Spleen but not tumor infiltration by dendritic and T cells is ... - Nature

Cancer Gene Therapy (2007) 14, 364–371 r

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ORIGINAL ARTICLE

Spleen but not tumor infiltration by dendritic and T cells is increased by intravenous adenovirus-Flt3 ligand injection JC Solheim1, AJ Reber1, AE Ashour2, S Robinson3, M Futakuchi2, SG Kurz2, K Hood2, RR Fields2, LR Shafer2, D Cornell4, S Sutjipto4, S Zurawski5, DM LaFace4, RK Singh2 and JE Talmadge2 1

Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska, USA; 2Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA; 3MD Anderson Cancer Center, The University of Texas, Houston, TX, USA; 4Canji Inc., San Diego, CA, USA and 5DNAX Research Institute, Palo Alto, CA, USA

Dendritic cell (DC) expansion is regulated by the hematopoietic growth factor fms-like tyrosine kinase 3 ligand (Flt3L). DCs are critical to the control of tumor growth and metastasis, and there is a positive correlation between intratumoral DC infiltration and clinical outcome. In this report, we first demonstrate that single intravenous (i.v.) injections of adenovirus (Adv)-Flt3L significantly increased splenic dendritic, B, T and natural killer (NK) cell numbers in both normal and mammary tumor-bearing mice. In contrast, the numbers of DCs and T cells infiltrating the tumors were not increased. Consistent with the minimal effect on immune cell infiltration, i.v. Adv-Flt3L injections had no therapeutic activity against orthotopic mammary tumors. In addition, we noted tumor and Adv-Flt3L expansion of Gr1 þ CD11b þ immature myeloid suppressor cells (IMSCs), which may inhibit the therapeutic efficacy of Adv-Flt3L-expanded DCs. Cancer Gene Therapy (2007) 14, 364–371. doi:10.1038/sj.cgt.7701018; published online 19 January 2007 Keywords: dendritic cell; Flt3L; mammary tumor; breast cancer; cytokine

Introduction

fms-like tyrosine kinase 3 ligand (Flt3L) stimulates the expansion and differentiation of hematopoietic progenitor and stem cells, and mobilizes them from the bone marrow (BM) into blood as well as into lymphoid and parenchymal tissues in mice and humans.1–9 Compared to control mice, Flt3L-treated mice have significantly increased frequencies and numbers of dendritic cell (DC) 1s (CD11c þ 11b), DC2s (CD11c þ 11b þ ) and plasmacytoid DCs (CD11c þ B220 þ ) in their spleens and peripheral blood (PB).7,10,11 Moreover, Flt3L administration expands the numbers of lymphoid lineage cells, including natural killer (NK) cells12–16 and B and T lymphocytes, particularly type 1 T cells.7,10 Several studies using mouse models have shown that Flt3L, administered either before or after tumor initiation, can inhibit tumor growth in vivo and act as a vaccine adjuvant.17–21 However, results from clinical studies on the effectiveness of Flt3L as a tumor vaccine adjuvant Correspondence: Dr JC Solheim, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA. E-mail: [email protected] Received 2 August 2006; accepted 4 November 2006; published online 19 January 2007

have been mixed.20,22,23 Patients with HER-2/neu-positive tumors, who were given Flt3L together with a HER-2/neu peptide vaccine, had an increased frequency of interferon-gamma (IFN-g)-secreting anti-HER-2/neu T-cell precursors.22 However, when Flt3L was given systemically as an adjuvant for a HER-2/neu peptide vaccine, only one of 20 patients developed a detectable antigen (Ag)-specific, T lymphocyte response.20 Flt3L has been reported to exhibit a paradoxical profile; in some cases, it has been found to expand DCs in the absence of a therapeutic response and to induce tolerance.24 Furthermore, in some studies when adenovirus-(Adv-) Flt3Lexpanded DCs were loaded with tumor peptides and used as a vaccine, no protection was observed. Instead, accelerated tumor growth occurred after challenge with a tumor expressing the relevant peptide.24 These, as well as other studies, have suggested that Flt3L, like other cytokines,25 can stimulate the expansion of immature myeloid suppressor cells (IMSCs). These cells have the potential to promote tumor escape from immune control by inhibiting adaptive immune responses, DC function, and infiltration of tumors with T cells, resulting in the suppression of T-cell control of tumor progression and growth.26 Given that the Flt3L protein requires repeated administration for DC expansion, we hypothesized that prolonged Flt3L production by a viral vector might result in improved therapeutic activity. In this study, we

Impact of adenovirus-Flt3 ligand on immune cells JC Solheim et al

assessed the immunopharmacologic and therapeutic activity of intravenous (i.v.)-injected Adv-Flt3L in clone (cl)-66 mammary tumor-bearing mice. We report here that Adv-Flt3L injection significantly expanded DCs and T cells in the spleens of cl-66 tumor-bearing mice (as well as normal mice), but had minimal therapeutic activity for mice bearing pre-existing tumors and did not increase DC and T-cell infiltration of primary cl-66 tumors. These observations suggest a local/regional immunosuppression associated with the tumor, which may be due to tumor and Adv-FLt3L expansion of IMSCs.

Materials and methods

Mice, cells and injections Female BALB/c mice, 6–8 weeks of age, were purchased from Charles River Laboratories (Wilmington, MA) and allowed to acclimate for at least 2 weeks before use. The Adv-FltL construct was made using a previously described Flt3L cDNA as an IgG1 Fc fusion.1 For experiments in which tumor-bearing mice were used, tumors were initiated by injection of cl-66 mammary adenocarcinoma cells in the mammary fat pad of BALB/c mice. The cl-66 tumor is a BALB/c-derived mammary adenocarcinoma that arose spontaneously and metastasizes to the lung and bone.27,28 Tumors were allowed to grow to a volume of B100 mm3 before the mice were injected i.v. with Adv-Flt3L, control Adv or phosphate– buffered saline (PBS). For some experiments, the treated mice were killed for harvesting of PB and/or spleens. PB differentials and blood, spleen and BM cell counts PB was collected into heparin from the retro-orbital sinuses of anesthetized mice. White blood cell (WBC) counts were performed on the blood with the System 9000 Hematology Series Cell Counter (Serono-Baker Diagnostics Inc., Allentown, PA). Spleens were removed, single-cell suspensions were prepared, and cell counts were determined with a Careside H-2000 Hematology Analyzer (Culver City, CA). Flow cytometric analysis After lysis of red blood cells (RBCs) in PB or splenic single-cell suspensions, 100 ml aliquots of WBCs (at a density of 5 106 cells/ml) were labeled by incubating the cells with antibodies (Abs) for 30 min. The Abs used were labeled with fluorescein isothiocyanate (FITC), biotin, Cy-Chrome or phycoerythrin (PE) (BD PharMingen, San Diego, CA). Biotinylated Abs were used in conjunction with allophycocyanin (APC)–streptavidin (Molecular Probes, Eugene, OR). For flow cytometric analysis of immune cells from murine tumors, the tumors from mice were resected and nonnecrotic tumor tissue was minced. The tumor samples were transferred to 15 ml tubes, pelleted by centrifugation at 300 g, and washed twice in sterile Hank’s balanced salt solution (HBSS). The samples were then re-suspended in freshly prepared HBSS containing collagenase (200 U/ml; Sigma-Aldrich, St Louis, MO) and deoxyribonuclease I (270 Ku/ml; Sigma-Aldrich, St Louis, MO, USA) and

incubated at 371C with continuous agitation for 1 h. Following collagenase and deoxyribonuclease I treatment, the cells were passed through a 70-mm nylon cell strainer (BD Biosciences, Franklin Lakes, NJ), washed one time in HBSS, and layered over Lympholyte-M (Cedarlane, Hornby, Ontario, canada). After centrifugation, mononuclear cells were collected from the interface, washed and adjusted to a concentration of 1 107 cells/ ml. Nonspecific binding was blocked with 1.5% normal mouse serum and the samples were stained with Abs for flow cytometric analysis. For staining of DC subsets, anti-CD11c (FITCconjugated), anti-CD11b (biotin-conjugated), anti-CD8 alpha (a) (Cy-Chrome-conjugated) and anti-B220 (CyChrome-conjugated) Abs were used (BD PharMingen, San Diego, CA and Molecular Probes, Eugene, OR). For NK cells, a DX5-FITC Ab (BD PharMingen) was used. For T-cell subsets, biotin-labeled anti-CD4 and CyChrome-labeled anti-CD8a were used (Molecular Probes and BD PharMingen). After staining, the cells were washed and fixed with 2% paraformaldehyde, and the data were acquired with a FACS Vantage (BD Immunocytometry Systems, San Jose, CA). Forward and side scatter values were collected on a linear scale, whereas the PE and FITC signals were collected on a 4-decade log scale. Overlaps of emission spectra were electronically compensated, and, using the threshold on forward scatter to eliminate debris, 30 000– 50 000 events were acquired. The frequency distributions of DC cell subsets were determined with Attractors 3.0 software (BD Immunocytometry Systems, San Jose, CA).

Lymphocyte proliferation Splenocytes from the mice were plated at 0.2 106 cells/ well and cultured with optimized concentrations of interleukin-2 (1000 IU/ml) or concanavalin A (Con A) (5 mg/ml). 3H-thymidine was added at 1 mCi/well 18 h before the cells were harvested. 3H-thymidine incorporation was quantified by scintillation counting as counts per minute. Control culture proliferation was subtracted from each group and mean stimulation indices were calculated. Statistical analysis SPSS for Windows (SPSS, Inc., Chicago, IL) was used with the Student’s t-test (two-tailed) to compare mean values. A P-value p0.05 was considered significant. For some experiments, the Mann–Whitney U-test was used for statistical analysis.

Results

Adv-Flt3L administration and expansion of splenic lymphocytes and DCs in normal mice Our preliminary experiments with Adv-Flt3L identified that the i.v. injection of 1 1011 viral particles (VPs) significantly increased spleen leukocyte numbers in a dose–dependent manner with no significant activity observed with 1 1010 VPs and intermediate activity at 3 1010. The responses of mice injected i.v. with control

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numbers of both CD4 þ and CD8 þ lymphocytes were observed on day 22. The delivery of two i.v. injections of Adv-Flt3L (day 22 and 6 before analysis) did not significantly increase any of the immune cell subsets examined relative to a single injection (Figure 1).

Adv (1 1011 VPs) or PBS revealed similar levels of splenic nucleated cells. The numbers of DC1s in the spleen were significantly increased following i.v. injection of 1 1011 VPs, as were the DC2 numbers by the injection of 5 1010 or 1 1011 Adv-Flt3L VPs. Thus, both DC1s and DC2s were significantly increased in the spleen by i.v. administration of 1011 Adv-Flt3L VPs.

Assessment of i.v. Adv-Flt3L treatment on mammary tumor growth Given the significant increases that we observed in the number of spleen cells (including DCs, T cells and NK cells) following i.v. Adv-Flt3L treatment, we examined the effect of i.v. Adv-Flt3L administration on tumor growth. Mice with orthotopic cl-66 mammary tumors were treated with one or two i.v. Adv-Flt3L or PBS injections 1 week apart. Therapy was initiated in mice with a median tumor volume of 230 mm3. However, no significant differences were observed in tumor growth with the injection of Adv-Flt3L as compared to PBS (Figure 2).

Kinetics of Adv-Flt3L effect on immune cell subsets To gain insight into the immune effectors regulated by the i.v. injection of Adv-Flt3L, we undertook a kinetic analysis of DC numbers in the spleen, after the administration of 1011 Adv-Flt3L VPs. CD11c þ CD11b DCs peaked 6 days after Adv-Flt3L injection (Figure 1), whereas DC2s (CD11c þ CD11b þ ) and plasmacytoid DCs (CD11c þ CD11bB220 þ ) peaked from 8 to 12 days (Figure 1 and results not shown). At the peak, eight-, six- and ninefold increases in splenic CD11c þ CD11b, CD11c þ CD11b þ and plasmacytoid DCs were observed, respectively. In this experiment, the levels of DCs returned to near baseline by day 22. We also examined lymphocyte subsets, observing increased numbers of B cells (B220 þ ) and NK cells (DX5 þ ) that peaked on day 12 post-injection and subsequently decreased. In contrast, the greatest

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Systemic effects of Adv-Flt3L administration on immune cells in tumor-bearing mice As the studies discussed above were undertaken using normal mice (Figure 1), the lack of a therapeutic effect (Figure 2) might be due to tumor-associated

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Cell Number # x 107 Figure 1 A single i.v. injection of Adv-Flt3L was found to expand multiple immune cell phenotypes in the spleen with a rapid expansion of DCs, IMSCs, B and NK cells and a later peak of T cells. BALB/c mice (4/group) were injected with 1 1011 Adv-Flt3L VPs at days 22, 16, 12 or 6, or both days 22 and 6. The mice were killed on day 0 and the spleen cells stained for flow cytometry. The mean7s.e.m. numbers of CD11c þ CD11b cells (DC1s), CD11c þ CD11b þ cells (DC2s), CD11c þ B220 þ cells (plasmacytoid DCs), Gr1 þ CD11b þ cells (IMSCs), and B, CD4 þ (T helper), CD8 þ (T cytotoxic) and NK cells, as well as the mean total spleen cells are shown in the figure. Values that are significantly different (Student’s t-test) from the corresponding value at day 0 are indicated by an asterisk.

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Impact of adenovirus-Flt3 ligand on immune cells JC Solheim et al 1800 Saline 1x Adv-Flt3L 1x Saline 2x Adv-Flt3L 2x

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Figure 2 Growth of orthotopic cl-66 tumors was not significantly delayed by i.v. Adv-Flt3L treatment. BALB/c mice with cl-66 tumors growing in the mammary fat pad (median of 230 mm3 volume) were injected i.v. with Adv-Flt3L VPs or with saline (PBS) either once or twice, 7 days apart. Changes in tumor volumes over time were monitored with calipers in two perpendicular directions. The volumes of the tumors (in mm3) were calculated utilizing the equation appropriate for a prolated sphere (volume ¼ width2 length/2). There was no significant difference in tumor volume.

immunosuppression. Therefore, we assessed whether Adv-Flt3L had immune augmenting activity in cl-66 mammary tumor-bearing mice (median tumor volume of 270 mm3) relative to activity in non-tumor-bearing mice. In both control and tumor-bearing mice, a significant increase in the spleen and PB WBC count was observed 9 days following Adv-Flt3L i.v. injection (results not shown). The DC subpopulations were also assessed in the spleens, using flow cytometry (Figure 3), with the demonstration that Adv-Flt3L injection significantly increased the numbers of CD11b þ CD11c þ and CD11bCD11c þ DCs in the spleen relative to salinetreated normal or control mice. In contrast, CD11c þ B220 þ plasmacytoid DCs were increased in Adv-Flt3Linjected control and tumor-bearing mice as compared to tumor-bearing control mice, but not non-tumor-bearing normal mice. In this study, a significant increase in IMSCs was observed following the injection of Adv-Flt3L in both the control and tumor-bearing mice. Interestingly, both IMSCs and CD11c þ CD11b DCs were significantly increased by Adv-Flt3L injection in the tumor-bearing mice compared to non-tumor-bearing mice. The number of B cells (B220 þ ) was also significantly increased by AdvFlt3L injection in normal mice, but not in tumor-bearing mice. In contrast, T-cell numbers in the spleens of normal and tumor-bearing mice were significantly increased compared to non-injected normal or tumor-bearing mice (Figure 3). Both CD4 þ and CD8 þ T cells were increased by the injection of Adv-Flt3L (results not shown).

Effect of Adv-Flt3L on immune cell infiltration of orthotopic tumors Because we observed increases in splenic DC subsets in mice treated with i.v. Adv-Flt3L, we investigated whether

there were also increased numbers of DCs around or within tumors after i.v. Adv-Flt3L administration. Routine hematoxylin and eosin (H&E) staining of paraffin sections and analysis of the margins of tumors from mice injected with Adv-Flt3L, Adv or PBS control did not reveal peritumoral inflammatory responses (data not shown). This was confirmed by flow analysis following collagenase and DNAse digestion of tumors and isolation of mononuclear leukocytes (Figure 4). No significant increases in the number of DCs or lymphocytes infiltrating the tumors were observed (Figure 4 and results not shown). It should be noted that 30–45% of the non-parenchymal cells (NPCs) in the tumors were DC2 cells (CD11b þ CD11c þ ), whereas CD11c þ CD11b þ , CD11c þ CD11b and CD11c þ B220 þ cells each represented approximately 5% of the infiltrating cells. The B, T and NK cells were also not increased and represented only minor populations in the tumor (p1%).

Discussion

In this study, we examined the immunopharmacology and therapeutic activity of i.v. injected Adv-Flt3L. The Adv vector was utilized to provide high and continuous levels of transgene expression in vivo, eliminating the need for multiple injections. Adv vectors have high transduction efficiency (infecting both replicating and differentiated cells), high levels of transgene expression and an absence of genomic DNA integration (thus posing little or no risk of insertional mutagenesis). Our studies showed that a single i.v. injection of 1 1011 Adv-Flt3L VPs induced a significant increase in splenic nucleated cells, predominantly of the myeloid lineage, although lymphocytes were also increased. However, despite the increase in immune effector cells, one or two i.v. injections of Adv-Flt3L have no significant effect on the growth of orthotopic cl-66 mammary tumors. There was a trend toward reduced tumor growth in mice that received two injections of Adv-Flt3L rather than PBS, but the difference in tumor growth was not statistically significant (Figure 2). To acquire insight into the lack of therapeutic activity by the i.v. administration of Adv-Flt3L, we analyzed the numbers and phenotypes of immune cells in the spleens and tumors of mice injected i.v. with Adv-Flt3L. In normal mice, DCs were significantly increased in the spleen following i.v. injection with 1 1011 Adv-Flt3L VPs. All DC phenotypes: splenic DC1s, DC2s, and plasmacytoid DCs peaked at levels six- to ninefold above baseline 6–12 days after Adv-Flt3L administration. The predominant DC subpopulation expanded by Adv-Flt3L injection was the DC2 subset. The DC subset secretes cytokines that may have skewed T cells away from the type 1 phenotype and induced a tolerant phenotype.29,30 Following Adv-Flt3L treatment, splenic B and NK cell numbers were also increased with kinetics similar to the DCs, whereas T-cell numbers continued to rise through at least day 22 following Adv-Flt3L injection. Thus, at a

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Average (+ sem) Cells per Spleen Figure 3 Splenic DCs, IMSCs, T cells and B cells in tumor-bearing mice were expanded by i.v. Adv-Flt3L treatment. BALB/c mice with orthotopic cl-66 mammary tumors (median of 270 mm3 volume) were injected i.v. with 1 1011 Adv-Flt3L VPs (TB Flt3L) or 1 1011 Adv VPs (TB Adv), or were control tumor-bearing mice (TB Ctrl), or age-matched control mice (Ctrl). Ten days later, the mice were killed, spleens harvested, and the cells stained for flow cytometry. Mean numbers of splenic CD11b þ CD11c þ cells (DC2s), CD11bCD11c þ cells (DC1s), CD11c þ B220 þ cells (plasmacytoid DCs), Gr1 þ CD11b þ cells (IMSCs), CD3 þ (T) lymphocytes, and B220 þ (B) lymphocytes are shown. *Significantly different from Ctrl, #significantly different from TB Ctrl mice, and &significantly different from Adv TB animals as determined by the Student’s t-test.

dose of 1 1011 VPs, i.v. Adv-Flt3L treatment was capable of inducing rapid elevations in multiple populations in the spleens of normal mice. In parallel with the increases in DC and T-cell subpopulations in the spleen of mice injected with Adv-Flt3L, the number of splenic Gr1 þ CD11b þ IMSCs was also significantly increased. These cells have potent T-cell suppressive activity via secretion of reactive oxygen species and direct T-cell toxicity.31 As with normal mice, i.v. administration of Adv-Flt3L to tumor-bearing mice caused a significant increase in spleen and PB cellularity relative to non-injected mice. Further, the increase in splenic DCs in tumor-bearing mice after i.v. Adv-Flt3L injection was of similar magnitude to that observed in normal mice injected with

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Adv-Flt3L. Indeed, in this study, the absolute numbers of CD11c þ CD11b DCs and Gr1 þ CD11b þ IMSCs in the spleens of tumor-bearing mice were significantly increased to levels greater than that observed in non-tumor-bearing mice. In several other studies using cl-66 tumor-bearing mice, we have also observed significant increases in the number of Gr1 þ CD11b þ IMSCs in the spleens as compared to non-tumor-bearing mice. However, this occurred only in mice with large tumors and was not observed in the present study with tumors having a median tumor volume of 270 mm3. In contrast to the spleen, the i.v. injection of Adv-Flt3L did not increase the frequency of immune cell subtype in the NPCs that we have examined from the tumor. This included an analysis of DCs, IMSCs, granulocytes, B,


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Frequency (average + sem) of NPCs in Tumors Figure 4 The frequency of intratumoral CD11c þ CD11b DC1s, CD11c þ CD11b þ DC2s, CD11c þ B220 þ plasmacytoid DCs, CD11c þ GR1 þ IMSCs, B220 þ B cells, CD4 þ T helper cells, CD8 þ T cytotoxic cells, and DX-5 þ NK cells were not significantly expanded by Adv-Flt3L treatment in BALB/c mice bearing orthotopic cl-66 mammary tumors. Mice were injected i.v. with 1 1011 Adv-Flt3L VPs (TB Flt3L), 1 1011 Adv VPs (TB Adv), or with PBS (saline). Ten days later, the mice were killed, the tumors collagenase dissociated, the NPCs isolated with Ficoll Hypaque, and the cells were stained for flow cytometry. The cell frequency was statistically analyzed using the Student’s t-test and no significant differences were observed.

NK and T cells within tumors following i.v. Adv-Flt3L injection. This was an unexpected observation, and we suggest that the lack of any change in the frequency of inflammatory cells infiltrating the tumors may be associated with high levels of nitric oxide synthase (NOS-2) activity that we have observed in the spleens and tumors of these tumor-bearing mice (results not shown). The lack of activity by Adv-Flt3L within the tumors is not due to a lack of virus infiltration, as distribution studies with quantitative real-time-polymerase chain reaction analysis of human Flt3L mRNA levels

have revealed virus infiltration of the tumors for 45 days following i.v. injection (results not shown). Overall, our findings suggest that cl-66 tumors do not inhibit Adv-Flt3L-induced expansion of immune cell populations in the spleen, although no increase in tumor infiltrating inflammatory cells is observed. The increase in splenic cell subpopulations included not only DCs, but also T cells and IMSCs. Therefore, the growth of cl-66 mammary tumor appears to inhibit local/regional infiltration of immune cells into the tumor, but not the expansion of T cells and DCs in the spleen of tumor-bearing mice.

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Acknowledgements

We gratefully acknowledge the assistance of the University of Nebraska Medical Center Cell Analysis Facility and Animal Facility. This work was supported by the Nebraska Research Initiative Programs in Molecular Therapeutics and Clinical Translation of Biotechnology (JET, RKS, and JCS), and by an Eppley Cancer Center Grant (JCS). AJR was supported by a DOD Breast Cancer Training Program postdoctoral fellowship (DAMD 17-00-1-0361), and AEA received support from a UNMC Graduate Studies Research Assistantship. References 1 Hannum C, Culpepper J, Campbell D, McClanahan T, Zurawski S, Bazan JF et al. Ligand for FLT3/FLK2 receptor tyrosine kinase regulates growth of haematopoietic stem cells and is encoded by variant RNAs. Nature 1994; 368: 643–648. 2 Lyman SD, James L, Vanden Bos T, de Vries P, Brasel K, Gliniak B et al. Molecular cloning of a ligand for the flt3/flk2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells. Cell 1993; 75: 1157–1167. 3 Gabbianelli M, Pelosi E, Montesoro E, Valtieri M, Luchetti L, Samoggia P et al. Multi-level effects of flt3 ligand on human hematopoiesis: expansion of putative stem cells and proliferation of granulomonocytic progenitors/monocytic precursors. Blood 1995; 86: 1661–1670. 4 Robinson S, Mosley RL, Parajuli P, Pisarev V, Sublet J, Ulrich A et al. Comparison of the hematopoietic activity of flt-3 ligand and granulocyte-macrophage colony-stimulating factor acting alone or in combination. J Hematother Stem Cell Res 2000; 9: 711–720. 5 Pulendran B, Banchereau J, Burkeholder S, Kraus E, Guinet E, Chalouni C et al. Flt3-ligand and granulocyte colonystimulating factor mobilize distinct human dendritic cell subsets in vivo. J Immunol 2000; 165: 566–572. 6 Morse MA, Nair S, Fernandez-Casal M, Deng Y, St Peter M, Williams R et al. Preoperative mobilization of circulating dendritic cells by Flt3 ligand administration to patients with metastatic colon cancer. J Clin Oncol 2000; 18: 3883–3893. 7 Parajuli P, Mosley RL, Pisarev V, Chavez J, Ulrich A, Varney M et al. Flt3 ligand and granulocyte-macrophage colony-stimulation factor preferentially expand and stimulate different dendritic cell and T cell subsets. Exp Hematol 2001; 29: 1185–1193. 8 Maraskovsky E, Daro E, Roux E, Teepe M, Maliszewski CR, Hoek J et al. In vivo generation of human dendritic cell subsets by Flt3 ligand. Blood 2000; 96: 878–884. 9 Mosca PJ, Hobeika AC, Colling K, Clay TM, Thomas EK, Caron D et al. Multiple signals are required for maturation of human dendritic cells mobilized in vivo with Flt3 ligand. J Leukoc Biol 2002; 72: 546–553. 10 Mosley RL, Parajuli P, Pisarev V, Chavez J, Meeks A, Steffel A et al. Flt3 ligand augmentation of T cell mitogenesis and expansion of type 1 effector/memory T cells. Int Immunopharmacol 2002; 2: 925–940. 11 Maraskovsky E, Brasel K, Teepe M, Roux ER, Lyman SD, Shortman K et al. Dramatic increase in the numbers of functionally mature. J Exp Med 1996; 184: 1953–1962. 12 Brasel K, McKenna HJ, Morrissey PJ, Charrier K, Morris AE, Lee CC et al. Hematologic effects of flt3 ligand in vivo in mice. Blood 1996; 88: 2004–2012.

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