Strategies for antigen choice and priming of dendritic cells influence the polarization and efficacy of antitumor T-cell responses in dendritic cell-based cancer vaccination - PubMed (original) (raw)

Strategies for antigen choice and priming of dendritic cells influence the polarization and efficacy of antitumor T-cell responses in dendritic cell-based cancer vaccination

Joanna Galea-Lauri et al. Cancer Immunol Immunother. 2004 Nov.

Abstract

Dendritic cells (DCs) primed with tumor antigens (Ags) can stimulate tumor rejection. This study was aimed at evaluating the polarization of T-cell responses using various DC Ag-priming strategies for vaccination purposes. DCs cocultured with irradiated "apoptotic" tumor cells, DC-tumor fusions, and DCs pulsed with freeze-thaw tumor lysate Ags served as Ag-primed DCs, with EG7 tumor cells (class II negative) expressing OVA as the model Ag. DCs loaded with class I- and class II-restricted OVA synthetic peptides served as controls. Primed DCs were assessed by the in vitro activation of B3Z OVA-specific CD8 T cells and the proliferation of OVA-specific CD8 and CD4 T cells from OT-I and OT-II TCR transgenic mice, respectively. In vivo responses were measured by tumor regression following treatment with Ag-primed DCs and by CTL assays. Quantification of IL-2, IL-4, IL-5, IFN-gamma, and TNF-alpha by cytometric bead array (CBA) assay determined the polarization of TH1/TH2 responses, whereas H-2 Kb/SIINFEKL tetramers monitored the expansion of OVA-specific T cells. DC-EG7 hybrids stimulated both efficient class I and class II OVA responses, showing that DC-tumor hybrids are also capable of class II cross-presentation. The hybrids also induced the most potent CTLs, offered the highest protection against established EG7 tumors and also induced the highest stimulation of IFN-gamma and TNF-alpha production. DCs cocultured with irradiated EG7 were also effective at inducing OVA-specific responses, however with slightly reduced potency to those evoked by the hybrids. DCs loaded with lysates Ags were much less efficient at stimulating any of the OVA-specific T-cell responses, showed very little antitumor protection, and stimulated a weak TH1 response, overbalanced by an IL-5 TH2 response. The strategy of Ag-loading clearly influences the ability of DCs to polarize T cells for a TH1/TH2 response and thus determines the outcome of the elicited immune response, during various vaccination protocols.

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Figures

Fig. 1

Fig. 1

A FACS profiles of day 7 bone marrow–derived DCs cultured in GM-CSF and low levels of IL-4, showing that the cells express high levels of H-2 Kb/Db, I-A, CD11c, B7.1, B7.2, ICAM-1, and modest levels of CD40 consistent with DC phenotype. B FACS profiles showing that DCs cultured without the addition of IL-4 express significant levels of the neutrophil marker GR-1 and macrophage marker F4-80, indicating that IL-4 is essential for DC differentiation

Fig. 2

Fig. 2

A FACS profiles of DCs after being pulsed with SIINFEKL peptide and stained with the mAb 25-D1.16 specific for the H-2 Kb /SIINFEKL complex. Graphs show that the relative staining of the cells (i.e., binding of SIINFEKL to the H-2 Kb) is proportional to the initial dose of the peptide used. B DCs pulsed with an irrelevant peptide are negative for 25-D1.16 staining, showing that this mAb is only specific for H-2 Kb/SIINFEKL. C FACS analysis of the EG7 cells shows significant expression of surface H-2 Kb/SIINFEKL compared with the parental line EL4. On comparison to the DCs pulsed with SIINFEKL in A, the physiological levels of SIINFEKL on the EG7 cells are estimated to be 1 nM

Fig. 3

Fig. 3

A Histogram profiles of DCs after coculture with tumor-derived lysate proteins with (dotted line) or without (solid line) the addition of albumin-FITC protein used as a tracer for protein uptake. As shown in the overlay histograms, 90% of the DCs are positive for FITC-protein indicating active protein uptake by the DCs. B FACS analysis of DCs after being pulsed with increasing doses of albumin-FITC showing that at this dose range the amount of albumin-FITC taken up by the DCs is proportional to the intial protein concentration

Fig. 4

Fig. 4

A Dot plot showing FACS analysis of DCs (labeled with MHC class II–FITC, FL1 channel) after the uptake of apoptotic cell bodies (labeled with PKH26 red dye, FL2 channel) after a 6-h coculture of the DCs with apoptotic EG7 cells. The DCs that have taken up the apoptotic cells are fluorescent in both channels (shown as blue pseudocolor dots). B Flourescence microscopy confirms the presence of apoptotic cell bodies (red) in the DCs. C Dot plot showing the successful removal by Ficoll centrifugation of any apoptotic EG7 cells that are not taken up by the DCs

Fig. 5A–F

Fig. 5A–F

FACS analysis for the detection and quantification of DC-EG7 hybrid cells. The fusion partners are detected by the unique expression of CD28 on EG7 cells marked by PE labeling in the FL2 channel (A) and Class II on DC marked by FITC labeling in the FL1 channel (B). Following PEG/DMSO treatment, a fusion efficiency of about 30% is obtained and the hybrid cells are detected as double fluorescent cells (shown as pseudoblue dots in D), which are not present in the control mixture (C). The hybrid cells are confirmed as double fluorescent by microscopy (white arrows). The successful removal of unfused EG7 cells by immunomagnetic depletion (E), and after fusion, the hybrid cells (marked as 29.6% of total DC), also express surface H-2 Kb/SIINFEKL which they obtained from the EG7 fusion partner (F)

Fig. 6

Fig. 6

A Activation of the B3Z T-cell hybridoma. B Proliferation of OT-I naïve T cells by DCs loaded with EG7-derived Ags, by various methods. DCs pulsed with SIINFEKL peptide served as positive control. The data shows that the methods used to load the DCs induce differential cross-presentation of OVA to the T cells; however, the fusion strategy gives the highest efficiency of T-cell activation, reflecting higher levels of H-2 Kb/SIINFEKL after loading. C Activation of naïve OT-II T cells by Ag-loaded DCs. The data show that the DC-EG7 hybrid cells and DCs cocultured with irradiated EG7 cells are much more efficient at cross-presentation of class II–OVA peptide and hence T-cell activation, than DCs pulsed with tumor lysate Ags

Fig. 7

Fig. 7

Induction of CTL activity in mice immunized by different Ag-loaded DCs. After immunization, the lymphocytes were cocultured with irradiated EG7 cells and tested for CTL activity against EG7 (solid squares) and parental EL4 cells (open diamonds). Unimmunized mice, or mice injected with a control mixture of DCs and EG7, EG7 alone, or peptide alone, failed to give any significant CTL activity. Mice immunized with DCs pulsed with SIINFEKL peptide gave high levels of CTLs specific against the target cells that expressed that Ag, i.e., EG7 and not EL4. Mice immunized with the different Ag-loaded DCs showed a differential potency in the induction of CTLs, with the DCs pulsed with lysates showing the weakest response and the hybrid cells giving the strongest response. Additionally, the CTLs induced by the mice immunized with DCs cocultured with apoptotic cells or the hybrid cells also showed a higher CTL response against the EG7 cells compared with the EL4 cells, indicating that some of the CTLs were OVA specific

Fig. 8

Fig. 8

FACS analysis of immunized mouse lymphocytes for the induction of Ag-specific T-cell response by the different Ag-loaded DCs. As shown in the figure, the only mice that induced an Ag-specific response (marked by tetramer staining of CD8+ T cells) were the mice immunized with DCs pulsed with SIINFEKL peptide (used as a positive control), mice immunized with DCs cocultured with apoptotic cells and DC-EG7 hybrid cells. Values represent percentage number of total cells that stained positive for CD8 and H-2 Kb/SIINFEKL tetramers

Fig. 9

Fig. 9

Analysis of TH1/TH2 response by the CBA assay. This assay shows the relative amounts of the different cytokines secreted by the immunized lymphocytes after immunization by different Ag-loaded DCs. IL-4 was not detected during the assay; however, raised IL-5 levels (TH2 marker) and very low or absent IL-2 (TH1 marker) were present in the same mice that lacked CTL activity, i.e., mice immunized with control mixture of DCs and EG7, EG7 alone, or peptide alone. High IL-2, low or absent IL-5, and high IFN-γ were all present in the mice which gave significant CTL responses

Fig. 10A–C

Fig. 10A–C

Effect of Ag-loaded DCs on survival and treatment of EG7 tumors using eight mice/group. A Survival curve of mice treated with different Ag-loaded DCs after establishment of EG7 tumor. The DC-EG7 hybrid cells offered the highest protection in the immunized mice with 50% of the mice being long-term tumor free. The mice immunized with DCs pulsed with tumor lysates offered the least protection for mice survival. B The graph shows the relative size of the tumors in the different immunized mice. The mice treated with DC-EG7 hybrids showed the smallest tumors, control mice reached 23-mm sized tumors and were all culled by day 21. The data show that the different Ag-loaded DCs offer different degrees of protection to the mice. C Each mouse is plotted as an individual to emphasize that in some of the immunized mice (for example, the mice treated with the DC-EG7 hybrids) some of the mice never form any tumors, and some tumors that form also regress, indicative of a potent anti-EG7 immune response

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