IFN-g Inhibits IL-4–Induced Type 2 Cytokine Expression byCD8 T Cells In Vivo and Modulates the Anti-Tumor Response
Simon H. Apte,*,† Penny Groves,*,† Stuart Olver,*,† Adriana Baz,*,†,1 Denise L. Doolan,*
Anne Kelso,*,†,2 and Norbert Kienzle*,†,3
Activation of naive CD8 T cells in vitro in the presence of IL-4 induces type 2 cytokine expression, loss of CD8 expression, and
reduced cytolytic potential. This represents a major shift from the canonical phenotype of effector CD8 T cells. It has not been
established, however, whether IL-4 can induce comprehensive type 2 cytokine expression by CD8 T cells in vivo, nor whether the
effects of IL-4 on type 2 cytokine production by CD8 T cells can be inhibited by IFN-g. Furthermore, disparate results have been
reported regarding the anti-tumor ability of type 2 polarized effector CD8 T cells, and the effects of IFN-g in this respect remain
unknown. To address these questions, wild-type or IFN-g–deficient OVA-specific CD8+ T cells were activated in RAG-22/2 gc2/2
recipients with control or IL-4–expressing OVA+ tumor cells, and then transferred to secondary recipients for tumor challenge.
Tumor-derived IL-4 induced the expression of type 2 cytokines and the transcription factor GATA-3 by responding CD8 T cells
while reducing their CD8 coreceptor expression and ability to eliminate a secondary tumor challenge. Each of these effects of
IL-4 was exaggerated in IFN-g–deficient, compared with wild-type, CD8 T cells. The results demonstrate that endogenous IFN-g
counteracts the induction of type 2 cytokines and the downregulation of both CD8 coreceptor levels and the anti-tumor response in
CD8 T cells exposed to IL-4 during activation in vivo. These findings may explain the anomalies in the reported functional
phenotype of type 2 polarized CD8 T cells. The Journal of Immunology, 2010, 185: 998–1004.
Exposure of CD8 T cells to IL-4 during activation inducesan intracellular signaling cascade that profoundlyaffects the phenotype and functional abilities of effector
cells (1). For example, IL-4 can enhance CD8 T cell expansionduring an immune response (2), modulate the functional avid-ity of the TCR by reducing CD8 coreceptor expression (3–7),promote CD8 T cell survival by upregulating the expressionof the anti-apoptotic molecule Bcl-xL (8), and induce thedevelopment of effector CD8 T cells that can express type 2 aswell as type 1 cytokines (6, 9, 10). Early work inmice showed thatIL-4 exposure induced the loss of CD8 expression and cytolyticfunction of CD8 T cells in vitro (6). This was followed by theobservation that some HIV patients with Job’s syndrome have
high numbers of CD8 T cells with reduced CD8 expression, type2 cytokine expression, and poor cytolytic function (11). How-ever, those observations contrasted with reports that type 2 cyto-kine polarization generates highly effective antitumor or cytol-ytic CD8 T cells without downregulation of CD8 expression(10, 12–14).IFN-g can act reciprocally to IL-4, counteracting an IL-4–induced
reduction in IFN-g production in vitro (10) and, as we have recentlyreported, IL-4–induced downregulation of CD8 expression on CD8T cells in vitro and in vivo (7). The relative amount of IFN-g ex-pressed by type 2 cytokine-expressing CD8 T cells can vary dramat-ically from negligible levels (6, 10) to an amount similar to thatproduced by conventional type 1 CD8 T cells (9). Differences inIFN-g expression or exposure may modulate the effects of IL-4exposure on CD8 T cells and help to explain the apparent discrep-ancies in their reported anti-tumor or cytolytic ability. WhetherIFN-g can reciprocally modulate the strength of IL-4–inducedtype 2 polarization and the anti-tumor function of CD8 T cellsin vivo has not been determined and, to the best of our knowledge,amurinemodel for comprehensive type 2 polarization ofCD8T cellsin vivo has not been reported. To address these questions, we havedeveloped a murine model of CD8 T cell activation in vivo in whichthe endogenous expression of IFN-g and IL-4 can be controlled (7).Our results indicate that both type 2 cytokine expression and anti-tumor function can be reciprocally modulated in vivo by IL-4 andIFN-g, and that reduced levels of CD8 expression serve asa phenotypic correlate for type 2 cytokine-expressing CD8 T cellswith poor anti-tumor function.
Materials and MethodsMice
Specific pathogen-free C57BL/6 (CD45.2) and B6.SJL/J-Ptprca (CD45.1)mice (Animal Resources Centre, Perth, Australia) were used at 6–9 wk ofage. RAG-22/2 gc2/2 (provided by A. Pinzon-Charry, Queensland Instituteof Medical Research [QIMR], Queensland, Australia), OT-I (243.2)(W. Health,Walter and Eliza Hall Institute ofMedical Research,Melbourne,
*Queensland Institute of Medical Research; and †Cooperative Research Centre forVaccine Technology, Brisbane, Queensland, Australia
2Current address: World Health Organization Collaborating Centre for Reference andResearch on Influenza, North Melbourne, Victoria, Australia.
3Current address: Arana Therapeutics, Macquarie Park, New South Wales, Australia.
Received for publication October 16, 2009. Accepted for publication May 6, 2010.
This work was supported by grants from the Cooperative Research Centre for Vac-cine Technology, the National Health and Medical Research Council of Australia,and the Cancer Council Queensland. The Cooperative Research Centre for VaccineTechnology was established and supported under the Australian Government’s Co-operative Research Centres Programme. S.H.A. was supported by an Australianpostgraduate award and by the Basil Shaw Fellowship of the Australian RotaryHealth Research Fund. D.L.D. is supported by a Pfizer Australia Senior ResearchFellowship.
Address correspondence and reprint requests to Dr. Simon H. Apte, QueenslandInstitute of Medical Research, Brisbane, Queensland 4006, Australia. E-mail address:[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: b2m, b2-microglobulin; CBA, cytometric beadarray; Gzmb, granzyme B; QIMR, Queensland Institute of Medical Research;ROG, repressor of GATA.
Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00
Australia), and OT-I3 IFN-g2/2 mice (G. Karupiah, John Curtin School ofMedical Research, Canberra, Australia) were bred at QIMR. All animalstudies were approved by the QIMR Animal Ethics Committee.
Naive CD8+ T cell preparation
CD8+ cells were enriched by positive selection from pooled cell suspen-sions from spleen and brachial, axillary, inguinal, and lumbar lymph nodesof untreated mice using MACS CD8 Microbeads (Miltenyi Biotec, Au-burn, CA); .85% of purified CD8+ cells displayed a naive (CD44low
CD62Lhigh) phenotype (7).
Activation of CD8+ T cells in vivo with IL-4–expressing orcontrol tumor cells
Generation of IL-4–expressing (EG7–IL-4+) or control vector only (EG7–IL-42) tumor cells was reported recently (7). Naive CD8+ T cells wereactivated in vivo by coinjection of 1.6 3 106 cells s.c. with 2.5 3 107
EG7–IL-4+ or EG7–IL-42 cells at the base of the tail in 200 ml saline.
Secondary tumor challenge with parental EG7 tumors
Va2+ T cells activatedwith IL-4–expressing or control tumorswere recoveredafter 7–9 d from host spleens and enumerated by flow cytometry to ascertainthe percentage of donor cells present. Cells adjusted to contain 253 103 Va2+
donor cells were coinjected with 103 106 EG7 cells at the base of the tail in200 ml saline into new RAG-22/2 gc2/2 hosts.
Tumor recovery and preparation
Tumors were recovered by surgical resection and individually weighed. Forpreparation of single-cell suspensions for flow cytometric analysis, thetumors were pooled according to group and passed through stainless steelmesh, followed by Ficoll-Pacque (AmershamBiosciences, Rydalmere, NewSouth Wales, Australia) separation.
FACS and analysis
Cells were incubated on ice with combinations of fluorochrome-conjugatedAb to CD8a (53-6.7) and isotype control (BioLegend, San Diego, CA), orAb to Va2 (B20.1) and isotype control (eBioscience, San Diego, CA). Cellswere washed and resuspended with 1 mg/ml propidium iodide (Calbiochem,San Diego, CA). CD8+ T cells were purified using a MoFlo cytometer(DakoCytomation, Glostrup, Denmark) with exclusion of dead cells basedon forward scatter and propidium iodide uptake. For analysis without sorting,a FACSCalibur was used with CellQuest version 3.1f software (BD Bio-sciences, San Jose, CA). Postacquisition data analyses were performed usingSummit Software V4.3 (DakoCytomation).
In vitro restimulation of in vivo activated T cells
Donor Va2+ T cells were enriched from spleen by FACS 7–9 d after in-jection and cultured at 2 3 104 cells/well in six-well plates coated withpurified Ab to CD3ε (145-2C11; 1 mg/ml), CD8a (53.6; 10 mg/ml), andCD11a (I21/7.7; 5 mg/ml) (anti-receptor Ab) (15) in 8 ml growth medium(modified DMEM, 50 mM 2-ME, and 216 mg/L L-glutamine), 10% heat-inactivated FCS, and 20 IU/ml human rIL-2 (National Institutes of HealthAIDS Research and Reference Reagent Program, Germantown, MD) (16).
Cytometric bead array
Following the restimulation period described above, culture supernatantswere collected and pooled from triplicate cultures, and cytokines wereanalyzed according to the manufacturer’s instructions using the cytometricbead array (CBA) mouse inflammation kit and mouse Th1/Th2 kit (BDBiosciences). Analysis was performed on a FACScan cytometer equippedwith CellQuest Pro and CBA software (BD Biosciences).
RNA preparation and real-time PCR analysis
RNAwas extracted by Nonidet P-40 hypotonic lysis of 3 3 103 cells, andcDNAwas prepared, as described (3). Triplicate cDNA samples were pre-pared from each culture, and each cDNA sample was assayed in duplicate.cDNA was quantified by real-time PCR using primers and probes for b2-microglobulin (b2m), IFN-g, and IL-4, as described previously (17), andcommercial primer/probe kits (Applied Biosystems, Foster City, CA), asfollows: IL-5 (ID number MM00439646-m1), IL-10 (ID numberMM00439616-m1), IL-13 (ID number MM00434204-m1), GATA-3 (IDnumber MM00484683-m1), and granzyme B (Gzmb; ID numberMM00442834-m1). Samples were amplified in a Corbett Rotor-Gene3000 (Corbett Research, Mortlake, Australia) for 40 cycles at 95˚C for
2 min, 95˚C for 5 s, and 60˚C for 30 s. All results are reported in b2munits (target gene copy number/b2m copy number) and represent themean of triplicate cDNA samples.
51Cr release assay
Cells of the EL4 cell line were incubated with Na51CrO4 (AmershamBiosciences) and 10 mg/ml OVA257–264 peptide SIINFEKL for 60 min at37˚C and washed twice. Labeled target cells (2–5 3 103) were incubatedfor 4–5 h at 37˚C with serial dilutions of T cells in 200 ml growth me-dium in round-bottom, 96-well plates. Harvested supernatants were driedonto 96-well solid Lumaplates (Packard Instrument, Meriden, CT), andradioactivity was counted in a TopCount microplate scintillation counter(Packard). Spontaneous lysis of target cells was typically ,10%, anddifferences in sample release, performed in triplicate, were within 5%.Total 51Cr release from target cells was obtained by lysis in 1% SDS. Thepercent specific lysis was calculated by the following formula: 100 3([sample cpm2 spontaneous release cpm]/[total release cpm2 spontane-ous release cpm]).
Statistical analyses
Data were evaluated by unpaired t test (Prism 4.02 software package; Graph-Pad, San Diego, CA). Values of p are indicated in the figures by the followingsymbols: NS,.0.05;p, 0.01–0.05;pp, 0.001–0.01;ppp,,0.001.
ResultsIn vivo activation of CD8 T cells with IL-4–secreting tumorsinduces type 2 polarization of CD8 T cells that is enhanced inthe absence of IFN-g
To determine whether IL-4–induced type 2 polarization of CD8T cells in vivo is inhibited by the presence of IFN-g, naive Va2+
CD8+ cells purified from OVA257–264-specific TCR-transgenicOT-I or OT-I 3 IFN-g2/2 mice were adoptively transferred intoRAG-22/2 gc2/2 hosts and activated with OVA-expressing tumorcells (EG7) transfected with an IL-4–expressing vector or controlvector. Prior to adoptive transfer, freshly purified cells from bothOT-I and OT-I 3 IFN-g2/2 mice were .99% CD8+ Va2+
(Supplemental Fig. 1A), and most cells of both genotypesdisplayed a naive (CD44lowCD62Lhigh) phenotype (SupplementalFig. 1B). Seven days after the adoptive transfer, the number ofdonor Va2+ cells recovered from host spleens was highest in thegroups exposed to IL-4+ tumors (Fig. 1A). The mean fluorescenceintensity of CD8 expression followed the hierarchy describedpreviously (7), in which CD8 expression by the recovered donorcells was increased by IFN-g and decreased by IL-4 (Fig. 1B).Most Va2+ donor cells displayed an activated (CD44high)phenotype and heterogeneous expression of CD62L, althoughthe percentage of CD62Llow cells was higher in the groupsexposed to IL-4 (Fig. 1C). This pattern of expression is similarto that reported previously for type 1 and type 2 polarized CD8T cells generated in vitro by activation with peptide-coated sple-nocytes in cytokine-polarizing conditions (18). Following over-night restimulation of purified Va2+ cells with anti-receptor Ab,upregulation of expression of mRNAs encoding the type 2 cyto-kines IL-4, IL-5, IL-10, and IL-13 was evident in both groupsexposed to IL-4–expressing tumors; however, expression ofmRNA for type 2 cytokines and the type 2 transcription factorGATA-3 was more prominent in the absence of IFN-g (Fig. 2A).No effect on repressor of GATA (ROG) mRNA was observed inany condition (data not shown). The pattern of type 2 polarizationwas maintained on the mRNA level after 6-d restimulation in vitroin nonpolarizing conditions (Fig. 2B), and was also evident at theprotein level in supernatant (Fig. 2C). In contrast to IFN-g+/+ cells,IFN-g2/2 cells not exposed to IL-4 expressed low levels ofIL-4 and IL-13 mRNA following the 6-d restimulation. The datademonstrate that IL-4–induced type 2 polarization of CD8 T cellsin vivo is modulated by endogenous IFN-g.
The CD8 T cell anti-tumor response is inhibited by induction oftype 2 cytokines or absence of IFN-g, and further inhibitedwhen both conditions exist
To test the in vitro lytic activity of effector cells generated in vivoby activation of IFN-g+/+ and IFN-g2/2 CD8 T cells with IL-4–secreting or control tumor cells, Va2+ donor cells were purifiedfrom recovered spleens by flow cytometry and restimulated withanti-receptor mAb for 6 d with IL-2. Their specific cytolytic ac-tivity was then assessed in a 51Cr release assay. Cytolytic activitywas reduced in cells exposed to IL-4 or devoid of IFN-g duringactivation and further reduced when both conditions were present(Fig. 3A). Expression of mRNA encoding the cytotoxic granuleprotein Gzmb followed the same pattern as cytolytic function (Fig.3B). The data indicate that the effects of IL-4 and IFN-g oncytolytic ability are mediated, at least in part, at the level ofexpression of a key cytolytic molecule.To test the ability of IL-4–exposed CD8 T cells to inhibit tumor
growth, and the contribution of endogenous IFN-g to this process,effector cells generated in vivo by activation of IFN-g+/+ and IFN-
g2/2 CD8 T cells with IL-4–secreting or control tumor cells weretransferred to new RAG-22/2 gc2/2 hosts along with parental EG7
tumor cells for secondary tumor challenge (Fig. 4A). Spleens and
tumors were recovered after 8 d. All groups that received donor
T cells displayed some inhibition of tumor growth when
compared with the tumor-only control group (Fig. 4B); however,
the anti-tumor activity of the IL-4–exposed IFN-g2/2 group (IFN-
g2/2 + IL-4) was significantly reduced when compared with all
other groups that received T cells. Antitumor activity did not differ
significantly between the groups that received IL-4–exposed IFN-
g+/+ or non–IL-4–exposed IFN-g2/2 cells; however, tumor control
in both of these groups was impaired when compared withmice that
received the non–IL-4–exposed IFN-g+/+ cells (IFN-g+/+ + Ctrl) in
which there was no tumor growth.After the secondary tumor challenge, fewer donor cells were
present in the spleens of mice that received IL-4–exposed cells,
whereas no difference was observed between the IFN-g+/+ and
IFN-g2/2 IL-4–exposed cells (Fig. 4C). Donor cells were present
in all pooled tumor samples and, similar to the spleens, the per-
FIGURE 1. IL-4 modulates CD8 effector cell number and CD8 expression. MACS-purified Va2+ CD8+ cells from naive OT-I (IFN-g+/+) or OT-I 3 IFN-
g2/2 (IFN-g2/2) mice were coinjected with EG7–IL-4+ (IL-4) or EG7–IL-42 (Ctrl) tumor cells into RAG-22/2 gc2/2 mice. After 7 d, splenocytes were
isolated and analyzed. A, The numbers of donor CD8+ T cells recovered from host spleens in a representative experiment are shown (mean and SD, n = 5,
except IFN-g2/2 only and IFN-g+/+ only groups, n = 2). B, Recovered splenocytes were stained with Ab to Va2 and CD8 and analyzed by flow cytometry.
The CD8 mean fluorescence intensity for cells within the gate is shown. C, Splenocytes gated on Va2 and costained for CD44 and CD62L. The percentage
of cells within the respective quadrant is shown.
1000 IFN-g MODULATES CD8 T CELL FUNCTION AND PHENOTYPE IN VIVO
centage of Va2+ donor cells present was lowest inmice that receivedthe IL-4–exposed IFN-g2/2 cells (Fig. 4D).The hierarchy of CD8 expression observed after the initial acti-
vation and recovery (Fig. 1B, above) was maintained in Va2+ cellsrecovered from the spleen following secondary tumor challenge (Fig.4E) and in pooled secondary tumor samples (Fig. 4F), suggestinga state of commitment to the differential levels of CD8 expressio-n in vivo. The data demonstrate a hierarchy in which the anti-tumorfunctionofCD8Tcells is significantly impairedbyeither exposure toIL-4 or the absence of IFN-g, and is further impaired when exposureto IL-4 is combined with the absence of IFN-g.
DiscussionIn this study, we present the first demonstration of comprehensivein vivo type 2 cytokine induction in CD8 T cells and the first
report showing that endogenous IFN-g inhibits type 2 cytokinepolarization of CD8 T cells in vivo. Furthermore, we show thatIFN-g and IL-4 reciprocally modulate cytolytic potential and anti-tumor function by CD8 T cells in vivo.Reciprocal regulation of type 2 polarization by IL-4 and IFN-g
is well characterized in CD4 T cells in vitro and in vivo. However,to the best of our knowledge, there are no conclusive in vitro orin vivo studies of this phenomenon in CD8 T cells. One previousreport used mAb to block IFN-g in the presence of IL-4 and founda corresponding reduction in expression of IFN-g; however, theeffect on type 2 cytokine expression was not conclusive (10).In this study, we present evidence that IL-4 and IFN-g alsoreciprocally regulate type 2 polarization of CD8 T cells in vivo.In CD4 T cells, reciprocal regulation is thought to occur bya mechanism in which IFN-gR signaling activates STAT1 and
FIGURE 2. IL-4 and IFN-g modulate type 2 cytokine expression by CD8 T cells in vivo. MACS purified Va2+ CD8+ cells from naive OT-I (IFN-g+/+) or
OT-I 3 IFN-g 2/2 (IFN-g 2/2) mice were coinjected with EG7-IL-4+ (IL-4) or EG7-IL-42 (Ctrl) tumor cells into RAG-22/2gc2/2 mice. After 7 d,
splenocytes were isolated and sorted by flow cytometry based on Va2 expression. The indicated mRNA species were assayed in sorted Va2+ cells after
overnight restimulation in vitro with anti-receptor Ab (A). In parallel, sorted Va2+ cells were restimulated with anti-receptor Ab and IL-2 for 6 d before
measurement of mRNA (B) and cytokine protein in pooled culture supernatants by CBA (C). .d or ,d indicates that the sample was above or below the
threshold of detection for the assay. Groups are compared using unpaired t test (see Materials and Methods).
consecutively the type 1 transcription factor T-bet (19), whereasIL-4R signaling activates STAT6 and consecutively the mastertype 2 transcription factor GATA-3 (20); ultimately, antagonismbetween T-bet and GATA-3 enhances the effective cytokine polar-ization. We have hypothesized previously that GATA-3 may havea role in the modulation of CD8 expression (7), and in this studywe show that GATA-3 is upregulated in CD8 T cells in the absenceof IFN-g and the presence of IL-4, suggesting that some of theunderlying mechanisms may be similar to those in CD4 T cells. Incontrast to CD4 cells, GATA-3 protein expression is specificallyinhibited in CD8 T cells by the zinc finger protein ROG (21, 22).In our hands, IFN-g does not alter ROG or T-bet mRNA expres-sion by CD8 T cells (data not shown). Interestingly, however,IFN-g has been shown to inhibit STAT6 phosphorylation inCD4 T cells, a step necessary for the induction of GATA-3 (23),and recent work has shown that IL-4 induces the phosphorylationof STAT6 in CD8 T cells (1), supporting the idea that the effects ofIL-4 and IFN-g on CD8 T cells are mediated via STAT6 andGATA-3.We found that in vivo activation of adoptively transferred IFN-
g+/+ or IFN-g2/2 OT-I CD8 T cells with IL-4–transfected tumorcells induced the expression of type 2 cytokines by the OT-I CD8T cells and this effect was enhanced in the absence of IFN-g.IL-4 exposure also increased the number of recoverable donorOT-I cells after 7 d when compared with cells activated with a con-trol-transfected tumor. This is consistent with recent evidence thatendogenously produced IL-4 contributes to CD8 T cell expansionduring an immune response (2). Another possible explanation, that
FIGURE 4. Absence of IFN-g impairs the anti-tumor response. Spleno-
cyte populations in Fig. 1B were normalized to Va2+ donor cell number and
coinjected with EG7 tumor cells into new RAG-22/2 gc2/2 hosts. After 8 d,
tumors were weighed and Va2+ CD8+ cells present in spleens and tumors
were analyzed by flow cytometry. A, Schema of experimental design. B, C,
and E, The indicated parameters for all mice in a representative experiment
are shown (mean and SD, n = 4). Groups are compared using unpaired t test
(see Materials and Methods). D and F, Tumor samples for each group were
pooled and analyzed by flow cytometry.
FIGURE 3. IL-4 and IFN-g modulate cytolytic potential of CD8 T cells.
Naive Va2+ CD8 T cells from OT-I (IFN-g+/+) or OT-I 3 IFN-g2/2 mice
were adoptively transferred into RAG-22/2 gc2/2 hosts and activated by
stimulation with either EG7–IL-4+ (IL-4) or EG7–IL-42 (Ctrl) tumor cells
expressing OVA protein. After 9 d, the donor cells were recovered from the
host spleens and sorted by flow cytometry-based Va2 expression. The
sorted cells were restimulated with anti-receptor Ab for 6 d with IL-2 and
then assessed for cytolytic potential in a cognate 51Cr release assay using
EL4 target cells coated with (+pep) or without (no pep) SIINFEKL peptide
(A). In separate experiments, restimulated cells were assessed for Gzmb
mRNA expression (B) (mean and SD, n = 3). Groups are compared using
unpaired t test (see Materials and Methods).
1002 IFN-g MODULATES CD8 T CELL FUNCTION AND PHENOTYPE IN VIVO
slower clearance of IL-4–expressing tumors led to more persistentCD8 T cell stimulation, was not supported as neither the IL-4–transfected nor the control-transfected tumor cells grew to a re-coverable mass. Somewhat paradoxically, we found that IFN-g+/+
or IFN-g2/2 OT-I cells activated in the presence of IL-4 and thentransferred to new hosts were significantly reduced in number inthe spleen following tumor challenge with the parental EG7 tumorline. One possible interpretation of this finding is that the IL-4–exposed donor cells did not survive as well as the non–IL-4–exposed cells; this notion is supported by recent evidence thatCD8 T cells activated in the presence of IL-4 become IL-4 de-pendent for survival (2). An alternate interpretation may be thatthe IL-4–exposed CD8 T cells have different homing abilities andsurvive in organs other than the spleen or lymph nodes or withinthe tumors. Indeed, donor OT-I cells were found within all pooledtumor samples, although, consistent with splenic recovery, theirfrequency was lowest within the tumors of mice that received theIL-4–exposed IFN-g2/2 cells. It is important to note that therecovered donor cell frequency was not a strict correlate oftumor growth: splenic donor cell recovery was high in the non–IL-4–exposed IFN-g2/2 group and low in the IL-4–exposed IFN-g+/+ group, and yet tumor growth was similar between thesegroups.We have previously shown that degranulation in response to
specificAg recognition byCD8T cells correlateswith levels of CD8expression (7). We show further in this study that Gzmb mRNAexpression and cytolytic activity in vitro and inhibition of tumorgrowth in vivo were all inversely proportional to the levels of CD8expression on the recovered CD8 T cells (compare Figs. 3A, 4Bwith 4E). The data are supported by earlier work that showed thatreduced CD8 expression and type 2 polarization were associatedwith reduced cytolytic potential (3, 6, 7, 11). It seems unlikely thatdifferences in CD8 expression alone are responsible for the im-paired anti-tumor responses, although they may directly affect theavidity of T cell-tumor interaction. The observation that CD8 ex-pression is similar in the non–IL-4–exposed IFN-g+/+ and IFN-g2/2
cells, whereas tumor control was maximal in the presence of IFN-g,suggests that IFN-g itself plays an important role in tumor control.This notion is consistent with other work showing that the anti-tumorefficacy of polarized CD8 T cells is in part dependent on effectoror host IFN-g (12, 24–26). However, others have reported thatnoncytolytic type 2 polarized IFN-g2/2 CD8 T cells were effectiveat curing metastases in a manner that was dependent on donorIL-4 and host CD8 T and NK cells (27). The latter studies did notdissect the differences in the direct anti-tumor activity of type 2polarized IFN-g+/+ and type 2 polarized IFN-g2/2 CD8 T cells.This was possible in our model, as the RAG-22/2 gc2/2 hosts aredevoid of CD8, NK, and NKT cells.Another potential role for IFN-g in vivo is to induce the
upregulation ofMHC-I on APCs or tumor cells. Indeed, others haveshown that MHC-I expression on the parental tumor line (EL4) wasincreased following a 48-h incubation with IFN-g in vitro (28). Thiseffect may have some bearing on the anti-tumor function in vivo.However, we think it unlikely that potentially reduced levels ofMHC-I expression in the absence of IFN-g in vivo have resultedin suboptimal priming and altered the effector activity of the in vivoactivated IFN-g2/2 cells for two reasons. First, similar numbers ofIFN-g+/+ and IFN-g2/2 cells were recovered following adoptivetransfer with tumor cells and all displayed a CD44high phenotype(Fig. 1), suggesting comparable activation efficiency. Second, ourdata in vitro argue against an association of CD8 downregulationwith suboptimal priming as cells progressively lost CD8 over timeand many cell divisions even in the presence of optimal concen-trations of immobilized anti-receptor Ab (4).
The reciprocal effects of IL-4 and IFN-g on the functional abili-ties of effector CD8 T cells could be profound. We have shown inthis study and in previous work (7) that these two cytokines candramatically alter the fundamental characteristics of CD8 T cellsin vivo, namely their cytokine profile, cytolytic potential, and TCRsensitivity via modulation of CD8 expression. Deviations in thecytolytic activity and cytokine profile of CD8 T cells could signif-icantly impair the immune response to cancer and chronic infec-tions. Indeed, there is evidence for some association with clinicaldisease. IL-4–expressing human CD8 T cells were first reported inCD8 T cell clones from patients with lepromatous leprosy (29) andhave also been reported in tuberculosis patients (30, 31), whereasIL-4–expressing CD8 T cells with reduced CD8 expression havebeen described in chronic B cell lymphocytic leukemia (32) andHIV (11).In summary, IFN-g and IL-4 reciprocally regulate type 2 polar-
ization of CD8 T cells in vivo modulating membrane expression ofthe CD8 molecule, the degree of type 2 polarization, and the anti-tumor potential of CD8 T cells. This work has important impli-cations for the design of vaccines and immunotherapies that seekto elicit an effective CD8 T cell response.
AcknowledgmentsWe thank Drs. William Heath, Guna Karupiah, and Alberto Pinzon-Charry
for the generous gifts of mice; Grace Chojnowski and Paula Hall for assis-
tance with cell sorting; Suzanne Cassidy and staff from the QIMR animal
facility for animal husbandry; and Kathy Buttigieg for technical contribu-
tions. The following reagent was obtained through the AIDS Research
and Reference Reagent Program, Division of AIDS, National Institute of Al-
lergy and Infectious Diseases, National Institutes of Health: human rIL-2
from Dr. Maurice Gately, Hoffmann-LaRoche (Nutley, NJ).
DisclosuresThe authors have no financial conflicts of interest.
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1004 IFN-g MODULATES CD8 T CELL FUNCTION AND PHENOTYPE IN VIVO
