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. 2013 Jul;27(7):1538-47.
doi: 10.1038/leu.2013.66. Epub 2013 Mar 1.

Generation of multi-leukemia antigen-specific T cells to enhance the graft-versus-leukemia effect after allogeneic stem cell transplant

G Weber  1 , U Gerdemann, I Caruana, B Savoldo, N F Hensel, K R Rabin, E J Shpall, J J Melenhorst, A M Leen, A J Barrett, C M Bollard
Affiliations

Generation of multi-leukemia antigen-specific T cells to enhance the graft-versus-leukemia effect after allogeneic stem cell transplant

G Weber et al. Leukemia. 2013 Jul.

Abstract

Adoptive immunotherapy with ex vivo expanded T cells is a promising approach to prevent or treat leukemia. Myeloid leukemias express tumor-associated antigens (TAA) that induce antigen-specific cytotoxic T lymphocyte (CTL) responses in healthy individuals. We explored the feasibility of generating TAA-specific CTLs from stem cell donors of patients with myeloid leukemia to enhance the graft-versus-leukemia effect after stem cell transplantation. CTL lines were manufactured from peripheral blood of 10 healthy donors by stimulation with 15mer peptide libraries of five TAA (proteinase 3 (Pr3), preferentially expressed antigen in melanoma, Wilms tumor gene 1 (WT1), human neutrophil elastase (NE) and melanoma-associated antigen A3) known to be expressed in myeloid leukemias. All CTL lines responded to the mix of five TAA and were multi-specific as assessed by interferon-γ enzyme-linked immunospot. Although donors showed individual patterns of antigen recognition, all responded comparably to the TAAmix. Immunogenic peptides of WT1, Pr3 or NE could be identified by epitope mapping in all donor CTL lines. In vitro experiments showed recognition of partially human leukocyte antigen (HLA)-matched myeloid leukemia blasts. These findings support the development of a single clinical grade multi-tumor antigen-specific T-cell product from the stem cell source, capable of broad reactivity against myeloid malignancies for use in donor-recipient pairs without limitation to a certain HLA-type.

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Conflict of interest statement

Conflict of Interest: The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Multi-antigen-specific CTL lines are reproducibly expanded from adult donors and CB samples. (a) Fold expansion of TAAmix-specific CTLs generated from 10 healthy individuals by repeated stimulation of PBMC with pepmix-pulsed DC (mean ± s.d.). (b) Phenotype of TAAmix-specific CTL lines after three stimulations (% of lymphocyte gate, and mean) showing balanced predominance of CD4+ and CD8+ T cells. (c) T-cell subsets with predominance of effector memory phenotype (% of CD3+ cells, and mean). (d) Recognition of individual target antigens in IFNγ-ELISpot assay by CTLs generated by TAAmix (left) versus single antigen (SA) stimulation (right). Percentage of CTL lines recognizing from one to five antigens are shown. (e) Mean spot counts ± s.e. in response to target antigen stimulation by IFNγ-ELISpot assay of TAAmix CTLs. (f) mean cytolytic activity ± s.e. of TAAmix CTL lines (n = 8) against peptide-pulsed autologous PHA-blasts (PHAB) in a carboxyfluorescein succinimidyl ester (CFSE)-based cytotoxicity assay at an effector-to-target ratio of 40:1. (g) Phenotype of six cord blood-derived CTL lines after three restimulations (% of lymphocyte gate, and mean). (h) Phenotype of T-cell subsets (% of CD3+ cells, and mean) of these cord blood-derived CTL lines. (i) Mean of INFγ spot counts ± s.e. in response to target antigen stimulation by ELISpot assay of six cord blood TAAmix CTL lines showing recognition of all antigens.
Figure 2
Figure 2
Functional assays in TAAmix-specific CTL lines in two donors. (a) Recognition of TAAmix and SAs by IFNγ-ELISpot (mean spot count ± s.d.) and (b) cytolytic activity (mean ± s.d.) in a CFSE-based cytotoxicity assay against peptide-pulsed target cells correlating with these results, with no lysis of unpulsed PHAB (dotted line). (c) Intracellular cytokine detection of peptide stimulated CTLs in the presence of CD28/CD49d, Brefeldin A and Monensin showing secretion of multiple cytokines, (IFNγ, TNFα, IL2) and CD154 and CD107a-expression as a marker of activation and degranulation, respectively, in both CD4+ and CD8+ T-cell populations. (d) Recognition of TAAmix and SAs in IFNγ-ELISpot assay (mean spot count ± s.d.) by TAAmix CTLs from a representative donor. (e) Specific lysis (mean ± s.d.) of peptide-pulsed autologous PHAB in a CFSE-based cytotoxicity assay. (f) Cytokine secretion by CD4+ and CD8+ T cells.
Figure 3
Figure 3
Recognition of primary AML blast cells by TAAmix CTL lines. (a) Coculture of TAAmix CTLs with partially HLA-matched AML blast sample no. 2761. Leukemia blasts were quantified by anti-CD33/CD34 costaining and CTLs by CD3 staining. Leukemia blasts were eliminated over time when cocultured with TAAmix CTLs, but remained at higher levels in culture when incubated with control CTLs (virus-specific CTLs) from the same donor. Analysis on day 0 and after 1 and 3 days of coculture. (b) Summary of coculture experiments of TAAmix CTLs (black symbols) generated from five donors with partially HLA-matched AML blast samples no. 2761 and no. 9460 and nonspecific control CTL lines derived from the same donors (open symbols); % remaining leukemia blasts ± s.d. on day 0, and after 1 and 3 days of coculture. (c) Production of IFNγ by TAAmix CTLs coincubated with partially HLA-matched AML blasts at a 1:1 ratio in ELISpot (mean spot counts ± s.e. of five CTL lines). (d) Relative inhibition of AML blast colony formation in a CFU assay (calculated in comparison to nonspecific CTLs generated from the same donor; mean ± s.d. of 5 CTL lines).
Figure 4
Figure 4
Characterization of a MHC class II-restricted WT1 epitope. (a) Epitope mapping for WT1 by testing recognition of peptide pools (A–U). Each pool contained 10 or 11 15mer peptides spanning the entire WT1 protein. Recognition of pools F and U indicating recognition of WT1 peptide no. 60, confirmed in (b) by testing WT1 no. 60 and flanking peptides. (c) Specific cytotolytic activity against WT1 no. 60-pulsed autologous PHAB (black line) compared with unpulsed PHAB (dotted line) in a CFSE-based cytotoxicity assay (mean ± s.d.). (d) Recognition of WT1 no. 60 was blocked by preincubation of peptide-pulsed target cells with anti-HLA class II antibody, in particular anti-HLA-DR, indicating a CD4+-restricted recognition of this peptide. (e) Recognition of WT1 no. 60 in IFNγ-ELISpot presented by a variety of HLA-DRβ1*04-positive and -negative antigen-presenting cells (APCs) compared with autologous APCs showing promiscuity in the recognition of WT1 no. 60 for several HLA-DRβ1*04 alleles (mean spot counts ± s.d.). (f) To test for recognition of primary leukemia blasts, CTLs were stimulated with HLA-DRβ1*04-positive and -negative myeloid blast cells and healthy donor PBMC in IFNγ-ELISpot assay (mean ± s.d.) and (g) intracellular cytokine detection; the predominant response was seen mainly in the CD4+ compartment.
Figure 5
Figure 5
Identification of a novel MHC class I-restricted NE peptide. (a) Epitope mapping for NE with the complete peptide library and minipools (A–P) by INFγ-ELISpot. (b) Peptide matrix for NE—each pool contains 8 15mer peptides. (c) Recognition of pools C and P, corresponding with recognition of peptide no. 59 in INFγ-ELISpot (mean ± s.d.). (d) Cytolytic activity of TAAmix CTLs against PHAB pulsed with NE no. 59 antigen or NE total pool (control: unpulsed PHAB, mean ± s.d.). (e) Blocking experiments with anti-HLA class I and II blocking antibodies using autologous peptide-pulsed PHAB as targets (mean ± s.d.). IFNγ-secretion was reduced to near background (unpulsed autologous PHAB) by preincubation with anti-HLA class I antibody, indicating MHC class I-restriction. (f) Intracellular cytokine staining of TAAmix CTLs stimulated with NE no. 59 showing predominant response in the CD8+ population.
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