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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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Related in: MedlinePlus

(A) shows the percentage of MIG+ cells within the CD14+7AAD-ve gate in volunteers vaccinated with the PFPFPM vaccine regimen (n=12). V3+7 are shown in black and V3+14 are shown in white. The line indicates the cut of for positivity, determined by 3 standard deviations above the mean of the media wells at the baseline time point. Five of 12 positive responses detected by the MIG ICS assay at V3+7 and 2/12 positive responses at V3+14. (B) shows the percentage of IFN-γ+ cells within CD3+ 7AAD-ve gate in volunteers vaccinated with the PFPFPM vaccine regimen. The responses at the V3+7 time point are shown in black, and V3+14 are shown in white. The line indicates the cut of for positivity, determined by 3 standard deviations above the mean of the media wells at the baseline time point. Two of 12 of the volunteers induced a positive IFN-γ ICS response at the V3+7 time point. The % of MIG+ cells detected by ICS above the media is significantly higher than the % of IFN-γ + cells detected by ICS above the media control at these time points (Wilcoxon Signed Ranks Test P=0.00039).
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fig5: (A) shows the percentage of MIG+ cells within the CD14+7AAD-ve gate in volunteers vaccinated with the PFPFPM vaccine regimen (n=12). V3+7 are shown in black and V3+14 are shown in white. The line indicates the cut of for positivity, determined by 3 standard deviations above the mean of the media wells at the baseline time point. Five of 12 positive responses detected by the MIG ICS assay at V3+7 and 2/12 positive responses at V3+14. (B) shows the percentage of IFN-γ+ cells within CD3+ 7AAD-ve gate in volunteers vaccinated with the PFPFPM vaccine regimen. The responses at the V3+7 time point are shown in black, and V3+14 are shown in white. The line indicates the cut of for positivity, determined by 3 standard deviations above the mean of the media wells at the baseline time point. Two of 12 of the volunteers induced a positive IFN-γ ICS response at the V3+7 time point. The % of MIG+ cells detected by ICS above the media is significantly higher than the % of IFN-γ + cells detected by ICS above the media control at these time points (Wilcoxon Signed Ranks Test P=0.00039).

Mentions: The optimised MIG ICS assay was then performed using PBMC from 12 volunteers vaccinated with the PFPFPM vaccine regimen. PBMC taken before the vaccination (D0), 7 days after the final vaccination (V3 + 7) and 14 days after the final vaccination (V3 + 14) were stimulated with a pool of TRAP peptides for a total of 12 h. The percentage of MIG+ cells within the CD14+ population is shown in Fig. 5A. Fig. 5B shows the antigen induced IFN-γ responses detected by ICS. A positive response in both the MIG ICS and the IFN-γ ICS was defined as more than 3 standard deviations above the mean of media alone at baseline (D0). For the IFN-γ ICS this value was 0.451% IFN-γ+ CD3+ cells, and for the MIG ICS it was 3.7%MIG+CD14+ cells. The majority of the IFN-γ ICS responses fell below the limits of detection of the assay (2 volunteers were positive at V3 + 7 time point). Five positive responses were detected by the MIG ICS assay at V3 + 7 and 2 positive responses at V3 + 14. The percentage of MIG+ cells detected by ICS above the media was also significantly higher than the percentage of IFN-γ + cells detected by ICS above the media control at these time points (Wilcoxon Signed Rank Test P = 0.00039).


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) shows the percentage of MIG+ cells within the CD14+7AAD-ve gate in volunteers vaccinated with the PFPFPM vaccine regimen (n=12). V3+7 are shown in black and V3+14 are shown in white. The line indicates the cut of for positivity, determined by 3 standard deviations above the mean of the media wells at the baseline time point. Five of 12 positive responses detected by the MIG ICS assay at V3+7 and 2/12 positive responses at V3+14. (B) shows the percentage of IFN-γ+ cells within CD3+ 7AAD-ve gate in volunteers vaccinated with the PFPFPM vaccine regimen. The responses at the V3+7 time point are shown in black, and V3+14 are shown in white. The line indicates the cut of for positivity, determined by 3 standard deviations above the mean of the media wells at the baseline time point. Two of 12 of the volunteers induced a positive IFN-γ ICS response at the V3+7 time point. The % of MIG+ cells detected by ICS above the media is significantly higher than the % of IFN-γ + cells detected by ICS above the media control at these time points (Wilcoxon Signed Ranks Test P=0.00039).
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fig5: (A) shows the percentage of MIG+ cells within the CD14+7AAD-ve gate in volunteers vaccinated with the PFPFPM vaccine regimen (n=12). V3+7 are shown in black and V3+14 are shown in white. The line indicates the cut of for positivity, determined by 3 standard deviations above the mean of the media wells at the baseline time point. Five of 12 positive responses detected by the MIG ICS assay at V3+7 and 2/12 positive responses at V3+14. (B) shows the percentage of IFN-γ+ cells within CD3+ 7AAD-ve gate in volunteers vaccinated with the PFPFPM vaccine regimen. The responses at the V3+7 time point are shown in black, and V3+14 are shown in white. The line indicates the cut of for positivity, determined by 3 standard deviations above the mean of the media wells at the baseline time point. Two of 12 of the volunteers induced a positive IFN-γ ICS response at the V3+7 time point. The % of MIG+ cells detected by ICS above the media is significantly higher than the % of IFN-γ + cells detected by ICS above the media control at these time points (Wilcoxon Signed Ranks Test P=0.00039).
Mentions: The optimised MIG ICS assay was then performed using PBMC from 12 volunteers vaccinated with the PFPFPM vaccine regimen. PBMC taken before the vaccination (D0), 7 days after the final vaccination (V3 + 7) and 14 days after the final vaccination (V3 + 14) were stimulated with a pool of TRAP peptides for a total of 12 h. The percentage of MIG+ cells within the CD14+ population is shown in Fig. 5A. Fig. 5B shows the antigen induced IFN-γ responses detected by ICS. A positive response in both the MIG ICS and the IFN-γ ICS was defined as more than 3 standard deviations above the mean of media alone at baseline (D0). For the IFN-γ ICS this value was 0.451% IFN-γ+ CD3+ cells, and for the MIG ICS it was 3.7%MIG+CD14+ cells. The majority of the IFN-γ ICS responses fell below the limits of detection of the assay (2 volunteers were positive at V3 + 7 time point). Five positive responses were detected by the MIG ICS assay at V3 + 7 and 2 positive responses at V3 + 14. The percentage of MIG+ cells detected by ICS above the media was also significantly higher than the percentage of IFN-γ + cells detected by ICS above the media control at these time points (Wilcoxon Signed Rank Test P = 0.00039).

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