from 6 different donors were stimulated with increasing concentrations of rhIFN-γ. Stimulation with rhIFN-γ induced MIG expression in a dose dependent manner (Fig. 4A and B). A significant number of MIG secreting cells can be detected above background from as little as 0.01 ng/ml rhIFN-γ. The standard deviation between the 6 individuals tested was small and the ability of CD14+ cells to respond to IFN-γ in PBMC cell culture is reproducible between individuals.


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)

Investigation into the amount of rhIFN-γ needed to induce a MIG ICS response. Increasing concentrations of rhIFN-γ were added to PBMC from 6 donors. The cells were then incubated for 6 hours before and after the addition of brefeldin A. The data are presented as the mean percentage of MIG+ cells within the CD14+ population ± standard deviation (SD) (fig. 4A). A significant difference in MIG secretion was seen between the media and 0.01 ng/ml rhIFN-γ (P=0.005 -Student’s paired T-test), and all the higher concentrations of rhIFN-γ. Dot plots of one volunteer’s MIG responses to rhIFN-γ are shown in figure 4B. Firstly dead cells were excluded with 7AAD dead cell marker. Secondly the CD14+ cells were then selected against forward scatter. The CD14+ population is shown in green in plot (i). The following plots (ii-vi) show the MIG production within the CD14+ population. Responses to media (ii), 0.0001 ng/ml rhIFN-γ (iii), 0.001 ng/ml rhIFN-γ (iv), 0.01 ng/ml rhIFN-γ (v) and 0.1 ng/ml rhIFN-γ (vi) are shown.
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fig4: Investigation into the amount of rhIFN-γ needed to induce a MIG ICS response. Increasing concentrations of rhIFN-γ were added to PBMC from 6 donors. The cells were then incubated for 6 hours before and after the addition of brefeldin A. The data are presented as the mean percentage of MIG+ cells within the CD14+ population ± standard deviation (SD) (fig. 4A). A significant difference in MIG secretion was seen between the media and 0.01 ng/ml rhIFN-γ (P=0.005 -Student’s paired T-test), and all the higher concentrations of rhIFN-γ. Dot plots of one volunteer’s MIG responses to rhIFN-γ are shown in figure 4B. Firstly dead cells were excluded with 7AAD dead cell marker. Secondly the CD14+ cells were then selected against forward scatter. The CD14+ population is shown in green in plot (i). The following plots (ii-vi) show the MIG production within the CD14+ population. Responses to media (ii), 0.0001 ng/ml rhIFN-γ (iii), 0.001 ng/ml rhIFN-γ (iv), 0.01 ng/ml rhIFN-γ (v) and 0.1 ng/ml rhIFN-γ (vi) are shown.
Mentions: It has been shown previously that MIG is induced by IFN-γ (Farber, 1990, 1993; Amichay et al., 1996). PBMC from 6 different donors were stimulated with increasing concentrations of rhIFN-γ. Stimulation with rhIFN-γ induced MIG expression in a dose dependent manner (Fig. 4A and B). A significant number of MIG secreting cells can be detected above background from as little as 0.01 ng/ml rhIFN-γ. The standard deviation between the 6 individuals tested was small and the ability of CD14+ cells to respond to IFN-γ in PBMC cell culture is reproducible between individuals.

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