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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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Antigen-specific T-cell responses observed post-harmonised IVS in Phase II The responses of 2 donors (4 and 5) to the viral and tumour-associated antigens EBV (a), FLU (b), WT1 (c) and HIV (d) were assessed by IFNγ ELISPOT assay and multimer staining post-harmonised IVS; ex vivo data (mean of triplicate) generated by the organising centre during pre-screening are also shown. The criteria for a positive response for ELISPOT and multimer staining are as described in the Materials and Methods and Supplementary MIATA Information, ELISPOT and Multimer Module 4B. Triplicates are shown for each centre except Centre B (post-IVS multimer staining donor 4) for which only two FCS files were supplied to the organising centre for central analysis. Centre A, blue; Centre B, red; Centre C, green; Centre D, orange; Centre E, pink; Centre F, purple. Grey dashed line denotes the threshold for a positive response in ex vivo ELISPOT (20 SFC/million); black dashed line denotes the threshold for a positive response in post-IVS ELISPOT (500 SFC/million). ND, not detected
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Fig3: Antigen-specific T-cell responses observed post-harmonised IVS in Phase II The responses of 2 donors (4 and 5) to the viral and tumour-associated antigens EBV (a), FLU (b), WT1 (c) and HIV (d) were assessed by IFNγ ELISPOT assay and multimer staining post-harmonised IVS; ex vivo data (mean of triplicate) generated by the organising centre during pre-screening are also shown. The criteria for a positive response for ELISPOT and multimer staining are as described in the Materials and Methods and Supplementary MIATA Information, ELISPOT and Multimer Module 4B. Triplicates are shown for each centre except Centre B (post-IVS multimer staining donor 4) for which only two FCS files were supplied to the organising centre for central analysis. Centre A, blue; Centre B, red; Centre C, green; Centre D, orange; Centre E, pink; Centre F, purple. Grey dashed line denotes the threshold for a positive response in ex vivo ELISPOT (20 SFC/million); black dashed line denotes the threshold for a positive response in post-IVS ELISPOT (500 SFC/million). ND, not detected

Mentions: All 6 centres detected EBV-specific T cells in both donors by post-IVS ELISPOT and multimer staining (Fig. 3a); a mean detection rate of 100 and 94 % by ELISPOT and 100 and 100 % by multimer staining was observed for donors 4 and 5, respectively (Table 1b). Mean EBV-specific responses were greatest in donor 4 with 18813 SFC/million (ELISPOT) and 48.8 % CD8+EBV-multimer+ T cells (multimer), compared to 7445 SFC/million and 22.1 % CD8+EBV-multimer+ for donor 5. Overall, a combined detection rate of 97 and 100 % was observed for ELISPOT and multimer staining, respectively.Fig. 3


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)

Antigen-specific T-cell responses observed post-harmonised IVS in Phase II The responses of 2 donors (4 and 5) to the viral and tumour-associated antigens EBV (a), FLU (b), WT1 (c) and HIV (d) were assessed by IFNγ ELISPOT assay and multimer staining post-harmonised IVS; ex vivo data (mean of triplicate) generated by the organising centre during pre-screening are also shown. The criteria for a positive response for ELISPOT and multimer staining are as described in the Materials and Methods and Supplementary MIATA Information, ELISPOT and Multimer Module 4B. Triplicates are shown for each centre except Centre B (post-IVS multimer staining donor 4) for which only two FCS files were supplied to the organising centre for central analysis. Centre A, blue; Centre B, red; Centre C, green; Centre D, orange; Centre E, pink; Centre F, purple. Grey dashed line denotes the threshold for a positive response in ex vivo ELISPOT (20 SFC/million); black dashed line denotes the threshold for a positive response in post-IVS ELISPOT (500 SFC/million). ND, not detected
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Antigen-specific T-cell responses observed post-harmonised IVS in Phase II The responses of 2 donors (4 and 5) to the viral and tumour-associated antigens EBV (a), FLU (b), WT1 (c) and HIV (d) were assessed by IFNγ ELISPOT assay and multimer staining post-harmonised IVS; ex vivo data (mean of triplicate) generated by the organising centre during pre-screening are also shown. The criteria for a positive response for ELISPOT and multimer staining are as described in the Materials and Methods and Supplementary MIATA Information, ELISPOT and Multimer Module 4B. Triplicates are shown for each centre except Centre B (post-IVS multimer staining donor 4) for which only two FCS files were supplied to the organising centre for central analysis. Centre A, blue; Centre B, red; Centre C, green; Centre D, orange; Centre E, pink; Centre F, purple. Grey dashed line denotes the threshold for a positive response in ex vivo ELISPOT (20 SFC/million); black dashed line denotes the threshold for a positive response in post-IVS ELISPOT (500 SFC/million). ND, not detected
Mentions: All 6 centres detected EBV-specific T cells in both donors by post-IVS ELISPOT and multimer staining (Fig. 3a); a mean detection rate of 100 and 94 % by ELISPOT and 100 and 100 % by multimer staining was observed for donors 4 and 5, respectively (Table 1b). Mean EBV-specific responses were greatest in donor 4 with 18813 SFC/million (ELISPOT) and 48.8 % CD8+EBV-multimer+ T cells (multimer), compared to 7445 SFC/million and 22.1 % CD8+EBV-multimer+ for donor 5. Overall, a combined detection rate of 97 and 100 % was observed for ELISPOT and multimer staining, respectively.Fig. 3

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