MiR‐15a/16 deficiency enhances anti‐tumor immunity of glioma‐infiltrating CD8+ T cells through targeting mTOR

MiR‐15a/16, a miRNA cluster located at chromosome 13q14, has been reported to act as an immune regulator in inflammatory disorders besides its aberrant expression in cancers. However, little is known about its regulation in tumor‐infiltrating immune cells. In our study, using an orthotropic GL261 mouse glioma model, we found that miR‐15a/16 deficiency in host inhibited tumor growth and prolonged mice survival, which might be associated with the accumulation of tumor‐infiltrating CD8+ T cells. More importantly, tumor‐infiltrating CD8+ T cells without miR‐15a/16 showed lower expression of PD‐1, Tim‐3 and LAG‐3, and stronger secretion of IFN‐γ, IL‐2 and TNF‐α than WT tumor‐infiltrating CD8+ T cells. Also, our in vitro experiments further confirmed that miR‐15a/16^?/? CD8+ T displayed higher active phenotypes, more cytokines secretion and faster expansion, compared to WT CD8+ T cells. Mechanismly, mTOR was identified as a target gene of miR‐15a/16 to negatively regulate the activation of CD8+ T cells. Taken together, these data suggest that miR‐15a/16 deficiency resists the exhaustion and maintains the activation of glioma‐infiltrating CD8+ T cells to alleviate glioma progression via targeting mTOR. Our findings provide evidence for the potential immunotherapy through targeting miR‐15a/16 in tumor‐infiltrating immune cells.     

Tumor-derived CD4+CD25+ Tregs Inhibit the Maturation and Antigen-Presenting Function of Dendritic Cells

CD4+CD25+regulatory T cells (Tregs) play a key role in regulation of immnue response and maintenance ofself-tolerance. Studies have found Tregs could suppress tumor-specific T cell-mediated immune response andpromote cancer progression. Depletion of Tregs can enhance antitumor immunity. Dendritic cells (DCs) areprofessional antigen-presenting cells and capable of activating antigen-specific immune responses, which makethem ideal candidate for cancer immunotherapy. Now various DC vaccines are considered as effective treatmentfor cancers. The aim of this study was to evaluate variation of Tregs in BALB/C mice with hepatocellular carcinomaand investigate the interaction between tumor-derived Tregs, effector T cells (Teff) and splenic DCs. We foundthe percentages of Tregs/CD4+ in the peripheral blood of tumor-bearing mice were higher than in normal mice.Tumor-derived Tregs diminished the up-regulation of costimulatory molecule expression on splenic DCs, evenin the presence of Teff cells and simultaneously inhibited IL-12 and TNF-α secretion by DCs.     

CD28 costimulation overcomes transforming growth factor-beta-mediated repression of proliferation of redirected human CD4+ and CD8+ T cells in an antitumor cell attack

The T-cell-mediated antitumor immune response is frequently repressed in the tumor environment by an immunologic barrier, the predominant mediators of which are thought to be interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta). We explored the effect of these cytokines on the individual T-cell effector functions on antigen engagement during an antitumor cell attack. Isolated CD4+ and CD8+ T cells were antigen-specifically redirected toward carcinoembryonic antigen (CEA)-positive tumor cells by expression of a recombinant T-cell receptor (immunoreceptor), which triggers T-cell activation via CD3zeta on binding to CEA. Immunoreceptor-activated T cells secrete IFN-gamma, proliferate, and lyse CEA+ but not CEA- tumor cells. Whereas IL-10 has no direct effect on immunoreceptor-triggered effector functions, TGF-beta represses proliferation of both CD4+ and CD8+ T cells but neither IFN-gamma secretion nor specific cytolytic activities. CD28 costimulation, however, overcomes TGF-beta-mediated repression in T-cell proliferation. Consequently, T cells redirected by a combined CD28-CD3zeta signaling immunoreceptor are largely resistant to TGF-beta-mediated repression. This is reflected in vivo by a more pronounced antitumor activity of T cells against TGF-beta-secreting tumors when redirected by a costimulatory CD28-CD3zeta than by a CD3zeta signaling immunoreceptor.     

Characterization of PD‐1 upregulation on tumor‐infiltrating lymphocytes in human and murine gliomas and preclinical therapeutic blockade

Blockade of the immune checkpoint molecule programmed‐cell‐death‐protein‐1 (PD‐1) yielded promising results in several cancers. To understand the therapeutic potential in human gliomas, quantitative data describing the expression of PD‐1 are essential. Moreover, due the immune‐specialized region of the brain in which gliomas arise, differences between tumor‐infiltrating and circulating lymphocytes should be acknowledged. In this study we have used flow cytometry to quantify PD‐1 expression on tumor‐infiltrating T cells of 25 freshly resected glioma cell suspensions (10 newly and 5 relapsed glioblastoma, 10 lower grade gliomas) and simultaneously isolated circulating T cells. A strong upregulation of PD‐1 expression in the tumor microenvironment compared to the blood circulation was seen in all glioma patients. Additionally, circulating T cells were isolated from 15 age‐matched healthy volunteers, but no differences in PD‐1 expression were found compared to glioma patients. In the murine GL261 malignant glioma model, there was a similar upregulation of PD‐1 on brain‐infiltrating lymphocytes. Using a monoclonal PD‐1 blocking antibody, we found a marked prolonged survival with 55% of mice reaching long‐term survival. Analysis of brain‐infiltrating cells 21 days after GL261 tumor implantation showed a shift in infiltrating lymphocyte subgroups with increased CD8+ T cells and decreased regulatory T cells. Together, our results suggest an important role of PD‐1 in glioma‐induced immune escape, and provide translational evidence for the use of PD‐1 blocking antibodies in human malignant gliomas.     

Expression of the B7-related molecule B7-H1 by glioma cells: a potential mechanism of immune paralysis

Human glioblastoma is a highly lethal tumor that is known for its immune inhibitory capabilities. B7-homologue 1 (B7-H1), a recently identified homologue of B7.1/2 (CD80/86), has been described to exert costimulatory and immune regulatory functions. We investigated the expression and the functional activity of B7-H1 in human glioma cells in vitro and in vivo. Although lacking B7.1/2 (CD80/86), all 12 glioma cell lines constitutively expressed B7-H1 mRNA and protein. Exposure to IFN-gamma strongly enhanced B7-H1 expression. Immunohistochemical analysis of malignant glioma specimens revealed strong B7-H1 expression in all 10 samples examined, whereas no B7-H1 expression could be detected on normal brain tissues. To elucidate the functional significance of glioma cell-related B7-H1 expression, we performed coculture experiments of glioma cells with alloreactive CD4+ and CD8+ T cells. Glioma-related B7-H1 was identified as a strong inhibitor of CD4+ as well as CD8+ T-cell activation as assessed by increased cytokine production (IFN-gamma, interleukin-2, and interleukin-10) and expression levels of the T-cell activation marker (CD69) in the presence of a neutralizing antibody against B7-H1 (mAb 5H1). B7-H1 expression may thus significantly influence the outcome of T-cell tumor cell interactions and represents a novel mechanism by which glioma cells evade immune recognition and destruction.     

Herpes simplex virus (HSV) amplicon-mediated codelivery of secondary lymphoid tissue chemokine and CD40L results in augmented antitumor activity

Development of effective antitumor immune responses depends on timely interactions of effector cells. A bimodal approach that involves coexpression of chemokines and costimulatory molecules within the tumor bed may elaborate a more optimal antitumor response. One candidate includes secondary lymphoid tissue chemokine (SLC), which promotes the colocalization of na?ve, nonpolarized memory T cells and dendritic cells (DCs) within lymph nodes and Peyer's patches. CD40L-mediated DC activation could induce maturation, enhance antigen presentation, and facilitate priming of the recruited na?ve T cells. To this end, the antitumor activity of SLC and CD40L expressed singly or in combination using the herpes simplex virus (HSV)-derived amplicon was examined in two murine models: A20, a B-cell lymphoma, and CT-26, an adenocarcinoma. Administration of amplicons encoding SLC (HSV-SLC) into s.c. tumors established previously resulted in heavy infiltration of CD4+ and CD8+ T cells, and DCs, and the generation of cytolytic T-cell activity. Combined transduction of either tumor with HSV-SLC and HSV-CD40L resulted in a more enhanced antitumor activity that was CD8+ T cell-dependent than observed with either vector alone. mRNA expression of the Th1 markers IFN-gamma, perforin, and interleukin 12 was detectable only in transduced regressing tumors. In addition to identifying a potent antitumor immune strategy, we show that amplicon-mediated SLC and CD40L delivery may mimic lymph node conditions necessary for priming na?ve T cells within the tumor bed, and demonstrate the importance of DC activation status on antigen presentation and cytokine expression for priming of newly recruited T cells.     

Depletion of peripheral macrophages and brain microglia increases brain tumor titers of oncolytic viruses

Clinical trials have proven oncolytic virotherapy to be safe but not effective. We have shown that oncolytic viruses (OV) injected into intracranial gliomas established in rodents are rapidly cleared, and this is associated with up-regulation of markers (CD68 and CD163) of cells of monocytic lineage (monocytes/microglia/macrophages). However, it is unclear whether these cells directly impede intratumoral persistence of OV through phagocytosis and whether they infiltrate the tumor from the blood or the brain parenchyma. To investigate this, we depleted phagocytes with clodronate liposomes (CL) in vivo through systemic delivery and ex vivo in brain slice models with gliomas. Interestingly, systemic CL depleted over 80% of peripheral CD163+ macrophages in animal spleen and peripheral blood, thereby decreasing intratumoral infiltration of these cells, but CD68+ cells were unchanged. Intratumoral viral titers increased 5-fold. In contrast, ex vivo CL depleted only CD68+ cells from brain slices, and intratumoral viral titers increased 10-fold. These data indicate that phagocytosis by both peripheral CD163+ and brain-resident CD68+ cells infiltrating tumor directly affects viral clearance from tumor. Thus, improved therapeutic efficacy may require modulation of these innate immune cells. In support of this new therapeutic paradigm, we observed intratumoral up-regulation of CD68+ and CD163+ cells following treatment with OV in a patient with glioblastoma.     

Glucocorticoid-induced tumor necrosis factor receptor stimulation enhances the multifunctionality of adoptively transferred tumor antigen-specific CD8+ T cells with tumor regression

We have reported for the first time the significance of effector T-cell multifunctionality in antitumor immunity, suggesting that the appearance of multifunctional/polyfunctional tumor-specific CD8⁺ T cells in vivo is a critical determinant of the success of antitumor immunotherapy, and a strategy to induce multifunctionality in effector cells is required for the successful immunotherapy of hosts with progressing tumor. Glucocorticoid-induced tumor necrosis factor receptor (GITR) stimulation has been shown to enhance antitumor immune response. However, its functional impact on adoptively transferred T cells remains unclear. Here, we analyzed the impact of GITR stimulation in vivo on the functional profiles of adoptively transferred CD8⁺ T cells specific for murine fibrosarcoma CMS5. GITR stimulation was found to enhance multifunctionality (interferon (IFN)-γ and tumor necrosis factor (TNF)-α production and CD107a mobilization as a degranulation marker) in transferred cells at the single-cell level. These cells exhibited upregulated expression of CD25 in draining lymph nodes and increased infiltration in tumor. Mice that received T-cell therapy with GITR stimulation showed reduced Foxp3⁺CD4⁺ T cells among tumor infiltrating lymphocytes and increased in vivo cytotoxic T lymphocytes (CTL) activity even with progressing tumor, resulting in enhanced tumor regression. These data strengthen the idea that effector T-cell multifunctionality is a sensitive immune correlate for successful immunotherapy against malignancy and provide an immunological rationale for effective T-cell therapy combined with GITR stimulation. ( Cancer Sci 2009; 100: 1317–1325)     

Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis

Decoy receptor 3 (DcR3) is a newly identified soluble protein that binds to CD95 ligand (CD95L) and inhibits its proapoptotic activity. Here we report that DcR3 is expressed by the majority of long-term and ex vivo malignant glioma cell lines as well as in human glioblastoma in vivo. Expression of DcR3 correlates with the grade of malignancy: 15 of 18 (83%) glioblastomas (WHO grade IV) but none of 11 diffuse astrocytomas (WHO grade II) exhibited DcR3 immunoreactivity. We also demonstrate that human malignant glioma cells engineered to release high amounts of DcR3 into the cell culture supernatant are protected from CD95L-induced apoptotic cell death. In contrast, DcR3 does not confer protection from the death ligand Apo2 ligand (TRAIL). Importantly, ectopic expression of DcR3 resulted in substantial differences in immune cell infiltration in the 9L rat gliosarcoma model. Thus, the infiltration of CD4+ and CD8+ T cells as well as microglia/macrophages into glioma was substantially decreased in DcR3-producing tumors compared with control tumors. Chemotaxis assays revealed that DcR3 counteracts the chemotactic activity of CD95L against microglial cells in vitro. These findings suggest that DcR3 may be involved in the progression and immune evasion of malignant gliomas.     

Interleukin-12 requires initial CD80-mediated T-cell activation to support immune responses toward human breast and ovarian carcinoma.

One possible reason for the poor immunogenicity of tumors is the induction of peripheral tolerance by tumor cells that fail to deliver costimulatory signals. Furthermore, T cells stimulated with wild-type tumor cells often fail to secrete cytokines. The present study has been undertaken to identify cytokines that cooperate with CD80 in T-cell activation in vitro toward human breast and ovarian carcinoma cell lines. Tumor cell-mediated T-lymphocyte activation was analyzed directly in allogeneic mixed lymphocyte/tumor cell cultures as proliferation and effector functions were assessed in cytotoxic T-cell assays. Interleukin-7 (IL-7) amplified the proliferative response toward CD80-transfected breast and ovarian carcinomas and stimulated predominantly CD4+ T lymphocytes. IL-12 represses the proliferative response of naive T cells but cooperates with CD80-mediated activation during secondary stimulations. In long-term T-cell cultures, IL-12 synergizes with CD80 expression to stimulate cytolytic CD8+ T-cell lines, which recognize a breast carcinoma line in a human histocompatibility leukocyte antigen-restricted manner. These studies illustrate that costimulation is necessary for tumor cells to function as alloantigen-presenting cells. Furthermore, when added after the priming of T cells with CD80-transfected tumor cells, IL-12 could be helpful in propagating sufficient T-cell numbers to be used in adoptive transfers during cellular immunotherapy.     

Intratumoral delivery of dendritic cells engineered to secrete both interleukin (IL)-12 and IL-18 effectively treats local and distant disease in association with broadly reactive Tc1-type immunity

Dendritic cells (DCs) were adenovirally engineered to constitutively and durably secrete the potent Th1-biasing cytokines interleukin (IL)-12 (AdIL12DC) and/or IL-18 (AdIL18DC) and evaluated for their ability to promote therapeutic antitumor immunity in murine sarcoma models. Injection of either AdIL12DC or AdIL18DC into day 7 CMS4 or MethA tumors resulted in tumor rejection or slowed tumor growth when compared with control cohorts. Importantly, intratumoral injection with DCs engineered to secrete both IL-12 and IL-18 (AdIL12/IL18DC) resulted in complete and the most acute rejection of any treatment group analyzed. This strategy was also effective in promoting the regression of contralateral, untreated tumors. Both CD4+ and CD8+ T cells were required for tumor rejection. CD8+ splenic T cells from mice treated with AdIL12/IL18DC produced the highest levels of IFN-gamma in response to tumor rechallenge in vitro and displayed the broadest repertoire of Tc1-type reactivity to acid-eluted, tumor-derived peptides among all treatment cohorts. This apparent enhancement in cross-presentation of tumor-associated epitopes in vivo may result from the increased capacity of engineered DCs to kill tumor cells, survive tumor-induced apoptosis, and present immunogenic MHC/tumor peptide complexes to T cells after intratumoral injection. In support of this hypothesis, cytokine gene-engineered DCs expressed higher levels of MHC and costimulatory molecules, as well as Fas ligand and membrane-bound tumor necrosis factor alpha, with the latter markers associated with elevated tumoricidal activity in vitro. Cytokine gene-engineered DCs appeared to have a survival advantage in situ when injected into tumor lesions, to be found in approximation with regions of tumor apoptosis, and to have the capacity to ingest apoptotic tumor bodies. These results support the ability of combined cytokine gene transfer to enhance multiple effector functions mediated by intralesionally injected DCs that may concertedly promote cross-priming and the accelerated immune-mediated rejection of tumors.     

Generation of tumor-specific T-lymphocytes by cross-priming with human dendritic cells ingesting apoptotic tumor cells

It has been suggested that dendritic cells (DCs) are capable of ingesting apoptotic tumor cells (ATCs) and presenting tumor-associated antigens to immune cells. We evaluated the potential of human DCs, which have ingested ATCs, to serve as a source of antigenic epitopes for presentation to T cells specific for PCI-13, a squamous cell carcinoma of the head and neck cell line. Immature DCs (DCimm) generated in the presence of interleukin 4 and granulocyte machrophage colony-stimulating factor from peripheral blood monocytes of HLA-A2+ healthy donors were incubated in the presence of ATCs. Uptake of ATCs by DCs was monitored by flow cytometry and confocal microscopy after 2-18 h of coincubation. When DCs were matured (DCmat) in the presence of proinflammatory cytokines, their capacity to uptake ATCs was reduced. Responses of PCI-13-specific CD8+ T cells to unmodified PCI-13 cells and to DCimm or DCmat +/- ATCs or +/- tumor lysates were tested in gamma-IFN enzyme-linked immunospot and cytotoxicity assays. Unmodified tumor cells were found to be the best stimulators of antitumor activity of the established T-cell line, and ATCs alone were minimally stimulatory. However, DCs that ingested ATCs were able to present tumor antigens to CTLs, and DCimm and DCmat were almost equally stimulatory. When DCs plus various tumor-derived preparations were used as antigen-presenting cells with autologous HLA-A2+ T cells obtained from normal donors, DCs that had ingested ATCs were more effective in generating CD8+ CTLs than tumor cells alone or DCs pulsed with tumor lysates. The results indicate that human DCs fed with ATCs and then matured effectively generated T cell-mediated antitumor responses in vitro.     

Induction of antitumor immunity using intercellular adhesion molecule 1 (ICAM-1) transfection in mouse glioma cells.

We investigated the role of intercellular adhesion molecule 1 (ICAM-1) expression in tumorigenicity of gliomas and the antitumor effects of glioma cells genetically engineered to express ICAM- 1. Mouse glioma cells transfected with ICAM-1 grew in T-cell deficient mice but not in syngeneic mice. Vaccination with ICAM-1-transfected tumor cells markedly inhibited the growth of subcutaneously inoculated gliomas but not gliomas located in the brain. In vivo antibody ablation study revealed that CD4+ T, CD8+ T and NK cells were all required to produce the antitumor effect of SR/ICAM- 1. In this study, we demonstrated the therapeutic potential of vaccination with ICAM-1-overexpressing tumor cells for the control of the tumor growth.     

CD2-SLFA3/T11TS interaction facilitates immune activation and glioma regression by apoptosis

OBJECTIVE: Exogenous application of T11TS/SLFA3 in glioma model had shown the regression of tumor load through immunopotentiation. The mechanistic module of this interaction on immunological synapse formation and resulting effect in glioma regression is searched for delineating immunotherapeutic efficacy of T11TS. METHODS: After purification of T11TS/SLFA3 from sheep erythrocytes the glycoprotein was characterized by SDS-PAGE analysis and glycoprotein staining. The modulatory effect of T11TS application on glioma animals were studied by CD2 and MHC class II expression on peripheral lymphocytes, PMN, macrophages and intracranial microglia by flowcytometric analysis. Finally apoptotic killing of brain cells were studied through annevin-V expression and measuring fragmented cytoplasmic DNA by ELISA. RESULTS: Compared to healthy normal counter-parts the CD2 receptor downregulation by ENU treatment in lymphocytes and PMN were upregulated with three consecutive doses of T11TS. Splenic antigen presenting macrophages and intracranial mocroglia had shown CD2 and MHC class II modulation simultaneously in their different subtypes. These receptor studies revealed significant boosting of the immune competent cells most effectively in second dose of T11TS whereas the third dose had some regulatory effect. Sharp increase of apoptosis in brain cells was found by phosphatidylserine externalization and presence of fragmented DNA in cytoplasm with application of T11TS in consecutive doses as a result of immune potentiation. CONCLUSION: Receptor studies revealed modulation of CD2 and MHC class II, two important constituents of immunological synapse successfully help to form the TCR-p-MHC complex and provide required co-stimulation for activation. Potentiated immune effector machinery was then directed to abrogate glioma by apoptosis signifying T11TS as an immunotherapeutic probe.     

CD4+FoxP3+ regulatory T cells gradually accumulate in gliomas during tumor growth and efficiently suppress antiglioma immune responses in vivo

The suppressive activity of regulatory T cells (Treg) has been implicated as an important factor limiting immune mediated destruction of tumor cells. However, not much is known about the presence and function of Treg within tumors. Here we show in a syngeneic murine glioma model a time-dependent accumulation of CD4+FoxP3+ Treg in brain tumors. Further analysis revealed a time-dependent upregulation of CD25, CTLA-4, GITR and CXCR4 on intratumoral CD4+FoxP3+ Treg during tumor growth. Moreover, freshly isolated intratumoral Treg were highly suppressive when tested directly ex vivo. Treatment with anti-CD25 monoclonal antibodies (mAbs) significantly reduced the number of these highly suppressive CD4+FoxP3+ cells within the growing tumor and provoked a CD4 and CD8 T cell dependent destruction of the glioma cells. Combining Treg depletion with administration of blocking CTLA-4 mAbs further boosted glioma-specific CD4+ and CD8+ effector T cells as well as antiglioma IgG2a antibody titers resulting in complete tumor eradication without any signs of autoimmunity. These data illustrate that intratumoral accumulation and activation of CD4+FoxP3+ Treg act as a dominant immune escape mechanism for gliomas and underline the importance of controlling tumor-infiltrating Treg in glioma immunotherapy.