HUMAN GENE THERAPY 15:21–34 (January 2004)© Mary Ann Liebert, Inc.

CCL3/MIP-1a Is a Potent Immunostimulator WhenCoexpressed with Interleukin-2 or Granulocyte-Macrophage

Colony-Stimulating Factor in a Leukemia/Lymphoma Vaccine

ANDREE ZIBERT,1 STEFAN BALZER,1 MANFRED SOUQUET,1 TRONG HUNG QUANG,1

CRISTINA PARIS-SCHOLZ,1 MARIE ROSKROW,2 and DAGMAR DILLOO1

ABSTRACT

Chemokines orchestrate trafficking of immune effector cells during inflammation. Here we demonstrate thatchemokines also serve to potentiate effector cell-mediated antineoplastic immune responses in vaccinationstrategies. As a critical mediator of inflammation, macrophage inflammatory protein 1a (CCL3/MIP-1a) at-tracts and stimulates both antigen-presenting and cytotoxic cells. In the A20 leukemia/lymphoma vaccinemodel, we explored the efficacy of MIP-1a in combination with interleukin-2 (IL-2) or granulocyte-macro-phage colony-stimulating factor (GM-CSF). After subcutaneous injection of the MIP-1a1IL-2 or MIP-1a1GM-CSF combination vaccine, focal but pronounced infiltrates of CD41 and CD81 T cells were observedat the vaccination sites. In mice with preestablished leukemia/lymphoma, survival is significantly improvedin animals treated with MIP-1a1GM-CSF- and MIP-1a1IL-2-secreting vaccines. Protection is superior inthe MIP-1a1GM-CSF group, with the effects of MIP-1a and GM-CSF being synergistic. In contrast, sup-pression of lymphoblast proliferation by single-immunogen vaccines secreting MIP-1a, GM-CSF, or IL-2 alonedoes not translate to improved survival. The systemic protective effects afforded by the MIP-1a1IL-2 or MIP-1a1GM-CSF combination are mediated by different effector cell populations. In the MIP-1a1IL-2 group,antineoplastic defense is mediated by CD81 T and NK cells, whereas in the MIP-1a1GM-CSF group CD41

T cells are involved in addition to CD81 cytotoxic T cells, underscoring that T cell help is critical for long-term protection. Thus combination of MIP-1a with different cytokines recruits different sets of effector cellsinto a potent antineoplastic immune response.

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OVERVIEW SUMMARY

In leukemia/lymphoma vaccine models, the antineoplasticefficacy of different combinations of cytokines and costim-ulatory surface molecules has been well documented. How-ever, stimulation of an effective response also depends onrecruitment of the relevant immune system cells to the vac-cination site. In inflammation, the chemokine macrophageinflammatory protein 1a (CCL3/MIP-1a) has been shownto attract and stimulate a wide variety of immune systemcells such as monocytes, macrophages, eosinophils, and ba-sophils, as well as T and B lymphocytes and natural killercells. Because of its pleiotropic mode of action, MIP-1a isalso an attractive candidate for vaccination strategies. In-

deed, superior systemic antineoplastic protection againstdisease progression can be induced by vaccines transgeni-cally expressing MIP-1a in combination with different cy-tokines. In mice with preestablished disease, MIP-1a whencoexpressed with IL-2 or GM-CSF significantly enhancesthe survival benefit mediated by either immunogen alone.

INTRODUCTION

TUMOR VACCINE STRATEGIES involving genetically modifiedcells have been employed for immunotherapy of cancer

(Tepper et al., 1989; Bubenik et al., 1990; Fearon et al., 1990;Gansbacher et al., 1990), and aim to compensate for the re-

1Clinic for Pediatric Oncology, Hematology, and Immunology, University Hospital Dusseldorf, D-40225 Dusseldorf, Germany.2GSF National Research Center for Environment and Health, D-85764 Munich, Germany.

duced immunogenicity of tumor cells (for review see Stripeckeet al., 2002; Todryk, 2002; Morris et al., 2003). In utilizing ge-netically modified tumor cells for vaccination two principalstrategies have been followed for induction of an antineoplas-tic immune response. The first approach directly targets im-mune effector cells by providing in trans the costimulatory sig-nals required for effective T cell stimulation. To this end, genetransfer of costimulatory surface molecules, such as CD80(Matulonis et al., 1995; Dunussi-Joannopoulos et al., 1996;Hirst et al., 1997; Mutis et al., 1998; Stripecke et al., 1998) andCD40 ligand (CD40L) (Dilloo et al., 1997a; Vereecque et al.,2000), or of cytokines that enhance effector cell responses, suchas interleukin 2 (IL-2), IL-4, IL-7, or IL-12, or tumor necrosisfactor a (TNF-a) (Blankenstein et al., 1991; Golumbek et al.,1991; Schmidt-Wolf et al., 1994; Schadendorf et al., 1995; Dil-loo et al., 1996; Levitsky et al., 1996), have successfully beenemployed. The second strategy in tumor vaccination is basedon improving tumor antigen presentation by professional anti-gen-presenting cells (APCs). In this context, granulocyte-mac-rophage colony-stimulating factor (GM-CSF) (Dranoff et al.,1993; Dunussi-Joannopoulos et al., 1998) has been shown toeffectively stimulate cross-priming events, thereby enhancinguptake, processing, and presentation of tumor antigens by bonemarrow-derived dendritic cells (Huang et al., 1994).

Regardless of the vaccination strategy used, effector cellsfirst must be recruited to the vaccination site for immunostim-ulation to occur. Lymphocyte trafficking is also critical for in-duction of systemic immunity. Because of their capacity to reg-ulate migration of specific leukocyte subsets, chemokines havemoved into the focus of immunological research and may alsoprove to be attractive candidates for tumor vaccine generation.In inflammatory responses, it is chemokines that attract the var-ious subsets of effector cells to the site of inflammation and in-duce migration of APCs and lymphocytes to secondary lym-phoid organs (Yoshie et al., 1997; Tedla et al., 1998; Yoshidaet al., 1998; Kirk et al., 2001). Similarly in tumor vaccine ap-proaches, we and others have shown that chemokines may serveto attract immune effector cells to the vaccine site, promotingthe encounter with the relevant tumor antigens and costimula-tory signals (Dilloo et al., 1996; Cao et al., 1998; Braun et al.,2000; Giovarelli et al., 2000; Biragyn et al., 2001; Nagai andMasuzawa, 2001; Nomura et al., 2001; Dunussi-Joannopouloset al., 2002). Yet, information about the efficacy of chemokine-induced therapeutic immunity in the setting of residual neo-plastic disease is sparse (Dilloo et al., 1996; Dunussi-Joannopoulos et al., 2002; Xia et al., 2002).

More than 50 members of the chemokine family and nearly20 receptors known to bind chemokines have now been iden-tified (Rollins, 1997). On the basis of the localization of sharedcysteine sequence elements (Cys–Cys) chemokines are dividedinto two major groups, the CC and CXC chemokines (Rollins,1997), with XCL1/lymphotactin (Ltpn) (Kelner et al., 1994)and CX3CL1/fractalkine (Bazan et al., 1997) being members ofthe C and CX3C groups, respectively. Although there is con-siderable overlap with regard to their chemotactic activity, in-dividual chemokines tend to target different combinations ofcellular subsets. Most CXC chemokines target neutrophils orfibroblasts, whereas some, such as CXCL8/IL-8 (Qin et al.,1996) and CXCL10/IP-10 (Romagnani et al., 2001), also acton T cells. In contrast, the CC chemokines such as macrophage

inflammatory protein 1a (CCL3/MIP-1a do not attract neu-trophils, but target monocytes and lymphocytes. Lymphotactin,the only member of the XC group, is a truly T and natural killer[NK] cell-specific chemokine (Kelner et al., 1994; Hedrick etal., 1997). We and others have previously shown that in a vac-cine approach for hematological malignancies, Ltpn stimulatesa protective antineoplastic immune response (Dilloo et al.,1996; Cairns et al., 2001). In this article we describe work aimedto extend the chemotactic activity of the vaccine to APCs.

Because of its pleiotropic mode of action, macrophage in-flammatory protein 1a (MIP-1a) seems to be of particular in-terest for vaccination strategies. MIP-1a exerts its chemotacticactivity on monocytes (Fahey et al., 1992; Uguccioni et al.,1995) and dendritic cells (Xu et al., 1996), as well as on CD41

and CD81 T cells (Schall et al., 1993; Taub et al., 1993; Rothet al., 1995), NK cells (Taub et al., 1995; Loetscher et al., 1996),eosinophils (Rot et al., 1992), and to a lesser extent on basophils(Alam et al., 1994) via the CCR1 and CCR5 receptors (Gao etal., 1993; Raport et al., 1996). In addition to its chemotacticactivity, MIP-1a also has activating properties and stimulatesboth monocyte (Fahey et al., 1992; Uguccioni et al., 1995) andNK cell activity (Taub et al., 1995). In a leukemia/lymphomavaccine approach we chose to complement the chemotactic ac-tivity of MIP-1a on cytotoxic effector cells as well as APCswith the stimulatory activity of cytokines targeting the samecell populations. Thus, we combined MIP-1a with IL-2 or GM-CSF with the aim of enhancing effector cell stimulation as wellas cross-priming events as the two main principles of tumorvaccination. This approach also recognizes the fact that in acuteleukemia combinations of immunostimulatory molecules are re-quired to eradicate preexisting disease, whereas for many solidtumors single-immunogen vaccination is sufficient to affordprotection (Dranoff et al., 1993; Hogge et al., 1998; Nagai etal., 1998; Nomura et al., 2001). By using this approach, wedemonstrated that in mice with a preexisting pre-B lym-phoblastic malignancy, vaccines secreting MIP-1a in combi-nation with IL-2 or GM-CSF, while recruiting different effec-tor cells into the immune response, significantly improvedsurvival, with the MIP-1a1GM-CSF combination affording su-perior long-term protection.

MATERIALS AND METHODS

Cell lines

The murine prelymphoblastic cell line A20 and the fi-brosarcoma cell line CL7.1 were obtained from the AmericanType Culture Collection (Manassas, VA). The A20 cell lineconsists of pre-B lymphoblastoid cells originally derived froma BALB/c mouse with a spontaneously growing reticulum tu-mor (Kim et al., 1979; Glimcher et al., 1982). Surface markerexpression, such as positivity for CD45R (B220), CD19, heatshock antigen, and surface immunoglobulin and negativity forCD43, indicates that A20 cells meet the criteria for pre-B lym-phoblastic differentiation. A20 cells disseminate rapidly in vivoto lymphoid organs as well as bone marrow (Levitsky et al.,1996). The A20 cell line was maintained in RPMI 1640 con-taining 10% fetal calf serum (FCS), penicillin (100 IU/ml),streptomycin (100 mg/ml), 2 mM L-glutamine (Biochrom,

ZIBERT ET AL.22

Berlin, Germany), and 0.05 mM 2-mercaptoethanol (Sigma,Deisenhofen, Germany); the CL7.1 mouse fibrosarcoma cellline was maintained in Dulbecco’s modified Eagle’s medium(D-glucose, 4.5 g/liter; Biochrom) with the same supplementsas listed above except for the omission of 2-mercaptoethanol.Cell lines were incubated at 37°C with 5% CO2 for A20 cellsand 10% CO2 for CL7.1 cells.

For generation of the cell lines CL7.1-neo, CL7.1-IL-2, andCL7.1-GM-CSF expressing the neomycin phosphotransferasegene (neo) alone or in combination with human IL-2 or murineGM-CSF Moloney leukemia virus-based retroviral vectors wereused as described previously (Dilloo et al., 1996). Moloney leu-kemia virus-based retroviral vectors were used as described pre-viously (Dilloo et al., 1996). For generation of MIP-1a-secret-ing CL7.1 cells, murine MIP-1a cDNA was first amplified bypolymerase chain reaction (PCR) with primers containing re-striction sites for SacII and XhoI (primer sequences: 59-AGC-TACCGCGGTCATGAAGGTCTCC-3 9 and 59-ATGATCTC-GAGGAATTCAGGCATT-3 9). After digestion at the SacII andXhoI sites, the PCR fragment was cloned into the same sites ofthe neo-expressing Harvey sarcomavirus-based vector HaTKN(Spencer et al., 1996). The resulting plasmid was transfected intothe producer cell line BOSC (Pear et al., 1993) for generation ofretroviral supernatant, which was collected from the transfectedcells at confluence and used for transduction of CL7.1 fibroblasts.After three rounds of transduction in the presence of Polybrene(6 mg/ml), fibroblasts were selected in G418 (1 mg/ml; GIBCO,Eggenstein, Germany) for several weeks. Cytokine/chemokineproduction of transduced CL7.1 cell lines was determined in su-pernatants by enzyme-linked immunosorbent assay (ELISA), us-ing the IL-2 Eli-pair (Diaclone, Besançon, France), or by mouseGM-CSF and MIP-1a Quantikine (R&D Systems, Abingdon,UK) assays. Transduced CL7.1 cells (106) produce IL-2 (8ng/ml), GM-CSF (2 ng/ml), or MIP-1a (35 ng/ml) in 24 hr.

Assessment of vaccination effects in vivo

In vivo assessment of antineoplastic protection mediated byMIP-1a1IL-2- or MIP-1a1GM-CSF-secreting vaccine cellswas performed in 11- to 13-week-old female BALB/cBYJ mice(Animal Center of the University of Dusseldorf, Dusseldorf,Germany), a syngeneic model system, as both A20 and CL7.1cells are derived from BALB/c mice. For leukemia/lymphomachallenge, the flanks of mice were injected subcutaneously with105 nonirradiated A20 cells. Application of 105 A20 cells sub-cutaneously results in outgrowth of a solid lymphoblastic massthat can be monitored by serial tumor measurements. In addi-tion, 24 days after subcutaneous challenge, pronounced diseasedissemination and significant lymph node enlargement andorganomegaly, with 1.5- and 2.5-fold enlargement of liver andspleen, are observed. The A20 challenge was followed on days4 and 14 by two subcutaneous vaccine injections. For vaccinepreparation, 2 3 105 CL7.1 fibroblasts expressing either the neogene alone as the control, or IL-2, MIP-1a, or GM-CSF in ad-dition to neo, were admixed with 105 A20 cells. A20 cells wereirradiated to 10 Gy, and CL7.1 fibroblasts were irradiated to 30Gy, before injection. Tumor growth at the site of leukemic chal-lenge was determined by measuring the largest perpendiculardiameters and is presented as square millimeters. Animals werekilled when tumor growth exceeded 450 mm2, their body weight

increased by more than 30%, or when ulceration or other signsof physical distress were noted. Mice that died of causes otherthan disease progression were excluded from the analysis.

In vivo T cell depletion

Mice were depleted of effector cells by intraperitoneal in-jection of monoclonal antibodies directed against CD4 (CD4.1)or CD8 (CD8.2) (hybridomas were generously provided by J.Mysliwietz, GSF National Research Center for Environmentand Health, Munich, Germany). Anti-asialo-GM1 antiserum(Wako, Neuss, Germany) was used to deplete mice of NK cells.One hundred micrograms of the monoclonal T cell antibodiesor 25 ml of the anti-asialo-GM1 antiserum was injected in 200ml of PBS 1 day before A20 challenge and twice weekly there-after for the entire duration of the experiment. Depletion effi-cacy was consistently .98% as verified by weekly flow cyto-metric analysis of peripheral blood cells. Other cellular subsetswere not affected.

Analysis of effector cell infiltrates in vaccine nodules

To assess infiltration of the vaccination site by immune ef-fector cells, nonirradiated vaccine cells were injected subcuta-neously in a separate experiment and mice were killed whenthe vaccine nodules reached discernible size. Vaccine noduleswere excised and frozen sections were stained with anti-CD4(CD4.1), anti-CD8 (CD8.2), anti-asialo-GM1 (Wako), and anti-CD11b (Mac-1) (BD Biosciences Pharmingen, San Diego, CA)antibodies. Detection of positive cells was performed accord-ing to a standard alkaline phosphatase–anti-alkaline phospha-tase (APAAP) protocol (Li et al., 1984) employing rabbit anti-rat immunoglobulin as the secondary antibody and rat APAAPcomplex (DakoCytomation, Neuss, Germany). Single-cell sus-pensions derived from tumor nodules were analyzed for T andNK cell or APC infiltration by flow cytometry analysis in aFACSCalibur (BD Biosciences Immunocytometry Systems,San Jose, CA). For analysis the same panel of fluorescein-la-beled antibodies used for analysis of splenocytes (see below)was employed in addition to the anti-mouse pan-NK cell anti-body (DX5) (BD Biosciences Pharmingen).

Determination of splenocyte subsets in vaccinatedmice by flow cytometry

Spleens were obtained from mice at autopsy. Single-cell sus-pensions were prepared. To determine expansion of cellularsubsets, splenocytes were analyzed by flow cytometry, em-ploying fluorescein-labeled antibodies anti-CD4 (L3T4), anti-CD8 (Ly-2), anti-CD80, anti-CD86 (B7-2), anti-CD3e, anti-MHC-II (I-AD), anti-CD11b, anti-CD11c, anti-Mac3 (BDBiosciences Pharmingen), or anti-NLDC (Cambrex Bio ScienceRockland, Rockland, ME) for immunostaining.

Assessment of cytotoxic activity in splenocytes

Single-cell suspensions were prepared from spleens of micekilled 2 weeks after the second vaccination. Splenocytes werecryopreserved for subsequent analysis of cytotoxic activity. Af-ter defrosting, splenocytes were restimulated with irradiatedA20 cells at a 1:10 ratio for 10 days at 37°C with addition of100 units of IL-2 on day 3. Cytotoxic activity was assessed in

MIP-1a LEUKEMIA/LYMPHOMA VACCINES 23

a standard chromium release assay (Chen et al., 2000). In brief,restimulated splenocytes were mixed with 51Cr-labeled A20 orYAC-1 target cells at an effector-to-target ratio of 1:5, 1:10,1:20, or 1:40 and incubated at 37°C for 4 hr. Released 51Cr ra-dioactivity was measured in a b counter. Specific lysis was cal-culated on the basis of triplicate measurements and expressedas a percentage of maximal 51Cr release according to the for-mula: [(cpmrelease 2 cpmspontaneous release)/(cpmmaximal release 2

cpmspontaneous release)] 3 100.

Statistical analysis

Statistical analysis for comparison between experimentalgroups was performed by x2 test and Wilcoxon test. A valueof p , 0.05 was considered to indicate a significant difference.

RESULTS

MIP-1a in combination with IL-2 or GM-CSFenhances effector cell infiltration to vaccination site

First we addressed the hypothesis that paracrine delivery ofMIP-1a in combination with IL-2 or GM-CSF enhances effec-tor cell infiltration to the vaccination site. For vaccine genera-tion, syngeneic fibroblasts genetically modified by retroviralvectors to express neo alone as a control, or MIP-1a, IL-2, orGM-CSF in addition to neo, were admixed with A20 cells. Inthis first experiment vaccine cells were injected subcutaneouslywithout prior irradiation. Developing vaccine nodules were ex-cised when their diameter reached 8–10 mm2. Tumors in con-trol mice (n 5 4) reached the target size earlier (day 15 6 0)than in mice injected with MIP-1a1IL-2- or MIP-1a1GM-CSF-expressing vaccines (day 25 6 3 and day 42 6 10, re-spectively), indicating that combined expression of MIP-1awith either IL-2 or GM-CSF significantly suppresses leukemiccell proliferation. Analysis of frozen tumor sections by im-munohistochemistry revealed sparse infiltrates of single CD41

and CD81 T cells in the control group. In contrast, tumors fromthe MIP-1a1IL-2 and MIP-1a1GM-CSF vaccination groupswere largely necrotic with focal but pronounced infiltrates ofboth CD41 and CD81 T cells (Fig. 1). We also analyzed sin-gle-cell suspensions of vaccination nodules for NK cell infil-trates. In the controls, no DX5-positive NK cells were detectedby flow cytometry whereas vaccine nodules obtained from micevaccinated with the MIP-1a1IL-2 or the MIP-1a1GM-CSFcombination contained 0.4–2.0% NK cells. With regard to APCinfiltration in mice vaccinated with the MIP-1a combinationswe found no enhancement of CD11c1 cell infiltration by im-munohistochemistry. Also, flow cytometry analysis of single-cell suspensions obtained from vaccine nodules revealed no increase in CD11b1, CD11c1, MAC-31, and NLDC1 cell in-filtrates beyond the degree of infiltration observed in the con-trols (data not shown).

Treatment with MIP-1a vaccines inhibitslymphoblastic tumor growth in mice withpreestablished disease

Next we assessed the efficacy of the combination vaccine inprotecting mice with preexisting leukemia/lymphoma against

disease progression. To mimic the clinical situation of residualdisease, mice were first challenged with 105 nonirradiated A20cells injected subcutaneously before vaccination on days 4 and14 after leukemic challenge. Vaccines consisted of irradiatedA20 cells admixed with irradiated fibroblasts expressing MIP-1a, IL-2, or GM-CSF alone or in combination. The controlgroup received a vaccine expressing neo only. Experimentswere performed with five to seven mice per vaccination group.Results of three independent experiments were summarized foranalysis of tumor growth and survival (Figs. 2 and 3). As anindicator for neoplastic cell proliferation, subcutaneous tumorgrowth was monitored by serial measurements. Vaccines ex-pressing MIP-1a, IL-2, or GM-CSF alone significantly sup-pressed tumor growth (p , 0.05) but the effect was moderate(Fig. 2). Of note, combining MIP-1a with IL-2 had no addi-tional inhibitory effect, whereas combining MIP-1a with GM-CSF synergistically suppressed tumor growth in comparisonwith single-immunogen vaccination (p , 0.01).

MIP-1a1IL-2 and MIP-1a1GM-CSF combinationvaccines improve survival

Both the MIP-1a1IL-2 and MIP-1a1GM-CSF combinationvaccines significantly improved survival (Fig. 3). Whereas noneof the control mice survived day 48 after leukemia/lymphomachallenge, 46 and 75% of mice receiving the MIP-1a1IL-2- orMIP-1a1GM-CSF-expressing vaccine, respectively, survivedbeyond this day. Survival rates for mice vaccinated with singleimmunogens were lower: only 29% for the MIP-1a group, 33%for the IL-2 group, and 30% for the GM-CSF group. Also, long-term survival of these mice was poor after single-immunogenvaccination, with only one long-term survivor per group. In con-trast, 31 and 58% of mice vaccinated with the MIP-1a1IL-2or the MIP-1a1GM-CSF combination, respectively, survivedbeyond day 70 (p , 0.03 and p , 0.001, respectively, in com-parison with control). Survival of mice vaccinated with theMIP-1a1GM-CSF combination was also significantly im-proved as compared with vaccination with single im-munomodulators alone (p , 0.01).

Activated T cell populations are expanded in spleensof mice treated with combination MIP-1a1IL-2 orMIP-1a1GM-CSF vaccine

To characterize effector cells involved in mediating the sys-temic antileukemic immune response, splenocyte subsets wereexamined by flow cytometry at autopsy (Fig. 4). In spleens ofmice receiving the MIP-1a1IL-2- or MIP-1a1GM-CSF-ex-pressing vaccine, an expansion of activated T lymphocytes ex-pressing CD86 or MHC-II, of CD41 helper T cells, and to alesser degree of CD81 cytotoxic T cells was observed as com-pared with the control group. A concomitant expansion of NKcells was not detected in spleens of either of the vaccinationgroups.

Except for a slight but discernible increase in CD11c1

NLDC1 cells after vaccination with the MIP-1a1GM-CSFcombination, with 1.5 6 0.6% of splenocytes expressing thesedendritic cell markers compared with 0.9 6 0.3% in the MIP-1a1IL-2 group and 0.8 6 0.2% in the controls, we have notobserved any quantitative APC modulation in spleens of vac-cinated mice. Yet, there was a distinct difference in the activa-

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MIP-1a LEUKEMIA/LYMPHOMA VACCINES 25

FIG. 1. MIP-1a in combination with IL-2 or GM-CSF enhances effector cell infiltration to the vaccination site. Mice were in-jected subcutaneously with the nonirradiated vaccine mixture of 105 A20 cells and 2 3 105 genetically modified CL7.1 fibro-blasts expressing either neo alone as the control or MIP-1a1IL-2 or MIP-1a1GM-CSF in combination. Vaccine nodules wereexcised and frozen sections were stained by the APAAP technique with monoclonal antibodies directed against CD41 or CD81

T cells (red).

tion state of CD11b1 APCs, with pronounced upregulation ofthe costimulatory molecules CD80 and CD86 as well as in-creased coexpression of the activation markers MAC-3 and I-AD (MHC-II) (Fig. 5). These changes were not detectable af-ter administration of any of the single-immunogen vaccines.Levels of CD11b1CD191 cells were low and did not changesignificantly between the groups. In comparison with enhancedactivation of APCs and effector cells in spleen, no such changeswere detected in draining lymph nodes at the time of analysis(data not shown).

Vaccination with MIP-1a1IL-2 or MIP-1a1GM-CSFcombination stimulates leukemia/lymphoma-specificcytotoxic responses

We assessed the cytotoxic capacity of effector cell popula-tions in the spleens of vaccinated mice. Vaccinations were ap-plied 4 and 10 days after subcutaneous leukemia/lymphomachallenge. On day 24, mice were killed and splenocytes wereisolated for in vitro analysis of cytotoxic responses. After oneround of in vitro restimulation with A20 cells in the presenceof IL-2, the cytotoxic activity of splenocytes was measuredagainst the A20 target cells to assess the leukemia/lymphoma-specific immune response or against YAC-1 target cells to as-sess natural killer (NK) cell activity (Fig. 6). Splenocytes frommice receiving the MIP-1a1IL-2 vaccine mediated significantlysis of A20 as well as YAC-1 target cells. In contrast, cyto-toxic activity induced by the MIP-1a1GM-CSF vaccine wasexclusively directed against A20 cells, with no activity againstthe NK-sensitive YAC-1 target cells.

Adoptive transfer experiments were performed with spleno-cytes obtained from mice vaccinated in the presence ofpreestablished lymphoblastic disease according to the scheduledescribed above. Recipient mice received 107 nonpurged sple-nocytes intravenously, concomitant with a subcutaneous chal-lenge of 2 3 105 A20 cells. Adoptive transfer of splenocytesobtained from donor mice with lymphoblastic disease havingreceived the control vaccine resulted in rejection of the A20 in-oculate in 47% of recipient mice. Splenocytes adoptively trans-ferred from mice vaccinated with MIP-1a1IL-2 or MIP-1a1GM-CSF further improved protection, with 64 and 63%long-term survivors, respectively.

Antineoplastic protection induced by MIP-1a1IL-2and MIP-1a1GM-CSF combination vaccines ismediated by different effector cell populations

To further delineate the role of T lymphocytes and NK cellsin mediating the antineoplastic immune response in mice vac-cinated with either the MIP-1a1IL-2 or MIP-1a1GM-CSFcombination, mice were depleted of these effector cell popula-tions by injection of antibodies directed against CD41, CD81,and NK cells. Antibodies were injected before leukemia/lym-phoma challenge and then weekly thereafter. As in previous ex-periments, tumors in the control group reached maximum sizebetween days 45 and 48, averaging 416 6 7 mm2, and none ofthe control mice survived beyond these days (Table 1). At209 6 83 mm2, tumors were only half as large after MIP-1a1IL-2 vaccination, and three of seven mice survived. Whenmice treated with the MIP-1a1IL-2 vaccine were depleted of

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FIG. 2. Treatment with MIP-1a vaccines inhibits leukemic tumor growth in mice with preestablished disease. Mice were chal-lenged subcutaneously with 105 A20 leukemic cells followed by two vaccinations on days 4 and 14 with irradiated vaccine con-sisting of 105 A20 cells and 2 3 105 genetically modified CL7.1 fibroblasts expressing either neo as the control, or MIP-1a, IL-2, or GM-CSF alone or in combination. Serial tumor measurements (mean 6 SE, in mm2) on individual days after leukemicchallenge are presented.

CD81 T cells tumors grew rapidly, reaching 322 6 6 mm2 ondays 45–48, and none of the mice survived beyond these days.NK cell-depleted mice died even more rapidly, with all but onemouse dead before day 42. In contrast, protection mediated bythe MIP-1a1IL-2 vaccine was maintained in mice depleted ofCD41 T cells, with tumor sizes of 235 6 65 mm2 and three ofseven mice surviving, as among nondepleted mice. These dataunderscore the observation (Fig. 6) that protection induced bythe MIP-1a1IL-2 vaccine is predominantly mediated by CD81

T and NK cells.In contrast, both CD41 and CD81 T cells are critically in-

volved in the antineoplastic immune response induced by theMIP-1a1GM-CSF vaccine. In vaccinated mice, depletion ofeither CD41 or CD81 T cells reversed the antiproliferative ef-fect mediated by the MIP-1a1GM-CSF vaccine. On the basisof our in vitro analysis (Fig. 6), NK cells seemed not to be in-volved in the vaccine response induced by the MIP-1a1GM-CSF combination, such that an NK cell-depleted group was notincluded. Although onset of tumor development was shifted to

earlier time points after depletion of CD41 and CD81 T cells(Fig. 7), the effects of T cell depletion on survival were not dis-cernible on day 48. Yet, only two of seven mice in each de-pletion group survived beyond day 70 compared with five ofseven mice surviving in the nondepleted MIP-1a1GM-CSFvaccination group (Table 1).

DISCUSSION

Immune responses are regulated by a complex cellular andhumoral network and it is chemokines that mediate the inter-actions between different immune effector cells. In a vaccina-tion strategy, we employed macrophage inflammatory protein1a (CCL3/MIP-1a) on the basis of the rationale that thispleiotropic chemokine may serve to recruit immune cells to thevaccination site, enhancing the probability for antigen-present-ing and cytotoxic cells to encounter the relevant antigens. Wechose GM-CSF and IL-2 to complement the immunomodula-

MIP-1a LEUKEMIA/LYMPHOMA VACCINES 27

FIG. 3. Treatment with MIP-1a1IL-2 and MIP-1a1GM-CSF combinationvaccines improves survival. Mice withpreestablished leukemic disease weretreated on days 4 and 14 with the irra-diated vaccine mixture of 105 A20 cellsand 2 3 105 genetically modified fibro-blasts expressing either neo as the con-trol, or MIP-1a, IL-2, or GM-CSF aloneor in combination. Each data point rep-resents the survival time of an individ-ual mouse.

FIG. 4. Activated T cell populations areexpanded in spleens of mice treated with thecombination MIP-1a1IL-2 or MIP-1a1GM-CSF vaccine. At autopsy, isolatedsplenocytes from mice treated with the neocontrol vaccine or the MIP-1a1IL-2 or MIP-1a1GM-CSF combination vaccine were an-alyzed by flow cytometry. T cell subpopula-tions are presented as a percentage of totalcells (median and range, n 5 4–7).

tory effect of MIP-1a on antigen-presenting and cytotoxic ef-fector cells, respectively. Indeed, in combination with GM-CSFor IL-2, the chemokine MIP-1a supports a protective antineo-plastic response against preestablished lymphoblastic disease.Just as MIP-1a does not need to be produced by the entire ef-fector cell population to mediate antibacterial defense (Cook etal., 1999), paracrine delivery of MIP-1a by secreting bystandercells in our model was sufficient to stimulate systemic anti-neoplastic immunity. Also, genetically modified bystander cellshave been successfully employed in a number of tumor vaccinestrategies (Tsai et al., 1993; Kim and Cohen, 1994; Tahara etal., 1994; Smythe et al., 1999) and have proven particularlyuseful for neoplasms that do not readily lend themselves to ge-netic modification, such as lymphoblastic malignancies (Dillooet al., 1996, 1997a).

In combination with GM-CSF or IL-2, secretion of MIP-1a

by genetically modified fibroblasts admixed with leukemia/lymphoma cells enhances effector cell recruitment to the vac-cination site. In both the MIP-1a1GM-CSF and MIP-1a1IL-2 groups, vaccination nodules were characterized by dense focal infiltrates of both CD41 and CD81 T cells (Fig. 1). Incontrast, T cell infiltrates in mice receiving the control vaccinewere sparse. Enhanced lymphocyte recruitment mediated bychemokine/cytokine combinations such as MIP-1a1GM-CSFor MIP-1a1IL-2 underscores previous results demonstratingthat chemokines in combination with cytokines augment T cellinfiltration (Dilloo et al., 1996; Nomura et al., 2001). Whenvaccination nodules obtained from mice of the MIP-1a1IL-2and MIP-1a1GM-CSF combination groups were subjected toflow cytometric analysis, an increase in NK cells was noted.With regard to infiltration of APCs at the vaccination site, onlya few studies document such tumor infiltrates after injection of

ZIBERT ET AL.28

FIG. 5. MIP-1a1IL-2 or MIP-1a1GM-CSF vaccines upregulate activation markers in splenic APCs. Splenocytes from micetreated with the neo control vaccine, single vaccines, or the MIP-1a1IL-2 or MIP-1a1GM-CSF combination vaccines were an-alyzed at autopsy by flow cytometry. APCs expressing costimulatory molecules and activation markers are presented as a per-centage of CD11b1 cells (mean 6 SE, n 5 5 or 6).

TABLE 1. TUMOR SIZE AND SURVIVAL AFTER IN VIVO DEPLETION OF EFFECTOR CELLS

Parameter No ControlVaccine cells measured depletion Anti-CD4 Anti-CD8 (Neo)

MIP-1a1IL-2 Tumor sizea 209 6 83 235 6 65 322 6 60 416 6 7(days 45–48)

MIP-1a1GM-CSF Tumor size 061 6 57 125 6 50 143 6 72 334 6 3(days 45–48)

MIP-1a1IL-2 Survivors 3/7 3/7 0/5 0/7(.48 days)

MIP-1a1GM-CSF Survivors 5/7 2/7 2/7 0/7(.70 days)

aIn square millimeters.

a chemokine-secreting vaccine (Nomura et al., 2001; Guo etal., 2003). Whereas MIP-1a also attracts monocytes and den-dritic cells (Uguccioni et al., 1995; Xu et al., 1996), an increasein localized APCs beyond the degree of infiltration seen in thecontrol group was no longer detectable in our study at the timewhen the nonirradiated vaccine cells had grown out to form anodule of discernible size. These findings may be explained bythe rapid and transient chemokinetic effects of MIP-1a andother chemokines on APCs (Nakashima et al., 1996; Lee et al.,2000; Nomura et al., 2001), which may no longer be discernibleat the time when effector cell infiltrates are pronounced. Also,as observed in our and other studies (Nomura et al., 2001;Dunussi-Joannopoulos et al., 2002), considerable APC infiltra-tion is already detectable at the vaccination site of the controlsin the absence of any transgenic chemokine expression.

In mice with preestablished lymphoblastic disease, vaccina-tion with either the MIP-1a1GM-CSF or MIP-1a1IL-2 com-bination resulted in significantly improved survival in compar-ison with controls. The effects of MIP-1a and GM-CSF weresynergistic, with superior protection in the MIP-1a1GM-CSFgroup. Although lymphoblastoid tumor growth was also inhib-ited in mice treated with vaccines secreting MIP-1a, GM-CSF,or IL-2 alone, suppression of neoplastic cell proliferation bysingle-immunogen vaccination did ultimately not translate toimproved survival (Fig. 3).

The systemic protective effects afforded by the combinationvaccines were mediated by different effector cell populationsdepending on whether IL-2 or GM-CSF was used to comple-

ment the chemotactic activity of MIP-1a. In the MIP-1a1IL-2 group, antineoplastic defense was mediated by CD81 T andNK cells. Preferential involvement of NK cells after MIP-1a1IL-2 vaccination was discernible in both the in vivo de-pletion experiments as well as in the in vitro cytotoxic assayswith splenocytes of vaccinated mice as effector cells. EnhancedNK cell function after MIP-1a1IL-2 vaccination may occurbecause the biological activity of MIP-1a extends beyondchemokinetic properties to the stimulation of cytotoxic effec-tor cells. MIP-1a alone induces the generation of NK cell-de-rived cytotoxic cells, termed chemokine-activated killer(CHAK) cells, which are different from IL-2-induced lym-phokine-activated (LAK) cells (Loetscher et al., 1996; Mag-hazachi et al., 1996). The combination of MIP-1a and IL-2therefore is a promising approach to effectively target the MHC-unrestricted cytotoxic pathway, and may be of benefit whenMHC expression on neoplastic target cells is low. Although IL-2 has been reported to provide T cell help and facilitate ex-pansion of leukemia/lymphoma-specific cytotoxic T cells, only31% of mice in the MIP-1a1IL-2 group survived long term.The importance of CD41 helper T cells for in vivo priming ofcytotoxic T lymphocytes has been well established (Keene andForman, 1982; Huang et al., 1994, 1996). Accordingly, survivalbenefit was superior in mice vaccinated with the MIP-1a1GM-CSF combination, which induced a CD41 as well as a CD81

T cell response. The significant contribution of CD41 T cellsin chemokine vaccines acting on effector cells and APCs is inkeeping with previous results (Braun et al., 2000; Cairns et al.,2001; Dunussi-Joannopoulos et al., 2002; Xia et al., 2002)demonstrating that systemic expansion of CD41 T cells is crit-ical to maintain a potent antineoplastic immune response (Dil-loo et al., 1996, 1997a).

Like the chemokine MIP-1a, the cytokine GM-CSF can at-tract APCs to tumor sites (Huang et al., 1994). GM-CSF alsostimulates tumor cell lysis (Grabstein et al., 1986), enhancesthe antigen-presenting capacity of monocytes (Coleman et al.,1988; Masucci et al., 1989), and supports maturation of den-dritic cells into potent activators of resting T cells (Romani etal., 1989). It is thought to be due to these biologic propertiesthat GM-CSF as a single immunogen has proven most effec-tive in solid tumor vaccines at stimulating a potent antineo-plastic immune response. In numerous solid tumor models (Dra-noff et al., 1993; Schmidt et al., 1995; Chatterjee et al., 1999;Suh et al., 1999; Kinoshita et al., 2001) and in early clinicaltrials (Jaffee et al., 1998, 2001; Soiffer et al., 1998; Simons etal., 1999; Kusumoto et al., 2001), GM-CSF-secreting vaccinecells have been shown to induce potent tumor-specific re-sponses. These antitumor responses are not restricted to CD81

T cells but involve both helper T cell type 1 (Th1) and Th2 re-sponses (Hung et al., 1998; Simons et al., 1999). Evidencepoints to cross-priming events as the underlying mechanism ofGM-CSF-facilitated antitumor immunity (Huang et al., 1994,1996). In cross-priming, it is not the genetically modified tu-mor vaccine cells themselves that serve to present the tumorantigen, but rather bone marrow-derived dendritic cells that areattracted to the vaccination site. Effective recruitment of APCsto the vaccination site, however, seems to be critically depen-dent on high levels of GM-CSF expression. Such high levelsof GM-CSF expression are readily achieved by viral gene trans-fer into solid organ tumor cells. In leukemia/lymphoma vac-

MIP-1a LEUKEMIA/LYMPHOMA VACCINES 29

FIG. 6. Vaccination with the MIP-1a1IL-2 or MIP-1a1GM-CSF combination stimulates antileukemic cytotoxicactivity. Splenocytes from mice receiving the MIP-1a1IL-2 orMIP-1a1GM-CSF combination vaccine were isolated 2 weeksafter the second vaccination, restimulated with irradiated A20cells, and analyzed in a standard chromium release assay. Re-stimulated effector cells were tested for cytotoxic activityagainst 51Cr-labeled A20 cells to assess the antileukemic im-mune response or against YAC-1 target cells to assess naturalkiller cell activity. Several ratios of effector to target cells areshown.

cines, however, these levels are more difficult to obtain. Pri-mary human acute lymphoblastic leukemia cells in particularare difficult to transduce with vectors currently available forclinical trial. Even with herpesviral or lentiviral vectors, re-ported GM-CSF levels reach only the lower nanogram range(Dilloo et al., 1997b; Stripecke et al., 2000). This may be oneof the reasons why, in vaccine models aiming to eradicatepreestablished leukemia, GM-CSF as a single immunogen hasproven comparatively ineffective. Only when vaccine cells wereselected for high-producing clones or vaccine cell numbersclearly exceeded the leukemic challenge dose have benefitsbeen observed (Levitsky et al., 1996; Hsieh et al., 1997; de Voset al., 1998; Dunussi-Joannopoulos et al., 1998; Strehl et al.,1999; Stripecke et al., 1999). In most models of acute leuke-mia, GM-CSF has therefore been combined with additional co-stimulation for induction of an effective antineoplastic immuneresponse (Dilloo et al., 1996; Nakazaki et al., 1998; Stripeckeet al., 1998). Here we chose to complement the stimulatory ef-fect of GM-CSF on APCs with the chemotactic activity of MIP-1a. Although we could not delineate any localized effects onAPCs at the vaccination site, a distinct qualitative difference inthe activation status of splenic APCs was detected after vacci-nations with the MIP-1a1IL2 or MIP-1a1GM-CSF combina-tion, which was not observed after single-immunogen vaccina-tions. Also, after vaccination with the MIP-1a1GM-CSFcombination a slight but discernible increase in CD11c1NLDC1

cells was noted. Of interest, this increase in APCs with DC dif-

ferentiation was observed only when MIP-1a was combined withGM-CSF and was not detectable in the MIP-1a1IL-2 vaccina-tion group, underscoring the role of APCs for antineoplastic ef-ficacy induced by the MIP-1a1GM-CSF vaccine.

Induction of tolerance toward leukemia/lymphoma antigens inmice with preestablished disease is a potential concern. Indeed,induction of T cell tolerance against an artificial tumor antigenexpressed by genetically modified A20 cells has been reported(Sotomayor et al., 2001). In our A20 model, adoptive transfer ofsplenocytes obtained from mice with preestablished diseasetreated with the control vaccines resulted in A20 rejection in halfof the recipients. Improved cytotoxic responses observed both invitro and in vivo with splenocytes obtained from mice vaccinatedwith the MIP-1a1IL-2 and MIP-1a1GM-CSF combinationsfurther indicate that any potential tolerizing effects in mice withestablished disease are effectively overcome. Considering the en-hanced activation status of APCs in vaccinated mice, the MIP-1a combination vaccines might thus provide the appropriatestimulatory environment for professional APCs to mediate anti-gen-specific T cell activation rather than tolerance.

Thus, the potential of MIP-1a in a tumor vaccine strategy isbased on its broad spectrum of biological activities, recruitingAPCs as well as cytotoxic effectors into the immune response.The pleiotropic activity of MIP-1a can be complemented byvarious cytokines to target both the afferent and efferent armsof the immunological defense, affording long-term protectionin mice with malignant pre-B lymphoblastic disease.

ZIBERT ET AL.30

FIG. 7. Antileukemic protection induced by the MIP-1a1GM-CSF combination vaccine is mediated by CD41 and CD81 Tcells. Mice with preestablished leukemic disease were treated with the neo control vaccine or the MIP-1a1GM-CSF combina-tion vaccine on days 4 and 14 after leukemic challenge. Mice were depleted in vivo of either CD41 or CD81 T cells by serialintraperitoneal injections of T cell-specific monoclonal antibodies. Tumor growth (mm2) after leukemic challenge is presentedfor individual mice.

ACKNOWLEDGMENTS

We are grateful to C. Krüseman for excellent technical as-sistance. This work was supported by a multicenter grant fromthe German National Ministry for Research and Technology,Strategies for Somatic Gene Transfer (research alliance of theuniversity medical centers at Dusseldorf, Essen, and Halle; co-ordinator, U. Göbel); project 4, Cytokine and Chemokine GeneTransfer into Malignant Hematopoietic Cells for the Genera-tion of a Leukemia Vaccine; and the parents’ support group,Elterninitiative Kinderkrebsklinik e.V.

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Address reprint requests to:Dr. Dagmar Dilloo

Universitätsklinikum DüsseldorfZentrum für Kinder und Jugendmedizin

Klinik für Kinder-Onkologie, Hämatologie, und ImmunologieMoorenstrasse 5

D-40225 Dusseldorf, Germany

E-mail: dilloo@uni-duesseldorf.de

Received for publication February 7, 2003; accepted after re-vision November 17, 2003.

Published online: December 16, 2003.

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