The Journal of Immunology
Immunoglobulin Fc Fragment Tagging Allows Strong
Activation of Endogenous CD4 T Cells To Reshape the Tumor
Milieu and Enhance the Antitumor Effect of Lentivector
Yuan Hong,* Yibing Peng,* Haiyan Xiao,* Michael Mi,†David Munn,*,‡and Yukai He*,x
A major problem with current cancer vaccines is that the induction of CD8 immune responses is rarely associated with antitumor
benefits, mainly owing to multiple immune suppressions in established tumor lesions. In this study, we investigated if and how
activation of endogenous CD4 T cells could be achieved to influence the suppressive tumor milieu and antitumor effect. We engi-
neereda lentivector (lv) to express a nominal fusion Ag composed of hepatitis B surface protein and IgG2aFc fragment (HBS-Fc-lv)
to increase the magnitude of CD8 response but, more importantly, to induce effective coactivation of CD4 T cells. We found that,
remarkably, immunization with HBS-Fc-lv caused significant regression of established tumors. Immunologic analysis revealed that,
compared with HBS-lv without Fc fragment, immunization with HBS-Fc-lv markedly increased the number of functional CD8 and
CD4 T cells and the level of Th1/Tc1-like cytokines in the tumor while substantially decreasing the regulatory T cell ratio. The
favorable immunologic changes in tumor lesions and the improvement of antitumor effects from HBS-Fc-lv immunization were
dependent on the CD4 activation, which was Fc receptor mediated. Adoptive transfer of CD4 T cells from the HBS-Fc-lv–
immunized mice could activate endogenous CD8 T cells in an IFN-g–dependent manner. We conclude that endogenous CD4
T cells can be activated by lv expressing Fc-tagged Ag to provide another layer of help—that is, creating a Th1/Tc1-like
proinflammatory milieu within the tumor lesion to boost the effector phase of immune responses in enhancing the antitumor
effect.The Journal of Immunology, 2012, 188: 4819–4827.
vaccines have been demonstrated to activate and expand tumor-
specific CD8 T cells. We (5–9) and others (10–12) have found
that recombinant lentivector (lv) can effectively stimulate CD8
responses, thanks to its efficient transduction of migrating skin
DCs that are able to directly prime naive CD8 T cells in the
draining lymph nodes (6, 13). However, despite these intensive
efforts and the induction of potent CD8 responses, the antitumor
effect of current cancer vaccines in treating established tumors is
limited (9, 14, 15). In contrast to CD8 T cells, the activation of
urrent tumor immunotherapies rely heavily on the in-
duction of CD8 immune responses. Peptide- (1), dendritic
cell- (DC) (2), and viral vector- (3, 4) based cancer
CD4 T cells is much more difficult (16), especially when the
production of effector cytokines by CD4 T cells is used as a cri-
terion of activation. We hypothesize that one possible reason for
the lack of significant antitumor benefit in treating established
tumors is due to ineffective coactivation of endogenous CD4
T cells by current cancer vaccines.
As a helper T cell, CD4 is widely recognized for its role in
assisting in the induction of CD8 responses by possibly “licensing”
DCs (17–21). In addition, the “postlicensing” role of CD4 T cells
at the effector phase is becoming increasingly appreciated (22).
Recent studies showed that the recruitment, proliferation, and
effector functions of CD8 T cells inside tumors or infected lesions
could be greatly enhanced by cotransfer of CD4 T cells (23–25).
Furthermore, several recent reports showed that some CD4 T cells
could directly kill tumor cells (26–29). However, in most, if not
all, of these studies, adoptive transfer of a large number of highly
selected TCR transgenic (Tg) CD4 T cells was used. At present,
it is not clear if the rare endogenous CD4 T cells can exercise
similar functions because most of the cancer vaccines, especially
those that are gene based, are not effective at activating CD4
T cells. Although proven to be very effective at stimulating CD8
responses, lv immunization has limited ability to activate endog-
enous CD4 T cells. Collins and colleagues (12) demonstrated that
CD4 T cells could be activated by lv immunization only in the
scenario in which adoptive transfer of a high number of exogenous
TCRTg OT-II CD4 T cells was used. In another study, Goold et al.
(30) demonstrated that a limited activation of endogenous OVA-
specific CD4 T cells was achieved after lv immunization when
OVA Ag was channeled to an MHC II-restricted pathway.
To improveCD4 activation, we recently studied various methods
and Ags for activating endogenous CD4 T cells and found that
*Immunology/Immunotherapy Program, Cancer Center, Medical College of Georgia,
Georgia Health Sciences University, Augusta, GA 30912;†Harvard Medical School,
Harvard University, Boston, MA 02115;‡Department of Pediatrics, Medical College
of Georgia, Georgia Health Sciences University, Augusta, GA 30912; andxDepart-
ment of Medicine, Medical College of Georgia, Georgia Health Sciences University,
Augusta, GA 30912
Received for publication December 8, 2011. Accepted for publication March 16,
This work was supported by National Institutes of Health Grant R01 CA16444 and
the Distinguished Investigator Fund from the Georgia Research Alliance (to Y. He).
Address correspondence and reprint requests to Dr. Yukai He, CN-4150,
Immunology/Immunotherapy Program, Cancer Center, Medical College of Georgia,
Georgia Health Sciences University, 1120 15th Street, Augusta, GA 30912. E-mail
The online version of this article contains supplemental material.
Abbreviations used in this article: DC, dendritic cell; FcRg, FcR g-chain; GrB,
granzyme B; HBsAg, hepatitis B virus surface Ag; HBS-Fc, HBsAg and mouse
IgG2a Fc fragment fusion Ag; HBS-Fc-lv, lentivector expressing HBS-Fc fusion
Ag; KO, knockout; lv, lentivector; qRT-PCR, quantitative RT-PCR; Tg, transgenic;
TIL, tumor-infiltrating lymphocyte; Treg, regulatory T cell; WT, wild-type.
tagging HBsAg with Ig Fc fragment in the lv immunization
platform could potently activate endogenous CD4 T cells to pro-
duce IFN-g and to enhance CD8 responses (31). In this report, we
studied the questions of if and how activation of endogenous CD4
T cells could be achieved to modulate the tumor milieu and to
improve the antitumor effect of lv immunization. We investigated
the antitumor effect of lv immunization and the immunologic
changes in the tumor lesions with or without CD4 coactivation.
We found that immunization with lv expressing Fc-tagged HBsAg
effectively activated both CD8 and CD4 responses and caused
regression of established HBsAg+B16 (B16-S) tumors. Immu-
nologic analysis revealed a significant increase of CD8 and CD4
T cells and preservation of their effector function in tumor lesions
when CD4 cells were activated. The level of Th1/Tc1-like cyto-
kines and chemokines was also markedly increased in tumor
lesions in the presence of CD4 activation. In contrast, the regu-
latory T cell (Treg) ratio was substantially decreased in the im-
munized tumor when CD4 cells were activated. These favorable
immunologic changes in the tumor lesions following lv immuni-
zation were dependent on CD4 activation, which was mediated by
FcgR. Using the adoptive transfer approach, we further discovered
that the vaccine-activated CD4 T cells could effectively activate
endogenous CD8 T cells in an IFN-g–dependent pathway. We
conclude that tagging tumor Ag with an Fc fragment offers an
effective way to activate endogenous CD4 and that the activated
CD4 T cells can improve the tumor milieu by increasing Th1/Tc1-
like proinflammatory cytokines and chemokines, reducing the Treg
ratio, and maintaining the effector function of tumor-infiltrating
lymphocytes (TILs), which together result in an enhanced antitu-
Materials and Methods
Cell lines, tumors, and mice
B16-F10 and 293T cells were obtained from American Type Culture
Collection. B16-S tumor cells were kindly provided by Dr. Jorg Reinmann
(32). IFN-g knockout (KO; IFN-g2/2) mice were purchased from The
Jackson Laboratory (Bar Harbor, ME). FcR common g-chain (FcRg) KO
mice were purchased from Taconic Farms (Germantown, NY). The com-
mon g-chain is associated with FcgR I, III, and FcεRI, which is essential
for FcgR assembly, signaling, and expression on the cell surface. C57BL/6
mice were purchased from the National Cancer Institute (Frederick, MD).
All the mice were housed under specific pathogen-free conditions in the
Laboratory Animal Services of Georgia Health Sciences University. Ani-
mal care protocols were approved by the Institutional Animal Care and
Use Committee of Georgia Health Sciences University.
To establish tumors, B16-S (5 3 105) or B16-F10 (2 3 105) cells were
inoculated s.c. into the shaved flank of C57BL/6 mice. Tumor growth was
monitored by measuring the perpendicular diameters three times per week.
The tumor mass was weighed at the end of experiments.
Lv and immunization
HBS-lv (expressing the HBsAg without Fc tagging) and HBS-Fc-lv
(expressing the HBsAg and mouse IgG2a Fc fragment fusion Ag [HBS-
Fc]) were constructed by replacing the TRP1 gene in TRP1-lv (9) with
the small S full gene of hepatitis B virus (ayw serotype) or the HBS-Fc
fusion gene (HBS-Fc) that contained the HBsAg and CH2-CH3 domains
(Fc fragment) of the mouse IgG 2a H chain. The entire amino acid
sequences of HBS-Fc protein were provided in Supplemental Fig. 1. Lv
preparation, concentration, and titration were conducted as previously de-
scribed (7). For immunization, 2.5 3 107transduction units of HBS-lv or
HBS-Fc-lv was injected in the footpad. For tumor treatment, all immuni-
zations were started on day 5, when the tumor lesions were clearly visible.
Preparation of single-cell suspension from tumor lesions
The mice were sacrificed; tumors were collected and weighed. Tumor
single-cell suspensions were prepared as previously reported (9). Briefly,
20–100 mg of each tumor was cut into small pieces and incubated at 37˚C
for 0.5 h in RPMI 1640 containing 1 mg/ml collagenase, 1 mg hyaluron-
idase, and 100 U DNase I. All enzymes were purchased from Sigma-
Aldrich (St. Louis, MO). Cells were then stained with various cocktails
of indicated Abs.
Analysis of tumor-infiltrating lymphocytes by flow cytometry
The Abs used in this study—aCD45, aCD90, aCD8, aCD4, aCD40L,
aCD107a, anti-granzyme B (GrB), anti-Foxp3, anti–IFN-g, and anti–TNF-
a—were purchased from BD Biosciences (San Diego, CA), BioLegend
(San Diego, CA), and eBioscience (San Diego, CA).
To measure cytokines, single-cell suspensions from peripheral blood,
spleen, or tumor were ex vivo stimulated for 4 h with 1 mg/ml HBsAg
peptide S190–197, identified previously by Schirmbeck et al. (33) (Gen-
Script, Piscataway, NJ), or 5 mg/ml whole HBsAg (Propsec, East Bruns-
wick, NJ) in the presence of GolgiStop (BD Biosciences). In some
experiments, the CD4 T cells were stimulated with PMA/Ionomycin
(leukocyte activation mixture; BD Biosciences). Intracellular staining of
IFN-g and TNF-a or GrB was performed (7). Alternatively, to measure
degranulation, Ab against CD107a was added to the ex vivo cell culture, as
described previously (34). After staining, the cell events were collected
using a FACSCanto system (BD Biosciences). Data were analyzed using
the FCS Express V3 software (De Novo Software, Ontario, Canada).
Tumor tissue total RNA was extracted using the RNA extraction kit from
using the Mouse Chemokines and Receptors RT2Profiler PCR Array from
SABiosciences (Frederick, MD), which can detect 89 different chemokines
and receptors based on the manufacturer’s recommendation. Those che-
mokines with significant changes were further verified with quantitative
RT-PCR (qRT-PCR), using primers derived from previous reports (35). To
detect cytokine expression in the tumor lesions, we also used qRT-PCR. To
determine levels of Foxp3, GrB, perforin, TNF-a, and IFN-g, qRT-PCR
was performed as described by Kohlmeyer et al. (36). qRT-PCR primers
for IL-17, IL-21, RoRgt, and GADPH were derived from a previous report
by Das et al. (37) and Tsujita et al. (38). qRT-PCR primers for other
cytokines or transcription factors were also derived from previous pub-
lications: T-bet (39), TGF-b (40), IL-6, IL-1b (41), IL-7 (42), IL-12 (43),
and IL-15 (44). Primers for IL-2 are designed and synthesized as follows:
IL-2 upstream: 59-CCCTTGCTAATCACTCCTCA-39; IL-2 downstream:
Wild-type (WT) C57BL/6 mice (Thy1.1) or IFN-g KO mice (Thy1.2
cogenic) were immunized with HBS-Fc-lv or HBS-lv. Two weeks after
immunization, total CD8 and CD4 T cells were isolated using anti-CD4
and anti-CD8 magnetic microbeads, as described by the manufacturer
(Miltenyi Biotec, Auburn, CA). Purified T cells were then injected into the
irradiated (low dose, 5 Gy) mice bearing 5-d B16-S tumors.
Data were analyzed using Student unpaired t test or ANOVAwith the Prism
software (GraphPad Prism, La Jolla, CA).
Fc tagging increases the CD8 as well as CD4 immune
responses of lv immunization that causes regression of
Ag–Ab immune complex (45–47) and recombinant DNA express-
ing Fc-tagged Ag (48, 49) were previously reported to enhance Ag-
specific T cell immune responses, possibly because they could be
more effectively captured by APCs via Fc receptors (50). We re-
cently found that lv expressing the HBS-Fc fusion Ag could ef-
fectively activate endogenous CD4 T cells in addition to enhancing
CD8 responses (31). To determine whether the Fc tag is indeed
required to enhance CD8 responses, and, more importantly, to in-
duce activation of endogenous CD4 T cells, we compared the
magnitude of CD8 and CD4 responses of HBS-lv and HBS-Fc-lv
immunization. Two weeks after immunization, peripheral blood
cells were restimulated ex vivo with either HBS190peptide or whole
HBsAg protein for 4 h before measuring the IFN-g level by in-
4820Fc TAGGING ACTIVATES ENDOGENOUS CD4 TO IMPROVE TUMOR MILIEU
tracellular staining. We found that, compared with HBS-lv, HBS-
Fc-lv immunization not only significantly increased the magnitude
of CD8 responses but also, more importantly, induced potent CD4
responses (Fig. 1). In contrast, HBS-lv (without Fc tag) immuni-
zation stimulated no measurable CD4 responses. Therefore, we
conclude that tagging the lv-encoded Ag with Fc fragment induces
To find out whether the enhanced Ag-specific CD8 and CD4 im-
mune responses are correlated with a better antitumor effect of lv
immunization, mice bearing established B16-S tumors of sizes 10–
15 mm2were treated with HBS-Fc-lv or HBS-lv immunization
(Fig. 2A). As shown in Fig. 2B, immunization with both HBS-lv
and HBS-Fc-lv could strongly inhibit B16-S tumor growth in
treated mice, compared with untreated controls. However, only the
tumors receiving HBS-Fc-lv immunization experienced substan-
tial regression and even complete eradication. During the peak of
the immune response period, the majority of B16-S tumors in
the group of mice treated with HBS-Fc-lv underwent regression.
Some of the tumors were completely eradicated (Fig. 2B). In
a summary of four experiments, ∼70–80% of well-established
B16-S tumors experienced shrinkage after HBS-Fc-lv immuniza-
tion, and complete regression was found in 5 of 20 tumor-bearing
mice. The tumor-free mice from HBS-Fc-lv treatment resisted fur-
ther challenge by not only B16-S tumor cells but also B16-F10 tu-
mor cells, strongly suggesting that the antitumor immune responses
had spread to other tumor-associated Ags. In contrast, even though
B16-S tumor growth was inhibited by HBS-lv immunization, no
tumor regression was observed. All mice in the HBS-lv–treated
group eventually succumbed to tumor growth. Thus, in the lv im-
munization platform, Fc tagging not only increases the magnitude
of CD8 responses but also induces potent CD4 responses, which
may contribute to the tumor regression observed in HBS-Fc-lv–
Fc tagging increases the ability of lv immunization to stimulate
a proinflammatory milieu within tumor lesions
Tumor lesions are characterized as indolent chronic inflammation
that can promote tumor growth (51). However, recent studies
demonstrate that Th1 cytokines in tumor lesions may make the
chronic tumor-promoting inflammation become immune stimu-
lating (52). Although cancer vaccines have been proved to stim-
ulate antitumor immune responses, it is not clear how immuni-
zation with cancer vaccines will affect inflammation status in
tumor lesions. The remarkable regression of established tumors by
HBS-Fc-lv immunization provides a strong rationale for analyzing
the inflammatory changes in tumor lesions. Using qRT-PCR, we
compared the levels of proinflammatory cytokines and chemo-
kines in tumor lesions after immunization with HBS-lv and HBS-
Fc-lv. We found that, compared with tumors without treatment,
those treated with HBS-Fc-lv had a 50- to 100-fold increase in
mRNA levels of proinflammatory cytokines such as IL-1b and
IL-6. At the same time, the Th1/Tc1 cytokines of IFN-g, perforin,
granzyme, TNF-a, and transcription factor T-bet in the tumor
lesions of HBS-Fc-lv–immunized mice increased by 50- to 200-
fold. In addition, cytokines for CD8 T cell survival, such as IL-2
and IL-7, were significantly increased in HBS-Fc-lv–immunized
tumors. But IL-15 was not obviously changed. Without Fc tag-
ging, HBS-lv immunization also increased the amount of proin-
flammatory and Th1/Tc cytokines in the tumor lesions, but to a
significantly lesser extent. In contrast, the RNA levels of Foxp3,
IL-17, and RORgt increased only slightly or remained unchanged
(Fig. 3). Thus, our data demonstrate that Fc tagging significantly
increases the effect of lv immunization on converting tumor lesions
into a Th1/Tc1-like immune stimulatory microenvironment.
Consistent with the Th1/Tc1-like cytokine changes, the che-
mokines responsible for attracting NK, DCs, Th1, and Tc1 cells
in the tumor lesions of HBS-Fc-lv–immunized mice increased as
much as 150-fold, compared with untreated tumor. Again, the lv
expressing the HBS-Fc fusion Ag demonstrated a much more
significant effect (Fig. 3). In contrast, the chemokine CCL22 (53)
for Treg recruitment was only slightly increased. These data
strongly indicate that HBS-Fc-lv immunization alters chemokine
levels such that innate immune effectors and T effectors, but not
Tregs, can be effectively recruited into tumor lesions, which may
CD4 T cell immune responses. C57BL/6 mice were immunized with either
HBS-lv or HBS-Fc-lv. Nonimmunized mice were used as control. Two
weeks later, HBsAg-specific CD8 and CD4 T cell responses in the pe-
ripheral blood were determined by intracellular staining of IFN-g after
brief stimulation ex vivo with S190–197peptide (for CD8 response) or whole
HBsAg (for CD4 response). Only CD8 or CD4 T cells were gated and
shown. Data from five mice in each group are summarized and presented.
The experiment was repeated three times with similar results.
Lv expressing Fc-tagged Ag elicits more potent CD8 and
B16-S tumors. (A) The experimental design of tumor treatment with lv
immunization. (B) The growth curve of lv-treated and control tumors.
Partial and complete tumor regressions were observed.
HBS-Fc-lv immunization results in regression of established
The Journal of Immunology4821
play an important role in reshaping the tumor milieu to become
less immune suppressive and more Th1/Tc1-like and immune
Fc tagging markedly increases tumor infiltration of functional
CD8 and CD4 T cells of lv immunization
The increase of chemokines in the tumor milieu following HBS-
Fc-lv immunization suggests that more T effectors can be recruited
to the tumor lesions. To test this hypothesis, we analyzed the cellu-
lar immune components in the B16-S tumor lesions after differ-
ent treatments. First, we counted the absolute number of CD4 and
CD8 TILs (Supplemental Fig. 2, Fig. 4). We found that HBS-Fc-lv
immunization induced significantly more CD8 T cell infiltration
compared with HBS-lv immunization (Fig. 4). More importantly,
only HBS-Fc-lv immunization significantly increased the number
of CD4 TILs in the tumor lesions, which is consistent with data
showing that only HBS-Fc-lv immunization could effectively ac-
tivate CD4 T cells (Fig. 1). When the Treg ratio was analyzed in
the tumor lesions, we found that HBS-Fc-lv reduced the Treg ratio
in tumor lesions more significantly than did HBS-lv immunization
(Supplemental Fig. 2, Fig. 4). Thus, we concluded that, in the lv
platform, Fc tagging significantly increases infiltration of CD4 and
CD8 T cells into tumor lesions and at the same time reduces the
Treg ratio. More specifically, the marked increase of CD4 TILs
and reduction of Treg ratio were observed only in the HBS-Fc-lv–
immunized tumor lesions.
In the next study, the effector function of CD8 and CD4 TILs
following HBS-Fc-lv and HBS-lv immunization was measured. As
demonstrated in Fig. 5, when compared with HBS-lv immuniza-
tion, HBS-Fc-lv immunization stimulated more CD8 TILs to
produce IFN-g in the tumor lesions (Fig. 5A). In addition, more
CD4 TILs produced IFN-g in the tumors treated with HBS-Fc-lv
(Fig. 5B). To examine the cytolytic function of CD8 TILs, we
stained for CD107a, the degranulation marker in response to Ag
stimulation, which can be used as a surrogate test of cytolytic
function (34). We found that similar to the results of IFN-g
staining, when compared with HBS-lv immunization, HBS-Fc-lv
immunization caused more CD8 TILs to be CD107a positive (Fig.
5C). Because the absolute number (Fig. 4) and effector function
(Fig. 5) of CD8 and CD4 TILs were significantly increased in the
HBS-Fc-lv–immunized tumors, the total number of functional
CD8 and CD4 TILs in the HBS-Fc-lv–immunized tumors should
be much higher. These data suggest that Fc tagging markedly
increases the number and function of CD4 and CD8 effector
T cells in the tumor lesions.
The immunologic changes in the tumor lesions and the
antitumor effect of HBS-Fc-lv immunization are dependent on
Using the two lv (HBS-lv and HBS-Fc-lv) that could differentially
activate CD4 T cells, we demonstrated in the above studies that
effective activation of CD4 T cells by HBS-Fc-lv immunization
may play an important role in increasing Th1/Tc1-like proin-
flammatory cytokines and functional effector T cell infiltration and
decreasing Treg ratio in the tumor lesions. However, in those
studies, the differences in the magnitude of CD8 responses may
also contribute to immunologic changes in the tumor lesions. To
examine the role of CD4 in immunologic changes in the tumor
milieu, it would be a good approach if only the activated effector
munization, total RNA was extracted from the tumor tissues of control and immunized mice (three tumor lesions in each group were combined together),
and qRT-PCR was performed, using primers for indicated cytokines, transcription factors, and chemokines. The result is presented as how many folds of
increase of cytokines or chemokines in the immunized tumor over the control tumors. Each sample was done in triplicate; the average and SD are shown.
The experiment was repeated three times with similar results.
Tumor lesion is skewed toward a Th1/Tc1-like immune stimulatory microenvironment after HBS-Fc-lv immunization. At 18 d after im-
Mice bearing 5-d tumors were immunized with either HBS-lv or HBS-Fc-lv, or left untreated. The tumor lesions were collected on days 17–20 after
immunization and analyzed for tumor-infiltrating CD8 and CD4 T cells and the Treg ratio. The absolute numbers of CD8 and CD4 T cells and the Treg
ratios in the tumor lesions of control and treated mice from a cohort of two studies are summarized.
HBS-Fc-lv immunization markedly increases tumor infiltration of CD4 and CD8 T cells and decreases the Treg ratio in the tumor lesions.
4822 Fc TAGGING ACTIVATES ENDOGENOUS CD4 TO IMPROVE TUMOR MILIEU
strategy is available at present, and the simultaneous depletion of
Tregs with anti-CD4 Ab will confound the analysis of the role of
effector CD4 T cells. To circumvent this problem, we used FcRg
KO mice because activation of CD4 T cells following HBS-Fc-lv
immunization was severely compromised, whereas the CD8 re-
sponse was not affected (31) (Fig. 6A), creating a scenario that
allowed us to study immunologic changes in the tumor milieu in
the absence of CD4 activation. The data showed that, following
HBS-Fc-lv immunization, the Treg ratio in the tumors of FcRg
KO mice was significantly higher than that in WT mice (Fig. 6B).
Furthermore, increase of Th1 cytokines in the tumor milieu after
lv immunization was significantly compromised in FcRg KO mice
(Fig. 6C). Consistent with the Foxp3 staining data (Fig. 6B), the
level of Foxp3 mRNA was significantly higher in the FcRg KO
tumor (Fig. 6C). Concurrently, the antitumor effect of HBS-Fc-lv
immunization in FcRg KO mice was also compromised (Fig. 6D).
These data suggest that CD4 activation has an important part in
increasing the Th1/Tc1 cytokines and decreasing the Treg ratio in
tumors and in enhancing the antitumor effect of HBS-Fc-lv im-
Adoptive transfer of CD4 T cells can activate endogenous CD8
TILs in the tumors and generate antitumor effects
To further examine the antitumor effect of CD4 and CD8 T cells
andtheir effectonendogenous CD8T cells inthe tumorlesions, we
conducted adoptive transfer experiments using vaccine-activated
tumors treated with HBS-Fc-lv possess better
effector function. (A) The effector function of
CD8 TILs was measured by intracellular
staining of IFN-g after brief ex vivo peptide
stimulation. Representative dot plots of CD8
TILs from HBS-lv– or HBS-Fc-lv–immunized
tumors are shown. Dot plots from control
tumors are not shown because only few CD8
TILs could be collected. Only the CD8 T cells
were gated and shown. Data of 5 mice are
summarized. (B) Effector cytokine production
by CD4 TILs is shown after stimulation with
PMA/Ionomycin. Only the CD4 T cells were
gated and shown. These experiments examin-
ing the TIL and their effector functions were
repeated three times with similar results. (C)
Degranulation of CD8 TILs was measured
by CD107a staining. A summary of data from
five tumors in each group is presented. This
experiment was repeated twice, with similar
CD4 and CD8 TILs in the
and FcRg KO mice were inoculated with B16-S tumor cells. At 5 d later, tumor-bearing mice were immunized with HBS-Fc-lv. (A) CD8 and CD4
responses in the peripheral blood were determined 2 wk after immunization by intracellular staining of IFN-g. The summary data of five mice are shown on
the right. (B) The Treg ratio in the tumor was analyzed 3 wk after tumor inoculation, and the data of five mice are summarized in the right column. (C)
Cytokines in the tumor lesions of WT and KO mice were analyzed by qRT-PCR. (D) The tumor growth curve and the tumor weight were recorded. Two
experiments were conducted, with similar results.
Immunologic changes in the tumors and the antitumor effect of HBS-Fc-lv immunization are dependent on CD4 activation and FcgR. WT
The Journal of Immunology 4823
CD4 and CD8 T cells. CD4 and CD8 T cells were isolated from
HBS-Fc-lv–immunized Thy1.1 congenic C57BL/6 mice and adop-
tively transferred into irradiated B16-S tumor-bearing Thy1.2
congenic mice (Fig. 7A). Two weeks later, the effector function of
TILs was analyzed. We found that adoptive transfer of activated
CD4 T cells could effectively activate endogenous CD8 TILs to
express GrB (Fig. 7B) and IFN-g (data not shown). In addition,
more endogenous CD8 T cells were recruited into tumor lesions
in the presence of activated CD4 T cells. In contrast, transfer of
activated CD8 T cells did not increase granzyme expression of
the endogenous CD8 TILs (Fig. 7B). The adopted exogenous CD8
as well as CD4 T cells were found to be capable of expressing
GrB in the tumor lesions (Fig. 7C). Adoptive transfer of CD4 or
CD8 T cells could effectively inhibit tumor growth, but low-dose
irradiation alone did not significantly affect tumor growth (Fig.
7D). Thus, adoptive transfer of both activated CD4 and CD8
T cells achieved similar antitumor effects in tumor-bearing mice, but
the mechanisms were different. The antitumor effect of CD4 was
mediated indirectly by activating endogenous CD8 T cells, whereas
exogenous CD8 T cells may directly kill tumor cells.
Then, we compared the antitumor activity of the CD4 T cells
from mice immunized with HBS-Fc-lv and HBS-lv, as the level
of CD4 activation was drastically different. We observed that, in
agreement with the finding that more CD4 T cells were activated in
the HBS-Fc-lv–immunized mice (Fig. 1), adoptive transfer of the
preactivated CD4 T cells from HBS-Fc-lv–immunized mice could
result in more infiltration of endogenous CD8 TILs that possessed
better effector function (Supplemental Fig. 3B), resulting in
stronger antitumor effect (Supplemental Fig. 3A).
IFN-g expression by CD4 T cells plays a critical role in the
CD4-mediated antitumor effect
With use of TCR Tg CD4 T cells, it was reported that IFN-g
produced by CD4 T cells played a critical role in helping recruit
CD8 T cells to virally infected lesions (25) and tumor lesions (23,
54). To assess the role of IFN-g in the CD4-mediated antitumor
effect, we examined the antitumor effect of CD4 T cells from WT
and IFN-g KO mice. First, the WT and IFN-g KO mice were
immunized with HBS-Fc-lv. Then, CD4 T cells from immunized
WT and IFN-g KO mice (Thy1.2) were isolated and adoptively
transferred into tumor-bearing mice (Thy1.1) to monitor their
antitumor effects and their activation of endogenous CD8 T cells
(Fig. 8A). On the basis of the TNF-a expression, a comparable
number of activated CD4 T cells were found in the WTand IFN-g
KO mice after HBS-Fc-lv immunization (Fig. 8B). The data from
the transfer experiment showed that, compared with WT CD4
T cells, adoptive transfer of CD4 T cells from the IFN-g KO mice
was incapable of activating endogenous CD8 T cells in the tumor
lesions (Fig. 8C). Furthermore, tumor infiltration of endogenous
CD8 T cells was also significantly decreased in mice treated with
adoptive transfer of CD4 T cells from IFN-g KO mice (Fig. 8C).
In agreement with the data, adoptive transfer of CD4 T cells from
IFN-g KO mice had severely compromised antitumor effect (Fig.
8D). These data suggest that the expression of IFN-g by vaccine-
induced CD4 T cells plays a vital part in activating endogenous
CD8 TILs and in mediating antitumor effect.
Cancer vaccine research, thus far, demonstrates that mere induc-
tion of CD8 T cell response has little correlation with success in
achieving antitumor effect. The lack of antitumor effect may be
in part due to the inability of most current cancer vaccines to si-
multaneously coactivate CD4 T cells. In this study, we asked
whether activation of endogenous CD4 T cells by lv immunization
would influence the tumor milieu and antitumor effect of cancer
vaccines and howit would accomplish this. Remarkably, we found
that strong activation of endogenous CD4 T cells and enhance-
ment of CD8 responses could be achieved by tagging the Ag with
the Fc fragment, which increased the antitumor effect of lv im-
munization, including regression of established tumors. This
Activated CD4 and CD8 T cell subsets were isolated from HBS-Fc-lv–immunized Thy1.1 congenic mice, and 10 million cells were adoptively transferred
into mice bearing 5-d B16-S tumor after a low-dose (5 Gy) irradiation. At 2 wk after adoptive transfer, the tumor lesions were collected and the GrB
expression of TILs was analyzed. (B) The GrB expression of endogenous CD8 TILs from mice that were transferred with activated CD4 or CD8 T cells.
Irradiated mice without adoptive transfer of T cells were used as control. A summary of data of total CD8 TILs and percentage of GrzB expression is also
presented. (C) The GrzB expression of exogenous adopted CD4 and CD8 TILs. A summary of data is presented on the right. (D) Tumor growth curve and
tumor weight. A summary of data from 5 mice is presented. Two experiments were conducted with similar results.
Adoptive transfer of CD4 T cells can activate endogenous CD8 TILs in tumors and generate antitumor effects. (A) The experimental scheme:
4824 Fc TAGGING ACTIVATES ENDOGENOUS CD4 TO IMPROVE TUMOR MILIEU
potent antitumor effect of Fc tagging in the lv platform is asso-
ciated with improved immunologic changes in the tumor milieu,
from substantial increase of TIL number and conversion of the
tumor milieu into a Th1/Tc1-like proinflammatory microenvi-
ronment to significant decrease of the Treg ratio. These favorable
changes in the tumor microenvironment and remarkable antitumor
effects of lv immunization were dependent on the activation of
endogenous CD4 T cells. IFN-g production plays a critical role in
CD4-mediated immunologic changes in the tumor milieu. Thus,
in addition to the well-recognized CD4 role in priming adaptive
immune responses, our findings suggest that activation of en-
dogenous CD4 helps the effector phase of antitumor immunity by
modulating the tumor milieu.
How does activation of endogenous CD4 T cells change the
tumor microenvironment? First, activation of CD4 may help ef-
fector T cell migration into tumor lesions and license DCs to
reactivate CD8 TILs. We found that IFN-g production by activated
CD4 T cells has a function in mediating tumor infiltration of
effector T cells into tumor lesions, as the CD8 infiltration was
markedly decreased after adoptive transfer of CD4 T cells from
IFN-g KO mice (Fig. 8C). These data are in agreement with pre-
vious findings, which show that adoptive transfer of TCR Tg CD4
T cells can help migration of CD8 T cell to infection site (25) and
tumor lesion (23) in an IFN-g–dependent manner. Therefore, it is
likely that, following activation by cancer vaccines, the activated
endogenous CD4 T cells may enter tumors and secrete IFN-g to
mediate chemokine production in the tumor stroma to attract more
CD8 T cells. After migrating into tumor lesions, CD8 T cells that
are activated in the lymphoid tissues need reactivation to execute
their effector function in the tumor milieu. In this process, the
CD40L on the activated CD4 T cells may license DCs to reac-
tivate CD8 T cells in the tumor milieu to produce more IFN-g.
The substantial increase of chemokines responsible for effector
T cell recruitment into the tumor lesions after lv immunization
supports this argument. Then, the CD4 and CD8 TILs secrete
more cytokines and turn the suppressive tumor lesion into a Th1/
Tc1-like immune stimulatory microenvironment.
Second, activation of CD4 T cells may help reduce the Tregratio
in tumor lesions. Tregs are a key component of immune sup-
pression in tumor lesions (55) and have been shown to inhibit
effector function. They are also associated with poor clinical
outcome (56, 57). The T effector/Treg ratio correlates with the
outcome of tumor immunotherapy (58, 59). Thus, reduction of
Tregs can be an effective way to increase the antitumor efficacy of
immunization (60). In our study, we found that activation of en-
dogenous CD4 T cells by lv immunization could markedly de-
crease the Treg ratio in tumor lesions. It is not clear how such
activation of endogenous CD4 T cells can accomplish this de-
crease. But, two recent studies showed that Th1/Th2 T cells could
inhibit peripheral Treg induction in vitro and in vivo (61) in an
IFN-g–dependent manner (62). Addition of exogenous IFN-g
markedly decreased Treg generation in vitro. Thus, it is possible
that induction of endogenous CD4 T cells to become Th1 cells
following HBS-Fc-lv immunization will reciprocally inhibit ex-
pansion of Tregs. Another possible mechanism for the decreased
Treg ratio following CD4 activation is the induction of Treg ap-
optosis via the Fas-FasL pathway (63). However, we were unable
to detect increased apoptosis of Tregs in the tumor after lv im-
munization (data not shown).
Recently, DCs were found to take up Ab–Ag immune complexes
efficiently via the FcgR to cross-prime CD8 T cells (47, 50). In
our study, we found that even though covalent Fc tagging could
significantly enhance CD8 T cell immune responses, FcgR may
not be critical in lv immunization because a similar magnitude of
CD8 responses could be induced in FcRg KO mice (Fig. 6A). A
possible explanation is that, following lv immunization, direct
presentation of Ag synthesized endogenously in the DCs is the
main mechanism for priming CD8 T cells (5, 6, 64). The increase
of CD8 responses by Fc tagging may result mainly from an in-
crease in Ag production (31). The reuptake of secreted Ag by DCs
in an autocrine or paracrine fashion may play only a minor role in
CD8 T cell activation. Thus, activation of CD8 T cells following
lv immunization is less dependent on FcgR-mediated cross-pre-
sentation. In contrast, CD4 activation predominantly relies on the
reuptake of foreign Ag to enter the MHC II-mediated Ag pro-
cessing and presentation pathway and thus depends on the FcgR-
mediated Ag reuptake. In agreement with this argument, a classic
paper by Celis and Chang (65) nearly 30 y ago showed that
in vitro proliferation of HBsAg-specific CD4 T cell clone induced
by HBsAg could be markedly enhanced by addition of HBsAb.
Although in our study, the role of humoral immunity is not ex-
amined, the increase of humoral response following immuniza-
IFN-g KO mice (Thy1.2) were immunized with HBS-Fc-lv. Two weeks later, activated CD4 T cells were isolated and 5 million cells were injected into
irradiated B16-S tumor-bearing mice (Thy1.1). Mice with irradiation only, without cell transfer, were used as control. (B) The activation of CD4 T cells by
HBS-Fc-lv immunization in both WT and IFN-g KO mice was determined by examining TNF-a expression. A similar percent of CD4 T cells of WT and
IFN-g KO mice produced TNF-a in response to Ag stimulation. (C) Two weeks after transfer, tumor lesions were collected and analyzed for IFN-g
production by the endogenous CD8 T cells. The absolute number of IFN-g–producing CD8 TILs of five mice in two experiments is summarized. (D) The
tumor weights were recorded when the mice were sacrificed at the end of the experiment.
IFN-g expression plays a critical role in the antitumor effect of CD4 T cell adoptive transfer. (A) The experimental design is shown. WTand
The Journal of Immunology4825
tion, as we recently observed (31), may also contribute to the
enhanced antitumor effect by targeting and opsonizing the tumor
In this study, using an HBsAg-expressing B16 tumor model,
we demonstrated that activation of endogenous CD4 T cells by ac-
tive immunization could markedly relieve immune suppression in
tumor lesions and substantially increase the antitumor effect.
However, the expression of foreign HBsAg by tumor cells per se
in the absence of immunization did not reduce the Treg ratio in
the tumor lesions, suggesting that expression of foreign Ag is not
sufficient to significantly change the tumor microenvironment.
More importantly, HBS-lv immunization incapable of activating
CD4 T cells did not significantly change the tumor milieu and
resulted in no regression of B16-S tumor. Thus, improvements of
the tumor milieu—that is, conversion of the tumor milieu into a
Th1/Tc1-like environment and reduction of the Treg ratio in the
tumor lesions by HBS-Fc-lv immunization—are related to the
vaccine’s ability to activate CD4 T cells. Whether these findings
made in the B16-S tumor model have general implications
requires further studies using other Ags and even self tumor Ags.
Such studies will be critical for the rational design of tumor
vaccines to effectively treat tumors not only by increasing effector
T cells but also by improving the tumor microenvironment
through activation of endogenous CD4 T cells.
We thank Dr. Jorg Reinmann, Institute for Medical Microbiology, Univer-
sity of Ulm, Ulm, Germany, for providing the HBsAg+B16-S tumor cell
line and Drs. Gang Zhou, Tracy McGaha, and Andy Mellor, Immunother-
apy Center, Georgia Health Sciences University, for helpful discussions.
The authors have no financial conflicts of interest.
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