ICOS promotes the function of CD4+ effector T cells during anti … · 2016. 5. 4. · Further, it...

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ICOS promotes the function of CD4+ effector T cells during anti-OX40 mediated tumor rejection

Todd C. Metzger1*, Hua Long1, Shobha Potluri1, Thomas Pertel1, Samantha L. Bailey-

Bucktrout1, John C. Lin1, Tihui Fu2, Padmanee Sharma2,3, James P. Allison2, Reid M.R.

Feldman1*

1Rinat Laboratories, Pfizer Inc., South San Francisco, CA 2Department of Immunology, 3Department of Genitourinary Medical Oncology, University of

Texas MD Anderson Cancer Center, Houston, TX

*Corresponding Authors: Todd Metzger, [email protected], Reid Feldman,

[email protected], Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San

Francisco, CA 94080, (650)615-7429

Disclosure of Potential Conflicts of Interest

J. Lin has ownership interest (including patents) in Pfizer Inc. P. Sharma serves on the scientific advisory boards of and has ownership interest in Jounce Therapeutics and Kite Pharmaceuticals, and consults for Bristol-Myers Squibb, GlaxoSmithKline, Amgen, and AstraZeneca. J.P. Allison serves on the scientific advisory boards of and has ownership interest in Jounce Therapeutics, Neon Therapeutics, and Kite Pharmaceuticals, is a licensor of intellectual property to Bristol-Myers Squibb, Jounce Therapeutics, and Merck, and receives royalties from Bristol-Myers Squibb and Merck. Pfizer provides research support to the M.D. Anderson Immunotherapy Platform as part of an alliance partnership agreement. No potential conflicts of interest were disclosed by other authors.

Running Title: ICOS promotes anti-OX40-mediated tumor immunity

Keywords: ICOS, costimulation, immunotherapy, OX40

Word Count: 2,999

Figures: 3

on July 21, 2021. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 4, 2016; DOI: 10.1158/0008-5472.CAN-15-3412

http://cancerres.aacrjournals.org/

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Abstract

ICOS is a T cell co-regulatory receptor that provides a costimulatory signal to T cells

during antigen-mediated activation. Antitumor immunity can be improved by ICOS-targeting

therapies, but their mechanism-of-action remains unclear. Here we define the role of ICOS

signaling in antitumor immunity using a blocking, non-depleting antibody against ICOS ligand

(ICOS-L). ICOS signaling provided critical support for the effector function of CD4+ Foxp3- T

cells during anti-OX40-driven tumor immune responses. By itself, ICOS-L blockade reduced

accumulation of intratumoral T regulatory cells (Treg), but it was insufficient to substantially

inhibit tumor growth. Further, it did not impede antitumor responses mediated by anti-4-1BB-

driven CD8+ T cells. We found that anti-OX40 efficacy, which is based in Treg depletion and to a

large degree on CD4+ effector T cell (Teff) responses, was impaired with ICOS-L blockade. In

contrast, the provision of additional ICOS signaling through direct ICOS-L expression by tumor

cells enhanced tumor rejection and survival when administered along with anti-OX40 therapy.

Taken together, our results showed that ICOS signaling during antitumor responses acts on both

Teff and Treg cells which have opposing roles in promoting immune activation. Thus, effective

therapies targeting the ICOS pathway should seek to promote ICOS signaling specifically in

effector CD4+ T cells by combining ICOS agonism and Treg depletion.




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Introduction

ICOS was originally identified as a marker of T cell activation (1), and has since been

found to have important roles in T cell proliferation and cytokine secretion (2). The role of

ICOS in supporting follicular T cell-dependent germinal center responses has been well

documented (3-5), but its contribution to the anti-tumor immune response remains unclear.

Initial studies found that ICOS-L transfection of tumor cells led to increased tumor rejection (6,

7) and resulted in subsequent immunity upon re-exposure to the same tumor (7). More recent

work has found that ICOS-L transfection of B16 tumor cells can promote enhanced anti-tumor

immunity when such cells are irradiated and used for vaccination alongside anti-CTLA-4

treatment (8), and experiments with wild-type or ICOS-/- tumor-bearing mice have shown a

requirement for ICOS signaling to support effective anti-CTLA-4 immunotherapy (9).

Additional studies have found that recombinant ICOS-L can also promote anti-tumor immune

responses (10, 11), although the strong FcR-binding affinity of some of these molecules makes it

difficult to distinguish whether they act through manipulation of ICOS signaling or direct

antibody-dependent cellular cytotoxicity (ADCC)-based depletion of ICOS+ populations.

However, ICOS can support both effector and regulatory populations (12), and in contrast to the

above studies, clinical observations suggest that ICOS promotion of immunosuppressive Tregs

may impair tumor immunity (13), while human melanoma cells have been shown to support the

induction of Tregs in vitro through ICOS-L expression (14). Here, we investigate the role of ICOS

in the context of CD4+ T cell-dependent anti-OX40 immunotherapy, and find a critical role for

ICOS in supporting CD4+ effector T cell function during anti-tumor immune responses.

Combination therapies with ICOS agonism and anti-OX40-based Treg depletion demonstrate the




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potential to enhance anti-OX40 efficacy through provision of additional ICOS signaling and

support the pairing of ICOS agonist therapies with Treg-depleting modalities for immunotherapy.

Materials and Methods

Mice and cell lines

7-8 week old female Balb/c mice were purchased (JAX #000651or Harlan 047) for CT26

tumor growth and flow cytometry experiments. Six week old C57BL/6 mice (Charles River)

were used for MB49 implantation. Balb/c.Foxp3-GFP mice (JAX #006769) were used for RNA

seq analysis. All Balb/c mice were maintained at an AAALAC-approved facility at Rinat, and

studies were conducted according to protocols approved by the Institutional Animal Care and

Use Committee of Rinat, Pfizer Inc.

CT26.wt cells were purchased from ATCC in 2014 and used with minimal passaging.

IMPACT testing for pathogens was performed at the Research Animal Diagnostic Laboratory.

No additional authentication was performed. The chemically-induced murine bladder carcinoma

MB49 cell line (15) was kindly provided by Dr. Ashish Kamat (MDACC) in 2008 and used

without further authentication. CT26.iICOS-L cells with stable integration of a tet-inducible

mouse ICOS-L expression cassette were generated with standard lentiviral transfection of

ATCC-derived CT26.wt cells as described in the Supplementary Materials and Methods. ICOS-

L expression was induced in vivo by oral gavage of doxycycline (2 mg / dose, in H2O), or

through doxycycline chow (Harlan Teklad, TD.01306).




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Tumor growth and treatment

For CT26 experiments, Balb/c mice were anesthetized and inoculated with 5 or 10 x 104

CT26.wt or CT26.iICOS-L cells as indicated by subcutaneous injection. Mouse health was

monitored by weight assessment and visual inspection. Tumor volumes were assessed by digital

calipers twice weekly (V = 0.5 L x W2), and mice were euthanized when tumors reached the

primary endpoint of 2000 mm3; date of euthanasia was used to generate survival plots.

Antibodies and dosing are further described in the Supplementary Materials and Methods.

Flow Cytometry

Spleens were prepared by manual dissociation followed by ACK lysis of erythrocytes.

Tumor-infiltrating lymphocytes were isolated by mincing tumors followed by digestion with an

enzyme cocktail (Miltenyi, 130-096-730) using a Miltenyi OctoDissociator. Single cell

suspensions were stained with LIVE/DEAD fixable Blue kit (L-23105, Life Technologies),

followed by addition of 2.4G2 Fc receptor block and surface antibodies as described in the

Supplementary Materials and Methods. Stained cells were fixed and permeabilized with the

eBioscience Foxp3 Buffer Set (00-5523-00), followed by intracellular staining for Foxp3 and/or

Ki67, data collection on a BD LSRII, and analysis by FlowJo (TreeStar Inc.).

Statistical Analysis

Dot plots show mean +/- SD; tumor growth curves display mean +/- SEM. All analyses

were performed on GraphPad Prism 6, and statistical significance was determined by unpaired

parametric t-tests with Welch’s correction unless otherwise indicated; p values less than 0.05

were considered significant.




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Results and Discussion

Agonist antibodies against TNF family receptors are known to promote tumor rejection in

mouse syngeneic tumor models (16), but the primary cellular targets of such therapies vary

between receptors: Evidence suggests that anti-OX40 therapy requires direct interaction with

CD4+ T cell populations to mediate its anti-tumor efficacy (17), while anti-4-1BB therapy

depends only on CD8+ T cell responses (18). Prior work has shown that, in addition to driving

signaling through the OX40 receptor, the anti-mouse OX40 clone OX86 also exerts its anti-

tumor effects in part through FcR engagement and Treg depletion (19). We obtained the OX86

sequence, re-formatted the anti-OX40 antibody to contain either a mouse IgG1 (OX86-g1) or

mouse IgG2a (OX86-g2a) Fc region, and confirmed that strong FcR engagement by OX86

enhances tumor regression, as OX86-g2a promoted more frequent tumor rejection than OX86-g1

in CT26 syngeneic tumor models (Fig. 1A). Furthermore, dramatic tumor Treg depletion was

observed following treatment of tumor-bearing mice with the mouse IgG2a variant of OX86,

while OX86-g1 did not significantly affect Treg frequencies within the tumor (Fig. 1B,

Supplementary Fig. S1). Lastly, we confirmed that CD4+ T cells play a critical role in driving

the anti-tumor efficacy of anti-OX40 by showing that CD4+ depletion (Supplementary Fig. S2)

significantly impaired the ability of anti-OX40 to promote survival of CT26 (Fig. 1C) and MB49

(Fig. 1D) tumor-bearing mice. An important role for CD8+ T cells was also observed in these

experiments. However, while CD8+ T cells may respond directly to OX40 signaling (20), a role

for CD4+ T cells is consistent with prior studies which have demonstrated that CD8+ T cell anti-

tumor responses require anti-OX40-driven CD4+ T cell help to carry out optimal anti-tumor

responses (21).




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We next sought to determine whether a combination immunotherapy strategy targeting

ICOS and OX40 might drive enhanced tumor efficacy, as ICOS is known to have a key role in

supporting the efficacy of Treg-depleting anti-CTLA-4 therapy (8, 9, 22, 23). We first examined

patterns of ICOS expression in mouse syngeneic tumors, and found that ICOS surface expression

was highly upregulated within the tumor microenvironment (Fig. 2A). Among tumor-resident T

cells, ICOS was expressed most highly by Tregs, but was also upregulated across CD8+ and CD4+

effector populations at the protein and transcript level (Fig. 2A, 2B) when compared to splenic T

cells. Anti-CTLA-4 can drive increased ICOS expression on T cells in clinical trials (24, 25),

and the upregulation of ICOS on peripheral T cells correlates with clinical responses to anti-

CTLA-4 (25). We therefore examined expression of ICOS on peripheral and CT26 tumor-

derived T cells from mice that had received treatment with either PBS or anti-OX40, and found

that OX40 stimulation alone was sufficient to drive an increase in the frequency of ICOS

expression among all peripheral T cell subsets (Fig. 2C, Supplementary Fig. S3). While ICOS

was already expressed at a high frequency in baseline CT26 tumors, we were also occasionally

able to observe an increase in ICOS expression among CD4+ effector T cells within the tumor as

well (Fig. 2D). Taken together, these results suggest that targeting the ICOS pathway may

further boost the efficacy of anti-OX40 therapy.

We next sought to understand the effects of modulating ICOS-dependent signaling. To

selectively perturb ICOS:ICOSL signaling we generated and used a novel rat anti-mouse ICOS-L

antibody reformatted to contain a mouse IgG1 Fc. Treatment of tumor-bearing mice with our

ICOS-L-blocking antibody resulted in a significant increase in ICOS surface expression across

all tumor-derived T cell subsets (Fig. 3A), suggesting that ICOS signaling is tightly regulated

within the tumor microenvironment. We found minimal evidence that blockade of ICOS-L alone




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was sufficient to alter the progression of the tumor immune response (Supplementary Fig. S4A),

although modestly lower tumor volumes were observed across multiple experiments

(Supplementary Fig. S4B). However, when we combined ICOS-L blockade with anti-OX40

therapy, we observed a significant inhibition of the ability of anti-OX40 treatment to promote

expansion of proliferating, ICOS+ CD4+ effector T cells (Fig. 3B), while the effect on Foxp3+

Tregs was less robust (Supplementary Fig. S4C). Both high and low FcR-binding variants of the

OX86 clone promoted ICOS-dependent expansion of ICOS+ Ki67+ Teff (Supplementary Fig.

S4D), suggesting that OX40 agonism was sufficient to drive CD4+ Teff expansion.

We next addressed whether the ability of ICOS-L blockade to inhibit CD4+ Teff

expansion would impair anti-OX40 therapy, and found that co-treatment of CT26 tumor-bearing

mice with anti-OX40 and ICOS-L blockade resulted in a significant reduction in overall survival

when compared with anti-OX40 alone (Fig. 3C). Importantly, ICOS-L blockade alone was

sufficient to drive reduction of tumor Tregs (Supplementary Fig. S4E), which were further

depleted by co-treatment with OX86g2a, suggesting that inhibition of anti-OX40 efficacy by

ICOS-L blockade is not a result of enhanced Treg-based immunosuppression, but instead derives

from impaired ICOS-dependent CD4+ Teff responses. In contrast to anti-OX40, an agonist

antibody against TNF family member 4-1BB which is able to drive extensive tumor regression in

the CT26 syngeneic tumor model (Supplementary Fig. S4F, S4G), did not promote expansion of

ICOS+ CD4+ effectors in either the presence or absence of ICOS-L blockade (Fig. 3B).

Furthermore, CD8+ T cell-dependent anti-4-1BB-mediated tumor rejection was not impaired by

ICOS-L blockade (Supplementary Fig. S4F, S4G), and ICOS-L blockade alone did not impair

IFN-γ production by tumor-derived CD8+ T cells (Supplementary Fig. S4H), suggesting that

ICOS signaling may be dispensable for CD8+ T cell-directed immunomodulators but required to




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support the function of CD4+ Teff dependent tumor rejection. Finally, the importance of ICOS

signaling in supporting anti-OX40-mediated anti-tumor efficacy led to us to address whether

provision of additional ICOS signaling could enhance the function of anti-OX40 therapies.

Towards this end, we created a modified version of the CT26 cell line with doxycycline-

inducible ICOS-L expression (CT26.iICOS-L) and confirmed that these modified cells expressed

ICOS-L in a doxycycline-controlled manner (Fig. 3D). While we had previously found that

ICOS-L blockade inhibited the activity of anti-OX40, we observed that provision of additional

ICOS ligand within the tumor microenvironment together with anti-OX40 promoted enhanced

tumor rejection and survival when compared with anti-OX40 treatment alone (Fig. 3D).

Importantly, doxycycline administration to mice bearing wild-type CT26 tumors did not alter the

effects of anti-OX40 treatment (Supplementary Fig. S4I), and rejection of tumors by dual OX40

treatment and ICOS-L induction continued to be mediated by both CD4+ and CD8+ Teff

responses (Supplementary Fig. S4J).

In summary, these results show that ICOS is highly upregulated within the tumor

microenvironment, and expressed across T cell subsets with opposing functions. Thus, simple

manipulation of ICOS signaling will likely have opposing effects of suppressive and activating

arms of the T cell response, and helps to explain the lack of anti-tumor efficacy by ICOS-L

blockade alone. However, our data suggests that depletion of Tregs in conjunction with ICOS

agonism may remove the potential for ICOS signaling to promote immunosuppressive Tregs

responses and allow ICOS agonism to act solely in promoting activity of CD4+ Teff. It should be

noted that within the tumor, ICOS is expressed most highly by tumor Tregs, and thus a single

ICOS agonist with strong Fc engagement may be sufficient to drive simultaneous ADCC-

mediated depletion of Tregs and agonist-based enhancement of Teff responses, a pattern of activity




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which seems to be achieved by OX86 therapy (19). Additionally, while the potential for

combination therapy of anti-ICOS and anti-CTLA-4 has been well described (8, 9), the results

from these studies also support combination of ICOS agonism with other Treg-depleting therapies

such as those targeting OX40 and GITR (26).

Author’s Contributions

Conception and design: T. Metzger, J. Lin, R. Feldman

Development of methodology: T. Metzger, R. Feldman, H. Long

Acquisition of data (provided animals, acquired and managed patients, provided facilities,

etc.): T. Metzger, H. Long, T. Pertel, T. Fu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational

analysis): T. Metzger, H. Long, S. Potluri, J. Lin, T. Fu, J. Allison, R. Feldman

Writing, review, and/or revision of the manuscript: T. Metzger, H. Long, S. Bailey-

Bucktrout, J. Lin, T. Fu, P. Sharma, R. Feldman

Administrative, technical, or material support (i.e., reporting or organizing data,

constructing databases): T. Metzger, T. Fu

Study Supervision: R. Feldman, S. Bailey-Bucktrout, P. Sharma, J. Allison, J. Lin

Acknowledgements

The authors thank Orla Cunningham at Pfizer Global Biotherapeutics (Dublin, Ireland) for the

generation and provision of in-house anti-ICOS-L antibodies.




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References

1. Hutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R, Anagnostopoulos I, et al.

ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28.

Nature 1999;397:263-6.

2. Simpson TR, Quezada SA, Allison JP. Regulation of CD4 T cell activation and effector

function by inducible costimulator (ICOS). Curr Opin Immunol 2010;22:326-32.

3. Tafuri A, Shahinian A, Bladt F, Yoshinaga SK, Jordana M, Wakeham A, et al. ICOS is

essential for effective T-helper-cell responses. Nature 2001;409:105-9.

4. Dong C, Juedes AE, Temann UA, Shresta S, Allison JP, Ruddle NH, et al. ICOS co-

stimulatory receptor is essential for T-cell activation and function. Nature 2001;409:97-101.

5. Yoshinaga SK, Whoriskey JS, Khare SD, Sarmiento U, Guo J, Horan T, et al. T-cell co-

stimulation through B7RP-1 and ICOS. Nature 1999;402:827-32.

6. Wallin JJ, Liang L, Bakardjiev A, Sha WC. Enhancement of CD8+ T cell responses by

ICOS/B7h costimulation. J Immunol 2001;167:132-9.

7. Liu X, Bai XF, Wen J, Gao JX, Liu J, Lu P, et al. B7H costimulates clonal expansion of,

and cognate destruction of tumor cells by, CD8(+) T lymphocytes in vivo. J Exp Med

2001.194:1339-48.

8. Fan X, Quezada SA, Sepulveda MA, Sharma P, Allison JP. Engagement of the ICOS

pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy. J Exp

Med 2014;211:715-25.

9. Fu T, He Q, Sharma P. The ICOS/ICOSL pathway is required for optimal antitumor

responses mediated by anti-CTLA-4 therapy. Cancer Res 2011;71:5445-54.




12

10. Zuberek K, Ling V, Wu P, Ma HL, Leonard JP, Collins M, et al. Comparable in vivo

efficacy of CD28/B7, ICOS/GL50, and ICOS/GL50B costimulatory pathways in murine

tumor models: IFNgamma-dependent enhancement of CTL priming, effector functions, and

tumor specific memory CTL. Cell Immunol 2003;225:53-63.

11. Ara G, Baher A, Storm N, Horan T, Baikalov C, Brisan E, et al. Potent activity of soluble

B7RP-1-Fc in therapy of murine tumors in syngeneic hosts. Int J Cancer 2003;103:501-7.

12. Burmeister Y, Lischke T, Dahler AC, Mages HW, Lam KP, Coyle AJ, et al. ICOS controls

the pool size of effector-memory and regulatory T cells. J Immunol 2008;180:774-82.

13. Faget J, Bendriss-Vermare N, Gobert M, Durand I, Olive D, Biota C, et al. ICOS-ligand

expression on plasmacytoid dendritic cells supports breast cancer progression by promoting

the accumulation of immunosuppressive CD4+ T cells. Cancer Res 2012;72:6130-41.

14. Martin-Orozco N, Li Y, Wang Y, Liu S, Hwu P, Liu YJ, et al. Melanoma cells express

ICOS ligand to promote the activation and expansion of T-regulatory cells. Cancer Res

2010;70:9581-90.

15. Summerhayes IC, Franks LM. Effects of donor age on neoplastic transformation of adult

mouse bladder epithelium in vitro. J Natl Cancer Inst 1979;62:1017-23.

16. Moran AE, Kovacsovics-Bankowski M, Weinberg AD. The TNFRs OX40, 4-1BB, and

CD40 as targets for cancer immunotherapy. Curr Opin Immunol 2013;25:230-7.

17. Piconese S, Valzasina B, Colombo MP. OX40 triggering blocks suppression by regulatory T

cells and facilitates tumor rejection. J Exp Med 2008;205:825-39.

18. Miller RE, Jones J, Le T, Whitmore J, Boiani N, Gliniak B, et al. 4-1BB-specific

monoclonal antibody promotes the generation of tumor-specific immune responses by direct

activation of CD8 T cells in a CD40-dependent manner. J Immunol 2002;169:1792-800.




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19. Bulliard Y, Jolicoeur R, Zhang J, Dranoff G, Wilson NS, Brogdon JL. OX40 engagement

depletes intratumoral Tregs via activating FcgammaRs, leading to antitumor efficacy.

Immunol Cell Biol 2014;92:475-80.

20. Bansal-Pakala P, Halteman BS, Cheng MH, Croft M. Costimulation of CD8 T cell

responses by OX40. J Immunol 2004;172:4821-5.

21. Song A, Song J, Tang X, Croft M. Cooperation between CD4 and CD8 T cells for anti-

tumor activity is enhanced by OX40 signals. Eur J Immunol 2007;37:1224-32.

22. Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, Arce F, et al. Fc-

dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-

CTLA-4 therapy against melanoma. J Exp Med 2013;210:1695-710.

23. Selby MJ, Engelhardt JJ, Quigley M, Henning KA, Chen T, Srinivasan M, et al. Anti-

CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of

intratumoral regulatory T cells. Cancer Immunol Res 2013;1:32-42.

24. Chen H, Liakou CI, Kamat A, Pettaway C, Ward JF, Tang DN, et al. Anti-CTLA-4 therapy

results in higher CD4+ICOShi T cell frequency and IFN-gamma levels in both

nonmalignant and malignant prostate tissues. Proc Natl Acad Sci U S A 2009;106 2729-34.

25. Carthon BC, Wolchok JD, Yuan J, Kamat A, Ng Tang DS, Sun J, et al. Preoperative CTLA-

4 blockade: tolerability and immune monitoring in the setting of a presurgical clinical trial.

Clin Cancer Res 2010;16:2861-71.

26. Bulliard Y, Jolicoeur R, Windman M, Rue SM, Ettenberg S, Knee DA, et al. Activating Fc

gamma receptors contribute to the antitumor activities of immunoregulatory receptor-

targeting antibodies. J Exp Med 2013;210:1685-93.




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Figure Legends

Figure 1. Anti-OX40 tumor immunotherapy requires both CD4+ and CD8+ T cell

responses. (A) Growth curves showing individual tumor volumes over time of CT26-bearing

mice randomized at day 9 post-inoculation and treated on days 9, 13, and 16 as indicated.

Representative of three independent experiments. (B) Frequency of Foxp3+ events among tumor-

derived CD4+ T cells at 17 days post-inoculation, taken from CT26-bearing mice treated 9, 12,

and 15 days post-inoculation as indicated. (C) Treatment schematic (top) and survival plots

(bottom), based on euthanasia when CT26 tumor volumes reached 2000 mm3 endpoint. p-

values for Tx (OX40) vs. all other curves are <0.01 by Log Rank (Mantel-Cox) tests. (D)

Treatment schematic (top) and survival plots (bottom) for MB49-bearing mice treated as

indicated. Tx vs. NK depl, ns; Tx vs. CD8 depl, p = 0.135; Tx vs. CD4 depl, p = 0.0026; Tx vs.

CD4 + CD8 depl, p = 0.0015.

Figure 2. Anti-OX40 treatment induces ICOS expression on CD4+ effector T cells. (A)

Flow cytometric analysis of ICOS expression among indicated populations isolated from the

spleen or tumor of CT26 tumor-bearing mice at two weeks post-implantation. Representative of

more than 5 independent experiments. (B) RNA seq analysis of Icos mRNA transcripts from T

and NK cell populations isolated from spleens and tumors of CT26-bearing Foxp3-GFP mice

implanted 2.5-3 weeks before tissue harvest. Tconv = CD4+Foxp3-, Treg = CD4+Foxp3+. (C)

Frequency of ICOS+ events among the indicated T cell subsets isolated from the spleen of CT26

tumor-bearing mice following one week of pre-treatment with either PBS alone or the indicated

anti-OX40 antibodies. (D) Frequency of ICOS+ events among tumor Tconv (left) and CD8+ T cells

(right) from mice following the treatment regimen in (C).




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Figure 3. ICOS signaling promotes anti-OX40-mediated anti-tumor efficacy. (A) Mean

fluorescent intensities (MFI) of ICOS staining on the indicated tumor-derived T cell subsets from

mice pre-treated with an isotype control (MOPC-21) or anti-ICOS-L. (B) Frequency of ICOS+

Ki67+ events among Foxp3- CD4+ Teff from the spleens of tumor-bearing mice 15 days post-

inoculation following i.p. treatment with the indicated antibodies at days 9 and 13.

Representative of two to four experiments per condition. (C) Pooled data from two separate

experiments showing overall survival of mice with indicated treatments. p = 0.0482 by two-

tailed chi2 contingency test of survival at 37 days post-inoculation between OX86g2a +/- isotype.

(D) Flow cytometric analysis of ICOS-L expression by the indicated cell populations after 16

hours of culture in vitro (left) and survival curves for CT26.iICOS-L-bearing mice (right)

randomized at day 10 post-inoculation and treated on days 10, 13, and 17 i.p. with isotype

control or OX86-g2a, and treated with doxycycline as indicated beginning on day 10. Results

are pooled from three independent experiments. *p<0.05,**p <0.01, ***p<0.001, ****p<0.0001.




Figure 1

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f Fox

p3-

Iso ICOS-L Iso ICOS-L Iso ICOS-L+ 4-1BB + OX86-g2a

****

**

nsns

**

Std IsotypeStd OX86g2aDox IsotypeDox OX86g2a

BA

C

CT26.iICOS-L 1 ug/mL DoxCT26.iICOS-L control mediaCT26.wt control media

% M

ax

ICOS-L

D

0

5000

15000

10000

Treg Tconv CD8+

Iso ICOS-L Iso ICOS-L Iso ICOS-L

*

***

**

ICO

S M

FI

0 20 400

50

100

Perc

ent s

urvi

val

*

0 20 40 600

50

100

days post tumor inoculation

Perc

ent s

urvi

val

OX86g2a +IsotypeOX86g2a +ICOS-L blockade

Isotype

ICOS-L blockade

*




Published OnlineFirst May 4, 2016.Cancer Res Todd C Metzger, Hua Long, Shobha Potluri, et al. anti-OX40 mediated tumor rejectionICOS promotes the function of CD4+ effector T cells during

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