Anti-leukemia T cells in AML

Figures & data

Table 1. T cell assays detecting antigen-specific T cells: Overview

	Assay
Patient #	Pentamer staining	ICS	CBA	IFN-γ ELISpot
1	(+) (INFL)	-	-	-
2	+ (PR3, WT1,INFL)	+(PR3: IFNγ+/TNFα-)	-	-
3	(+) (INFL)	-	+ (INFL:TNFα only)	-
4	+ (WT1)	-	-	-
5	+ (INFL)	+ (INFL:IFNγ+/ TNFα+) (PR3: IFNγ+/ TNFα-)	-	(+) (INFL)
6	-	-	-	-
7	n.d.	n.d.	-	-
8	n.d.	+ (WT1:TNF-α+/ IFNγ-) (PR3:TNF-α+/ IFNγ-)	(+) (INFL: TNF-α only) (WT1: TNF-α only)	(+) (INFL)

Antigen-specific T cell responses against PR3, WT1 and INFL as measured by different T cell assays (pentamer staining, ICS, CBA and IFN-γ ELISpot). -, negative, not detectable over background levels; (+), detectable over background level, but not meeting our predefined cut-off for specificity (see material and methods); +, positive, detectable and meeting our criteria for peptide-specific responses as defined in the material and methods section. CBA, cytometric bead array; ICS, intracytoplasmatic cytokine staining; INFL, Influenza-A; IFN-γ, Interferon-γ; n.d. not determined; PR3, proteinase-3; TNF-α, Tumor necrosis factor-α; WT1, Wilms´ tumor protein- 1.

Table 2. Cytokine secretion profile of antigen-specific T cells: Overview

	Cytokine profile of antigen-specific T cells
Patient #	Time after SCT (months)	IFN-γ^+/TNF-α^+	IFN-γ^+/ TNF-α^-	TNF-α^+/ IFN-γ^-	Pentamer^+/ Cytokine^- (a)
Antigen recognized
1	1	-	-	-	-
2	15	-	PR3 (‡)	-	WT1, INFL(§)
3	6	-	-	INFL(#)	-
4	2	-	-	-	WT1(§)
5	7	INFL(§Δ‡)	PR3 (‡)	-	-
6	9	-	-	-	-
7	2	-	-	-	-
8	2	-	-	WT1, PR3(‡)	-

Antigen-specific T cell responses against PR3, WT1 and INFL with four different cytokine secretion patterns in patients with AML after SCT. (a) indicating detection of pentamer+ T cells without peptide-specific cytokine secretion in any of the functional T cell assays (ICS, CBA and ELISpot). §, Pentamer; ‡, intracytoplasmatic cytokine staining (ICS); #, cytometric bead array (CBA); Δ IFN-γ ELISpot. INFL, Influenza-A; IFN-γ, Interferon-γ; SZT, stem cell transplantation; PR3, proteinase-3; TNF-α, Tumor necrosis factor-α; WT1, Wilms´ tumor protein-1.

Figure 1. Intracytoplasmatic cytokine staining (ICS). Intracytoplasmatic cytokine staining (IFN-γ and TNF-α) of CD8⁺ T cells, after in vitro stimulation with peptide- pulsed PBMC (patient # 8). Control, irrelevant HLA-A2+ peptide.

Figure 2. Cytometric bead array (CBA) and IFN-γ ELISpot in patients with TNF-α+/ IFN-γ- CD8⁺ T cells. TNF-α+/ IFN-γ- antigen-specific T cell responses in patients #3 and #8 as measured by CBA and IFN-γ ELISpot. *, peptide-specific cytokine secretion; **, detectable over background, but not meeting our predefined cut-off for specificity. INFL, Influenza-A; IFN-γ, Interferon-γ; PR3, proteinase-3; TNF-α, Tumor necrosis factor-α; WT1, Wilms´ tumor protein- 1.