Todd D. Armstrong

Recruitment of latent pools of high-avidity CD8+ T cells to the antitumor immune response

A major barrier to successful antitumor vaccination is tolerance of high-avidity T cells specific to tumor antigens. In keeping with this notion, HER-2/neu (neu)-targeted vaccines, which raise strong CD8+ T cell responses to a dominant peptide (RNEU420-429) in WT FVB/N mice and protect them from a neu-expressing tumor challenge, fail to do so in MMTV-neu (neu-N) transgenic mice. However, treatment of neu-N mice with vaccine and cyclophosphamide-containing chemotherapy resulted in tumor protection in a proportion of mice. This effect was specifically abrogated by the transfer of neu-N–derived CD4+CD25+ T cells. RNEU420-429-specific CD8+ T cells were identified only in neu-N mice given vaccine and cyclophosphamide chemotherapy which rejected tumor challenge. Tetramer-binding studies demonstrated that cyclophosphamide pretreatment allowed the activation of high-avidity RNEU420-429-specific CD8+ T cells comparable to those generated from vaccinated FVB/N mice. Cyclophosphamide seemed to inhibit regulatory T (T reg) cells by selectively depleting the cycling population of CD4+CD25+ T cells in neu-N mice. These findings demonstrate that neu-N mice possess latent pools of high-avidity neu-specific CD8+ T cells that can be recruited to produce an effective antitumor response if T reg cells are blocked or removed by using approaches such as administration of cyclophosphamide before vaccination.

Mesothelin-specific CD8+ T Cell Responses Provide Evidence of In Vivo Cross-Priming by Antigen-Presenting Cells in Vaccinated Pancreatic Cancer Patients

Tumor-specific CD8+ T cells can potentially be activated by two distinct mechanisms of major histocompatibility complex class I–restricted antigen presentation as follows: direct presentation by tumor cells themselves or indirect presentation by professional antigen-presenting cells (APCs). However, controversy still exists as to whether indirect presentation (the cross-priming mechanism) can contribute to effective in vivo priming of tumor-specific CD8+ T cells that are capable of eradicating cancer in patients. A clinical trial of vaccination with granulocyte macrophage–colony stimulating factor–transduced pancreatic cancer lines was designed to test whether cross-presentation by locally recruited APCs can activate pancreatic tumor-specific CD8+ T cells. Previously, we reported postvaccination delayed-type hypersensitivity (DTH) responses to autologous tumor in 3 out of 14 treated patients. Mesothelin is an antigen demonstrated previously by gene expression profiling to be up-regulated in most pancreatic cancers. We report here the consistent induction of CD8+ T cell responses to multiple HLA-A2, A3, and A24-restricted mesothelin epitopes exclusively in the three patients with vaccine-induced DTH responses. Importantly, neither of the vaccinating pancreatic cancer cell lines expressed HLA-A2, A3, or A24. These results provide the first direct evidence that CD8 T cell responses can be generated via cross-presentation by an immunotherapy approach designed to recruit APCs to the vaccination site.

A Vascular Endothelial Growth Factor Receptor-2 Inhibitor Enhances Antitumor Immunity through an Immune-Based Mechanism

Purpose: Given the complex tumor microenvironment, targeting multiple cellular components may be the most effective cancer treatment strategy. Therefore, we tested whether antiangiogenic and immune-based therapy might synergize by characterizing the activity of DC101, an antiangiogenic monoclonal antibody specific for vascular endothelial growth factor receptor-2 (VEGF-R2), alone and with HER-2/neu (neu)–targeted vaccination. Experimental Design: Neu-expressing breast tumors were measured in treated nontolerant FVB mice and immune-tolerant neu transgenic (neu-N) mice. Neu-specific and tumor cell–specific immune responses were assessed by intracellular cytokine staining, ELISPOT, and CTL assays. Results: DC101 decreased angiogenesis and increased tumor cell apoptosis. Although DC101 increased serum levels of the immunosuppressive cytokine VEGF, no evidence of systemic immune inhibition was detected. Moreover, DC101 did not impede the influx of tumor-infiltrating lymphocytes. In FVB mice, DC101 inhibited tumor growth in part through a T cell–dependent mechanism, resulting in both increased tumor-specific CD8+ T cells and tumor regression. Combining DC101 with neu-specific vaccination accelerated tumor regression, augmenting the lytic activity of CD8+ cytotoxic T cells. In tolerant neu-N mice, DC101 only delayed tumor growth without inducing frank tumor regression or antigen-specific T-cell activation. Notably, mitigating immune tolerance by inhibiting regulatory T cell activity with cyclophosphamide revealed DC101-mediated augmentation of antitumor responses in vaccinated neu-N mice. Conclusions: This is the first report of DC101-induced antitumor immune responses. It establishes the induction of tumor-specific T-cell responses as one consequence of VEGF-R2 targeting with DC101. These data support the development of multitargeted cancer therapy combining immune-based and antiangiogenic agents for clinical translation.

OX40 Costimulation Synergizes with GM-CSF Whole-Cell Vaccination to Overcome Established CD8+ T Cell Tolerance to an Endogenous Tumor Antigen

T cell costimulation via OX40 is known to increase CD4+ T cell expansion and effector function and enhances the development of T cell memory. OX40 costimulation can also prevent, and even reverse, CD4+ T cell anergy. However, the role of OX40 in CD8+ T cell function is less well defined, particularly in the setting of immune tolerance. To determine the effects of OX40 costimulation on the induction of the host CD8+ T cell repertoire to an endogenous tumor Ag, we examined the fate of CD8+ T cells specific for the immunodominant rat HER-2/neu epitope, RNEU420–429, in FVB MMTV-neu (neu-N) mice, which express rat HER-2/neu protein in a predominantly mammary-restricted fashion. We show that the RNEU420–429-specific T cell repertoire in neu-N mice expands transiently after vaccination with a neu-targeted GM-CSF-secreting whole-cell vaccine, but quickly declines to an undetectable level. However, OX40 costimulation, when combined with GM-CSF-secreting tumor-targeted vaccination, can break established CD8+ T cell tolerance in vivo by enhancing the expansion, and prolonging the survival and effector function of CD8+ T cells specific for RNEU420–429. Moreover, we demonstrate that OX40 expression is up-regulated on both CD4+ and CD8+ T cells shortly after administration of a GM-CSF expressing vaccine. These studies highlight the increased efficacy of OX40 costimulation when combined with a GM-CSF-secreting vaccine, and define a new role for OX40 costimulation of CD8+ T cells in overcoming tolerance and boosting antitumor immunity.

Allergies and the risk of pancreatic cancer: A meta-analysis with review of epidemiology and biological mechanisms

Previous reports suggest that allergic disorders may protect against various types of cancer, but the association between history of allergy and pancreatic cancer risk has not been well studied. We did a systematic review and meta-analysis of published studies to evaluate the association of any type, and specific types, of allergy and the risk of pancreatic cancer. We did a comprehensive literature search using MEDLINE, PUBMED, and the ISI Web of Science databases to identify potential relevant case-control and cohort studies. Pooled relative risks (RR) and 95% confidence intervals (95% CI) were calculated using the fixed- and random-effects model. Fourteen population-based studies (4 cohort and 10 case-control studies) with a total of 3,040 pancreatic cancer cases fulfilled our inclusion criteria. A history of allergy was associated with a reduced risk of pancreatic cancer (RR, 0.82; 95% CI, 0.68-0.99). The risk reduction was stronger for allergies related to atopy (RR, 0.71; 95% CI, 0.64-0.80), but not for asthma (RR, 1.01; 95% CI, 0.77-1.31). There was no association between allergies related to food or drugs and pancreatic cancer (RR, 1.08; 95% CI, 0.74-1.58). Overall, there was no evidence of publication bias. Allergies, in particular those related to atopy, seem to be associated with a decreased risk of pancreatic cancer. The hyperactive immune system of allergic individuals may, therefore, in some way lead to increased surveillance and protect against pancreatic cancer development.

Antibody association with HER-2/neu–targeted vaccine enhances CD8+ T cell responses in mice through Fc-mediated activation of DCs

The pathogenic nature of cancer is attributed, at least in part, to the ability of tumors cells to induce systemic and local mechanisms of immune tolerance. However, we previously reported that tumor-free survival in up to 100% of tolerized HER-2/neu transgenic mice can be achieved by administration of neu-specific mAb concurrently with a HER-2/neu-expressing, GM-CSF-secreting whole cell vaccine. In this report, we show that one mechanism of improved antitumor activity induced by the combination of these 2 neu-targeted interventions was enhanced Fc-mediated activation of APCs. Specifically, in vivo studies demonstrated localization of radiolabeled neu-specific mAb at the vaccine site. Subsequently, increased accumulation of neu-specific mAb at the vaccine-draining lymph node correlated with increased vaccine cell uptake by DCs in vivo. This led to enhancement of CD8(+) neu-specific T cell function in terms of proliferation, cytokine production, and central memory development. Thus, the administration of a neu-specific mAb with a neu-targeted GM-CSF-secreting tumor vaccine enhanced induction of neu-specific CD8(+) T cells through Fc-mediated activation of DCs. This multimodality attack on the same tumor antigen may have the potential to overcome tolerance to self antigens and weaken the immunosuppressive networks within the tumor microenvironment.

Paclitaxel enhances early dendritic cell maturation and function through TLR4 signaling in mice

Subclinical doses of Paclitaxel (PTX) given 1day prior to a HER-2/neu (neu)-targeted, granulocyte-macrophage colony stimulating factor (GM-CSF)-secreting whole-cell vaccine enhances neu-specific T cell responses and slows neu(+) tumor growth in tolerized HER-2/neu (neu-N) mice. We demonstrate that co-administration of PTX and Cyclophosphamide (CY) synergizes to slow tumor growth, and that in vitro, DC precursors exposed to PTX before LPS maturation results in greater co-stimulatory molecule expression, IL-12 production, and the ability to induce CD8(+) T cells with enhanced lytic activity against neu(+) tumors. PTX treatment also enhances maturation marker expression on CD11c(+) DCs isolated from vaccine-draining lymph nodes. Ex vivo, these DCs activate CD8(+) T cells with greater lytic capability than DCs from vaccine alone-treated neu-N mice. Finally, PTX treatment results in enhanced antigen-specific, IFN-gamma-secreting CD8(+) T cells in vivo. Thus, administration of PTX with a tumor vaccine improves T cell priming through enhanced maturation of DC.

Trafficking of High Avidity HER-2/neu-Specific T Cells into HER-2/neu-Expressing Tumors after Depletion of Effector/Memory-Like Regulatory T Cells

Background Cancer vaccines are designed to activate and enhance cancer-antigen-targeted T cells that are suppressed through multiple mechanisms of immune tolerance in cancer-bearing hosts. T regulatory cell (Treg) suppression of tumor-specific T cells is one barrier to effective immunization. A second mechanism is the deletion of high avidity tumor-specific T cells, which leaves a less effective low avidity tumor specific T cell repertoire available for activation by vaccines. Treg depleting agents including low dose cyclophosphamide (Cy) and antibodies that deplete CD25-expressing Tregs have been used with limited success to enhance the potency of tumor-specific vaccines. In addition, few studies have evaluated mechanisms that activate low avidity cancer antigen-specific T cells. Therefore, we developed high and low avidity HER-2/neu-specific TCR transgenic mouse colonies specific for the same HER-2/neu epitope to define the tolerance mechanisms that specifically affect high versus low avidity tumor-specific T cells. Methodology/Principal Findings High and low avidity CD8+ T cell receptor (TCR) transgenic mice specific for the breast cancer antigen HER-2/neu (neu) were developed to provide a purified source of naïve, tumor-specific T cells that can be used to study tolerance mechanisms. Adoptive transfer studies into tolerant FVB/N-derived HER-2/neu transgenic (neu-N) mice demonstrated that high avidity, but not low avidity, neu-specific T cells are inhibited by Tregs as the dominant tolerizing mechanism. High avidity T cells persisted, produced IFNγ, trafficked into tumors, and lysed tumors after adoptive transfer into mice treated with a neu-specific vaccine and low dose Cy to deplete Tregs. Analysis of Treg subsets revealed a Cy-sensitive CD4+Foxp3+CD25low tumor-seeking migratory phenotype, characteristic of effector/memory Tregs, and capable of high avidity T cell suppression. Conclusion/Significance Depletion of CD25low Tregs allows activation of tumor-clearing high avidity T cells. Thus, the development of agents that specifically deplete Treg subsets should translate into more effective immunotherapies while avoiding autoimmunity.

Cytokine modified tumor vaccines

The immune system can recognize tumors, but may be actively tolerized to tumors during the tumorigenesis process. The identity of most tumor antigens remains unknown, but the number is growing due to new techniques. Most clinical trials using genetically modified tumor vaccines have shown immunological responses (DTH), but few clinical responses. Certain chemotherapeutic agents may enhance the immune effects of genetically modified tumor vaccines.

A STING Agonist Given with OX40 Receptor and PD-L1 Modulators Primes Immunity and Reduces Tumor Growth in Tolerized Mice

The efficacy and immune dynamics of STING modulation in the toleragenic tumor microenvironment were examined. Combining a STING agonist, PD-L1 blockade, and OX40R stimulation created an inflamed tumor microenvironment that recruited T cells and activated tumor-specific immunity. Stimulator of interferon genes (STING) signaling induces IFNβ production by intratumoral dendritic cells (DC), driving T-cell priming and recruitment into the tumor microenvironment (TME). We examined to what extent preexisting antigen-specific tolerance influenced the efficacy of in situ delivery of a potent STING-activating cyclic dinucleotide (CDN), ADU S-100, against established HER-2+ breast tumors. ADU S-100 induced HER-2–specific CD8+ T-cell priming and durable tumor clearance in 100% of nontolerant parental FVB/N mice. In contrast, ADU S-100 did not sufficiently prime HER-2–specific CD8+ T cells in tolerant neu/N mice, resulting in only delayed tumor growth and tumor clearance in 10% of the mice. No differences in IFNβ production, DC priming, or HER-2–specific CD8+ T-cell trafficking were detected between FVB/N and neu/N mice. However, activation and expansion of HER-2–specific CD8+ T cells were defective in neu/N mice. Immune cell infiltrates of untreated tumor-bearing neu/N mice expressed high numbers of PD1 and OX40 receptors on their CD8+ T cells, and PD-L1 was highly expressed on both myeloid and tumor cells. Modulating PD-L1 and OX40 receptor signaling combined with intratumoral ADU S-100 administration enhanced HER-2–specific CD8+ T-cell activity, clearing tumors in 40% of neu/N mice. Thus, intratumoral STING agonists could potently prime tumor antigen–specific CD8+ T-cell responses, and adding PD-L1 blockade and OX40 receptor activation can overcome antigen-enforced immune tolerance to induce tumor regression. Cancer Immunol Res; 5(6); 468–79. ©2017 AACR.