PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1+CD38hi cells and anti-PD-1 resistance.

Understanding resistance to antibody to programmed cell death protein 1 (PD-1; anti-PD-1) is crucial for the development of reversal strategies. In anti-PD-1-resistant models, simultaneous anti-PD-1 and vaccine therapy reversed resistance, while PD-1 blockade before antigen priming abolished therapeutic outcomes. This was due to induction of dysfunctional PD-1+CD38hi CD8+ cells by PD-1 blockade in suboptimally primed CD8 cell conditions induced by tumors. This results in erroneous T cell receptor signaling and unresponsiveness to antigenic restimulation. On the other hand, PD-1 blockade of optimally primed CD8 cells prevented the induction of dysfunctional CD8 cells, reversing resistance. Depleting PD-1+CD38hi CD8+ cells enhanced therapeutic outcomes. Furthermore, non-responding patients showed more PD-1+CD38+CD8+ cells in tumor and blood than responders. In conclusion, the status of CD8+ T cell priming is a major contributor to anti-PD-1 therapeutic resistance. PD-1 blockade in unprimed or suboptimally primed CD8 cells induces resistance through the induction of PD-1+CD38hi CD8+ cells that is reversed by optimal priming. PD-1+CD38hi CD8+ cells serve as a predictive and therapeutic biomarker for anti-PD-1 treatment. Sequencing of anti-PD-1 and vaccine is crucial for successful therapy.

Author Correction: PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1+CD38hi cells and anti-PD-1 resistance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Androgen receptor activity in T cells limits checkpoint blockade efficacy.

Immune checkpoint blockade has revolutionized the field of oncology, inducing durable anti-tumour immunity in solid tumours. In patients with advanced prostate cancer, immunotherapy treatments have largely failed1-5. Androgen deprivation therapy is classically administered in these patients to inhibit tumour cell growth, and we postulated that this therapy also affects tumour-associated T cells. Here we demonstrate that androgen receptor (AR) blockade sensitizes tumour-bearing hosts to effective checkpoint blockade by directly enhancing CD8 T cell function. Inhibition of AR activity in CD8 T cells prevented T cell exhaustion and improved responsiveness to PD-1 targeted therapy via increased IFNγ expression. AR bound directly to Ifng and eviction of AR with a small molecule significantly increased cytokine production in CD8 T cells. Together, our findings establish that T cell intrinsic AR activity represses IFNγ expression and represents a novel mechanism of immunotherapy resistance.

Restored Thymic Output after Androgen Blockade Participates in Antitumor Immunity.

The thymus is a hormone-sensitive organ, which involutes with age in response to production of sex steroids. Thymic involution leads to a decrease in the generation of recent thymic emigrants (RTEs), resulting in a reduced response to immune challenges such as cancer. Interestingly, the standard of care for prostate cancer patients is androgen deprivation therapy (ADT), which leads to thymic regeneration and an increase in thymic output. It remains unknown whether these newly produced T cells can contribute to the antitumor immune response. This study defines the kinetics of thymic regeneration in response to ADT in mice, determining that thymic epithelial cell proliferation is critical for the increase in RTE output. Using a mouse model to track RTE in vivo, we demonstrate that these newly generated RTEs can traffic to tumors, where they become activated and produce effector cytokines at levels similar to more mature T cells. Collectively, these data suggest that RTEs produced from ADT-induced thymic regeneration could be harnessed for the antitumor immune response.

Oncolytic VSV Primes Differential Responses to Immuno-oncology Therapy.

Vesicular stomatitis virus encoding the IFNß transgene (VSV-IFNß) is a mediator of potent oncolytic activity and is undergoing clinical evaluation for the treatment of solid tumors. Emerging preclinical and clinical data suggest treatment of tumors with oncolytic viruses may sensitize tumors to checkpoint inhibitors and increase the anti-tumor immune response. New generations of immuno-oncology molecules including T cell agonists are entering clinical development and could be hypothesized to enhance the activity of oncolytic viruses, including VSV-IFNß. Here, we show that VSV-IFNß exhibits multiple mechanisms of action, including direct cell killing, stimulation of an innate immune response, recruitment of CD8 T cells, and depletion of T regulatory cells. Moreover, VSV-IFNß promotes the establishment of a CD8 T cell response to endogenous tumor antigens. Our data demonstrate a significant enhancement of anti-tumor function for VSV-IFNß when combined with checkpoint inhibitors, but not OX40 agonists. While the addition of checkpoint inhibitors to VSV-IFNß generated robust tumor growth inhibition, it resulted in no increase in viral replication, transgene expression, or immunophenotypic changes beyond treatment with VSV-IFNß alone. We hypothesize that tumor-specific T cells generated by VSV-IFNß retain activity due to a lack of immune exhaustion when checkpoint inhibitors were used.

CEA/CD3-bispecific T cell-engaging (BiTE) antibody-mediated T lymphocyte cytotoxicity maximized by inhibition of both PD1 and PD-L1.

Bispecific T cell-engaging (BiTE) antibodies recruit polyclonal cytotoxic T cells (CTL) to tumors. One such antibody is carcinoembryonic antigen (CEA) BiTE that mediates T cell/tumor interaction by simultaneously binding CD3 expressed by T cells and CEA expressed by tumor cells. A widely operative mechanism for mitigating cytotoxic T cell-mediated killing is the interaction of tumor-expressed PD-L1 with T cell-expressed PD-1, which may be partly reversed by PD-1/PD-L1 blockade. We hypothesized that PD-1/PD-L1 blockade during BiTE-mediated T cell killing would enhance CTL function. Here, we determined the effects of PD-1 and PD-L1 blockade during initial T cell-mediated killing of CEA-expressing human tumor cell lines in vitro, as well as subsequent T cell-mediated killing by T lymphocytes that had participated in tumor cell killing. We observed a rapid upregulation of PD-1 expression and diminished cytolytic function of T cells after they had engaged in CEA BiTE-mediated killing of tumors. T cell cytolytic activity in vitro could be maximized by administration of anti-PD-1 or anti-PD-L1 antibodies alone or in combination if applied prior to a round of T cell killing, but T cell inhibition could not be fully reversed by this blockade once the T cells had killed tumor. In conclusion, our findings demonstrate that dual blockade of PD-1 and PD-L1 maximizes T cell killing of tumor directed by CEA BiTE in vitro, is more effective if applied early, and provides a rationale for clinical use.

The immune microenvironment: a major player in human cancers.

Cancer is a major public health issue and figures among the leading causes of death in the world. Cancer development is a long process, involving the mutation, amplification or deletion of genes and chromosomal rearrangements. The transformed cells change morphologically, enlarge, become invasive and finally detach from the primary tumor to metastasize in other organs through the blood and/or lymph. During this process, the tumor cells interact with their microenvironment, which is complex and composed of stromal and immune cells that penetrate the tumor site via blood vessels and lymphoid capillaries. All subsets of immune cells can be found in tumors, but their respective density, functionality and organization vary from one type of tumor to another. Whereas inflammatory cells play a protumoral role, there is a large body of evidence of effector memory T cells controlling tumor invasion and metastasis. Thus, high densities of memory Th1/CD8 cytotoxic T cells in the primary tumors correlate with good prognosis in most tumor types. Tertiary lymphoid structures, which contain mature dendritic cells (DC) in a T cell zone, proliferating B cells and follicular DC, are found in the tumor stroma and they correlate with intratumoral Th1/CD8 T cell and B cell infiltration. Eventually, tumors undergo genetic and epigenetic modifications that allow them to escape being controlled by the immune system. This comprehensive review describes the immune contexture of human primary and metastatic tumors, how it impacts on patient outcomes and how it could be used as a predictive biomarker and guide immunotherapies.

Tumour-retained activated CCR7+ dendritic cells are heterogeneous and regulate local anti-tumour cytolytic activity.

Tumour dendritic cells (DCs) internalise antigen and upregulate CCR7, which directs their migration to tumour-draining lymph nodes (dLN). CCR7 expression is coupled to an activation programme enriched in regulatory molecule expression, including PD-L1. However, the spatio-temporal dynamics of CCR7+ DCs in anti-tumour immune responses remain unclear. Here, we use photoconvertible mice to precisely track DC migration. We report that CCR7+ DCs are the dominant DC population that migrate to the dLN, but a subset remains tumour-resident despite CCR7 expression. These tumour-retained CCR7+ DCs are phenotypically and transcriptionally distinct from their dLN counterparts and heterogeneous. Moreover, they progressively downregulate the expression of antigen presentation and pro-inflammatory transcripts with more prolonged tumour dwell-time. Tumour-residing CCR7+ DCs co-localise with PD-1+CD8+ T cells in human and murine solid tumours, and following anti-PD-L1 treatment, upregulate stimulatory molecules including OX40L, thereby augmenting anti-tumour cytolytic activity. Altogether, these data uncover previously unappreciated heterogeneity in CCR7+ DCs that may underpin a variable capacity to support intratumoural cytotoxic T cells.

Mediation of antitumor activity by AZD4820 oncolytic vaccinia virus encoding IL-12.

Oncolytic viruses are engineered to selectively kill tumor cells and have demonstrated promising results in early-phase clinical trials. To further modulate the innate and adaptive immune system, we generated AZD4820, a vaccinia virus engineered to express interleukin-12 (IL-12), a potent cytokine involved in the activation of natural killer (NK) and T cells and the reprogramming of the tumor immune microenvironment. Testing in cultured human tumor cell lines demonstrated broad in vitro oncolytic activity and IL-12 transgene expression. A surrogate virus expressing murine IL-12 demonstrated antitumor activity in both MC38 and CT26 mouse syngeneic tumor models that responded poorly to immune checkpoint inhibition. In both models, AZD4820 significantly upregulated interferon-gamma (IFN-γ) relative to control mice treated with oncolytic vaccinia virus (VACV)-luciferase. In the CT26 study, 6 of 10 mice had a complete response after treatment with AZD4820 murine surrogate, whereas control VACV-luciferase-treated mice had 0 of 10 complete responders. AZD4820 treatment combined with anti-PD-L1 blocking antibody augmented tumor-specific T cell immunity relative to monotherapies. These findings suggest that vaccinia virus delivery of IL-12, combined with immune checkpoint blockade, elicits antitumor immunity in tumors that respond poorly to immune checkpoint inhibitors.

Efficacy and pharmacodynamic effect of anti-CD73 and anti-PD-L1 monoclonal antibodies in combination with cytotoxic therapy: observations from mouse tumor models.

CD73 is a cell surface 5'nucleotidase (NT5E) and key node in the catabolic process generating immunosuppressive adenosine in cancer. Using a murine monoclonal antibody surrogate of Oleclumab, we investigated the effect of CD73 inhibition in concert with cytotoxic therapies (chemotherapies as well as fractionated radiotherapy) and PD-L1 blockade. Our results highlight improved survival in syngeneic tumor models of colorectal cancer (CT26 and MC38) and sarcoma (MCA205). This therapeutic outcome was in part driven by cytotoxic CD8 T-cells, as evidenced by the detrimental effect of CD8 depleting antibody treatment of MCA205 tumor bearing mice treated with anti-CD73, anti-PD-L1 and 5-Fluorouracil+Oxaliplatin (5FU+OHP). We hypothesize that the improved responses are tumor microenvironment (TME)-driven, as suggested by the lack of anti-CD73 enhanced cytopathic effects mediated by 5FU+OHP on cell lines in vitro. Pharmacodynamic analysis, using imaging mass cytometry and RNA-sequencing, revealed noteworthy changes in specific cell populations like cytotoxic T cells, B cells and NK cells in the CT26 TME. Transcriptomic analysis highlighted treatment-related modulation of gene profiles associated with an immune response, NK and T-cell activation, T cell receptor signaling and interferon (types 1 & 2) pathways. Inclusion of comparator groups representing the various components of the combination allowed deconvolution of contribution of the individual therapeutic elements; highlighting specific effects mediated by the anti-CD73 antibody with respect to immune-cell representation, chemotaxis and myeloid biology. These pre-clinical data reflect complementarity of adenosine blockade with cytotoxic therapy, and T-cell checkpoint inhibition, and provides new mechanistic insights in support of combination therapy.

Rapid functional impairment of natural killer cells following tumor entry limits anti-tumor immunity.

Immune cell dysfunction within the tumor microenvironment (TME) undermines the control of cancer progression. Established tumors contain phenotypically distinct, tumor-specific natural killer (NK) cells; however, the temporal dynamics, mechanistic underpinning and functional significance of the NK cell compartment remains incompletely understood. Here, we use photo-labeling, combined with longitudinal transcriptomic and cellular analyses, to interrogate the fate of intratumoral NK cells. We reveal that NK cells rapidly lose effector functions and adopt a distinct phenotypic state with features associated with tissue residency. NK cell depletion from established tumors did not alter tumor growth, indicating that intratumoral NK cells cease to actively contribute to anti-tumor responses. IL-15 administration prevented loss of function and improved tumor control, generating intratumoral NK cells with both tissue-residency characteristics and enhanced effector function. Collectively, our data reveals the fate of NK cells after recruitment into tumors and provides insight into how their function may be revived.

Safety and Tolerability of MEDI0562, an OX40 Agonist mAb, in Combination with Durvalumab or Tremelimumab in Adult Patients with Advanced Solid Tumors.

PURPOSE: Combination therapies targeting immunologic checkpoints have shown promise in treating multiple tumor types. We report safety and tolerability of MEDI0562, a humanized IgG1K OX40 mAb, in combination with durvalumab (anti-PD-L1), or tremelimumab (anti-CTLA-4), in adult patients with previously treated advanced solid tumors. PATIENTS AND METHODS: In this phase I, multicenter, open-label study, patients received escalating doses of MEDI0562 (2.25, 7.5, or 22.5 mg) every 2 weeks in combination with durvalumab (1,500 mg) or tremelimumab (75 or 225 mg) every 4 weeks, intravenously, until unacceptable toxicity or progressive disease. Tumor assessments were performed every 8 weeks. The primary objective was to evaluate safety and tolerability. RESULTS: Among the 27 and 31 patients who received MEDI0562 + durvalumab or MEDI0562 + tremelimumab, 74.1% and 67.7% reported a treatment-related adverse event (AE), and 22.2% and 19.4% experienced a treatment-emergent AE that led to discontinuation, respectively. The MTD of MEDI0562 + durvalumab was 7.5 mg MEDI0562 + 1,500 mg durvalumab; the maximum administered dose of MEDI0562 + tremelimumab was 22.5 mg MEDI0562 + 225 mg tremelimumab. Three patients in the MEDI0562 + durvalumab arm had a partial response. The mean percentage of Ki67+CD4+ and Ki67+CD8+ memory T cells increased by >100% following the first dose of MEDI0562 + durvalumab or tremelimumab in all dose cohorts. A decrease in OX40+FOXP3 regulatory T cells was observed in a subset of patients with available paired biopsies. CONCLUSIONS: Following dose escalation, moderate toxicity was observed in both treatment arms, with no clear efficacy signals demonstrated.

In vivo labeling reveals continuous trafficking of TCF-1+ T cells between tumor and lymphoid tissue.

Improving the efficacy of immune checkpoint therapies will require a better understanding of how immune cells are recruited and sustained in tumors. Here, we used the photoconversion of the tumor immune cell compartment to identify newly entering lymphocytes, determine how they change over time, and investigate their egress from the tumor. Combining single-cell transcriptomics and flow cytometry, we found that while a diverse mix of CD8 T cell subsets enter the tumor, all CD8 T cells retained within this environment for more than 72 h developed an exhausted phenotype, revealing the rapid establishment of this program. Rather than forming tumor-resident populations, non-effector subsets, which express TCF-1 and include memory and stem-like cells, were continuously recruited into the tumor, but this recruitment was balanced by concurrent egress to the tumor-draining lymph node. Thus, the TCF-1+ CD8 T cell niche in tumors is highly dynamic, with the circulation of cells between the tumor and peripheral lymphoid tissue to bridge systemic and intratumoral responses.

Tertiary Lymphoid Structure-B Cells Narrow Regulatory T Cells Impact in Lung Cancer Patients.

The presence of tertiary lymphoid structures (TLS) in the tumor microenvironment is associated with better clinical outcome in many cancers. In non-small cell lung cancer (NSCLC), we have previously showed that a high density of B cells within TLS (TLS-B cells) is positively correlated with tumor antigen-specific antibody responses and increased intratumor CD4+ T cell clonality. Here, we investigated the relationship between the presence of TLS-B cells and CD4+ T cell profile in NSCLC patients. The expression of immune-related genes and proteins on B cells and CD4+ T cells was analyzed according to their relationship to TLS-B density in a prospective cohort of 56 NSCLC patients. We observed that tumor-infiltrating T cells showed marked differences according to TLS-B cell presence, with higher percentages of naïve, central-memory, and activated CD4+ T cells and lower percentages of both immune checkpoint (ICP)-expressing CD4+ T cells and regulatory T cells (Tregs) in the TLS-Bhigh tumors. A retrospective study of 538 untreated NSCLC patients showed that high TLS-B cell density was even able to counterbalance the deleterious impact of high Treg density on patient survival, and that TLS-Bhigh Treglow patients had the best clinical outcomes. Overall, the correlation between the density of TLS-Bhigh tumors with early differentiated, activated and non-regulatory CD4+ T cell cells suggest that B cells may play a central role in determining protective T cell responses in NSCLC patients.

Activity of murine surrogate antibodies for durvalumab and tremelimumab lacking effector function and the ability to deplete regulatory T cells in mouse models of cancer.

Preclinical studies of PD-L1 and CTLA-4 blockade have relied heavily on mouse syngeneic tumor models with intact immune systems, which facilitate dissection of immunosuppressive mechanisms in the tumor microenvironment. Commercially developed monoclonal antibodies (mAbs) targeting human PD-L1, PD-1, and CTLA-4 may not demonstrate cross-reactive binding to their mouse orthologs, and surrogate anti-mouse antibodies are often used in their place to inhibit these immune checkpoints. In each case, multiple choices exist for surrogate antibodies, which differ with respect to species of origin, affinity, and effector function. To develop relevant murine surrogate antibodies for the anti-human PD-L1 mAb durvalumab and the anti-human CTLA-4 mAb tremelimumab, rat/mouse chimeric or fully murine mAbs engineered for reduced effector function were developed and compared with durvalumab and tremelimumab. Characterization included determination of target affinity, in vivo effector function, pharmacokinetic profile, and anti-tumor efficacy in mouse syngeneic tumor models. Results showed that anti-PD-L1 and anti-CTLA-4 murine surrogates with pharmacologic properties similar to those of durvalumab and tremelimumab demonstrated anti-tumor activity in a subset of commonly used mouse syngeneic tumor models. This activity was not entirely dependent on antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis effector function, or regulatory T-cell depletion, as antibodies engineered to lack these features showed activity in models historically sensitive to checkpoint inhibition, albeit at a significantly lower level than antibodies with intact effector function.

Selective targeting and potent control of tumor growth using an EphA2/CD3-Bispecific single-chain antibody construct.

The EphA2 receptor tyrosine kinase is frequently overexpressed and functionally altered in malignant cells and thus provides opportunities for selective targeting of tumor cells. We describe here the development of a novel, bispecific single-chain antibody (bscAb) referred to as bscEphA2xCD3. This molecule simultaneously targets EphA2 on tumor cells and the T-cell receptor/CD3 complex on T cells and possesses structural and functional characteristics of the recently developed BiTE technology. An EphA2-specific single-chain antibody was selected for recognition of an epitope that is preferentially exposed on malignant cells based on the concept of epitope exclusion; this was fused to a CD3-specific single-chain antibody to generate bscEphA2xCD3. The resultant bscAb redirected unstimulated human T cells to lyse EphA2-expressing tumor cells both in vitro and in vivo. In separate experiments, efficient tumor cell lysis was achieved in vitro at drug concentrations

Automated image analysis of NSCLC biopsies to predict response to anti-PD-L1 therapy.

BACKGROUND: Immune checkpoint therapies (ICTs) targeting the programmed cell death-1 (PD1)/programmed cell death ligand-1 (PD-L1) pathway have improved outcomes for patients with non-small cell lung cancer (NSCLC), particularly those with high PD-L1 expression. However, the predictive value of manual PD-L1 scoring is imperfect and alternative measures are needed. We report an automated image analysis solution to determine the predictive and prognostic values of the product of PD-L1+ cell and CD8+ tumor infiltrating lymphocyte (TIL) densities (CD8xPD-L1 signature) in baseline tumor biopsies. METHODS: Archival or fresh tumor biopsies were analyzed for PD-L1 and CD8 expression by immunohistochemistry. Samples were collected from 163 patients in Study 1108/NCT01693562, a Phase 1/2 trial to evaluate durvalumab across multiple tumor types, including NSCLC, and a separate cohort of 199 non-ICT- patients. Digital images were automatically scored for PD-L1+ and CD8+ cell densities using customized algorithms applied with Developer XD™ 2.7 software. RESULTS: For patients who received durvalumab, median overall survival (OS) was 21.0 months for CD8xPD-L1 signature-positive patients and 7.8 months for signature-negative patients (p = 0.00002). The CD8xPD-L1 signature provided greater stratification of OS than high densities of CD8+ cells, high densities of PD-L1+ cells, or manually assessed tumor cell PD-L1 expression ≥25%. The CD8xPD-L1 signature did not stratify OS in non-ICT patients, although a high density of CD8+ cells was associated with higher median OS (high: 67 months; low: 39.5 months, p = 0.0009) in this group. CONCLUSIONS: An automated CD8xPD-L1 signature may help to identify NSCLC patients with improved response to durvalumab therapy. Our data also support the prognostic value of CD8+ TILS in NSCLC patients who do not receive ICT. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01693562 . Study code: CD-ON-MEDI4736-1108. Interventional study (ongoing but not currently recruiting). Actual study start date: August 29, 2012. Primary completion date: June 23, 2017 (final data collection date for primary outcome measure).

Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein.

Ligation of OX40 (CD134, TNFRSF4) on activated T cells by its natural ligand (OX40L, CD252, TNFSF4) enhances cellular survival, proliferation, and effector functions such as cytokine release and cellular cytotoxicity. We engineered a recombinant human OX40L IgG4P Fc fusion protein termed MEDI6383 that assembles into a hexameric structure and exerts potent agonist activity following engagement of OX40. MEDI6383 displayed solution-phase agonist activity that was enhanced when the fusion protein was clustered by Fc gamma receptors (FcγRs) on the surface of adjacent cells. The resulting costimulation of OX40 on T cells induced NFκB promoter activity in OX40-expressing T cells and induced Th1-type cytokine production, proliferation, and resistance to regulatory T cell (Treg)-mediated suppression. MEDI6383 enhanced the cytolytic activity of tumor-reactive T cells and reduced tumor growth in the context of an alloreactive human T cell:tumor cell admix model in immunocompromised mice. Consistent with the role of OX40 costimulation in the expansion of memory T cells, MEDI6383 administered to healthy nonhuman primates elicited peripheral blood CD4 and CD8 central and effector memory T-cell proliferation as well as B-cell proliferation. Together, these results suggest that OX40 agonism has the potential to enhance antitumor immunity in human malignancies. Mol Cancer Ther; 17(5); 1024-38. ©2018 AACR.

Transcutaneous immunization induces mucosal CTLs and protective immunity by migration of primed skin dendritic cells.

Transcutaneous immunization (TCI), the application of vaccines on the skin, induces robust systemic and mucosal antibodies in animal models and in humans. The means by which mucosal immune responses to vaccine antigens are elicited by TCI has not been well characterized. We examined the effect of TCI with an HIV peptide vaccine on the induction of mucosal and systemic CTL responses and protective immunity against mucosal challenge with live virus in mice. Robust HIV-specific CTL responses in the spleen and in the gut mucosa were detected after TCI. The responses were dependent upon the addition of an adjuvant and resulted in protection against mucosal challenge with recombinant vaccinia virus encoding HIV gp160. Although it is clear that adjuvant-activated DCs migrated mainly to draining lymph nodes, coculture with specific T cells and flow cytometry studies with DCs isolated from Peyer's patches after TCI suggested that activated DCs carrying skin-derived antigen also migrated from the skin to immune-inductive sites in gut mucosa and presented antigen directly to resident lymphocytes. These results and previous clinical trial results support the observation that TCI is a safe and effective strategy for inducing strong mucosal antibody and CTL responses.

Combination therapy with an OX40L fusion protein and a vaccine targeting the transcription factor twist inhibits metastasis in a murine model of breast cancer.

OX40 is a costimulatory receptor that potentiates proliferation, survival, memory formation, and effector function of CD4+ and CD8+ T-cells, while overcoming the suppressive activity of regulatory T-cells (Tregs). Here, we explored the combination of an OX40L fusion protein (OX40L-FP) with a poxvirus-based cancer vaccine (MVA-Twist-TRICOM) to inhibit tumor metastasis in the 4T1 murine breast cancer model. Contrary to the single agent treatments, the combination therapy significantly decreased the number of metastatic colonies per lung and prolonged survival. Depletion studies demonstrated that these effects were mediated by both CD4+ and CD8+ T-cells. The combination therapy a) increased the total number of T-cells in the CD4+Foxp3- population and the CD4+ central and effector memory subsets within the lung, spleen, and draining lymph node, b) enhanced infiltration of CD4+ T-cells into metastatic areas of the lung, and (c) increased the number of functional CD8+ T-cells that produced IFNγ and TNFα. The combination therapy also promoted the development of KLRG1-CD127+ memory precursor CD8+ T-cells, while reducing those with a KLRG1+ terminally differentiated phenotype. Moreover, the combination of OX40L-FP and vaccine induced greater CD4+ and CD8+ Twist-specific responses. In addition, Tregs isolated from mice receiving the combination were also less immunosuppressive in ex-vivo proliferation assays than those from the OX40L-FP and MVA-Twist-TRICOM monotherapy groups. Such results provide the rationale to combine co-stimulatory agonists with cancer vaccines for the treatment of tumor metastasis.