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Advances in Brief |
Tumor Immunology Laboratory [A. A. O. E., M. W. J. S., A. J. d. B., C. G. F., G. J. A.] and Departments of Nuclear Medicine [O. C. B., W. J. C. O.] and Medical Oncology [C. J. A. P.], University Hospital Nijmegen St. Radboud, 6525 EX Nijmegen, the Netherlands
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Materials and Methods |
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Reagents.
Cells are cultured in Iscove’s modified DMEM with glutamax supplemented with 10% heat-inactivated FCS (Life Technologies, Inc., Breda, the Netherlands), 50 ßM ß-mercaptoethanol, antibiotics, and antimycotics (Life Technologies, Inc.), unless mentioned otherwise. Murine recombinant GM-CSF (rmGM-CSF) and murine recombinant IL-4 (rmIL-4) were kindly provided by Dr. G. Zurawski (DNAX Research Institute, Palo Alto, CA). The following monoclonal antibodies were used: CD45R/B220 (RA3–6B2), CD4 (MT-4), CD8 (LYT-2), I-Ab (17/227), and the activating anti-CD40 monoclonal antibody FGK-45 (kindly provided by Dr. Rolink, Basel Institute for Immunology, Basel, Switzerland; Ref. 12
). The mTRP2 (VYDFFVWL) peptide and irrelevant peptide OVA (SIINFEKL) were synthesized with a free COOH terminus by F-moc peptide chemistry using ABIMED Multiple Synthesizer or by T-boc chemistry on a Biosearch SAM2 peptide synthesizer. The peptides were >90% pure, as indicated by high-performance liquid chromatography. Peptides were dissolved in DMSO and stored at - 20°C.
DC Culture.
DCs were generated according to Inaba et al. (4)
, with modifications. Briefly, femurs were dissected, placed in 70% alcohol for 1 min, and washed with PBS. Marrow was flushed and passed through nylon mesh to remove debris. After washing, lymphocytes, granulocytes, and I-A-positive cells were removed by immunomagnetic depletion against the CD45R, CD4, CD8, and I-A-antigens. Remaining cells were cultured overnight, and the nonadherent cells were seeded at 2 x 105 cells/ml and 4 ml/well in the presence of 20 ng/ml rmGM-CSF and rmIL-4 in 6-well plates (Costar, Badhoevedorp, the Netherlands). On day 4, the cultures were refreshed by adding 1 ml of culture medium supplemented with GM-CSF and IL-4 (10 ng/ml). At day 7, nonadherent and loosely adherent proliferating DC aggregates were collected and replated in fresh medium, cytokines (1 x 106 cells/ml), and 500 ßl of hybridoma supernatant of activating anti-CD40 antibody FGK 45.
[111In]oxinate Labeling, Administration, Gamma Camera Imaging and Biodistribution Analysis.
Bone marrow DCs were labeled with [111In] oxinate (Mallinckrodt Medical, Petten, the Netherlands) in 0.1 M Tris-HCl (pH 7.0) for 20 min at room temperature. Cells were washed three times with PBS, and the labeling efficiency was calculated as the percentage of the activity that remained associated with the cell pellet. To determine the stability in vitro, DC samples were kept in medium without cytokines at 37°C. After different time periods, cells were spun down, the supernatant was transferred to other tubes, cell pellets were resuspended in the same volume, and the emitted radioactivity was counted in the gamma counter. The percentage of cell-associated radioactivity was calculated. For the in vivo studies, mice, five per experimental group, were given injections either s.c. (abdomen or thighs), i.p., or i.v. in the lateral tail vein with 1 x 106 DC (30–50 ßCi) in 200 ßl of PBS/mouse. At different time points, mice were anesthetized with oxygen/nitrous oxide/ethrane (ICI Farma, Rotterdam, The Netherlands), and images were recorded with a gamma camera (Siemens Orbiter; Siemens Inc., Hoffmann Estate, IL) equipped with parallel-hole medium-energy collimator. Images were obtained (minimum of 100.000 acquired counts) and were digitally stored in a 256 x 256 matrix. For tissue biodistribution, groups of five mice were analyzed at different time points after injection of the 111In-labeled DCs. Blood samples, lungs, liver, kidneys, spleen, thymus, small intestine, mediastinal tissue, and lymph nodes (brachial, lumbal, inguinal, popliteal, and mesenterial) were collected, weighed, and counted in the gamma counter. To correct for radioactivity decay, injection standards were counted simultaneously. The measured activity in tissues and samples was expressed as the percentage of ID or as percentage of ID/0.1 gram tissue (%ID/0.1 g). All values are expressed as mean ± SE.
Microscopic Autoradiography.
Lymphoid organs were dissected 24 h after i.v. and s.c. injection of living or gluteraldehyde-fixed (0.1%; 10 min at room temperature) [111In]oxinate-labeled DCs (250–300 ß Ci/1 x 106 DC), fixed in Unifix and embedded in paraffin. Deparaffinized sections were dipped in LM1 photographic emulsion (Amersham, Buck, United Kingdom) and exposed for 2–3 weeks at 4°C. After exposure, the sections were developed and poststained with hematoxylin. Parallel series of sections stained directly with H&E were included for better appreciation of morphology.
Vaccination with Peptide-pulsed DCs.
On days 8 or 9, DCs were harvested and loaded with 10–20 ßM peptide and 5 ßg/ml ß2microglobuline (Cymbus Biotechnology LTD, Canpro Scientific, Veenendaal, the Netherlands) in Optimem (Life Technologies, Inc.; 2–3 x 106cells/ml). After loading, DCs were washed in saline, and 3–4 x 105 DC/0.1 ml of saline were injected. Peptide-pulsed DCs were injected twice with a 2-week interval. Two weeks after the second vaccination, mice were challenged s.c. with 1 x 105 live B16 melanoma cells in 0.1 ml of saline in each flank. The size of growing tumors was measured every 2–3 days using microcalipers. The murine melanoma cell line B16 (subline F10) was grown as described by Fidler (13)
. The murine thymoma cell line EL-4 (American Type Culture Collection, Manassas, VA) was cultured in Iscove’s medium (Life Technologies, Inc.) supplemented with 5% FCS and 50 ßl ß-mercaptoethanol.
CTL Culture and Chromium Release Assay.
After tumor challenge, spleens were isolated from tumor-bearing and protected mice, and 3 x 106 single-cell splenocytes were restimulated in an upright T25 flask (10 ml; Costar) with 1 x 106 irradiated (25 Gy) peptide-loaded lipopolysaccharide blasts [1 x 106/ml splenocytes cultured for 3 days in the presence of 25 ßg/ml lipopolysaccharide (Salmonella typhosa; Sigma) and 7 ßg/ml Dextran-sulfate in a T75-flask (Costar), and loaded with 100 ß M peptide + 5 ßg/ml human ß2m (Cymbus)]. Bulk CTLs were isolated after a 7-day restimulation by density gradient centrifugation (Lympholite-M; Cedarlane Laboratories, Sanbio, Uden, the Netherlands) and were used as effectors in a chromium release assay, performed as described previously (14)
. When used in a chromium release assay, B16 tumor cells were pretreated with recombinant rat IFN-
, 50–100 units/ml, for 48 h.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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, the percentage of cell-associated radioactivity is still >75% after 8 h, 50% after 20 h, and is decreased to 25% after 48 h. As shown in Fig. 1B
, the observed decrease in cell-associated radioactivity in time corresponds to the loss of viable DCs. This loss of viable cells is likely due to apoptosis of the mature DC in vitro and does not occur as a consequence of the 111In-labeling (Fig. 1B)
. These findings demonstrate that in vitro-generated bone marrow-derived DCs can be efficiently labeled with [111In]oxinate (85–90%), which is in line with the previous observation that freshly isolated spleen-derived DCs can be labeled with [111In] chloride (>65% efficiency; 15). The observation that the radioactivity stays associated with viable cells for up to 48 h allows usage of 111In-labeled DCs to study migration and homing in vivo.
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, as well as by total body imaging in time (Fig. 2A)
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, i.v. injection of 111In-labeled DCs resulted in the immediate accumulation in capillary rich, well perfused organs like the liver, lung, spleen, and kidneys. As expected, a rapid decrease in the amount of radioactivity was observed in the lungs in time. After 48 h, most of the detectable dose was located in the liver, spleen, and kidneys, resulting in a "dome-shaped" area, as shown in Fig. 2A
. To determine the DC biodistribution more precisely, different organs (five mice/group) were dissected and the emitted radioactivity was measured at each time point. As shown in Fig. 2B
, 50–60% of the ID could be recovered from the dissected organs and is similar at each time point. The results further confirmed the bioimage data, revealing the decrease of radioactivity in the lungs and the accumulation in the liver, spleen, and kidneys (Fig. 2C)
. To correct for weight differences (liver, 
1 g; popliteal lymph node, 0.001 g) and to visualize the accumulation of DCs in small organs, like lymph nodes, the obtained values were also adjusted to 0.1 g of tissue. As shown in Fig. 2D
, the highest amount of radioactivity relative to tissue weight is present in the spleen. However, still hardly any radioactivity is contained in the small lymph nodes. Collectively, the data indicate that 111In-labeled DCs are well suited to follow DC migration in vivo and that, after i.v. injection, DCs accumulate preferentially in the spleen, whereas the amount of radioactivity recovered from lymph nodes is very low, even 48 h after injection.
The Route of Administration Affects the Accumulation of DCs in T-cell Areas of Lymph Nodes.
Because of their capacity to induce immune responses, DCs loaded with tumor antigens are used to vaccinate against tumors in mice, as well as in man. However, it is not clear which route of administration of DCs is preferred. Therefore, we investigated the biodistribution of DCs in relation to the route of administration (e.g., i.v., s.c., and i.p. injection of 111In-labeled DCs). This analysis revealed (Fig. 3A)
, that the amount of radioactivity present in the lungs, spleen, and liver at 48 h after s.c. and i.p. injection was low in comparison to i.v. administration, whereas the amount of radioactivity present in kidneys seemed to be relatively independent of the route of administration and constant in time. The relatively low amount of radioactivity recovered from the dissected organs was due to the fact that a substantial amount of radioactivity (55– 35%) is contained at the injection site after i.p. and s.c. application. Analysis of the s.c. injection site by H&E staining and by autoradiography revealed that the radioactivity containing cells were located in the s.c. fat tissue and were >90% viable (Fig. 3F
and data not shown). However, in time, a marked accumulation of radioactivity (%ID) in the draining lymph nodes was observed after s.c. application (Fig. 3B)
, whereas intermediate amounts could be detected after i.p. application (Fig. 3B)
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and autoradiography (Fig. 3, C and D)
. As shown in Fig. 3C
, the 111In-labeled DCs specifically home to the paracortical area of the lymph node. In contrast, s.c.-injected gluteraldehyde-fixed 111In-labeled DCs (Fig. 3D)
did not reach the T-cell areas, indicating that the observed migration into the T-cell areas (Fig. 3C)
is specific. Collectively, these data demonstrate that in contrast to i.v.-injected DCs, s.c.-administrated DCs specifically accumulate in the draining lymph node and migrate into the T-cell area where they are known to interact with T cells to initiate an immune response.
s.c. DC Vaccination Leads to Improved Survival in the B16 Tumor Model.
It has been well established that i.v. administration of antigen-pulsed DCs can protect mice from a subsequent lethal challenge with relatively immunogenic tumors, like in the B16-OVA tumor model (19)
. To study the effect of the route of DC administration in other models, we used the poorly immunogenic and fully autologous B16 tumor model and compared i.v. and s.c. vaccination with DCs loaded with the recently identified mouse melanocyte differentiation antigen TRP-2-derived T-cell epitope VYDFFVWL, which is endogenously presented by the B16 tumor cells (11)
. As shown in Fig. 4, A and C
, after a lethal B16 tumor challenge, no difference was observed in tumor growth (Fig. 4A)
and survival (Fig. 4C)
between mice vaccinated via the i.v. route with DCs loaded with either the mTRP-2 peptide or an irrelevant peptide. However, after s.c. DC administration, a minor but reproducible delay in tumor outgrowth (Fig. 4B)
was observed. In the same experiment, one of four mice was protected against the B16 tumor challenge (Fig. 4D)
. In a total of three independent experiments, 5 of 15 mice remained tumor-free after s.c. DC vaccination for more than 50 days, whereas 0 of 10 mice survived after i.v. vaccination. Subsequent CTL analysis of i.v.-vaccinated tumor-bearing mice (Fig. 4E)
versus s.c.-vaccinated tumor-free mice (Fig. 4F)
further confirmed the presence of strong anti-TRP-2 CTL reactivity in s.c.-, but not i.v.-, vaccinated mice. Collectively, these data implicate that in the fully autologous B16/mTRP2 DC vaccination model, delivery of DCs via the s.c. route is preferred over i.v. administration and that the observed difference correlates with more efficient accumulation of DCs in lymph nodes as well as increased CTL reactivity in vitro after s.c. vaccination.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Because lymph nodes are believed to be the location where DCs encounter naive T cells to efficiently induce an immune response, we investigated whether the observed homing patterns do influence the outcome of vaccinations in the poorly immunogenic B16 mouse melanoma tumor model. Whereas all i.v.-vaccinated mice developed tumors at the same rate as control mice, a delay in tumor growth and an enhanced survival were observed in s.c.-vaccinated mice. Analysis of the CTL response in tumor-free, s.c.-vaccinated mice versus i.v.-vaccinated mice demonstrated a highly increased reactivity of anti-TRP-2 CTLs in s.c.-vaccinated mice. Therefore, these data suggest that the s.c. route of DC vaccination results in the induction of a more efficient antitumor immune response in the fully autologous B16 melanoma model.
We would like to note, however, that in more immunogenic tumor models, the i.v. route of DC administration can also result in tumor protection (19) . Similarly, we did not observe differences between s.c. and i.v. injection in the B16-OVA-model (data not shown). One major difference between the aforementioned model is usage of a highly immunogenic foreign antigen that is artificially expressed in the tumor cells versus the differentiation antigen TRP-2, which is endogenously expressed by melanocytes and the B16 tumor cells. In addition, the T-cell epitope derived from foreign antigens like OVA bind with high affinity to the presenting MHC class I molecule, whereas the self-mouse TRP-2-derived epitope has an intermediate to low affinity as has been observed previously for the peptide epitopes identified in human melanocyte differentiation antigens (11) . The difference between the models might be explained by the stability of the MHC-peptide complex in combination with the time required for the DCs to migrate to the lymph nodes. As s.c. vaccination leads to a rapid accumulation of DCs in the lymph nodes, the magnitude of the immune response induced by DCs carrying low-affinity binding peptides may be higher after s.c. vaccination, relative to i.v. vaccination. As peptide-pulsed DCs are currently used in clinical trials, it may be considered, especially in vaccination studies in which peptides with moderately MHC binding are used, to administer DC s.c. Alternatively, peptide-loaded DCs may be injected directly into the lymph node, which has been demonstrated to initiate a potent antimelanoma immune response (9) . Whether optimal vaccination with DCs expressing a tumor antigen for a longer period (e.g., after protein loading or transfection) is also critically dependent on the route of administration remains to be established.
In summary, our findings show that the distribution of DCs to lymphoid tissues is dependent on the route of vaccination. The efficiency of DCs administered s.c. to induce an immune response is higher when compared with i.v.-injected DCs in the autologous B16 model and correlates with the preferential accumulation of DCs in lymph nodes after s.c. injection. This tumor model, in combination with 111In-labeled DCs, will provide a valuable tool to further investigate the in vivo behavior of DCs in vaccination systems. It allows semiquantitative analysis of the amount of labeled cells that accumulate in tissues and to find ways to efficiently target DCs into secondary lymphoid tissues.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the European Community, ERB FMRX CT960053. 
2 To whom requests for reprints should be addressed, at Department of Tumor Immunology, University Hospital Nijmegen St. Radboud, Philips van Leydenlaan 25, 6525 EX Nijmegen, the Netherlands. Phone: 31-24-3617600; Fax: 31-24-3540339; E-mail: g.adema{at}dent.kun.nl
3 The abbreviations used are: DC, dendritic cell; TRP, tyrosinase-related protein; p.i., postinjection; In, indium; APC, antigen-presenting cell; GM-CSF, granulocyte macrophage colony-stimulating factor; ID, injected dose; IL, interleukin.
Received 2/15/99. Accepted 6/ 1/99.
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 G. Parmiani, C. Castelli, P. Dalerba, R. Mortarini, L. Rivoltini, F. M. Marincola, and A. Anichini |
) and used to vaccinate mice either i.v. (A, C, and E) or s.c. (B, D, and F). After challenge with a lethal dose of B16 F10, the mean (± SE) tumor size (A and B) and survival (C and D) in time are indicated for a representative experiment with five mice/experimental group. Cytotoxicity of bulk CTLs, obtained after a single restimulation of splenocytes isolated from i.v.-vaccinated tumor-bearing mice (E) and s.c.-vaccinated tumor-free mice (F), against EL-4 target cells loaded with an irrelevant peptide (
). Specific lysis (%) at different E:T ratios performed in triplicate are depicted.