Cyclin-A1 represents a new immunogenic targetable antigen expressed in acute myeloid leukemia stem cells with characteristics of a cancer-testis antigen - PubMed (original) (raw)

. 2012 Jun 7;119(23):5492-501.

doi: 10.1182/blood-2011-07-365890. Epub 2012 Apr 23.

Ravindra Majeti, Thomas Schmitt, Derek Stirewalt, Ulrich Keilholz, Keith R Loeb, Brent Wood, Yongiae E Choi, Marie Bleakley, Edus H Warren, Michael Hudecek, Yoshiki Akatsuka, Irving L Weissman, Philip D Greenberg

Affiliations

Cyclin-A1 represents a new immunogenic targetable antigen expressed in acute myeloid leukemia stem cells with characteristics of a cancer-testis antigen

Sebastian Ochsenreither et al. Blood. 2012.

Abstract

Targeted T-cell therapy is a potentially less toxic strategy than allogeneic stem cell transplantation for providing a cytotoxic antileukemic response to eliminate leukemic stem cells (LSCs) in acute myeloid leukemia (AML). However, this strategy requires identification of leukemia-associated antigens that are immunogenic and exhibit selective high expression in AML LSCs. Using microarray expression analysis of LSCs, hematopoietic cell subpopulations, and peripheral tissues to screen for candidate antigens, cyclin-A1 was identified as a candidate gene. Cyclin-A1 promotes cell proliferation and survival, has been shown to be leukemogenic in mice, is detected in LSCs of more than 50% of AML patients, and is minimally expressed in normal tissues with exception of testis. Using dendritic cells pulsed with a cyclin-A1 peptide library, we generated T cells against several cyclin-A1 oligopeptides. Two HLA A*0201-restricted epitopes were further characterized, and specific CD8 T-cell clones recognized both peptide-pulsed target cells and the HLA A*0201-positive AML line THP-1, which expresses cyclin-A1. Furthermore, cyclin-A1-specific CD8 T cells lysed primary AML cells. Thus, cyclin-A1 is the first prototypic leukemia-testis-antigen to be expressed in AML LSCs. The pro-oncogenic activity, high expression levels, and multitude of immunogenic epitopes make it a viable target for pursuing T cell-based therapy approaches.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Systematic approach to identify potential target genes in AML LSCs based on 3′IVT expression microarray data. The microarray panel was created combining data files from 5 independent studies. Datasets were filtered for present calls in more than 10% of the samples and overall variability (reject probe sets with an SD/mean < 2). Hierarchical cluster analysis was performed to confirm clustering in accordance to sample biology. Target candidates were identified by mathematical filtering followed by visual inspection of the respective expression patterns. Targeted sequences of the probe sets were then analyzed to make sure that they actually represented a coding mRNA sequence. In case of several probe sets representing the same gene, expression patterns of all probe sets were inspected for consistency. Finally, target candidates, which were known to be membrane bound or secreted, were rejected.

Figure 2

Figure 2

Identification of cyclin-A1 as target candidate by in silico expression analysis and RT-PCR quantification. (A) Seven candidate target genes predicted in silico: Heatmap of normalized probe sets. Each row represents one probe set, and each column represents one sample dataset. Red represents increased levels of expression; and blue, decreased levels of expression. The darkest shade of red/blue represents average expression ± 3 SDs. (B-C) Model-based expression of probe set 205899_at representing cyclin-A1. (B) Expression in AML LSCs compared with HSCs/CD34+ BM mononuclear cells, PBMCs, and nonhematopoietic somatic tissues. The dashed line represents the cut-off value of 69.2 (mean plus 3 SDs of the HSC samples). *P < .001 (explorative). (C) Expression in AML LSCs and corresponding blasts (P = .297). (D) Cyclin-A1 expression quantified by quantitative RT-PCR. The dashed line represents the cut-off value of 0.048 (mean plus 3 SDs of the BM samples). (E) Immunofluorescence staining of cyclin-A1 in primary AML samples (representative example). Shown is a healthy BM (top row) and primary leukemic blasts (bottom row). Cells were stained with 4,6-diamidino-2-phenylindole (DAPI; left) and indirectly for cyclin-A1 (AlexaFluor-488, right). Negative controls without primary antibody and nonspecific isotype showed no fluorescence in both samples (not shown). Images were captured using a Nikon Eclipse E800 microscope. (F) Intracellular FACS staining of cyclin-A1 in primary AML blasts (representative example). BM mononuclear cells of a patient with cyclin-A1-expressing AML after gating out doublets in forward scatter (FSC)-area/height (A/H) and SSC-A/H plots. Top row: gating on blasts based on SSC and CD45 expression. Bottom row: gating on CD33-positive cells. Histograms show cyclin-A1 (black line) and isotype control (shaded) in the blast population.

Figure 3

Figure 3

Reverse immunology strategy for epitope mapping in cyclin-A1 using ICS for IFN-γ. (A) After 4 stimulations with the peptide library, T-cell lines from both donors consisted of more than 60% specific cells (gated on CD8+ cells). (B) The cell lines were subsequently tested against 20 peptide pools with each 15-mer being represented in 2 different pools in the peptide matrix. Shaded rows/columns indicate pools with more than 10% IFN-γ-positive cells from each donor. (C) Peptides, which tested positive in both of its pools (> 10% specificity, marked black), were further analyzed as individual peptides. Black columns represent the peptides for which the minimal immunogenic AA sequence from the initial 15-mer was determined.

Figure 4

Figure 4

HLA A*0201-restricted epitope 341-351. Mapping the minimal immunogenic AA sequence and HLA stabilization. (A) The position of the different peptides in cyclin-A1. IFN-γ ICS of T-cell line (donor 2264) stimulated by autologous LCLs pulsed with the 10-mer 1, 10-mer 2, and 11-mer. Only pulsing with the 11-mer results in IFN-γ production in the T-cell line. (B) HLA stabilization assay: both 10-mer 1 and the 11-mer peptide stabilize HLA A*0201 on T2 cells. Negative controls are T2 cells pulsed with an irrelevant 15-mer (shaded).

Figure 5

Figure 5

T-cell clones recognize endogenous processed epitopes 227-235 and 341-351. (A) Expression of cyclin-A1 in several myeloid cell lines quantified by quantitative RT-PCR. (B) Clones 2196.D9 and D11a (both specific for epitope 227-235) and clone 2264.E30 (specific for epitope 341-351) were tested for apoptosis induction in the THP-1 cell line using a caspase-3 assay. As negative controls, targets alone (shaded) and clone 2264.A1 specific for epitope 118-127 (HLA B*4001 restricted, THP-1 is B*4001-negative) were used. As a positive control, targets were incubated with 4μM camptothecin. The histograms correspond to the gates on the PKH26-prelabeled target cells.

Figure 6

Figure 6

CD8 T-cell clone 2196.D11b exhibits specific cytotoxic activity for cyclin-A1–expressing primary AML samples. (A) Four-hour caspase-3 assay. Specific caspase-3 cleavage is shown after subtraction of spontaneous caspase-3 activation, and determined relative to staurosporine-induced cleavage as 100%. (B) Four-hour 51Cr release assay at a range of E:T ratio with the same effector and target cells. H001 and 1690-59 are HLA A*0201 positive, and R10009 and R50400 are A*0201-negative.

Similar articles

Cited by

References

    1. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997;3(7):730–737. - PubMed
    1. Lapidot T, Sirard C, Vormoor J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994;367(6464):645–648. - PubMed
    1. Blair A, Hogge DE, Sutherland HJ. Most acute myeloid leukemia progenitor cells with long-term proliferative ability in vitro and in vivo have the phenotype CD34(+)/CD71(−)/HLA-DR. Blood. 1998;92(11):4325–4335. - PubMed
    1. Cornelissen JJ, van Putten WL, Verdonck LF, et al. Results of a HOVON/SAKK donor versus no-donor analysis of myeloablative HLA-identical sibling stem cell transplantation in first remission acute myeloid leukemia in young and middle-aged adults: benefits for whom? Blood. 2007;109(9):3658–3666. - PubMed
    1. Yanada M, Matsuo K, Emi N, Naoe T. Efficacy of allogeneic hematopoietic stem cell transplantation depends on cytogenetic risk for acute myeloid leukemia in first disease remission: a metaanalysis. Cancer. 2005;103(8):1652–1658. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources