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C5a in Tumor Progression and the Tumor
Opposing Roles for Complement Component
Huang-ge Zhang and Jun Yan
Chunjian Qi, Yihua Cai, Xiaoling Hu, Deep Aggarwal,
Lacey Gunn, Chuanlin Ding, Min Liu, Yunfeng Ma,
2012; 189:2985-2994; Prepublished online 22
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The Journal of Immunology
by guest on January 13, 2016
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The Journal of Immunology
Opposing Roles for Complement Component C5a in Tumor
Progression and the Tumor Microenvironment
Lacey Gunn,*,†,1Chuanlin Ding,*,1Min 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: 2985–2994.
eral levels by C regulatory proteins, such as CD55 (2, 3). CD55
(decay-accelerating factor) is expressed on nearly all cells of the
body and overexpressed on tumor cells. It is responsible for the
accelerated dissolution of the C3 and C5 convertases and elimi-
nates release of anaphylatoxins (2). C activation component C5a is
a potent immune mediator, activating immune cells upon inter-
action with the C5a receptor (C5aR), resulting in oxidative bursts
in neutrophils, phagocytosis augmentation, and enzymatic granule
release, as well as vasodilation of local blood vessels to enhance
immune cell entry and exchange of cells and factors from the
circulation into the local tissue (4). C5a can be quickly degraded
to C5a desArg by carboxypeptidases available abundantly in tis-
sues and serum (5).
omplement (C) is an important component of the immune
system, and activation of the C cascade occurs via three
pathways (1). The C cascade is highly regulated at sev-
Translational research efforts support a positive role for C5a in
anti-tumor mAb therapy. Yeast-derived b-glucan polysaccharide
adjuvant therapy has been demonstrated to prime C receptor 3
(CR3) on neutrophils for enhanced tumor killing (6). C activation
during b-glucan therapy is critical for efficacy, because it leads to
the release of C5a responsible for recruiting b-glucan–primed
neutrophils (7). Expression of CD55 by human ovarian carcinoma
SKOV-3 inhibits the release of C5a, thus limiting therapeutic
efficacy of combined b-glucan with anti-Her2/neu Ab treatment
in vivo (8). In addition, in vitro studies involving human breast
cancer cells demonstrated improvement in tumor cell killing by
neutrophils following treatment of cells with anti-Her2/neu mAb
fused to either C5a or C5a desArg (9). Furthermore, murine
mammary sarcoma EMT6 cells transfected with mouse C5a,
which were capable of producing low levels of C5a and inducing
minimal migration of J774 macrophage cells in vitro, had the most
substantial reduction in tumor growth and regression as compared
with control EMT6 cells in vivo (10). Contrary to these findings,
a more recent study demonstrated a protumorigenic role of C5a
generated via the classical pathway within the tumor microenvi-
ronment (11). In this TC-1 murine cervical cancer model, C5a was
shown to recruit significantly more myeloid-derived suppressor
cells (MDSCs) and to enhance the production of immunosup-
pressive reactive oxygen species and reactive nitrogen species by
these cells resulting in the inhibition of the antitumor-specific
CD8+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 that
the local C5a concentration within the tumor microenvironment is
critical in determining its role in tumor progression.
Materials and Methods
C5a transfection and mouse tumor models
Murine lymphoma RMA and human ovarian carcinoma SKOV-3 cells from
the American Type Culture Collection (Manassas, VA) were transfected
with secreting murine C5a, or empty vector DNA plasmids provided by Dr.
*Division of Hematology/Oncology, Department of Medicine, James Graham Brown
Cancer Center, University of Louisville, Louisville, KY 40202;†Department of Mi-
crobiology and Immunology, University of Louisville School of Medicine, Louis-
ville, KY 40202; and
Medical University, Changzhou No. 2 People’s Hospital, Changzhou 213003, China
‡Department of Oncology, Affiliated Hospital of Nanjing
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 Health
Grants R01 CA86412 and R01 CA150947 and the Kentucky Lung Cancer Research
Board (to J.Y.).
Address correspondence and reprint requests to Dr. Jun Yan, Tumor Immunobiology
Program, James Graham Brown Cancer Center, Clinical and Translational Research
Building, Room 319, University of Louisville, 505 South Hancock Street, Louisville,
KY 40202. E-mail address: email@example.com
The online version of this article contains supplemental material.
Abbreviations used in this article: C, complement; C5aR, C5a receptor; CR3, C
receptor 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, regulatory
T cell; VEGF, vascular endothelial growth factor.
by guest on January 13, 2016
F. Liang (Veterinary School of Medicine, Louisiana State University, Baton
Rouge, 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 by
C5a ELISA (BD Biosciences, Bedford, MA), and chemotaxis assay was
performed using murine macrophage J774 cells. Positively transfected
cells, identified by ELISA, were plated in a limiting dilution assay to
isolate single clones expressing C5a for injection and were then passaged
in vivo and, following excision, were rescreened by ELISA and immu-
nohistochemistry (IHC) for C5a expression. In vitro tumor cell growth
rates were measured in real time, using the Acea system (Acea Bio-
sciences, San Diego, CA) as described previously (15). Each cell line was
plated in quadruplicate in the ACEA 16-well plate. The cell index was
recorded to reveal tumor cell growth.
All mice used for in vivo studies were approved and treated following
requirements of the Institutional Animal Care and Use Committee of the
University of Louisville. Studies were performed on both SCID mice 6–10
wk 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 106C5a-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 mice
were implanted s.c. with RMA-C5a (3CF4), RMA-C5a (1474), or RMA-
CVA1 cells (2 3 105/mouse). Tumor growth was monitored by caliper
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 detection
Ab (BD Biosciences) was applied at a concentration of 2 mg/ml. Assays
were developed with tetramethylbenzidine conductivity substrate (BioFX
Laboratories, Owings Mills, MD), and the OD value was measured at
IHC and immunofluorescent staining
Frozen tumor tissue sections were fixed in ice-cold acetone. Appropriate
blocking steps were performed on tissue samples including 3% hydrogen
peroxide, 3% BSA in 13 PBS, and avidin/biotin blocking kit (Vector
Laboratories Burlingame, CA). Biotinylated rat anti-mouse C5a Ab (BD
Biosciences) was added at 2 mg/ml. Slides were incubated in the humidity
chamber overnight at 4˚C. Streptavidin–HRP (Vector Laboratories) was
prepared and applied to slides. Slides were rinsed and then incubated with
the 3-amino-9-ethylcarbazole substrate solution (Vector Laboratories).
Slides were then counterstained with hematoxylin. Images were acquired
with Aperio ScanScope digital scanners (Aperio, Vista, CA). For immu-
nofluorescent (IF) staining, slides were stained with DX5 Alexa Fluor 647
or F4/80 allophycocyanin (BioLegend, San Diego, CA) with appropriate
isotype controls. Slides were washed, and DAPI (Invitrogen) nuclear stain
was added. Images were acquired by Leica TCS SP5 confocal microscopy
system (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 used
in various cytotoxicity experiments. Single-cell suspensions prepared from
naive SCID mouse spleens were added to a flask and cultured briefly for
macrophage adherence. Nonadherent cells that contained mostly NK cells
and 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 from
SKOV-3 C5a and SKOV-3 CV tumors were added to wells containing the
growing SKOV-3 WT cells in the presence or absence of SCID mouse
nonadherent leukocytes. NK cells were also purified from naive SCID
mouse spleens using the EasySep magnetic beads kit (StemCell Technol-
ogies, Vancouver, BC, Canada). The percentage of cytotoxicity was cal-
culated as described previously (15).
Excised tumor masses were minced and mixed with digestion buffer to
prepare single-cell suspensions. Cells from tumor, tumor-draining lymph
nodes (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 with
PMA/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 following
fluorochrome-conjugated mAbs were used: Gr-1, CD11b, F4/80, DX5,
NK1.1, CD4, CD8, IFN-g, IL-17, and Foxp3 (BioLegend). Cells were
collected 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 and
weighed. Tumor tissues with similar weight were placed in 1 ml TRIzol
reagent (Invitrogen) and kept at 280˚C. Sorted cells were also placed in
TRIzol. RNAs were isolated using a Qiagen RNeasy kit, according to the
manufacturer’s instructions (Qiagen, Valencia, CA). A set amount of RNA
was also used to control for differences in tumor size. After reverse
transcription into cDNA with a Reverse Transcription kit (Bio-Rad, Her-
cules, CA), quantitative real-time PCR (qRT-PCR) was then performed
on Bio-Rad MyiQ single-color RT-PCR detection system using SYBR
Green Supermix (Bio-Rad), and gene-specific primers were summarized in
the Supplemental Table I. Gene expression levels were normalized to
GAPDH housekeeping gene, and data were represented as fold differences
by the 22ΔΔCtmethod, where ΔCt = Cttarget gene2 CtGAPDHand ΔΔCt =
Cytospin and stain
Sorted cells were applied to the Shandon premade cuvettes (Shandon,
Pittsburgh, PA) and slide and spun in Shandon Cytospin 3 centrifuge. After
spinning, cells were fixed to the slide with methanol and stained with
Protocol 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) were
purified by microbead separation (AutoMACS; Miltenyi Biotec). Macro-
phages were harvested from peritoneal cavity and purified with F4/80
microbeads. Macrophages were cultured with varying concentrations of
C5a (R&D Systems) for 24 h and then cocultured with CD4 T cells (2:1
ratio) in the presence of OVA (15 mg/ml) for an additional 3 d. For reg-
ulatory T cell (Treg) induction assay, macrophages were cocultured with
CD4 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 GolgiPlug
for 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 graphs
and analyzing data collected from in vitro and in vivo studies. Following
C5a ELISA, the standard curve was graphed, a linear regression test was
performed, and sample concentrations were extrapolated from the standard
curve. Analysis of tumor growth significance and significance between the
percentages of infiltrating cells or cytokine-secreting cells and qRT-PCR
data analysis used the Student t test or two-way ANOVA.
C5a-expressing tumor cells have significantly reduced growth
in SCID mice
The human ovarian adenocarcinoma cell line, SKOV-3, has been
shown to overexpress the C regulatory protein CD55 (8). CD55
accelerated inhibition of C5a release and has been demonstrated to
result in diminished tumor infiltration of b-glucan–primed neu-
trophils (8). To determine whether C5a expression could enhance
the recruitment of innate leukocytes to the tumor microenviron-
ment, eliminating the negative effects of CD55, SKOV-3 cells
were transfected with mouse C5a or CV. SKOV-3 WT cells were
confirmed 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 not
shown), suggesting that C5a secreted from transfected tumor cells
was functionally active.
To determine whether the transfection of C5a introduced any
growth disparities on cells, in vitro growth kinetics was monitored.
2986ROLE OF C5a IN TUMOR PROGRESSION
by guest on January 13, 2016
Both C5a- and CV-transfected clones displayed an initial in vitro
growth enhancement over SKOV-3 WT cells but was no longer
grow equally well (Supplemental Fig. 1B). Selected clones were
then used to observe their in vivo tumor growth. Using the SCID-
immunocompromised/SKOV-3 tumor model was beneficial on two
fronts. 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 human
carcinoma that overexpresses CD55 (8), resulting in the inhibition
of C activation at the C3 and C5 convertase step, eliminating local
C5a release. All tumor cell lines demonstrated similar initial
in 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 31
and 38 d for three separate experiments, C5a-expressing tumors
weighed significantly less than both CVand WT tumors (Fig. 1B).
Enhanced infiltration of innate immune cell subsets in SKOV-3
Innate immune cells have been shown to be important to mount an
antitumor response (7, 16, 17) as well as play an important role
in sustaining the immunosuppressive environment and angiogenic
switch 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 in
circulating DX5+NK cells from spleen and peripheral blood
samples and a surprising decrease in splenic Gr-1+CD11b+cells
compared with naive SCID (data not shown). Tumors were then
examined for the role of C5a in enhancing the migration of in-
nate leukocytes into the tumor tissue. Flow cytometric analysis
revealed that there was no difference in the percentage of Gr-1+
CD11b+cells infiltrating the tumor (Fig. 2A). However, C5a
tumors 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 enhancing
the infiltration of NK cells and macrophages in tumor, two distinct
subsets of innate immune cells that have been shown to be im-
portant in tumor immunity.
Tumor microenvironment analysis reveals a significant
decrease in the production of protumorigenic factors
Next,we examined pro- and antitumorigenic gene levelsto identify
alterations 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: inducible
NO synthase (iNOS), TGF-b, IL-6, IL-10, IL-12, IFN-g, granzyme
B, or perforin (Fig. 3A; data not shown). However, the mRNA
levels of vascular endothelial growth factor (VEGF), arginase, and
TNF-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 CD11b2tumor cells
were analyzed (Fig. 3B). These data indicated the activity of C5a
resulted in reduction of VEGF from tumor cells. In contrast, iNOS
mRNA level was significantly lower in C5a-expressing SKOV-3
tumor-infiltrating leukocytes (Fig. 3B). Isolation of F4/80+infil-
trates followed by qRT-PCR demonstrated no difference in the
case 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 macrophage
The presence of C5a renders naive innate leukocytes more
cytotoxic to tumor cells and decreases Gr-1+CD11b+cell
In vitro studies were enlisted to determine whether C5a could
enhance the cytotoxicity of tumor cells by naive innate leukocytes.
Compared with SKOV-3 CV cells, SKOV-3 C5a cells were killed at
a significantly higher percentage by the naive leukocytes (Fig. 4A).
In addition, NK cells from naive mice had significantly higher
killing activity for SKOV-3 C5a tumor cells as compared with
SKOV-3 CV cells (Fig. 4B). These results indicate C5a has acti-
vating potential of naive neutrophils and/or NK cells for superior
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 of
tumor cells. Nonadherent leukocytes from naive SCID mice
showed significant level of cytotoxicity against SKOV-3 tumor
cells. The addition of Gr-1+CD11b+cells from either SKOV-3 C5a
or SKOV-3 CV tumors significantly inhibited cytotoxic activity
(Fig. 4C). However, Gr-1+CD11b+cells from SKOV-3 C5a tumors
were significantly less suppressive. In the absence of the naive
leukocytes, neither tumor-isolated Gr-1+CD11b+cells led to
SKOV-3 WT tumor cell destruction. They actually promoted tu-
mor cell growth in vitro. The images of the Gr-1+CD11b+cells
demonstrated nearly all these cells were morphologically similar
to neutrophils (Fig. 4D).
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.
In vivo growth of SKOV-
The Journal of Immunology 2987
by guest on January 13, 2016
C5a-expressing tumor cells have significantly accelerated
growth in immunocompetent mice
Although the SCID mouse model allows us to study C5a effect
on innate immune cells, these mice lack adaptive T/B cells. To
determine C5a-expressing tumor growth in immunocompetent
host, 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 than
CV-transfected cells (RMA-CVA1) from all three independent
experiments (Fig. 5A; data not shown). The frequency of Gr-1+
CD11b+MDSCs was significantly higher in RMA-3CF4 spleen
compared with RMA-CVA1 although no difference was observed
in TDLN and tumors (Fig. 5B). Innate immune cells including F4/
80+macrophages and NK1.1+NK cells were largely unchanged
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 the
RMA-3CF4 spleen and TDLN-infiltrating CD8+T cells produced
IFN-g, although a similar percentage of IFN-g–producing CD8+
T cells was observed in the tumor. In addition, the percentage of
CD4+T cells from RMA-3CF4 spleen and tumor-producing IFN-g
displayed a slightly different pattern. Significantly more of the
splenic CD4+T cells produced IFN-g; however, significantly less
tumor-infiltrating CD4+T cells produced IFN-g when compared
with 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 decreases
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 and
RMA-1474 tumor-bearing mice (Fig. 6C). T cell infiltration pat-
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.
Enhanced innate immune cell infiltration in SKOV-3 cells expressing C5a. (A) Percentage of Gr-1+CD11b+cells in SKOV-3 C5a tumor
2988ROLE OF C5a IN TUMOR PROGRESSION
by guest on January 13, 2016
terns also differed between the groups of RMA tumors. Percen-
tages of infiltrating CD4+and CD8+T cells were significantly
increased in the spleen but significantly decreased in TDLN in
RMA-1474 tumor-bearing mice. In addition, splenic Treg were
comparable in RMA-1474 versus CVA1 tumor-bearing mice
(Supplemental Fig. 2B). Although there were significantly fewer
of the T cell subsets infiltrating RMA-3CF4 tumors with high levels
of C5a, no differencewas observed in the percentage of infiltration of
tumors between CVA1 and RMA-1474 with low C5a (Fig. 6D, 6E).
Similar to RMA-3CF4 T cell populations, a significantly greater
percentage of CD4+and CD8+T cells produced IFN-g in the spleen
and TDLN of RMA-1474 tumor-bearing mice.
C5a drives Th1 and Treg differentiation via
C5a has been previously demonstrated to stimulate Th17 cell
differentiation 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,
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 CD11b2tumor 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.
The altered tumor microenvironment by C5a. (A) Total tumor samples were collected, and RNAs were extracted. qRT-PCR data revealed the
The Journal of Immunology 2989
by guest on January 13, 2016
C5a at the higher level (500 ng/ml) significantly decreased Th1
differentiation (Fig. 7A). In contrast, increasing concentrations of
C5a gradually decreased Treg induction (Fig. 7C). C5a at 100 and
300 ng/ml significantly decreased Treg induction, whereas C5a at
the higher concentration significantly promoted Treg induction
(Fig. 7C). These effects were completely abrogated in C5aR-
deficient mice (data not shown). No difference was observed for
Th17 cell differentiation (Fig. 7B). These data suggest that C5a-
mediated Th1 and Treg differentiation appear to be bell shaped.
Thus far, the role of C5a in the tumor microenvironment has been
C5a release from the tumor resulted in reduced tumor growth (10)
or C5a-enhanced immune suppressive cells and supported tumor
growth (11). We have demonstrated in this study in the SKOV-3
xenograft model support of a proimmunogenic, antitumor role for
C5a released in the tumor microenvironment. C5a is acting on host
immune cells and indirectly on tumor cells to alter the cytokine
milieu and enhance tumor infiltration and cytotoxic function of in-
nate immune effector cells. The C5a effect in the immunocompetent
model appears to be concentration dependent. High C5a levels
stimulate tumor growth with significantly decreased infiltration of
CD4+and CD8+T cells, whereas low levels of C5a withinthe tumor
concentration 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 dramatic
changes occur in the tumor microenvironment. Because SKOV-3
tumor cells lack C5aR expression, the reduced in vivo growth of
SKOV-3 tumor cells expressing C5a is likely due to the responses
by host innate immune cells. As demonstrated by the in vitro
cytotoxicity assay, the C5a secreted from tumor cells enhances
the effector functions of neutrophils and NK cells in vitro and
renders them more cytotoxic to the tumor cells. Similarly, a recent
study showed that C5a–C5aR interaction enhanced NK cell IFN-g
production in sepsis (22). C5a release in the tumor also enhances
the recruitment of innate immune cells to the tumor. Enhanced
recruitment of the DX5+CD11b+NK cells into the tumor is ben-
eficial because of the tumor cytotoxic and immune enhancing
potential of NK cells (23, 24). In solid tumors, NK cell penetration
is noted as a positive prognostic factor, but most solid tumors
demonstrate inferior NK cell infiltration (23). In a C5a-expressing
tumor, macrophages are also significantly increased. Macrophages
play a dominant role in influencing other immune cells and tumor
growth depending on phenotype (25, 26). Two extreme ends of
macrophage polarization have been characterized, based on the
stimulatory factors and products released by the cells: M1 (anti-
tumorigenic) and M2 (protumorigenic) macrophages (25, 27). As
shown in the current study, macrophages from C5a-expressing
tumor 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, the
abundant expression of C5aR on these cells renders them most
sensitive to local C5a concentrations. Indeed, Gr-1+CD11b+cells
from SKOV-3 C5a tumor have less immune-suppressive effect as
compared with those from SKOV-3 CV tumor. Thus, local C5a
may promote innate immune cell traffic into tumor, and once
immune cells are recruited, C5a can activate and enhance the
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.
C5a promotes cytotoxicity of SKOV-3 tumor cells, whereas Gr-1+CD11b+cells from SKOV-3 C5a tumors are significantly less immuno-
2990 ROLE OF C5a IN TUMOR PROGRESSION
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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.
C5a-expressing lymphoma cells have significantly enhanced tumor progression. (A) WT C57BL/6 mice were injected with C5a-expressing
The Journal of Immunology2991
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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 micewere 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.
Low C5a-expressing lymphoma cells have significantly decreased tumor progression. (A) C5a levels of RMA cells transfected with C5a or CV.
2992 ROLE OF C5a IN TUMOR PROGRESSION
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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 CV
tumors: arginase, iNOS, VEGF, and TNF-a. Arginase and iNOS
are 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 is
the tumor cells and not the immune-infiltrating CD11b+cells that
express significantly more VEGF. Because of the lack of C5aR
expression on tumor cells, the mechanism of C5a is indirect.
A recent study also demonstrated that C5a negatively regulates
neovascularization and angiogenesis via secretion of soluble
VEGF receptor 1 (33).
On the contrary, C5a-expressing tumor cells in immunocom-
petent mice showed more complex results. High C5a release in the
tumor microenvironment promotes tumor progression, which is
similar as TC-1 tumor model (11). The most striking finding from
this model is the overall decreased frequencies of CD4+and CD8+
T cells in the spleen, TDLN, and tumor in high C5a-expressing
tumor-bearing mice. Although MDSCs significantly accumulated
in the spleen, MDSCs have been shown to downregulate L-
selectin expression on CD4+and CD8+T cells, thus decreasing
their homing to sites where they should be activated (34, 35). The
decreased 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 decreased
in TDLN but equivalent in tumor. The C system has recently
demonstrated to be critical in regulating adaptive T cell responses
(36). C activation can regulate CD4+Th1, Treg, and Th17 cell
differentiation (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 augment
IFN-g production by CD8+T cells at distant sites. However, IFN-
g–producing CD4+T cells are differentially regulated by C5a
because tumor-infiltrating IFN-g–producing CD4+T cells are
significantly decreased in high C5a-expressing tumors, whereas no
difference is observed in C5a low-expressing tumor. This is
further supported by the in vitro CD4+T cell differentiation
experiments showing C5a at 100 and 300 ng/ml promotes Th1
responses, 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-expressing
tumor-bearing mice while percentages of splenic Tregs are com-
parable in C5a low tumor-bearing animals as compared with CV
We thus hypothesize that tumor growth outcomes may differ
tremendously because of C5a concentration levels in the local tumor
of infiltrating cells or enhancement of an inflammatory setting to
perpetuate tumor growth, angiogenesis, and suppression of the an-
titumor T cell infiltration. This may be analogous to sepsis, during
which an overactivated C system (e.g., the release of high levels of
C5a) disables innate immune cells, decreasing phagocytic function
and 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 stimulates
a more powerful antitumor immune response. However, quantita-
tion of the chemoattractant C5a and its quick degradation by
enzymes in the environment complicate pinpointing the critical
concentration threshold of C5a. This is particularly important in the
setting of invivo anti-tumor mAb therapy. In addition, the C5a levels
could be very different in the current model system where tumors
continuously secrete C5a, whereas under natural conditions, C5a is
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.
C5a regulates CD4 T cell differentiation. Peritoneal macrophages were stimulated with varying concentrations of C5a (0–500 ng/ml) for 24 h
The Journal of Immunology 2993
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primarily produced through local complement fixation. More work
needs to be done to determine whether the current findings are
relevant for an in vivo immunotherapeutic setting. In addition, the
differences observed in the two model systems need to be rec-
onciled because of different tumor types, mouse strains, and the
presence or absence of the adaptive immune system. Nevertheless,
the evidence generated from previous studies (10, 11) and the
current work support the hypothesis that C5a concentration holds
the key in determining the response generated in the tumor.
The authors have no financial conflicts of interest.
1. Huber-Lang, M., J. V. Sarma, F. S. Zetoune, D. Rittirsch, T. A. Neff,
S. R. McGuire, J. D. Lambris, R. L. Warner, M. A. Flierl, L. M. Hoesel, et al.
2006. Generation of C5a in the absence of C3: a new complement activation
pathway. Nat. Med. 12: 682–687.
2. Spendlove, I., J. M. Ramage, R. Bradley, C. Harris, and L. G. Durrant. 2006.
Complement decay accelerating factor (DAF)/CD55 in cancer. Cancer Immunol.
Immunother. 55: 987–995.
3. Yan, J., D. J. Allendorf, B. Li, R. Yan, R. Hansen, and R. Donev. 2008. The role
of membrane complement regulatory proteins in cancer immunotherapy. Adv.
Exp. Med. Biol. 632: 159–174.
4. Guo, R. F., and P. A. Ward. 2005. Role of C5a in inflammatory responses. Annu.
Rev. Immunol. 23: 821–852.
5. Ehrnthaller, C., A. Ignatius, F. Gebhard, and M. Huber-Lang. 2011. New insights
of an old defense system: structure, function, and clinical relevance of the
complement system. Mol. Med. 17: 317–329.
6. Liu, J., L. Gunn, R. Hansen, and J. Yan. 2009. Combined yeast-derived beta-
glucan with anti-tumor monoclonal antibody for cancer immunotherapy. Exp.
Mol. Pathol. 86: 208–214.
7. Allendorf, D. J., J. Yan, G. D. Ross, R. D. Hansen, J. T. Baran, K. Subbarao,
L. Wang, and B. Haribabu. 2005. C5a-mediated leukotriene B4-amplified neu-
trophil chemotaxis is essential in tumor immunotherapy facilitated by anti-tumor
monoclonal antibody and beta-glucan. J. Immunol. 174: 7050–7056.
8. Li, B., D. J. Allendorf, R. Hansen, J. Marroquin, D. E. Cramer, C. L. Harris, and
J. Yan. 2007. Combined yeast b-glucan and antitumor monoclonal antibody
therapy requires C5a-mediated neutrophil chemotaxis via regulation of decay-
accelerating factor CD55. Cancer Res. 67: 7421–7430.
9. Fuenmayor, J., K. Perez-Vazquez, D. Perez-Witzke, M. L. Penichet, and
R. F. Montano. 2010. Decreased survival of human breast cancer cells expressing
HER2/neu on in vitro incubation with an anti-HER2/neu antibody fused to C5a
or C5a desArg. Mol. Cancer Ther. 9: 2175–2185.
10. Kim, D. Y., C. B. Martin, S. N. Lee, and B. K. Martin. 2005. Expression of
complement protein C5a in a murine mammary cancer model: tumor regression
by interference with the cell cycle. Cancer Immunol. Immunother. 54: 1026–
11. Markiewski, M. M., R. A. DeAngelis, F. Benencia, S. K. Ricklin-Lichtsteiner,
A. Koutoulaki, C. Gerard, G. Coukos, and J. D. Lambris. 2008. Modulation of
the antitumor immune response by complement. Nat. Immunol. 9: 1225–1235.
12. Markiewski, M. M., and J. D. Lambris. 2009. Unwelcome complement. Cancer
Res. 69: 6367–6370.
13. Markiewski, M. M., and J. D. Lambris. 2009. Is complement good or bad for
cancer patients? A new perspective on an old dilemma. Trends Immunol. 30:
14. Ostrand-Rosenberg, S. 2008. Cancer and complement. Nat. Biotechnol. 26:
15. Li, B., D. J. Allendorf, R. Hansen, J. Marroquin, C. Ding, D. E. Cramer, and
J. Yan. 2006. Yeast beta-glucan amplifies phagocyte killing of iC3b-opsonized
tumor cells via complement receptor 3-Syk-phosphatidylinositol 3-kinase path-
way. J. Immunol. 177: 1661–1669.
16. Hicks, A. M., G. Riedlinger, M. C. Willingham, M. A. Alexander-Miller, C. Von
Kap-Herr, M. J. Pettenati, A. M. Sanders, H. M. Weir, W. Du, J. Kim, et al. 2006.
Transferable anticancer innate immunity in spontaneous regression/complete
resistance mice. Proc. Natl. Acad. Sci. USA 103: 7753–7758.
17. Nausch, N., and A. Cerwenka. 2008. NKG2D ligands in tumor immunity. On-
cogene 27: 5944–5958.
18. Nozawa, H., C. Chiu, and D. Hanahan. 2006. Infiltrating neutrophils mediate the
initial angiogenic switch in a mouse model of multistage carcinogenesis. Proc.
Natl. Acad. Sci. USA 103: 12493–12498.
19. Qian, B. Z., J. Li, H. Zhang, T. Kitamura, J. Zhang, L. R. Campion, E. A. Kaiser,
L. A. Snyder, and J. W. Pollard. 2011. CCL2 recruits inflammatory monocytes to
facilitate breast-tumour metastasis. Nature 475: 222–225.
20. Hashimoto, M., K. Hirota, H. Yoshitomi, S. Maeda, S. Teradaira, S. Akizuki,
P. Prieto-Martin, T. Nomura, N. Sakaguchi, J. Ko ¨hl, et al. 2010. Complement
drives Th17 cell differentiation and triggers autoimmune arthritis. J. Exp. Med.
21. Fang, C., X. Zhang, T. Miwa, and W. C. Song. 2009. Complement promotes the
development of inflammatory T-helper 17 cells through synergistic interaction
with Toll-like receptor signaling and interleukin-6 production. Blood 114: 1005–
22. Fusakio, M. E., J. P. Mohammed, Y. Laumonnier, K. Hoebe, J. Ko ¨hl, and
J. Mattner. 2011. C5a regulates NKT and NK cell functions in sepsis. J.
Immunol. 187: 5805–5812.
23. Chan, C. J., D. M. Andrews, and M. J. Smyth. 2008. Can NK cells be a thera-
peutic target in human cancer? Eur. J. Immunol. 38: 2964–2968.
24. Smyth, M. J., Y. Hayakawa, K. Takeda, and H. Yagita. 2002. New aspects of
natural-killer-cell surveillance and therapy of cancer. Nat. Rev. Cancer 2: 850–
25. Qian, B. Z., and J. W. Pollard. 2010. Macrophage diversity enhances tumor
progression and metastasis. Cell 141: 39–51.
26. Ma, J., L. Liu, G. Che, N. Yu, F. Dai, and Z. You. 2010. The M1 form of tumor-
associated macrophages in non-small cell lung cancer is positively associated
with survival time. BMC Cancer 10: 112.
27. Solinas, G., S. Schiarea, M. Liguori, M. Fabbri, S. Pesce, L. Zammataro,
F. Pasqualini, M. Nebuloni, C. Chiabrando, A. Mantovani, and P. Allavena. 2010.
Tumor-conditioned macrophages secrete migration-stimulating factor: a new
marker for M2-polarization, influencing tumor cell motility. J. Immunol. 185:
28. Gabrilovich, D. I., and S. Nagaraj. 2009. Myeloid-derived suppressor cells as
regulators of the immune system. Nat. Rev. Immunol. 9: 162–174.
29. Corzo, C. A., T. Condamine, L. Lu, M. J. Cotter, J. I. Youn, P. Cheng, H. I. Cho,
E. Celis, D. G. Quiceno, T. Padhya, et al. 2010. HIF-1a regulates function and
differentiation of myeloid-derived suppressor cells in the tumor microenviron-
ment. J. Exp. Med. 207: 2439–2453.
30. Hanahan, D., and R. A. Weinberg. 2000. The hallmarks of cancer. Cell 100: 57–
31. Chen, W., T. Tang, J. Eastham-Anderson, D. Dunlap, B. Alicke, M. Nannini,
S. Gould, R. Yauch, Z. Modrusan, K. J. DuPree, et al. 2011. Canonical hedgehog
signaling augments tumor angiogenesis by induction of VEGF-A in stromal
perivascular cells. Proc. Natl. Acad. Sci. USA 108: 9589–9594.
32. Liu, Y., Z. P. Han, S. S. Zhang, Y. Y. Jing, X. X. Bu, C. Y. Wang, K. Sun,
G. C. Jiang, X. Zhao, R. Li, et al. 2011. Effects of inflammatory factors on
mesenchymal stem cells and their role in the promotion of tumor angiogenesis in
colon cancer. J. Biol. Chem. 286: 25007–25015.
33. Langer, H. F., K. J. Chung, V. V. Orlova, E. Y. Choi, S. Kaul, M. J. Kruhlak,
M. Alatsatianos, R. A. Deangelis, P. A. Roche, P. Magotti, et al. 2010.
Complement-mediated inhibition of neovascularization reveals a point of con-
vergence between innate immunity and angiogenesis. Blood 116: 4395–4403.
34. Hanson, E. M., V. K. Clements, P. Sinha, D. Ilkovitch, and S. Ostrand-Rosen-
berg. 2009. Myeloid-derived suppressor cells down-regulate L-selectin expres-
sion on CD4+and CD8+T cells. J. Immunol. 183: 937–944.
35. Ostrand-Rosenberg, S. 2010. Myeloid-derived suppressor cells: more mecha-
nisms for inhibiting antitumor immunity. Cancer Immunol. Immunother. 59:
36. Carroll, M. C. 2004. The complement system in regulation of adaptive immunity.
Nat. Immunol. 5: 981–986.
37. Peng, Q., K. Li, H. Patel, S. H. Sacks, and W. Zhou. 2006. Dendritic cell syn-
thesis of C3 is required for full T cell activation and development of a Th1
phenotype. J. Immunol. 176: 3330–3341.
38. Strainic, M. G., J. Liu, D. Huang, F. An, P. N. Lalli, N. Muqim, V. S. Shapiro,
G. R. Dubyak, P. S. Heeger, and M. E. Medof. 2008. Locally produced com-
plement fragments C5a and C3a provide both costimulatory and survival signals
to naive CD4+T cells. Immunity 28: 425–435.
39. Lalli, P. N., M. G. Strainic, M. Yang, F. Lin, M. E. Medof, and P. S. Heeger.
2008. Locally produced C5a binds to T cell-expressed C5aR to enhance effector
T-cell expansion by limiting antigen-induced apoptosis. Blood 112: 1759–1766.
40. Weaver, D. J., Jr., E. S. Reis, M. K. Pandey, G. Ko ¨hl, N. Harris, C. Gerard, and
J. Ko ¨hl. 2010. C5a receptor-deficient dendritic cells promote induction of Treg
and Th17 cells. Eur. J. Immunol. 40: 710–721.
41. Fang, C., T. Miwa, H. Shen, and W. C. Song. 2007. Complement-dependent
enhancement of CD8+T cell immunity to lymphocytic choriomeningitis virus
infection in decay-accelerating factor-deficient mice. J. Immunol. 179: 3178–
42. Raedler, H., M. Yang, P. N. Lalli, M. E. Medof, and P. S. Heeger. 2009. Primed
CD8+T-cell responses to allogeneic endothelial cells are controlled by local
complement activation. Am. J. Transplant. 9: 1784‑1795.
43. Ward, P. A. 2004. The dark side of C5a in sepsis. Nat. Rev. Immunol. 4: 133–142.
2994ROLE OF C5a IN TUMOR PROGRESSION
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