Blockade of CTLA-4 and PD-1 Enhances Adoptive T-cell Therapy Efficacy in an ICOS-Mediated Manner.

Adoptive transfer of tumor-reactive T cells (ACT) has led to modest clinical benefit in the treatment of solid tumors. Failures with this therapy are primarily due to inadequate infiltration and poor function of adoptively transferred cells in the tumor microenvironment. To improve the efficacy of ACT, we combined ACT with dual blockade of CTLA-4 and PD-1. Treatment with anti-CTLA-4 plus anti-PD-1 compared with monotherapy resulted in durable antitumor responses, enhanced effector function of ACT, utilizing PMEL-1 transgenic (Tg+) CD8+ T cells, and improved survival. Using PMEL-1ICOS-/- mice, we showed that deletion of the inducible T-cell costimulator (ICOS) receptor abolished the therapeutic benefits, with selective downregulation of Eomesodermin (Eomes), interferon gamma (IFNγ), and perforin. Higher expression of IFNγ and Eomes was noted in human ICOShi CD8+ T cells compared with ICOSlow counterparts. Together, our data provide direct evidence that ACT combined with immune-checkpoint therapy confers durable antitumor responses, which largely depended on CD8+ T-cell-intrinsic expression of ICOS. Our study provides a foundation of testing combinatorial therapy of ACT of CD8 T cells and dual blocking of CTLA-4 and PD-1 in patients with melanoma.

Cancer vaccine formulation dictates synergy with CTLA-4 and PD-L1 checkpoint blockade therapy.

Anticancer vaccination is a promising approach to increase the efficacy of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death ligand 1 (PD-L1) checkpoint blockade therapies. However, the landmark FDA registration trial for anti-CTLA-4 therapy (ipilimumab) revealed a complete lack of benefit of adding vaccination with gp100 peptide formulated in incomplete Freund's adjuvant (IFA). Here, using a mouse model of melanoma, we found that gp100 vaccination induced gp100-specific effector T cells (Teffs), which dominantly forced trafficking of anti-CTLA-4-induced, non-gp100-specific Teffs away from the tumor, reducing tumor control. The inflamed vaccination site subsequently also sequestered and destroyed anti-CTLA-4-induced Teffs with specificities for tumor antigens other than gp100, reducing the antitumor efficacy of anti-CTLA-4 therapy. Mechanistically, Teffs at the vaccination site recruited inflammatory monocytes, which in turn attracted additional Teffs in a vicious cycle mediated by IFN-γ, CXCR3, ICAM-1, and CCL2, dependent on IFA formulation. In contrast, nonpersistent vaccine formulations based on dendritic cells, viral vectors, or water-soluble peptides potently synergized with checkpoint blockade of both CTLA-4 and PD-L1 and induced complete tumor regression, including in settings of primary resistance to dual checkpoint blockade. We conclude that cancer vaccine formulation can dominantly determine synergy, or lack thereof, with CTLA-4 and PD-L1 checkpoint blockade therapy for cancer.

Interdependent IL-7 and IFN-γ signalling in T-cell controls tumour eradication by combined α-CTLA-4+α-PD-1 therapy.

Combination therapy with α-CTLA-4 and α-PD-1 has shown significant clinical responses in different types of cancer. However, the underlying mechanisms remain elusive. Here, combining detailed analysis of human tumour samples with preclinical tumour models, we report that concomitant blockade of CTLA-4 and PD-1 improves anti-tumour immune responses and synergistically eradicates tumour. Mechanistically, combination therapy relies on the interdependence between IL-7 and IFN-γ signalling in T cells, as lack of either pathway abrogates the immune-boosting and therapeutic effects of combination therapy. Combination treatment increases IL-7Rα expression on tumour-infiltrating T cells in an IFN-γ/IFN-γR signalling-dependent manner, which may serve as a potential biomarker for clinical trials with immune checkpoint blockade. Our data suggest that combining immune checkpoint blockade with IL-7 signalling could be an effective modality to improve immunotherapeutic efficacy. Taken together, we conclude that combination therapy potently reverses immunosuppression and eradicates tumours via an intricate interplay between IFN-γ/IFN-γR and IL-7/IL-7R pathways.

ICOS Promotes the Function of CD4+ Effector T Cells during Anti-OX40-Mediated Tumor Rejection.

ICOS is a T-cell coregulatory 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, nondepleting 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. Furthermore, it did not impede antitumor responses mediated by anti-4-1BB-driven CD8(+) T cells. We found that anti-OX40 efficacy, which is based on 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. Cancer Res; 76(13); 3684-9. ©2016 AACR.

CD4 T cells require ICOS-mediated PI3K signaling to increase T-Bet expression in the setting of anti-CTLA-4 therapy.

The transcription factor T-bet controls the Th1 genetic program in T cells for effective antitumor responses. Anti-CTLA-4 immunotherapy elicits dramatic antitumor responses in mice and in human patients; however, factors that regulate T-bet expression during an antitumor response mediated by anti-CTLA-4 remain to be elucidated. We were the first to report that treatment with anti-CTLA-4 led to an increase in the frequency of T cells expressing inducible costimulator (ICOS). In both treated patients and mice, our data revealed that CD4(+)ICOS(hi) T cells can act as effector T cells, which produce the Th1 cytokine IFN-γ. We also showed in a small retrospective analysis that an increased frequency of CD4(+)ICOS(hi) T cells correlated with better clinical outcome and the absence of ICOS or its ligand (ICOSL) in mouse models led to impaired tumor rejection. Here, we show that CD4(+)ICOS(hi) T cells from anti-CTLA-4-treated patients had an increase in signaling via the phospoinositide-3-kinase (PI3K) pathway and an increase in expression of T-bet. An ICOS-specific siRNA transfected into human T cells led to diminished PI3K signaling and T-bet expression. Therefore, we hypothesized that ICOS, and specifically ICOS-mediated PI3K signaling, was required for T-bet expression. We conducted studies in ICOS-deficient and ICOS-YF mice, which have a single amino acid change that abrogates PI3K signaling by ICOS. We found that ICOS-mediated PI3K signaling is required for T-bet expression during an antitumor response elicited by anti-CTLA-4 therapy. Our data provide new insight into the regulation of T-bet expression and suggest that ICOS can be targeted to improve Th1 antitumor responses.

Identification of human regulatory T cells in the setting of T-cell activation and anti-CTLA-4 immunotherapy on the basis of expression of latency-associated peptide.

UNLABELLED: Effector and regulatory T cells (Treg) share multiple markers that make it difficult to discern differences in these populations in humans. The transcription factor FoxP3 has been shown to identify Tregs. However, the detection of FoxP3 requires cell permeabilization, thereby preventing isolation of viable Tregs. Subsequently, the extracellular marker CD127 was established for the identification of Tregs. However, these studies were not conducted in the setting of immunotherapy. Here, we conducted studies to analyze CD127 and FoxP3 expression on T cells before and after in vitro activation as well as in the setting of patients treated with antibodies directed against cytotoxic T-lymphocyte antigen-4 (CTLA-4). We show that latency-associated peptide (LAP), as opposed to CD127, was capable of identifying Tregs after in vitro activation as well as after treatment with anti-CTLA-4. Therefore, we propose that LAP should be used as a marker of Tregs for immune monitoring studies in patients treated with active immunotherapy such as anti-CTLA-4. SIGNIFICANCE: Tregs play an important role in human diseases, including cancer and autoimmunity; however, it has been difficult to study these cells because of a lack of an appropriate marker. Here, we propose LAP as a marker that can be used to identify Tregs in patients treated with immunotherapy, thereby permitting isolation of these cells for functional studies and for ex vivo expansion.

The ICOS/ICOSL pathway is required for optimal antitumor responses mediated by anti-CTLA-4 therapy.

The anti-CTL-associated antigen 4 (anti-CTLA-4) antibody ipilimumab is the first agent to show improved survival in a randomized phase III trial that enrolled patients with metastatic melanoma. Studies are ongoing to identify mechanisms that elicit clinical benefit in the setting of anti-CTLA-4 therapy. We previously reported that treated patients had an increase in the frequency of T cells expressing the inducible costimulator (ICOS) molecule, a T-cell-specific molecule that belongs to the CD28/CTLA-4/B7 immunoglobulin superfamily. ICOS and its ligand (ICOSL) have been shown to play diverse roles in T-cell responses such as mediating autoimmunity as well as enhancing the development/activity of regulatory T cells. These seemingly opposing roles have made it difficult to determine whether the ICOS/ICOSL pathway is necessary for antitumor responses. To determine whether the ICOS/ICOSL pathway might play a causal role in the antitumor effects mediated by anti-CTLA-4, we conducted studies in ICOS-sufficient and ICOS-deficient mice bearing B16/BL6 melanoma. We show that ICOS(+) T cells comprised a population of Th1 cytokine producing and tumor antigen-specific effector cells. Furthermore, in the absence of ICOS, antitumor T-cell responses elicited by anti-CTLA-4 are significantly diminished, thereby impairing tumor rejection. Our findings establish that the ICOS/ICOSL pathway is necessary for the optimal therapeutic effect of anti-CTLA-4, thus implicating this pathway as a target for future combinatorial strategies to improve the efficacy of anti-CTLA-4 therapy.

Modulation of Notch signaling by antibodies specific for the extracellular negative regulatory region of NOTCH3.

The Notch pathway regulates the development of many tissues and cell types and is involved in a variety of human diseases, making it an attractive potential therapeutic target. This promise has been limited by the absence of potent inhibitors or agonists that are specific for individual human Notch receptors (NOTCH1-4). Using an unbiased functional screening, we identified monoclonal antibodies that specifically inhibit or induce activating proteolytic cleavages in NOTCH3. Remarkably, the most potent inhibitory and activating antibodies bind to overlapping epitopes within a juxtamembrane negative regulatory region that protects NOTCH3 from proteolysis and activation in its resting autoinhibited state. The inhibitory antibodies revert phenotypes conveyed on 293T cells by NOTCH3 signaling, such as increased cellular proliferation, survival, and motility, whereas the activating antibody mimics some of the effects of ligand-induced Notch activation. These findings provide insights into the mechanisms of Notch autoinhibition and activation and pave the way for the further development of specific antibody-based modulators of the Notch receptors, which are likely to be of utility in a wide range of experimental and therapeutic settings.

Toll-like receptor 8-mediated reversal of CD4+ regulatory T cell function.

CD4+ regulatory T (Treg) cells have a profound ability to suppress host immune responses, yet little is understood about how these cells are regulated. We describe a mechanism linking Toll-like receptor (TLR) 8 signaling to the control of Treg cell function, in which synthetic and natural ligands for human TLR8 can reverse Treg cell function. This effect was independent of dendritic cells but required functional TLR8-MyD88-IRAK4 signaling in Treg cells. Adoptive transfer of TLR8 ligand-stimulated Treg cells into tumor-bearing mice enhanced anti-tumor immunity. These results suggest that TLR8 signaling could play a critical role in controlling immune responses to cancer and other diseases.

Functional characterization of EBV-encoded nuclear antigen 1-specific CD4+ helper and regulatory T cells elicited by in vitro peptide stimulation.

CD4(+) helper and regulatory T (Treg) cells play important but opposing roles in regulating host immune responses against cancer and other diseases. However, very little is known about the antigen specificity of CD4(+) Treg cells. Here we describe the generation of a panel of EBV-encoded nuclear antigen 1 (EBNA1)-specific CD4(+) T-cell lines and clones that recognize naturally processed EBNA1-P(607-619) and -P(561-573) peptides in the context of HLA-DQ2 and HLA-DR11, -DR12, and -DR13 molecules, respectively. Phenotypic and functional analyses of these CD4(+) T cells revealed that they represent EBNA1-specific CD4(+) T helper as well as Treg cells. CD4(+) Treg cells do not secrete interleukin (IL)-10 and transforming growth factor beta cytokines but express CD25, the glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), and Forkhead Box P3 (Foxp3), and are capable of suppressing the proliferative responses of naive CD4(+) and CD8(+) T cells to stimulation with mitogenic anti-CD3 antibody. The suppressive activity of these CD4(+) Treg cells is mediated via cell-cell contact or in part by a cytokine-dependent manner. Importantly, these Treg cells suppress IL-2 secretion by CD4(+) effector T cells specific for either EBNA1 or a melanoma antigen, suggesting that these CD4(+) Treg cells induce immune suppression. These observations suggest that the success of peptide-based vaccines against EBV-associated cancer and other diseases may likely depend upon our ability to identify antigens/peptides that preferentially activate helper T cells and/or to design strategies to regulate the balance between CD4(+) helper and Treg cells.

A unique mucin immunoenhancing peptide with antitumor properties.

Implantation of DA-3 mammary tumor cells into BALB/c mice results in tumor growth, metastatic lesions, and death. These cells were transfected with genes encoding for either the transmembrane (DA-3/TM) or secreted (DA-3/sec) form of human mucin 1 (MUC1). Although the gene for the secreted form lacks the transmembrane and cytoplasmic domains, the 5' sequences of these mucins are identical; however, the gene for the secreted mucin isoform ends with a sequence encoding for a unique 11 amino acid peptide. The DA-3/TM or DA-3 cells transfected with the neomycin vector only (DA-3/neo) have the same in vivo growth characteristics as the parent cell line. In contrast, DA-3/sec cells fail to grow when implanted in immunocompetent BALB/c animals. DA-3/sec cells implanted in nude mice resulted in tumor development verifying the tumorigenic potential of these cells. Pre-exposure of BALB/c mice to DA-3/sec cells afforded protection against challenge with DA-3/TM or DA-3/neo mammary tumors and the unrelated tumors K7, an osteosarcoma, and RENCA, a renal cell carcinoma. Partial protection against subsequent tumor challenges was also achieved by substituting the 11 amino acid peptide found only in the secreted MUC1 isoform, for the live DA-3/sec cells. Notably, the efficacy of this peptide is not strain restricted because it also retarded the growth of Lewis lung carcinoma cells in C57 BL/6 mice. These findings reveal that a unique peptide present in the secreted MUC1 has immunoenhancing properties and may be a potential agent for use in immunotherapy.

Critical role of EBNA1-specific CD4+ T cells in the control of mouse Burkitt lymphoma in vivo.

CD4+ T cells play important roles in orchestrating host immune responses against cancer and infectious diseases. Although EBV-encoded nuclear antigen 1-specific (EBNA1-specific) CD4+ T cells have been implicated in controlling the growth of EBV-associated tumors such as Burkitt lymphoma (BL) in vitro, direct evidence for their in vivo function remains elusive due to the lack of an appropriate experimental BL model. Here, we describe the development of a mouse EBNA1-expressing BL tumor model and the identification of 2 novel MHC H-2I-A(b)-restricted T cell epitopes derived from EBNA1. Using our murine BL tumor model and the relevant peptides, we show that vaccination of mice with EBNA1 peptide-loaded DCs can elicit CD4+ T cell responses. These EBNA1-specific CD4+ T cells recognized peptide-pulsed targets as well as EBNA1-expressing tumor cells and were necessary and sufficient for suppressing tumor growth in vivo. By contrast, EBNA1 peptide-reactive CD8+ T cells failed to recognize tumor cells and did not contribute to protective immunity. These studies represent what we believe to be the first demonstration that EBNA1-specific CD4+ T cells can suppress tumor growth in vivo, which suggests that CD4+ T cells play an important role in generating protective immunity against EBV-associated cancer.

Evidence for the presentation of major histocompatibility complex class I-restricted Epstein-Barr virus nuclear antigen 1 peptides to CD8+ T lymphocytes.

The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated tumors, making it an important target for immunotherapy. However, evidence for major histocompatibility complex (MHC) class I-restricted EBNA1 peptides endogenously presented by EBV-transformed B and tumor cells remains elusive. Here we describe for the first time the identification of an endogenously processed human histocompatibility leukocyte antigen (HLA)-B8-restricted EBNA1 peptide that is recognized by CD8+ T cells. T cell recognition could be inhibited by the treatment of target cells with proteasome inhibitors that block the MHC class I antigen processing pathway, but not by an inhibitor (chloroquine) of MHC class II antigen processing. We also demonstrate that new protein synthesis is required for the generation of the HLA-B8 epitope for T cell recognition, suggesting that defective ribosomal products (DRiPs) are the major source of T cell epitopes. Experiments with protease inhibitors indicate that some serine proteases may participate in the degradation of EBNA1 DRiPs before they are further processed by proteasomes. These findings not only provide the first evidence of the presentation of an MHC class I-restricted EBNA1 epitope to CD8+ T cells, but also offer new insight into the molecular mechanisms involved in the processing and presentation of EBNA1.

Identification of HLA-DP3-restricted peptides from EBNA1 recognized by CD4(+) T cells.

The EBV-encoded nuclear antigen 1 (EBNA1) is required for the maintenance and replication of the viral episome in EBV-transformed human B-lymphoblastoid cell lines. It is expressed in all EBV-associated tumors, making it a potentially important target for immunotherapy. However, this promise has not been realized, because an endogenously processed MHC class I-restricted T-cell epitope remains to be identified, and relatively little is known about MHC class II-restricted helper epitopes in the molecule. In this report, we identify a T-cell peptide derived from EBNA1 that is recognized by CD4(+) T cells. More importantly, EBNA1-specific, HLA-DP3-restricted CD4(+) T cells are capable of recognizing MHC class II-matched Burkitt's lymphoma cells, autologous peripheral blood mononuclear cells loaded with the purified EBNA1 protein, as well as target cells transfected with Ii-EBNA1 cDNA. These new findings demonstrate that EBNA1 is processed endogenously and presented to T cells by MHC class II molecules, and, hence, may be useful to incorporate into cancer vaccines to enhance antitumor immunity against EBV-associated tumors.

Induction of CD4(+) T cell-dependent antitumor immunity by TAT-mediated tumor antigen delivery into dendritic cells.

Dendritic cell-based (DC-based) immunotherapy represents a promising approach to the prevention and treatment of many diseases, including cancer, but current strategies have met with only limited success in clinical and preclinical studies. Previous studies have demonstrated that a TAT peptide derived from the HIV TAT protein has the ability to transduce peptides or proteins into various cells. Here, we describe the use of TAT-mediated delivery of T cell peptides into DCs to prolong antigen presentation and enhance T cell responses. While immunization of mice with DCs pulsed with an antigenic peptide derived from the human TRP2 protein generated partial protective immunity against B16 tumor, immunization with DCs loaded with a TAT-TRP2 peptide resulted in complete protective immunity, as well as significant inhibition of lung metastases in a 3-day tumor model. Although both DC/TRP2 and DC/TAT-TRP2 immunization increased the number of TRP2-specific CD8(+) T cells detected by K(b)/TRP2 tetramers, T cell activity elicited by DC/TAT-TRP2 was three- to tenfold higher than that induced by DC/TRP2. Furthermore, both CD4(+) and CD8(+) T cells were required for antitumor immunity demonstrated by experiments with antibody depletion of subsets of T cells, as well as with various knockout mice. These results suggest that a TAT-mediated antigen delivery system may have important clinical applications for cancer therapy.