CTL Activity Research Articles

Characterization of a water-soluble polysaccharide from Boletus edulis and its antitumor and immunomodulatory activities on renal cancer in mice

A polysaccharide (BEP, Mw=113,432Da) was purified from Boletus edulis, which had a backbone consisting of (1→6)-linked-α-d-glucopyranosyl, (1→2,6)-linked-α-d-galactopyranosyl, (1→6)-linked-α-d-galactopyranosyl, and (1→3)-linked-α-d-rhamnopyranosyl residues, which were branched at O-2 position of (1→2,6)-linked-α-d-galactopyranosyl residue with a single terminal (1→)-linked-α-l-arabinofuranosyl residue. After 32 days’ BEP administration to Renca tumor bearing mice, the tumor mass of Renca transplanted in mice was significantly repressed. Furthermore, BEP could significantly increase the spleen and thymus indices, stimulate splenocytes proliferation, augment NK cell and CTL activities in spleen, and promote the secretion of the cytokines IL-2 and TNF-α in Renca tumor bearing mice. Meanwhile oral administration of BEP (100 and 400mg/kg) restored all the altered hematological and biochemical parameters of tumor-bearing mice to normal levels. Thus, these data demonstrate that BEP possesses potential immunomodulatory activity and might be employed as effective therapeutic agents for the prevention of renal caner.

Vaccination with a HSV-2 UL24 mutant induces a protective immune response in murine and guinea pig vaginal infection models

The rational design and development of genetically attenuated HSV-2 mutant viruses represent an attractive approach for developing both prophylactic and therapeutic vaccines for genital herpes. Previously, HSV-2 UL24 was shown to be a virulence determinant in both murine and guinea pig vaginal infection models. An UL24-βgluc insertion mutant produced syncytial plaques and replicated to nearly wild type levels in tissue culture, but induced little or no pathological effects in recipient mice or guinea pigs following vaginal infection. Here we report that immunization of mice or guinea pigs with high or low doses of UL24-βgluc elicited a highly protective immune response. UL24-βgluc immunization via the vaginal or intramuscular routes was demonstrated to protect mice from a lethal vaginal challenge with wild type HSV-2. Moreover, antigen re-stimulated splenic lymphocytes harvested from immunized mice exhibited both HSV-2 specific CTL activity and IFN-γ expression. Humoral anti-HSV-2 responses in serum were Th1-polarized (IgG2a>IgG1) and contained high-titer anti-HSV-2 neutralizing activity. Guinea pigs vaccinated subcutaneously with UL24-βgluc or the more virulent parental strain (186) were challenged with a heterologous HSV-2 strain (MS). Acute disease scores were nearly indistinguishable in guinea pigs immunized with either virus. Recurrent disease scores were reduced in UL24-βgluc immunized animals but not to the same extent as those immunized with strain 186. In addition, challenge virus was not detected in 75% of guinea pigs subcutaneously immunized with UL24-βgluc. In conclusion, disruption of the UL24 gene is a prime target for the development of a genetically attenuated live HSV-2 vaccine.

Co-delivery of LIGHT expression plasmid enhances humoral and cellular immune responses to HIV-1 Nef in mice

The immunogenicity and efficacy of a DNA vaccine can be greatly enhanced when a gene adjuvant is used. LIGHT, a member of TNF superfamily, can function as a costimulatory molecule for human naïve T cells to proliferate and can be a potential gene adjuvant. In the current study, the eukaryotic expression plasmid pcDNA-nef was constructed by inserting a full-length nef gene into pcDNA3.1(+), and an in vitro transfection experiment suggested that the nef gene could be expressed successfully in mammalian cells. BALB/c mice were immunized with HIV-1 nef DNA vaccine plasmids alone or in combination with LIGHT expression plasmids, and the specific humoral and cellular immune responses were measured. The data showed that HIV-1 nef DNA vaccine plasmids could induce anti-Nef antibodies, Nef-specific lymphocyte proliferation and CTL activity, whereas stronger specific immune responses were induced in mice when co-immunizing with HIV-1 nef DNA vaccine plasmids and LIGHT expression plasmids, suggesting that the eukaryotic expression vector encoding HIV-1 nef is capable of inducing specific immune responses towards HIV-1 Nef and that LIGHT could be considered as a gene adjuvant for HIV-1 DNA vaccination.

CTLA-4 Blockade Enhances Antitumor Immunity of Intratumoral Injection of Immature Dendritic Cells into Irradiated Tumor in a Mouse Colon Cancer Model

Dendritic cells (DCs)-based cancer immunotherapy has been used various strategies to inhibit immune suppressive mechanisms. CD25 antibodies and cyclophosphamide are well-studied immunomodulators through inhibition of regulatory T cells (Treg) and a blockade the immune-checkpoint molecule, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) was recently targeted for immunomodulation. We used anti-CTLA-4 antibody, which is known to induce effective antitumor immunity by facilitating tumor-specific T-cell activation and suppressing Treg cells, as useful immunomodulator to provide a potentiating effect in the intratumoral injection of immature DCs (iDCs) into the irradiated tumor (IR/iDC). Ionizing radiation (IR) was applied at a dose of 10 Gy to the tumor on the right thigh of mice. Then, iDCs were intratumorally injected into the irradiated tumor. Anti-CTLA-4 antibody (100 µg/mouse) was administered intraperitoneally to mice on the same day with every iDCs injection. The growth of distant tumors was inhibited by IR/iDC and this effect was significantly augmented by combination treatment of anti-CTLA-4 antibody. Furthermore, the survival rate of tumor-bearing mice improved more by the combination treatment of anti-CTLA-4 antibody and IR/iDC compared with other groups. It was related to the increased tumor-specific interferon-γ-secreting T cells and CTL activity. Therefore, our results demonstrated that immunomodulator such as anti-CTLA-4 antibody enhances antitumor immunity of intratumoral injection of iDCs into irradiated tumor and suggested a new strategy to get more clinical benefits for cancer treatment.

Combination therapy targeting toll like receptors 7, 8 and 9 eliminates large established tumors.

BackgroundThe TLR7/8 agonist 3M-052 and the TLR9 agonist CpG ODN both trigger innate immune responses that support the induction of tumor-specific immunity. Previous studies showed that these agonists used individually could improve the survival of mice challenged with small tumors but were of limited therapeutic benefit against large/advanced tumors.MethodsNormal mice were challenged with syngeneic tumors. Once these tumors reached clinically detectable size (500–800 mm3) they were treated by intra-tumoral injection with 3M-052 and/or CpG ODN. Anti-tumor immunity and tumor growth were evaluated.ResultsThe co-delivery of agonists targeting TLRs 7, 8 and 9 increased the number and tumoricidal activity of tumor infiltrating CTL and NK cells while reducing the frequency of immunosuppressive MDSC. The combination of 3M-052 plus CpG ODN (but not each agent alone) eradicated large primary tumors and established long-term protective immunity.ConclusionThe combination of agonists targeting TLRs 7/8 and 9 represents a significant improvement in cancer immunotherapy.

Combined Stimulation of IL-2 and 4-1BB Receptors Augments the Antitumor Activity of E7 DNA Vaccines by Increasing Ag-Specific CTL Responses

Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer.

HPV16 CTL Epitope Peptide-activated Dendritic Cell and Natural Killer Co-culture for Therapy of Cervical Cancer in an Animal Model

There is increasing evidence that natural killer (NK) cells play an important role in antitumor immunity following dendritic cell (DC) vaccination. Little is known, however, about the optimal stimulation of DCs by epitopes and NK interactions for cytotoxicity in tumors. In this study, DC cells activated by the HPV16E7.49-57 epitope and LPS were co-cultured with NK cells in vitro, and then used ot immunize mice to study CTL activity of TC-1, which constitutively expresses HPV16E6E7, with an LDH release assay. Cytotoxicity in mice immunized with DC loaded with epitope HPVE7.49-57 vaccine co-cultured with NK was enhanced significantly (p<0.01). In conclusion, talk-across between DC and NK cells enhances their functions, also improving cytotoxicity againsttumor cells, suggesting that activated DC-NK by epitopes has potential application for cancer-specific immuno-cellular therapy.

Intramuscular electroporation of a P1A-encoding plasmid vaccine delays P815 mastocytoma growth

This study aimed to construct DNA vaccines encoding the mouse P1A tumor antigen and to generate a protective immune response against the P815 mastocytoma, as a model for vaccines against human MAGE-type tumor antigens. DNA vaccines were constructed and delivered to mice by intramuscular electroporation before tumor challenge. Immunization with a plasmid coding for the full-length P1A significantly delayed tumor growth and mice survived at least 10days longer than untreated controls. 10% of the mice completely rejected the P815 tumors while 50% of them showed a regression phase followed by tumor regrowth. Mice immunized by electroporation of a P1A35–43 minigene-encoding plasmid failed to reject tumor and even delay tumor growth. The P1A35–43-encoding plasmid was modified and helper epitope sequences were inserted. However, these modified plasmids were not able to improve the response against P815 mastocytoma. Consistent with these results, a 12-fold higher CTL activity was observed when the plasmid coding for full-length P1A was delivered as compared to the plasmid encoding the P1A35–43 epitope. Our results demonstrated that electroporation is an efficient method to deliver DNA vaccines against P815 and suggested the superiority of full-length as compared to minigene constructs for DNA vaccines.

Immunotherapy of cancer via mediation of cytotoxic T lymphocytes by methionine enkephalin (MENK)

The aim of this study was to investigate the immunological mechanisms by which synthetic methionine enkephalin (MENK) exerts therapeutic effects on tumor growth. Our findings in vivo or in vitro show that MENK treatment either in vivo or in vitro could up-regulate the percentages of CD8+T cells, induce markers of activated T cells, increased cytotoxic activity against mouse S180 tumor cells and increase secretion of IFNγ. In addition, the adoptively transferred CD8+T cells, after either in vitro or in vivo treatment with MENK, result in significantly increased survival of S180 tumor-bearing mice and significant shrinkage in tumor growth. Opioid receptors are detected on normal CD8+T cells and exposure to MENK leads to increased expression of opioid receptors. Interaction between MENK and the opioid receptors on CD8+T cells appears to be essential for the activation of CTL, since the addition of naltrexone (NTX), an opioid receptor antagonist, significantly inhibits all of the effects of MENK. The evidence obtained indicates that the MENK-induced T cell signaling is associated with a significant up-regulation of Ca2+ influx into the cytoplasm and the translocation of NFAT2 into nucleus, and these signaling effects are also inhibited by naltrexone.

IL-7 enhancement of antigen-driven activation/expansion of HIV-1-specific cytotoxic T lymphocyte precursors (CTLp).

CD8+ cytotoxic T lymphocytes are an important component in the immunologic control of human viral diseases. IL-7, a stromal cell-derived cytokine, has been demonstrated to enhance both anti-tumour and anti-viral CTL as well as lymphokine-activated killer (LAK) activity. We studied the ability of IL-7 to support the activation and the growth of in vitro antigen-specific CTL precursors (CTLp) present in peripheral blood mononuclear cells (PBMC) from HIV-infected patients. Results from these studies demonstrate that inclusion of IL-7 in a vaccinia/HIV-1 vector-based stimulation strategy greatly augmented overall CTL reactivities, whereas addition of IL-7 to unstimulated cultures failed to induce any significant anti-viral cytolytic activity. In four of six patients, HIV-specific lytic activities were significantly higher in cultures stimulated with antigen plus IL-7 compared with in vitro stimulation (IVS) with antigen alone. Cytotoxic activity was principally mediated by CD8+ effector cells, and CD3+/CD8+ cell expansion was increased by 2.7-fold in the presence of IL-7. In PBMC from seronegative donors, IL-7 enhanced anti-vaccinia CTL activities with less effect on cell proliferation. Furthermore, anti-gag CTL frequencies determined by limiting dilution analysis were increased by 2- and 10-fold in two asymptomatic patients following IVS plus IL-7 compared with antigen stimulation alone. Cytofluorimetric analysis revealed that IL-7 preferentially expanded CD8 memory cells (CD45RO+) and CD8+ lymphocytes expressing activation molecules. IL-7 was also able to support the growth of CD4+ lymphocytes, while having no effect on natural killer (NK)/K lymphocytes. Taken together, these data suggest that IL-7 acts cooperatively with the antigen supporting in vitro maturation of CTLp into functional cytotoxic effectors. Thus IL-7 may be an important biologic entity to consider as part of future immune-based therapies in which ex vivo expansion of antigen-driven CTL is an important determinant.

CD137 Expression Identifies Leukemia Specific CTL After In Vitro Priming Of Cord Blood T Cells

IntroductionWe have previously reported that cord blood T cells (CBT) can be expanded ∼ 2 log after 2 weeks in culture following stimulation with CD3/CD28 co-stimulatory beads and exogenous IL2 + IL7. The expanded progeny retained the starting naive phenotype typical of unmanipulated cord blood T cells (Cancer Research, Post-expansion, CD137, CD40L and perforin expression remains minimal and these cells demonstrate no cytotoxicity against an immunogenic lymphoblastoid cell line (IM9) or against transplant recipient PHA blasts. However, when primed against (IM9) or IFN-γ treated myeloid leukemia cell lines in the presence of IL12, IL7 and IL15, after 3 weeks of restimulation, lineage restricted cytotoxic activity was demonstrable (Cancer Research, ). Recently, we have demonstrated that IL15 alone can support the priming and expansion of CTL (abs 414, ASBMT 2013, manuscript in preparation) from CD3/CD28 expanded antigen inexperienced CB T cells. HypothesisCD137 (4-1BB) is a member of the TNFR- family and is expressed on activated T cells. 4-1BB ligation and signaling augments the survival, proliferation and lytic function of activated effectors. Expression of CD137 on activated T cells reflects recent activation. Therefore it may identify antigen-specific CTL. Adoptive T cell therapy with a restricted TCRVβ repertoire compared to bulk, unselected CTL pools is preferable to exclude potentially GVHD-causing clones in the HLA-mismatched cord blood transplant setting. Here we tested the hypothesis that de novo CD137 expression may identify leukemia-specific CTL precursors and that CD137 selection step could enrich for CTL clones in the priming culture. MethodsIM9-specific CTL was generated from pre-expanded, >98% pure T cells using weekly stimulations with irradiated targets and IL15. After 3 weeks, cytotoxicity was tested against fresh IM9 leukemia cells in a Europium labeling assay. CD137 expressing cells were sorted on FACS Aria II (BD Biosciences). CD137- and CD137+ cells were re-tested for cytotoxicity and TCRβ CDR3 spectratyping was done to identify their clonality. RT2 profilerTM PCR array human Th1 & Th2 responses (SABiosciences) was employed to contrast the sorted populations for differential gene expression ResultsAfter 3 weeks of priming/expansion, 2-3 % of the CTL were CD137+. However, after overnight re-stimulation, CD137 expression increased to a range of 12-39% of all T cells (Figure-1). The cytotoxicity of bulk cells in the culture segregated into the CD137+ cells after FACS sort (Figure-2). Amongst the 23 TCR Vβ families tested, distinct oligoclonal expansion of Vβ7, Vβ5.1 and Vβ21 were observed in the CD137+ population in contrast with CD137- T cells from the same culture, where the 3 Vβ families retained Gaussian-like, multiple clonal distributions (Figure-3). Notably, despite the different genetic background of starting CBT, the IM9 specific CTL activity appears to reside in the same CTL clones based on the virtually identical TCR Vβ clonality. This finding suggests that CTL targets have the greater influence on shaping CTL repertoir and not the genetic background (HLA, etc) of CBT cells. While post-sort RT qPCR analysis demonstrate significantly increased message for CCR4, EBI3, TNFRSF9 and TNFSF4 genes, CD40LG, IL2, IL6, IL7R, SFTPD and SPP1 expressions were significantly decreased in CD137+ cells. ConclusionIL15 without other exogenous cytokines supports the in vitro priming and expansion of polyclonal CTL against leukemia cell lines from expanded cord blood T cells. Cytotoxicity dominates in the CD137 positive subset, therefore CD137 positive selection can be used to isolate and enrich leukemia specific CTLs that retain a predictable TCRVβ repertoire shaped by the leukemic target. These findings will have great influence on attaining clinically applicable leukemia-specific CTL protocols.▪▪▪ Disclosures:No relevant conflicts of interest to declare.

Therapeutic activity of a combination of immunostimulatory monoclonal antibodies (anti-B7-H1, CD137 and OX40) on a c-myc-driven spontaneous transgenic model of hepatocellular carcinoma

Several immunostimulatory monoclonal antibodies (ISmAbs) that derepress and unleash antitumor immune responses are showing efficacy in cancer clinical trials. MAbs anti-B7-H1 (PD-L1) block a critical inhibitory pathway for T cells, while antibodies anti-CD137 and OX40 provide intense T cell costimulation. Documenting efficacy of immunotherapies on spontaneous tumors arising in oncogene transgenic mice is considered more predictive than experiments on transplanted tumors. A combination of these ISmAbs has been tested on a transgenic mouse model of spontaneous primary liver cancer in which c-myc drives transformation and the tumor cells express ovalbumin as a surrogate transgenic antigen. The induced tumor lymphocyte infiltrates and immune mechanisms of action were studied. The triple combination of mAbs clearly extended survival of mice bearing hepatocellular carcinomas (HCC) and synergized with adoptive T cell therapy with activated TCR-transgenic T cells that recognize OVA. Mice undergoing therapy showed a clear increase in the tumor tissue infiltration by activated and blastic CD8 T lymphocytes expressing the ISmAb-targeted receptors. The triple combination of ISmAbs did not result in enhanced OVA-specific CTL activity but other antigens in cell lines derived from such HCC were recognized by the elicited tumor infiltrating T lymphocytes. Indeed adoptive transfer of OT-1 cells to tumor bearing mice resulted in their tolerization, unless the triple mAb therapy was instigated. Our results emphasize the role of combinational immunotherapy approaches including ISmAbs to impact on agressive and highly T cell-tolerizing hepatocellular carcinomas.

Abstract PL07-02: The Renaissance of immunotherapy.

Abstract Bringing down the walls of defense The recent results of clinical trials with various anti-PD-1/PDL-1 monoclonal antibodies have converted many non-believers into believers of the enormous potential of immunomodulatory agents to gain control over some, if not many, solid tumors. Tackling immune suppression, seems to be a much more powerful intervention than short-lived immune-activating strategies and the current axioma of "inhibiting the inhibitor" and "breaking tolerance" appears to be a particularly powerful approach. This was first demonstrated by the success of anti-CTLA4 molecules which “prevent the downregulation of CTL activity and/or reawaken immuneresponses that have gone silent.” The focus these days is very much on anti-PD1 and anti-PDL1 monoclonal antibodies that can prevent the downregulation of CTL activity at the tumor site because of PDL1 expression on tumor cells. Taking down very effective walls of defense is essential to give immune-therapeutic strategies a chance. Immune-stimulation alone with T-cell agonists or cytokines or vaccines is rather ineffective when defense mechnisms are intact centrally (lymph nodes: mostly CTLA4) and peripherally (tumor site: mostly PDL1). Moreover anti-CTLA4 and anti-PD1/PDL1 can synergize in boosting an effective immuneresponse leading to long lasting responses in high percentages of patients. The concept of a clinical cure emerges, as chronic control of metastatic disease is an emerging reality in melanoma, with immuno-combos. It is clear that checkpoint inhibitors open the door for the effective use of agonists, such as OX40, 4BB-1 and CD137 and for synergy with cytokines such as IL2 and IFN. Key role of the immune system in various combination approaches The immune system is the key-component of long term responses, not only in immunotherapeutic approaches. There is an increasing body of evidence that patients with long term responses or “clinical cure” conditions after chemotherapy or targeted-agent therapies depend on immunogenic cell death mechanisms, (re)activation of the immune system and the presence of prominent immune-infiltrates in residual tumor lesions. Chemotherapeutics and targeted agents can lead to immunogenic or immune-tolerizing cell death, the first being associated with prolonged responses and control or even cure, the latter being associated with failure and death. Examples for immunogenic chemotherapeutics are anthracyclines, oxaliplatine, ionizing radiation and cyclophosphamide, whereas other chemotherapeutics (cisplatinum, mitomycin C, etoposide) have been found to induce unresponsiveness. Therapeutics that cause immunogenic cell death The molecular cues underlying this immunogenic cell death have been partly unveiled. Cell demise should be accompanied by an ER stress response which promotes calreticuline exposure and phagocytosis of dead cells by phagocytes. Next, autophagy is mandatory for ATP release and inflammatory monocytes to become dendritic cells (DC) priming T cell responses. Finally, HMGB1 release from the nucleus is indispensable to engage TLR4 on DC to accelerate the processing and antigen presentation to T lymphocytes By eliciting anticancer effector and memory T cells, immunogenic cell death contributes to the success of chemotherapy.Immunological off target effects have also been described for targeted agents and radiation therapy. Conclusions Clinical cures and eventually possibly a complete cancer cure becomes possible if we respect one basic principle: successful therapies require the induction / re-induction of anticancer immune responses. Renaissance is here. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL07-02. Citation Format: Alexander M. Marie Eggermont. The Renaissance of immunotherapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PL07-02.

Deletion of naïve T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers inhibits cognate CTL responses and alters immunodominance

Alloreactive T-cell responses directed against minor histocompatibility (H) antigens, which arise from diverse genetic disparities between donor and recipient outside the MHC, are an important cause of rejection of MHC-matched grafts. Because clinically significant responses appear to be directed at only a few antigens, the selective deletion of naïve T cells recognizing donor-specific, immunodominant minor H antigens in recipients before transplantation may be a useful tolerogenic strategy. We have previously demonstrated that peptide-MHC class I tetramers coupled to a toxin can efficiently eliminate specific TCR-transgenic T cells in vivo. Here, using the minor histocompatibility antigen HY as a model, we investigated whether toxic tetramers could inhibit the subsequent priming of the two H2-Db-restricted, immunodominant T-cell responses by deleting precursor CTL. Immunization of female mice with male bone marrow elicited robust CTL activity against the Uty and Smcy epitopes, with Uty constituting the major response. As hypothesized, toxic tetramer administration prior to immunization increased survival of cognate peptide-pulsed cells in an in vivo CTL assay, and reduced the frequency of corresponding T cells. However, tetramer-mediated decreases in either T-cell population magnified CTL responses against the non-targeted epitope, suggesting that Db-Uty+ and Db-Smcy+ T cells compete for a limited common resource during priming. Toxic tetramers conceivably could be used in combination to dissect manipulate CD8+ T-cell immunodominance hierarchies, and to prevent the induction of donor-specific, minor H antigen CTL responses in allotransplantation.

Targeting FSTL1 Prevents Tumor Bone Metastasis and Consequent Immune Dysfunction

Bone metastasis greatly deteriorates the quality of life in patients with cancer. Although mechanisms have been widely investigated, the relationship between cancer bone metastasis and antitumor immunity in the host has been much less studied. Here, we report a novel mechanism of bone metastasis mediated by FSTL1, a follistatin-like glycoprotein secreted by Snail(+) tumor cells, which metastasize frequently to bone. We found that FSTL1 plays a dual role in bone metastasis-in one way by mediating tumor cell invasion and bone tropism but also in a second way by expanding a population of pluripotent mesenchymal stem-like CD45(-)ALCAM(+) cells derived from bone marrow. CD45(-)ALCAM(+) cells induced bone metastasis de novo, but they also generated CD8(low) T cells with weak CTL activity in the periphery, which also promoted bone metastasis in an indirect manner. RNA interference-mediated attenuation of FSTL1 in tumor cells prevented bone metastasis along with the parallel increase in ALCAM(+) cells and CD8(low) T cells. These effects were accompanied by heightened antitumor immune responses in vitro and in vivo. In clinical specimens of advanced breast cancer, ALCAM(+) cells increased with FSTL1 positivity in tumor tissues, but not in adjacent normal tissues, consistent with a causal connection between these molecules. Our findings define FSTL1 as an attractive candidate therapeutic target to prevent or treat bone metastasis, which remains a major challenge in patients with cancer.

Accessory Cells of the Microenvironment Protect Multiple Myeloma from T-Cell Cytotoxicity through Cell Adhesion-Mediated Immune Resistance

Cellular immunotherapy frequently fails to induce sustained remissions in patients with multiple myeloma, indicating the ability of multiple myeloma cells to evade cellular immunity. Toward a better understanding and effective therapeutic modulation of multiple myeloma immune evasion mechanisms, we here investigated the role of the tumor microenvironment in rendering multiple myeloma cells resistant to the cytotoxic machinery of T cells. Using a compartment-specific, bioluminescence imaging-based assay system, we measured the lysis of luciferase-transduced multiple myeloma cells by CD4(+) or CD8(+) CTLs in the presence versus absence of adherent accessory cells of the bone marrow microenvironment. We simultaneously determined the level of CTL activation by measuring the granzyme B release in culture supernatants. Bone marrow stromal cells from patients with multiple myeloma and healthy individuals, as well as vascular endothelial cells, significantly inhibited the lysis of multiple myeloma cells in a cell-cell contact-dependent manner and without substantial T-cell suppression, thus showing the induction of a cell adhesion-mediated immune resistance (CAM-IR) against CTL lysis. Further analyses revealed that adhesion to accessory cells downregulated Fas and upregulated the caspase-3 inhibitor survivin in multiple myeloma cells. Reconstitution of Fas expression with bortezomib enhanced the CTL-mediated lysis of multiple myeloma cells. Repressing survivin with the small-molecule YM155 synergized with CTLs and abrogated CAM-IR in vitro and in vivo. These results reveal the cell adhesion-mediated induction of apoptosis resistance as a novel immune escape mechanism and provide a rationale to improve the efficacy of cellular therapies by pharmacologic modulation of CAM-IR.

A VLP library of C-terminally truncated Hepatitis B core proteins: correlation of RNA encapsidation with a Th1/Th2 switch in the immune responses of mice.

An efficient pBR327- and Ptrp-based E. coli expression system was used to generate a large-scale library of virus like particles (VLP) formed by recombinant hepatitis B virus (HBV) core (HBc) protein derivatives. To construct the library, the gene of HBc protein of the genotype D/subtype ayw2 virus was gradually truncated from the 3`-end and twenty-two HBc variants (with truncation up to 139 aa) were expressed at high levels. The proteins were purified by salt precipitation and gel filtration. Background RNA binding was observed for VLPs formed by HBc1-149, which lacked all C-terminal Arg blocks, and the addition of three Arg residues (HBc1-152) only slightly increased RNA binding. The presence of two Arg blocks (proteins HBc1-162 and HBc1-163) resulted in approximately half of the typical level of RNA binding, and the presence of three blocks (protein HBc1-171) led to approximately 85% of the typical level of binding. Only a small increase in the level of RNA binding was found for the HBc1-175 VLPs, which contained all four Arg blocks but lacked the last 8 aa of the full-length HBc protein. VLPs containing high levels of RNA had higher antigenicity according to an ELISA with anti-HBc mAbs than the VLPs formed by HBc variants without C-terminal Arg blocks and lacking RNA. The results indicate that the VLPs were stabilised by nucleic acids. The immunogenicity in BALB/c mice was comparable for VLPs formed by different HBc proteins, but a clear switch from a Th1 response to a Th2 response occurred after the loss of encapsidated RNA. We did not observe significant differences in lymphocyte proliferation in vitro for the tested VLP variants; however, the loss of RNA encapsidation correlated with a decreased level of IFN-γ induction, which is a measure of the potential CTL activity of immunogens.

Wnt signaling inhibits CTL memory programming

Induction of functional CTLs is one of the major goals for vaccine development and cancer therapy. Inflammatory cytokines are critical for memory CTL generation. Wnt signaling is important for CTL priming and memory formation, but its role in cytokine-driven memory CTL programming is unclear. We found that wnt signaling inhibited IL-12-driven CTL activation and memory programming. This impaired memory CTL programming was attributed to up-regulation of eomes and down-regulation of T-bet. Wnt signaling suppressed the mTOR pathway during CTL activation, which was different to its effects on other cell types. Interestingly, the impaired memory CTL programming by wnt was partially rescued by mTOR inhibitor rapamycin. In conclusion, we found that crosstalk between wnt and the IL-12 signaling inhibits T-bet and mTOR pathways and impairs memory programming which can be recovered in part by rapamycin. In addition, direct inhibition of wnt signaling during CTL activation does not affect CTL memory programming. Therefore, wnt signaling may serve as a new tool for CTL manipulation in autoimmune diseases and immune therapy for certain cancers.

Immunogenicity of a Multiepitope Plasmid DNA Encoding T and B Lymphocyte Epitopes from Latent Membrane Protein 2 (LMP2) of Epstein-Barr Virus as a Vaccine in Mice

Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associating with several malignant diseases. Latent membrane protein 2 (LMP2) of EBV is considered to be an ideal candidate for immunotherapy or prophylactic EBV vaccine. We designed a LMP2 multiepitope containing T and B-cell epitope-rich peptides and constructed a recombinant plasmid containing mammalian codonoptimization EBV LMP2 multiepitope (pcDNA3.1+/EBV-LMP2 multiepitope). After pcDNA3.1+/EBV-LMP2 multiepitope was transfected into COS-7 cells, significant expression of the multiepitope in COS-7 cells was confirmed by RT-PCR and immunofluorescence assay. Western blot analysis indicated that serum from immunized mice could be discerned by the EBV-LMP2 protein and the EBV-LMP2 multiepitope specifically. The plasmid DNA of EBV-LMP2 multiepitope induced high levels anti-EBV membrane protein and anti-EBV LMP2 multiepitope IgG in mice. T lymphocytes from spleen of immunized mice showed a strong CTL activity. The present study suggested that plasmid DNA encoding EBV LMP2 multiepitope capable of stimulating enough cellular and humoral immunity could have potential for preventing or controlling EBV infection and EBV associated disease in mice.

Japanese Encephalitis Virus Generated Neurovirulence, Antigenicity, and Host Immune Responses

In response to a JE virus attack, infected body cells start secretion of different cytokines and activate innate immune response. Virus starts neuronal invasion by entering into nerve cells and inflecting the central nervous system. It avoids exposure of body’s natural immunity and generates neurotrophic effects. Virus causes acute susceptibility to CNS and establishes encephalitis syndrome that results in very high fatality in children. In survivors, JEV inhibits the growth and proliferation of NCPs and imposes permanent neuronal disorders like cognitive, motor, and behavioral impairments. However, body cells start TCR mediated interactions, to recognize viral antigens with class I MHC complex on specific target cells, and operate mass killing of virus infected cells by increased CTL activity. Thus, both cell mediated and antibody interactions plays a central role in protection against JEV. In the present review article virus generated neurovirulence, antigenicity, and host immune responses are described in detail. More emphasis is given on diagnosis, clinical care, and active immunization with well-designed potential antiflavivirus vaccines. Further, for achieving an elite success against JEV, global eradication strategies are to be needed for making vaccination program more responsible and effective in endemic areas.