of February 10, 2018.This information is current as

MicroenvironmentC5a in Tumor Progression and the Tumor Opposing Roles for Complement Component

Huang-ge Zhang and Jun YanChunjian Qi, Yihua Cai, Xiaoling Hu, Deep Aggarwal, Lacey Gunn, Chuanlin Ding, Min Liu, Yunfeng Ma,

ol.1200846http://www.jimmunol.org/content/early/2012/08/22/jimmun

published online 22 August 2012J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2012 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

Opposing Roles for Complement Component C5a in TumorProgression and the Tumor Microenvironment

Lacey Gunn,*,†,1 Chuanlin Ding,*,1 Min Liu,* Yunfeng Ma,* Chunjian Qi,*,‡

Yihua Cai,* Xiaoling Hu,* Deep Aggarwal,* Huang-ge Zhang,*,† and Jun Yan*,†

Promoting complement (C) activation may enhance immunological mechanisms of anti-tumor Abs for tumor destruction. However,

C activation components, such as C5a, trigger inflammation, which can promote tumor growth. We addressed the role of C5a on

tumor growth by transfecting both human carcinoma and murine lymphoma with mouse C5a. In vitro growth kinetics of C5a,

control vector, or parental cells revealed no significant differences. Tumor-bearing mice with C5a-transfected xenografted tumor

cells had significantly less tumor burden as compared with control vector tumors. NK cells and macrophages infiltrated C5a-

expressing tumors with significantly greater frequency, whereas vascular endothelial growth factor, arginase, and TNF-a pro-

duction were significantly less. Tumor-bearing mice with high C5a-producing syngeneic lymphoma cells had significantly accel-

erated tumor progression with more Gr-1+CD11b+ myeloid cells in the spleen and overall decreased CD4+ and CD8+ T cells in the

tumor, tumor-draining lymph nodes, and the spleen. In contrast, tumor-bearing mice with low C5a-producing lymphoma cells had

a significantly reduced tumor burden with increased IFN-g–producing CD4+ and CD8+ T cells in the spleen and tumor-draining

lymph nodes. These studies suggest concentration of local C5a within the tumor microenvironment is critical in determining its

role in tumor progression. The Journal of Immunology, 2012, 189: 000–000.

Complement (C) is an important component of the immunesystem, and activation of the C cascade occurs via threepathways (1). The C cascade is highly regulated at sev-

eral levels by C regulatory proteins, such as CD55 (2, 3). CD55(decay-accelerating factor) is expressed on nearly all cells of thebody and overexpressed on tumor cells. It is responsible for theaccelerated dissolution of the C3 and C5 convertases and elimi-nates release of anaphylatoxins (2). C activation component C5a isa potent immune mediator, activating immune cells upon inter-action with the C5a receptor (C5aR), resulting in oxidative burstsin neutrophils, phagocytosis augmentation, and enzymatic granulerelease, as well as vasodilation of local blood vessels to enhanceimmune cell entry and exchange of cells and factors from thecirculation into the local tissue (4). C5a can be quickly degradedto C5a desArg by carboxypeptidases available abundantly in tis-sues and serum (5).

Translational research efforts support a positive role for C5a inanti-tumor mAb therapy. Yeast-derived b-glucan polysaccharideadjuvant therapy has been demonstrated to prime C receptor 3(CR3) on neutrophils for enhanced tumor killing (6). C activationduring b-glucan therapy is critical for efficacy, because it leads tothe release of C5a responsible for recruiting b-glucan–primedneutrophils (7). Expression of CD55 by human ovarian carcinomaSKOV-3 inhibits the release of C5a, thus limiting therapeuticefficacy of combined b-glucan with anti-Her2/neu Ab treatmentin vivo (8). In addition, in vitro studies involving human breastcancer cells demonstrated improvement in tumor cell killing byneutrophils following treatment of cells with anti-Her2/neu mAbfused to either C5a or C5a desArg (9). Furthermore, murinemammary sarcoma EMT6 cells transfected with mouse C5a,which were capable of producing low levels of C5a and inducingminimal migration of J774 macrophage cells in vitro, had the mostsubstantial reduction in tumor growth and regression as comparedwith control EMT6 cells in vivo (10). Contrary to these findings,a more recent study demonstrated a protumorigenic role of C5agenerated via the classical pathway within the tumor microenvi-ronment (11). In this TC-1 murine cervical cancer model, C5a wasshown to recruit significantly more myeloid-derived suppressorcells (MDSCs) and to enhance the production of immunosup-pressive reactive oxygen species and reactive nitrogen species bythese cells resulting in the inhibition of the antitumor-specificCD8+ T cell response and increased tumor burden (11). Thus,the role of C5a in the tumor remains controversial (12–14).In the current study, we addressed the role of C5a on tumor

growth by transfecting tumor cells with mouse C5a. We found thatthe local C5a concentration within the tumor microenvironment iscritical in determining its role in tumor progression.

Materials and MethodsC5a transfection and mouse tumor models

Murine lymphoma RMA and human ovarian carcinoma SKOV-3 cells fromthe American Type Culture Collection (Manassas, VA) were transfectedwith secreting murine C5a, or empty vector DNA plasmids provided by Dr.

*Division of Hematology/Oncology, Department of Medicine, James Graham BrownCancer Center, University of Louisville, Louisville, KY 40202; †Department of Mi-crobiology and Immunology, University of Louisville School of Medicine, Louis-ville, KY 40202; and ‡Department of Oncology, Affiliated Hospital of NanjingMedical University, Changzhou No. 2 People’s Hospital, Changzhou 213003, China

1L.G. and C.D. contributed equally to this work.

Received for publication March 16, 2012. Accepted for publication July 22, 2012.

This work was supported by research funding from National Institutes of HealthGrants R01 CA86412 and R01 CA150947 and the Kentucky Lung Cancer ResearchBoard (to J.Y.).

Address correspondence and reprint requests to Dr. Jun Yan, Tumor ImmunobiologyProgram, James Graham Brown Cancer Center, Clinical and Translational ResearchBuilding, Room 319, University of Louisville, 505 South Hancock Street, Louisville,KY 40202. E-mail address: jun.yan@louisville.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: C, complement; C5aR, C5a receptor; CR3, Creceptor 3; CV, control vector; IF, immunofluorescent; IHC, immunohistochemistry;iNOS, inducible NO synthase; MDSC, myeloid-derived suppressor cell; qRT-PCR,quantitative real-time PCR; TDLN, tumor-draining lymph node; Treg, regulatoryT cell; VEGF, vascular endothelial growth factor.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200846

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F. Liang (Veterinary School of Medicine, Louisiana State University, BatonRouge, LA). Transfection using the Lipofectamine reagent (Invitrogen,Carlsbad, CA) was performed, according to the manufacturer’s guidelines.Supernatants from wells of confluent G418-selected cells were screened byC5a ELISA (BD Biosciences, Bedford, MA), and chemotaxis assay wasperformed using murine macrophage J774 cells. Positively transfectedcells, identified by ELISA, were plated in a limiting dilution assay toisolate single clones expressing C5a for injection and were then passagedin vivo and, following excision, were rescreened by ELISA and immu-nohistochemistry (IHC) for C5a expression. In vitro tumor cell growthrates were measured in real time, using the Acea system (Acea Bio-sciences, San Diego, CA) as described previously (15). Each cell line wasplated in quadruplicate in the ACEA 16-well plate. The cell index wasrecorded to reveal tumor cell growth.

All mice used for in vivo studies were approved and treated followingrequirements of the Institutional Animal Care and Use Committee of theUniversity of Louisville. Studies were performed on both SCID mice 6–10wk of age purchased from Taconic (Germantown, NY) or Harlan Labo-ratories (Fox Chase Cancer Center, Philadelphia, PA) and wild-type (WT)C57BL/6 mice (National Cancer Institute, Frederick, MD). For the SKOV-3 tumor model, 7–10 3 106 C5a-transfected or control vector (CV)-transfected SKOV-3 cells were injected s.c. with Matrigel (BD Bio-sciences). For the murine lymphoma RMA model, female C57BL/6 micewere implanted s.c. with RMA-C5a (3CF4), RMA-C5a (1474), or RMA-CVA1 cells (2 3 105/mouse). Tumor growth was monitored by calipermeasurement.

C5a ELISA

ELISA plates were coated with 2 mg/ml purified rat anti-mouse C5a mAb(BD Biosciences) for overnight at 4˚C. Culture supernatants from trans-fected cells were added, and the biotinylated rat anti-mouse C5a detectionAb (BD Biosciences) was applied at a concentration of 2 mg/ml. Assayswere developed with tetramethylbenzidine conductivity substrate (BioFXLaboratories, Owings Mills, MD), and the OD value was measured at450 nm.

IHC and immunofluorescent staining

Frozen tumor tissue sections were fixed in ice-cold acetone. Appropriateblocking steps were performed on tissue samples including 3% hydrogenperoxide, 3% BSA in 13 PBS, and avidin/biotin blocking kit (VectorLaboratories Burlingame, CA). Biotinylated rat anti-mouse C5a Ab (BDBiosciences) was added at 2 mg/ml. Slides were incubated in the humiditychamber overnight at 4˚C. Streptavidin–HRP (Vector Laboratories) wasprepared and applied to slides. Slides were rinsed and then incubated withthe 3-amino-9-ethylcarbazole substrate solution (Vector Laboratories).Slides were then counterstained with hematoxylin. Images were acquiredwith Aperio ScanScope digital scanners (Aperio, Vista, CA). For immu-nofluorescent (IF) staining, slides were stained with DX5 Alexa Fluor 647or F4/80 allophycocyanin (BioLegend, San Diego, CA) with appropriateisotype controls. Slides were washed, and DAPI (Invitrogen) nuclear stainwas added. Images were acquired by Leica TCS SP5 confocal microscopysystem (Leica Microsystems, Buffalo Grove, IL).

In vitro cytotoxicity

In vitro cytotoxicity assay was performed using the Acea system as de-scribed previously (15). Several innate immune cell populations were usedin various cytotoxicity experiments. Single-cell suspensions prepared fromnaive SCID mouse spleens were added to a flask and cultured briefly formacrophage adherence. Nonadherent cells that contained mostly NK cellsand neutrophils were collected for cytotoxicity studies. These innate im-mune cells were added at an E:T cell ratio of 20:1. To examine Gr-1+

CD11b+ myeloid cell inhibitory activity, Gr-1+CD11b+ cells sorted fromSKOV-3 C5a and SKOV-3 CV tumors were added to wells containing thegrowing SKOV-3 WT cells in the presence or absence of SCID mousenonadherent leukocytes. NK cells were also purified from naive SCIDmouse spleens using the EasySep magnetic beads kit (StemCell Technol-ogies, Vancouver, BC, Canada). The percentage of cytotoxicity was cal-culated as described previously (15).

Flow cytometry

Excised tumor masses were minced and mixed with digestion buffer toprepare single-cell suspensions. Cells from tumor, tumor-draining lymphnodes (TDLN), and the spleen were blocked with CD16/32 FcgR mAb.Surface marker staining on the prepared single-cell suspensions was per-formed on ice. For intracellular staining, the cells were stimulated withPMA/ionomycin in the presence of GolgiPlug for 4 h. The staining was

performed using Foxp3 staining buffer set (eBioscience or BioLegend,San Diego, CA), according to the manufacturer’s protocol. The followingfluorochrome-conjugated mAbs were used: Gr-1, CD11b, F4/80, DX5,NK1.1, CD4, CD8, IFN-g, IL-17, and Foxp3 (BioLegend). Cells werecollected on FACSCalibur, followed by analysis using FloJo software(Tree Star, Ashland, OR).

Quantitative real-time PCR

Small portions of excised tumors were removed with a scalpel blade andweighed. Tumor tissues with similar weight were placed in 1 ml TRIzolreagent (Invitrogen) and kept at 280˚C. Sorted cells were also placed inTRIzol. RNAs were isolated using a Qiagen RNeasy kit, according to themanufacturer’s instructions (Qiagen, Valencia, CA). A set amount of RNAwas also used to control for differences in tumor size. After reversetranscription into cDNA with a Reverse Transcription kit (Bio-Rad, Her-cules, CA), quantitative real-time PCR (qRT-PCR) was then performedon Bio-Rad MyiQ single-color RT-PCR detection system using SYBRGreen Supermix (Bio-Rad), and gene-specific primers were summarized inthe Supplemental Table I. Gene expression levels were normalized toGAPDH housekeeping gene, and data were represented as fold differencesby the 22ΔΔCt method, where ΔCt = Cttarget gene 2 CtGAPDH and ΔΔCt =ΔCtinduced 2 ΔCtreference.

Cytospin and stain

Sorted cells were applied to the Shandon premade cuvettes (Shandon,Pittsburgh, PA) and slide and spun in Shandon Cytospin 3 centrifuge. Afterspinning, cells were fixed to the slide with methanol and stained withProtocol Hema 3 solution I and II (Fisher Diagnostics, Middletown, VA).Cells were analyzed by microscopy under 320 and 340 magnification.Images were captured using the Nikon Eclipse E400.

T cell differentiation assay

Naive CD4 T cells from OT-II OVA TCR transgenic mice (Taconic) werepurified by microbead separation (AutoMACS; Miltenyi Biotec). Macro-phages were harvested from peritoneal cavity and purified with F4/80microbeads. Macrophages were cultured with varying concentrations ofC5a (R&D Systems) for 24 h and then cocultured with CD4 T cells (2:1ratio) in the presence of OVA (15 mg/ml) for an additional 3 d. For reg-ulatory T cell (Treg) induction assay, macrophages were cocultured withCD4 T cells in the presence of OVA and varying amounts of C5a for 4 d.Cells were restimulated with PMA/ionomycin in the presence of GolgiPlugfor 4 h. Intracellular IFN-g, IL-17, or Foxp3 staining was performed.

Graphing and statistical analysis

Prism 5.0 (GraphPad Software, San Diego, CA) was used in creating graphsand analyzing data collected from in vitro and in vivo studies. FollowingC5a ELISA, the standard curve was graphed, a linear regression test wasperformed, and sample concentrations were extrapolated from the standardcurve. Analysis of tumor growth significance and significance between thepercentages of infiltrating cells or cytokine-secreting cells and qRT-PCRdata analysis used the Student t test or two-way ANOVA.

ResultsC5a-expressing tumor cells have significantly reduced growthin SCID mice

The human ovarian adenocarcinoma cell line, SKOV-3, has beenshown to overexpress the C regulatory protein CD55 (8). CD55accelerated inhibition of C5a release and has been demonstrated toresult in diminished tumor infiltration of b-glucan–primed neu-trophils (8). To determine whether C5a expression could enhancethe recruitment of innate leukocytes to the tumor microenviron-ment, eliminating the negative effects of CD55, SKOV-3 cellswere transfected with mouse C5a or CV. SKOV-3 WT cells wereconfirmed not to express C5a by ELISA and IHC, whereas SKOV-3 C5a tumors had abundant C5a deposition (Supplemental Fig.1A). In vitro chemotaxis assay indicated supernatants from SKOV-3 C5a tumor cells enhanced migration of J774 cells (data notshown), suggesting that C5a secreted from transfected tumor cellswas functionally active.To determine whether the transfection of C5a introduced any

growth disparities on cells, in vitro growth kinetics was monitored.

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Both C5a- and CV-transfected clones displayed an initial in vitrogrowth enhancement over SKOV-3 WT cells but was no longerapparent after 12 h of culture; all three cell lines were determined togrow equally well (Supplemental Fig. 1B). Selected clones werethen used to observe their in vivo tumor growth. Using the SCID-immunocompromised/SKOV-3 tumor model was beneficial on twofronts. It permitted for focus on the effect of C5a on innate leu-kocyte infiltration and functional activity of these cells in the tu-mor, exclusively, which were hypothesized to be the main targets.In addition, the model allowed for study of an aggressive humancarcinoma that overexpresses CD55 (8), resulting in the inhibitionof C activation at the C3 and C5 convertase step, eliminating localC5a release. All tumor cell lines demonstrated similar initialin vivo growth; however, beginning around day 24 postinjection,SKOV-3 C5a tumors revealed significant reduction in tumor pro-gression (Fig. 1A). Upon excision at an endpoint time between 31and 38 d for three separate experiments, C5a-expressing tumorsweighed significantly less than both CVand WT tumors (Fig. 1B).

Enhanced infiltration of innate immune cell subsets in SKOV-3C5a-expressing tumors

Innate immune cells have been shown to be important to mount anantitumor response (7, 16, 17) as well as play an important rolein sustaining the immunosuppressive environment and angiogenicswitch promoting tumor growth and metastasis (18, 19). Expres-sion of C5a from the tumor environment may harness the anti-tumor response of these cells. We found a slight increase incirculating DX5+ NK cells from spleen and peripheral bloodsamples and a surprising decrease in splenic Gr-1+ CD11b+ cellscompared with naive SCID (data not shown). Tumors were thenexamined for the role of C5a in enhancing the migration of in-nate leukocytes into the tumor tissue. Flow cytometric analysisrevealed that there was no difference in the percentage of Gr-1+

CD11b+ cells infiltrating the tumor (Fig. 2A). However, C5atumors showed an increased percentage of infiltrating DX5+

CD11b+ NK cells (Fig. 2B) and F4/80+CD11b+ subsets of mac-rophages (Fig. 2C). Taken together, C5a appears to be enhancingthe infiltration of NK cells and macrophages in tumor, two distinctsubsets of innate immune cells that have been shown to be im-portant in tumor immunity.

Tumor microenvironment analysis reveals a significantdecrease in the production of protumorigenic factors

Next, we examined pro- and antitumorigenic gene levels to identifyalterations in the tumor microenvironment as a result of C5a ex-

pression. When the total tumor samples were analyzed by qRT-PCR, many genes evaluated did not show a difference: inducibleNO synthase (iNOS), TGF-b, IL-6, IL-10, IL-12, IFN-g, granzymeB, or perforin (Fig. 3A; data not shown). However, the mRNAlevels of vascular endothelial growth factor (VEGF), arginase, andTNF-a were significantly lower in C5a-expressing tumors (Fig.3A). To further delineate the source of VEGF and iNOS influ-enced by C5a, both CD11b+ leukocytes and CD11b2 tumor cellswere analyzed (Fig. 3B). These data indicated the activity of C5aresulted in reduction of VEGF from tumor cells. In contrast, iNOSmRNA level was significantly lower in C5a-expressing SKOV-3tumor-infiltrating leukocytes (Fig. 3B). Isolation of F4/80+ infil-trates followed by qRT-PCR demonstrated no difference in thecase of VEGF, iNOS, TNF-a, or IL-12 (data not shown); how-ever, F4/80+ cells infiltrating SKOV-3 C5a tumors made signifi-cantly less arginase (Fig. 3C), suggesting an antitumor macrophagephenotype.

The presence of C5a renders naive innate leukocytes morecytotoxic to tumor cells and decreases Gr-1+CD11b+ cellinhibitory activity

In vitro studies were enlisted to determine whether C5a couldenhance the cytotoxicity of tumor cells by naive innate leukocytes.Compared with SKOV-3 CV cells, SKOV-3 C5a cells were killed ata significantly higher percentage by the naive leukocytes (Fig. 4A).In addition, NK cells from naive mice had significantly higherkilling activity for SKOV-3 C5a tumor cells as compared withSKOV-3 CV cells (Fig. 4B). These results indicate C5a has acti-vating potential of naive neutrophils and/or NK cells for superioreffector function.As shown in Fig. 2A, the frequency of Gr-1+CD11b+ cells was

not significantly altered in a C5a-expressing tumor. Next, we ex-amined the inhibitory activity of tumor-infiltrating Gr-1+CD11b+

cells on naive, nonadherent leukocytes-mediated cytotoxicity oftumor cells. Nonadherent leukocytes from naive SCID miceshowed significant level of cytotoxicity against SKOV-3 tumorcells. The addition of Gr-1+CD11b+ cells from either SKOV-3 C5aor SKOV-3 CV tumors significantly inhibited cytotoxic activity(Fig. 4C). However, Gr-1+CD11b+ cells from SKOV-3 C5a tumorswere significantly less suppressive. In the absence of the naiveleukocytes, neither tumor-isolated Gr-1+CD11b+ cells led toSKOV-3 WT tumor cell destruction. They actually promoted tu-mor cell growth in vitro. The images of the Gr-1+CD11b+ cellsdemonstrated nearly all these cells were morphologically similarto neutrophils (Fig. 4D).

FIGURE 1. In vivo growth of SKOV-

3 tumor cells in SCID mice. (A) In vivo

growth of SKOV-3 C5a and controls

revealed a significant reduction in tu-

mor growth of SKOV-3 C5a. Following

s.c. injection of SCID mice with SKOV-

3 tumor cell lines (n = 20, 16, and 8 for

SKOV-3 C5a, CV, and WT, respec-

tively), tumor growth was monitored by

measuring two perpendicular diameters

every 2–4 d. *p , 0.05, **p , 0.01,

***p , 0.001. (B) Tumor weight mea-

surement when all animals were sacri-

ficed. **p , 0.01, ***p , 0.001.

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C5a-expressing tumor cells have significantly acceleratedgrowth in immunocompetent mice

Although the SCID mouse model allows us to study C5a effecton innate immune cells, these mice lack adaptive T/B cells. Todetermine C5a-expressing tumor growth in immunocompetenthost, murine lymphoma RMA cells with or without C5a expres-sion were implanted in WT C57BL/6 mice. Surprisingly, C5a-expressing tumors (RMA-3CF4) grew significantly faster thanCV-transfected cells (RMA-CVA1) from all three independentexperiments (Fig. 5A; data not shown). The frequency of Gr-1+

CD11b+ MDSCs was significantly higher in RMA-3CF4 spleencompared with RMA-CVA1 although no difference was observedin TDLN and tumors (Fig. 5B). Innate immune cells including F4/80+ macrophages and NK1.1+ NK cells were largely unchanged(Fig. 5B).Strikingly, the frequency of both CD4+ and CD8+ T cells from

the spleen, TDLN, and tumor was significantly lower in C5a-expressing tumor-bearing mice as compared with CV tumor-bearing mice (Fig. 5C, 5D). However, significantly more of theRMA-3CF4 spleen and TDLN-infiltrating CD8+ T cells producedIFN-g, although a similar percentage of IFN-g–producing CD8+

T cells was observed in the tumor. In addition, the percentage ofCD4+ T cells from RMA-3CF4 spleen and tumor-producing IFN-gdisplayed a slightly different pattern. Significantly more of thesplenic CD4+ T cells produced IFN-g; however, significantly less

tumor-infiltrating CD4+ T cells produced IFN-g when comparedwith CVA1 controls. In addition, splenic Tregs were significantly

increased in C5a-expressing tumor-bearing mice (Supplemental

Fig. 2A) as compared with CVA1 animals. Taken together, RMA-

3CF4 tumor-bearing mice had overall significantly lower per-

centages of infiltrating CD4+ and CD8+ T cells in the spleen,

TDLN, and tumor, with an increased percentage of these subsets

producing IFN-g in the spleen and TDLN by CD8+ T cells and in

the spleen by CD4+ T cells but decreased IFN-g production by

tumor-infiltrating CD4+ T cells.

Low C5a production from tumor cells significantly decreasestumor growth

Given the contradictory data generated from the different models,we noted C5a concentrations detected from SKOV-3 versus RMA

cells differed. RMA-3CF4 cells secreted higher levels of C5a as

compared with SKOV-3 C5a cells (data not shown). Next, we

examined whether C5a levels from tumor cells have any impact on

the tumor growth. As shown in Fig. 6A, RMA-1474 cells secreted

low level of C5a, which is comparable to SKOV-3 C5a. These

cells had significantly delayed tumor progression as compared

with RMA-CVA1 (Fig. 6B), similar to the SKOV-3 model.In contrast to RMA-3CF4 tumor, Gr-1+CD11b+ MDSC fre-

quency was not significantly changed between RMA-CVA1 andRMA-1474 tumor-bearing mice (Fig. 6C). T cell infiltration pat-

FIGURE 2. Enhanced innate immune cell infiltration in SKOV-3 cells expressing C5a. (A) Percentage of Gr-1+CD11b+ cells in SKOV-3 C5a tumor

and SKOV-3 CV tumor was not significantly altered. (B) Increased percentage of DX5+CD11b+ NK cells were found to infiltrate SKOV-3 C5a tumors

in vivo, as seen by flow cytometry and IF staining. *p , 0.05. (C) Increased percentage of F4/80+CD11b+ macrophages in SKOV-3 C5a tumors as

determined by flow cytometry and IF staining. *p , 0.05, **p , 0.01. Representative data from SKOV-3 C5a (n = 10) and SKOV-3 CV (n = 9) tumors

are shown. Scale bar, 50 mm.

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terns also differed between the groups of RMA tumors. Percen-tages of infiltrating CD4+ and CD8+ T cells were significantlyincreased in the spleen but significantly decreased in TDLN inRMA-1474 tumor-bearing mice. In addition, splenic Treg werecomparable in RMA-1474 versus CVA1 tumor-bearing mice(Supplemental Fig. 2B). Although there were significantly fewerof the T cell subsets infiltrating RMA-3CF4 tumors with high levelsof C5a, no difference was observed in the percentage of infiltration oftumors between CVA1 and RMA-1474 with low C5a (Fig. 6D, 6E).Similar to RMA-3CF4 T cell populations, a significantly greaterpercentage of CD4+ and CD8+ T cells produced IFN-g in the spleenand TDLN of RMA-1474 tumor-bearing mice.

C5a drives Th1 and Treg differentiation viaconcentration-dependent manner

C5a has been previously demonstrated to stimulate Th17 celldifferentiation and trigger autoimmune arthritis and experimental

autoimmune encephalomyelitis (20, 21). We next examined

whether Th cell differentiation mediated by C5a was also con-

centration dependent. To this end, naive OVA TCR transgenic

CD4 T cells were cultured with macrophages in the presence of

varying levels of C5a. As shown in Fig. 7A, C5a at the concen-

trations of 100 and 300 ng/ml significantly promoted Th1 cell

differentiation as revealed by more IFN-g production. However,

FIGURE 3. The altered tumor microenvironment by C5a. (A) Total tumor samples were collected, and RNAs were extracted. qRT-PCR data revealed the

downregulation of VEGF, arginase, and TNF-a mRNA levels in C5a-expressing tumors. *p , 0.05, **p , 0.01. (B) Sorted CD11b+ innate immune cells

and CD11b2 tumor cells were performed for qRT-PCR analysis. Data indicate that VEGF mRNA level is significantly decreased in C5a-expressing tumor

cells, whereas the CD11b+ cells sorted from SKOV-3 C5a have significantly lower levels of iNOS mRNA. *p , 0.05. (C) The SKOV-3 C5a-infiltrating

F4/80+ macrophages expressed significantly lower levels of arginase mRNA. *p , 0.05.

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C5a at the higher level (500 ng/ml) significantly decreased Th1differentiation (Fig. 7A). In contrast, increasing concentrations ofC5a gradually decreased Treg induction (Fig. 7C). C5a at 100 and300 ng/ml significantly decreased Treg induction, whereas C5a atthe higher concentration significantly promoted Treg induction(Fig. 7C). These effects were completely abrogated in C5aR-deficient mice (data not shown). No difference was observed forTh17 cell differentiation (Fig. 7B). These data suggest that C5a-mediated Th1 and Treg differentiation appear to be bell shaped.

DiscussionThus far, the role of C5a in the tumor microenvironment has beeninconclusive, with previously published studies demonstrating eitherC5a release from the tumor resulted in reduced tumor growth (10)or C5a-enhanced immune suppressive cells and supported tumorgrowth (11). We have demonstrated in this study in the SKOV-3xenograft model support of a proimmunogenic, antitumor role forC5a released in the tumor microenvironment. C5a is acting on hostimmune cells and indirectly on tumor cells to alter the cytokinemilieu and enhance tumor infiltration and cytotoxic function of in-nate immune effector cells. The C5a effect in the immunocompetentmodel appears to be concentration dependent. High C5a levelsstimulate tumor growth with significantly decreased infiltration ofCD4+ and CD8+ T cells, whereas low levels of C5a within the tumormicroenvironment decrease tumor progression. Therefore, local C5aconcentration is critical in determining its role in tumor progression.In the SKOV-3 xenograft model, the effect of C5a release in the

tumor elicits minimal changes in the periphery, and dramaticchanges occur in the tumor microenvironment. Because SKOV-3tumor cells lack C5aR expression, the reduced in vivo growth of

SKOV-3 tumor cells expressing C5a is likely due to the responsesby host innate immune cells. As demonstrated by the in vitrocytotoxicity assay, the C5a secreted from tumor cells enhancesthe effector functions of neutrophils and NK cells in vitro andrenders them more cytotoxic to the tumor cells. Similarly, a recentstudy showed that C5a–C5aR interaction enhanced NK cell IFN-gproduction in sepsis (22). C5a release in the tumor also enhancesthe recruitment of innate immune cells to the tumor. Enhancedrecruitment of the DX5+CD11b+ NK cells into the tumor is ben-eficial because of the tumor cytotoxic and immune enhancingpotential of NK cells (23, 24). In solid tumors, NK cell penetrationis noted as a positive prognostic factor, but most solid tumorsdemonstrate inferior NK cell infiltration (23). In a C5a-expressingtumor, macrophages are also significantly increased. Macrophagesplay a dominant role in influencing other immune cells and tumorgrowth depending on phenotype (25, 26). Two extreme ends ofmacrophage polarization have been characterized, based on thestimulatory factors and products released by the cells: M1 (anti-tumorigenic) and M2 (protumorigenic) macrophages (25, 27). Asshown in the current study, macrophages from C5a-expressingtumor have significantly low mRNA levels of arginase, suggest-ing an M1 phenotype. Although the frequency of Gr-1+CD11b+

cells is similar in SKOV3 C5a and SKOV-3 CV tumor, theabundant expression of C5aR on these cells renders them mostsensitive to local C5a concentrations. Indeed, Gr-1+CD11b+ cellsfrom SKOV-3 C5a tumor have less immune-suppressive effect ascompared with those from SKOV-3 CV tumor. Thus, local C5amay promote innate immune cell traffic into tumor, and onceimmune cells are recruited, C5a can activate and enhance thecytotoxic functions.

FIGURE 4. C5a promotes cytotoxicity of SKOV-3 tumor cells, whereas Gr-1+CD11b+ cells from SKOV-3 C5a tumors are significantly less immuno-

suppressive. (A) SKOV-3 C5a and CV tumor cells were cultured overnight, and the following day, nonadherent leukocytes from naive SCID mice as effector

cells were added at a ratio of 20:1 (E:T). After 16 h of coculture, the percentage of cytotoxicity was calculated (n = 6). Data indicate that effector cells kill

significantly more SKOV-3 C5a cells than SKOV-3 CV cells. **p , 0.01. (B) Similarly, purified NK cells from naive SCID mice were added to SKOV-3

C5a or CV tumor cells in vitro, and the percentage of cytotoxicity was determined following 24 h coculture. *p , 0.05. (C) SKOV-3 tumor cells were

cocultured with nonadherent leukocytes as effector cells (20:1) in the presence or absence of Gr-1+CD11b+ cells sorted from CV- or C5a-transfected tumor

(1:1) or without effectors but with sorted Gr-1+CD11b+ cells from tumor. The innate leukocytes demonstrated effective cytotoxicity of SKOV-3 tumor cells

and cytotoxicity was significantly decreased in the presence of Gr-1+CD11b+ cells sorted from tumors. However, Gr-1+CD11b+ cells from SKOV-3 C5a

tumors were significantly less suppressive. **p , 0.01. (D) Cytospin and stain of the Gr-1+CD11b+ cells sorted from the SKOV-3 tumors. Images were

acquired at 320 (left) and 340 (right) magnification.

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FIGURE 5. C5a-expressing lymphoma cells have significantly enhanced tumor progression. (A) WT C57BL/6 mice were injected with C5a-expressing

lymphoma RMA-3CF4 cells or RMA CVA1 cells (n = 10), and tumor growth was recorded. Data are shown as mean 6 SEM. ***p , 0.001. (B) The

Spleen, TDLN, and tumor from tumor-bearing mice were prepared for single-cell suspensions. Cells were then stained with Gr-1, CD11b, F4/80, or NK1.1.

Representative dot plots and summarized data are shown. (C) Cells were stimulated with PMA/ionomycin and surface stained with CD8 and IFN-g in-

tracellularly. Representative dot plots (cells were gated on the CD8+ T cells), summarized IFN-g–producing CD8+ T cells, and total CD8+ T cells are

shown. (D) Cells were stimulated with PMA/ionomycin and surface stained with CD4 and IFN-g intracellularly. Representative dot plots (cells were gated

on the CD4+ T cells), summarized IFN-g–producing CD4+ T cells, and total CD4+ T cells are shown.

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FIGURE 6. Low C5a-expressing lymphoma cells have significantly decreased tumor progression. (A) C5a levels of RMA cells transfected with C5a or CV.

Data indicate that the RMA 3CF4 clone secreted higher levels of C5a than the RMA-1474 clone. (B) WT C57BL/6 mice were injected with low C5a-expressing

lymphoma RMA-1474 cells (n = 10) or RMA CVA1 cells (n = 15), and tumor growth was recorded. Data are shown as mean 6 SEM. *p, 0.05. (C) Cells from

spleen and tumor from tumor-bearing mice were stained with Gr-1 and CD11b. Representative dot plots and summarized data are shown. (D) Cells were stimulated

with PMA/ionomycin and surface stained with CD8 and IFN-g intracellularly. Representative dot plots (cells were gated on the CD8+ T cells), summarized IFN-g–

producing CD8+ T cells, and total CD8+ T cells are shown. (E) Cells were stimulated with PMA/ionomycin and surface stained with CD4 and IFN-g intra-

cellularly. Representative dot plots (cells were gated on the CD4+ T cells), summarized IFN-g–producing CD4+ T cells, and total CD4+ T cells are shown.

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C5a also leads to events that can alter a local tumor environ-ment. Significant changes in four important tumor and immune-regulating factors exist between SKOV-3 C5a and SKOV-3 CVtumors: arginase, iNOS, VEGF, and TNF-a. Arginase and iNOSare essential for MDSC-mediated immune-suppressive effect (28,29). VEGF and its role in angiogenesis and tumor neovascu-larization are exploited by the tumor (30‑32). We show that it isthe tumor cells and not the immune-infiltrating CD11b+ cells thatexpress significantly more VEGF. Because of the lack of C5aRexpression on tumor cells, the mechanism of C5a is indirect.A recent study also demonstrated that C5a negatively regulatesneovascularization and angiogenesis via secretion of solubleVEGF receptor 1 (33).On the contrary, C5a-expressing tumor cells in immunocom-

petent mice showed more complex results. High C5a release in thetumor microenvironment promotes tumor progression, which issimilar as TC-1 tumor model (11). The most striking finding fromthis model is the overall decreased frequencies of CD4+ and CD8+

T cells in the spleen, TDLN, and tumor in high C5a-expressingtumor-bearing mice. Although MDSCs significantly accumulatedin the spleen, MDSCs have been shown to downregulate L-selectin expression on CD4+ and CD8+ T cells, thus decreasingtheir homing to sites where they should be activated (34, 35). Thedecreased CD4+ and CD8+ T cells in high C5a-expressing tumor-bearing mice could be due to the accumulated MDSCs in spleen.This is also supported by the data generated from low C5a-expressing tumors where MDSCs are not significantly altered,and CD4+ and CD8+ T cells are increased in spleen and decreasedin TDLN but equivalent in tumor. The C system has recentlydemonstrated to be critical in regulating adaptive T cell responses(36). C activation can regulate CD4+ Th1, Treg, and Th17 celldifferentiation (20, 21, 37–40) as well as CD8+ T cell immunity(41, 42). Indeed, IFN-g–producing CD8+ T cells are significantly

increased in the spleen and TDLN in both high and low C5a-expressing tumor-bearing mice, suggesting C5a can augmentIFN-g production by CD8+ T cells at distant sites. However, IFN-g–producing CD4+ T cells are differentially regulated by C5abecause tumor-infiltrating IFN-g–producing CD4+ T cells aresignificantly decreased in high C5a-expressing tumors, whereas nodifference is observed in C5a low-expressing tumor. This isfurther supported by the in vitro CD4+ T cell differentiationexperiments showing C5a at 100 and 300 ng/ml promotes Th1responses, whereas C5a at the higher concentration (500 ng/ml)inhibits Th1 and promotes Treg differentiation. In addition,splenic Treg are significantly increased in high C5a-expressingtumor-bearing mice while percentages of splenic Tregs are com-parable in C5a low tumor-bearing animals as compared with CVtumor-bearing mice.We thus hypothesize that tumor growth outcomes may differ

tremendously because of C5a concentration levels in the local tumormicroenvironment. High C5a levels may lead to either overactivationof infiltrating cells or enhancement of an inflammatory setting toperpetuate tumor growth, angiogenesis, and suppression of the an-titumor T cell infiltration. This may be analogous to sepsis, duringwhich an overactivated C system (e.g., the release of high levels ofC5a) disables innate immune cells, decreasing phagocytic functionand resulting in an overall immunosuppressive state (43). Con-versely, low levels of C5a may enhance infiltration of immune cells,and upon entry into the environment, C5a at low levels stimulatesa more powerful antitumor immune response. However, quantita-tion of the chemoattractant C5a and its quick degradation byenzymes in the environment complicate pinpointing the criticalconcentration threshold of C5a. This is particularly important in thesetting of in vivo anti-tumor mAb therapy. In addition, the C5a levelscould be very different in the current model system where tumorscontinuously secrete C5a, whereas under natural conditions, C5a is

FIGURE 7. C5a regulates CD4 T cell differentiation. Peritoneal macrophages were stimulated with varying concentrations of C5a (0–500 ng/ml) for 24 h

and then cocultured with naive CD4 OVA transgenic T cells in the presence of OVA for 3 d. Cells were then stained intracellularly with IFN-g (A) and

IL-17A (B). For Treg induction assay, macrophages were cocultured with naive CD4 OVA transgenic T cells in the presence of OVA with varying con-

centrations of C5a (0–500 ng/ml) for 4 d. Cells were stained intracellularly with Foxp3 (C). Representative dot plots and summarized data are shown.

Cells were gated on the CD4+ T cells. Data are representative of at least three independent experiments.

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primarily produced through local complement fixation. More workneeds to be done to determine whether the current findings arerelevant for an in vivo immunotherapeutic setting. In addition, thedifferences observed in the two model systems need to be rec-onciled because of different tumor types, mouse strains, and thepresence or absence of the adaptive immune system. Nevertheless,the evidence generated from previous studies (10, 11) and thecurrent work support the hypothesis that C5a concentration holdsthe key in determining the response generated in the tumor.

DisclosuresThe authors have no financial conflicts of interest.

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