In the FVB/N HER-2/<Emphasis Type="Italic">neu</Emphasis> transgenic mouse both peripheral and central tolerance limit the immune response targeting HER-2/neu induced by <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>-based vaccines

Listeria monocytogenes-based vaccines for HER-2/neu are capable of breaking tolerance in FVB/N rat HER-2/neu transgenic mice. The growth of implanted NT-2 tumors, derived from a spontaneously occurring tumor in the FVB/N HER-2/neu transgenic mouse, was significantly slower in these mice following vaccination with a series of L. monocytogenes-based vaccines for HER-2/neu. Mechanisms of T cell tolerance that exist in these transgenic mice include the absence of functional high avidity anti-HER-2/neu CD8⁺ T cells and the presence of CD4⁺CD25⁺ regulatory T cells. The in vivo depletion of these regulatory T cells resulted in the slowing in growth of tumors even without the treatment of mice with an anti-HER-2/neu vaccine. The average avidities of responsive CD8⁺ T cells to six of the nine epitopes in HER-2/neu we examined, four of which were identified in this study, are shown here to be of a lower average avidity in the transgenic mice versus wild type FVB/N mice. In contrast, the average avidity of CD8⁺ T cells to three epitopes that showed the lowest avidity in the wild-type mice did not differ between wild type and transgenic mice. This study demonstrates the ability of L. monocytogenes-based vaccines to impact upon tolerance to HER-2/neu in FVB/N HER-2/neu transgenic mice and further defines some of the aspects of tolerance in these mice.     

Immunotherapy with dendritic cells pulsed with tumor-derived gp96 against murine lung cancer is effective through immune response of CD8+ cytotoxic T lymphocytes and natural killer cells

Background and purpose Immunization with heat shock proteins, gp96, elicits specific protective immunity against parent tumors. However, it is marginally effective as a therapeutic tool against established tumors. In the present study, we evaluated the efficacy and mechanism of immunotherapy with bone marrow-derived dendritic cells (DCs) pulsed with tumor-derived gp96 against murine lung cancer. Methods Mice were transplanted subcutaneously with ovalbumin (OVA)-transfected Lewis Lung Cancer (LLC-OVA) cells and immunized with gp96 derived from LLC-OVA, DCs, or DCs pulsed with gp96 derived from LLC-OVA. Results The antitumor effect was significantly enhanced in the mice immunized with DCs pulsed with gp96 derived from LLC-OVA, compared to mice immunized with gp96 or DCs (P < 0.05). The antitumor effect was significantly dependent on natural killer (NK) cells and CD8⁺ cells and partially dependent on CD4⁺ cells. Analysis by laser confocal microscopy demonstrated that gp96 was shown on the cell surface at 15 min, and after 30 min internalized in the endosomes and not in the endoplasmic reticulum or lysosomes. OVA-specific⁺ CD8⁺ cells were more readily recruited into the draining lymph nodes and higher CD8⁺ cytotoxic T cell activity against LLC-OVA was observed in splenocytes from mice immunized with DCs pulsed with gp96 derived from LLC-OVA. Re-challenge of the surviving mice with LLC-OVA tumors after the initial tumor inoculation showed dramatic retardation in tumor growth. Conclusion In conclusion, immunotherapy of DCs pulsed with tumor-derived gp96 against murine lung cancer is effective through immune response of CD8⁺ cytotoxic T lymphocytes and NK cells.     

Immune Adjuvant Activity of Pre-Resectional Radiofrequency Ablation Protects against Local and Systemic Recurrence in Aggressive Murine Colorectal Cancer

Purpose

While surgical resection is a cornerstone of cancer treatment, local and distant recurrences continue to adversely affect outcome in a significant proportion of patients. Evidence that an alternative debulking strategy involving radiofrequency ablation (RFA) induces antitumor immunity prompted the current investigation of the efficacy of performing RFA prior to surgical resection (pre-resectional RFA) in a preclinical mouse model.

Experimental Design

Therapeutic efficacy and systemic immune responses were assessed following pre-resectional RFA treatment of murine CT26 colon adenocarcinoma.

Results

Treatment with pre-resectional RFA significantly delayed tumor growth and improved overall survival compared to sham surgery, RFA, or resection alone. Mice in the pre-resectional RFA group that achieved a complete response demonstrated durable antitumor immunity upon tumor re-challenge. Failure to achieve a therapeutic benefit in immunodeficient mice confirmed that tumor control by pre-resectional RFA depends on an intact adaptive immune response rather than changes in physical parameters that make ablated tumors more amenable to a complete surgical excision. RFA causes a marked increase in intratumoral CD8⁺ T lymphocyte infiltration, thus substantially enhancing the ratio of CD8⁺ effector T cells: FoxP3⁺ regulatory T cells. Importantly, pre-resectional RFA significantly increases the number of antigen-specific CD8⁺ T cells within the tumor microenvironment and tumor-draining lymph node but had no impact on infiltration by myeloid-derived suppressor cells, M1 macrophages or M2 macrophages at tumor sites or in peripheral lymphoid organs (i.e., spleen). Finally, pre-resectional RFA of primary tumors delayed growth of distant tumors through a mechanism that depends on systemic CD8⁺ T cell-mediated antitumor immunity.

Conclusion

Improved survival and antitumor systemic immunity elicited by pre-resectional RFA support the translational potential of this neoadjuvant treatment for cancer patients with high-risk of local and systemic recurrence.     

Prevention of lymph node metastases by adoptive transfer of CD4+ T lymphocytes admixed with irradiated tumor cells

CD4⁺8^– T lymphocytes with potent antitumor activity in vivo were obtained in peritoneal exudate cells by immunizing mice with irradiated MM48 tumor cells admixed with OK-432. These immune CD4⁺ T cells were used in adoptive immunotherapy for prevention of lymph node metastases after removal of the primary tumor. Complete cure of metastases was obtained by adoptive transfer of CD4⁺ T cells admixed with irradiated MM48 tumor cells, but not by CD4⁺ T cells alone. To analyze the curative effect of admixing tumor cells on the prevention of metastases, a model of 1-day tumor inoculated with macrophages was used. Administration of immune CD4⁺ T cells alone resulted in the regression of local tumor in more than half of the mice, although all of them eventually died of lymph node metastases. On the other hand, adoptive transfer of immune CD4⁺ T cells plus irradiated tumor cells resulted in the complete regression of local tumors in all the mice, which survived without any sign of metastasis. The curative effect of the immune CD4⁺ T cells obtained by admixing irradiated tumor cells was tumor-specific. Macrophages induced by OK-432 (tumoricidal), implanted together with tumor, assisted tumor regression more than did macrophages elicited by proteose peptone (nontumoricidal) in the same adoptive transfer system. Administration of recombinant interleukin-2 instead of stimulant tumor cells did not enhance, but rather eliminated the constitutive antitumor activity of CD4⁺ T cells. On the other hand, exogenous recombinant interleukin-1 was more effective in the enhancement of antitumor activity of the CD4⁺ T cells as compared with stimulant tumor cell administration. In this case, the activating states of macrophages at the implanted tumor site had no influence on the therapeutic efficacy. A possible role of macrophages for induction of tumor-specific cytotoxic T cells that were mediated by tumor-specific CD4⁺ T cells is discussed.     

UV‐induced somatic mutations elicit a functional T cell response in the YUMMER1.7 mouse melanoma model

Human melanomas exhibit relatively high somatic mutation burden compared to other malignancies. These somatic mutations may produce neoantigens that are recognized by the immune system, leading to an antitumor response. By irradiating a parental mouse melanoma cell line carrying three driver mutations with UVB and expanding a single‐cell clone, we generated a mutagenized model that exhibits high somatic mutation burden. When inoculated at low cell numbers in immunocompetent C57BL/6J mice, YUMMER1.7 (Yale University Mouse Melanoma Exposed to Radiation) regresses after a brief period of growth. This regression phenotype is dependent on T cells as YUMMER1.7 tumors grow significantly faster in immunodeficient Rag1^?/? mice and C57BL/6J mice depleted of CD4 and CD8 T cells. Interestingly, regression can be overcome by injecting higher cell numbers of YUMMER1.7, which results in tumors that grow without effective rejection. Mice that have previously rejected YUMMER1.7 tumors develop immunity against higher doses of YUMMER1.7 tumor challenge. In addition, escaping YUMMER1.7 tumors are sensitive to anti‐CTLA‐4 and anti‐PD‐1 therapy, establishing a new model for the evaluation of immune checkpoint inhibition and antitumor immune responses.     

Manganese is critical for antitumor immune responses via cGAS-STING and improves the efficacy of clinical immunotherapy

CD8⁺ T cell-mediated cancer clearance is often suppressed by the interaction between inhibitory molecules like PD-1 and PD-L1, an interaction acts like brakes to prevent T cell overreaction under normal conditions but is exploited by tumor cells to escape the immune surveillance. Immune checkpoint inhibitors have revolutionized cancer therapeutics by removing such brakes. Unfortunately, only a minority of cancer patients respond to immunotherapies presumably due to inadequate immunity. Antitumor immunity depends on the activation of the cGAS-STING pathway, as STING-deficient mice fail to stimulate tumor-infiltrating dendritic cells (DCs) to activate CD8⁺ T cells. STING agonists also enhance natural killer (NK) cells to mediate the clearance of CD8⁺ T cell-resistant tumors. Therefore STING agonists have been intensively sought after. We previously discovered that manganese (Mn) is indispensable for the host defense against cytosolic dsDNA by activating cGAS-STING. Here we report that Mn is also essential in innate immune sensing of tumors and enhances adaptive immune responses against tumors. Mn-insufficient mice had significantly enhanced tumor growth and metastasis, with greatly reduced tumor-infiltrating CD8⁺ T cells. Mechanically, Mn²⁺ promoted DC and macrophage maturation and tumor-specific antigen presentation, augmented CD8⁺ T cell differentiation, activation and NK cell activation, and increased memory CD8⁺ T cells. Combining Mn²⁺ with immune checkpoint inhibition synergistically boosted antitumor efficacies and reduced the anti-PD-1 antibody dosage required in mice. Importantly, a completed phase 1 clinical trial with the combined regimen of Mn²⁺ and anti-PD-1 antibody showed promising efficacy, exhibiting type I IFN induction, manageable safety and revived responses to immunotherapy in most patients with advanced metastatic solid tumors. We propose that this combination strategy warrants further clinical translation.Subject terms: Pattern recognition receptors, Immunosurveillance     

Reduced naïve CD8+ T‐cell priming efficacy in elderly adults

Aging is associated with impaired vaccine efficacy and increased susceptibility to infectious and malignant diseases. CD8⁺ T‐cells are key players in the immune response against pathogens and tumors. In aged mice, the dwindling naïve CD8⁺ T‐cell compartment is thought to compromise the induction of de novo immune responses, but no experimental evidence is yet available in humans. Here, we used an original in vitro assay based on an accelerated dendritic cell coculture system in unfractioned peripheral blood mononuclear cells to examine CD8⁺ T‐cell priming efficacy in human volunteers. Using this approach, we report that old individuals consistently mount quantitatively and qualitatively impaired de novo CD8⁺ T‐cell responses specific for a model antigen. Reduced CD8⁺ T‐cell priming capacity in vitro was further associated with poor primary immune responsiveness in vivo. This immune deficit likely arises as a consequence of intrinsic cellular defects and a reduction in the size of the naïve CD8⁺ T‐cell pool. Collectively, these findings provide new insights into the cellular immune insufficiencies that accompany human aging.     

Promoting effect of Antrodia camphorata as an immunomodulating adjuvant on the antitumor efficacy of HER-2/neu DNA vaccine

It is well known that DNA vaccines induce protective humoral and cell-mediated immune responses in several animal models. Antrodia camphorata (AC) is a unique basidiomycete fungus of the Polyporaceae family that only grows on the aromatic tree Cinnamomum kanehirai Hayata (Lauraceae) endemic to Taiwan. Importantly, AC has been shown to be highly beneficial in the treatment and prevention of cancer. The goal of this study was to investigate whether AC is able to augment the antitumor immune properties of a HER-2/neu DNA vaccine in a mouse model in which p185neu is overexpressed in MBT-2 tumor cells. Compared with the mice that received the HER-2/neu DNA vaccine alone, co-treatment with AC suppressed tumor growth and extended the survival rate. This increase in the antitumor efficacy was attributed to the enhancement of the Th1-like cellular immune response by the HER-2/neu DNA vaccine–AC combination. Evidence for this came from the marked increase in the IFN-γ mRNA expression in CD4⁺ T cells in the draining inguinal lymph nodes, an increase in the number of functional HER-2/neu-specific CTLs, and the increased tumor infiltration of both CD4⁺ and CD8⁺ T cells, depletion of which abolishes the antitumor effect of the HER-2/neu DNA vaccine–AC therapy. Our results further indicate that the treatment of mice with AC enhanced DC activation and production of Th1-activating cytokines (e.g. IL-12, and IFN-α) in the draining lymph nodes, which were sufficient to directly stimulate T cell proliferation and higher IFN-γ production in response to ErbB2. Overall, these results clearly demonstrate that AC represents a promising immunomodulatory adjuvant that could enhance the therapeutic potency of HER-2/neu DNA vaccines in cancer therapy.     

Ontak reduces the immunosuppressive tumor environment and enhances successful therapeutic vaccination in HER-2/<Emphasis Type="Italic">neu</Emphasis>-tolerant mice

Disrupting tumor-mediated mechanisms suppressing host immunity represents a novel approach to tumor immunotherapy. Depletion of regulatory T cells (Tregs) increases endogenous anti-tumor immunity and the efficacy of active immunotherapy in experimental tumor models. HLA-A2.1/HLA-DR1 (A2.1/DR1) × BALB- neuT ⁺ (neuT ⁺) triple transgenic mice represent an improvement over neuT ⁺ mice for evaluating vaccination regimens to overcome tolerance against HER-2/neu. We questioned whether depletion of Tregs with Denileukin diftitox (Ontak) enhances the efficacy of a therapeutic vaccine consisting of HER-2(85–94) (p85) CTL and HER-2(776–790) (p776) Th peptides against the growth of TUBO.A2 transplantable tumor in male A2.1/DR1 × neuT ⁺ Tg mice. While the therapeutic vaccine primed the tumor-reactive CD8⁺ CTLs and CD4⁺ effector T lymphocytes (Teffs) compartment, inducing activation, tumor infiltration, and tumor rejection or delay in tumor growth, treatment with Ontak 1 day prior to vaccination resulted in enhanced CD4⁺ and CD8⁺ T-cell-mediated vaccine-specific immune responses in the periphery. This was closely associated with greater infiltration and a striking change in the intratumor balance of Tregs and vaccine-specific CTLs/Teffs that directly correlated with markedly enhanced antitumor activity. The data suggest that Tregs control both CD4⁺ and CD8⁺ T-cell activity within the tumor, emphasize the importance of the intratumor ratio of vaccine-specific lymphocytes to Tregs, and demonstrate significant inversion of this ratio and correlation with tumor rejection during Ontak/vaccine immunotherapy.     

Neuropilin-1 Expression Is Induced on Tolerant Self-Reactive CD8+ T Cells but Is Dispensable for the Tolerant Phenotype

Establishing peripheral CD8⁺ T cell tolerance is vital to avoid immune mediated destruction of healthy self-tissues. However, it also poses a major impediment to tumor immunity since tumors are derived from self-tissue and often induce T cell tolerance and dysfunction. Thus, understanding the mechanisms that regulate T cell tolerance versus immunity has important implications for human health. Signals received from the tissue environment largely dictate whether responding T cells become activated or tolerant. For example, induced expression and subsequent ligation of negative regulatory receptors on the surface of self-reactive CD8⁺ T cells are integral in the induction of tolerance. We utilized a murine model of T cell tolerance to more completely define the molecules involved in this process. We discovered that, in addition to other known regulatory receptors, tolerant self-reactive CD8⁺ T cells distinctly expressed the surface receptor neuropilin-1 (Nrp1). Nrp1 was highly induced in response to self-antigen, but only modestly when the same antigen was encountered under immune conditions, suggesting a possible mechanistic link to T cell tolerance. We also observed a similar Nrp1 expression profile on human tumor infiltrating CD4⁺ and CD8⁺ T cells. Despite high expression on tolerant CD8⁺ T cells, our studies revealed that Nrp1 had no detectable role in the tolerant phenotype. Specifically, Nrp1-deficient T cells displayed the same functional defects as wild-type self-reactive T cells, lacking in vivo cytolytic potential, IFNγ production, and antitumor responses. While reporting mostly negative data, our findings have therapeutic implications, as Nrp1 is now being targeted for human cancer therapy in clinical trials, but the precise molecular pathways and immune cells being engaged during treatment remain incompletely defined.     

Intratumoral interleukin-2/agonist CD40 antibody drives CD4<Superscript>+</Superscript>-independent resolution of treated-tumors and CD4<Superscript>+</Superscript>-dependent systemic and memory responses

Targeting interleukin-2 (IL-2) and/or agonist anti-CD40 antibody (Ab) into tumors represents an effective vaccination strategy that avoids systemic toxicity and resolves treated-site tumors. Here, we examined IL-2 and/or anti-CD40 Ab-driven local versus systemic T cell function and the installation of T cell memory. Single tumor studies showed that IL-2 induced a potent CD4⁺ and CD8⁺ T cell response that was limited to the draining lymph node and treated-site tumor, and lymph node tumor-specific CD8⁺ T cells did not upregulate CD44. A two-tumor model showed that while IL-2-treated-site tumors resolved, distal tumors continued to grow, implying limited systemic immunity. In contrast, anti-CD40 Ab treatment with or without IL-2 expanded the systemic T cell response to non-draining lymph nodes, and distal tumors resolved. Tumor-specific T cells in lymph nodes of anti-CD40 Ab ± IL-2-treated mice upregulated CD44, demonstrating activation and transition to effector/memory migratory cells. While CD40-activated CD4⁺ T cells were not required for eradicating treated-site tumors, they, plus CD8⁺ T cells, were crucial for removing distal tumors. Rechallenge/depletion experiments showed that the effector/memory phase required the presence of previously CD40/IL-2-activated CD4⁺ and CD8⁺ T cells to prevent recurrence. These novel findings show that different T cell effector mechanisms can operate for the eradication of local treated-site tumors versus untreated distal tumors and that signaling through CD40 generates a whole of body, effector/memory CD4⁺ and CD8⁺ T cell response that is amplified and prolonged via IL-2. Thus, successful immunotherapy needs to generate collaborating CD4⁺ and CD8⁺ T cells for a complete long-term protective cure.     

Tumor-secreted miR-214 induces regulatory T cells: a major link between immune evasion and tumor growth

An increased population of CD4⁺CD25^highFoxp3⁺ regulatory T cells (Tregs) in the tumor-associated microenvironment plays an important role in cancer immune evasion. However, the underlying mechanism remains unclear. Here we observed an increased secretion of miR-214 in various types of human cancers and mouse tumor models. Tumor-secreted miR-214 was sufficiently delivered into recipient T cells by microvesicles (MVs). In targeted mouse peripheral CD4⁺ T cells, tumor-derived miR-214 efficiently downregulated phosphatase and tensin homolog (PTEN) and promoted Treg expansion. The miR-214-induced Tregs secreted higher levels of IL-10 and promoted tumor growth in nude mice. Furthermore, in vivo studies indicated that Treg expansion mediated by cancer cell-secreted miR-214 resulted in enhanced immune suppression and tumor implantation/growth in mice. The MV delivery of anti-miR-214 antisense oligonucleotides (ASOs) into mice implanted with tumors blocked Treg expansion and tumor growth. Our study reveals a novel mechanism through which cancer cell actively manipulates immune response via promoting Treg expansion.     

Tumor-specific CD4+ T cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory T cells

The important role of tumor-specific cytotoxic CD8⁺ T cells is well defined in the immune control of the tumors, but the role of effector CD4⁺ T cells is poorly understood. In the current research, we have used a murine retrovirus-induced tumor cell line of C57BL/6 mouse origin, namely FBL-3 cells, as a model to study basic mechanisms of immunological control and escape during tumor formation. This study shows that tumor-specific CD4⁺ T cells are able to protect against virus-induced tumor cells. We show here that there is an expansion of tumor-specific CD4⁺ T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth. Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8⁺ T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4⁺ T cells and increases FV-specific CD4⁺ T-cell effector and cytotoxic responses leading to the complete tumor regression. Therefore, the capacity to reject tumor acquired by tumor-reactive CD4⁺ T cells largely depends on the direct suppressive activity of Tregs. We suggest that a cytotoxic CD4⁺ T-cell immune response may be induced to enhance resistance against oncovirus-associated tumors.     

Enhanced Antitumor Immunity Contributes to the Radio-Sensitization of Ehrlich Ascites Tumor by the Glycolytic Inhibitor 2-Deoxy-D-Glucose in Mice

Two-deoxy-D-glucose (2-DG), an inhibitor of glycolysis differentially enhances the radiation and chemotherapeutic drug induced cell death in cancer cells in vitro, while the local tumor control (tumor regression) following systemic administration of 2-DG and focal irradiation of the tumor results in both complete (cure) and partial response in a fraction of the tumor bearing mice. In the present studies, we investigated the effects of systemically administered 2-DG and focal irradiation of the tumor on the immune system in Ehrlich ascites tumor (EAT) bearing Strain “A” mice. Markers of different immune cells were analyzed by immune-flow cytometry and secretary cytokines by ELISA, besides monitoring tumor growth. Increase in the expression of innate (NK and monocytes) and adaptive CD4⁺cells, and a decrease in B cells (CD19) have been observed after the combined treatment, suggestive of activation of anti-tumor immune response. Interestingly, immature dendritic cells were found to be down regulated, while their functional markers CD86 and MHC II were up regulated in the remaining dendritic cells following the combination treatment. Similarly, decrease in the CD4⁺ naïve cells with concomitant increase in activated CD4⁺ cells corroborated the immune activation. Further, a shift from Th2 and Th17 to Th1 besides a decrease in inflammatory cytokines was also observed in the animals showing complete response (cure; tumor free survival). This shift was also complimented by respective antibody class switching followed by the combined treatment. The immune activation or alteration in the homeostasis favoring antitumor immune response may be due to depletion in T regulatory cells (CD4⁺CD25⁺FoxP3⁺). Altogether, these results suggest that early differential immune activation is responsible for the heterogenous response to the combined treatment. Taken together, these studies for the first time provided insight into the additional mechanisms underlying radio-sensitization by 2-DG in vivo by unraveling its potential as an immune-modulator besides direct effects on the tumor.     

Elimination of Metastatic Melanoma Using Gold Nanoshell-Enabled Photothermal Therapy and Adoptive T Cell Transfer

Ablative treatments such as photothermal therapy (PTT) are attractive anticancer strategies because they debulk accessible tumor sites while simultaneously priming antitumor immune responses. However, the immune response following thermal ablation is often insufficient to treat metastatic disease. Here we demonstrate that PTT induces the expression of proinflammatory cytokines and chemokines and promotes the maturation of dendritic cells within tumor-draining lymph nodes, thereby priming antitumor T cell responses. Unexpectedly, however, these immunomodulatory effects were not beneficial to overall antitumor immunity. We found that PTT promoted the infiltration of secondary tumor sites by CD11b⁺Ly-6G/C⁺ myeloid-derived suppressor cells, consequently failing to slow the growth of poorly immunogenic B16-F10 tumors and enhancing the growth of distant lung metastases. To exploit the beneficial effects of PTT activity against local tumors and on antitumor immunity whilst avoiding the adverse consequences, we adoptively transferred gp100-specific pmel T cells following PTT. The combination of local control by PTT and systemic antitumor immune reactivity provided by adoptively transferred T cells prevented primary tumor recurrence post-ablation, inhibited tumor growth at distant sites, and abrogated the outgrowth of lung metastases. Hence, the combination of PTT and systemic immunotherapy prevented the adverse effects of PTT on metastatic tumor growth and optimized overall tumor control.     

The influence of cyclophosphamide on antitumor immunity in mice bearing late-stage tumors

Spleen cells from mice bearing late-stage methylcholanthrene-induced tumor did not show any tumor activity when mixed with tumor cells in Winn's assay. Treatment of these mice with cyclophosphamide (CY) induced a tumor-inhibitory activity in spleen, occurring on day 7 after treatment, reaching its maximum on day 11 and disappearing by day 21. This antitumor activity could not be induced in control, tumor-free or T-deficient tumor-bearing mice. CY-induced tumor-inhibitory activity was immunologically specific, and mediated by Thy-1⁺, L3T4^–, Ly-2⁺ cells. Contrary to spleen cells from untreated tumor-bearing mice, spleen cells from CY-treated tumor-bearing mice did not suppress the antitumor activity of immune spleen cells in Winn's assay. However, in contrast to immune spleen cells, CY-induced tumor-inhibitory cells did not manifest antitumor activity when transferred systemically (i. v.) into T-cell-deficient tumor-bearing mice. Even more, spleen cells from CY-pretreated mice, harvested 7–15 days after the drug administration, partially suppressed the antitumor activity of concomitantly transferred spleen cells from specifically immune mice. Nevertheless, CY-pretreated mice manifested concomitant immunity, i.e. these mice exhibited higher resistance to a second inoculum of the same tumor than did nontreated mice or even mice with excised primary tumor.     

Gene silencing of TGF-β1 enhances antitumor immunity induced with a dendritic cell vaccine by reducing tumor-associated regulatory T cells

Active immunotherapy and cancer vaccines that promote host antitumor immune responses promise to be effective and less toxic alternatives to current cytotoxic drugs for the treatment of cancer. However, the success of tumor immunotherapeutics and vaccines is dependent on identifying approaches for circumventing the immunosuppressive effects of regulatory T (Treg) cells induced by the growing tumor and by immunotherapeutic molecules, including Toll-like receptor (TLR) agonists. Here, we show that tumors secrete high concentrations of active TGF-β1, a cytokine that can convert naive T cells into Foxp3⁺ Treg cells. Silencing TGF-β1 mRNA using small interfering RNA (siRNA) in tumor cells inhibited active TGF-β1 production in vitro and restrained their growth in vivo. Prophylactic but not therapeutic administration of TGF-β1 siRNA reduced the growth of CT26 tumors in vivo. Furthermore, suppressing TGF-β1 expression at the site of a tumor, using siRNA before, during and after therapeutic administration of a TLR-activated antigen-pulsed dendritic cell vaccine significantly reduced the growth of B16 melanoma in mice. The protective effect of co-administering TGF-β1 siRNA with the DC vaccine was associated with suppression of CD25⁺Foxp3⁺ and CD25⁺IL-10⁺ T cells and enhancement of tumor infiltrating CD4 and CD8 T cells. Our findings suggest that transient suppression of TGF-β1 may be a promising approach for enhancing the efficacy of tumor vaccines in humans.     

Immune parameters of mice bearing human head and neck cancer

A xenogeneic human head and neck squamous cell carcinoma (HNSCC) model in immunocompetent mice was evaluated for its requirement of cyclosporine for progressive tumor growth. Tumor growth and T cell functions were assessed in mice receiving cyclosporine treatment for various lengths of time. Tumor cells were injected s.c. on day 1 and cyclosporine was injected i.p. daily on days 1, 1–7, 1–14, 1–21, or for the entire 28 days of tumor growth. All mice developed tumors. These tumors were confirmed to be squamous carcinomas of human origin histologically and by positive staining for human MHC class I antigen expression. Tumors were largest in mice that received cyclosporine for days 1–21 or days 1–28. Increased tumor size was associated with increased serum levels of tumor-reactive antibodies, an increased intratumoral frequency of CD4⁺ and CD8⁺ cells, but a diminished production of interleukin-2 (IL-2) by the tumor infiltrate. Also correlating with increasing tumor size was splenomegaly, a decline in the frequency, but not the absolute levels, of splenic CD4⁺ and CD8⁺ cells, and a diminished capacity to proliferate in response to concanavalin A and to be stimulated to secrete IL-2. The HNSCC tumors contributed to the immune decline since T cell functions were more depressed in the tumor bearers than in control mice receiving only cyclosporine treatment. These results demonstrate that human HNSCC tumor xenografts can grow in mice even with limited cyclosporine treatment, and that the survival of these xenografts may, in part, be due to a tumor-induced decline in select T cell functions.This study was supported by the Medical Research Service of the Department of Veterans Affairs, by grants CA-45080 and CA-48080 from the National Institutes of Health     

A B-cell lymphoma vaccine using a depot formulation of interleukin-2 induces potent antitumor immunity despite increased numbers of intratumoral regulatory T cells

Therapeutic vaccination holds great potential as complementary treatment for non-Hodgkin’s lymphoma. Here, we report that a therapeutic whole cell vaccine formulated with IL-2 adsorbed onto aluminum hydroxide as cytokine-depot formulation elicits potent antitumor immunity and induces delayed tumor growth, control of tumor dissemination and longer survival in mice challenged with A20-lymphoma. Therapeutic vaccination induced higher numbers of tumor’s infiltrating lymphocytes (CD4⁺ and CD8⁺ T cells and NK cells), and the production of IFN-γ and IL-4 by intratumoral CD4⁺ T cells. Further, strong tumor antigen-specific cellular responses were detected at systemic level. Both the A20-derived antigenic material and the IL-2 depot formulation were required for induction of an effective immune response that impacted on cancer progression. All mice receiving any form of IL-2, either as part of the vaccine or alone as control, showed higher numbers of CD4⁺CD25^+/highFoxp3⁺ regulatory T cells (Treg) in the tumor, which might have a role in tumor progression in these animals. Nevertheless, for those animals that received the cytokine as part of the vaccine formulation, the overall effect was improved immune response and less disseminated disease, suggesting that therapeutic vaccination overcomes the potential detrimental effect of intratumoral Treg cells. Overall, the results presented here show that a simple vaccine formulation, that can be easily prepared under GMP conditions, is a promising strategy to be used in B-cell lymphoma and may have enough merit to be tested in clinical trials.     

Anti-tumor activity of dendritic cells transfected with mRNA for receptor for hyaluronan-mediated motility is mediated by CD4+ T cells

Receptor for hyaluronan-mediated motility (RHAMM) is overexpressed in various tumors with high frequency, and was recently identified as an immunogenic antigen by serologic screening of cDNA expression libraries. In this study, we explored whether RHAMM is a potential target for dendritic cell (DC) immunotherapy. We constructed a plasmid for transduction of in vitro-transcribed mRNAs into DCs to efficiently transport the intracellular protein RHAMM into MHC class II compartments by adding a late endosomal/lysosomal sorting signal to the RHAMM gene. Immunization of mice with modified RHAMM mRNA-transfected DCs (DC/RHAMM) induced killing activity against RHAMM-positive tumor cells in splenocytes. To examine whether CD4⁺ and/or CD8⁺ T cells were required for this antitumor immunity, an anti-CD4 or anti-CD8 antibody was administered to mice after immunization with DC/RHAMM. Depletion of CD4⁺ T cells significantly diminished the induction of tumor cell-killing activity in splenocytes, whereas CD8⁺ T cell depletion had no effect. We then investigated the therapeutic effect of DC/RHAMM in a 3-day tumor model of EL4. DC/RHAMM was administered to mice on days 3, 7 and 10 after EL4 tumor inoculation. The treatment markedly inhibited tumor growth compared to control DCs. Moreover, antibody-mediated depletion of CD4⁺ T cells completely abrogated the therapeutic effect of DC/RHAMM, whereas depletion of CD8⁺ T cells had no effect. The results of this preclinical study indicate that DCs transfected with a modified RHAMM mRNA targeted to MHC class II compartments can induce CD4⁺ T cell-mediated antitumor activity in vivo.