Intratumoral activation of 41BB co-stimulatory signals enhances CD8 T cell expansion and modulates tumor-infiltrating myeloid cells

Abstract

The activation of 41BB co-stimulatory signals by agonistic antibodies enhances the expansion and function of tumor-infiltrating lymphocytes (TILs) for treating cancer patients with adoptive cell therapy (ACT). However, the impact of 41BB agonism is not limited to enhancing the activity of T cells and the mechanism by which additional activation of this co-stimulatory axis in tumor-associated myeloid cells is poorly understood. Here, we describe that the intratumoral administration of 41BB agonistic antibodies led to increases in CD8 T cell infiltration followed by tumor regression in murine models. We found that granulocytes and monocytes rapidly replaced macrophages and dendritic cells in tumors following administration of anti-41BB antibodies. Overall, myeloid cells from anti-41BB treated tumors had an improved capacity to stimulate T cells in comparison to control treated tumors. In human co-culture systems, we demonstrated that the agonism of the 41BB-41BBL axis enhanced co-stimulatory signals and effector functions among antigen presenting cells and autologous TILs. Overall, these findings suggest that the effect of 41BB agonistic antibodies are supported by additional co-stimulatory signals from tumor-associated myeloid cells leading to enhanced TIL expansion and function.

Introduction

Activation of 41BB (CD137) co-stimulatory signals is an effective means to enhance the expansion and function of tumor-infiltrating lymphocytes (TILs) from primary tumor fragments for the purpose of preparing cells for adoptive cell therapy (ACT) (1). Recently, it has been identified that the direct injection of 41BB agonistic antibodies into tumors can mount potent immune responses against local and distant untreated tumors (2). Moreover, strategies to engineer therapeutics that selectively activate 41BB within tumors have demonstrated feasibility in mouse models providing support for advancement of these therapies to clinical trials (3–5). Overall, the targeting of 41BB within tumors can effectively increase T cell proliferation and promote the eradication of tumor cells both in vitro and in vivo.

41BB is widely known as a co-stimulatory molecule expressed by T cells, however nearly all subsets of immune cells express 41BB (6). Previous studies identified that 41BB and 41BB ligand (41BBL) play critical, yet context-dependent roles in myeloid cell development and function (7, 8). In particular, the knockout of 41BB promotes the accumulation of myeloid cells under steady-state conditions, while triggering 41BB activation in dendritic cells (DCs) enhanced their capacity to stimulate T cell in vitro (7, 9). While accumulating evidence in mice has suggested that both 41BB and 41BBL are critical for directly regulating the function of myeloid cells, little is known about how this receptor-ligand axis potentiates myeloid-mediated anti-tumor immune responses in humans. Given that the importance of the inflammatory context in 41BB-41BBL signaling, a deeper understanding of 41BB-41BBL signaling in human myeloid cells, particularly in the context of tumor-mediated inflammation, is needed (7, 10).

In human biological systems, 41BBL acts as a maturation factor for monocytes, promoting the expression of co-stimulatory molecules and cytokines, including IL-12, IL-6, IL-8, TNF, and M-CSF (11). The stimulation of 41BBL with 41BB protein induces reverse signaling in monocytes, triggering their maturation to DCs (12). Although 41BB-41BBL bidirectional signaling between T cells and APCs has been shown to promote effector immune responses, it remains unclear how the context of inflammation within human tumors influence this process. At our institution, treatment of melanoma patients using ACT with TIL has resulted in a 38% overall response rate (13, 14). Moreover, 41BB agonists are currently being explored for the ability to enhance TIL expansion for the use ACT (NCT02652455). Hence, the development of therapeutics that exploits immunologic mechanisms to boost ex vivo TIL expansion can greatly benefit from an enhanced understanding of how a supportive immune microenvironment promotes anti-tumor immune functions. The work outlined in this study highlights the importance of triggering co-stimulatory signals on T cells and how augmenting the interactions of 41BB-41BBL bidirectional signals provided by antigen presenting cells (APCs) ultimately provides support for the improvement of TIL expansion from primary tumor fragments and the promotion of anti-tumor immune responses in vivo.

Methods

Human TIL specimens and tumor digest preparation

Preparation of TIL was performed as previously described (13). Briefly, surgically resected tumors were minced to 1mm fragments and placed into individual wells of a 24 well plate containing 6000IU/mL IL-2 (aldesleukin, Prometheus Laboratories). TILs were expanded for up to 5 weeks and then tested for IFN-gamma production in co-cultures with autologous tumor cell lines or cryopreserved tumor digest cell suspensions. IFN-gamma⁺ TILs underwent a rapid expansion protocol (REP) and were then cryopreserved in 90% human serum with 10% dimethyl sulfoxide (DMSO). A fully human IgG4 monoclonal agonistic anti–41BB antibody (α41BB mAb;BMS-663513, urelumab) was a kind gift from Bristol-Myers Squibb. α41BB was added with media containing 6000IU/mL IL-2 to tumor fragments at the initiation of TIL expansion. Thereafter, TILs were fed every 3–4 days with media containing 6000IU/mL IL-2 only. Cyropreserved TILs were thawed and rested in media containing 3000IU/mL IL-2 for 3–4 days before being subjected to further stimulation and co-culture conditions. For the preparation of tumor digests, the remaining tumor tissue was suspended in digestion media containing collagenase type II and type IV, hyaluronidase, and DNAse I (all from Fisher Scientific) and then subjected to GentleMACS dissociation (Miltenyi Biotec). Tumor digest cell suspensions were incubated at 37°C in a rocking water bath for 1hr and then filtered with 100μM cell strainers to remove large cellular debris.

Isolation and culture of human myeloid cells from peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMCs) were obtained from the whole blood of healthy donor volunteers or apheresis product from melanoma patients. For whole blood, PBMCs were prepared by Ficoll-Pacque (GE Healthcare) and then cryopreserved. Thawed PBMCs were labeled with CD11b microbeads human and mouse (Miltenyi Biotec, 130-49-601) for magnetic activated cell sorting. Purity of CD11b⁺ cells was >90%. For healthy donor myeloid cells, 2.5×10⁵ cells were cultured in 6 well plates coated with 10μg/mL α41BB or 10μg/mL 41BB-Fc (R&D Systems) in media alone or media containing 100ng/mL human GMCSF (Peprotech) for 3 days. For the generation of APCs, CD11b⁺ cells from the pheresis product of a melanoma patient were cultured with 10μg/mL immobilized 41BB-Fc for 3 days. Adherent cells were dissociated using Accutase (STEMCELL Technologies) and gentle pipetting. Cell lysate from an autologous melanoma cell line was prepared by suspending cells at 30×10⁶/mL in PBS and exposure to repeated and alternating temperatures (solid CO₂ ice and 37°C water). Five cycles of alternating temperature exposure at 5-minute intervals were performed. Three cell equivalents of tumor cell lysate were added to 1×10⁶ 41BB-Fc conditioned APCs in media containing 100ng/mL GMCSF and incubated overnight. Tumor-lysate pulsed APCs were then dissociated using Accutase and gentle pipetting before co-culture with TILs as described below. Cytokines in supernatants were measured by LEGENDplex HU Essential Immune Response Panel (BioLegend) and acquired via BD FACSCelesta.

Human TIL co-culture assays

Thawed autologous tumor cell suspensions were added to 96-well round-bottom plates at a 1:1 ratio with TILs and cultured for 24hrs. Anti-human HLA-A,B,C (W6/32, BioLegend) was added to tumor cells at a concentration of 10μg/mL and incubated for 1hr at 37°C before adding TILs to respective wells. Anti-human CD3 (OKT3, Ortho Biotech Inc., Bridgewater, NJ) was immobilized on the bottom of wells at 5μg/mL. Supernatants were collected after 24hrs of TIL-tumor digest co-cultures and IFN-gamma was measured in supernatants via IFN-gamma ELISA (BD Biosciences). For TIL co-cultures with autologous tumor cell lines or autologous tumor-lysate pulsed APCs, supernatants were collected after 72hrs of cultures. Prior to co-culture, autologous tumor cell lines were subjected to X-ray irradiation at a dose of 2×10⁴ rad. TILs were co-cultured at a 10:1 ratio with irradiated tumor cells or tumor-lysate pulsed APCs. Where indicated, soluble 10μg/mL α41BB or anti-41BBL (5F4, BioLegend) was added to TIL co-cultures. Cytokines in supernatants were measured by LEGENDplex HU Essential Immune Response Panel (BioLegend) and acquired via BD FACSCelesta. Proliferation was measured by ³H thymidine uptake (1μCi added per well) during the final 18hrs of co-culture.

Detection of cytokines from primary melanomas

Tumor digest cell suspensions were prepared as described above. One million cells were seeded in 48 well plates containing media with or without 6000IU/mL IL-2 in combination with soluble α41BB (10μg/mL). After 24hrs, cell-free supernatants were collected and stored at −80°C until ready for analysis. Cytokines in supernatants were measured by LEGENDplex HU Essential Immune Response Panel (BioLegend) and acquired via BD FACSCelesta.

Flow cytometry

Mouse spleens and tumors were harvested under sterile conditions. Spleens were homogenized by applying pressure to tissue on 100μm cell strainers. Single-cell suspensions were prepared, and red blood cells were removed using red blood cell lysis buffer (BioLegend). The resulting suspension was passed through a 70μm cell strainer and washed once with PBS. Mouse tumor cell suspensions were prepared by enzymatic digestion with media (Hank’s Balanced Salt Solution, Life Technologies) containing 1mg/mL collagenase IV, 0.1mg/mL DNAaseI, and 2.5 U/mL hyaluronidase (all from Sigma Aldrich) and then subjected to GentleMACS dissociation (Miltenyi Biotec). Tumor digest cell suspensions were incubated at 37°C in a rocking water bath for 1hr. Red blood cells were removed using red blood cell lysis buffer (BioLegend) and then cell suspensions were filtered with 100μM cell strainers to remove large cellular debris. Cells were resuspended in to a concentration of 0.5–1×10⁶ cells/mL for flow cytometric analysis in FACS Buffer containing PBS, 5% fetal bovine serum, 1mM Ethylenediaminetetraacetic acid (EDTA) (Sigma Aldrich), and 0.1% sodium azide (Sigma Aldrich). Cell viability was measured by staining cell suspensions with ZombieNIR (BioLegend). Prior to surface staining, cells were incubated with Fc Shield (TonboBiosciences) for murine specimens and Fc Blocker (Miltenyi Biotec) for human specimens. For surface staining of murine specimens, cells were stained in FACS buffer with the following antibodies: CD3 (145–2C11), CD4 (GK1.5), CD8 (53–6.7), CD11b (M1/70), Ly6G (1A8), Ly6C (HK1.4), F4/80 (BM8), CD11c (N418), MHCII (M5/114.15.2), CD80 (16–10A1), CD86 (GL-1), PD-1 (29F.1A12) (all from BioLegend); 41BB (17B5–1H1, Miltenyi Biotec). For intracellular cytokine detection, cells were incubated for 18hrs with 1X Brefeldin A (BioLegend), stained with cell surface antibodies, subjected to fixation and permeabilization via Fixation and Permeabilization Solution Kit (BD Biosciences), and then stained anti-mouse antibodies against IL-12p40/p70 (BD Biosciences), IL-6 (MP5–20F3), IL-10 (JES5–16E3), TNFα (MP6-XT22) (all from BioLegend). For human specimens, cell surface staining was conducted with the following antibodies: CD3 (145–2C11), CD4 (RPA-T4), CD8 CD11c (Bly6), CD14 (MoP9), CD15 (HI98), CD11b (ICRF44), HLA-DR/DP/DQ (Tu39), CD86 (all from BD Biosciences); PD-L1 (29E-2A3), 41BBL (5F4), 41BB (4B4–1), CD45 (2D1) (from BioLegend); CD141 (AD5–14H12) (Miltenyi Biotec). Cells were acquired by FACS Celesta (BD Biosciences), and the data were analyzed with FlowJo (Tree Star).

Mouse models

Female C57BL/6 mice (6–8 weeks old) were purchased from Charles River Laboratories (Wilmington, MA). OT-I mice (originally obtained from Jackson Laboratories) were bred and housed at the Animal Research Facility of the H. Lee Moffitt Cancer Center and Research Institute. Mice were humanely euthanized by CO₂ inhalation and secondary cervical dislocation according to the American Veterinary Medical Association Guidelines. Mice were observed daily and were humanely euthanized if a solitary subcutaneous tumor exceeded 400 cm² in area or mice showed signs referable to metastatic cancer.

Murine cell lines and in vivo treatment

B16 melanoma, Panc02 pancreatic cancer, MC38 colorectal cancer cell lines (all obtained from ATCC), were cultured in complete media (CM): RPMI media supplemented with 10% heat-inactivated FBS, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM fresh L-glutamine, 100 mg/ml streptomycin, 100 U/ml penicillin, 50 mg/ml gentamicin, 0.5 mg/ml fungizone (all from Life Technologies, Rockville, MD), and 0.05 mM 2-ME (Sigma-Aldrich, St. Louis, MO). B16 melanoma with pAc-neo-OVA plasmid (B16-OVA) was maintained in media with 0.8mg/mL G418 as previously described (15). To generate the ovalbumin (OVA) expressing fluorescent Panc02 cell line, cells were exposed to supernatants containing a lentiviral vector comprised of a fluorescent ZsGreen (ZsG) protein and OVA. Upon successful transfection, ZsGreen^hi tumor cells were subjected to FACS using BD FACSAria. OVA-ZsGreen^hi tumor cells were passaged in vitro 4 times whereby OVA expression was validated by staining for H2-K^b bound to SIINFEKL peptide (25-D1.16, BioLegend). The cell lines tested negative for mycoplasma contamination. All cell lines were passaged less than 10 times after initial revival from frozen stocks. All cell lines were validated in core facilities prior to use. Tumor cells (1×10⁵) were implanted subcutaneously in the flank of mice. When tumors reached ~25mm², 75μg of InVivoPlus anti-mouse 41BB (clone LOB12.3) or rat IgG1 isotype control, anti-horseradish peroxidase (both from BioXCell), were injected in 50μL volume intratumorally. Injections were repeated twice weekly until experimental endpoint. In some experiments, anti-mouse 41BB (clone LOB12.3) or rat IgG1 isotype control, anti-horseradish peroxidase were injected with 300μg of antibody twice weekly until experimental endpoint. For CD8 T cell depletion, 300μg of InVivoPlus anti-mouse CD8α (BioXCell) were injected intraperitoneally twice weekly for the duration of the experiment. CD8 T cell depletion was initiated prior to treatment with isotype or α41BB antibodies.

Tumor-myeloid cell co-culture with OT-I T cells

Myeloid cells were isolated from MC38 tumors after treatment with isotype or α41BB antibodies using EasySep Mouse CD11b Positive Selection Kit II (STEMCELL Technologies). CD8 T cells were isolated from the spleens of OT-I mice using EasySep Mouse CD8 T cell Isolation Kit (STEMCELL Technologies). OT-I T cells were labeled with CellTrace Violet (Invitrogen) prior to co-culture. OT-I T cells were co-cultured with myeloid cells in media containing 1μg/mL OVA_(257–264) peptide with or without neutralizing antibodies for IL-10 (JES5–2A5) or IL-12-p75 (R2–9A5) (both from BioXCell) at a concentration of 10μg/mL each. Cells and supernatants were harvested after 72hrs incubation. IFN-gamma in supernatants were measured by (Mouse IFN-gamma Quantikine ELISA Kit, R&D Systems).

Isolation of murine TILs

TILs from mice with MC38 tumors were isolated after treatment with isotype or α41BB antibodies using EasySep Mouse CD90.2 Positive Selection Kit II (STEMCELL Technologies). TILs were cultured in round-bottom 96 well plates with immobilized anti-CD3 antibodies (145–2C11 BD Biosciences) at a concentration of 5μg/mL or with irradiated tumor cell lines. MC38 or irrelevant target B16 tumor cells were exposed to X-ray irradiation at a dose of 2×10⁴ rad and cultured with CD90.2⁺ TILs at a 1:10 (target:TIL) ratio for 48hrs. Supernatants were collected and IFN-gamma was measured by (Mouse IFN-gamma Quantikine ELISA Kit, R&D Systems).

Statistical analysis

Graphs were generated using GraphPad Prism software. Graphs represent mean values with SEM. P values were calculated in each respective figure where statistical tests were indicated. For mouse-tumor growth studies, tumor growth curves are shown as mean with SEM and significance was determined by 2-way ANOVA and Sidak’s multiple comparison’s test. Mice were randomized after tumor cell implantation into respective treatment groups. Tumors were measured with Vernier calipers. Experimental groups were blinded to the operator throughout the duration of the experiment. For all other experiments, data were compared using either an unpaired 2-tailed Student’s t-test corrected for multiple comparisons by a Bonferroni adjustment or Welch’s correction. *=P<0.05; **=P<0.01; ***=P<0.001; ****=P<0.0001.

Study Approval

Studies were performed under approved Institutional Review Board (IRB) laboratory protocols at the H. Lee Moffitt Cancer Center (Tampa, FL). TIL, PBMC, and autologous tumors were collected from melanoma patients or PBMC from lung tumor patients as part of TIL ACT clinical trials. All samples were de-identified prior to use in research studies. All patients signed approved consent forms. All animal experiments were approved by the University of South Florida Institutional Animal Care and Use Committee and performed in accordance with the U.S. Public Health Service policy and National Research Council guidelines.

Results

Intratumoral 41BB activation leads to tumor regression in multiple models

To determine the efficacy of 41BB agonists, we validated that systemic treatment via intraperitoneal (i.p.) administration led to tumor growth delay and complete regressions in mice with established MC38 tumors (Fig. 1A, C). We observed that intratumoral (i.t.) administration with anti-41BB (α41BB) antibodies exhibited comparable efficacy to the i.p. route of administration by which the tumor growth kinetics and the frequency of complete regressions were similar (Fig. 1B, D). We validated these results in two additional tumor models bearing the model ovalbumin antigen, Panc02-ZsGOVA (Fig. 1E, G) and B16-OVA (Fig. 1F, H). Intratumoral treatment of α41BB led to significant growth delay and tumor regression in mice with Panc02-ZsGOVA tumors (Fig. 1E) and B16-OVA tumors (Fig. 1F) compared to control mice that received i.t. isotype antibodies. Moreover, the survival of mice treated with i.t. α41BB was significantly enhanced in both models (Fig. 1G–H). In mice with MC38 tumors, i.t. α41BB led to complete regressions in approximately 30% of treated mice (Fig. 1D). Likewise, 70% of mice with Panc02-ZsGOVA tumors (Fig. 1G) or B16-OVA tumors (Fig. 1H) had no evidence of tumor growth after tumor inoculation (44 days and 70 days, respectively). This suggested that presence of a highly immunogenic antigen, such as OVA, could potently direct local anti-tumor immune responses after i.t. treatment with α41BB. Together, these data demonstrate that the intratumoral treatment with 41BB agonistic antibodies is a feasible approach to induce tumor regression in mice.

FIGURE 1.

Intratumoral treatment with agonistic 41BB antibodies promotes tumor regression in multiple mouse tumor models. A and C, Mice with MC38 tumors were treated with α41BB via intraperitoneal administration. A, Tumor growth summary. C, Tumor growth in individual mice. B and D, Mice with MC38 tumors were treated with α41BB antibodies via intratumoral administration. B, Tumor growth summary. D, Tumor growth in individual mice. E, Panc02-ZsGOVA tumor growth in mice that received intratumoral treatment with isotype or α41BB antibodies. F, B16-OVA tumor growth in mice that received intratumoral treatment with isotype or α41BB antibodies. G and H, Survival curves of mice from E and F. One of two representative experiments are shown.

Intratumoral treatment with a 41BB agonist increases CD8 T cell infiltration

We determined that within one week after the initial treatment with i.t. α41BB, the size of tumors was significantly reduced in mice with MC38 tumors (Fig. 2A) and Panc02-ZsGOVA tumors (Fig. 2B). The reduction in tumor size in response to i.t. α41BB treatment was associated with an increase of CD8 T cell infiltration in both tumor models compared to mice treated with isotype antibodies (Fig. 2C–D). However, the frequency of CD4 TILs was unchanged between mice treated with isotype or α41BB (Fig. 2C–D). We next determined that the increase of CD8 T cells in MC38 tumors was required for the anti-tumor efficacy because the depletion of CD8 T cells prior to the start of i.t. α41BB treatment abrogated the reduction of tumor growth (Fig. 2E). In contrast, the depletion of CD8 T cells had no effect in mice that received i.t. treatment with isotype antibodies indicating that basal anti-tumor CD8 T cell responses are ineffective against MC38 tumors (Fig. 2E). Not only was the presence of CD8 T cells necessary for the reduction of tumor growth, we found that TILs isolated from α41BB treated tumors exhibited higher IFN-γ production in response to CD3 stimulation or co-culture with irradiated MC38 tumor cells (Fig. 2F). Conversely, TILs from isotype treated tumors were successfully stimulated with CD3 antibodies but failed to produce IFN-γ in cultures with MC38 tumor cells (Fig. 2F). These results demonstrate that i.t. 41BB agonism can rejuvenate CD8 T cell responses leading to an improvement of anti-tumor immune responses.

FIGURE 2.

Intratumoral treatment with agonistic 41BB antibodies increases CD8 T cell infiltration. A and B, Mass of MC38 and Panc02-ZsGOVA tumors harvested 7 days after initial α41BB treatment. C and D, Frequency of CD4 and CD8 TILs in MC38 and Panc02-ZsGOVA tumors 7 days after initial α41BB treatment. E, Mice with MC38 tumors were treated with isotype or α41BB antibodies in combination with CD8 depleting antibodies. Tumor growth is shown (n=5 mice per group). F, TILs were isolated from MC38 tumors treated with intratumoral isotype or α41BB antibodies. TILs were cultured with immobilized anti-CD3 antibody or co-cultured with irradiated MC38 or B16 tumor lines for 48hrs. IFN-γ was measured from supernatants. One of two representative experiments are shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Intratumoral 41BB activation remodels the tumor immune microenvironment

We demonstrated that the triggering of 41BB co-stimulation via i.t. treatment with α41BB converted tumors to a more T cell inflamed environment (Fig. 2). Indeed, the ratio of CD11b⁺ myeloid cells relative to CD8⁺ T cells was significantly reduced in tumors that received α41BB treatment compared to mice that received control isotype antibodies (Fig. 3A). In contrast to control tumors whereby the majority of CD45⁺ leukocytes were CD11b⁺MHCII⁺F480⁻CD11c⁻ myeloid cells and CD11b⁺F480⁺Ly6C⁻ tumor-associated macrophages (TAMs), we found that MC38 tumors treated with i.t. α41BB had a dramatic reduction in these myeloid cell populations (Fig. 3B). Likewise, α41BB treatment decreased the frequency of CD11b⁺MHCII⁺F480⁻CD11c⁺ cells (DCs). In isotype-treated tumors, CD11b⁺F480⁻Ly6C⁺Ly6G⁻ monocytes and CD11b⁺F480⁻Ly6C⁺Ly6G⁺ polymorphonuclear cells (PMNs), presumably monocytic- and PMN- myeloid derived suppressor cells (MDSCs), comprised <10% of CD45⁺ cells. Conversely, the reduction of TAMs and other myeloid cell populations coincided with significant increases of monocytes and PMNs in tumors that received treatment with α41BB (Fig. 3B). Nevertheless, the changes in myeloid cell frequency in response to α41BB treatment were associated with an increased abundance of CD80⁺CD86⁺ DCs, TAMs, and monocytes (Fig. 3C). Furthermore, DCs, TAMs, monocytes, and PMNs significantly upregulated CD80 and/or CD86 in tumors treated with α41BB compared to isotype controls (Fig. 3D–G). Despite the loss of classical APCs within the tumor microenvironment, the increase in CD80 and CD86 among all tumor-associated myeloid cells may support anti-tumor T cell responses after intratumoral administration of 41BB agonists.

FIGURE 3.

Remodeling of the immune microenvironment after intratumoral administration of α41BB antibodies. B, Percentage of myeloid cell subsets in MC38 tumors. Tumors were harvested 7 days after initial antibody treatment. (n=5 mice/group). C, Percentage of CD80 and CD86 double positive myeloid subsets is increased after α41BB treatment. D and F, Representative histograms for CD80 (F) and CD86 (G) gated on indicated myeloid cell subset. Gray=FMO, Black=Isotype, Red= α41BB. E and G, Fold change in CD80 (H) and CD86 (I) expression in myeloid cell subsets from MC38 tumors. (n= 9–10 mice/group). E, G, Data is a summation of two independent experiments. A-C, One of two independent experiments are shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Intratumoral α41BB alters the immune stimulatory capacity of myeloid cells

We further evaluated changes to intratumoral myeloid cells by examining the production of cytokines after i.t. α41BB. We harvested tumors from isotype and α41BB treated mice 7 days after treatment initiation and cultured the cells overnight. We found that myeloid cells from tumors produced IL-6 and TNF-alpha, but no difference was observed between cells from α41BB or isotype tumors (Fig. 4A, D). In contrast, DCs and TAMs had an elevated expression of IL-10, while CD11b⁺Gr-1⁺ cells exhibited a reduced production of IL-10 (Fig. 4B). Similarly, DCs and TAMs exhibited an increased production of IL-12 (Fig. 4C). While we observed changes in cytokine expression among myeloid cell subsets, the frequency of DCs and TAMs were significantly reduced by α41BB treatment (Fig. 3B). Consistent with these data, the number of cytokine-producing DCs and TAMs were largely reduced in cultured tumor cell digests of α41BB treated tumors in comparison to isotype treated tumors (Fig. 4E–H). Concordantly, the number of cytokine-producing monocytes and PMNs (CD11b⁺Gr-1⁺ cells) were significantly elevated in α41BB treated tumors (Fig. 4E–H). Because IL-10 and IL-12 are key regulators in T cell priming and activation by myeloid cells, we next evaluated the capacity of intratumoral myeloid cells to stimulate T cells after treatment. We found that OT-I T cells produced more IFN-γ in co-cultures with myeloid cells from α41BB treated tumors both with and without in vitro IL-10 neutralization (Fig. 4J). Upon examination of the phenotype of OT-I T cells after co-culture, we observed that T cells co-cultured with myeloid cells from α41BB-treated tumors had an elevated expression of cell surface 41BB compared to T cells cultured with myeloid cells from isotype-treated tumors (Fig. 4K–M). Moreover, the in vitro neutralization of IL-10, but not IL-12, enhanced the expression of 41BB in OT-I T cells from co-cultures with tumor myeloid cells, suggesting that myeloid cell derived IL-10 restricted the expression of 41BB (Fig. 4K–M). Similar to the increase of 41BB expression, IL-10 neutralization resulted in an increased expression of PD-1 in co-cultures with myeloid cells from isotype tumors (Supplemental Fig. S1). Thus, i.t. α41BB treatment enhances the ability of tumor-myeloid cells to potentiate T cell responses.

FIGURE 4.

Intratumoral anti-41BB treatment alters myeloid immunostimulatory capacity. A-D, Intracellular cytokine staining from myeloid cells from MC38 tumors treated with isotype or α41BB antibodies. (A) IL-6, (B) IL-10, (C) IL-12, (D) TNF-α. (n=7–10 mice/group). E-H, The number of cytokine producing cells were determined by back-calculating the percentage of myeloid cell subsets relative to the total number of cells from each mouse tumor. I, Experimental design for (J-M). J-M, CD8⁺ OT-I T cells after 72hrs with peptide stimulation with or without culture with myeloid cells from isotype or anti-41BB treated tumors. J, IFN-γ was measured in supernatants from OT-I : myeloid cell co-cultures incubated with or without IL-10 neutralizing antibodies. K and L, 41BB expression in OT-I T cells after 72hrs of co-culture with or without IL-10 or IL-12 neutralizing antibodies. Percentage of 41BB positive OT-I T cells (K) and expression level (L). M, Representative histogram of 41BB expression. (I-T) Each data point represents of pool of CD11b⁺ cells collected from 3–4 individual mouse tumors. (n=13–16 individual mice/group). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

41BB agonistic antibodies promote ex vivo human TIL expansion

We have previously shown that the addition of 41BB agonistic antibodies enhances the expansion and function of melanoma TILs from primary tumor fragments in vitro (16). We obtained two melanoma specimens and attempted to expand TILs from tumor fragments placed in media containing IL-2 or IL-2 in combination with α41BB. In Patient 1, TILs expanded in cultures with IL-2+α41BB (5/6 fragments), while no expansion occurred in cultures with IL-2 only (0/6 fragments) (Fig. 5A). Similarly, an enhancement of TIL expansion was observed in Patient 2 among fragments grown with IL-2 and α41BB (23/24 fragments) compared to TILs grown in IL-2 alone (12/24 fragments). Moreover, the number of TIL expanded per fragment was greater in cultures containing IL-2 and α41BB compared to IL-2 alone conditions (Fig. 5B). Among fragments grown in IL-2 only, the distribution of CD4⁺ TILs and CD8⁺ TILs were approximately equal. In contrast, the combination of IL-2+α41BB almost exclusively promoted the expansion of CD8⁺ TILs (Fig. 5C). Because the yield of TILs from IL-2 alone cultures was relatively low, we pooled together TILs from fragments that exhibited the best expansion at the end of the culture. Similarly, we chose TILs expanded from 6 individual fragments grown in IL-2+α41BB that yielded the highest number of cells. We then co-cultured the selected TILs with autologous tumor digest and determined the magnitude of IFN-γ production. IFN-γ was detected and effectively blocked with MHC-I blocking antibodies in TILs grown in IL-2 alone and in 3/6 selected TILs grown in IL-2 and α41BB (Fig. 5D). Notably, the abundance of IFN-γ was higher in co-cultures with TILs expanded with IL-2 and α41BB compared to TILs grown in IL-2 only (Fig. 5D).

FIGURE 5.

41BB agonism enhances the growth of TILs from primary human melanomas. A and B, Melanoma tumor fragments were grown in media containing IL-2 or IL-2 in combination with α41BB. Patient 1 (A) and Patient 2 (B). The number of fragments to successfully grow TILs are indicated above each plot. C, Frequency of CD4 and CD8 TILs expanded from tumor fragments from Patient 2. D, TILs from Patient 2 were co-cultured with autologous tumor digests with or with MHC-I blocking antibodies for 24hrs. IFN-γ was measured in supernatants. E, Tumor digest from Patient 1 was analyzed for immune infiltrates. Outer ring represents the frequency of CD45+ cells among the total live cells. Inner pie chart represents the proportions of immune cell subsets. Cell subset frequency is indicated on pie chart and adjacent to the indicated cell subset on the right. F-G, Fresh tumor digests from Patient 1 (F) and Patient 2 (G) were cultured at 1×10⁶ cells/mL in indicated media overnight. Comparison of cytokines for IL-2 vs IL-2+α41BB are shown. *p<0.05, **p<0.01, ***p<0.001. Significance was determined by two-tailed t-test (D) or 2-way ANOVA with Dunnett’s multiple comparisons (F-I).

To better understand the contribution of myeloid cells in the process of ex vivo TIL expansion, we evaluated the frequency of leukocyte populations in a fresh tumor sample from melanoma Patient 1. We found that 17.1% of all live cells were CD45⁺, which 73% of CD45⁺ cells consisted of CD11b⁺CD11c⁺CD14⁺HLA-DR⁺ myeloid cells. Approximately 15% of CD45⁺ cells were CD4⁺ and CD8⁺ T cells and the remainder consisted of a variety of myeloid cell subsets (Fig. 5E). We next evaluated the production of cytokines in fresh tumor digests from Patient 1 and Patient 2. Fresh tumor cell digests were cultured overnight in IL-2 alone or in combination with α41BB. Tumors produced vast amounts of CCL2, IL-6, IL-8, IL-1β, IL-10, and TGF-β. In response to IL-2 and IL-2 in combination with α41BB, we detected an increased production of CXCL10 and IFN-γ in comparison to unstimulated tumor digests (Fig. 5F–G). Moreover, a trend consistent with an increase of CXCL10 and IFN-γ were observed when tumors were cultured with IL-2 and α41BB compared to IL-2 alone (Fig. 5H). Collectively, these data demonstrate that the augmentation of ex vivo TIL expansion via IL-2 and 41BB stimulation is associated with increases in proinflammatory cytokine production.

Stimulation of the 41BB-41BBL axis alters myeloid cell phenotype and function

Next, we dissected how myeloid cells facilitate ex vivo TIL expansion via 41BB stimulation. First, we observed that CD11b⁺ myeloid cells within a fresh melanoma tumor lacked the expression of 41BB (Fig. 6A). Next, we phenotyped peripheral blood myeloid cells and found that CD11b⁺ cells in PBMCs expressed HLA-DR/DP/DQ, CD14, CD86, CD11c, and low levels of PD-L1 and CD141 (Fig. 6B). Moreover, these cells lacked expression of 41BB, but did express 41BBL (Fig. 6B–C). Since the activation of 41BBL in monocytes is known to promote the maturation to DCs (17, 18), and 41BB expression was poorly expressed by myeloid cells, we examined how the stimulation of 41BBL could differ in activating myeloid cells in comparison to a 41BB agonistic antibody. We stimulated myeloid cells with immobilized α41BB to agonize 41BB or immobilized 41BB protein (41BB-Fc) to agonize 41BBL. The viability of donor myeloid cells was greatly reduced in unstimulated cultures or under stimulation with α41BB alone. In contrast, stimulation with 41BB-Fc alone maintained cell viability similar to that of cultures containing GMCSF (positive control) or GMCSF in combination with α41BB or 41BB-Fc (Fig. 6D). This suggested that activation of 41BBL, but not 41BB, was sufficient to maintain the survival of myeloid cells and that 41BB(L) signals did not augment cell viability in the presence of GMCSF. Compared to pre-cultured cells, myeloid cells upregulated 41BBL expression, but not 41BB expression, when incubated with a GMCSF maturation stimuli (Fig. 6E). In addition to maintaining myeloid cell viability, the stimulation with 41BB-Fc reduced the expression of CD14, while enhancing the expression of PD-L1, CD141, 41BBL, and CD86 compared to pre-culture myeloid cells (Fig. 6F). Likewise, GMCSF-stimulated cells exhibited similar phenotypic changes to 41BB-Fc treated cells, however, GMCSF failed to upregulate CD86 (Fig. 6G). The increase in CD86 expression was highly consistent between all donors (Fig. 6H). In contrast to 41BB-Fc or GMCSF stimulation, α41BB alone failed to maintain myeloid viability (Fig. 6D) and the phenotype was similar to myeloid cells cultured in media alone (Fig. 6F). Likewise, the addition of α41BB or 41BB-Fc failed to augment cell surface marker expression when combined with GMCSF (Supplemental Fig. S2). While increases in CD86 and 41BBL expression are indicative of an enhanced co-stimulatory capacity, we further interrogated the impact of 41BB-41BBL activation in myeloid cells by assessing cytokine production. We found that IL-4 and CCL2 expression was potently induced by GMCSF and to a lesser extent by α41BB and 41BB-Fc (Fig. 6I–J). Similarly, IL-1β, IL-6, and IL-8 production was amplified by stimulation with GMCSF or 41BB-Fc, but not α41BB (Fig. 6K–M). CXCL10 expression was reduced by 41BB-Fc stimulation but maintained with GMCSF or α41BB alone (Fig. 6N). In addition, myeloid cells readily produced IL-2 and TGF-β, but the culture conditions maintained or modestly increased the production of these cytokines (Fig. 6O–P). In large part, 41BB activation alone via α41BB or its addition to GMCSF stimulus had little effect on cell viability, the expression of cell surface markers, or induction of cytokine expression (Fig. 6D–P, Supplemental Fig. S2). Hence, it is possible that 41BB agonism on myeloid cells provides a weaker stimulus in comparison to reverse signaling through 41BBL.

FIGURE 6.

Activation of 41BB and 41BBL alter myeloid cell phenotype and function. A, 41BB expression in myeloid cells from a fresh melanoma sample. B, Phenotype of CD11b⁺ cells from healthy donor PBMCs. C, Representative dot plots for 41BBL and 41BB expression on CD11b⁺ cells from PBMCs (left) or tumor myeloid cells (right). D, Viability of sorted healthy donor myeloid cells were determined at pre-culture and after 3 days culture with immobilize urelumab, immobilized 41BB-Fc with or without GMCSF. E, Representative histogram for 41BB and 41BBL expression on healthy donor myeloid cells. Gray=fluorescence minus one (FMO), Black=pre-culture, Red=3 days culture with GMCSF. F, Histograms showing the expression of cell surface markers before and after culture with indicated conditions for one representative donor. Each condition is indicated on the far left. G, Heatmap representing the fold change in MFI for respective cell surface markers comparing cell culture conditions to media alone control for 3 individual donor cells. H, Fold change of CD86 expression normalized to media control. I-P, Supernatants from donor myeloid cell cultures were collected after 3 days incubation with indicated conditions. Each line represents myeloid cells from an individual donor.

The stimulation of TILs is potentiated by 41BBL on APCs

The agonistic 41BB antibody urelumab does not compete with the binding of 41BBL to 41BB, thereby preserving native 41BBL-mediated co-stimulation (19). Consistent with our data in Figure 6, the maturation of monocytes via 41BBL reverse signaling leads to an increased potential to prime T cells characterized by increased CD86 expression and enhanced production of IL-6 and IL-8 (11). Hence, we hypothesized that the enhancement of ex vivo TIL expansion by 41BB agonists may be aided by additional co-stimulation mediated by myeloid 41BBL. To determine this, we generated APCs from CD11b⁺ cells isolated from the pheresis product of a melanoma patient. The myeloid cells were incubated with immobilized 41BB-Fc for 3 days, collected, and then pulsed with autologous tumor lysate for 24hrs in the presence of GMCSF to generate 41BBL-conditioned APCs (41BBL APCs). First, we examined if α41BB could augment the production of cytokines in TIL co-cultures with autologous tumor or pulsed APCs. A variety of cytokines were detected in cultures containing TILs stimulated with αCD3 or co-cultures of TILs with autologous tumor cells. In particular, the combined stimulation of TILs with αCD3 and α41BB increased the production CXCL10 and IFN-γ (Fig. 7A). As expected, IFN-γ and TNFα were induced in TIL co-cultures with tumor cells, which were effectively reduced by MHC-I blockade (Fig. 7B). Next, we determined the cytokine profile in TIL co-cultures with autologous APCs. When cultured alone, the 41BBL APCs produced high amounts of CCL2 and IL-8, and modest amounts of IL-2, TGF-β, and IL-1β, which was not impacted by additional stimulation with soluble α41BB (Fig. 7C). In comparison to unstimulated TILs and cultures with 41BBL APCs only, CXCL10, IL-2, IFN-γ, IL-1β, TGF-β, and IL-6 were elevated when TILs were cultured with APCs, which the production of some cytokines was augmented by the addition of agonistic α41BB and/or α41BBL blocking antibodies (α41BBL) (Fig. 7D). CXCL10 was only detected TIL-APC co-cultures, which suggested that cell-cell contact facilitated the production of CXCL10. Accordingly, we found that the production of CXCL10 and IL-2 was only augmented by 41BBL blockade, which was enhanced when 41BBL blocking was combined with α41BB (Fig. 7E). Similarly, we observed that CXCL10 was elevated in Panc02-ZsGOVA tumors taken from mice treated with i.t. α41BB (Supplemental Fig. S3). In human TIL-APC co-cultures, the addition of α41BB alone, 41BBL blockade alone, and/or the combination increased IFN-γ, IL-1β, and TGF-β. Likewise, IL-6 was absent in all conditions except in TIL-APC co-cultures with the addition of α41BB in combination with 41BBL blockade (Fig. 7E). Next, we demonstrated that TILs from this patient readily proliferated in co-cultures with αCD3 or irradiated autologous tumor cells compared to basal proliferation. The combination of α41BB with αCD3 enhanced TIL proliferation but was reduced when 41BBL antibodies were present in culture. However, the addition of α41BB and/or α41BBL did not alter TIL proliferation in cultures with tumor cells. In parallel, we co-cultured TILs with autologous 41BBL APCs pulsed with tumor lysate in combination with soluble α41BBL and/or α41BB. 41BBL APCs induced the proliferation of TILs, which was negatively impacted by 41BBL blockade alone or in combination with α41BB, indicating that the blockade of 41BBL dampened TIL proliferation and that additional co-stimulation with α41BB was not sufficient to reverse this effect. Together, these results demonstrate that myeloid 41BBL can contribute to the effect of 41BB agonists characterized by enhanced TIL proliferation and production of cytokines.

FIGURE 7.

41BBL expression on APCs alters the capacity to prime TILs. TILs from a melanoma patient were cultured with αCD3, autologous tumor cells at a 1:1 ratio, or tumor-lysate pulsed 41BBL APCs at a 1:10 ratio for 72hrs. A and B, Heatmap representing cytokine abundance in supernatants from cell cultures were collected at 72hrs. Numerical values are indicated for each parameter with its respective condition. Cytokine production by TIL stimulated with αCD3 +/− urelumab (A). Cytokines in TIL-tumor co-cultures +/− α41BB or MHC-I blocking antibodies (B). C, 41BBL APCs were generated and then pulsed with autologous tumor lysate in the presence of GMCSF. Pulsed APCs were then seeded in culture wells with or without α41BB. D, Cytokines were measured in the supernatants of TIL-APC co-cultures incubated with α41BB and/or α 41BBL. TIL only condition is the same data from (B); APCs only from (C). Statistics are indicated for cytokines that are higher than both TILs alone and APCs alone conditions. E, Fold change in cytokine induction vs. TIL+APCs in co-cultures from (D). Dotted line represents the basal induction of cytokines in TIL-APC co-cultures. F, TIL proliferation in co-cultures was determined in the final 18hrs of the culture by ³H thymidine incorporation in the presence of soluble α41BB and/or soluble α41BBL. Dotted line represents basal TIL proliferation without additional stimulation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Significance was determined by two-tailed t-test or 2-way ANOVA with Dunnett’s multiple comparisons. ND = not detected.

Discussion

Overall, 41BB agonists are potent immune stimulators, but their translational potential has been heavily restricted by the onset of severe adverse events (20). Significant advancement in the development of tumor-selective 41BB agonists to limit or even eliminate any 41BB-related adverse events has revitalized the therapeutic feasibility of targeting 41BB in humans (4, 5). While we did not evaluate toxicity in mice that received intratumoral injections of α41BB, we did not observe any overt toxicity during the administered treatment regimen. Overall, our data support that stimulating 41BB in tumors is a feasible approach to promote anti-tumor immune responses (Fig. 1, Fig. 2). Consistent with other reports, the activity of 41BB agonists in our hands was not independent of changes to the myeloid-tumor immune milieu (21, 22). The anti-tumor activity of 41BB agonists is greatly reduced in the absence of BATF3-dependent DCs, suggesting that 41BB agonists may act directly on the myeloid compartment to promote the eradication of tumors in mice (21). Indeed, we observed that treatment with 41BB agonists in mice led to increases in monocytes and PMNs coincided by a depletion of macrophages and DCs (Fig. 3A–B). Furthermore, myeloid cells from α41BB-treated tumors induced the upregulation of 41BB on CD8⁺ T cells and enhanced the production of IFN-γ (Fig. 4J–N). Hence, it is possible that myeloid cells contribute to the efficacy of 41BB activation in vivo by inducing the upregulation of surface 41BB on T cells. We show that IL-10 was essential for restricting both the production of IFN-γ and the expression 41BB on T cells (Fig. 4J, L). While DCs and TAMs increased their production of IL-10 and IL-12 in response to 41BB agonism (Fig. 4B–C), the i.t. administration of α41BB simultaneously promoted the accumulation of monocytes and PMNs that produced IL-6, IL-12, IL-10, and TNF-α (Fig. 4E–H). Thus, both the proportionality and function of distinct intratumoral myeloid cells are likely relevant factors in driving anti-tumor immune responses elicited by 41BB agonists.

It has been described that species differences exist between mouse and human myeloid cells in response to 41BBL signaling (23). While, we did not evaluate the role of 41BBL in murine models, the data we present in human cell culture systems provide relevance for the role of 41BBL in the ability of myeloid cells to potentiate T cell responses. Importantly, the upregulation of co-stimulatory markers, such as CD80 and/or CD86, in both mouse and human myeloid cells were consistent after exposure to 41BB or 41BBL stimulation. This suggests that myeloid-mediated co-stimulation could be enhanced in the context of 41BB agonism, leading to the potentiation of TIL expansion and function (Fig. 3C–G, Fig. 6G–H) (16). In this study, we found that 41BB agonism, contrary to 41BBL stimulation via 41BB-Fc, had little effect in augmenting human myeloid cell phenotypes and cytokine production (Fig. 6). This, perhaps, was not surprising because CD11b⁺ cells expressed little to no 41BB on their cell surface (Fig. 6A–C). Concordantly, the stimulation of myeloid cells with a 41BB agonist alone failed to sustain myeloid cell viability, increase CD86 expression, or provide other maturation stimuli even when combined with GMCSF (Fig. 6D–H, Supplemental Fig. S2). Moreover, the addition of α41BB to tumor-lysate pulsed APCs did not significantly alter basal cytokine production (Fig. 7C). Hence, the 41BB agonist used in these experiments appears to be a weak stimulator of myeloid cells at best. In mice, the engagement of 41BBL on myeloid cells by its cognate receptor, 41BB, restricts the accumulation of IL-12⁺ cDCs and TAMs within tumors, leading to a diminished ability to control tumor growth (22). In a contradictory manner, 41BB knockout mice exhibit remarkably similar anti-tumor immune responses to mice treated with agonistic 41BB antibodies, which supports the hypothesis that the lack of interaction between 41BB with 41BBL on myeloid cells promotes anti-tumor immune responses. However, the evidence we provide in this study demonstrates that reverse 41BBL signaling can promote the immunostimulatory capacity of monocytes and APCs in humans. Consistent with previous reports in human cells (18, 24), we show that the induction of reverse 41BBL signaling in human monocytes via 41BB-Fc promoted the expression of co-stimulatory markers CD86 and 41BBL, while simultaneously increasing the production of IL-8, IL-6, IL-1β, CCL2, and IL-4 (Fig. 6I–P). Thus, differences among species and experimental murine tumor models likely contribute to the contrasting findings in reports investigating the role 41BB-41BBL co-stimulatory axis in anti-tumor immunity. Together, these results demonstrate that the 41BB agonists may not act directly on human myeloid cells alone to promote the co-stimulatory capacity of APCs. Rather the activation of 41BBL, and potential bidirectional signaling between myeloid cells and T cells, were responsible for providing efficient maturation stimuli to enhance the capacity of APCs to prime T cells.

In contrast to other 41BB agonistic antibodies, urelumab facilitates the cross-linking of 41BBL to 41BB, suggesting that bidirectional signaling orchestrated by 41BBL⁺ cells could augment the agonistic activity of α41BB (19, 25). We provide evidence here that the activation of 41BBL can contribute to the expansion of TILs stimulated with 41BB agonists because the proliferation of TILs cultured with 41BBL-conditioned APCs was reduced when 41BBL was blocked, even in the presence of urelumab (α41BB) (Fig. 7F). Intriguingly, CXCL10 was elevated in TIL-APC co-cultures when 41BBL was blocked. Consistent with this data, the production of CXCL10 was reduced in donor myeloid cells conditioned with 41BB-Fc (Fig. 6N), suggesting that the stimulation of 41BBL on myeloid cells represses CXCL10 expression which may have been relieved when blocking antibodies were present in TIL-APC co-cultures. Moreover, we acknowledge that IFN-γ is a known inducer of CXCL10, a maturation factor for DCs (26), and can enhance the ability of 41BBL-APCs to prime cytotoxic T cell responses (27). Hence, it is possible that the induction of CXCL10 could have been indirectly promoted in the presence of α41BB through an increased abundance of IFN-γ. While, the blockade of 41BBL can prevent the induction of T cell proliferation through interaction with its cognate receptor, we cannot rule out the possibility that the production of cytokines, such as IL-6, IL-1β, and TGF-β by APCs may also have an impact on TIL proliferation (Fig. 7D–E). However, the cellular origin and the specific activity of these cytokines on TIL proliferation and function remains unclear. Hence, future studies need to determine the role of 41BB-41BBL induced cytokines, including CXCL10, IL-6, IL-1β, and TGF-β and their impact on both, the expansion of TILs and the activity of 41BB agonists.

In our previous report, the addition of α41BB was associated with enhanced TIL expansion and the modulation of tumor-resident DC phenotypes characterized by the upregulation of CD80, CD86, and MHCII (16). We conclusively demonstrated that human myeloid cells upregulated co-stimulatory markers CD86 and 41BBL and proinflammatory cytokines in response to 41BBL stimulation, but not in response to 41BB agonists. Moreover, tumor-lysate pulsed APCs that were matured via reverse 41BBL signaling effectively primed TILs. Together, our findings provide feasibility that 41BB-41BBL bidirectional signaling between immune cells can be exploited to enhance to the expansion and function of TILs.

Key Points.

• Intratumoral 41BB agonism induces tumor regression in mouse models.

• 41BB agonism potentiates myeloid-mediated co-stimulation in tumors.

• Activation of 41BBL, but not 41BB, in human APCs promotes co-stimulatory responses.