Induction Of Effective Anti-tumor Immunity Research Articles

Targeting myeloid-derived suppressor cells in combination with tumor cell vaccination predicts anti-tumor immunity and breast cancer dormancy: an in silico experiment

Among the different breast cancer subsets, triple-negative breast cancer (TNBC) has the worst prognosis and limited options for targeted therapies. Immunotherapies are emerging as novel treatment opportunities for TNBC. However, the surging immune response elicited by immunotherapies to eradicate cancer cells can select resistant cancer cells, which may result in immune escape and tumor evolution and progression. Alternatively, maintaining the equilibrium phase of the immune response may be advantageous for keeping a long-term immune response in the presence of a small-size residual tumor. Myeloid-derived suppressor cells (MDSCs) are activated, expanded, and recruited to the tumor microenvironment by tumor-derived signals and can shape a pro-tumorigenic micro-environment by suppressing the innate and adaptive anti-tumor immune responses. We recently proposed a model describing immune-mediated breast cancer dormancy instigated by a vaccine consisting of dormant, immunogenic breast cancer cells derived from the murine 4T1 TNBC-like cell line. Strikingly, these 4T1-derived dormant cells recruited fewer MDSCs compared to aggressive 4T1 cells. Recent experimental studies demonstrated that inactivating MDSCs has a profound impact on reconstituting immune surveillance against the tumor. Here, we developed a deterministic mathematical model for simulating MDSCs depletion from mice bearing aggressive 4T1 tumors resulting in immunomodulation. Our computational simulations indicate that a vaccination strategy with a small number of tumor cells in combination with MDSC depletion can elicit an effective immune response suppressing the growth of a subsequent challenge with aggressive tumor cells, resulting in sustained tumor dormancy. The results predict a novel therapeutic opportunity based on the induction of effective anti-tumor immunity and tumor dormancy.

Integrative epigenetic analysis reveals AP-1 promotes activation of tumor-infiltrating regulatory T cells in HCC.

Regulatory T (Treg) cells that infiltrate human tumors exhibit stronger immunosuppressive activity compared to peripheral blood Treg cells (PBTRs), thus hindering the induction of effective antitumor immunity. Previous transcriptome studies have identified a set of genes that are conserved in tumor-infiltrating Treg cells (TITRs). However, epigenetic profiles of TITRs have not yet been completely deciphered. Here, we employed ATAC-seq and CUT&Tag assays to integrate transcriptome profiles and identify functional regulatory elements in TITRs. We observed a global difference in chromatin accessibility and enhancer landscapes between TITRs and PBTRs. We identified two types of active enhancer formation in TITRs. The H3K4me1-predetermined enhancers are poised to be activated in response to tumor microenvironmental stimuli. We found that AP-1 family motifs are enriched at the enhancer regions of TITRs. Finally, we validated that c-Jun binds at regulatory regions to regulate signature genes of TITRs and AP-1 is required for Treg cells activation in vitro. High c-Jun expression is correlated with poor survival in human HCC. Overall, our results provide insights into the mechanism of AP-1-mediated activation of TITRs and can hopefully be used to develop new therapeutic strategies targeting TITRs in liver cancer treatment.

Cytokine Therapeutics in Cancer Immunotherapy: Design and Development

In this article, recent advances in the application of cytokine therapeutics in cancer, as single agents or in combination with other immunotherapies, are reviewed. Additionally, critical characteristics that govern cytokine exposure-response properties are discussed. Immunotherapy has revolutionized the traditional approach to treating cancer. Targeted therapies, via modulation of the host immune responses, have elevated the expectations for a beneficial impact on patients’ lives. Novel drugs that modulate various steps in the cancer-immunity cycle are now available with an impact on both the magnitude and duration of effect relative to traditional chemotherapeutic approaches. Due to cancer heterogeneity, induction of effective anti-tumor immunity requires the orchestration of several critical events, and hence, combination approaches are being explored. Combination immunotherapy is anticipated to enhance the durability and efficacy profiles of otherwise single agents that can benefit from the “synergistic” effects from the engagement of two or more pathways, simultaneously. As cytokines regulate many aspects of innate and adaptive immunity, application of cytokines in cancer immunotherapy has attracted considerable attention. Application of cytokines in the treatment of cancer is limited by their pleiotropic effects, toxicity at required clinical doses, and unfavorable pharmacokinetic properties. Recent advances in protein engineering have now made it possible to improve the undesirable properties of these biologics. Additionally, the recent breakthroughs in the treatment of cancer, using immunotherapies, have renewed interest in evaluating cytokines and their variants in combination regiments to improve immunity against malignant cells.

Selectively hampered activation of lymph node-resident dendritic cells precedes profound T cell suppression and metastatic spread in the breast cancer sentinel lymph node

BackgroundImmune regulated pathways influence both breast cancer (BrC) development and response to (neo)adjuvant chemotherapy. The sentinel lymph node (SLN), as the first metastatic site, is also the first site where BrC-induced suppression of immune effector subsets occurs. Since intricate knowledge of the phenotypic and functional status of these immune effector subsets is lacking, we set out to map the immune landscape of BrC SLN.MethodsViable LN cells from BrC SLN (n = 58) were used for detailed flowcytometry-assisted mapping of the immune landscape of BrC SLN in a comparative analysis with healthy (i.e. prophylactic mastectomy-derived) axillary lymph nodes (HLN, n = 17). Findings were related to clinicopathological characteristics.ResultsOur data show that BrC-induced immune suppression in tumor-involved SLN, as evidenced by increased Treg and MDSC rates as well as by a generalized state of T cell anergy, coincides with hampered activation of LN-resident (LNR) dendritic cell (DC) subsets rather than of migratory DC subsets. Importantly, suppression of these LN-resident DC subsets preceded profoundly disabled T cell effector functions in tumor-involved SLN. Furthermore, we provide evidence that the suppressed state of LNR-cDC is not only related to nodal involvement but is also related to high-risk breast cancer subtypes that lack expression of hormone receptors and may be a negative predictor of disease-free survival.ConclusionThese data thus provide new insights in the mechanisms underlying loco-regional immune suppression induced by BrC and how these relate to clinical outcome. They identify the LNR-cDC subset as a pivotal regulatory node in cellular immune suppressive pathways and therefore as a promising therapeutic target to combat immune suppression and secure the induction of effective antitumor immunity, e.g. in combination with neo-adjuvant chemotherapy.

Combination Strategies to Optimize Efficacy of Dendritic Cell-Based Immunotherapy.

Dendritic cells (DCs) are antigen-presenting cells (APCs) that are essential for the activation of immune responses. In various malignancies, these immunostimulatory properties are exploited by DC-therapy, aiming at the induction of effective anti-tumor immunity by vaccination with ex vivo antigen-loaded DCs. Depending on the type of DC-therapy used, long-term clinical efficacy upon DC-therapy remains restricted to a proportion of patients, likely due to lack of immunogenicity of tumor cells, presence of a stromal compartment, and the suppressive tumor microenvironment (TME), thereby leading to the development of resistance. In order to circumvent tumor-induced suppressive mechanisms and unleash the full potential of DC-therapy, considerable efforts have been made to combine DC-therapy with chemotherapy, radiotherapy or with checkpoint inhibitors. These combination strategies could enhance tumor immunogenicity, stimulate endogenous DCs following immunogenic cell death, improve infiltration of cytotoxic T lymphocytes (CTLs) or specifically deplete immunosuppressive cells in the TME, such as regulatory T-cells and myeloid-derived suppressor cells. In this review, different strategies of combining DC-therapy with immunomodulatory treatments will be discussed. These strategies and insights will improve and guide DC-based combination immunotherapies with the aim of further improving patient prognosis and care.

Abstract A51: Intradermal delivery of polymeric nanoparticle based vaccine formulation using a hollow microneedle system

Abstract Introduction: Nanoparticle (NP)-based anticancer vaccine delivery systems provide a number of advantages including spatial repetitive display of the antigen, size dependent lymphatic and intracellular trafficking, and the depot effect of persistence and sustained release of antigens/adjuvants. However, the optimal NP delivery mode and route of administration need additional investigation. To achieve reproducible intradermal PLGA NP vaccine delivery using an in-vivo rat model, we used a hollow microneedle array system (1). The profile of the resulting humoral and cellular immune responses was compared to that resulting from conventional intramuscular (IM) injection of the same formulation. Methods: Immunostimulatory Nanoparticles (ISNPs) loaded with imiquimod and monophosphoryl Lipid A (MPL-A) were created using emulsion- solvent evaporation technique. All vaccine formulations for the in vivo study contained 2 mg of ISNPs as the adjuvant component. Antigen was given either in the form of soluble OVA solution or OVA encapsulated PLGA NPs at equivalent doses of OVA (16 µg). Two doses of vaccine (prime and boost) were administered to Crl:CD(SD) rats three weeks apart via IM or ID delivery. ID delivery was enabled by a plastic, hollow microneedle array with 18 microneedles, each <1mm in length, similar to those referenced previously (1). Blood collection was carried out three weeks after each dose. Serially-diluted serum was incubated with OVA coated plate for IgG1/IgG2a titer test by ELISA. Spleens were harvested at the endpoint of the experiment (three weeks after the booster dose) for splenocyte isolation. The T cell response was determined by pulsing the splenocyte culture with OVA antigen using a Mabtech rat Interferon gamma ELISpot kit. Results and Discussion: ISNPs promoted high antigen-specific antibody response in both IM and ID vaccination groups. Compared to the soluble form, antigen encapsulated in NPs triggered significantly stronger IgG1 antibody response three weeks after priming dose regardless of administration route. Not only did the NP formulation demonstrate faster kinetics in mounting antibody response, but the antibody avidity, determined by Urea ELISA, was also better in the OVA NP formulation when compared to the soluble OVA formulation, indicating quicker progress of antibody affinity maturation. However, three weeks after the boost, IgG1 level of all groups were comparable, reaching a titer plateau. In contrast to the IgG1 response, antigen OVA encapsulated in NPs and delivered intradermally via the microneedles resulted in the highest IgG2a titer among all experimental groups at both time points after both priming and booster doses. IgG2a is widely considered a surrogate biomarker for Th1 response, while IgG1 is a biomarker for Th2 response. The relative strength of Th1/Th2 response could be assessed from IgG1/IgG2a ratio (2). Among the different groups, ID delivery of the OVA NP formulation demonstrated the highest relative strength of Th1 response. This result was further confirmed by the secretion of Th1 cytokine Interferon gamma by splenocytes upon antigen recall response. These data suggest that for a NP vaccine carrier, the skin is a vaccination priming locus that enhances Th1 response, which is considered essential for effective induction of anti-tumor immunity. Conclusion: A polymer-based NP vaccine formulation delivered intradermally via hollow microneedles elicited robust humoral and cellular immunity. When compared to IM injection, the unique combination of the polymeric NP formulation and the ID route of administration led to an optimal Th1 immune response.

Chemotherapy enhances cross-presentation of nuclear tumor antigens.

Cross-presentation of tumor antigen is essential for efficient priming of naïve CD8+ T lymphocytes and induction of effective anti-tumor immunity. We hypothesized that the subcellular location of a tumor antigen could affect the efficiency of cross-presentation, and hence the outcome of anti-tumor responses to that antigen. We compared cross-presentation of a nominal antigen expressed in the nuclear, secretory, or cytoplasmic compartments of B16 melanoma tumors. All tumors expressed similar levels of the antigen. The antigen was cross-presented from all compartments but when the concentration was low, nuclear antigen was less efficiently cross-presented than antigen from other cellular locations. The efficiency of cross-presentation of the nuclear antigen was improved following chemotherapy-induced tumor cell apoptosis and this correlated with an increase in the proportion of effector CTL. These data demonstrate that chemotherapy improves nuclear tumor antigen cross-presentation and could be important for anti-cancer immunotherapies that target nuclear antigens.

Effective induction of anti-tumor immunity using p5 HER-2/neu derived peptide encapsulated in fusogenic DOTAP cationic liposomes co-administrated with CpG-ODN

Cationic liposomes have been used as efficient antigen delivery systems for cancer vaccination. The current study has investigated whether the incorporation of the helper-fusogenic lipid dioleoylphosphatidylethanolamine (DOPE) in cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)–cholesterol enhances the cytosolic delivery of p5 HER-2/neu derived peptide (p5) and promotes cytotoxic T lymphocytes (CTL) response. The p5, which is a very hydrophobic peptide, was encapsulated into liposomes by using three different methods and characterized for their colloidal properties. A chaotropic loading method using 7M urea provided the highest encapsulation yields. Mice were first immunized with encapsulated p5 in liposomes composed of either DOTAP–cholesterol or DOTAP–cholesterol–DOPE, alone or co-administered with CpG-ODN, as an immunoadjuvant, then, inoculated with a subcutaneous injection of TUBO tumor cells. Results obtained from enzyme-linked immunospot, cytotoxicity and intracellular cytokine assays as well as tumor sizes and animal survival analysis demonstrated that p5 encapsulated in DOTAP–cholesterol–DOPE liposomes co-administered with CpG-ODN greatly enhanced the cytotoxic T lymphocytes response and highly inhibited the tumor progression. The outperformance of DOTAP–cholesterol–DOPE liposomes+CpG-ODN was found to be attributed to its capability in induction of both CD8+ and CD4+ responses. This formulation could be a suitable vaccine candidate against Her2 positive cancers and merits further investigations.

Anti-CCR4 mAb selectively depletes effector-type FoxP3+CD4+regulatory T cells, evoking antitumor immune responses in humans

CD4(+) Treg cells expressing the transcription factor FOXP3 (forkhead box P3) are abundant in tumor tissues and appear to hinder the induction of effective antitumor immunity. A substantial number of T cells, including Treg cells, in tumor tissues and peripheral blood express C-C chemokine receptor 4 (CCR4). Here we show that CCR4 was specifically expressed by a subset of terminally differentiated and most suppressive CD45RA(-)FOXP3(hi)CD4(+) Treg cells [designated effector Treg (eTreg) cells], but not by CD45RA(+)FOXP3(lo)CD4(+) naive Treg cells, in peripheral blood of healthy individuals and cancer patients. In melanoma tissues, CCR4(+) eTreg cells were predominant among tumor-infiltrating FOXP3(+) T cells and much higher in frequency compared with those in peripheral blood. With peripheral blood lymphocytes from healthy individuals and melanoma patients, ex vivo depletion of CCR4(+) T cells and subsequent in vitro stimulation of the depleted cell population with the cancer/testis antigen NY-ESO-1 efficiently induced NY-ESO-1-specific CD4(+) T cells. Nondepletion failed in the induction. The magnitude of the responses was comparable with total removal of FOXP3(+) Treg cells by CD25(+) T-cell depletion. CCR4(+) T-cell depletion also augmented in vitro induction of NY-ESO-1-specific CD8(+) T cells in melanoma patients. Furthermore, in vivo administration of anti-CCR4 mAb markedly reduced the eTreg-cell fraction and augmented NY-ESO-1-specific CD8(+) T-cell responses in an adult T-cell leukemia-lymphoma patient whose leukemic cells expressed NY-ESO-1. Collectively, these findings indicate that anti-CCR4 mAb treatment is instrumental for evoking and augmenting antitumor immunity in cancer patients by selectively depleting eTreg cells.

In situ immune modulation therapy with a novel chimeric chaperone generates a potent antitumor response (P4271)

Abstract The immunosuppressive tumor microenvironment (TME) represents a major hurdle for cancer immunotherapy. In the present study, we evaluate the feasibility of conditioning the immune environment in the tumor sites for induction of effective antitumor immunity using a novel immune modulator Flagrp170. This chaperone-based immune modulating agent is created by integrating a pathogen (i.e., flagellin)-derived ‘danger’ signal into Grp170, the largest chaperone resident in the endoplasmic reticulum. This chimeric chaperone molecule is highly efficient in inducing functional activation of dendritic cells via engaging the NF-κB and MAPK signaling pathways, and concurrently facilitating cross-presentation of tumor antigen. Intratumoral delivery of adenoviruses encoding Flagrp170 provokes a strong tumor-specific cytotoxic T lymphocyte response, leading to profound inhibition of tumor growth and metastasis. In situ Flagrp170 therapy preferentially drives the Th1 polarization of the TME, as evidenced by marked elevation of IL-12 and IFN-γ as well as increased tumor infiltration by CD8+ cells. Antibody depletion and blocking assays reveal that CD8+ cells and IFN-γ are essential for enhanced antitumor activity of Flagrp170. Our data demonstrate that this modified chaperone represents a promising immune modulating agent that may be utilized to potentially reverse immunosuppression in the TME and improve immunotherapeutic response to metastatic malignancies.

HLA Class II Disparity Is Necessary and Sufficient for Induction of Effective Anti-Tumor Immunity by Donor Lymphocyte Infusion in a NOD/Scid Mouse Model for Human Acute Lymphoblastic Leukemia

Abstract 648Donor lymphocyte infusion (DLI) can be a curative treatment for patients with relapsed hematological malignancies after HLA matched allogeneic stem cell transplantation (alloSCT). However, curative responses in patients with acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia in lymphoid blastic phase (CML-BP) are infrequent after HLA matched DLI. This may be partly explained by the poor immunogenicity of these malignancies, since we previously demonstrated efficient induction of Graft-versus-Leukemia (GvL) immune responses in vitro and in vivo upon modification of ALL and CML-BP cells into leukemic antigen presenting cells (APC). Leukemic-APC may be particularly relevant for efficient generation of GvL immune responses after HLA matched DLI, since T cells recognizing allo-antigens in matched HLA molecules are known to reside in the naïve T cell compartment. In contrast, T cells recognizing allo-antigens in mismatched HLA molecules reside in the memory T cell compartment as well. Since memory T cells can also be activated by non-professional APC, HLA mismatched alloSCT and DLI may particularly be considered as a treatment modality for induction of GvL reactivity against poorly immunogenic malignancies. However, T cell responses across HLA barriers can induce severe Graft-versus-Host Disease (GvHD). Mismatched HLA class I molecules, which are broadly expressed on all nucleated cells, are frequent targets of alloreactive T cells. Since HLA class II molecules are predominantly expressed on hematopoietic cells, HLA class II mismatched alloSCT and DLI may more selectively induce GvL reactivity without inducing severe GvHD. In this study, we investigated the in vivo immunogenicity of established B-ALL or CML-BP by comparing the anti-tumor responses after fully HLA matched versus HLA class II mismatched DLI in a NOD/scid mouse model.Mice engrafted with primary B-ALL and CML-BP were treated with DLI from HLA matched (12/12 match) or HLA class II mismatched, but HLA class I matched donors. In mice engrafted with B-ALL or CML-BP, treatment with HLA matched DLI induced expansion of human CD4+ and CD8+ T cells in peripheral blood, but leukemic cells were only delayed in growth, and not eliminated. In contrast, after HLA class II mismatched DLI, leukemic cells rapidly disappeared upon emergence of human CD4+ and CD8+ T cells in peripheral blood. To analyze the specificity of the T cells, we clonally isolated CD4+ and CD8+ T cells from bone marrow and spleens of mice after treatment with DLI. All T cell clones were tested for recognition of patient leukemic cells, donor EBV transformed B cells (EBV-LCL) and murine bone marrow derived dendritic cells in IFNg ELISA. Isolated CD8+ and CD4+ T cell clones recognized either patient leukemic cells or murine cells, indicating that the T cell clones were either leukemia-reactive or xeno-reactive. After HLA matched DLI, only 2 of the 106 CD4+ T cell clones, and none of the 183 CD8+ T cell clones, recognized patient leukemic cells. The majority of isolated CD4+ and CD8+ T cell clones were xeno-reactive, as demonstrated by specific recognition of murine bone marrow derived dendritic cells, or non-reactive against any of the tested target cells. In contrast, after HLA class II mismatched DLI, 95 of the 322 CD4+ T cell clones specifically recognized patient leukemic cells. These leukemia-reactive CD4+ T cell clones were shown to be restricted by the mismatched HLA-DRB3, -DQB1 and –DPB1 alleles of the patient. None of the 49 CD8+ T cell clones were leukemia-reactive, but a significant number of CD8+ T cell clones and remaining CD4+ were xeno-reactive.In conclusion, our data show that HLA class II mismatched, but HLA class I matched, DLI is far more effective in inducing anti-tumor reactivity as compared to HLA matched DLI, whereas the in vivo capacity of both DLI's to induce allo-immune reactivity based on the induction of xeno-reactive T cells was similar. Our study emphasizes the necessity of HLA class II disparity for efficient in vivo induction of HLA class II mediated anti-tumor immunity against poorly immunogenic B-ALL and CML-BP in NOD/scid mice. We therefore hypothesize that use of HLA class II mismatched as compared to HLA matched alloSCT and DLI, despite an increased risk for GvHD, may improve the outcome for patients with HLA class II positive high risk acute lymphoblastic leukemia. Disclosures:No relevant conflicts of interest to declare.

A peptide epitope derived from the cancer testis antigen HOM-MEL-40/SSX2 capable of inducing CD4⁺ and CD8⁺ T-cell as well as B-cell responses.

Antigen-derived HLA class I-restricted peptides can generate specific CD8(+) T-cell responses in vivo and are therefore often used as vaccines for patients with cancer. However, only occasional objective clinical responses have been reported suggesting the necessity of CD4(+) T-cell help and possibly antibodies for the induction of an effective anti-tumor immunity in vivo. The SSX2 gene encodes the cancer testis antigen (CTA) HOM-MEL-40/SSX2, which is frequently expressed in a wide spectrum of cancers. Both humoral and cellular immune responses against SSX2 have been described making SSX2 an attractive candidate for vaccine trials. SYFPEITHI algorithm was used to predict five pentadecamer peptides with a high binding probability for six selected HLA-DRB1 subtypes (*0101, *0301, *0401, *0701, *1101, *1501) which are prevalent in the Caucasian population. Using peripheral blood cells of 13 cancer patients and 5 healthy controls, the HOM-MEL-40/SSX2-derived peptide p101-111 was identified as an epitope with dual immunogenicity for both CD4(+) helper and cytotoxic CD8(+) T cells. This epitope also reacted with anti-SSX2 antibodies in the serum of a patient with breast cancer. Most remarkably, SSX2/p101-111 simultaneously induced specific CD8, CD4, and antibody responses in vitro. p101-111 is the first CTA-derived peptide which induces CD4(+), CD8(+), and B-cell responses in vitro. This triple-immunogenic peptide represents an attractive vaccine candidate for the induction of effective anti-tumor immunity.

Vaccines for glioblastoma and high-grade glioma

Vaccination by administering tumor antigen plus cell-free or cellular adjuvant has garnered hope for more effective, less toxic therapy for patients with malignant brain tumors including glioblastoma multiforme. To determine if this approach demonstrates ample clinical promise, all published reports of vaccination for glioma were evaluated. These reports suggest vaccination is associated with low toxicity and favorable clinical outcomes. The possibility of selection bias is evident in many published vaccine trials, but several of the more recent ones appropriately attempt to account for bias. Effective induction of antitumor immunity is consistently observed, and, in the latest trials, correlates with significant clinical improvement.

TLR2-Dependent Induction of IL-10 and Foxp3+CD25+CD4+ Regulatory T Cells Prevents Effective Anti-Tumor Immunity Induced by Pam2 Lipopeptides In Vivo

16 S-[2,3-bis(palmitoyl)propyl]cysteine (Pam2) lipopeptides act as toll-like receptor (TLR)2/6 ligands and activate natural killer (NK) cells and dendritic cells (DCs) to produce inflammatory cytokines and cytotoxic NK activity in vitro. However, in this study, we found that systemic injection of Pam2 lipopeptides was not effective for the suppression of NK-sensitive B16 melanomas in vivo. When we investigated the immune suppressive mechanisms, systemic injection of Pam2 lipopeptides induced IL-10 in a TLR2-dependent manner. The Pam2 lipopeptides increased the frequencies of Foxp3+CD4+ regulatory T (T reg) cells in a TLR2- and IL-10- dependent manner. The T reg cells from Pam2-lipopeptide injected mice maintained suppressor activity. Pam2 lipopeptides, plus the depletion of T reg with an anti-CD25 monoclonal antibody, improved tumor growth compared with Pam2 lipopeptides alone. In conclusion, our data suggested that systemic treatment of Pam2 lipopeptides promoted IL-10 production and T reg function, which suppressed the effective induction of anti-tumor immunity in vivo. It is necessary to develop an adjuvant that does not promote IL-10 and T reg function in vivo for the future establishment of an anti-cancer vaccine.

Allogeneic effector/memory Th-1 cells impair FoxP3+ regulatory T lymphocytes and synergize with chaperone-rich cell lysate vaccine to treat leukemia

AbstractTherapeutic strategies combining the induction of effective antitumor immunity with the inhibition of the mechanisms of tumor-induced immunosuppression represent a key objective in cancer immunotherapy. Herein we demonstrate that effector/memory CD4+ T helper-1 (Th-1) lymphocytes, in addition to polarizing type-1 antitumor immune responses, impair tumor-induced CD4+CD25+FoxP3+ regulatory T lymphocyte (Treg) immunosuppressive function in vitro and in vivo. Th-1 cells also inhibit the generation of FoxP3+ Tregs from naive CD4+CD25−FoxP3− T cells by an interferon-γ–dependent mechanism. In addition, in an aggressive mouse leukemia model (12B1), Th-1 lymphocytes act synergistically with a chaperone-rich cell lysate (CRCL) vaccine, leading to improved survival and long-lasting protection against leukemia. The combination of CRCL as a source of tumor-specific antigens and Th-1 lymphocytes as an adjuvant has the potential to stimulate efficient specific antitumor immunity while restraining Treg-induced suppression.

Administration route‐dependent induction of antitumor immunity by interferon‐alpha gene transfer

Type I interferon (IFN) protein is a cytokine with pleiotropic biological functions that include induction of apoptosis, inhibition of angiogenesis, and immunomodulation. We have demonstrated that intratumoral injection of an IFN-alpha-expressing adenovirus effectively induces cell death of cancer cells and elicits a systemic tumor-specific immunity in several animal models. On the other hand, reports demonstrated that an elevation of IFN in the serum following an intramuscular delivery of a vector is able to activate antitumor immunity. In this study, we compared the intratumoral and systemic routes of IFN gene transfer with regard to the effect and safety of the treatment. Intratumoral injection of an IFN-alpha adenovirus effectively activated tumor-responsive lymphocytes and caused tumor suppression not only in the gene-transduced tumors but also in distant tumors, which was more effective than the intravenous administration of the same vector. The expression of co-stimulatory molecules on CD11c(+) cells isolated from regional lymph nodes was enhanced by IFN gene transfer into the tumors. Systemic toxicity such as an elevation of hepatic enzymes was much lower in mice treated by intratumoral gene transfer than in those treated by systemic gene transfer. Our data suggest that the intratumoral route of the IFN vector is superior to intravenous administration, due to the effective induction of antitumor immunity and the lower toxicity.

DCVax®-Brain and DC vaccines in the treatment of GBM

Background: DCVax®-Brain (Northwest Biotherapeutics, Inc., Bethesda, MD, USA) is a personalized treatment for brain tumors. Its approach of administering autologous tumor antigen-bearing dendritic cells (DCs) has garnered hope for more effective and less toxic therapy for patients with malignant brain tumors including glioblastoma multiforme (GBM). DCVax-Brain composition and efficacy are not fully disclosed, although sponsors claim it is poised to critically test clinical DC vaccine efficacy in GBM patients. Objective: This review examines the efficacy of DC vaccine therapy in treating GBM patients. Review question: To determine if the approach of DC vaccination followed by DCVax-Brain shows ample clinical promise in GBM patients. Search strategy: All published reports of DC vaccination for GBM and press releases regarding DCVax-Brain findings were evaluated. Critical appraisal of reports and summary of outcomes: Published DC vaccine trials for high-grade glioma patients suggest favorable clinical outcomes not easily ascribed to non-treatment parameters. Evidence of possible selection bias exists in many reports, but efforts to account for this are evident in the most recent publications. Conclusion: DC vaccine trials provide evidence of low toxicity in GBM patients and effective induction of antitumor immunity in the latest publications correlate with clinical improvements. Preliminary reports on DCVax-Brain clinical outcomes seem to follow these trends.

Effect of Lipofectin on Antigen-presenting Function and Anti-tumor Activity of Dendritic Cells

Dendritic cells (DC) are professional antigen-presenting cells in the immune system and can induce T cell response against virus infections, microbial pathogens, and tumors. Therefore, immunization using DC loaded with tumor-associated antigens (TAAs) is a powerful method of inducing anti-tumor immunity. For induction of effective anti-tumor immunity, antigens should be efficiently introduced into DC and presented on MHC class I molecules at high levels to activate antigen-specific CD8 + T cells. We have been exploring methods for loading exogenous antigens into APC with high efficiency of Ag presentation. In this study, we tested the effect of the cationic liposome (Lipofectin) for transferring and loading exogenous model antigen (OVA protein) into BM-DC. Methods: Bone marrow-derived DC (EM-DC) were incubated with OVA-Lipofectin complexes and then co-cultured with B3Z cells. B3Z activation, which is expressed as the amount of β-galactosidase induced by TCR stimulation, was determined by an enzymatic assay using β-gal assay system. C57BL/6 mice were immunized with OVA-pulsed DC to monitor the in vivo vaccination effect. After vaccination, mice were inoculated with EG7-OVA tumor cells. Results: BM-DC pulsed with OVA-Lipofectin complexes showed more efficient presentation of OVA-peptide on MHC class I molecules than soluble OVA-pulsed DC. OVA-Lipofectin complexes-pulsed DC pretreated with an inhibitor of MHC class I-mediated antigen presentation, brefeldin A, showed reduced ability in presenting OVA peptide on their surface MHC class I molecules. Finally, immunization of OVA-Lipofectin complexes-pulsed DC protected mice against subsequent tumor challenge. Conclusion: Our data provide evidence that antigen-loading into DC using Lipofectin can promote MHC class I- restricted antigen presentation. Therefore, antigen-loading into DC using Lipofectin can be one of several useful tools for achieving efficient induction of antigen-specific immunity in DC-based immunotherapy.

Effective induction of antitumor immunity by immunization with plasmid DNA encoding TRP-2 plus neutralization of TGF-beta.

Plasmid DNA vaccine is an appealing cancer immunotherapy. However, it is a weak immunogen and immunization with plasmid DNA encoding self-antigens, such as melanoma-associated antigens, could not induce antitumor immunity because of tolerance. In this study, we investigated the feasibility of using a plasmid DNA encoding Xenopus laevis transforming growth factor-beta 5 (aTGF-beta5) as an immunogen to induce neutralizing antibodies against murine TGF-beta1 (mTGF-beta1) and thus enhance the efficacy of plasmid DNA vaccine encoding murine tyrosinase-related protein 2 (mTRP-2) through neutralization of TGF-beta. The results showed that immunization with aTGF-beta5 resulted in the generation of mTGF-beta1-neutralizing antibodies, and immunization with a combination of aTGF-beta5 and mTRP-2 induced specific cytotoxic T lymphocytes (CTLs). On the contrary, immunization with mTRP-2 alone could not elicit the CTL response. Moreover, immunization of C57BL/6 wild-type mice with a combination of aTGF-beta5 and mTRP-2 induced the protective and therapeutic antitumor immunity to B16F10 melanoma, whereas the antitumor activity was abrogated in both CD4-deficient mice and CD8-deficient mice on the C57BL/6 background. Our results indicate that immunization with aTGF-beta5 is capable of breaking immune tolerance and induces mTGF-beta1-neutralizing antibodies. Neutralization of TGF-beta can enhance the efficacy of DNA vaccine encoding mTRP-2 and the induction of antitumor immunity by this immunization strategy is associated with CD4+ and CD8+ T cells.

The N-terminal flanking region of the invariant chain peptide augments the immunogenicity of a cryptic "self" epitope from a tumor-associated antigen.

The N-terminal flanking region of the invariant chain peptide termed CLIP appears to have superagonistic properties interacting with the T cell receptor and the MHC class II molecule at or near the binding site for the bacterial superantigen Staphylococcal enterotoxin B (SEB). The present studies explored the hypothesis that the N-terminal segment of CLIP can augment the immunogenicity of cryptic "self" tumor-associated antigens. A chimeric construct of an MHC class II binding peptide from the c-erb oncogene (Her-2/neu) containing the N-terminal flanking region of CLIP elicited potent antitumor activity against a Her-2/neu-positive tumor in a rat model system. Comparatively, the unmodified parent peptide was ineffective. The induction of effective antitumor immunity, however, required presentation of the chimeric peptide construct on irradiated tumor cells or the peptide construct in concert with a Her-2/neu MHC class I-restricted peptide from Her-2/neu. As revealed by adoptive transfer studies, effective protective antitumor immunity in this setting required the CD4 T helper subset. Additionally, in vitro analysis revealed that immunization with the parent peptide resulted in a weak immune response to the unmodified peptide consisting of both type 1 (IL-2, IFN-gamma) and type 2 (IL-4, IL-10) cytokine-producing cells analyzed by RT-PCR (qualitative and quantitative) and by limiting dilution assay. Comparatively, immunization with the chimeric construct elicited a potent immune response to the parent peptide with predominantly type 1 cytokine-producing cells. Taken together, the results suggest that immunization with the chimeric Her-2/neu peptide induced protective antitumor immunity. Associated with this immunization strategy was the enhancement of a type 1 cytokine response.