Reducing background in MHC tetramer assays: Characterization of a new

commercially available MHC tetramer for the violet laser – Brilliant!

Cheryl A. Guyre1, Peter Reinhold1, Kohei Narumiya2, Paul K. Wallace3, Dalin Pan3, Alan J. Bergeron4, Jacqueline Y. Channon4, Kazuyuki Atarashi1, Marc Delcommenne2, MBL International, 1Woburn, MA 01801, USA and 2Des Plaines, IL 60016 USA;

3Department of Flow & Image Cytometry, Roswell Park Cancer Institute, Buffalo, NY 14263 USA; 4DartLab: Immunoassay and Flow Cytometry Shared Resource at the Geisel School of Medicine at Dartmouth, Lebanon, NH 03766 USA

Figure 4. CD8 Ab clone choice and proper titration is critical for reducing artifact in mouse H-2 Kb tetramer staining. OT-I transgenic splenocytes were stained simultaneously with CD8-FITC Ab and tetramer. Anti-murine CD8 clone 53.6-7, but not clone KT15, led to some dual positive (CD8+Tet+) staining even when an irrelevant tetramer, H-2 Kb TRP2, was used. Tetramer performance improved with decreased Ab concentration.

BACKGROUND

  MHC tetramer reagents, comprised of biotinylated MHC:peptide monomers complexed with a fluorochrome-labeled streptavidin, have been used for detection of antigen-specific T cells by flow cytometry for nearly two decades (Altman, 1996).

  Commercial sources of MHC multimers are still generally limited to phycoerythrin (PE) and allophycocyanin (APC). While FITC-labeled multimers are also available, there is a need for alternative and brighter fluors to expand detection options off the 405 nm laser in multicolor flow panels.

  MBL International (MBLI) has developed high quality tetramer reagents, incorporating patented technology to reduce non-specific background, in combination with Brilliant Violet™ 421 (BV421)-conjugated streptavidin.

  We find that BV421-tetramers perform as well as or better than PE and APC tetramers to detect rare antigen-specific T cells with minimal background.

  Using BV421-labeled H-2Kb OVA (SIINFEKL) tetramers, we explored the interaction of two different murine CD8 antibody clones with mouse H-2Kb tetramers on cells isolated from the spleen and lymph nodes of OT-I transgenic mice.

Figure 2. Detection of OVA-specific T cells in OT-I transgenic splenocytes. Similar percentages of antigen-specific T cells were detected in OT-I transgenic splenocytes stained individually (A) or simultaneously (B) with H-2 Kb OVA (SIINFEKL) tetramers labeled with PE, APC, or BV421. Tetramer signal to noise ratios for OVA tetramer were 15.6, 31.0, and 15.9 for PE, APC, and BV421, respectively when tetramers were used individually (A), versus 9.5, 21.1, 9.7 when used simultaneously, reflecting the competition of the three tetramers to the same target (B). Regions were set based on TRP2 irrelevant tetramer (C).

CONCLUSIONS

  Brilliant Violet™ 421 tetramers display low background fluorescence and a signal:noise ratio favorable for rare events detection.

  In both human and mouse systems, BV421 tetramers detected similar percentages of antigen-specific T cells as the well-studied iTag™ PE and APC tetramer conjugates, originally developed at Beckman Coulter and now manufactured at MBLI.

  BV421-tetramer signal was stable on fixed cells for at least five days. Background staining was similar to PE and cleaner than APC tetramers.

  BV421 tetramers can be used in conjunction with PE and APC tetramers in the same sample.

  Anti-murine CD8 clone KT15 does not contribute to non-TCR-specific artifact observed with the more common 53.6-7 clone. CD8 antibody titration is recommended for best performance with murine H-2 Kb tetramers.

REFERENCES

Altman JD, Moss PH, Goulder PJR, Barouch DH, McHeyzer W, Bell JI, McMichael AJ, and Davis MM. 1996. Phenotypic analysis of antigen-specific T lymphocytes. Science 274:94-96.

Altman JD, Davis MM. 2003. MHC-peptide tetramers to visualize antigen-specific T cells. Curr Protoc Immunol. 17(17.3 Suppl 53): 23.

Daniels MA, Jameson SC. 2000. Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers. J Exp Med. 191:335–46.

Holman PO, Walsh ER and Jameson SC. 2005. Characterizing the impact of CD8 antibodies on Class I MHC multimer binding. J Immunol. 174:3986-3991.

McMichael AJ, and O ‘Callaghan CA. 1998. A new look at T cells. J. Exp. Med. 187:1367-1371.

Skinner PJ, Daniels MA, Schmidt CS, Jameson SC, and Haase AT. 2000. In situ tetramer staining of antigen-specific T cells in tissues. J. Immunol. 165:613-617.

Wooldridge L, Hutchinson SL, Choi EM et al. 2003. Anti-CD8 antibodies can inhibit or enhance peptide–MHC class I (pMHCI) multimer binding: this is paralleled by their effects on CTL activation and occurs in the absence of an interaction between pMHCI and CD8 on the cell surface. J Immunol. 171:6650–60.

Wooldridge L, Scriba TJ, Milicic A, Laugel B, Gostick E, Price DA, Phillips RE, Sewell AK. 2006. Anti-co-receptor antibodies profoundly affect staining with peptide–MHC class I and class II tetramers. Eur J Immunol. 36:1847–55.

Wooldridge L, Lissina A, Cole DK, van den Berg HA, Price DA, and Sewell AK. 2009. Tricks with tetramers: how to get the most from multimeric peptide–MHC. Immunology. 126:147-64.

Gating Strategies and Data Analyses

CD8

Irrelevant TCR HLA/peptide

complex

Fluorochrome

CD8

Specific TCR α-3

mutation

HLA/peptide complex

Fluorochrome

MBLI Class I Tetramer Wild-Type Class I Tetramer

Materials and Methods Whole Blood (lyse-wash) 1.  Add tetramer, CD8-FITC, and CD3-PC7 to each flow tube. 2.  Add 200 µL of EDTA-anti-coagulated whole blood. 3.  Vortex gently and incubate 30 minutes @ RT, protected from light. 4.  Lyse with 2 mL of VersaLyse™ (Beckman Coulter) supplemented with 0.2% formaldehyde. 5.  Incubate at least 10 minutes @ RT, protected from light. 6.  Centrifuge tubes at 150 x g for 5 minutes. 7.  Wash with 3 mL of FACS buffer and reconstitute with 0.5 mL of 0.1% formaldehyde in PBS.

Cell suspension 1.  Add tetramer and CD8-FITC to each flow tube. 2.  Add 100 µL cells (1x106 human PBMC*, 5x105 OT-I splenocytes, or 2x105 Mart1-Jurkat cells). 3.  Vortex gently and incubate at least 10 minutes @RT, protected from light. 4.  Wash with 3 mL of FACS buffer and reconstitute with 0.5 mL of 0.1% formaldehyde in PBS. *PBMC were first stained with Zombie Yellow™ (BioLegend) for viability gating.

Acquisition and Analysis   Samples were acquired on Gallios™(Beckman Coulter) with parameters for double discrimination enabled.

(BV421 tetramer performance was additionally validated on FACSCanto™ and LSRII (BD Biosciences). Data not shown.)   Twenty-five thousand CD3 events (blood); 250,000 ungated events (PBMCs); 50,000 ungated events (OT-I

cells); or 10,000 ungated events (Mart-1 Jurkat cells) were acquired on high flow rate.   Analysis was done using Kaluza™ software (Beckman Coulter).

Human Tetramers   Negative Tetramer   HLA-A*02:01 Mart1 (ELAGIGILTV)   HLA-A*02:01 EBV (GLCTLVAML)

Mouse Tetramers   H-2 Kb OVA (SIINFEKL)   H-2 Kb TRP2 (SVYDFFVWL)

Target cells   PBMC or whole blood from donors positive or negative for Epstein Barr Virus (EBV)   Jurkat cells expressing the TCR specific for Mart1 peptide in context of HLA-A*02:01   OT-I transgenic splenocytes expressing the TCR specific for OVA peptide in the context of H-2 Kb

A.

B.

C.

Figure 1. Detection of EBV-specific T cells in donor PMBCs. Similar percentages of antigen-specific T cells were detected in PMBCs from an EBV+ donor stained with HLA-A*02:01 EBV (GLCTLVAML) tetramer labeled with APC or BV421. Tetramer signal to noise ratios for EBV tetramer was 25.5 for APC and 23.1 for BV421. Fewer background events were observed in the BV421 tetramer sample compared with the APC tetramer sample.

PE APC BV421 Neg

BV421

APC

EBV

0

5

10

15

20

25

5 µg/mL 0.5 µg/mL 0.05 µg/mL

Sign

al/N

oise

Final tetramer concentration

Day 0 Day 5

Figure 3. BV421 signal is stable for at least 5 days. Jurkat cells stably expressing the TCR for Mart1 peptide in the context of HLA-A*02:01 were stained with CD8-FITC and Mart1-BV421 (ELAGIGILTV) tetramer. Cells were fixed and acquired immediately post-fixation. Leftover samples were re-acquired five days later. Adjustments were needed for FSC/SSC gating, as predicted, but BV421 fluorescence was stable. The average signal/noise ratio (+/- SD) for three replicates is shown, as well as representative dot plots.

Figure 5. BV421 tetramers detect rare populations of antigen-specific T cells in whole blood. Blood from an EBV+ donor was diluted into EBV- blood and stained with CD3-PC7, CD8-FITC, and HLA-A*02:01 EBV (GLCTLVAML) or Negative tetramer-BV421. EBV tetramer positive cells were detected in samples diluted as far as 1:8, with a limit of detection of ~0.08% of CD3+ cells or 0.2% of CD8hi cells.

Irrelevant Tetramer OVA Tetramer Irrelevant Tetramer OVA Tetramer

10 µL Ab

2 µL Ab

0.4 µL Ab

Anti-mouse CD8 clone 53-6.7 Anti-mouse CD8 clone KT15

Blood Sample

µg/mL Tet final

CD8hiTet+ MFI

CD8hiTet- MFI Signal:Noise

CD8hiTet+ CV

% of CD3+ cells

% of CD8hi cells

EBV Tet events

Neg Tet events

Neat EBV+ 1.25 52.6 0.61 85.9 64.9 0.53 3.63 689 4 2.5 61.1 0.63 97.3 59.9 0.51 3.54 676 8 5 60.0 0.64 93.2 61.3 0.53 3.69 683 19

EBV+ 1:2 1.25 54.6 0.57 96.5 61.0 0.30 0.93 192 2.5 58.9 0.59 99.7 59.7 0.24 0.74 148 5 65.0 0.61 105.9 60.8 0.30 0.91 191

EBV+ 1:4 1.25 44.4 0.56 79.6 77.0 0.13 0.34 70 2.5 48.5 0.60 80.6 68.8 0.12 0.30 60 5 50.5 0.60 84.6 85.1 0.14 0.36 73

EBV+ 1:8 1.25 60.8 0.57 106.0 72.4 0.09 0.22 42 2.5 40.9 0.59 69.1 74.6 0.08 0.20 40 5 26.6 0.62 42.8 105.6 0.11 0.26 54

EBV+ 1:16 1.25 28.6 0.58 49.5 105.5 0.03 0.08 16 2.5 40.4 0.62 64.7 95.3 0.04 0.09 18 5 4.6 0.62 7.4 119.2 0.06 0.13 27

Neat EBV- 1.25 2.2 0.59 3.8 47.4 0.01 0.01 3 0 2.5 2.3 0.59 3.9 17.4 0.02 0.04 8 8 5 3.4 0.63 5.4 143.7 0.04 0.08 18 13

Neat EBV+ blood EBV+ blood diluted 1:8

Cells

Blood