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Many antigens recognized by tumor-reactive cytotoxic CD8+ T cells are self-antigens. Tyrosinase-related protein 2 (TRP-2) is a melanogenic enzyme expressed by both melanocytes and melanomas that is reported to be a candidate melanoma rejection antigen. To study the role of self-reactive CD8+ T cells in tumor immunity and autoimmunity, we generated mice which bear a T cell receptor transgene (TCR Tg) specific for the TRP-2(180-188) epitope. TRP-2 TCR Tg mice did not spontaneously develop depigmentation despite systemic expression of TRP-2 in the skin. Peripheral T cells from these TCR Tg mice exhibited a naïve phenotype and proliferated in response to TRP-2 in vitro. In addition, transfer of in vitro-activated Tg T cells reduced B16 pulmonary tumor burden, but not subcutaneous tumors. We next sought to determine the in vivo responses of the Tg T cells to endogenous and tumor-derived TRP-2. Adoptive transfer of naïve TCR Tg T cells into wild-type C57BL/6 mice, in combination with a TRP-2-pulsed dendritic cell vaccine, induced proliferation of the Tg T cells and resulted in migration of the Tg T cells into a subcutaneous B16 melanoma tumor. Although these tumor-infiltrating Tg T cells remained reactive against TRP-2, they did not reduce growth of the primary subcutaneous tumor; similarly, these in vivo-primed effector cells had no significant effect on growth of pulmonary nodules. These data demonstrate that despite in vivo priming, tumor-infiltrating T cells may fail to reduce tumor burden. Determining the basis for the inability of the tumor micro-environment to sustain effective anti-tumor responses will be critical for designing newer, more potent anti-tumor immunotherapies.

Our laboratory is interested in how immunogenicity may be modulated in vivo in order to better design more effective immunotherapeutics against cancer. Our main approach is to use a facultative intracellular bacterium, Listeria monocytogenes, which has the unusual ability to live and grow in the cytoplasm of the cell and is thus an excellent vector for targeting passenger antigens to the major histocompatibility complex (MHC) class I pathway of antigen processing with the generation of authentic CTL epitopes. We have used this approach to target tumor antigens expressed on breast, melanoma and cervical cancer. We are also exploring the role of Listerial virulence factors in potentiating adaptive immune responses by activating innate immunity. Specifically, we are using these proteins as adjuvants for B cell lymphomas.

Most of the human tumor-associated antigens (TAAs) characterized thus far are derived from nonmutated “self”-proteins. Numerous strategies have been developed to break tolerance to TAAs, combining various forms of antigens with different vectors and adjuvants. However, no study has yet determined how to select epitopes within a given TAA to induce the highest antitumor effector response. We addressed this question by evaluating in HLA-A*0201-transgenic HHD mice the antitumor vaccination efficacy of high- and low-affinity epitopes from the naturally expressed murine telomerase reverse transcriptase (mTERT). Immunity against low-affinity epitopes was induced with heteroclitical variants. We show here that the CTL repertoire against high-affinity epitopes is partially tolerized, while that against low-affinity epitopes is composed of frequent CTLs with high avidity. The high-affinity p797 and p545 mTERT epitopes are not able to protect mice from a lethal challenge with the mTERT-expressing EL4-HHD tumor. In contrast, mice developing CTL responses against the p572 and p988 low-affinity epitopes exhibit potent antitumor immunity and no sign of autoimmune reactivity against TERT-expressing normal tissues. Our results strongly argue for new TAA epitope selection and modification strategies in antitumor immunotherapy applications in humans.

Background

As tumor antigen-specific CD4+ T cells can mediate strong therapeutic anti-tumor responses in melanoma patients we set out to establish a comprehensive screening strategy for the identification of tumor-specific CD4+ T cell epitopes suitable for detection, isolation and expansion of tumor-reactive T cells from patients.

Methods and Findings

To scan the human melanoma differentiation antigens TRP-1 and TRP-2 for HLA-DRB1*0301-restricted CD4+ T cell epitopes we applied the following methodology: Splenocytes of HLA-DRB1*0301-transgenic mice immunized with recombinant adenovirus encoding TRP-1 (Ad5.TRP-1) or TRP-2 (Ad5.TRP-2) were tested for their T cell reactivity against combinatorial TRP-1- and TRP-2-specific peptide libraries. CD4+ T cell epitopes thus identified were validated in the human system by stimulation of peripheral blood mononuclear cells (PBMC) from healthy donors and melanoma patients. Using this strategy we observed that recombinant Ad5 induced strong CD4+ T cell responses against the heterologous tumor antigens. In Ad5.TRP-2-immunized mice CD4+ T cell reactivity was detected against the known HLA-DRB1*0301-restricted TRP-260–74 epitope and against the new epitope TRP-2149–163. Importantly, human T cells specifically recognizing target cells loaded with the TRP-2149–163-containing library peptide or infected with Ad5.TRP-2 were obtained from healthy individuals, and short term in vitro stimulation of PBMC revealed the presence of epitope-reactive CD4+ T cells in melanoma patients. Similarly, immunization of mice with Ad5.TRP-1 induced CD4+ T cell responses against TRP-1-derived peptides that turned out to be recognized also by human T cells, resulting in the identification of TRP-1284–298 as a new HLA-DRB1*0301-restricted CD4+ T cell epitope.

Conclusions

Our screening approach identified new HLA-DRB1*0301-restricted CD4+ T cell epitopes derived from melanoma antigens. This strategy is generally applicable to target antigens of other tumor entities and to different HLA class II molecules even without prior characterization of their peptide binding motives.

Tumor vaccination using tumor associated antigen primed dendritic cells (DCs) is in clinical trials. Investigators are using patients’ own immune system to activate T-cells against recurrent or metastatic tumors. Following vaccination of DCs or attenuated tumor cells, clinical as well as radiological improvements have been noted due to migration and accumulation of Cytotoxic T-cells (CTLs). CTLs mediated tumor cell killing resulted in extended survival in clinical trails and in preclinical models. Besides administration of primed DCs or attenuated or killed tumors cells to initiate the generation of CTLs, investigators have started making genetically altered T-cells (CTLs) to target specific tumors and showed in vivo migration and accumulation in the implanted or recurrent tumors by different imaging modalities. Our groups also showed the utilization of both in vivo and in vitro techniques to make CTLs against glioma and used them as imaging probes to determine the sites of tumors. In this short review, current status of vaccination therapy against glioma and utilization of CTLs as in vivo imaging probes to determine the sites of tumors and differentiate recurrent glioma from radiation necrosis will be discussed.

The infusion of TIL586 along with interleukin-2 into the autologous patient with metastatic melanoma resulted in the objective regression of tumor. A gene encoding a tumor antigen recognized by TIL586 was previously isolated and shown to encode gp75 or TRP-1. Here we report that TRP-2 was identified as a second tumor antigen recognized by a HLA-A31– restricted CTL clone derived from the TIL586 cell line. The peptide LLPGGRPYR epitope was subsequently identified from the coding region of TRP-2 based on studies of the recognition of truncated TRP-2 cDNAs and the HLA-A31 binding motif. This epitope peptide was capable of sensitizing target cells for lysis by a CTL clone at 1 nM peptide concentration. Although some modified peptides could be recognized by the CTL clone, none were found to be better recognized by T cells than the parental peptide. Like other melamona differentiation antigens, TRP-2 was only expressed in melanoma, melanocytes, and retina, but not in other human tissues tested.

The development of cancer vaccines requires approaches to induce expansion and functional differentiation of tumor antigen-specific cytotoxic T lymphocyte (CTL) effectors which posses cytolytic capability and produce cytokines. Efficient induction of such cells is hindered by the poor immunogenicity of tumor antigens and by the poor transduction efficiency of dendritic cells (DCs) with current nonreplicating vectors. We have investigated the use of influenza A virus, a potent viral inducer of CTLs, as a vector expressing the immunodominant HER-2 CTL epitope KIF (E75). For this purpose, an attenuated influenza A/PR8/34 virus with a truncated nonstructural (NS1) gene was generated containing the E75 epitope in its neuraminidase protein (KIF-NS virus). Stimulation of peripheral blood mononuclear cells from healthy donors and of tumor-associated lymphocytes from ovarian and breast cancer patients with DCs infected with KIF-NS virus (KIF-NS DC) induced CTLs that specifically recognized the peptide KIF and HER-2-expressing tumors in cytotoxicity assays and secreted gamma interferon (IFN-γ) and interleukin-2 at recall with peptide. Priming with KIF-NS DCs increased the number of E75+ CD45RO+ cells by more than 10-fold compared to nonstimulated cells. In addition, KIF-NS virus induced high levels of IFN-α in DCs. This is the first report demonstrating induction of human epitope-specific CTLs against a tumor-associated antigen with a live attenuated recombinant influenza virus vector. Such vectors may provide a novel approach for tumor antigen delivery, lymphocyte activation, and differentiation in human cancer vaccine development.

Live, attenuated strains of many bacteria that synthesize and secrete foreign antigens are being developed as vaccines for a number of infectious diseases and cancer. Bacterial-based vaccines provide a number of advantages over other antigen delivery strategies including low cost of production, the absence of animal products, genetic stability and safety. In addition, bacterial vaccines delivering a tumor-associated antigen (TAA) stimulate innate immunity and also activate both arms of the adaptive immune system by which they exert efficacious anti-tumor effects. Listeria monocytogenes and several strains of Salmonella have been most extensively studied for this purpose. A number of attenuated strains have been generated and used to deliver antigens associated with infectious diseases and cancer. Although both bacteria are intracellular, the immune responses invoked by Listeria and Salmonella are different due to their sub-cellular locations. Upon entering antigen-presenting cells by phagocytosis, Listeria is capable of escaping from the phagosomal compartment and thus has direct access to the cell cytosol. Proteins delivered by this vector behave as endogenous antigens, are presented on the cell surface in the context of MHC class I molecules, and generate strong cell-mediated immune responses. In contrast, proteins delivered by Salmonella, which lacks a phagosomal escape mechanism, are treated as exogenous antigens and presented by MHC class II molecules resulting predominantly in Th2 type immune responses. This fundamental disparity between the life cycles of the two vectors accounts for their differential application as antigen delivery vehicles. The present paper includes a review of the most recent advances in the development of these two bacterial vectors for treatment of cancer. Similarities and differences between the two vectors are discussed.

Vaccines that target blood-feeding disease vectors, such as mosquitoes and ticks, have the potential to protect against the many diseases caused by vector-borne pathogens. We tested the ability of an anti-tick vaccine derived from a tick cement protein (64TRP) of Rhipicephalus appendiculatus to protect mice against tick-borne encephalitis virus (TBEV) transmitted by infected Ixodes ricinus ticks. The vaccine has a “dual action” in immunized animals: when infested with ticks, the inflammatory and immune responses first disrupt the skin feeding site, resulting in impaired blood feeding, and then specific anti-64TRP antibodies cross-react with midgut antigenic epitopes, causing rupture of the tick midgut and death of engorged ticks. Three parameters were measured: “transmission,” number of uninfected nymphal ticks that became infected when cofeeding with an infected adult female tick; “support,” number of mice supporting virus transmission from the infected tick to cofeeding uninfected nymphs; and “survival,” number of mice that survived infection by tick bite and subsequent challenge by intraperitoneal inoculation of a lethal dose of TBEV. We show that one dose of the 64TRP vaccine protects mice against lethal challenge by infected ticks; control animals developed a fatal viral encephalitis. The protective effect of the 64TRP vaccine was comparable to that of a single dose of a commercial TBEV vaccine, while the transmission-blocking effect of 64TRP was better than that of the antiviral vaccine in reducing the number of animals supporting virus transmission. By contrast, the commercial antitick vaccine (TickGARD) that targets only the tick's midgut showed transmission-blocking activity but was not protective. The 64TRP vaccine demonstrates the potential to control vector-borne disease by interfering with pathogen transmission, apparently by mediating a local cutaneous inflammatory immune response at the tick-feeding site.

Synopsis

Blood-sucking vectors such as mosquitoes and ticks transmit hundreds of micro-organisms that cause diseases like malaria and Lyme disease. Controlling so many diseases is an enormous challenge. A new idea is to make vaccines against the vectors rather than against all the individual disease agents they carry. The authors examined this hypothesis using a vaccine prepared from tick cement. This cement is secreted by ticks to help them attach to a human or animal to feed. A mouse model was used in which mice were infested with ticks infected with tick-borne encephalitis virus (TBEV), the most important vector-borne virus in Europe and northern Asia. The control mice developed fatal encephalitis and died about a week after being bitten by the infected tick. By contrast, the tick cement vaccine gave protection similar to the level seen in mice immunized with a single shot of the commercial TBEV vaccine for humans. However, a commercial tick vaccine used to control cattle ticks did not protect the mice. The authors' tick cement vaccine appeared to work by causing a cellular immune response in the skin where ticks were feeding. These results show that it is feasible to produce a vaccine against a tick that protects against the disease agent it transmits.

“Self” melanocyte differentiation antigens are potential targets for specific melanoma immunotherapy. Vaccination against murine tyrosinase-related protein (TRP)-1/gp75 was shown recently to cause melanoma rejection, which was accompanied by autoimmune skin depigmentation (vitiligo). To further explore the linkage between immunotherapy and autoimmunity, we studied the response to vaccination with a related antigen, TRP-2. i.m. inoculation of plasmid DNA encoding murine trp-2 elicited antigen-specific CTLs that recognized the B16 mouse melanoma and protected the mice from challenge with tumor cells. Furthermore, mice bearing established s.c. B16 melanomas rejected the tumor upon vaccination with a recombinant vaccinia virus encoding trp-2. Depletion experiments showed that CD8+ lymphocytes and natural killer cells were crucial for the antitumor activity of the trp-2-encoding vaccines. Mice that rejected the tumor did not develop generalized vitiligo, indicating that protective immunity can be achieved in the absence of widespread autoimmune aggression.

Purpose

A variety of cancers, including malignant gliomas, overexpress transforming growth factor-β (TGF-β), which helps tumors evade effective immune surveillance through a variety of mechanisms, including inhibition of CD8+ cytotoxic T lymphocytes (CTL) and enhancing the generation of regulatory T (Treg) cells. We hypothesized that inhibition of TGF-β would improve the efficacy of vaccines targeting glioma-associated antigen (GAA)-derived CTL epitopes by reversal of immunosuppression.

Experimental Design

Mice bearing orthotopic GL261 gliomas were treated systemically with a TGF-β neutralizing monoclonal antibody, 1D11, with or without subcutaneous (s.c.) vaccinations of synthetic peptides for GAA-derived CTL epitopes, GARC-1 (77-85) and EphA2 (671-679) emulsified in incomplete Freund's adjuvant.

Results

Mice receiving the combination regimen exhibited significantly prolonged survival compared with mice receiving either 1D11 alone, GAA-vaccines alone or mock-treatments alone. TGF-β neutralization enhanced the systemic induction of antigen-specific CTLs in glioma-bearing mice. Flow cytometric analyses of brain infiltrating lymphocytes revealed that 1D11 treatment suppressed phosphorylation of Smad2, increased GAA-reactive/interferon (IFN)-γ-producing CD8+ T cells, and reduced CD4+/FoxP3+ Treg cells in the glioma microenvironment. Neutralization of TGF-β also up-regulated plasma levels of interleukin (IL)-12, macrophage inflammatory protein-1α and IFN-inducible protein-10, suggesting a systemic promotion of type-1 cytokine/chemokine production. Furthermore, 1D11 treatment up-regulated plasma IL-15 levels and promoted the persistence of GAA-reactive CD8+ T cells in glioma-bearing mice.

Conclusions

These data suggest that systemic inhibition of TGF-β by 1D11 can reverse the suppressive immunological environment of orthotopic tumor-bearing mice both systemically and locally, thereby enhancing the therapeutic efficacy of GAA-vaccines.

Targeting tumors using cancer vaccine-therapeutics has several advantages including the induction of long-term immunity, prime boost strategies for additional treatments and reduced side effects compared to conventional chemotherapeutics. However, one problem in targeting tumor antigens directly is that this can lead to antigen loss or immunoediting. We hypothesized that directing the immune response to a normal cell type required for tumor growth and survival could provide a more stable immunotherapeutic target. We thus examined the ability of an anti-angiogenesis, Listeria monocytogenes (Lm) based vector to deliver extracellular and intracellular fragments of the mouse VEGFR2/Flk-1 molecule, Lm-LLO-Flk-E1 and Lm-LLO-Flk–I1 respectively, in an autochthonous model for Her-2/neu+ breast cancer. We found that these vaccines could cause epitope spreading to the endogenous tumor protein Her-2/neu and significantly delay tumor onset. However, tumors that grew out overtime accumulated mutations in the Her-2/neu molecule near or within CTL epitopes. We show here for the first time how an anti-angiogenesis immunotherapy can be used to delay the onset of a spontaneous tumor through epitope spreading and determine a possible mechanism of how immunoediting of an endogenous tumor protein can allow for tumor escape and outgrowth in an autochthonous mouse model for Her-2/neu+ breast cancer.

Background

This study evaluated the safety and immune responses to an autologous dendritic cell vaccine pulsed with class I peptides from tumor-associated antigens (TAA) expressed on gliomas and overexpressed in their cancer stem cell population (ICT-107).

Methods

TAA epitopes included HER2, TRP-2, gp100, MAGE-1, IL13Rα2, and AIM-2. HLA-A1- and/or HLA-A2-positive patients with glioblastoma (GBM) were eligible. Mononuclear cells from leukapheresis were differentiated into dendritic cells, pulsed with TAA peptides, and administered intradermally three times at two-week intervals.

Results

Twenty-one patients were enrolled with 17 newly diagnosed (ND-GBM) and three recurrent GBM patients and one brainstem glioma. Immune response data on 15 newly diagnosed patients showed 33 % responders. TAA expression by qRT-PCR from fresh-frozen tumor samples showed all patient tumors expressed at least three TAA, with 75 % expressing all six. Correlations of increased PFS and OS with quantitative expression of MAGE1 and AIM-2 were observed, and a trend for longer survival was observed with gp100 and HER2 antigens. Target antigens gp100, HER1, and IL13Rα2 were downregulated in recurrent tumors from 4 HLA-A2+ patients. A decrease in or absence of CD133 expression was seen in five patients who underwent a second resection. At a median follow-up of 40.1 months, six of 16 ND-GBM patients showed no evidence of tumor recurrence. Median PFS in newly diagnosed patients was 16.9 months, and median OS was 38.4 months.

Conclusions

Expression of four ICT-107 targeted antigens in the pre-vaccine tumors correlated with prolonged overall survival and PFS in ND-GBM patients. The goal of targeting tumor antigens highly expressed on glioblastoma cancer stem cells is supported by the observation of decreased or absent CD133 expression in the recurrent areas of gadolinium-enhanced tumors.

We have performed a detailed analysis of the recognition of melanoma Ags by the tumor-infiltrating lymphocytes (TIL) 1790, isolated from a patient who experienced a dramatic tumor regression following immunization with peptides from the gp100, MART-1, and tyrosinase Ags. This TIL was found to recognize HLA-A2-restricted CTL epitopes in tyrosinase-related protein (TRP)-2 (clone MR7) and NY-ESO-1 (clone M8). These epitopes were the same as the previously identified nonapeptide TRP-2: 180–188, and the overlapping NY-ESO-1 peptides, obtained by using lymphocytes from in vitro stimulation. We also cloned a previously unknown TRP-2 mRNA isoform (TRP-2-6b) that contained two novel exons alternatively spliced from the sixth intron between exons 6 and 7 of TRP-2 mRNA. The isoform encoded an HLA-A2-restricted antigenic epitope recognized by TIL clone MB4. An immunologic analysis of the patient’s PBMC obtained before treatment showed the presence of high reactivity against NY-ESO-1 and both TRP-2 Ags, but not the Ags used for immunization. Because immune response against these Ags was less pronounced, it is possible that NY-ESO-1, TRP-2, and TRP-2-6b may be of importance in the generation of CTL-mediated tumor destruction and may have played a role in the dramatic tumor regression seen in this patient.

Vesicles comprised of the ether glycerolipids of the archaeon Methanobrevibacter smithii (archaeosomes) are potent adjuvants for evoking CD8+ T cell responses. We therefore explored the ability of archaeosomes to overcome immunologic tolerance to self-antigens. Priming and boosting of mice with archaeosome-antigen evoked comparable CD8+ T cell response and tumor protection to an alternate boosting strategy utilizing live bacterial vectors for antigen delivery. Vaccination with melanoma antigenic peptides TRP181-189 and Gp10025-33 delivered in archaeosomes resulted in IFN-γ producing antigen-specific CD8+ T cells with strong cytolytic capability and protection against subcutaneous B16 melanoma. Targeting responses against multiple antigens afforded prolonged median survival against melanoma challenge. Entrapment of multiple peptides within the same vesicle or admixed formulations were both effective at evoking CD8+ T cells against each antigen. Melanoma-antigen archaeosome formulations also afforded therapeutic protection against established B16 tumors when combined with depletion of T-regulatory cells. Overall, we demonstrate that archaeosome adjuvants constitute an effective choice for formulating cancer vaccines.

Efficient vaccination against infectious agents and tumors depends on specific antigen targeting to dendritic cells (DCs). We report here that biosafe coronavirus-based vaccine vectors facilitate delivery of multiple antigens and immunostimulatory cytokines to professional antigen-presenting cells in vitro and in vivo. Vaccine vectors based on heavily attenuated murine coronavirus genomes were generated to express epitopes from the lymphocytic choriomeningitis virus glycoprotein, or human Melan-A, in combination with the immunostimulatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). These vectors selectively targeted DCs in vitro and in vivo resulting in vector-mediated antigen expression and efficient maturation of DCs. Single application of only low vector doses elicited strong and long-lasting cytotoxic T-cell responses, providing protective antiviral and antitumor immunity. Furthermore, human DCs transduced with Melan-A-recombinant human coronavirus 229E efficiently activated tumor-specific CD8+ T cells. Taken together, this novel vaccine platform is well suited to deliver antigens and immunostimulatory cytokines to DCs and to initiate and maintain protective immunity.

IMPORTANCE

Vaccination against infectious agents has protected many individuals from severe disease. In addition, prophylactic and, most likely, also therapeutic vaccination against tumors will save millions from metastatic disease. This study describes a novel vaccine approach that facilitates delivery of viral or tumor antigens to dendritic cells in vivo. Concomitant immunostimulation via the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) was achieved through delivery by the same viral vector. Single immunization with only low doses of coronavirus-based vaccine vectors was sufficient to elicit (i) vigorous expansion and optimal differentiation of CD8+ T cells, (ii) protective and long-lasting antiviral immunity, and (iii) prophylactic and therapeutic tumor immunity. Moreover, highly efficient antigen delivery to human DCs with recombinant human coronavirus 229E and specific stimulation of human CD8+ T cells revealed that this approach is exceptionally well suited for translation into human vaccine studies.

How tumor-infiltrating lymphocytes (TILs) that are tumor-specific but functionally tolerant persist in the antigen-expressing tumor tissue is largely unknown. We have previously developed a modified TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model where prostate cancer cells express the T cell epitope SIYRYYGL (SIY) recognized by CD8 T cells expressing the 2C TCR (referred to as TRP-SIY mice). In TRP-SIY mice, activated 2C T cells rapidly become tolerant following infiltration into the prostate tumor. In this study, we show that tolerant 2C T cells persist in the prostate tumor of TRP-SIY mice by proliferating slowly in a tumor-dependent, but antigen-, interleukin (IL)-7- and IL-15-independent manner. We also show that disappearance of 2C T cells from the lymphoid organs of TRP-SIY mice are due to antigen-induced T cell contraction rather than altered trafficking or generalized T cell depletion in the mice. Finally we show that clonal T cells unreactive to SIY are equally capable of persisting in the prostate tumor. These findings suggest that while functional tolerance of TILs is induced by antigen, persistence of tolerant TILs in the tumor tissue is mediated by a novel mechanism: slow proliferation independent of antigen and homeostatic cytokines. These results also allow CD8 T cell survival in the tumor environment to be compared with T cell survival in chronic infection.

Several immunotherapeutic approaches rely on antigen-specific T-cells. Restrictions in the T-cell receptor (TCR) repertoire were reported as indicator of anti-tumor cytotoxic T-lymphocyte (CTL) response in various tumor entities. It is unclear yet whether a TCR restriction in peripheral blood mirrors the tumor compartment. We compared the expression of TCR Vβ-families for the quantification of TCR repertoire alterations in blood and tissue samples from patients with colorectal carcinoma. Blood samples from patients with colorectal carcinoma and healthy volunteers and tissue samples of normal colonic mucosa and colorectal carcinoma were analyzed. Relative Vβ-family quantification was performed based on quantitative reverse transcribed PCR. Standard deviation and average mean of the single families were determined. Two variables describing the degree of Vβ-repertoire restriction were defined. Forty-eight blood samples and 37 tissue samples were analyzed. TCR repertoire restriction was higher in blood of tumor patients than in blood of healthy controls (p < 0.05). No difference in the degree of TCR repertoire restriction was found between carcinoma and unaffected colon tissue. We found no corresponding elevated TCR families among the different compartments blood, normal colon, and carcinoma tissue of the same patient. In conclusion, we observed a repertoire restriction in peripheral blood as well as in tumor tissue of cancer patients. However, in tumor tissue, repertoire alterations were comparable to normal mucosa, suggesting compartment-specific TCR distribution rather than alterations due to tumor-T-cell interaction questioning the presence of highly restricted clonal T-cell expansions in colorectal cancer as they have been described in other, assumingly more immunogenic tumor entities.

Tumor vaccination using tumor-associated antigen-primed dendritic cells (DCs) is in clinical trials. Investigators are using patients’ own immune systems to activate T-cells against recurrent or metastatic tumors. Following vaccination of DCs or attenuated tumor cells, clinical as well as radiological improvements have been noted due to migration and accumulation of cytotoxic T-cells (CTLs). CTLs mediated tumor cell killing resulted in extended survival in clinical trails and in preclinical models. Besides administration of primed DCs or attenuated or killed tumors cells to initiate the generation of CTLs, investigators have started making genetically altered T-cells (CTLs) to target specific tumors and showed in vivo migration and accumulation in the implanted or recurrent tumors using different imaging modalities. Our groups have also showed the utilization of both in vivo and in vitro techniques to make CTLs against glioma and used them as imaging probes to determine the sites of tumors. In this short review, the current status of vaccination therapy against glioma and utilization of CTLs as in vivo imaging probes to determine the sites of tumors and differentiate recurrent glioma from radiation necrosis will be discussed.

Epitope selection is an important consideration in the design of cancer vaccines, but factors impacting selection are not fully understood. We compared the immune response to peptides and glycopeptides from the common human tumor antigen MUC1, a mucin that is coated with O-linked carbohydrates in its variable number of tandem repeats (VNTR) region. MUC1 expressed on tumor cells is characteristically underglycosylated , creating peptide and glycopeptide neoepitopes that are recognized by the immune system. The response to VNTR peptides is weaker in MUC1 transgenic mice (MUC1-Tg mice) than in wild type (WT) mice, whereas the response to VNTR glycopeptides is equally strong in both strains. Thus, glycopeptides appear to be recognized as foreign, while peptides, although immunogenic, are perceived as self. To explore this further, we generated MUC1 peptide and glycopeptide-specific TCR transgenic mice and studied the function of their CD4 T cells when adoptively transferred into MUC1-Tg or WT mice. Peptide-specific T cell precursors were not centrally deleted in MUC1-Tg mice and did not acquire a T regulatory (Treg) phenotype. However, their response to the cognate peptide was reduced in MUC1-Tg mice compared to WT mice. In contrast, glycopeptide-specific CD4 T cells responded equally well in both hosts, and when simultaneously activated also enhanced the peptide-specific T cell responses. Our data show that the immune system differentially recognizes various epitopes of tumor-associated antigens either as self or as foreign, and this controls the strength of anti-tumor immunity. This represents an important consideration for designing safe and effective cancer vaccines.

Although tumors express potentially immunogenic tumor-associated antigens (TAAs), cancer vaccines often fail because of inadequate antigen delivery and/or insufficient activation of innate immunity. The engineering of non-pathogenic bacterial vectors to deliver TAAs of choice may provide an efficient way of presenting TAAs in an immunogenic form. In this study, we used genes of Salmonella Pathogenicity Island 2 (SPI2) to construct a novel cancer vaccine, where a TAA, survivin was fused to SseF effector protein and placed under control of SsrB, the central regulator of SPI2 gene expression. This construct uses the type III secretion system (T3SS) of Salmonella and allows preferential delivery of tumor antigen into the cytosol of antigen-presenting cells for optimal immunogenicity. In a screen of a panel of attenuated strains of Salmonella we found that a double-attenuated strain of Salmonella typhimurium, MvP728 (purD/htrA) was not toxic to mice and effectively expressed and translocated survivin protein inside cytosol of murine macrophages. We also found that a ligand for CD1d-reactive Natural Killer T (NKT) cells, α-Glucuronosylceramide (GSL1) enhanced MvP728-induced IL-12 production in human DCs and that in vivo co-administration of a NKT ligand with MvP728-Llo or MvP728-survivin enhanced effector-memory CTL responses. Furthermore, combined use of MvP728-survivin with GSL1 produced anti-tumor activity in mouse models of CT26 colon carcinoma and orthotopic DBT glioblastoma. Therefore, the use of TAA delivery via SPI-2-regulated T3SS of Salmonella and NKT ligands as adjuvants may provide a foundation for new cancer vaccines.

Background

Microorganisms and plants are able to produce tryptophan. Enzymes catalysing the last seven steps of tryptophan biosynthesis are encoded in the canonical trp operon. Among the trp genes are most frequently trpA and trpB, which code for the alpha and beta subunit of tryptophan synthase. In several prokaryotic genomes, two variants of trpB (named trpB1 or trpB2) occur in different combinations. The evolutionary history of these trpB genes is under debate.

Results

In order to study the evolution of trp genes, completely sequenced archeal and bacterial genomes containing trpB were analysed. Phylogenetic trees indicated that TrpB sequences constitute four distinct groups; their composition is in agreement with the location of respective genes. The first group consisted exclusively of trpB1 genes most of which belonged to trp operons. Groups two to four contained trpB2 genes. The largest group (trpB2_o) contained trpB2 genes all located outside of operons. Most of these genes originated from species possessing an operon-based trpB1 in addition. Groups three and four pertain to trpB2 genes of those genomes containing exclusively one or two trpB2 genes, but no trpB1. One group (trpB2_i) consisted of trpB2 genes located inside, the other (trpB2_a) of trpB2 genes located outside the trp operon. TrpA and TrpB form a heterodimer and cooperate biochemically. In order to characterise trpB variants and stages of TrpA/TrpB cooperation in silico, several approaches were combined. Phylogenetic trees were constructed for all trp genes; their structure was assessed via bootstrapping. Alternative models of trpB evolution were evaluated with parsimony arguments. The four groups of trpB variants were correlated with archeal speciation. Several stages of TrpA/TrpB cooperation were identified and trpB variants were characterised. Most plausibly, trpB2 represents the predecessor of the modern trpB gene, and trpB1 evolved in an ancestral bacterium.

Conclusion

In archeal genomes, several stages of trpB evolution, TrpA/TrpB cooperation, and operon formation can be observed. Thus, archeal trp genes may serve as a model system for studying the evolution of protein-protein interactions and operon formation.

The development of effective anti-tumor immune responses is normally constrained by low avidity, tumor-specific cytotoxic T lymphocytes (CTLs) which are unable to eradicate the tumor. Strategies to rescue anti-tumor activity of low avidity melanoma-specific CTLs in vivo may improve immunotherapy efficacy. To boost the in vivo effectiveness of low avidity CTLs we immunized mice bearing lung melanoma metastases with artificial Antigen Presenting Cells (aAPC), made by covalently coupling pepMHC-Ig dimers and B7.1-Ig molecules to magnetic beads. aAPC treatment induced significant tumor reduction in a mouse telomerase antigen system and complete tumor eradication in a mouse TRP-2 antigen system, when low avidity CTLs specific for these antigens were adoptively transferred. In addition, in an in vivo treatment model of subcutaneous melanoma, aAPC injection also augmented the activity of adoptively transferred CTLs and significantly delayed tumor growth. In vivo tumor clearance due to aAPC administration correlated with in situ proliferation of the transferred CTL. In vitro studies showed that aAPC effectively stimulated cytokine release, enhanced CTL-mediated lysis and TCR down-regulation in low avidity CTLs. Therefore, in vivo aAPC administration represents a potentially novel approach to improve cancer immunotherapy.

Tumors express embryonic carbohydrate antigens called tumor-associated carbohydrate antigens (TACA). TACA-containing glycopeptides are appealing cytotoxic T cell (CTL)-based vaccines to prevent or treat cancer because the same sugar moieties are expressed in a variety of tumors, rendering a vaccination strategy applicable in a large population. Here we demonstrate that by using glycopeptides with high affinity for the major histocompatibility complex and glycosylated in a position corresponding to a critical T cell receptor (TcR) contact, it is possible to induce anti-TACA CTL in vivo. In the current study we show that designer glycopeptides containing the Thomsen-Freidenreich (TF) antigen (β-Gal-[1→3]-α-GalNAc-O-serine) are immunogenic in vivo and generate TF-specific CTL capable of recognizing a variety of tumor cells in vitro including a MUC1-expressing tumor. The fine specificity of the TF-specific CTL repertoire indicates that the TcR recognize the glycosylated amino acid residue together with TF in a conventional major histocompatibility complex class I–restricted fashion. These results have high potential for immunotherapy against a broad range of tumors.

It is established that mutations in viral antigenic epitopes, or antigenic drifts, allow viruses to escape recognition by both Ab’s and T lymphocytes. It is unclear, however, whether tumor cells can escape immune recognition via antigenic drift. Here we show that adoptive therapy with both monoclonal and polyclonal transgenic CTLs, specific for a natural tumor antigen, P1A, selects for multiple mutations in the P1A antigenic epitope. These mutations severely diminish T cell recognition of the tumor antigen by a variety of mechanisms, including modulation of MHC:peptide interaction and TCR binding to MHC:peptide complex. These results provide the first evidence for tumor evasion of T cell recognition by antigenic drift, and thus have important implications for the strategy of tumor immunotherapy.