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. 2012 Oct 1;1(7):1095-1103.
doi: 10.4161/onci.20954.

Detecting T-cell Reactivity to Whole Cell Vaccines: Proof of Concept Analysis of T-cell Response to K562 Cell Antigens in CML Patients

Free PMC article

Detecting T-cell Reactivity to Whole Cell Vaccines: Proof of Concept Analysis of T-cell Response to K562 Cell Antigens in CML Patients

Ana Brusic et al. Oncoimmunology. .
Free PMC article


BCR-ABL(+) K562 cells hold clinical promise as a component of cancer vaccines, either as bystander cells genetically modified to express immunostimulatory molecules, or as a source of leukemia antigens. To develop a method for detecting T-cell reactivity against K562 cell-derived antigens in patients, we exploited the dendritic cell (DC)-mediated cross-presentation of proteins generated from apoptotic cells. We used UVB irradiation to consistently induce apoptosis of K562 cells, which were then fed to autologous DCs. These DCs were used to both stimulate and detect antigen-specific CD8(+) T-cell reactivity. As proof-of-concept, we used cross-presented apoptotic influenza matrix protein-expressing K562 cells to elicit reactivity from matrix protein-reactive T cells. Likewise, we used this assay to detect increased anti-CML antigen T-cell reactivity in CML patients that attained long-lasting clinical remissions following immunotherapy (donor lymphocyte infusion), as well as in 2 of 3 CML patients vaccinated with lethally irradiated K562 cells that were modified to secrete high levels of granulocyte macrophage colony-stimulating factor (GM-CSF). This methodology can be readily adapted to examine the effects of other whole tumor cell-based vaccines, a scenario in which the precise tumor antigens that stimulate immune responses are unknown.


Figure 1. Schema for developing a cross-presentation assay for measuring immunoreactivity to K562 cells.
Figure 2. High stable rates of apoptosis of K562 cells occur upon UVB irradiation (400 mJ). (A) K562 cells were cultured for 15 h after irradiation with UVB and then stained with AnnexinV and PI. Early apoptotic cells were defined as AnnexinV+ and PI-, and late apoptotic cells were described as double positive. Dot plots are representative of a minimum of 5 experiments. (B) Average results of AnnexinV and PI staining of K562 cells over 24 h following UVB irradiation at 0–500 mJ. All results shown are based on the average of a minimum of three experiments.
Figure 3. DCs effectively phagocytose apoptotic K562 cells. Immature DCs labeled in green (with PKH2) and apoptotic K562 cells labeled in red (with PKH26) were co-cultured at either 4°C (top) or 37°C (bottom) for 24 h at a 1:9 ratio. Shown are FACS results for the total population (A), and cells gated on the DC population (B). Dot plots are representative of 3 experiments.
Figure 4. Apoptotic K562 cells when presented on DCs can stimulate antigen-specific immunity against either introduced or native antigen. (A) K562 cells were transfected to express M1 (see inset) and then irradiated with 400 mJ UVB to induce apoptosis. Following irradiation, these cells were co-cultured with iDCs for 24 h and matured using a cytokine cocktail for 24 h. On ELISpot, we used CD8+ T cells alone as a negative control, M1-pulsed DCs and PHA as positive controls, and apoptotic M1-K562 and DCs alone on CD8+ T-cells to establish background reactivity. There was a significant increase in reactivity of apoptotic M1-K562 when cross-presented by DCs compared with apoptotic M1-K562 cells alone (*p = 0.04) or DC cross-presented apoptotic K562 cells (p = 0.04). Significance was determined using the two-tailed Student’s t test. All experiments were performed in duplicate wells, and mean spot-forming cells per 100,000 CD8+ T cells are reported. Error bars represent two standard deviations. The ELISpot image is representative of more than 3 experiments, while the bar graph displays average results from four experiments, with standard deviation as shown. (B) T cells from pre- and post-DLI patients were tested on ELISpot against CML antigens in the form of apoptotic K562 cross-presented on DCs, and results were reported as spots per 100,000 CD8+ T cells (minus background, defined as reactivity of apoptotic K562 cells alone or autologous DCs alone on CD8+ T cells); p = 0.08 (Student’s t test). Three patients treated with imatinib were tested as comparison, and showed no reactivity. Results are shown as means with error bars representing two standard deviations.
Figure 5. Skin reactions characterized by erythema, swelling and warmth occurred commonly at the immunization site, following sc/id injection of irradiated GM-CSF secreting K562 cells. These reactions were transient, lasting 1–5 d following vaccination.
Figure 6. Immunologic monitoring of CML patients subjected to therapeutic vaccination with irradiated GM-CSF-secreting K562 cells. Reactivity to K562 antigens by cross-presentation assay (measured by ELISpot and shown on bottom row) in relation to: timing of vaccination (indicated by arrows, top row); to absolute WBC count (top row); molecular response, measured by %BCR-ABL/GUS transcript in PBMC (mid row); and ratio of CD8+ cells to regulatory T cells (Tregs) (defined as FOXP3+/CD25+), measured by flow cytometry (bottom row).

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