B7-H1–Dependent Sex-Related Differences in Tumor ... Journal of Immunology B7-H1–Dependent Sex-Related Differences in Tumor Immunity and Immunotherapy Responses Pei-Yi Lin,* Lishi

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ResponsesTumor Immunity and Immunotherapy

Dependent Sex-Related Differences in−B7-H1

Lieping Chen, Bin Zhang, Tahiro Shin and Tyler J. CurielBahar, Mark J. Kious, Carolina B. Livi, Shawna R. Wall,Ludwig, Ratna K. Vadlamudi, Vincent J. Hurez, Rumana Pei-Yi Lin, Lishi Sun, Suzanne R. Thibodeaux, Sara M.

http://www.jimmunol.org/content/185/5/2747doi: 10.4049/jimmunol.1000496August 2010;

2010; 185:2747-2753; Prepublished online 4J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2010 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

B7-H1–Dependent Sex-Related Differences in Tumor Immunityand Immunotherapy Responses

Pei-Yi Lin,* Lishi Sun,* Suzanne R. Thibodeaux,* Sara M. Ludwig,* Ratna K. Vadlamudi,†

Vincent J. Hurez,* Rumana Bahar,* Mark J. Kious,* Carolina B. Livi,*

Shawna R. Wall,* Lieping Chen,‡ Bin Zhang,* Tahiro Shin,* and Tyler J. Curiel*

CD4+CD25+Foxp3+ regulatory T cells (Tregs) are immunopathogenic in cancers by impeding tumor-specific immunity. B7-

homologue 1 (B7-H1) (CD274) is a cosignaling molecule with pleiotropic effects, including hindering antitumor immunity. In this

study, we demonstrate sex-dependent, B7-H1–dependent differences in tumor immunity and response to immunotherapy in

a hormone-independent cancer, murine B16 melanoma. Antitumor immunity was better in B7-H12/2 females versus males as

a result of reduced regulatory T cell function in the B7-H12/2 females, and clinical response following B7-H1 blockade as tumor

immunotherapy was significantly better in wild-type females than in males, owing to greater B7-H1 blockade-mediated reduction

of Treg function in females. Wild-type female Tregs expressed significantly lower B7-H1 versus males but were insensitive to

estrogen in vitro. Female B7-H12/2 Tregs were exquisitely sensitive to estrogen-mediated functional reduction in vitro, suggesting

that B7-H1 effects occur before terminal Treg differentiation. Immune differences were independent of known B7-H1 ligands.

Sex-dependent immune differences are seldom considered in designing immune therapy or interpreting immunotherapy treat-

ment results. Our data demonstrate that sex is an important variable in tumor immunopathogenesis and immunotherapy

responses through differential Treg function and B7-H1 signaling. The Journal of Immunology, 2010, 185: 2747–2753.

Women generally exhibit more robust immunity thanmen postinfection (1) and increased allograft rejection(2) and experience a generally greater risk for auto-

immunity (3). Perhaps because estrogens are anti-inflammatory(3), studies of male–female immune differences tend to focus oninflammatory pathways, such as through TLRs (4, 5).B7-homologue 1 (B7-H1) is a cosignaling molecule abundantly

expressed on APCs and other immune cells (6). It contributes totumor immune evasion (7–9) and to induced T regulatory cell (Treg)function (10, 11). We found that B7-H1–mediated Treg function ismodulated in an estrogen-dependent manner; therefore, we exam-ined sex-dependent Treg functional differences in cancer giventhe central role that Tregs play in tumor immunopathology (10,12–14). We hypothesized that B7-H1 signals would differentiallyaffect female versus male tumor immunity and that response toB7-H1 blockade as cancer immunotherapy (9) would consequently

be more effective in females. We tested hypotheses using B16 mel-anoma, a well-described, transplantable tumor without known hor-monal influences on its growth or induced immunity, and whichresponds favorably to immunotherapy (15). B16 lacks a Y chromo-some (16); thus, immunity to it is not influenced by minor sex-related antigenic differences. Premenopausal women have a greatermelanoma risk comparedwith age-matchedmen, but this trend laterreverses, such that men .50 y old have a greater melanoma riskcompared with age-matched women. Many factors aside from im-munity, including hormonally controlled genetic repair mecha-nisms, could play roles in these sex-associated disparities (17).We showed that B7-H12/2 females resisted syngeneic B16

melanoma tumor better than males as a result of reduced Tregfunction, which allowed the development of superior antitumorimmunity. Strikingly, anti–B7-H1 blockade was significantly moreclinically effective in wild-type (WT) females than in WT malesas a result of greater female B7-H1 blockade-mediated reduction inTreg function. B7-H1 expression on naive WT female Tregs wassignificantly lower than in naive males, but it did not alter Tregsuppression in the presence of estrogen in vitro. By contrast, femaleB7-H12/2 Tregs were exquisitely sensitive to estrogen-mediatedreduction in suppression. Effects are not dependent on programmeddeath-1 (PD-1) or CD80, the known ligands of B7-H1 (18), sug-gesting a novel B7-H1 signaling pathway. These data demonstratean unexpected B7-H1–dependent, sex-related difference in Tregfunction that causes sex-dependent, B7-H1–mediated differencesin tumor immunity and immunotherapy responses.

Materials and MethodsMice

All mice were on the C57/BL6 (BL6) background. WT mice were purchasedfrom the National Cancer Institute (Bethesda, MD). CD802/2 and MHCclass I-restricted OVA-specific TCR transgenic (OT-I) mice were purchasedfrom The Jackson Laboratory (Bar Harbor, ME). B7-H12/2, PD-12/2, andFoxp3-internal ribosome entry site-monomeric red fluorescent protein (FIR)

*Department of Medicine and Cancer Therapy and Research Center and †Depart-ment of Obstetrics and Gynecology, The University of Texas Health Science Center atSan Antonio, San Antonio, TX 78229; and ‡Department of Oncology, The Johns HopkinsUniversity School of Medicine, Baltimore, MD 21287

Received for publication February 12, 2010. Accepted for publication June 26, 2010.

This work was supported by National Institutes of Health Grants CA105207,FD003118, 5P30CA54174; the Voelcker, Hayes, and Greehey Trusts; the FannyE. Rippel Foundation; the Holly Beach Public Library Association; the Texas STARSProgram; and the Owens Foundation.

Address correspondence and reprint requests to Dr. Tyler J. Curiel, Cancer Therapy andResearch Center, 2040 Babcock Road, Suite 201, San Antonio, TX 78229-3900. E-mailaddress: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this paper: B7-H1, B7-homologue 1; DC, dendritic cell; DLN,draining lymph node; DT, denileukin diftitox; E2, estradiol; Eff, effector T cell; FIR,Foxp3-internal ribosome entry site-monomeric red fluorescence protein; mTOR,mammalian target of rapamycin; OT-I, MHC class I-restricted OVA-specific TCRtransgenic mice; OVAp, dendritic cell loaded with SIINFEKL peptide; PD-1,programmed death-1; SPL, spleen; TDLN, tumor-draining lymph node; Treg, regu-latory T cell; WT, wild-type.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

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micewere gifts fromLiepingChen (The JohnsHopkinsUniversity, Baltimore,MD), Tasuku Honjo (KyotoUniversity, Sakyo-ku, Kyoto, Japan), and RichardA. Flavell (Yale University, New Haven, CT), respectively. All mice werehoused under specific pathogen-free conditions and used at 6–10 wk of age.

Abs

Anti-CD45RB (16A), anti–CTLA-4 (UC10-4F10-11), anti–glucocorticoid-induced TNFR (DTA-1), anti–IFN-g (XMG1.2), anti-CD25 (PC61), anti-CD4 (GK1.5), anti-CD3 (500A2), anti-CD11c (HL3), anti–B7-H1 (MIH5),and matched isotype control Abs were from BD Pharmingen (San Diego,CA). Anti-Foxp3 (FJK-16a), anti-CD62L (MEL14), anti–IL-10 (JES5-16E3),anti-granzyme B (16G6), and respective matched isotype control Abs werefrom eBioscience (San Diego, CA). Anti-CD8a (5H10) and control isotypeAbs were from Caltag Laboratories (Burlingame, CA). Polyclonal anti–neuropilin-1 and monoclonal anti–TGF-b Abs were purchased from R&DSystems (Minneapolis, MN). PE-conjugated OVA-specific pentamers werepurchased from ProImmune (Oxford, U.K.). Intracellular staining was per-formed according to the manufacturer’s instructions. Data were acquired onan LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed usingFlowJo software (Tree Star, Ashland, OR).

Cell lines and tumor induction

B16F10 melanoma, an X chromosome monozygotic tumor (16), was pur-chased from the American Type Culture Collection (Manassas, VA). ID8epithelial ovarian carcinoma was the gift of George Coukos (University ofPennsylvania, Philadelphia, PA). B16F10 was transfected with a plasmidencoding GFP-OVA. OVA expression was confirmed by ELISA (Supplemen-tal Fig. 1). A total of 1.25 3 105 B16 tumor cells in 0.2 ml sterile PBS wereinjected into sterilized flanks intradermally with a 27-gauge needle. Intrader-mal tumor size was measured using Vernier calipers and confirmed by his-tologic analysis. Tumor volume was calculated as width3 (length)2 3 0.5. Atotal of 1 3 107 ID8 cells were injected into the peritoneum. Ascites wasnoted as .30% weight gain from baseline. Mice were sacrificed by CO2

asphyxiation.

Treg suppression

A total of 3 3 104 flow cytometry-purified CD4+CD252 (effector) T cellsfrom naive mice were incubated for 9 min at 37˚C in 10 mM CFSE(Invitrogen, Carlsbad, CA) in PBS and resuspended in RPMI 1640 me-dium. Sorted CD4+RFP+ T cells from FIR mice or CD4+CD25hi T cellsfrom all other mice (Tregs) were added, in indicated ratios, with Dyna-beads Mouse CD3/CD28 T Cell Expander (Invitrogen) beads (1 bead:1effector T cell). After 72 h, the percentage of Treg suppression was cal-culated as 12 [(effector T cell proliferation without Treg2 effector T cell

with Treg)/effector T cell without Treg] 3 100%. In some cultures, wereplaced FBS with 10% charcoal dextran-treated FBS (estrogen-free;Hyclone, Logan, UT) alone or supplemented with 1027 or 1028 M 17b-estradiol (E2; Sigma-Aldrich, St. Louis, MO), with or without the estrogenreceptor antagonist ICI182,780 (19) (Tocris Cookson, Bristol, U.K.) at1026 M.

Dendritic cell isolation. Spleen and tumor-draining lymph nodes (TDLNs)were harvested aseptically, and dendritic cells (DCs) were isolated as de-scribed (20). Briefly, 100 mg/ml Liberase (research grade, Roche, SanFrancisco, CA) and 0.2 mg/ml DNAse (Roche) mild digestion was appliedto release DCs from murine TDLN fragments at 37˚C for 30 min. CD11c+

cells were then positively selected ($90% purity by FACS) using EasySepmouse CD11c positive selection kits (StemCell Technologies, Vancouver,British Columbia, Canada). A total of 25,000 CD11c+ DCs were pulsed withSIINFEKL (10 ng/ml) or nothing for 1 h and incubated with 50,000 CFSE-labeled, sex-matched OT-I cells for 72 h. CFSE dilution and IFN-g ex-pression were assessed by flow cytometry, gating on CD3+ cells.

Adoptive cell transfers

One third of the total cells in OT-I mouse spleens were labeled with CFSE, asdescribed above, and injected i.v. into mice. The next day, B16 tumor wasgiven, as described above, and the mice were sacrificed 4 d later to assessOT-I cell proliferation by flow cytometry. Cells from FIR mice were sortedas CD4+RFP+ or CD4+RFP2 in the CD3 gate, and 2 3 106 cells per mousewere given i.v. in PBS. B16 tumor was given the next day. At sacrifice,spleen cells were analyzed by flow cytometry or were sorted for ex vivofunctional tests.

In vivo treatments

Endotoxin-free anti–B7-H1 Ab (10F.9G2) or isotype control IgG2b Ab(both from BioLegend, San Diego, CA) was administered i.p. at 200mg/mouse every 3 d starting 1 d before tumor challenge and until sacrifice.Denileukin diftitox (DT; Eisai, Research Triangle Park, NC) was given at 5mg/mouse twice weekly, starting 4 d after tumor challenge until sacrifice.

Statistical analysis

Data are expressed as mean 6 SEM. The Student t test or ANOVA wasperformed, as appropriate, with two-tailed p , 0.05 considered significant.

ResultsB16 cells engineered to express OVA (OVA+ B16 is hereinafter“B16”; see Supplemental Fig. 1 and Materials and Methods) grewequally in WT males and females, but in support of our hypothesis,

FIGURE 1. B7-H12/2 females resist B16 better

than males through superior antitumor immunity

independent of PD-1 or CD80. A, Groups of five

mice were challenged with 125,000 B16 cells/

flank. Tumor growth was measured with Vernier

calipers. B, Tumor-specific immunity was assessed

as proliferation of CFSE-labeled, adoptively trans-

ferred OT-I cells using flow cytometry 4 d after

transfer. C, Groups of five mice each were chal-

lenged with B16, as above. D, Treg (CD4+CD25hi

T cell) function was tested in groups of five naive

mice each in vitro. DLN, draining lymph node;

SPL, spleen.

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they grew significantly slower in B7-H12/2 females versus B7-H12/2

males (p = 0.037) and versusWT females or males (Fig. 1A). Minormale histocompatibility Ags cannot explain these results becauseB16 is X chromosome monochromatic and lacks a Y chromosome(16). Tumor (OVA)-specific T cells were not detected in untreatedB16-bearing WT mice of either gender (data not shown). Supportingan immune-based mechanism for differential tumor growth, wedetected CD8+ tumor-specific T cells in vivo in three of threeB7-H12/2 females versus zero of three B7-H12/2 males (Fig. 1B,Supplemental Fig. 2). Supporting microenvironment-specific effects,tumor-specific CD8+ T cells were generated in TDLNs but notspleen (Fig. 1B). PD-1 and CD80 are the two known B7-H1 ligands(18). However, there was no significant difference in tumor growth(Fig. 1C) or Treg function (Fig. 1D, Supplemental Fig. 3) in WTversus PD-12/2 or CD802/2 mice of either sex, suggesting a novelB7-H1 ligand mediating Treg effects.Naive B7-H12/2males and females have comparable numbers of

phenotypic CD4+CD25+Foxp3+ Tregs, although Treg function issignificantly reduced in vitro and in vivo in naive B7-H12/2 fe-males (P.-Y. Lin, L. Sun, V. Hurez, S. Thibodeaux, R. Vadlamudi,M. Kious, C. Livi, R. Bahar, M.E. Wierman, R.R Tekmal, A. Pierce,B.J. Daniel, and T.J. Curiel, submitted for publication). To testreduced Treg function as a mechanism for improved immunity inB7-H12/2 females, we depleted Tregs in tumor-bearing mice usingthe IL-2–diphtheria fusion toxin DT (21). DT equally depletedphenotypic CD4+CD25hiFoxp3+ Tregs and CD4+CD25hi T cells

expressing other molecules associated with Treg function (12, 14),including CTLA-4, glucocorticoid-induced TNFR, and neuropilin-1,in both sexes in spleen and TDLNs (Fig. 2A, Supplemental Fig. 4).Despite equal DT-mediated phenotypic Treg depletion, DT reducedTreg function inB7-H12/2males,with no effect on lowbaselineTregfunction in B7-H12/2 females (Fig. 2B). Treg functional depletionwas accompanied by significantly reduced tumor growth (Fig. 2C)and improved tumor-specific immunity (Fig. 2D, SupplementalFig. 5) in B7-H12/2 males, but not females, supporting reducedTreg function as the basis for female B7-H12/2 tumor resistanceand improved antitumor immunity. To test possible differentialsusceptibility to Treg-mediated regulation of female B7-H12/2

effector T cells in the tumor microenvironment, we sorted Tregsfrom tumor-bearing B7-H12/2 females and tested their capacityto suppress B7-H12/2CD4+CD252 effector T cells from naive ortumor-bearing B7-H12/2 females. B7-H12/2CD4+CD252 effectorT cells from naive or tumor-bearing mice were equally susceptibleto regulation by tumor-associated B7-H12/2 Tregs (Fig. 2E). Thesedata support the concept that reduced susceptibility of tumoreffector cells to Treg-mediated suppression did not contribute todifferential sex-based immune and clinical responses to tumor, aswell as demonstrate that effector cell B7-H1 is dispensable fortheir regulation by Tregs.To test possible functional differences between female and male

B7-H12/2Tregs,we assessedgranzymeB, IL-10, andTGF-b expres-sion but found them comparable in mean fluorescence intensity and

FIGURE 2. Defective Treg function in B7-H12/2 females contributes to increased tumor resistance and superior antitumor immunity. Groups of five

mice were challenged with 125,000 B16 cells/flank, treated with DT 5 mg or PBS twice a week beginning 4 d later, and sacrificed 17 d after tumor

challenge. Effects of DT on phenotypic Treg depletion in SPL and DLN (A), Treg (CD4+CD25hi T cell) function (B), and tumor growth (C) were

determined. D, Effects of DT on tumor-specific immunity was assessed by OT-I cell proliferation using flow cytometry as for Fig. 1B. E, Tregs were

obtained from spleens of B16-bearing B7-H12/2 females and tested for regulation of B7-H12/2CD4+CD252 Eff from naive or tumor-bearing females. F,

Granzyme B, IL-10, and TGF-b expression gated on CD3+CD4+CD25+Foxp3+ cells in B16-bearing mice. n = 6/group. G, OT-I cell proliferation and IFN-g

expression induced by CD11c+ DCs obtained from spleens and DLNs of B16-bearing mice 7 d after tumor challenge. n = 3/group. Control mice received

DCs with no peptide. DLN, draining lymph node; Eff, effector T cell; OVAp, DC loaded with SIINFEKL peptide; SPL, spleen.

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in the percentage of positive cells among CD3+CD4+CD25+Foxp3+

cells (Fig. 2F). To test the possibility that altered APC function couldexplain sex-based immune differences, we obtained CD11c+ DCsfrom B16-bearing mice 7 d after tumor challenge and assessedtheir ability to activate OT-I cell proliferation and IFN-g expression(Fig. 2G). Nontumor microenvironmental (spleen) CD11c+ DCsfrom tumor-bearing B7-H12/2males and females activated OT-I cellproliferation identically. DCs from female TDLNs activatedOT-I cells statistically significantly better than male DCs, althoughthe magnitude of the difference was probably not biologicallyrelevant. Female B7-H12/2 spleen DCs activated OT-I cell IFN-gsignificantly better than did male spleen APCs, whereas IFN-gactivation from TDLNs using DCs from either sex was indis-tinguishable. Together, these data suggest that female B7-H12/2

DCs may function slightly better versus male B7-H12/2 DCs, butthe net effect on tumor immunity may not be very significant.To investigate further whether defective Tregs contributed

to improved antitumor immunity in B7-H12/2 females, we adop-tively transferred RFP+CD4+ T cells from female homozygousFIR mice into B7-H12/2 females and challenged them with B16.RFP+CD4+ T cell recipients exhibited significantly faster tumorgrowth (Fig. 3A), together with impaired tumor-specific immunityand CD8+ T cell IFN-g production (Fig. 3B), compared withtumor-bearing littermates receiving CD4+RFP2 (non-Treg) T cells.CD4+RFP+ cell recipients had greater percentages of CD4+Foxp3+

T cells compared with CD4+RFP2 cell recipients (Fig. 3C), consis-tent with increased Treg content. CD4+RFP+ cells recovered from

tumor-bearing CD4+RFP+ cell recipients (transferred Tregs) weresignificantly more suppressive compared with CD4+CD25+RFP2

T cells (endogenous Tregs; Fig. 3D), demonstrating that transferred(WT) Treg function was superior to endogenous (B7-H12/2) Tregfunction and was maintained in the female B7-H12/2 tumorenvironment. By comparing Treg function in tumor-bearing micewith or without RFP+ cell transfer, we further demonstrated thatadoptively transferred functional Tregs did not boost Treg functionin endogenous Tregs (compare RFP2 [endogenous] Treg functionin RFP+ recipients in Fig. 3D with endogenous Treg function intumor-bearing B7-H12/2 females not receiving any cell transfers inFig. 3E), suggesting that female B7-H12/2 Tregs resist infectioustolerance (22). Some transferred CD4+RFP2 cells converted intofunctionally suppressive CD4+RFP+ Tregs in vivo in tumor-bearingB7-H12/2 female recipients (Fig. 3D), demonstrating that inducibleTreg (23) conversion occurred in the absence of host B7-H1 but to anextent ineffective in significantly altering tumor growth or antitu-mor immunity in these studies. Together, these data are consistentwith a causal role for Tregs in mediating the immune and clinicaleffects observed. B16 cells also express B7-H1 (Supplemental Fig.6), which could affect Treg differentiation or function (10). How-ever, Treg function in tumor-bearing B7-H12/2 females remainedsignificantly lower than in tumor-bearing B7-H12/2 males (Fig. 3E),which was also comparable to the relative differences in Tregs fromnaive B7-H12/2 males and females (P.-Y. Lin et al., submitted forpublication). Altogether, these data establish defective Treg functionas a basis for sex-dependent differential tumor immunity in a B7-H1–

FIGURE 3. Adoptively transferred, functional female WT Tregs reverse immune and clinical differences in B7-H12/2 females. RFP+ or RFP2CD4+

T cells were sorted from female FIR mice and transferred into B7-H12/2 females. A, Mice were challenged with B16 the day after transfer. At sacrifice 14

d after tumor challenge, flow cytometry was used to detect CD8+ T cell IFN-g production (B) and tumor-specific CD8+ T cells (OVA pentamer stain) or (C)

Foxp3 expression in CD4+CD25hi spleen cells. The percentage positive gated events is shown. D, Functional status of endogenous, transferred, or converted

Tregs in B16-bearing mice was tested 15 d after adoptive transfer. E, Endogenous Treg (CD4+CD25hi T cell) function was tested 14 d after B16 tumor

challenge in mice not receiving adoptive cell transfers. F, Treg function 28 d after i.p. ID8 tumor challenge with 103 106 ID8 cells. G, Ascites development

(.30% weight increase) in mice challenged with ID8 tumor.

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dependent manner. We next challengedWTor B7-H12/2 females (n =12) with syngeneic ID8 epithelial carcinoma by i.p. injection. Con-sistent with results using B16, Treg function remained defective inB7-H12/2 females (Fig. 3F, Supplemental Fig. 7) following ID8 tu-mor challenge. Also consistent with reduced Treg function in B7-H12/2 females, tumor ascites, a clinical surrogate for tumor burden,following ID8 challenge developed more slowly than in WT females(Fig. 3G), confirming that sex-based differences in tumor Treg functionand tumor survival are not specific to B16 melanoma nor to the in-tradermal anatomic compartment.Based on the preceding data, we hypothesized that WT females

would benefit more than males from B7-H1 blockade as tumor im-munotherapy. We tested this hypothesis by treating B16 tumor-bearing WT males or females with anti–B7-H1 Ab. Strikingly,but consistent with data from tumor-bearing B7-H12/2 mice(Fig. 1), anti–B7-H1 Ab reduced tumor growth to a greater degreein WT females compared with males (Fig. 4A). Supporting ourproposed mechanism, anti–B7-H1 Ab treatment reduced Tregfunction significantly greater in tumor-bearing WT females com-pared with WT males (Fig. 4B). Anti–B7-H1 treatment augmentedantitumor immunity in WT males and females, consistent with thetreatment effects seen (Fig. 4A), but the relative increase in tumor-specific T cell prevalence was greater in anti–B7-H1–treatedfemales, as were their absolute numbers and relative increasefollowing B7-H1 blockade (Fig. 4C), consistent with the enhancedtreatment effect in females. Because B7-H1 blockade affectedmales and females differently, we next tested for sex-dependentB7-H1 expression differences. WT naive female CD4+CD25hi

Tregs expressed significantly lower B7-H1 versus WT males, al-though this difference was unaffected by 1028 M (physiologicconcentration) estrogen (Fig. 4D). We then tested B7-H1 expres-sion in tumor-bearing WT males and females (Fig. 4E). Surpris-ingly, WT male and female Treg B7-H1 expression in tumor wasequivalent (and also equivalent to naive male WT). Thus, tumorfactors likely augment female B7-H1 Treg expression as we pre-viously reported for tumor DC B7-H1 expression (7). Finally, tohelp understand why female B7-H12/2 Tregs were functionallydefective, we incubated them with 1028 M E2, which completelyabolished their function.The estrogen receptor antagonist ICI182,780rescued estrogen-mediated reduction of Treg suppression (Fig. 4F),demonstrating that estrogen receptor signaling is involved. By con-trast, femaleWTTreg function was not significantly affected by 1028

M (Fig. 4F) or supraphysiologic 1027 M (data not shown) E2.

DiscussionInterest in immune therapy for cancer is resurging as the resultof a better understanding of the underlying immune dysfunctionthat must be corrected for improved clinical efficacy (12). None-theless, advances in some areas have lagged. Notably, there is littlepublished regarding potential sex-based differences in antitumorimmunity or potential sex-based differential responses to tumor im-munotherapy. Such differences are likely to exist based on the well-known sexual dimorphisms in immunity in males and females (3).We studied potential sexual dimorphisms using B16 melanoma

because it has no known hormonal influences on its growth orinduced immunity (15). We studied anti–B7-H1 Ab treatment as

FIGURE 4. Differential sex-dependent B7-H1 responses contribute to sex-dependent differences in tumor immunity. Groups of five mice each were

challenged with 125,000 B16 cells/flank, treated with anti–B7-H1 or isotype control Abs (200 mg/mouse) starting 1 d before tumor challenge and every 2 d

thereafter, and sacrificed on day 16 after tumor challenge. A, Tumor growth was measured by Vernier calipers. B, Tregs (CD4+CD25hi T cells) were sorted

from spleen and tested for function. C, Splenocytes were analyzed for tumor-specific CD8+ T cells by flow cytometry using OVA-specific pentamers.

aB7-H1 or isotype refer to in vivo treatments in panels B and C. D, B7-H1 expression in naive WT mice gated on CD3+CD4+CD25hi Tregs cultured without

or with 1028 M E2 for 48 h (n = 6/group). E, B7-H1 expression in B16-bearing WT mice gated on CD3+CD4+CD25hi Tregs (n = 6/group). F, Female B7-

H12/2 Tregs from naive mice were tested for in vitro Treg without or with 1028 M E2 and without or with 1026 M ICI182,780 (an estrogen receptor

antagonist) (n = 6/group).

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a known antitumor immunotherapeutic agent (9) and establishedthat B7-H1 blockade was more effective in treating B16 mela-noma in WT females versus males, which was due, in part, tothe greater ability of anti–B7-H1 Ab to reduce Treg function inWT females. We recently showed that B7-H1 seems to desensitizeTregs to estrogen-mediated functional reduction through alteringTreg mammalian target of rapamycin (mTOR) and phosphataseand tensin homolog signals (P.-Y. Lin et al., submitted for publi-cation). However, because males derived some benefit fromB7-H1 blockade, factors in addition to inhibited Treg functionare likely also involved in treatment effects.Based on the sexually dimorphic response to B7-H1 blockade, in-

cluding significantly reduced Treg function in B7-H12/2 females,we hypothesized that female B7-H12/2 mice would resist atransplantable tumor challenge better than males, by virtue ofimproved antitumor immunity. Our data support this concept byshowing that B16 grew more slowly in B7-H12/2 femalesbecause they had superior antitumor immunity. This resistance totumor growth was lost upon transfer of functional Tregs into B7-H12/2 females, reducing tumor-specific immunity. These data es-tablish reduced Treg function as a basis for improved antitumorimmunity in females in the presence of deficient B7-H1 signals.Female B7-H12/2 DCs in tumor functioned slightly better com-pared with male B7-H12/2DCs in activating tumor-specific immu-nity, but the net effect on tumor immunity may not be very signif-icant based on the relatively small magnitudes of differences.Nonetheless, we cannot exclude improved DC performance as con-tributing to improved female antitumor immunity in B7-H1 defi-ciency. However, because functional Treg transfer into B7-H12/2

females recapitulated the reduced antitumor immunity observedin B7-H12/2 and WT males, improved DC perfor-mance in B7-H12/2 females could be due to their reduced Treg function, withreduced capacity, thereby, to degrade DC function in tumors (14).Anti–B7-H1 Ab significantly reduced tumor growth in WT

males and WT females bearing B16 melanoma, consistent withits ability to reduce Treg function in males and females andconsistent with known beneficial anti–B7-H1 effects on otherelements of antitumor immunity (9). Nonetheless, anti–B7-H1Ab reduced Treg function significantly greater in WT femalesversus WT males in vivo, consistent with sex-dependent B7-H1signaling. Strikingly, E2 essentially eliminated female B7-H12/2 Treg function in vitro, whereas E2 had no discernibleeffect on WT Treg function. These data suggest that E2 effectscan be directly on Tregs and that effects likely occur prior toterminal Treg differentiation. We recently demonstrated thatTregs differentiating in the presence of E2 in a B7-H1–deficientenvironment are highly sensitive to E2-mediated functional inhi-bition (P.-Y. Lin et al., submitted for publication), which accountsfor these observations. Prior work demonstrated that PD-1 block-ade is beneficial to antitumor immunity (24). Nonetheless,PD-12/2 mice did not phenocopy the sexually dimorphic re-sponses we now demonstrate. Thus, PD-1 effects may not ex-hibit sexual dimorphism as does B7-H1, or not, in this setting;thus, PD-1 blockade could benefit a different subset of tumorsor clinical scenarios. We previously reported that B7-H1 sig-nals in the tumor environment induce T cell IL-10 and reduceT cell IFN-g, inhibiting antitumor immunity (7). The effects werenot through known B7-H1 receptors. Thus, B7-H1 was reported toexert detrimental effects in tumor that are not through knownreceptors, as we extend in the present work. Our data also suggestthat known beneficial effects of anti–PD-1 in tumors may notalways involve ligation with B7-H1. Additional factors aside fromdifferential Treg effects could also help to explain the sexuallydimorphic differences, but they remain to be discovered.

A sex-dependent difference in response to tumor immunotherapybased on a sex-dependent difference in Treg function predicts thatmales and females will respond differently to certain immunothera-pies, a fact not incorporated into most current trial designs (3). Forexample, because depleted Tregs in tumor regenerate rapidly (25),our data suggest that combining Treg depletion with B7-H1 block-ade could be more beneficial as antitumor immunotherapy infemales versus males, or when B7-H1 blockade plus Treg deple-tion are combined with an E2 agonist. Our use of transplantabletumors demonstrated important proofs of principle regarding an-titumor immunity and tumor immunotherapy. Studies using au-tochthonous tumor will yield additional information regardingpotential sexual dimorphism in antitumor immune surveillance.For example, antiestrogens reduce male risk for hepatocarcinomain a mouse model for immune tumor surveillance, attributed toanti-inflammatory effects (4).Our data finally suggest a rethinking of the modes of action

of certain agents. For example, antiestrogens, long used to treathormone-sensitive tumors, also affect Treg function (26, data pre-sented in this study). Thus, Treg effects as an additional mecha-nism of action of antiestrogens should be explored further. TheB7-H1–associated Treg defect we report depends on estrogen-dependent sexually dimorphic differential mTOR and phosphataseand tensin homolog Treg signaling (P.-Y. Lin et al., submitted forpublication). Thus, mTOR inhibitors, now undergoing significantclinical trials as anticancer agents, in addition to uses as immunemodulators in autoimmunity, graft rejection, and other indications(27, 28), likewise warrant a reanalysis for potential sexually di-morphic optimal uses.

AcknowledgmentsWe thank Ai-Jie Liu, Dakshyani Lomada, and Xiuhua Sun for technical as-

sistance.

DisclosuresThe authors have no financial conflicts of interest.

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