Dynamics of central and peripheral immunomodulation in a murine glioma model

All animal care and experiments involved with this work follow internationally recognized guidelines and are overseen by the Columbia University Institutional Animal Care and Use Committee (Columbia University IACUC Protocol # AC-AAAA6721).

Several studies have shown that PDGF-expressing retrovirus will induce the formation of glial tumors in mice [31, 32]. Our lab has generated a murine glioma model, similar to our rat model [33], by injecting PDGF-expressing retrovirus into the white matter of adult BALB/c mice (CEM, unpublished). Our reasons for use of this model are several. Use of a syngeneic tumor model such as ours is essential to appropriately characterize the interactions of the tumor with the immune system, as syngeneity confers MHC expression recognizable by the animals' adaptive immune systems, as well as avoids induction of an immune response to exogenous foreign antigen. Histologically, these tumors closely resemble GBM, demonstrating palisading necrosis, vascular proliferation, and migration and infiltration by the tumor cells. Considering the high frequency of immune cells at the borders of these tumors, these infiltrative characteristics make this a particularly strong model for gaining insight into the interactions between GBM and the immune system. We have shown that these tumor cells can be isolated and injected into naïve mice for several generations, producing consistent tumor formation.

Briefly, a total of 1 × 10⁵ tumor cells were injected into the right frontal white matter of 13 adult BALB/c mice. All mice were female, between 7 and 8 weeks old, and purchased from Jackson Laboratory (Bar Harbor, ME). In the first series of experiments, 4 mice were injected with tumor cells and then analyzed at 10 dpi. In a subsequent, more comprehensive series of experiments, 13 mice were analyzed at 13 dpi (n = 4), 26 dpi (n = 4), and at 29 dpi upon exhibiting clinical tumor morbidity (n = 1) or at 40 dpi (n = 4); the mouse that became symptomatic at 29 dpi was included with the animals studied at 40 dpi. Mice were anesthetized with ketamine and xylazine, sacrificed, and spleens and brains were harvested.

Fresh spleens were dissociated into single cell suspensions through a 70 μm filter in culture medium (MEM alpha medium with 2ME, Penicillin/Streptomycin/Amphotericin, and 10% FBS). Culture medium and supplies were purchased from Invitrogen (Carlsbad, CA). Cells were stimulated with 5 nM PMA, 1 mM Ionomycin, and 0.1% monensin solution (eBioscience, San Diego, CA) and incubated at 37°C for 9 hours.

Tumors were dissected and mechanically as well as enzymatically dissociated with trypsin, papain, and DNAse. Samples were filtered through 70 μm mesh to create single cell suspensions. These suspensions were purified using a sucrose gradient at 2000 rpm for 20 min to remove neurons and blood, and resuspended. Cells were stimulated with 1 μM LPS and 0.1% monensin and incubated at 37°C for 7.5 hrs.

Splenocytes and tumor cell preparations were washed with PBS/2% FBS and 0.01% monensin, spun again, and resuspended in 100 μl of this medium. Cell surface staining was performed at room temperature, in the dark, for 20 min. Splenocytes were stained using FITC-conjugated anti-mouse CD8, PerCP-conjugated anti-mouse CD3, and Pacific Blue-conjugated anti-mouse CD4 monoclonal antibodies. Tumor cell preparations were stained using PE-conjugated anti-mouse CD11b, PerCP-conjuaged anti-mouse CD45, APC-conjugated anti-mouse CD3, Pacific Blue-conjugated anti-mouse CD4, and Pacific Blue-conjugated anti-mouse CD3 monoclonal antibodies. Cells were washed once in PBS/2% FBS and 0.1% monensin, then resuspended in 200 μL of Fix/Perm solution (BD Bioscience, San Jose, CA) at 4°C for 30 min. They were then washed twice in permeabilization buffer (BD Bioscience, saponin, FBS), and stained for cytokines and FoxP3 at 4°C for 30 min. For intracellular stainining, splenocytes were stained using PE-FoxP3, APC-IFN-γ, and APC-IL-10 antibodies. Tumor cell preparations were stained using APC-conjugated anti-mouse TNF-a and APC-conjugated anti-mouse IL-10 antibodies. The cell preparations were then washed twice in Perm buffer (BD Bioscience) and resuspended in PBS/2% FBS. All antibodies for intracellular and cell surface staining were purchased from eBioscience (San Diego, CA), except for PerCP-conjugated anti-mouse CD45, which was purchased from BD Bioscience (San Jose, CA).

Multiparameter flow cytometry data were collected on an LSR II (BD Biosciences) and analyzed using FlowJo version 8.7.1 (Tree Star, Ashland, OR). As lymphocytes are a large proportion of the splenocyte preparation, there is a reliable and recognizable cluster with the expected forward and side scatter properties, around which the initial gates were created. However, due to the relatively small population of TILs within the tumor/CNS preparation, serial dilutions of splenocytes with tumor preparations were made to derive a lymphocyte gate in tumor preparations. This gate was then applied to the pure tumor preparations for all TIL analyses.

In separate BALB/c mice injected with the same tumor cells in the same fashion outlined above, tumors were grown until tumor morbidity was apparent. Mice were then anesthetized with ketamine and xylazine, sacrificed, perfused with PBS, then PBS with 4% paraformaldehyde. Brains were harvested and kept in 4% paraformaldehyde for 24 hours, then stored in increasing concentrations of glucose in PBS up to 30%. The brains were then frozen, sliced, and mounted on glass slides. For indirect immunofluorescence labeling, sections were pre-incubated with buffered blocking solution (PBS containing 10% horse serum, 0.5% triton and NaN₃ in dilution of 1:500) for 1 hr at room temperature. Sections were subsequently incubated with rat anti-mouse CD11b primary antibody (eBioscience, San Diego, CA) diluted 1:100 in buffered blocking solution at room temperature overnight. Subsequently, sections were thoroughly washed (5 × 3 mins) in PBS and incubated in solutions of rhodamine-conjugated goat anti-rat IgG secondary antibody (Jackson Immunological, West Grove, PA) in buffered blocking solution for 1 hr at room temperature. After incubation, sections were thoroughly washed again in PBS (5 × 3 mins) and incubated with Hoescht stain for five minutes for nuclear visualization. Sections were washed in PBS again and rinsed with distilled water.

Where appropriate, means of proportions were compared using two-tailed Student's t-tests with unequal variances. For analyses of small proportions (< 0.05), logarithmically transformed data were analyzed using one-way ANOVA, and parameters with statistically significant ANOVA results were analyzed further using two-tailed Student's t-tests. P-values less than 0.05 were considered significant for all analyses.