Excerpt from MACS&more Vol 17 1/2016. Kenji Miki¹, Koji Nagaoka¹, Hermann Bohnenkamp², Takayuki Yoshimoto³, Ryuji Maekawa¹*, and Takashi Kamigaki⁴
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1 Excerpt from MCS&more Vol 17 1/2016 Dendritic cells pulsed with induce both OV-specific CD4 + and CD8 + T cells and cause antitumor effects in a mouse model of lymphoma Kenji Miki¹, Koji Nagaoka¹, Hermann ohnenkamp², Takayuki Yoshimoto³, Ryuji Maekawa¹, and Takashi Kamigaki⁴ ¹ Medinet Medical Ititute, MEDINET Co., Ltd., Tokyo, Japan; ² Miltenyi iotec GmbH, ergisch Gladbach, Germany; ³ Ititute of Medical Science, Tokyo Medical University, Tokyo, Japan; ⁴ Seta Clinic Group, Shin-Yokohama, Japan Miltenyi iotec provides products and services worldwide. Visit to find your nearest Miltenyi iotec contact. Unless otherwise specifically indicated, Miltenyi iotec products and services are for research use only and not for therapeutic or diagnostic use. MCS, the MCS logo, and are registered trademarks or trademarks of Miltenyi iotec GmbH. Copyright 2016 Miltenyi iotec GmbH. ll rights reserved.
2 Dendritic cells pulsed with induce both OV-specific CD4 + and CD8 + T cells and cause antitumor effects in a mouse model of lymphoma Kenji Miki¹, Koji Nagaoka¹, Hermann ohnenkamp², Takayuki Yoshimoto³, Ryuji Maekawa¹, and Takashi Kamigaki⁴ ¹ Medinet Medical Ititute, MEDINET Co., Ltd., Tokyo, Japan ² Miltenyi iotec GmbH, ergisch Gladbach, Germany ³ Ititute of Medical Science, Tokyo Medical University, Tokyo, Japan ⁴ Seta Clinic Group, Shin-Yokohama, Japan not shown Introduction Dendritic cell (DC)-based vaccines hold great promise for cancer immunotherapy. DCs are pulsed with peptides and subsequently used as antigen-presenting cells to induce an antitumor respoe in vivo through the activation of T cells. Thus far, mostly epitope-specific peptides with 8 10 amino acids in length have been used to generate DC vaccines, which however activate only CD8 + T cells. Moreover, available single epitope-specific peptides are restricted to MHCI or MHCII and therefore activate either CD8 + or CD4 + T cells. Here we used a Pool, which coists mainly of 15-mer peptides covering the complete sequence of the target antigen, in this case ovalbumin (OV), with an 11 amino acid overlap. We show that DCs pulsed with this peptide pool induced both OV-specific CD8 + and CD4 + T cell respoes and caused strong antitumor effects in a mouse model of lymphoma. Material and methods DC generation and antigen loading DCs were generated from mouse (C57L/6) bone marrow cells cultured in the presence of 20 ng/ml GM-CSF for 10 days. Subsequently, DCs were maturated in the presence of 10 ng/ ml GM-CSF, 10 ng/ml IL-4, and 1 µg/ml LPS. DCs were loaded with antigen by pulsing with peptide pool (Miltenyi iotec) for 4 hours. (OV) peptides with I-b (MHCII)-restricted (OV ) and H-2Kb (MHCI)-restricted (OV ) epitopes were used as positive controls. ll peptides were used at the final concentration of 2 µg/ml. Following the incubation, cells were washed with medium to remove excess peptides. E.G7 DCs T cell isolation T cells were obtained from splee of OT-II or OT-I mice carrying a tragenic OVspecific T cell receptor (TCR). CD4 + and CD8 + T cells were isolated with the CD4 + T Cell Isolation Kit, mouse and the CD8a + T Cell Isolation Kit, mouse, respectively (both from Miltenyi iotec). T cell priming capacity of DCs To test the capacity of DCs to induce T cell proliferation, the DCs pulsed with or the I-b- or H-2Kb-restricted OV peptides were cocultured with CFSElabeled or. The ratio of DCs to T cells was 1:20. Untreated DCs were used as a negative control. fter 1 to 4 days of coculture, T cell proliferation was determined by flow cytometry using absolute (I- b ) (H-2K b ) (I- b + H-2K b ) OV tetramer assay Serum IgG detection by ELIS Measurement of tumor weight C57L/ Figure 1 Timeline for the evaluation of antitumor effects of DCs. Numbers indicate the days after tumor induction with E.G7 cells. Vol 17 1/2016 MCS & more 7
3 count beads. Moreover, proliferation of T cells was analyzed on day 3 by a CFSE dilution assay. On day 2, concentratio of IL-2 and IFN-γ in the culture supernatants were measured by ELIS. ssessment of antitumor effects of DCs in a mouse model of lymphoma The timeline for tumor induction, immunization with DCs, and evaluation of antitumor effects by DCs is outlined in figure 1. Tumors were induced in C57L/6 mice on day 0 by subcutaneously injecting 5 10⁵ E.G7-OV tumor cells. E.G7-OV cells are derivatives of EL4 mouse lymphoma cells modified to express and secrete OV cotitutively. Therefore, these cells can be recognized by pulsed DCs. fter 4 days, the mice were injected with 3 10⁵ DCs that were either left untreated or pulsed with or MHCII (I-b)- restricted or MHCI (H-2Kb)-restricted OV peptides or both peptides in combination. The tumor volume was measured with a micrometer calliper at various time points until day 14. On day 14, six mice were sacrificed to measure tumor size by weight. Evaluation of DC-induced immune respoes in a mouse model On day 11, serum from three mice was examined for the presence of OV-specific IgG by ELIS. Moreover, the numbers of OVspecific CD8 + cytotoxic T cells were determined by flow cytometry. To this end, resected tumors were dissociated into single-cell suspeio by treatment with collagenase. Subsequently, cells were analyzed by flow cytometry for H-2K b OV tetramer + CD8 + T cells. Cell numbers were normalized to the weight of tumors to allow for direct comparison of the individual mice. Results OV peptide pulsed DCs induce CD4 + and CD8 + T cells to proliferate and secrete pro-inflammatory cytokines Mature DCs have the capacity to induce T cell proliferation. To test whether the OV peptide pulsed DCs can exert this function, we cocultured the DCs with CD4 + or CD8 + T cells carrying an OV-specific TCR and measured the numbers of T cells after various time points. C IL-2 level (ng/ml) IFN-γ level (ng/ml) T cell number ( 10⁵) Relative cell number Time (days) no antigen peptide (I- b ) no antigen peptide (I- b ) 10⁰ 10¹ 10² 10³ CFSE (I- b ) (I- b ) IL-2 IFN-γ Figure 2 Capacity of DCs to induce T cell proliferation and secretion of proinflammatory cytokines. DCs loaded with or the MHCII (I- b ) or MHCI (H-2Kb) restricted OV peptides were cocultured with isolated CD4 + or CD8 + T cells on day 0. T cell numbers were determined by flow cytometry at various time points (; n = 3; mea±sd; Student s t-test; : non-significant at p 5). On day 3, T cell proliferation was assessed by flow cytometric CFSE dilution assay (). Concentratio of IL-2 and IFN-γ in the supernatant of the coculture were determined on day 2 (C; n = 3; mea±sd; Student s t-test; : non-significant at p 5). T cell number ( 10⁵) IL-2 level (ng/ml) IFN-γ level (ng/ml) Relative cell number ⁰ 10¹ 10² 10³ CFSE Time (days) no antigen peptide (H-2K b ) no antigen peptide (H-2K b ) (H-2K b ) (H-2K b ) 8 MCS & more Vol 17 1/2016
4 3000 Tumor volume (mm³) volume with all pulsed DCs. DCs pulsed with or the two MHCIand MHCII-restricted OV peptides led to the largest reduction in tumor size by about 90%, followed by the MHCI-restricted peptide (80% reduction). The inhibitory effect of DCs pulsed with the MHCII-restricted peptide was the smallest, leading to a reduction in tumor size by about 50% (fig. 3). Similar results were obtained for the tumor weight ten days after immunization (fig. 3). Tumor weight (mg) Time after tumor inoculation (days) (I- b ) (H-2K b ) (I- b + H-2K b ) (I- b ) (H-2K b ) (I- b + H-2K b ) Figure 3 Inhibition of tumor growth by OV-pulsed DCs. C57L/6 mice were injected with E.G7 cells on day 0. () Tumor volume was measured with a micrometer calliper at various time points after immunization with DCs that were loaded with or the MHCII (I-b) and/or MHCI (H-2Kb) restricted OV peptides. () t day 14 after injection of tumor cells, i.e., day 10 after DC immunization, mice were sacrificed and the tumor size was measured in weight. ( and ; n = 6; mea±sd; Student s t-test; : non-significant at p 5; p < 5. DCs loaded with induced an increase in the numbers of both CD4 + and CD8 + T cells from day 2 onward, similarly to the respective MHCII or MHCIrestricted peptides. DCs that were not loaded with any antigen did not induce T cell proliferation (fig. 2). T cell proliferation was confirmed with the CFSE dilution assay on day 3 (fig. 2). Likewise, DCs loaded with or the MHCI- or MHCII-restricted OV peptides induced the secretion of IL-2 and IFN-γ, in contrast to DCs that were not loaded with antigen. Cytokine secretion was measured on day 2 (fig. 2C). OV peptide pulsed DCs inhibit tumor growth in vivo in a mouse model of lymphoma Cells from the OV-expressing mouse lymphoma cell line E.G7 were injected into C57L/6 mice on day 0 to induce tumor growth (fig. 3). fter four days the mice were immunized by injection of DCs pulsed with or the MHCI- or MHCII-restricted OV peptides, or both OV peptides in combination. s a negative control, mice were injected with untreated DCs. Six days after immunization the DCs pulsed with OV antigen already caused a slight decrease in tumor size compared to the negative control (fig. 3). Ten days after immunization there was a significant decrease in tumor OV peptide pulsed DCs induce immune respoes in a mouse model of lymphoma The tumors induced by injection of E.G7 cells were also analyzed for OV-specific cytotoxic CD8 + T cells (CTLs) with a tetramer assay (fig. 4). The numbers of CTLs were normalized to the tumor weight, which allows for the direct comparison of the individual mice. DCs pulsed with or the combination of both MHCI- and MHCIIrestricted peptides led to the highest relative numbers of OV-specific CTLs, whereas the MHCI-restricted peptide resulted in a significantly lower number of OV-specific CTLs. No OV-specific CTLs were detectable in tumors from mice treated with control DCs or DCs pulsed with the MHCII-restricted peptide (fig. 4). We also analyzed the relative titers of OVspecific IgG in the serum. The results were similar to the results from CTL enumeration, except that DCs pulsed with the MHCIIrestricted peptide also induced IgG production, just like the MHCI-restricted peptide (fig. 4). Conclusion pulsed DCs induced both CD4 + and CD8 + T cell respoes in vitro, similar to the MHCII- and MHCIrestricted peptides. Immunization of mice bearing OVexpressing tumors with DCs that were previously pulsed with or the MHCI-restricted peptide led to infiltration of OV-specific CD8 + CTLs into the tumor. In contrast, no CTLs were detectable in the tumors from mice that were treated with DCs pulsed with the MHCII-restricted peptide. This directly reflects the greater inhibition of tumor Vol 17 1/2016 MCS & more 9
5 Number of intratumor OD CTLs per mg of tumor150 (I- b ) (H-2K b ) (I- b + H-2K b ) (I- b ) (H-2K b ) (I- b + H-2K b ) Figure 4 Infiltration of CTLs into tumors and production of OV-specific IgG by DCs pulsed with OV peptides. C57L/6 mice were injected with E.G7 cells on day 0. On day 11, i.e., 7 days after immunization, single-cell suspeio prepared from tumor tissue were analyzed for OV-specific CTLs by a flow cytometric tetramer assay () and the serum was tested for OV-specific IgG by ELIS (). CTL numbers were normalized to tumor weight. n = 3; mea ± SD; Student s t-test; : non-significant at p 5; 1 p < 5; p < 1. growth by the DCs pulsed with or the MHCI-restricted peptide. pulsed DCs could induce OV-specific CD4 + T cell respoe in vivo, which resulted in the efficient production of OV-specific IgG. pulsed DCs could induce both MHCII- and MHCI-restricted T cell respoes in vivo. Therefore, antitumor effect and OV-specific CTL induction by pulsed DCs were stronger compared to MHCI- or MHCIIrestricted peptide-pulsed DCs. Respoes elicited by -pulsed DCs may be not restricted to MHCI or MHCII. MCS Product research grade Order no CD4 + T Cell Isolation Kit, mouse CD8a + T Cell Isolation Kit, mouse Mouse GM-CSF premium grade Mouse IL-4 premium grade For additional Pools, visit Order numbers are provided for 100 μg sizes. For different quality grades and additional package sizes, visit Unless otherwise specifically indicated, Miltenyi iotec products and services are for research use only and not for therapeutic or diagnostic use. 10 MCS & more Vol 17 1/2016
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