Expanding DC In Vivo With Flt3L Followed by Administration of a Tumor Associated Antigen and TLR Agonist

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3.1.1. Preparation and Use of Flt3-L

1. Expansion of DC with Flt3-L:

a. Flt3-L is reconstituted in sterile PBS at 50 |g/mL. Fill syringe with Flt3-L, expel any air bubbles and inject 200 ||L into the peritoneum of 6- to 8-wk-old

Table 1

Overview of the Animal Models in Which the Flt3-L/Antigen/CpG Combination Strategy has Been Successfully Used for Tumor Therapy

Tumor Immunodominant cell line Tumor dose Tumor antigen Mouse strain Relevant MHC CTL epitope Reference

Table 1

Overview of the Animal Models in Which the Flt3-L/Antigen/CpG Combination Strategy has Been Successfully Used for Tumor Therapy

Tumor Immunodominant cell line Tumor dose Tumor antigen Mouse strain Relevant MHC CTL epitope Reference

B16-OVA

1 x 104

Ovalbumin

C57Bl/6

H2b

SIINFEKL (H-2Kb)

15

(MO5)

B16

1 x 104

TRP2 1Jxri 180-188

C57Bl/6

H2b

TRP2 180-188 (H-2Kb)

17

- 1 x 105

CT26

1 x 106

AH1 peptide (SPSYVYHQF)

Balb/c

H2d

AH1 (SPSYVYHQF) (H-2Ld)

17

MC38.CEA

2 x 106

CEA

CEA-tg mice

H2b

Not identified

Therapeutic Effects of Flt3-L + Antigen + CpG on Survival of Tumor-Bearing Mice Compared to Control Treatments

Flt3-ligand therapy Percent survival following tumor therapy

Tumor antigen Tumor cells + antigen Flt3-L + CpG + antigen + antigen PBS Reference

Table 3

Therapeutic Effects of Intratumoral CCL20 Injections + CpG on Survival of B16-Bearing Mice Compared to Control Treatments

CCL20 injections CCL20 transduction

Table 3

Therapeutic Effects of Intratumoral CCL20 Injections + CpG on Survival of B16-Bearing Mice Compared to Control Treatments

CCL20 injections CCL20 transduction

Tumor cells

Therapy

Percent tumor-free after 30 d

Therapy

Percent survival (100 d)

Reference

B16

i.t. CCL20

50%

CCL20

40%

16

+ 5 x CPG

+ 5 x CPG

i.t. CCL20

20%

CCL20

0%

16

+ 5 x ODN

+ 5 x ODN

PBS

0%

Mock + 5 x CPG 0%

16

CT26

i.t. CCL20

20%

CCL20

100%

16

PBS

5%

PBS

0%

16

animals (five per treatment group). Mice receiving the same volume of PBS should be used as controls. Each mouse should be individually marked in the ear, so that it is later possible to correlate tumor growth and induction of immunity with vaccine efficacy. b. In vivo expansion of DC by Flt3-L requires the continuous presence of the growth factor for at least 1 wk. Therefore, mice should be injected with 200 |L of Flt3-L (10 |g) once a day for a period of 7-10 d.

2. Preparation of antigen and DC activator:

a. Each mouse in the vaccination group should receive 300 | g of the antigen TRP2 (see Note 3) and 30 |g CpG subcutaneously (s.c.) the day after the last Flt3-L injection. CpG (or control oligonucleotide) and tumor antigen should be mixed and diluted to their final concentration of 300 |g/mL CpG and 3 mg/mL TRP2 peptide in sterile PBS directly before use. It is important to inject a mixture of tumor antigen and TLR-agonist rather than injecting them separately, as CpG should activate the same DC that process the antigen for presentation to T cells.

b. Mix gently by inverting the tubes.

c. Fill a 1-mL syringe attached to a 27.5-gauge needle with antigen/CpG mixture (no air bubbles should be present) and inject 100 |L subcutaneously into the left flank.

3.1.2. Tumor Challenge

1. Maintenance of the B16 melanoma: B16 cells are maintained in RPMI 1640 supplemented with 10% FBS, 5 mM glutamine, penicillin, and streptomycin. B16 cells grow in an adherent fashion in tissue culture flasks and need to be passaged at a 1:10 ratio approximately twice per week (see Notes 4 and 5). As B16 is relatively unstable and tumorigenicity may change during culture, it is best to freeze cells on a regular basis. Such cells are best used for tumor experiments one or two passages after thawing.

2. Preparation of tumor cells for injection: B16 cells should be used when the cells are in exponential growth phase. Optimal tumor dose load may vary between 1 x 104 and 5 x 105 cells depending on the experiment and culture conditions, and should be determined in preliminary experiments. For tumor prevention experiments, a tumor inoculation of 1 x 105 B16 cells represents a good starting point.

a. Aspirate medium from tissue culture flask with B16 cells (<50% density).

b. Wash once with PBS to remove FBS.

c. Add 3 mL 1X trypsin solution to the flask, incubate for 5 min at 37°C or until the cells start to detach from the flask.

d. Add 10 mL of culture medium to inactivate the trypsin, dislodge cells from flask by pipetting, and transfer cells into 50-mL centrifuge vial. Wash cells once with culture medium.

e. Resuspend the cells in ice-cold sterile PBS and filter through a 40-|im nylon mesh to remove any aggregates.

f. Determine viable cell counts and dilute the cells with sterile PBS to a final concentration of 1 x 106 B16 cells/mL of PBS. The cells should be kept on ice during transfer to the animal facility.

3. Injection of B16 melanoma cells:

a. The right flank of mice should be shaved prior to tumor inoculation to facilitate injection and monitoring of tumor growth.

b. All experimental groups should be inoculated identically. Therefore, great care should be taken that the syringe is filled with a homogenous cell suspension and that no air bubbles are present. The cells should be mixed just before filling of the syringe by inverting the tube several times and should be mixed between injections by carefully inverting the syringe. B16 cell suspensions tend to become more concentrated near the plunger. Therefore, the last 50-100 |L should be discarded.

c. Each mouse should be injected s.c. with 100 ||L of B16 cell suspension into the shaved part of the right flank. A defined swelling/bleb should be visible after correct injection at this site.

4. Monitoring of tumor growth:

a. Once the tumor becomes visible, tumor growth should be monitored three times a week. As s.c. B16 tumors are easily palpable, the tumor diameter can be directly measured using a caliper. Either tumor area or tumor size can be used to monitor tumor growth. The tumor area is calculated as the product of two perpendicular diameters multiplied by 0.8 and tumor size represents the product of the largest diameter multiplied by 0.52 and the square of the smallest diameter.

b. Another way of determining the efficacy of the vaccine is to compare survival of the mice. All mice whose tumors reach a certain diameter (usually 20 mm) or show signs of distress should be euthanized.

3.2. Vaccination of B16 Melanoma-Bearing Mice With a Mixture of TRP2 Peptide and CpG Following DC Expansion With Flt3-L

1. For evaluation of antitumor activity, mice receive a s.c. injection of 1 x 104 B16 cells in 100 ||L PBS into the right flank as described in Subheading 3.2.3. Because of the aggressive growth of B16 tumors it is necessary to reduce the number of tumor cells to 1 x 104 cells to allow the vaccine sufficient time to induce an antitumor immune response.

2. Flt3-L treatment is begun 3 d after tumor inoculation. Ten micrograms of Flt3-L (50 |g/mL in PBS) per mouse is administered daily over a course of 9 d by intraperitoneal (i.p.) injection (similar to Subheading 3.2.2.). At day 12 after tumor inoculation (or 1 d after the last Flt3-L injection), mice receive a single injection of a solution containing 30 |g CpG and 300 |g TRP2 peptide s.c. into the left flank. Use of contralateral injection sites ensures that differences in tumor growth are due to induction of systemic immune responses rather than local inflammation (see Note 5).

3. Tumor growth should be measured by determining the perpendicular diameters with a caliper three times a week.

3.3. Recruiting DC to B16 Tumors With CCL20 Chemokine Followed by Intratumoral Injection of CpG

1. Inoculate three groups of male C57Bl/6 mice with 5 x 104 B16 cells s.c. into the right flank (tumor dose may need to be adapted depending on the particular clone of B16 used). The injection area should be shaved before tumor inoculation to mark the injection site.

2. Recruitment of DC is induced by intratumoral injections of CCL20 for a period of 3 wk starting 1 d after tumor challenge (see Note 7). At the time of the first CCL20 injection, the tumor is not yet visible. Therefore, it is critical to mark the area of tumor inoculation. The recombinant CCL20 protein is diluted in PBS to a final concentration of 3.3 |g/mL and 30 |L of this solution should be administered directly into the tumor. It is best to use small syringes such as a tuberculine syringe with low retention volume for this purpose.

3. Tumor growth should be monitored at least twice a week. As repeated injections may induce local inflammation at the injection site, tumor-bearing mice receiving intratumoral PBS injection should serve as control. Induction of systemic immunity may also be evaluated by rechallenge of mice with 5 x 104 B16 tumor cells in the opposite flank.

4. In contrast to more immunogenic tumors such as CT26, increasing intratumoral DC numbers in B16 tumors is not sufficient to induce systemic antitumor immunity. In this case, the DC need to receive external activation signals to overcome inhibitory signals provided by the tumor. For activation of recruited DC, inject mice with 80 |g CpG (or control oligonucleotide) in 30 |L of PBS into the tumor on day 7 after tumor inoculation. This procedure should be repeated every 3 d for a total of five injections (d 7, 10, 13, 16, and 19).

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