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Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs.

Tungatt K, Bianchi V, Crowther MD, Powell WE, Schauenburg AJ, Trimby A, Donia M, Miles JJ, Holland CJ, Cole DK, Godkin AJ, Peakman M, Straten PT, Svane IM, Sewell AK, Dolton G - J. Immunol. (2014)

Bottom Line: In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity.Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents.We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, Wales, United Kingdom;

No MeSH data available.


Related in: MedlinePlus

Ex vivo staining and detection of T cells is improved by the addition of an anti-fluorochrome and conjugated secondary Ab to standard pMHC multimer staining protocols. (A) A T cell line primed with GILGFVFTL peptide from the influenza virus (flu) was treated with PKI and stained with cognate HLA-A2 PE-conjugated cognate and control (HLA-A2-RLGPTLMCL from MG50 protein) tetramers (Tet), alone or in combination with anti-PE unconjugated 1° Ab ± a PE-conjugated 2° Ab. (B) TILs from an HLA-A2+ metastatic melanoma patient were treated with PKI and stained with HLA-A2–ELAGIGILTV (Melan-A) or HLA-A2–ALWGPDPAAA (PPI) PE-conjugated tetramers and Abs as in (A). (C) The staining described in (B) was performed on TILs that had been cultured with autologous tumor for 5 d. (D) HLA-A2+ PBMCs taken directly ex vivo were incubated ± PKI and stained with HLA-A2–ELAGIGILTV or HLA-A2–ILAKFLHWL (hTERT) PE-conjugated tetramers or dextramers (Dex) and Abs as described in (A). Samples were minimally stained for viability, CD3, and CD8, with CD14 and CD19 also being stained in (C). The tetramer+ cells are expressed as a percentage of total cells (A and B) or CD8+ cells (C) and the MFIs are shown (inset).
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fig05: Ex vivo staining and detection of T cells is improved by the addition of an anti-fluorochrome and conjugated secondary Ab to standard pMHC multimer staining protocols. (A) A T cell line primed with GILGFVFTL peptide from the influenza virus (flu) was treated with PKI and stained with cognate HLA-A2 PE-conjugated cognate and control (HLA-A2-RLGPTLMCL from MG50 protein) tetramers (Tet), alone or in combination with anti-PE unconjugated 1° Ab ± a PE-conjugated 2° Ab. (B) TILs from an HLA-A2+ metastatic melanoma patient were treated with PKI and stained with HLA-A2–ELAGIGILTV (Melan-A) or HLA-A2–ALWGPDPAAA (PPI) PE-conjugated tetramers and Abs as in (A). (C) The staining described in (B) was performed on TILs that had been cultured with autologous tumor for 5 d. (D) HLA-A2+ PBMCs taken directly ex vivo were incubated ± PKI and stained with HLA-A2–ELAGIGILTV or HLA-A2–ILAKFLHWL (hTERT) PE-conjugated tetramers or dextramers (Dex) and Abs as described in (A). Samples were minimally stained for viability, CD3, and CD8, with CD14 and CD19 also being stained in (C). The tetramer+ cells are expressed as a percentage of total cells (A and B) or CD8+ cells (C) and the MFIs are shown (inset).

Mentions: We next compared pMHC multimer staining of a T cell line, TILs, and PBMC samples using the following conditions: 1) pMHC multimer alone (test 1, Fig. 1); 2) pMHC multimer + anti-PE unconjugated 1° Ab (control 1, Fig. 1); and 3) pMHC multimer + the 1° Ab + anti-Ab PE-conjugated 2° Ab (test 2, Fig. 1) (Fig. 5). Fig. 5A shows classic tetramer staining of an HLA-A2–restricted influenza matrix-specific T cell line. As expected, the cognate CD8+ T cells in this antiviral line stain well with tetramer. Nevertheless, inclusion of a 1° Ab during staining almost doubled the MFI and resulted in recovery of a ∼25% greater population of cells. Further inclusion of a 2° Ab resulted in a further minor increase in both MFI and percent population recovered. We next applied the same conditions in the presence of PKI for staining of HLA-A2–ELAGIGILTV-specific cells in TILs expanded from a melanoma lesion (Fig. 5B). A total of 2.3% of the cells in these TILs stained with Melan-A–specific pMHC tetramer. The size of this population almost doubled when 1° Ab was included in the protocol. The population recovered increased from 3.9 to 4.9% when a 2° Ab was also included. In an independent assay using the same TILs, the Melan-A specific T cell population segregated into two clean populations when 1° and 2° Abs were included with tetramer (Supplemental Fig. 1A). The VB6G4.24 T cell clone shown in Fig. 4A was cloned from these TILs and is effective at killing patient autologous tumors. This clone does not stain with pMHC tetramer (Fig. 4A), so we assume that this clone is one of the T cell clonotypes that fails to stain using tetramer alone in the presence of PKI in Fig. 5B. Importantly, staining can be recovered when 1° Ab and 1° + 2° Abs were included in the staining protocol. Enhanced tetramer staining was also seen when tumor-specific T cells were relatively abundant. The aforementioned TILs were enriched for Melan-A–specific cells by coculture with autologous tumor for 5 d. Twice as many cells were stained with Melan-A tetramers when 1° and 2° Abs (9.4% versus 18.9%) were included, which represents a considerable increase in the number of T cells being detected (Fig. 5C). Thus, pMHC tetramer staining in the absence of Ab stabilization can fail to recover effective cognate CD8+ T cells resulting in a large underestimation of the size of an Ag-specific T cell population. This large underestimation of effective, Ag-specific CD8+ T cells with pMHC I tetramer is in accordance with a previous study that showed that most Ag-specific CD4+ T cells could not be detected by pMHC II tetramer staining of ex vivo samples (13).


Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs.

Tungatt K, Bianchi V, Crowther MD, Powell WE, Schauenburg AJ, Trimby A, Donia M, Miles JJ, Holland CJ, Cole DK, Godkin AJ, Peakman M, Straten PT, Svane IM, Sewell AK, Dolton G - J. Immunol. (2014)

Ex vivo staining and detection of T cells is improved by the addition of an anti-fluorochrome and conjugated secondary Ab to standard pMHC multimer staining protocols. (A) A T cell line primed with GILGFVFTL peptide from the influenza virus (flu) was treated with PKI and stained with cognate HLA-A2 PE-conjugated cognate and control (HLA-A2-RLGPTLMCL from MG50 protein) tetramers (Tet), alone or in combination with anti-PE unconjugated 1° Ab ± a PE-conjugated 2° Ab. (B) TILs from an HLA-A2+ metastatic melanoma patient were treated with PKI and stained with HLA-A2–ELAGIGILTV (Melan-A) or HLA-A2–ALWGPDPAAA (PPI) PE-conjugated tetramers and Abs as in (A). (C) The staining described in (B) was performed on TILs that had been cultured with autologous tumor for 5 d. (D) HLA-A2+ PBMCs taken directly ex vivo were incubated ± PKI and stained with HLA-A2–ELAGIGILTV or HLA-A2–ILAKFLHWL (hTERT) PE-conjugated tetramers or dextramers (Dex) and Abs as described in (A). Samples were minimally stained for viability, CD3, and CD8, with CD14 and CD19 also being stained in (C). The tetramer+ cells are expressed as a percentage of total cells (A and B) or CD8+ cells (C) and the MFIs are shown (inset).
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fig05: Ex vivo staining and detection of T cells is improved by the addition of an anti-fluorochrome and conjugated secondary Ab to standard pMHC multimer staining protocols. (A) A T cell line primed with GILGFVFTL peptide from the influenza virus (flu) was treated with PKI and stained with cognate HLA-A2 PE-conjugated cognate and control (HLA-A2-RLGPTLMCL from MG50 protein) tetramers (Tet), alone or in combination with anti-PE unconjugated 1° Ab ± a PE-conjugated 2° Ab. (B) TILs from an HLA-A2+ metastatic melanoma patient were treated with PKI and stained with HLA-A2–ELAGIGILTV (Melan-A) or HLA-A2–ALWGPDPAAA (PPI) PE-conjugated tetramers and Abs as in (A). (C) The staining described in (B) was performed on TILs that had been cultured with autologous tumor for 5 d. (D) HLA-A2+ PBMCs taken directly ex vivo were incubated ± PKI and stained with HLA-A2–ELAGIGILTV or HLA-A2–ILAKFLHWL (hTERT) PE-conjugated tetramers or dextramers (Dex) and Abs as described in (A). Samples were minimally stained for viability, CD3, and CD8, with CD14 and CD19 also being stained in (C). The tetramer+ cells are expressed as a percentage of total cells (A and B) or CD8+ cells (C) and the MFIs are shown (inset).
Mentions: We next compared pMHC multimer staining of a T cell line, TILs, and PBMC samples using the following conditions: 1) pMHC multimer alone (test 1, Fig. 1); 2) pMHC multimer + anti-PE unconjugated 1° Ab (control 1, Fig. 1); and 3) pMHC multimer + the 1° Ab + anti-Ab PE-conjugated 2° Ab (test 2, Fig. 1) (Fig. 5). Fig. 5A shows classic tetramer staining of an HLA-A2–restricted influenza matrix-specific T cell line. As expected, the cognate CD8+ T cells in this antiviral line stain well with tetramer. Nevertheless, inclusion of a 1° Ab during staining almost doubled the MFI and resulted in recovery of a ∼25% greater population of cells. Further inclusion of a 2° Ab resulted in a further minor increase in both MFI and percent population recovered. We next applied the same conditions in the presence of PKI for staining of HLA-A2–ELAGIGILTV-specific cells in TILs expanded from a melanoma lesion (Fig. 5B). A total of 2.3% of the cells in these TILs stained with Melan-A–specific pMHC tetramer. The size of this population almost doubled when 1° Ab was included in the protocol. The population recovered increased from 3.9 to 4.9% when a 2° Ab was also included. In an independent assay using the same TILs, the Melan-A specific T cell population segregated into two clean populations when 1° and 2° Abs were included with tetramer (Supplemental Fig. 1A). The VB6G4.24 T cell clone shown in Fig. 4A was cloned from these TILs and is effective at killing patient autologous tumors. This clone does not stain with pMHC tetramer (Fig. 4A), so we assume that this clone is one of the T cell clonotypes that fails to stain using tetramer alone in the presence of PKI in Fig. 5B. Importantly, staining can be recovered when 1° Ab and 1° + 2° Abs were included in the staining protocol. Enhanced tetramer staining was also seen when tumor-specific T cells were relatively abundant. The aforementioned TILs were enriched for Melan-A–specific cells by coculture with autologous tumor for 5 d. Twice as many cells were stained with Melan-A tetramers when 1° and 2° Abs (9.4% versus 18.9%) were included, which represents a considerable increase in the number of T cells being detected (Fig. 5C). Thus, pMHC tetramer staining in the absence of Ab stabilization can fail to recover effective cognate CD8+ T cells resulting in a large underestimation of the size of an Ag-specific T cell population. This large underestimation of effective, Ag-specific CD8+ T cells with pMHC I tetramer is in accordance with a previous study that showed that most Ag-specific CD4+ T cells could not be detected by pMHC II tetramer staining of ex vivo samples (13).

Bottom Line: In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity.Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents.We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, Wales, United Kingdom;

No MeSH data available.


Related in: MedlinePlus