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Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8(+) T cells with detection by ELISPOT and HLA-multimer staining.

Chudley L, McCann KJ, Coleman A, Cazaly AM, Bidmon N, Britten CM, van der Burg SH, Gouttefangeas C, Jandus C, Laske K, Maurer D, Romero P, Schröder H, Stynenbosch LF, Walter S, Welters MJ, Ottensmeier CH - Cancer Immunol. Immunother. (2014)

Bottom Line: Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %.We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS.In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.

View Article: PubMed Central - PubMed

Affiliation: Cancer Sciences Unit, Faculty of Medicine, Experimental Cancer Medicine Centre, Southampton General Hospital, University of Southampton, Tremona Road, Southampton, SO16 6YD, UK, ll4@soton.ac.uk.

ABSTRACT
Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.

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Correlation of ex vivo and post-IVS and ELISPOT and multimer responses in Phase I and II The responses of all donors (1–5), where appropriate, were assessed for the existence of significant (<0.01 %) correlation: Phase I, ex vivo ELISPOT with post-IVS ELISPOT (a); ex vivo multimer with post-IVS multimer (b); ex vivo ELISPOT with ex vivo multimer (c); Phase I and II, post-IVS ELISPOT with post-IVS multimer (d). Correlations were evaluated using the Pearson’s correlation coefficient (R) and the coefficient of determination (R2), as shown on the bottom right of each graph. Significance testing used the Student’s t test and a confidence level of 99 %
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Fig4: Correlation of ex vivo and post-IVS and ELISPOT and multimer responses in Phase I and II The responses of all donors (1–5), where appropriate, were assessed for the existence of significant (<0.01 %) correlation: Phase I, ex vivo ELISPOT with post-IVS ELISPOT (a); ex vivo multimer with post-IVS multimer (b); ex vivo ELISPOT with ex vivo multimer (c); Phase I and II, post-IVS ELISPOT with post-IVS multimer (d). Correlations were evaluated using the Pearson’s correlation coefficient (R) and the coefficient of determination (R2), as shown on the bottom right of each graph. Significance testing used the Student’s t test and a confidence level of 99 %

Mentions: Phase I ex vivo and post-IVS responses detected by both ELISPOT and multimer staining were evaluated for the existence of any correlation using R2 and significance testing at 99 % confidence (Fig. 4 and Supplementary Table 7). Overall, a significant (P < 0.0001) positive correlation was observed between ex vivo ELISPOT and post-IVS ELISPOT, but was associated with a weak R2 value of 0.3312 (Fig. 4a). Upon dissection and examination at the level of individual donors, significance of this correlation was only maintained for donor 2 (R2 = 0.5411 and P = 0.0027). There was no correlation observed between ex vivo multimer and post-IVS multimer or between ex vivo ELISPOT and ex vivo multimer (Fig. 4b, c). Examination of Phase I and II post-IVS ELISPOT and multimer responses revealed a significant (P < 0.0001) positive correlation with an R2 value of 0.5113 (Fig. 4d).Fig. 4


Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8(+) T cells with detection by ELISPOT and HLA-multimer staining.

Chudley L, McCann KJ, Coleman A, Cazaly AM, Bidmon N, Britten CM, van der Burg SH, Gouttefangeas C, Jandus C, Laske K, Maurer D, Romero P, Schröder H, Stynenbosch LF, Walter S, Welters MJ, Ottensmeier CH - Cancer Immunol. Immunother. (2014)

Correlation of ex vivo and post-IVS and ELISPOT and multimer responses in Phase I and II The responses of all donors (1–5), where appropriate, were assessed for the existence of significant (<0.01 %) correlation: Phase I, ex vivo ELISPOT with post-IVS ELISPOT (a); ex vivo multimer with post-IVS multimer (b); ex vivo ELISPOT with ex vivo multimer (c); Phase I and II, post-IVS ELISPOT with post-IVS multimer (d). Correlations were evaluated using the Pearson’s correlation coefficient (R) and the coefficient of determination (R2), as shown on the bottom right of each graph. Significance testing used the Student’s t test and a confidence level of 99 %
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4209099&req=5

Fig4: Correlation of ex vivo and post-IVS and ELISPOT and multimer responses in Phase I and II The responses of all donors (1–5), where appropriate, were assessed for the existence of significant (<0.01 %) correlation: Phase I, ex vivo ELISPOT with post-IVS ELISPOT (a); ex vivo multimer with post-IVS multimer (b); ex vivo ELISPOT with ex vivo multimer (c); Phase I and II, post-IVS ELISPOT with post-IVS multimer (d). Correlations were evaluated using the Pearson’s correlation coefficient (R) and the coefficient of determination (R2), as shown on the bottom right of each graph. Significance testing used the Student’s t test and a confidence level of 99 %
Mentions: Phase I ex vivo and post-IVS responses detected by both ELISPOT and multimer staining were evaluated for the existence of any correlation using R2 and significance testing at 99 % confidence (Fig. 4 and Supplementary Table 7). Overall, a significant (P < 0.0001) positive correlation was observed between ex vivo ELISPOT and post-IVS ELISPOT, but was associated with a weak R2 value of 0.3312 (Fig. 4a). Upon dissection and examination at the level of individual donors, significance of this correlation was only maintained for donor 2 (R2 = 0.5411 and P = 0.0027). There was no correlation observed between ex vivo multimer and post-IVS multimer or between ex vivo ELISPOT and ex vivo multimer (Fig. 4b, c). Examination of Phase I and II post-IVS ELISPOT and multimer responses revealed a significant (P < 0.0001) positive correlation with an R2 value of 0.5113 (Fig. 4d).Fig. 4

Bottom Line: Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %.We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS.In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.

View Article: PubMed Central - PubMed

Affiliation: Cancer Sciences Unit, Faculty of Medicine, Experimental Cancer Medicine Centre, Southampton General Hospital, University of Southampton, Tremona Road, Southampton, SO16 6YD, UK, ll4@soton.ac.uk.

ABSTRACT
Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.

Show MeSH