Interleukin-4 impairs granzyme-mediated cytotoxicity of Simian virus 40 large tumor antigen-specific CTL in BALB/c mice

In this report we analyzed the impact of interleukin-4 (IL-4) on tumor-associated simian virus 40 (SV40) large T-antigen (TAg)-specific CD8⁺ cytotoxic T cells during rejection of syngeneic SV40 transformed mKSA tumor cells in BALB/c mice. Strikingly, challenge of naïve mice with low doses of mKSA tumor cells revealed a CD8⁺ T cell-dependent prolonged survival time of naïve IL-4^?/? mice. In mice immunized with SV40 TAg we observed in IL-4^?/? mice, or in wild type mice treated with neutralizing anti-IL-4 monoclonal antibody, a strongly enhanced TAg-specific cytotoxicity of tumor associated CD8⁺ T cells. The enhanced cytotoxicity in IL-4^?/? mice was accompanied by a significant increase in the fraction of CD8⁺ tumor associated T-cells expressing the cytotoxic effector molecules granzyme A and B and in granzyme B-specific enzymatic activity. The data suggest that endogenous IL-4 can suppress the generation of CD8⁺ CTL expressing cytotoxic effector molecules especially when the antigen induces only a very weak CTL response.     

MHC-class I-restricted CD4 T cells: a nanomolar affinity TCR has improved anti-tumor efficacy in vivo compared to the micromolar wild-type TCR

Clinical studies with immunotherapies for cancer, including adoptive cell transfers of T cells, have shown promising results. It is now widely believed that recruitment of CD4⁺ helper T cells to the tumor would be favorable, as CD4⁺ cells play a pivotal role in cytokine secretion as well as promoting the survival, proliferation, and effector functions of tumor-specific CD8⁺ cytotoxic T lymphocytes. Genetically engineered high-affinity T-cell receptors (TCRs) can be introduced into CD4⁺ helper T cells to redirect them to recognize MHC-class I-restricted antigens, but it is not clear what affinity of the TCR will be optimal in this approach. Here, we show that CD4⁺ T cells expressing a high-affinity TCR (nanomolar K _d value) against a class I tumor antigen mediated more effective tumor treatment than the wild-type affinity TCR (micromolar K _d value). High-affinity TCRs in CD4⁺ cells resulted in enhanced survival and long-term persistence of effector memory T cells in a melanoma tumor model. The results suggest that TCRs with nanomolar affinity could be advantageous for tumor targeting when expressed in CD4⁺ T cells.     

Aged mice develop protective antitumor immune responses with appropriate costimulation

There is a clear decrease in CD8(+) T cell effector function with aging, a loss once thought to be intrinsic to the CD8(+) T cells. Recent studies suggest, however, that this decline may be a consequence of altered stimulatory signals within the aged lymphoid microenvironment. In this study, we compared the immune responses of young and old mice against the BM-185 pre-B cell lymphoma expressing enhanced GFP (EGFP) as a surrogate tumor Ag. Young animals develop protective immune responses when immunized with BM-185-EGFP, but aged mice do not and ultimately succumb to the tumor. However, expression of CD80 (B7.1) on the BM-185-EGFP (BM-185-EGFP-CD80) results in rejection of the tumor by both young and old animals. Additionally, injection of BM-185-EGFP-CD80 cells in young mice promotes the development of long-lasting memory responses capable of rejecting BM-185 wild-type tumors. Aged animals similarly injected did not develop antitumor memory responses. Interestingly, old animals immunized with the BM-185-EGFP-CD80 cells plus injections of the agonist anti-OX40 mAb did develop long-lasting memory responses capable of rejecting the BM-185 wild-type tumors with the same vigor as the young animals. We show that old mice have the capacity to develop strong antitumor responses and protective memory responses as long as they are provided with efficient costimulation. These results have important implications for the development of vaccination strategies in the elderly, indicating that the aged T cell repertoire can be exploited for the induction of tumor immunity.     

CD4<Superscript>+</Superscript> T cell response against a non-tumor antigen is unaffected in melanoma-bearing mice

The tumor microenvironment is complex and creates an immunosuppressive network to tolerize tumor-specific immune responses; however, little information is available regarding the response against non-tumor antigens in tumor-bearing individuals. The goal of the present study was to evaluate if tumor burden could influence a CD4⁺ T cell response against a soluble protein, not expressed by the tumor, in the absence of in vitro stimulation. Using an experimental system in which we can compare CD4⁺ T cell responses to the Ea antigen when it is either expressed by B16F10 melanoma cells (B16EaRFP cells) or is an exogenous, non-tumor antigen (soluble EaRFP protein), in immunizations of B16F10 tumor-bearing mice, we observed that the tumor can modulate the CD4⁺ T cell-specific response to the antigen when it is expressed by the tumor cells. TEa cells proliferated poorly and produced less IFN-γ in mice bearing B16F10 melanoma expressing Ea peptide, and tumor growth was impervious to this response. However, in mice bearing 7 days B16F10 tumors, not expressing the Ea antigen, priming of TEa cells was similar to that observed in tumor-free mice, based on the total number of cells recovered and proliferation assessed by CFSE dilution after EaRFP immunization. We also investigated if tumor burden could influence recall responses of already differentiated effector cells. We immunized mice with EaRFP antigen and after a few days injected B16F10 cells. After 10 days of tumor growth, we challenged the mice with the non-tumor antigen. We found that the number of TEa cells producing IFN-γ in tumor-bearing mice was not different compared to tumor-free mice. No differences in antigen presentation, assessed by YAe antibody staining, were verified in the draining lymph node of these two groups. Collectively, our data indicate that tumor burden does not affect immune responses to non-tumor antigens. These results have important implications in the design of anti-cancer therapy.     

CpG-Oligodeoxynucleotides activate tyrosinase-related protein 2–specific T lymphocytes but do not lead to a protective tumor-specific memory response

Purpose: Peritumoral CpG-oligodeoxynucleotide (ODN) treatment has been successful in tumor mouse models expressing strong antigens to induce activation of tumor-specific CD8⁺ T lymphocytes which contribute to the control of tumor growth. To get near to clinical reality, the tumor-specific CD8⁺ response was investigated in mice bearing the weakly immunogenic B16 melanoma tumor and using the melanocyte differentiation tyrosinase-related protein 2 (TRP-2) as a tracking antigen. Methods: The expansion and activation of TRP-2–specific T lymphocytes by CpG-ODNs was analyzed by tetramer staining and IFN- production assays, while the activity of these cells in both memory and primary response was evaluated in vivo. Results: After CpG-ODN treatment, the number of TRP-2 tetramer-stained CD8⁺ T lymphocytes was not significantly modified, but these cells produced higher levels of interferon (IFN-) in response to the antigen than those from untreated mice. Mice possessing these activated T lymphocytes, when evaluated for their antitumor memory response, showed marginal protection against intravenous (i.v.) and subcutaneous (s.c.) tumor rechallenge. These cells were not crucial for the control of primary tumor growth since strong reduction of subcutaneous tumor was observed after CpG-ODN treatment in both CD8⁺ T cell depleted or nondepleted mice. On the contrary, NK cell depletion markedly reduced CpG-ODN-induced tumor growth inhibition. Conclusions: Altogether, these data indicate the CpG treatment activates tumor-reactive effector CD8⁺ T lymphocytes, but, paralleling recent clinical observations, our model indicates that the mere activation of antitumor T cells is insufficient to result in a clinical response.Abbreviations CpG unmethylated CpG dinucleotides - ODNs oligodeoxynucleotides - TLR9 toll-like receptor 9 - TRP-2 tyrosinase-related protein 2     

Introduction of 65 kDa antigen of Mycobacterium tuberculosis to cancer cells enhances anti-tumor effect of BCG therapy

Bacillus Calmette Guerin (BCG) immunotherapy has anti-tumorigenic effects against bladder cancer. To improve the efficacy of BCG therapy, we introduced the gene encoding the 65 kDa heat shock protein (hsp) of Mycobacterium tuberculosis into a mouse malignant melanoma cell line (B16). An expression vector harboring the 65 kDa antigen gene was transfected into B16 using Lipofectamine, then expression of the antigen was confirmed by RT-PCR and Western blotting. Several cell lines expressing 65 kDa antigen were established (B16/65 kDa). We also established a control cell line transfected with the vector alone (B16/con). All cell lines (B16, B16/con, B16/65 kDa) were injected intraperitoneally into syngeneic mice with or without BCG prior immunization and the development of tumor ascites was examined. To analyze the mechanism of the anti-tumor effect, CD4 T cells or CD8 T cells were depleted in vivo by administering the corresponding monoclonal antibody. B16/65k Da expressed the 65 kDa hsp of M. tuberculosis. The tumor growth of B16/65 kDa was slightly retarded in naive mice, but significantly inhibited by BCG. The anti-tumor effect was totally abrogated in mice deficient in CD4 T cells, suggesting that CD4 T cells are involved in this process. The 65 kDa hsp of M. tuberculosis was expressed after gene transduction in a malignant melanoma cell line and significantly enhanced the anti-tumor effect of BCG immunotherapy. CD4 T cells play an important role in this anti-tumor effect.     

Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations

T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19⁺ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2^nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR⁺ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1⁺ tumors. Moreover, such cells could eliminate ROR1⁺ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR⁺ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.     

Allele-specific expression of the mouse B-cell surface protein CD72 on T cells

 CD72 is a 45 000 M _r mouse B-cell surface glycoprotein involved in B-cell proliferation and differentiation. Expression of mouse CD72 is thought to be restricted to the B-cell lineage. We recently demonstrated that the monoclonal antibodies K10.6 and B9.689, previously defined as recognizing the mouse lymphocyte alloantigens Ly-19.2 and Ly-32.2, respectively, recognize specific alleles of CD72. Early studies using antibody-mediated cytotoxicity assays demonstrated that K10.6 and B9.689 react with B cells, several T-cell lines, and a subset of peripheral T cells. These findings led us to consider the possibility that CD72 might also be expressed on a subset of T cells. In this report we demonstrate that CD72 is constitutively expressed on a fraction of peripheral T cells isolated from strains of mice expressing the CD72 ^b allele, but not the CD72 ^a or CD72 ^c alleles. Three days after activating T cells with concanavalin A or plate-bound CD3-specific mAb, CD72 is expressed on a larger fraction of peripheral T cells as well as a fraction of thymocytes from mouse strains expressing the CD72 ^b allele. CD72 is expressed on both the CD4⁺ and CD8⁺ thymocyte and peripheral T-cell subsets. No CD72 expression is detected on activated thymocytes or peripheral T cells from mouse strains expressing the CD72 ^a or CD72 ^c alleles. Expression of CD72 ^b on peripheral T cells was confirmed by northern blot analysis demonstrating CD72 mRNA expression. These results demonstrate that CD72 expression is not restricted to B lineage cells in mouse strains expressing the CD72 ^b allele; instead, a population of T lineage cells in these mice also expresses CD72. Received: 18 June 1996 / Revised: 17 September 1996     

Small numbers of residual tumor cells at the site of primary inoculation are critical for anti-tumor immunity following challenge at a secondary location

Luciferase-transduced B16 murine melanoma cells (luc-B16) inoculated in ear skin do not form tumors but prevent tumor formation by luc-B16 cells injected into the footpad. To determine the requirements for such immunity, we followed the fate of luc-B16 cells following ear injection. Surprisingly, small numbers of viable luc-B16 cells were detected in tumor-free mouse skin for up to 60 days post-inoculation. After 1 week, the number of Foxp3⁺CD4⁺CD25⁺ T cells (along with foxp3 mRNA expression) increased rapidly in the injected ear skin. Residual tumor cells in ears were reduced in mice treated with anti-CD25 mAb and in CD4-deficient mice, but increased in CD8-deficient mice. Strikingly, the loss of luc-B16 cells in the ear skin, either spontaneously or following amputation of the injected ear, resulted in significantly enhanced tumor formation by parental and luciferase-expressing B16 cells after footpad injection. These studies suggest that small numbers of tumor cells (possibly regulated by CD4⁺CD25⁺ regulatory T cells expressing Foxp3) are required for effective host anti-tumor responses at alternate inoculation sites.     

Skin tumor responsiveness to interleukin-2 treatment and CD8 Foxp3+ T cell expansion in an immunocompetent mouse model

Recombinant human interleukin-2 (rhIL-2) therapy is approved for treating patients with advanced melanoma yet significant responses are observed in only 10–15% of patients. Interleukin-2 induces Foxp3 expression in activated human CD8 T cells in vitro and expands circulating CD8 Foxp3+ T cells in melanoma patients. Employing IL-2 responsive (B16-F1, B16-BL6, JB/MS, MCA-205) and nonresponsive (JB/RH, B16-F10) subcutaneous tumor mouse models, we evaluated CD8 Foxp3+ T cell distribution and changes in response to rhIL-2 (50,000 U, i.p. or s.q., twice daily for 5 days). In tumor-free mice and subcutaneous tumor-bearing mouse models, CD8 Foxp3+ T cells were a rare but naturally occurring cell subset. Primarily located in skin-draining lymph nodes, CD8 Foxp3+ T cells expressed both activated T cell (CD28⁺, CD44⁺) and Treg (CTLA4⁺, PD1^lo/var, NKG2A^+/var) markers. Following treatment with rhIL-2, a dramatic increase in CD8 Foxp3+ T cell prevalence was observed in the circulation and tumor-draining lymph nodes (TD.LNs) of animals bearing IL-2 nonresponsive tumors, while no significant changes were observed in the circulation and TD.LNs of animals bearing IL-2 responsive tumors. These findings suggest expansion of CD8 Foxp3+ T cell population in response to rhIL-2 treatment may serve as an early marker for tumor responsiveness to immunotherapy in an immune competent model. Additionally, these data may provide insight to predict response in patients with melanoma undergoing rhIL-2 treatment.     

Intratumoral delivery of vector mediated IL-2 in combination with vaccine results in enhanced T cell avidity and anti-tumor activity

Systemic IL-2 is currently employed in the therapy of several tumor types, but at the price of often severe toxicities. Local vector mediated delivery of IL-2 at the tumor site may enhance local effector cell activity while reducing toxicity. To examine this, a model using CEA-transgenic mice bearing established CEA expressing tumors was employed. The vaccine regimen was a s.c. prime vaccination with recombinant vaccinia (rV) expressing transgenes for CEA and a triad of costimulatory molecules (TRICOM) followed by i.t. boosting with rF-CEA/TRICOM. The addition of intratumoral (i.t.) delivery of IL-2 via a recombinant fowlpox (rF) IL-2 vector greatly enhanced anti-tumor activity of a recombinant vaccine, resulting in complete tumor regression in 70–80% of mice. The anti-tumor activity was shown to be dependent on CD8⁺ cells and NK1.1⁺. Cellular immune assays revealed that the addition of rF-IL-2 to the vaccination therapy enhanced CEA-specific tetramer⁺ cell numbers, cytokine release and CTL lysis of CEA⁺ targets. Moreover, tumor-bearing mice vaccinated with the CEA/TRICOM displayed an antigen cascade, i.e., CD8⁺ T cell responses to two other antigens expressed on the tumor and not the vaccine: wild-type p53 and endogenous retroviral antigen gp70. Mice receiving rF-IL-2 during vaccination demonstrated higher avidity CEA-specific, as well as higher avidity gp70-specific, CD8⁺ T cells when compared with mice vaccinated without rF-IL-2. These studies demonstrate for the first time that the level and avidity of antigen specific CTL, as well as the therapeutic outcome can be improved with the use of i.t. rF-IL-2 with vaccine regimens.     

Induced pluripotent stem cell‐derived myeloid cells expressing OX40 ligand amplify antigen‐specific T cells in advanced melanoma

Immune checkpoint inhibitors improved the survival rate of patients with unresectable melanoma. However, some patients do not respond, and variable immune‐related adverse events have been reported. Therefore, more effective and antigen‐specific immune therapies are urgently needed. We previously reported the efficacy of an immune cell therapy with immortalized myeloid cells derived from induced pluripotent stem cells (iPS‐ML). In this study, we generated OX40L‐overexpressing iPS‐ML (iPS‐ML‐Zsgreen‐OX40L) and investigated their characteristics and in vivo efficacy against mouse melanoma. We found that iPS‐ML‐Zsgreen‐OX40L suppressed the progression of B16‐BL6 melanoma, and prolonged survival of mice with ovalbumin (OVA)‐expressing B16 melanoma (MO4). The number of antigen‐specific CD8⁺ T cells was higher in spleen cells treated with OVA peptide‐pulsed iPS‐ML‐Zsgreen‐OX40L than in those without OX40L. The OVA peptide‐pulsed iPS‐ML‐Zsgreen‐OX40L significantly increased the number of tumor‐infiltrating T lymphocytes (TILs) in MO4 tumor. Flow cytometry showed decreased regulatory T cells but increased effector and effector memory T cells among the TILs. Although we plan to use allogeneic iPS‐ML in the clinical applications, iPS‐ML showed the tumorgenicity in the syngeneic mice model. Incorporating the suicide gene is necessary to ensure the safety in the future study. Collectively, these results indicate that iPS‐ML‐Zsgreen‐OX40L therapy might be a new method for antigen‐specific cancer immunotherapy.     

Antitumor activity of a self-adjuvanting glyco-lipopeptide vaccine bearing B cell,CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cell epitopes

Molecularly defined synthetic vaccines capable of inducing both antibodies and cellular anti-tumor immune responses, in a manner compatible with human delivery, are limited. Few molecules achieve this target without utilizing external immuno-adjuvants. In this study, we explored a self-adjuvanting glyco-lipopeptide (GLP) as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. A prototype B and T cell epitope-based GLP molecule was constructed by synthesizing a chimeric peptide made of a CD8⁺ T cell epitope, from ovalbumin (OVA_257–264) and an universal CD4⁺ T helper (Th) epitope (PADRE). The resulting CTL–Th peptide backbones was coupled to a carbohydrate B cell epitope based on a regioselectively addressable functionalized templates (RAFT), made of four α-GalNAc molecules at C-terminal. The N terminus of the resulting glycopeptides (GP) was then linked to a palmitic acid moiety (PAM), obviating the need for potentially toxic external immuno-adjuvants. The final prototype OVA-GLP molecule, delivered in adjuvant-free PBS, in mice induced: (1) robust RAFT-specific IgG/IgM that recognized tumor cell lines; (2) local and systemic OVA_257–264-specific IFN-γ producing CD8⁺ T cells; (3) PADRE-specific CD4⁺ T cells; (4) OVA-GLP vaccination elicited a reduction of tumor size in mice inoculated with syngeneic murine MO5 carcinoma cells and a protection from lethal carcinoma cell challenge; (5) finally, OVA-GLP immunization significantly inhibited the growth of pre-established MO5 tumors. Our results suggest self-adjuvanting glyco-lipopeptide molecules as a platform for B Cell, CD4⁺, and CD8⁺ T cell epitopes-based immunotherapeutic cancer vaccines. Both I. Bettahi and G. Dasgupta have contributed equally to this work.     

A20 Controls Macrophage to Elicit Potent Cytotoxic CD4+ T Cell Response

Emerging evidence indicates that CD4⁺ T cells possess cytotoxic potential for tumor eradication and perforin/granzyme-mediated cytotoxicity functions as one of the important mechanisms for CD4⁺ T cell-triggered cell killing. However, the critical issue is how the cytotoxic CD4⁺ T cells are developed. During the course of our work that aims at promoting immunostimulation of APCs by inhibition of negative regulators, we found that A20-silenced Mф drastically induced granzyme B expression in CD4⁺ T cells. As a consequence, the granzyme-highly expressing CD4⁺ T cells exhibited a strong cytotoxic activity that restricted tumor development. We found that A20-silenced Mф activated cytotoxic CD4⁺ T cells by MHC class-II restricted mechanism and the activation was largely dependent on enhanced production of IFN-γ.     

Immortalization of human T lymphocytes by oncogenes

Immortalized human T cell lines were established by cotransfecting c-Ha-ras and c-myc oncogenes to lymph node lymphocytes. The cell lines kept growing for 3 months after establishment without a decrease in growth rate. The cells did not require interleukin-2(IL-2) for their growth, but addition of IL-2 stimulated the growth of these cells. Flow cytometric analysis revealed that these cells were T cells expressing CD4 or CD8 antigens. A CD4 positive (CD4⁺) cell line produced IL-6, indicating that the cell line belongs to helper T cells. The CD8 positive (CD8⁺) cell line possessed cytotoxicity to tumor cells, indicating that the cell line were killer T cells. Both cell lines were able to proliferate in serum-free medium indefinitely.     

Signaling through CD40 enhances cytotoxic T lymphocyte generation by CD8+ T cells from mice bearing large tumors

Recent studies have demonstrated the importance of CD40/CD154 (CD40L) interactions for the generation of cell-mediated antitumor immune responses. Here we show that signaling via CD40 (through the use of the activating anti-CD40 mAb, 1C10) can actually promote the in vitro generation of CTL activity by CD8⁺ splenic T cells from mice bearing a large MOPC-315 tumor. Anti-CD40 mAb had to be added at the initiation of the stimulation cultures of tumor-bearing splenic cells in order to realize fully its potentiating activity for cytotoxic T lymphocyte (CTL) generation, suggesting that signaling through CD40 is important at the inductive stage of antitumor cytotoxicity. Moreover, anti-CD40 mAb was found to enhance the expression of the B7-2 (CD86) and, to a lesser extent, the B7-1 (CD80) costimulatory molecules on B220⁺ cells (i.e., B cells), and B7-2 and, to a lesser extent, B7-1 molecules played an important role in the potentiating effect of anti-CD40 mAb for CTL generation by tumor-bearer splenic cells. Furthermore, B220⁺ cells were found to be essential for the potentiating effect of anti-CD40 mAb, as depletion of B220⁺ cells at the inductive stage completely abrogated the ability of anti-CD40 mAb to enhance CTL generation. Thus, signaling through CD40 enhances CTL generation by CD8⁺ T cells from tumor-bearing mice by a mechanism that involves the up-regulation of B7-2 and, to a lesser extent, B7-1 expression on B220⁺ cells. Received: 23 December 1998 / Accepted: 22 February 1999     

Induction of antigen-specific immune responses against malignant brain tumors by intramuscular injection of sindbis DNA encoding gp100 and IL-18

We constructed pSin-SV40-HDV-SV40pA, an improved Sindbis DNA expression vector, and evaluated the potential of this vector system for brain tumor therapy. We investigated whether immunizing mice with xenogeneic DNA encoding human gp100 and mouse IL-18 would enhance the antitumor responses. To study the immune mechanisms involved in tumor regression, we examined tumor growth in B16-gp100-implanted brain tumor models using T-cell subset-depleted and IFN-gamma-neutralized mice. Hugp100/mIL-18 vaccination was also investigated for its antitumor effects against the wild-type murine B16 tumor, which expresses the murine gp100 molecule. Genetic immunization using plasmid pSin 9001 DNA codelivery of human gp100 and mouse IL-18 resulted in enhanced protective and therapeutic effects on the malignant brain tumors. The antitumor and protective effects were mediated by both CD4(+)/CD8(+) T cells and IFN-gamma. Vaccination with hugp100/mIL-18 conferred a significant survival merit to wild-type B16 tumor-harboring mice. Immunogene therapy with the improved Sindbis virus vector expressing xenogeneic gp100 and syngeneic IL-18 may be an excellent approach for developing a new treatment protocol. Thus, the Sindbis DNA system may represent a novel approach for the treatment of malignant brain tumors.     

Hyper‐reactive cloned mice generated by direct nuclear transfer of antigen‐specific CD4+ T cells

T‐cell receptor (TCR)‐transgenic mice have been employed for evaluating antigen‐response mechanisms, but their non‐endogenous TCR might induce immune response differently than the physiologically expressed TCR. Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen‐specific CD4⁺ T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre‐rearranged TCRα (rTα) and TCRβ (rTβ) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic‐type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTβ is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter‐regulated antigen‐specific TCR are a unique animal model with allergic predisposition for investigating CD4⁺ T‐cell‐mediated pathogenesis and cellular commitment in immune diseases.     

Induction of HCA587-Specific Antitumor Immunity with HCA587 Protein Formulated with CpG and ISCOM in Mice

HCA587 (also known as MAGE-C2) is a “cancer-testis” antigen highly expressed in a number of malignancies with unique immunological properties, making it a promising target for tumor immunotherapy. In this report, we demonstrated that HCA587 protein, when formulated with adjuvants CpG–containing oligodeoxynucleotides (CpG ODN) and ISCOM, was capable of inducing a potent cellular and humoral immune response as indicated by the presence of a large number of HCA587-specific, IFN-γ-producing CD4⁺ T cells and high levels of HCA587-specific antibodies. More importantly, vaccination with HCA587 conferred protection against challenge with HCA587-expressing B16 melanoma in prophylactic and therapeutic settings. In analysis of the mechanisms underlying the protective effect, we showed that the vaccination was followed by enhanced accumulation of tumor-infiltrating lymphocytes (TILs) with enrichment of conventional CD4⁺ T cells but reduced representation of Treg cells. Further, the antitumor effect was largely abrogated in mice either depleted of CD4⁺ T cells or deficient for IFN-γ. These results indicate that HCA587 protein vaccine possesses evident antitumor activity in a mouse model and holds promise for treatment of human cancers.     

Neem Leaf Glycoprotein Activates CD8+ T Cells to Promote Therapeutic Anti-Tumor Immunity Inhibiting the Growth of Mouse Sarcoma

In spite of sufficient data on Neem Leaf Glycoprotein (NLGP) as a prophylactic vaccine, little knowledge currently exists to support the use of NLGP as a therapeutic vaccine. Treatment of mice bearing established sarcomas with NLGP (25 µg/mice/week subcutaneously for 4 weeks) resulted in tumor regression or dormancy (Tumor free/Regressor, 13/24 (NLGP), 4/24 (PBS)). Evaluation of CD8⁺ T cell status in blood, spleen, TDLN, VDLN and tumor revealed increase in cellular number. Elevated expression of CD69, CD44 and Ki67 on CD8⁺ T cells revealed their state of activation and proliferation by NLGP. Depletion of CD8⁺ T cells in mice at the time of NLGP treatment resulted in partial termination of tumor regression. An expansion of CXCR3⁺ and CCR5⁺ T cells was observed in the TDLN and tumor, along with their corresponding ligands. NLGP treatment enhances type 1 polarized T-bet expressing T cells with downregulation of GATA3. Treg cell population was almost unchanged. However, T∶Treg ratios significantly increased with NLGP. Enhanced secretion/expression of IFNγ was noted after NLGP therapy. In vitro culture of T cells with IL-2 and sarcoma antigen resulted in significant enhancement in cytotoxic efficacy. Consistently higher expression of CD107a was also observed in CD8⁺ T cells from tumors. Reinoculation of sarcoma cells in tumor regressed NLGP-treated mice maintained tumor free status in majority. This is correlated with the increment of CD44^hiCD62L^hi central memory T cells. Collectively, these findings support a paradigm in which NLGP dynamically orchestrates the activation, expansion, and recruitment of CD8⁺ T cells into established tumors to operate significant tumor cell lysis.