Materials and Reagents

Falcon^® round-bottom 5 ml polypropylene tubes (Corning, Falcon^®, catalog number: 352063 )
Falcon^® round-bottom 5 ml polystyrene tubes with a 35 µm Cell-Strainer cap (Corning, Falcon^®, catalog number: 352235 )
APC-conjugated antigen-loaded MHC II tetramers can be obtained through the NIH Tetramer Core Facility, Emory University, USA. The tetramers are provided at a concentration in the 1-1.5 mg/ml range, in aliquots of 200 µl
APC-conjugated MHC II tetramers loaded with an irrelevant peptide (usually the CLIP peptide: PVSKMRMATPLLMQA can be obtained through the NIH Tetramer Core Facility, Emory University, USA)
Mouse-anti-human CD3 APC-eFluor^® 780 (clone UCHT1) (Affymetrix, eBioscience, catalog number: 47-0038-42 )
Mouse-anti-human CD4 BD Horizon^TM PE-CF594 (clone RPA-T4) (BD, BD Biosciences, catalog number: 562281 )
Mouse-anti-human CD8 Brilliant Violet 785^TM (clone RPA-T8) (BioLegend, catalog number: 301045 )
Mouse-anti-human CD14 VioGreen^® (clone TÜK4) (Miltenyi Biotec, catalog number: 130-096-875 )
Mouse-anti-human CD20 VioGreen^® (clone LT20) (Miltenyi Biotec, catalog number: 130-096-904 )
Fixable Viability Dye eFluor 506^® (Affymetrix, eBioscience, catalog number: 65-0866-14 )
16% paraformaldehyde (PFA) solution (Electron Microscopy Sciences, catalog number: 15710 )
Phosphate buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010023 )
RPMI 1604 GlutaMAX I^TM (Thermo Fisher Scientific, Gibco^TM, catalog number: 61870044 )
HyClone^TM fetal bovine serum (FBS) (South America), research grade (GE Healthcare, catalog number: SV30160.03 )
Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
1 M HEPES buffer (Dominique Dutscher SAS, catalog number: P05-01100P )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3912 )
Sodium azide (NaN3) 5% (w/v) (VWR, BDH^®, catalog number: BDH7465-2 )
Human AB serum (PAN-Biotech, catalog number: P30-2901 )
Complete RPMI (see Recipes)
FACS buffer (see Recipes)
Tetramer labeling buffer (see Recipes)
FACS sorting buffer (see Recipes)

Equipment

Benchtop centrifuge (Thermo Fisher Scientific, model: Sorvall^TM Legend^TM XTR )
Benchtop microcentrifuge (Eppendorf, model: 5254 R )
BD LSRFortessa^TM cell analyzer (BD, BD Biosciences, model: BD LSRFortessa^TM Cell Analyzer )
BD FACSAria II^TM flow cytometer (BD, BD Biosciences, model: BD FACSAria II^TM Cell Sorter )

Software

FACSDiva^TM version 8.0 (BD)
FlowJo^TM version 10.2 (FlowJo, LLC)

Procedure

Ex vivo tetramer labeling of CD4⁺ T cells from HIV infected patients

PBMC from HIV infected samples are either thawed from frozen vials or freshly processed from whole blood in a biosafety level-3 environment.
15-50 x 10⁶ cells are dispatched into 5 ml polypropylene tubes (5 x 10⁶ cells per tube) and washed once with 2 ml complete RPMI at 600 x g for 7 min at 4 °C.
Notes:
1. A wash entails the addition of the indicated volume of the specified washing buffer followed by centrifugation and careful removal of the supernatant.
2. A minimum of 5 x 10⁶ PBMC is required per tetramer labeling; the same number of PBMC is required for the control tetramer labeling. When scaling up, it is preferable to process multiple identical sample tubes in parallel, so that the amount of cells required for the control tube remains constant at 5 x 10⁶ PBMC.
3. Individual tubes containing 0.5 x 10⁶ cells in 50 μl FACS buffer should be prepared at this stage to be used as single stained compensation controls. These tubes should be kept at 4 °C for the duration of the tetramer labeling until the point of addition of cell surface antibodies.
Supernatants are removed and the cell pellets are re-suspended in 100 μl pre-chilled tetramer labeling buffer and placed on ice.
Solutions of MHC II tetramers loaded with either a validated HIV-1 peptide, or an irrelevant control peptide (CLIP) are spun at full speed for 5 min at 4 °C to remove protein aggregates.
Note: Spinning the MHC II tetramer solution before usage reduces background labeling.
APC-conjugated tetramers are added to the cell suspension at 1 μg per 10⁶ cells (i.e., 5 µg per tube).
Important: One tube containing 5 x 10⁶ cells has to be labeled with tetramers loaded with an irrelevant peptide (CLIP) and used as a negative control for tetramer labeling.
Tetramer labeling is performed at 0-4 °C for 60 min, protected from light.
Note: Staining under cold conditions contributes to reduction of background; however, these conditions are appropriate only for the detection of high-avidity pMHC/TCR interactions.
A mixture of antibodies targeting cell surface markers is added to each tube as follows (v/10⁶ cells): 1 μl CD3 APC-eFluor 780; 0.5 μl CD4 PE-CF594; 0.5 μl CD8 BV 785; 3 μl CD14-VioGreen; 3 μl CD20-VioGreen and 0.5 μl Fixable Viability Dye eFluor 506.
Notes:
1. Additional antibodies for cell surface phenotyping can be added at this point.
2. At this point, tubes containing 0.5 x 10⁶ cells in 50 μl FACS buffer are singly labeled with individual antibodies to be used as compensation controls. For the AmCyan-dump channel (including CD14-VioGreen; CD20-VioGreen and Fixable Viability Dye eFluor 506), a single tube containing 0.5 x 10⁶ cells in 50 μl FACS buffer is labeled with 1.5 μl CD14-VioGreen.
Cells are further incubated with the antibody mixture for 30 min at 4 °C in the dark.
For flow cytometric analysis
1. Labeled cells are washed twice with (pre-chilled) FACS buffer and fixed with 2% PFA diluted in PBS.
  Note: Fixation entails complete removal of the supernatants following centrifugation and resuspension in 400 µl of the fixation solution.
2. All cells in the tube are acquired on a flow cytometer (LSR Fortessa).
For cell sorting
1. Labeled cells are washed once with FACS sorting buffer and the cell concentration is adjusted to 10 x 10⁶ cells per ml in FACS sorting buffer.
2. Cells are filtered using a Cell-Strainer cap to obtain a single-cell suspension. Subsequently, all cells are acquired on a cell sorter (FACSAria II) installed in a microbiological safety cabinet.
Viable tetramer-labeled CD4⁺ T cells are visualized in the viable CD3⁺, CD4⁺, CD14^-, CD20^-, CD8^- lymphocyte gate (see gating strategy, Figure 1A).
Gates are set according to the negative control, consisting in CLIP-loaded tetramer labeled CD4⁺ T cells (Figure 1B).

Figure 1. Ex vivo detection of viable HIV-specific CD4⁺ T cells from patient PBMC. A. Gating strategy: The Dump channel includes the following antibodies: CD14-VioGreen, CD20-VioGreen and viability dye-eFluor 506. B. FACS plots depicting CD4⁺ T cells from an HIV-1 infected patient labeled with an HLA DRB5*0101 tetramer loaded either with a control CLIP peptide [PVSKMRMATPLLMQA] (left) or with an immunodominant HIV-1 Gag peptide [FRDYVDRFYKTLRAEQASQE] (right).

Data analysis

Flow cytometry data was analyzed with the Flowjo v10.2 software (FlowJo, LLC.).

Recipes

Complete RPMI
440 ml RPMI 1640
50 ml FBS
5 ml penicillin-streptomycin
5 ml HEPES
FACS buffer (For 1 L)
982 ml 1x PBS
10 g BSA
18 ml 5% (w/v) sodium azide
Tetramer labeling buffer (1 ml)
850 μl RPMI (no FBS)
150 μl human AB serum
FACS sorting buffer (50 ml)
49.5 ml 1x PBS
0.5 ml FBS

Acknowledgments

This protocol was adapted from Benati et al. (2016). We acknowledge funding from Agence Nationale de la Recherche sur le SIDA et les Hépatites Virales (ANRS EP36-8) and Agence Nationale de la Recherche (ANR PD1VAX).

References

Benati, D., Galperin, M., Lambotte, O., Gras, S., Lim, A., Mukhopadhyay, M., Nouel, A., Campbell, K. A., Lemercier, B., Claireaux, M., Hendou, S., Lechat, P., de Truchis, P., Boufassa, F., Rossjohn, J., Delfraissy, J. F., Arenzana-Seisdedos, F. and Chakrabarti, L. A. (2016). Public T cell receptors confer high-avidity CD4 responses to HIV controllers. J Clin Invest 126(6): 2093-2108.
Seth, N., Kaufmann, D., Lahey, T., Rosenberg, E. S. and Wucherpfennig K. W. (2005). Expansion and contraction of HIV-specific CD4 T cells with short bursts of viremia, but physical loss of the majority of these cells with sustained viral replication. J Immunol 175(10): 6948-58.