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.
BackgroundThe suppressive nature of immune cells in the tumor microenvironment plays a major role in regulating anti-tumor immune responses. Our previous work demonstrated that a soluble factor from tumor cells is able to induce a suppressor phenotype (SP) in human CD8+ T cells typified by loss of CD27/CD28 expression and acquisition of a potent suppressor function. The present study hypothesized that the soluble mechanism that is inducing the SP in CD8+ T cells are tumor-derived exosomes (TDEs).MethodsMembrane vesicles and TDEs from multiple head and neck cancer cell line’s conditioned growth media were isolated by ultracentrifugation and precipitation, respectively. Human purified CD3+CD8+ T cells were assessed for their induction of the T cell SP by flow cytometry identifying loss of CD27/CD28 expression and in vitro suppression assays. Furthermore, the T cell SP was characterized for the attenuation of IFN-γ production. To delineate exosomal proteins contributing to T cell SP, mass spectrometry was used to identify unique proteins that were present in TDEs. CRISPR/Cas9 knockout constructs were used to examine the role of one of these proteins, galectin-1. To assess the role of exosomal RNA, RNA purified from TDEs was nucleofected into CD8+ T cells followed by suppression analysis.ResultsUsing fractionated conditioned growth media, factors >200 kDa induced CD8+ T cell SP, which was determined to be an exosome by mass spectrometry analysis. Multiple head and neck cancer-derived cell lines were found to secrete T cell SP-inducing exosomes. Mass spectrometry analysis revealed that an immunoregulatory protein, galectin-1 (Gal-1), was expressed in those exosomes, but not in TDEs unable to induce T cell SP. Galectin-1 knockout cells were found to be less able to induce T cell SP. Furthermore, RNA purified from the T cell SP-inducing exosomes were found to partially induce the SP when transfected into normal CD8+ T cells.ConclusionsFor the first-time, TDEs have been identified to induce a SP in CD8+ T cells and their mode of action may be synergistic effects from exosomal proteins and RNA. One protein in particular, galectin-1, appears to play a significant role in inducing T cell SP. Therefore, tumor-derived immunosuppressive exosomes are a potential therapeutic target to prevent T cell dysfunction and enhance anti-tumor immune responses.
Subclinical doses of Paclitaxel (PTX) given 1 day prior to a HER-2/neu (neu)-targeted, granulocytemacrophage colony stimulating factor (GM-CSF)-secreting whole-cell vaccine enhances neuspecific 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 DC's from vaccine alone-treated neu-N mice. Finally, PTX treatment results in enhanced antigen-specific, IFN-γ-secreting CD8 + T cells in vivo. Thus, administration of PTX with a tumor vaccine improves T cell priming through enhanced maturation of DC.
Although senescence in oncogenesis has been widely studied, little is known regarding the role of this process in chemotherapy resistance. Thus, from the standpoint of enhancing and improving cancer therapy, a better understanding of the molecular machinery involved in chemotherapy-related senescence is paramount. We show for the first time that Mcl-1, a Bcl-2 family member, plays an important role in preventing chemotherapy-induced senescence (CIS ؉ cells conferred resistance to CIS and promoted tumor outgrowth. In summary, our data reveal that Mcl-1 can inhibit CIS in both a p53-dependent and -independent manner in vitro and in vivo and that this Mcl-1-mediated inhibition can enhance tumor growth in vivo.
Purpose We have previously reported that many types of tumors are able to induce changes in human T cells that lead to the acquisition of suppressive function as well as phenotypic alterations resembling those found in senescent T cells. In the present study we find a role for IL-7 in protecting T cells from these changes, and further define involved signaling pathways. Experimental Design We evaluated the ability of IL-7 treatment to prevent the gain of suppressive function and phenotypic alterations in human T cells after a short co-culture with tumor cells in vitro. We then used inhibitors of components of the PI3K/AKT pathway and siRNA knock-down of Mcl-1 and Bim to evaluate the role of these signaling pathways in IL-7 protection. Results We found that IL-7 inhibits CD27/CD28 loss and maintains proliferative capacity, IL-2 production, and reduced suppressive function. IL-7’s protective ability depended on activation of the PI3K/AKT pathway, which inhibited activation of glycogen synthase kinase 3β (GSK3β) which subsequently prevented the phosphorylation and loss of Mcl-1. We further demonstrated a key role for Mcl-1 in that its knock-down or inhibition abrogated the effects of IL-7. Additionally, knock-down of the Mcl-1 binding partner and pro-apoptotic protein Bim protected T cells from these dysfunctional alterations. Conclusion These observations confirm the role for Bcl-2 family members in cytokine signaling, and suggest that IL-7 treatment in combination with other immunotherapies could lead to new clinical strategies to maintain normal T cell function and reduce tumor-induced generation of dysfunctional and suppressor T cells.
Immune-checkpoint blockade enhances antitumor responses against cancers. One cancer type that is sensitive to checkpoint blockade is squamous cell carcinoma of the head and neck (SCCHN), which we use here to study limitations of this treatment modality. We observed that CD8 þ tumor-infiltrating lymphocytes (TILs) in SCCHN and melanoma express excess immune checkpoints components PD-1 and Tim-3 and are also CD27 À /CD28 À , a phenotype we previously associated with immune dysfunction and suppression. In ex vivo experiments, patients' CD8 þ TILs with this phenotype suppressed proliferation of autologous peripheral blood T cells. Similar phenotype and function of TILs was observed in the TC-1 mouse tumor model. Treatment of TC-1 tumors with anti-PD-1 or anti-Tim-3 slowed tumor growth in vivo and reversed the suppressive function of multi-checkpoint þ CD8 þ TIL. Similarly, treatment of both human and mouse PD-1 þ Tim-3 þ CD8 þ TILs with anticheckpoint antibodies ex vivo reversed their suppressive function. These suppressive CD8 þ TILs from mice and humans expressed ligands for PD-1 and Tim-3 and exerted their suppressive function via IL10 and close contact. To model therapeutic strategies, we combined anti-PD-1 blockade with IL7 cytokine therapy or with transfer of antigen-specific T cells. Both strategies resulted in synergistic antitumor effects and reduced suppressor cell function. These findings enhance our understanding of checkpoint blockade in cancer treatment and identify strategies to promote synergistic activities in the context of other immunotherapies.
2019) Combination PD-1 blockade and irradiation of brain metastasis induces an effective abscopal effect in melanoma, OncoImmunology, 8:1, e1507669, ABSTRACT Nearly half of melanoma patients develop brain metastases during the course of their disease. Despite advances in both localized radiation and systemic immunotherapy, brain metastases remain difficult to treat, with most patients surviving less than 5 months from the time of diagnosis. While both treatment regimens have individually shown considerable promise in treating metastatic melanoma, there is interest in combining these strategies to take advantage of potential synergy. In order to study the ability of local radiation and anti-PD-1 immunotherapy to induce beneficial anti-tumor immune responses against distant, unirradiated tumors, we used two mouse models of metastatic melanoma in the brain, representing BRAF mutant and non-mutant tumors. Combination treatments produced a stronger systemic anti-tumor immune response than either treatment alone. This resulted in reduced tumor growth and larger numbers of activated, cytotoxic CD8 + T cells, even in the unirradiated tumor, indicative of an abscopal effect. The immune-mediated effects were present regardless of BRAF status. These data suggest that irradiation of brain metastases and anti-PD-1 immunotherapy together can induce abscopal anti-tumor responses that control both local and distant disease. ARTICLE HISTORY
Background:Mcl-1 is a prosurvival gene critical for chemotherapy resistance. However, its antisenescence properties are poorly characterized. Results: Through mutagenesis of Mcl-1 in functional assays, we identified a loop domain required for inhibiting senescence. Conclusion: An internal loop domain of Mcl-1 is responsible for antisenescence functions. Significance: Our study provides additional targets within Mcl-1 that can enhance senescence-inducing cancer treatments.
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