The absence of invariant chain in MHC II cancer vaccines enhances the activation of tumor-reactive type 1 CD4<Superscript>+</Superscript> T lymphocytes

Activation of tumor-reactive T lymphocytes is a promising approach for the prevention and treatment of patients with metastatic cancers. Strategies that activate CD8⁺ T cells are particularly promising because of the cytotoxicity and specificity of CD8⁺ T cells for tumor cells. Optimal CD8⁺ T cell activity requires the co-activation of CD4⁺ T cells, which are critical for immune memory and protection against latent metastatic disease. Therefore, we are developing “MHC II” vaccines that activate tumor-reactive CD4⁺ T cells. MHC II vaccines are MHC class I⁺ tumor cells that are transduced with costimulatory molecules and MHC II alleles syngeneic to the prospective recipient. Because the vaccine cells do not express the MHC II-associated invariant chain (Ii), we hypothesized that they will present endogenously synthesized tumor peptides that are not presented by professional Ii⁺ antigen presenting cells (APC) and will therefore overcome tolerance to activate CD4⁺ T cells. We now report that MHC II vaccines prepared from human MCF10 mammary carcinoma cells are more efficient than Ii⁺ APC for priming and boosting Type 1 CD4⁺ T cells. MHC II vaccines consistently induce greater expansion of CD4⁺ T cells which secrete more IFNγ and they activate an overlapping, but distinct repertoire of CD4⁺ T cells as measured by T cell receptor Vβ usage, compared to Ii⁺ APC. Therefore, the absence of Ii facilitates a robust CD4⁺ T cell response that includes the presentation of peptides that are presented by traditional APC, as well as peptides that are uniquely presented by the Ii⁻ vaccine cells.     

MHC class II and CD80 tumor cell–based vaccines are potent activators of type 1 CD4<Superscript>+</Superscript> T lymphocytes provided they do not coexpress invariant chain

We are developing vaccines that activate tumor-specific CD4⁺ T cells. The cell-based vaccines consist of MHC class I⁺ tumor cells that are genetically modified to express syngeneic MHC class II and costimulatory molecules. Previous studies demonstrated that treatment of mice with established tumors with these vaccines resulted in regression of solid tumors, reduction of metastatic disease, and increased survival time. Optimal vaccines will prime naïve T cells and activate T cells to tumor peptides derived from diverse subcellular compartments, since potential tumor antigens may reside in unique cellular locales. To determine if the MHC class II / costimulatory molecule vaccines fulfill these conditions, the vaccines have been tested for their ability to activate antigen-specific, naïve, transgenic CD4⁺ T lymphocytes. MHC class II⁺CD80⁺ vaccine cells were transfected with hen eggwhite lysozyme targeted to the cytosol, nuclei, mitochondria, or endoplasmic reticulum, and used as antigen-presenting cells to activate I-A^k–restricted, lysozyme-specific CD4⁺ 3A9 transgenic T cells. Regardless of the cellular location of lysozyme, the vaccines stimulated release of high levels of IFN- and IL-2. If the vaccines coexpressed the MHC class II accessory molecule invariant chain, then IFN- and IL-2 release was significantly reduced. These studies demonstrate that in the absence of invariant chain the MHC class II and CD80 tumor cell vaccines (1) function as antigen-presenting cells to activate naïve, tumor-specific CD4⁺ cells to endogenously synthesized tumor antigens; (2) polarize the activated CD4⁺ T cells toward a type 1 response; and (3) present epitopes derived from varied subcellular locales.Abbreviations APC antigen-presenting cells - CIITA MHC class II transactivator - CytoHEL HEL targeted to cytoplasm - ER endoplasmic reticulum - ErHEL HEL targeted to ER - HEL hen eggwhite lysozyme - 3A9 HEL_46–61–specific, I-A^k–restricted TCR - Hph hygromycin - Ii invariant chain - MAb monoclonal antibody - MitoHEL HEL targeted to mitochondria - NucHEL HEL targeted to nucleus - Puro puromycin - TG transgenic - Zeo Zeocin     

Major Histocompatibility Complex Class II+ Invariant Chain Negative Breast Cancer Cells Present Unique Peptides that Activate Tumor-specific T Cells from Breast Cancer Patients

The major histocompatibility complex (MHC) class II-associated Invariant chain (Ii) is present in professional antigen presenting cells where it regulates peptide loading onto MHC class II molecules and the peptidome presented to CD4⁺ T lymphocytes. Because Ii prevents peptide loading in neutral subcellular compartments, we reasoned that Ii⁻ cells may present peptides not presented by Ii⁺ cells. Based on the hypothesis that patients are tolerant to MHC II-restricted tumor peptides presented by Ii⁺ cells, but will not be tolerant to novel peptides presented by Ii⁻ cells, we generated MHC II vaccines to activate cancer patients'' T cells. The vaccines are Ii⁻ tumor cells expressing syngeneic HLA-DR and the costimulatory molecule CD80. We used liquid chromatography coupled with mass spectrometry to sequence MHC II-restricted peptides from Ii⁺ and Ii⁻ MCF10 human breast cancer cells transfected with HLA-DR7 or the MHC Class II transactivator CIITA to determine if Ii⁻ cells present novel peptides. Ii expression was induced in the HLA-DR7 transfectants by transfection of Ii, and inhibited in the CIITA transfectants by RNA interference. Peptides were analyzed and binding affinity predicted by artificial neural net analysis. HLA-DR7-restricted peptides from Ii⁻ and Ii⁺ cells do not differ in size or in subcellular location of their source proteins; however, a subset of HLA-DR7-restricted peptides of Ii⁻ cells are not presented by Ii⁺ cells, and are derived from source proteins not used by Ii⁺ cells. Peptides from Ii⁻ cells with the highest predicted HLA-DR7 binding affinity were synthesized, and activated tumor-specific HLA-DR7⁺ human T cells from healthy donors and breast cancer patients, demonstrating that the MS-identified peptides are bonafide tumor antigens. These results demonstrate that Ii regulates the repertoire of tumor peptides presented by MHC class II⁺ breast cancer cells and identify novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.Cancer vaccines are a promising tool for cancer treatment and prevention because of their potential for inducing tumor-specific responses in conjunction with minimal toxicity for healthy cells. Cancer vaccines are based on the concept that tumor cells synthesize multiple peptides that are potential immunogens, and that with the appropriate vaccine protocol, these peptides will activate an efficacious antitumor response in the patient. Much effort has been invested in identifying and testing tumor-encoded peptides, particularly peptides presented by major histocompatibility complex (MHC)¹ class I, molecules capable of activating CD8⁺ T-cells that directly kill tumor cells (1, 2). Fewer studies have been devoted to identifying MHC class II-restricted peptides for the activation of tumor-reactive CD4⁺ T-cells despite compelling evidence that Type 1 CD4⁺ T helper cells facilitate the optimal activation of CD8⁺ T-cells and the generation of immune memory, which is likely to be essential for protection from metastatic disease.Activation of CD4⁺ T cells requires delivery of a costimulatory signal plus an antigen-specific signal consisting of peptide bound to an MHC II molecule. Most cells do not express MHC II or costimulatory molecules, so CD4⁺ T cells are typically activated by professional antigen presenting cells (APC), which endocytose exogenously synthesized antigen and process and present it in the context of their own MHC II molecules. This processing and presentation process requires Invariant chain (Ii), a molecule that is coordinately synthesized with MHC II molecules and prevents the binding and presentation of APC-encoded endogenous peptides (3, 4). As a result, tumor-reactive CD4⁺ T cells are activated to tumor peptides generated by the antigen processing machinery of professional APC, rather than peptides generated by the tumor cells. Because of the potential discrepancy in peptide generation between professional APC and tumor cells, and the critical role of Ii in preventing the presentation of endogenous peptides, we have generated “MHC II cancer vaccines” that consist of Ii⁻ tumor cells transfected with syngeneic MHC class II and CD80 genes. We reasoned that MHC II⁺Ii⁻CD80⁺ tumor cells may present a novel repertoire of MHC II-restricted tumor peptides that are not presented by professional APC, and therefore may be highly immunogenic. Once activated, CD4⁺ T cells produce IFNγ and provide help to CD8⁺ T cells and do not need to react with native tumor cells. Therefore, the MHC II vaccines have the potential to activate CD4⁺ Th1 cells that facilitate antitumor immunity. In vitro (5) and in vivo (5–7) studies with mice support this conclusion. In vitro studies with human MHC II vaccines further demonstrate that the absence of Ii facilitates the activation of MHC II-restricted tumor-specific CD4⁺ type 1 T cells of HLA-DR-syngeneic healthy donors and cancer patients, and that the vaccines activate CD4⁺ T cells with a distinct repertoire of T cell receptors (8–12). A critical negative role for Ii is also supported by studies of human acute myelogenous leukemia (AML). High levels of class II-associated invariant chain peptide (CLIP), a degradation product of Ii, by leukemic blasts is associated with poor patient prognosis (13, 14), whereas down-modulation of CLIP on AML cells increases the activation of tumor-reactive human CD4⁺ T cells (14, 15).We have now used mass spectrometry to identify MHC II-restricted peptides from MHC II⁺Ii⁻ and MHC II⁺Ii⁺ human breast cancer cells to test the concept that the absence of Ii facilitates the presentation of unique immunogenic MHC II-restricted peptides. We report here that a subset of MHC II-restricted peptides from HLA-DR7⁺ breast cancer cells are unique to Ii⁻ cells and are derived from source proteins not used by Ii⁺ cells. Ii⁻ peptides have high binding affinity for HLA-DR7 and activate tumor-specific T-cells from the peripheral blood of healthy donors and breast cancer patients. This is the first study to compare the human tumor cell MHC II peptidome in the absence or presence of Ii and to demonstrate that MHC II⁺Ii⁻ tumor cells present novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.     

Tumor-specific CD4+ T cells are activated by "cross-dressed" dendritic cells presenting peptide-MHC class II complexes acquired from cell-based cancer vaccines

Tumor cells that constitutively express MHC class I molecules and are genetically modified to express MHC class II (MHC II) and costimulatory molecules are immunogenic and have therapeutic efficacy against established primary and metastatic cancers in syngeneic mice and activate tumor-specific human CD4+ T lymphocytes. Previous studies have indicated that these MHC II vaccines enhance immunity by directly activating tumor-specific CD4+ T cells during the immunization process. Because dendritic cells (DCs) are considered to be the most efficient APCs, we have now examined the role of DCs in CD4+ T cell activation by the MHC II vaccines. Surprisingly, we find that DCs are essential for MHC II vaccine immunogenicity; however, they mediate their effect through "cross-dressing." Cross-dressing, or peptide-MHC (pMHC) transfer, involves the generation of pMHC complexes within the vaccine cells, and their subsequent transfer to DCs, which then present the intact, unprocessed complexes to CD4+ T lymphocytes. The net result is that DCs are the functional APCs; however, the immunogenic pMHC complexes are generated by the tumor cells. Because MHC II vaccine cells do not express the MHC II accessory molecules invariant chain and DM, they are likely to load additional tumor Ag epitopes onto MHC II molecules and therefore activate a different repertoire of T cells than DCs. These data further the concept that transfer of cellular material to DCs is important in Ag presentation, and they have direct implications for the design of cancer vaccines.     

Immunotherapy with vaccines combining MHC class II/CD80+ tumor cells with interleukin-12 reduces established metastatic disease and stimulates immune effectors and monokine induced by interferon γ

 Because they are difficult to treat, animal models of widespread, established metastatic cancer are rarely used to test novel immunotherapies. Two such mouse models are used in this report to demonstrate the therapeutic efficacy and to probe the mechanisms of a novel combination immunotherapy consisting of the cytokine interleukin-12 (IL-12) combined with a previously described vaccine based on MHC class II, CD80-expressing cells. BALB/c mice with 3-week established primary 4T1 mammary carcinomas up to 6 mm in diameter and with extensive, spontaneous lung metastases show a significant reduction in lung metastases following a 3-week course of immunotherapy consisting of weekly injections of the cell-based vaccine plus injections of IL-12 three times per week. C57BL/6 mice with 7-day established intravenous B16 melF10 lung metastases show a similar response following immunotherapy with IL-12 plus a vaccine based on B16 MHC class II, CD80-expressing cells. In both systems the combination therapy of cells plus IL-12 is more effective than IL-12 or the cellular vaccine alone, although, in the 4T1 system, optimal activity does not require MHC class II and CD80 expression in the vaccine cells. The cell-based vaccines were originally designed to activate tumor-specific CD4⁺ T lymphocytes specifically and thereby provide helper activity to tumor-cytotoxic CD8⁺ T cells, and IL-12 was added to the therapy to facilitate T helper type 1 lymphocyte (Th1) differentiation. In vivo depletion experiments for CD4⁺ and CD8⁺ T cells and natural killer (NK) cells and tumor challenge experiments in beige/nude/XID immunodeficient mice demonstrate that the therapeutic effect is not exclusively dependent on a single cell population, suggesting that T and NK cells are acting together to optimize the response. IL-12 may also be enhancing the immunotherapy via induction of the chemokine Mig (monokine induced by interferon γ), because reverse PCR experiments demonstrate that Mig is present in the lungs of mice receiving therapy and is most likely synthesized by the tumor cells. These results demonstrate that the combination therapy of systemic IL-12 and a cell-based vaccine is an effective agent for the treatment of advanced, disseminated metastatic cancers in experimental mouse models and that multiple effector cell populations and anti-angiostatic factors are likely to mediate the effect. Received: 15 October 1999 / Accepted: 24 November 1999     

Lung cancer patients’ CD4<Superscript>+</Superscript> T cells are activated in vitro by MHC II cell-based vaccines despite the presence of myeloid-derived suppressor cells

BACKGROUND: Advanced non-small cell lung cancer (NSCLC) remains an incurable disease. Immunotherapies that activate patients' T cells against resident tumor cells are being developed; however, these approaches may not be effective in NSCLC patients due to tumor-induced immune suppression. A major cause of immune suppression is myeloid-derived suppressor cells (MDSC). Because of the strategic role of CD4(+) T lymphocytes in the activation of cytotoxic CD8(+) T cells and immune memory, we are developing cell-based vaccines that activate tumor-specific CD4(+) T cells in the presence of MDSC. The vaccines are NSCLC cell lines transfected with costimulatory (CD80) plus major histocompatibility complex class II (MHC II) genes that are syngeneic to the recipient. The absence of invariant chain promotes the presentation of endogenously synthesized tumor antigens, and the activation of MHC II-restricted, tumor-antigen-specific CD4(+) T cells. METHODS: Potential vaccine efficacy was tested in vitro by priming and boosting peripheral blood mononuclear cells from ten NSCLC patients who had varying levels of MDSC. CD4(+) T cell activation was quantified by measuring Type 1 and Type 2 cytokine release. RESULTS: The vaccines activated CD4(+) T cells from all ten patients, despite the presence of CD33(+)CD11b(+) MDSC. Activated CD4(+) T cells were specific for NSCLC and did not cross-react with tumor cells derived from non-lung tissue or normal lung fibroblasts. CONCLUSIONS: The NSCLC vaccines activate tumor-specific CD4(+) T cells in the presence of potent immune suppression, and may be useful for the treatment of patients with NSCLC.     

Modification of tumor cells by a low dose of Newcastle disease virus

Summary Augmented tumor-specific T cell responses were observed against the high metastatic murine lymphoma variant ESb when using as immunogen ESb tumor cells that had been modified by infection with a low dose of Newcastle disease virus (NDV). Such virus-modified inactivated tumor cells (ESb-NDV) were potent tumor vaccines when applied postoperatively for active specific immunotherapy of ESb metastases. We demonstrate here that immune spleen cells from mice immunized with ESb-NDV contain enhanced immune capacity in both the CD4⁺, CD8^– and the CD4^–, CD8⁺ T cell compartments to mount a secondary-tumor-specific cytotoxic T cell response in comparison with immune cells from mice immunized with ESb. ESb-NDV immune CD4⁺, CD8^– helper T cells also produced more interleukin 2 after antigen stimulation than the corresponding ESb immune cells. There was no participation of either CD4⁺ or CD8⁺ virus-specific cells in the augmented response. The specificity of the T cells for the tumor-associated antigen remaind unchanged. Thus, there is the paradox that the virus-mediated augmentation of the tumor-specific T cell response in this system involves increased T helper activity but does not involve the recognition of viral epitopes as potential new helper determinants.Abbreviations CTL cytolytic T lymphocytes - IL-2 interleukin 2 - rIL-2 recombinant IL-2 - mAb monoclonal antibody - NDV Newcastle disease virus - SSC syngeneic spleen cell     

Invariant chain and the MHC class II cytoplasmic domains regulate localization of MHC class II molecules to lipid rafts in tumor cell-based vaccines

Cell-based tumor vaccines, consisting of MHC class I+ tumor cells engineered to express MHC class II molecules, stimulate tumor-specific CD4+ T cells to mediate rejection of established, poorly immunogenic tumors. Previous experiments have demonstrated that these vaccines induce immunity by functioning as APCs for endogenously synthesized, tumor-encoded Ags. However, coexpression of the MHC class II accessory molecule invariant chain (Ii), or deletion of the MHC class II cytoplasmic domain abrogates vaccine immunogenicity. Recent reports have highlighted the role of lipid microdomains in Ag presentation. To determine whether Ii expression and/or truncation of MHC class II molecules impact vaccine efficacy by altering MHC class II localization to lipid microdomains, we examined the lipid raft affinity of MHC class II molecules in mouse M12.C3 B cell lymphomas and SaI/A(k) sarcoma vaccine cells. Functional MHC class II heterodimers were detected in lipid rafts of both cell types. Interestingly, expression of Ii in M12.C3 cells or SaI/A(k) cells blocked the MHC class II interactions with cell surface lipid rafts. In both cell types, truncation of either the alpha- or beta-chain decreased the affinity of class II molecules for lipid rafts. Simultaneous deletion of both cytoplasmic domains further reduced localization of class II molecules to lipid rafts. Collectively, these data suggest that coexpression of Ii or deletion of the cytoplasmic domains of MHC class II molecules may reduce vaccine efficacy by blocking the constitutive association of MHC class II molecules with plasma membrane lipid rafts.     

Mathematical model of a personalized neoantigen cancer vaccine and the human immune system

Cancer vaccines are an important component of the cancer immunotherapy toolkit enhancing immune response to malignant cells by activating CD4⁺ and CD8⁺ T cells. Multiple successful clinical applications of cancer vaccines have shown good safety and efficacy. Despite the notable progress, significant challenges remain in obtaining consistent immune responses across heterogeneous patient populations, as well as various cancers. We present a mechanistic mathematical model describing key interactions of a personalized neoantigen cancer vaccine with an individual patient’s immune system. Specifically, the model considers the vaccine concentration of tumor-specific antigen peptides and adjuvant, the patient’s major histocompatibility complexes I and II copy numbers, tumor size, T cells, and antigen presenting cells. We parametrized the model using patient-specific data from a clinical study in which individualized cancer vaccines were used to treat six melanoma patients. Model simulations predicted both immune responses, represented by T cell counts, to the vaccine as well as clinical outcome (determined as change of tumor size). This model, although complex, can be used to describe, simulate, and predict the behavior of the human immune system to a personalized cancer vaccine.     

Immunization with a GM3 ganglioside nanoparticulated vaccine confers an effector CD8<Superscript>+</Superscript> T cells-mediated protection against melanoma B16 challenge

Preventive immunotherapy is an attractive strategy for patients at a high risk of having cancer. The success of prophylactic cancer vaccines would depend on the selection of target antigens that are essential for tumour growth and progression. The overexpression of GM3 ganglioside in murine and human melanomas and its important role in tumour progression makes this self antigen a potential target for preventive immunotherapy of this neoplasm. We have previously shown that preventive administration of a GM3-based vaccine to C57BL/6 mice elicited the rejection of the GM3 positive-B16 melanoma cells in most of the animals. Despite the crucial role of cellular immune response in tumour protection, the involvement of T cells in anti-tumour immunity of ganglioside vaccines is not described. Here, we examined the mechanisms by which this immunogen confers tumour protection. We have found that induction of anti-GM3 IgG antibodies correlated with tumour protection. Surprisingly, CD8⁺ T cells, but not NK1.1⁺ cells, are required in the effector phase of the antitumour immune response. The depletion of CD4⁺ T cells during immunization phase did not affect the anti-tumour activity. In addition, T cells from surviving-immunized animals secreted IFNγ when were co-cultured with IFNα-treated B16 melanoma cells or DCs pulsed with melanoma extract. Paradoxically, in spite of the glycolipidic nature of this antigen, these findings demonstrate the direct involvement of the cellular immune response in the anti-tumour protection induced by a ganglioside-based vaccine. Grant support: Center of Molecular Immunology, Elea Laboratories and Recombio.     

Cancer immunotherapy by antisense suppression of Ii protein in MHC-class-II-positive tumor cells

This study was aimed at creating a more effective tumor cell vaccine by suppressing Ii protein in the presence of MHC class II molecules within a cancer cell. Absence of the Ii protein, which normally blocks the antigenic-peptide-binding site of MHC class II molecules at synthesis in the endoplasmic reticulum, presumably increases the range of cancer-related epitopes presented to CD4⁺ helper T cells. Effective suppression of Ii protein was achieved with an antisense, phosphorothioate oligonucleotide, which was selected on the basis of (1) the RNase H activation assay, (2) an assay for Ii protein suppression, and (3) a test for potency with respect to the extent of base sequence (“sequence walking”). The SaI murine sarcoma, which is MHC-class-I⁺ and MHC-class-II⁻, Ii-protein⁻, upon transfection with genes for either interferon γ or the MHC class II transactivator, came to express MHC class II molecules and Ii protein. In each line of transfected tumor cells, the antisense oligonucleotide profoundly suppressed Ii protein in 35%–55% cells, without affecting expression of MHC class II molecules. Inoculation of mice with such Ii-protein-suppressed tumor vaccine cells, after either formaldehyde fixation or X-irradiation, led to much greater protection against challenge with the parental SaI sarcoma than did inoculation with untreated cells. This approach to cancer cell vaccination can be applied in a wide range of human tumors. Received: 22 June 1999 / Accepted: 28 July 1999     

CD4+ T cell-mediated cytotoxicity is associated with MHC class II expression on malignant CD19+ B cells in diffuse large B cell lymphoma

Diffuse large B cell lymphoma (DLBCL) is a common B cell malignancy with approximately 30% of patients present relapsed or refractory disease after first-line therapy. Research of further treatment options is needed. Cytotoxic CD4⁺ T cells express cytolytic molecules and have potential antitumor function. Here, we showed that the CD19⁺ cells from DLBCL patients presented significantly reduced expression of MHC II molecules than those from healthy controls. Three years after the first-line treatment, patients that presented relapsed disease had significantly lower MHC II expression on their CD19⁺ cells than patients who did not show recurrence. Examining cytotoxic CD4⁺ T cells show that DLBCL patients presented significantly elevated frequencies of granzyme A-, granzyme B-, and/or perforin-expressing cytotoxic CD4⁺ T cells. Also, frequency of cytotoxic CD4⁺ T cells in DLBCL patients was positively correlated with the MHC II expression level. Subsequently, the cytotoxic potential of CD4⁺ T cells against autologous CD19⁺ cells was investigated. We found that the cytotoxic potential of CD4⁺ T cells was highest in MHC II-high, intermediate in MHC II-mid, and lowest in MHC II-low patients. The percentage of MHC II-expressing viable CD19⁺ cells presented a significant reduction after longer incubation with cytotoxic CD4⁺ T cells, suggesting that cytotoxic CD4⁺ T cells preferentially eliminated MHC II-expressing CD19⁺ cells. Blocking MHC II on CD19⁺ cells significantly reduced the cytolytic capacity of CD4⁺ T cells. Despite these discoveries, the frequency of cytotoxic CD4⁺ T cells did not predict the clinical outcome of DLBCL patients. Together, these results demonstrated that cytotoxic CD4⁺ T cells presented an MHC II-dependent cytotoxic potential against autologous CD19⁺ cells and could potentially represent a future treatment option for DLBCL.     

Adjuvant IL-15 does not enhance the efficacy of tumor cell lysate-pulsed dendritic cell vaccines for active immunotherapy of T cell lymphoma

There has been a recent interest in using IL-15 to enhance antitumor activity in several models because of its ability to stimulate CD8⁺ T cell expansion, inhibit apoptosis and promote memory T cell survival and maintenance. Previously, we reported that C6VL tumor lysate-pulsed dendritic cell vaccines significantly enhanced the survival of tumor-bearing mice by stimulating a potent tumor-specific CD8⁺ T cell response. In this study, we determined whether IL-15 used as immunologic adjuvant would augment vaccine-primed CD8⁺ T cell immunity against C6VL and further improve the survival of tumor-bearing mice. We report that IL-15 given after C6VL lysate-pulsed dendritic cell vaccines stimulated local and systemic expansion of NK, NKT and CD8⁺ CD44^hi T cells. IL-15 did not, however, augment innate or cellular responses against the tumor. T cells from mice infused with IL-15 following vaccination did not secrete increased levels of tumor-specific TNF-α or IFN-γ or have enhanced C6VL-specific CTL activity compared to T cells from recipients of the vaccine alone. Lastly, IL-15 did not enhance the survival of tumor-bearing vaccinated mice. Thus, while activated- and memory-phenotype CD8⁺ T cells were dramatically expanded by IL-15 infusion, vaccine-primed CD8⁺ T cell specific for C6VL were not significantly expanded. This is the first account of using IL-15 as an adjuvant in a therapeutic model of active immunotherapy where there was not a preexisting pool of tumor-specific CD8⁺ T cells. Our results contrast the recent studies where IL-15 was successfully used to augment tumor-reactivity of adoptively transferred transgenic CD8⁺ T cells. This suggests that the adjuvant potential of IL-15 may be greatest in settings where it can augment the number and activity of preexisting tumor-specific CD8⁺ T cells.     

Superior In Vitro Stimulation of Human CD8+ T-Cells by Whole Virus versus Split Virus Influenza Vaccines

Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8⁺ T-cell responses, because they are mainly directed against conserved internal influenza proteins thereby presumably mediating cross-protection against circulating seasonal as well as emerging pandemic virus strains. Our study showed that influenza whole virus vaccines of major seasonal A and B strains activated DC more efficiently than those of pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains. In contrast, influenza split virus vaccines had a low ability to activate DC, regardless which strain was investigated. We also observed that whole virus vaccines stimulated virus-specific CD8⁺ memory T cells much stronger compared to split virus counterparts, whereas both vaccine formats activated CD4⁺ Th cell responses similarly. Moreover, our data showed that whole virus vaccine material is delivered into the cytosolic pathway of DC for effective activation of virus-specific CD8⁺ T cells. We conclude that vaccines against seasonal and pandemic (-like) influenza strains that aim to stimulate cross-reacting CD8⁺ T cells should include whole virus rather than split virus formulations.     

Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity

Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4⁺ and CD8⁺ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8⁺ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8⁺ T cell epitope enhance subdominant pathogen-specific CD8⁺ T cell responses. More specifically, CD8⁺ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8⁺ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T cell epitope responses induced by vaccination is not always advantageous for host immunity.     

Generation of tumor-specific cytotoxic T lymphocytesin vivo by combined treatment with inactivated tumor cells and recombinant interleukin-2

In order to search for a new therapy that would maximize the effect of interleukin-2 (IL-2) in evoking antitumor immunity in vivo, the therapeutic effect of a combination of mitomycin-C(MMC)-treated tumor cells and recombinant IL-2 was examined for its induction of antitumor activity against established melanoma metastasis. In C57BL/6 mice intravenously (i. v.) injected with B16 melanoma cells on day 0, the combined treatment with an intraperitoneal (i. p.) injection of MMC-treated melanoma cells on day 6 and 2500 U rIL-2 (twice daily) on days 7 and 8 markedly reduced the number of pulmonary metastases. This antitumor activity was more effective than that in untreated controls and mice that were injected with MMC-treated melanoma cells alone or rIL-2 alone. When the i. p. injection of MMC-treated tumor cells was replaced by other syngeneic tumor cells, antitumor activity against metastatic melanoma was not induced. The antitumor activity induced by this treatment increased in parallel with an increase in the dose of rIL-2 injected. In contrast, an i. p. injection of soluble tumor-specific antigens alone could induce only a marginal level of antitumor activity, and this activity was not augmented by subsequent i. p. injections of rIL-2. In vivo treatment with anti-CD8 monoclonal antibody (mAb), but not with anti-CD4 mAb or anti-asialo-GM1 antibody, abrogated the antitumor activity induced by this combined therapy. This suggests that the antitumor effect was dependent on CD8⁺ T cells. Lung-infiltrating lymphocytes from mice that had been i. v. injected with melanoma cells 11 days before and were treated with this combined therapy, showed melanoma-specific cytolytic activity. This combined therapy also showed significant antitumor activity against subcutaneously inoculated melanoma cells. These results demonstrate that the combined therapy of an i. p. injection of MMC-treated tumor cells and subsequent and consecutive i. p. administration of rIL-2 increases antitumor activity against established metastatic melanoma by generating tumor-specific CD8⁺ CTL in vivo.     

Prime-boost vaccination with rBCG/rAd35 enhances CD8⁺ cytolytic T-cell responses in lesions from Mycobacterium tuberculosis-infected primates

To prevent the global spread of tuberculosis (TB) infection, a novel vaccine that triggers potent and long-lived immunity is urgently required. A plasmid-based vaccine has been developed to enhance activation of major histocompatibility complex (MHC) class I–restricted CD8+ cytolytic T cells using a recombinant Bacille Calmette-Guérin (rBCG) expressing a pore-forming toxin and the Mycobacterium tuberculosis (Mtb) antigens Ag85A, 85B and TB10.4 followed by a booster with a nonreplicating adenovirus 35 (rAd35) vaccine vector encoding the same Mtb antigens. Here, the capacity of the rBCG/rAd35 vaccine to induce protective and biologically relevant CD8⁺ T-cell responses in a nonhuman primate model of TB was investigated. After prime/boost immunizations and challenge with virulent Mtb in rhesus macaques, quantification of immune responses at the single-cell level in cryopreserved tissue specimen from infected organs was performed using in situ computerized image analysis as a technological platform. Significantly elevated levels of CD3⁺ and CD8⁺ T cells as well as cells expressing interleukin (IL)-7, perforin and granulysin were found in TB lung lesions and spleen from rBCG/rAd35-vaccinated animals compared with BCG/rAd35-vaccinated or unvaccinated animals. The local increase in CD8⁺ cytolytic T cells correlated with reduced expression of the Mtb antigen MPT64 and also with prolonged survival after the challenge. Our observations suggest that a protective immune response in rBCG/rAd35-vaccinated nonhuman primates was associated with enhanced MHC class I antigen presentation and activation of CD8+ effector T-cell responses at the local site of infection in Mtb-challenged animals.     

Ubiquitin-fusion degradation pathway: a new strategy for inducing CD8 cells specific for mycobacterial HSP65

The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8⁺ T cells. In this study, we exploited UPS to induce CD8⁺ T cells specific for mycobacterial HSP65 (mHSP65), one of the leading vaccine candidates against infection with Mycobacterium tuberculosis. A chimeric DNA termed pU-HSP65 encoding a fusion protein between murine ubiquitin and mHSP65 was constructed, and C57BL/6 (B6) mice were immunized with the DNA using gene gun bombardment. Mice immunized with the chimeric DNA acquired potent resistance against challenge with the syngeneic B16F1 melanoma cells transfected with the mHSP65 gene (HSP65/B16F1), compared with those immunized with DNA encoding only mHSP65. Splenocytes from the former group of mice showed a higher grade of cytotoxic activity against HSP65/B16F1 cells and contained a larger number of granzyme B- or IFN-γ-producing CD8⁺ T cells compared with those from the latter group of mice.     

Chemically engineered glycan-modified cancer vaccines to mobilize skin dendritic cells

Dendritic cell (DC)–targeting vaccines show great promise in increasing antitumor immunity. Glycan-engineered vaccines facilitate both DC targeting and increased uptake by DCs for processing and presentation to CD4⁺ and CD8⁺ T cells to induce tumor-specific T-cell responses. However, the complexity of various DC subsets in skin tissues, expressing different glycan-binding receptors that can mediate vaccine uptake or drainage of vaccines via lymphatics directly to the lymph node–resident DCs, complicates the success of vaccines. Moreover, the influx of inflammatory immune cells to the site of vaccination, such as monocytes that differentiate to DCs and coexpress glycan-binding receptors, may contribute to the strength of DC-targeting glycovaccines for future clinical use.     

Tumor antigen presentation: changing the rules

 Cell-based tumor vaccines have been developed on the basis of the hypothesis that tumor cells can be genetically modified to present antigen to T lymphocytes directly. Contrary to expectations, cross-priming is the predominant pathway for activation of tumor-specific CD8⁺ T cells, while direct presentation of antigen dominates activation of tumor-specific CD4⁺ T cells. These results pose interesting paradoxes for the generation of immune responses, and have definite implications for the development of anti-cancer vaccines. Received: 10 October 1997 / Accepted 10 January 1998