Immunophenotyping of human CD4⁺ and CD8⁺ T cell differentiation and exhaustion using flow cytometry (panel B)

MBTP 59 = Miltenyi Biotec-tested panel 59

This application protocol describes the analysis of human CD4⁺ and CD8⁺ T cell differentiation and exhaustion markers after T cell stimulation. To demonstrate this state-of-the-art panel for T cell differentiation as well as T cell exhaustion of both CD4 T cells and CD8 T cells, PBMCs were stimulated for 96 hours using Miltenyi Biotec’s T Cell TransAct™, human. The state of differentiation and exhaustion was defined by CD279 (PD-1), CD38, HLA-DR, and CD223 expression.

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Protocol

Staining protocol

Surface staining

Volumes given below are for up to 10⁶ nucleated cells. When working with fewer than 10⁶ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×10⁶ nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).

Cell number was determined.
Cells were centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
A staining cocktail with all antibodies was prepared according to the dilutions mentioned in the respective data sheets.
100 µL staining cocktail was added to the cells.
Cells were resuspended and incubated for 10 minutes in the dark at room temperature .
Cells were washed by addition of 1 mL of PEB buffer and centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
Cells were resuspended in 100 µL of PEB buffer. 1 µL of 7-AAD Staining Solution was added and incubated for 5 minutes in the dark at room temperature right before flow cytometric analysis using the MACSQuant® Analyzer 10.

Tested antibody panel

Antibody panel for T cell differentiation and exhaustion

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
7-AAD*	–	Viability	–	655–730 (blue)
CD279 (PD-1)	REA1165	T cell differentiation and exhaustion	Vio® Bright B515	525/50 (blue)
CD4*	REA623	Helper T cells	VioGreen™	525/50 (violet)
CD8*	REA734	Cytotoxic T cells	APC-Vio 770	750 LP (red)
CD223	REA351	T cell differentiation and exhaustion	APC	655–730 (red)
CD3	REA613	T cells	VioBlue®	450/50 (violet)
CD38	REA572	T cell differentiation and exhaustion	PE	585/40 (blue)
HLA-DR	REA805	T cell differentiation and exhaustion	PE-Vio 770	750 LP (blue)

* Alternative CE-IVD/ASR reagents available.

Gating strategy

Figure 1: Gating strategy showing the identification of viable, single CD4⁺ and CD8⁺ T cells in human PBMCs. Cells were gated in an FSC-H and FSC-A dot plot to eliminate doublets (A). Lymphocytes were selected by a SSC^low gate which also excluded debris (B). Dead cells were excluded by gating on 7-AAD⁻ cells (C). CD4⁺ T cells were identified by expression of CD3 and CD4 (D). CD8⁺ T cells were identified by expression of CD3 and CD8 (E).

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Figure 2: Gating strategy for the analysis of human CD4⁺ T cell differentiation and exhaustion 96 hours after stimulation.
Among CD4⁺ T cells, the state of differentiation and exhaustion was defined by CD279 (PD-1) (A), CD38 (B), HLA-DR (C), and CD223 expression (D).

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Figure 3: Gating strategy for the analysis of human CD8⁺ T cell differentiation and exhaustion 96 hours after stimulation.
Among CD8⁺ T cells, the state of differentiation and exhaustion was defined by CD279 (PD-1) (A), CD38 (B), HLA-DR (C), and CD223 expression (D).

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CD244.1 Antibody, anti-mouse, PE-Vio
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CD244.1 Antibody, anti-mouse, Vio
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REA524

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130-108-002

MHC Class II (I-Ab) Antibody, anti-mouse, Biotin, REAfinity™

REA528

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MHC Class II (I-Ab) Antibody, anti-mouse, APC, REAfinity™

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FC, MICS, IF, IHC

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130-108-008

I-Aq Antibody, anti-mouse, Biotin, REAfinity™

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130-108-012

AD5-17F6

100 µg in 100 µL

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Materials

Reagent and instrument requirements

Antibody panel for T cell differentiation and exhaustion

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
7-AAD*	–	Viability	–	655–730 (blue)
CD279 (PD-1)	REA1165	T cell differentiation and exhaustion	Vio® Bright B515	525/50 (blue)
CD4*	REA623	Helper T cells	VioGreen™	525/50 (violet)
CD8*	REA734	Cytotoxic T cells	APC-Vio 770	750 LP (red)
CD223	REA351	T cell differentiation and exhaustion	APC	655–730 (red)
CD3	REA613	T cells	VioBlue®	450/50 (violet)
CD38	REA572	T cell differentiation and exhaustion	PE	585/40 (blue)
HLA-DR	REA805	T cell differentiation and exhaustion	PE-Vio 770	750 LP (blue)

* Alternative CE-IVD/ASR reagents available.

Additional reagents used

PEB buffer: PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA
Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343), MACSQuant X (# 130-105-100) or MACSQuant Analyzer 16 (# 130-109-803)
(Optional) T Cell TransAct, human (# 130-111-160)
(Optional) MACSQuant Calibration Beads (# 130-093-607)
(Optional) MACS® Comp Bead Kit, anti-REA (# 130-104-693)

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7-AAD Staining Solution

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HLA-DR Antibody, anti-human, PE-Vio
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CD4 Antibody, anti-human, VioGreen™, REAfinity™

REA623

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CD38 Antibody, anti-human, PE, REAfinity™

REA572

FC, MICS, IF, IHC

100

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CD3 Antibody, anti-human, VioBlue
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CD279 (PD1) Antibody, anti-human, Vio
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REA1165

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in 200 µL (1)

EUR 435,00

130-119-567

CD223 Antibody, anti-human, APC, REAfinity™

REA351

FC, MICS, IF, IHC

100

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in 200 µL (1)

EUR 315,00

Immunophenotyping of human CD4+ and CD8+ T cell differentiation and exhaustion using flow cytometry (panel B)

MBTP 59 = Miltenyi Biotec-tested panel 59

Protocol

Materials

Protocol

Surface staining

Antibody panel for T cell differentiation and exhaustion

Gating strategy

Materials

Antibody panel for T cell differentiation and exhaustion

Additional reagents used

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Immunophenotyping of human CD4⁺ and CD8⁺ T cell differentiation and exhaustion using flow cytometry (panel B)