Fig 1.
A schematic overview for measuring multimer quality by IP-FCM.
(A-B) Polyvalent mouse Ig is covalently linked to CML beads, then incubated with a tetramer specific, mouse secondary capture mAb. (C) These bead:secondary mAb conjugates are incubated with fluorescently labeled multimers to capture fluorescently labeled complexes containing MHC moieties expressing conformational antibody epitopes. (D) The binding of tetramers to antibody coated beads is assessed semi-quantitatively by flow cytometry.
Table 1.
Antibody specificities for the conformational dependent clones utilized to assess the structural integrity of both murine and human multimer reagents.
Fig 2.
The IP-FCM platform can accommodate multiple antibody isotypes in the secondary capture phase.
Antibodies of varying isotype, specific for conformational epitopes on a mouse class I tetramer were used to probe a human and mouse class I tetramer. The mouse tetramer was detected with comparable efficiency using all four isotypes tested.
Fig 3.
Fine specificity of folded H-2Kb tetramers.
Kb tetramers expressing the peptides SIINFEKL, SIINFEQL, and SIYR were assessed with a panel of negative control (B22-249.R1), cross reactive, anti-hB2M (L368), allele-specific, anti-H2-K (Y-3), and peptide-dependent/allele-specific antibodies, anti-Kb:SIINFEKL (25D1.16 and 22C5.9).
Fig 4.
Characterizing functional reactivity of Db:VP2 tetramers.
Three independently produced batches of Db:VP2 tetramers were studied to assess their ability to measure the antigen specific T cell response following TMEV infection. Brain infiltrating leukocytes were isolated from TMEV infected mice as described [12]. (A) Flow cytometric assessment with batch 1, 2, and 3 of Db:VP2 tetramers in BILs from TMEV infected mice. (B) Batch differences in detectable percentages of Db:VP2/CD8 positive BILs from infected mice. (C) Corresponding fluorescence of Db:VP2 tetramers following capture with the Db specific antibody, 28-14-8.
Fig 5.
Visualizing variability in human tetramer reagents using IP-FCM.
(A) Flow cytometric assessment of commercial batches of human tetramers using antibodies detecting conformational determinants expressed by folded MHC class I molecules. (B) Western blot quantification of monomeric heavy chains present in the tetramer reagents analyzed in panel A. (C) Absence of significant correlation between heavy chain concentration and detection of correctly folded class I molecules in the tetramers measured by IP-FCM. (D) Flow cytometric assessment of commercial batches of human pentamers by IP-FCM. (E) Comparison of two batches of HLA-A2:surviving peptide with a correctly folded commercial standard. Top row, examples of flow cytometry characterization of tetramer confonformation; bottom row, quantification of triplicate estimates with tight error estimates. (F) Negative control mouse heavy-chain tetramer showing absence of reactivity with human-specific heavy chain antibodies. (G) Detection of survivin-specific T cells in HLA-A2 transgenic mice using HLA-A2:surviving tetramer reagent and an irrelevant HLA-A2:TRP2 tetramer control.
Fig 6.
Correlation of structure and function of tetramers capture analysis.
(A) Three batches of Db:VP2 tetramer were used to assess antigen-specific CD8+ T cells from the same sample of brain infiltrating lymphocytes elicited by infection with intracranial infection with DA strain TMEV. The correlations between the efficacy of detecting antigen specific CD8 cells (from Fig 4) with the MFI using antibodies specific for β2M (L368, P = 0.296) or heavy chain (B22.249.R1 and 28-14-8 P = 0.0065) calculated using GraphPad Prism 6 are shown. (B) Seven HLA-A2 tetramers assembled with distinctive peptides were assayed with four different capture antibodies. Highly significant correlations were observed between the abilities of the heavy chain allele-specific antibody, BB7.2, and each of the pan-HLA antibodies, L368, W6/32, and MB40.5, to capture each of the tetramers (P < 0.001 for each case). No correlation was found with mouse H-2 specific antibody in the capture assay. (C) Three tetramers (HLA-A2:irrelevant peptide “-”, HLA-A2:survivin old and new from Fig 5E) were assayed using the four HLA-A2 binding antibodies BB7.2, MB40.5, W6/32, and L368. The positive control tetramer HLA-A2 (-), previously shown to bind robustly to each of the HLA-A2 capture antibodies was (Fig 5A), was used as a standard and compared to the HLA-A2:survivin tetramers. The abilities of the antibodies to capture the HLA-A2 (-) standard was highly correlated with their abilities to capture the new batch of HLA-A2:survivin tetramer (P = 0.0065), whereas there was no significant correlation in the comparison with the old batch (P = 0.296). Comparison of the two survivin tetramers revealed a significant difference in the curves, illustrating that these two reagents are different, despite their similar abilities to bind the β2M specific antibody L368.