Tumor-derived Antigens Research Articles

Contribution du système immunitaire à l’efficacité des chimiothérapies anticancéreuses

For over 40 years, four therapeutic modalities, namely surgery, radiotherapy, chemotherapy and hormone therapy have formed the core of anticancer treatments. Their mode of action is thought to involve a direct cytotoxic action on tumor cells. Recently, the discovery of tumor-associated immunosuppression and tumor immunosurveillance has led to cancer being reconsidered not only as an organ disease but also as a host disease. This new concept is supported by the recent discovery of the immunogenic effects of tumor cell death induced by a variety of cytotoxic drugs. This work describes a new pathway of tumor-derived antigen presentation mediated by the alarmin HMGB1 (released by dying tumor cells in response to chemo/radiotherapy) and by TLR4 on dendritic cells. In this model, TLR4 recognizes? tumor-derived antigens, leading to T cell activation and to the induction of an antitumor immune response. Accordingly, we show that breast cancer patients bearing a loss-of-function mutation of the TLR4 receptor have shorter disease-free survival, confirming the major role of the immune system in the response to cytotoxic treatments. The response to chemotherapy and/or radiotherapy may thus combine both direct cytotoxic effects and the development of long-term antitumor immunity. We anticipate that these new results will have major impact on cancer management.

Inefficient presentation of tumor-derived antigen by tumor-infiltrating dendritic cells

Inefficient presentation of tumor-derived antigen by tumor-infiltrating dendritic cells

Inefficient presentation of tumor-derived antigen by tumor-infiltrating dendritic cells.

Transplantable B16 melanoma is widely used as a tumor model to investigate tumor immunity. We wished to characterize the leukocyte populations infiltrating B16 melanoma tumors, and the functional properties of tumor-infiltrating dendritic cells (TIDC). We used the B16 melanoma cell line expressing ovalbumin protein (OVA) to investigate the phenotype and T cell stimulatory capacity of TIDC. The majority of leukocytes in B16 melanoma were macrophages, which colocalized with TIDCs, B and T cells to the peripheral area of the tumor. Both myeloid and plasmacytoid DC populations were present within tumors. Most of these DCs appeared immature, but about a third expressed a mature phenotype. TIDCs did not present tumor-derived antigen, as they were unable to induce the proliferation of tumor-specific CD4+ and CD8+ T cells in vitro unless in the presence of specific peptides. Some presentation of tumor-derived antigen could be demonstrated in the tumor-draining lymph node using in vivo proliferation assays. However, while proliferation of CD8+ T cells was reproducibly demonstrated, no proliferation of CD4+ T cells was observed. In summary, our data suggest that DCs in tumors have limited antigen-presenting function. Inefficient antigen presentation extends to the tumor-draining lymph node, and may affect the generation of antitumor immune responses.

The tumor‐draining lymph node as an immune‐privileged site

Lymph nodes that lie immediately downstream of tumors [tumor-draining lymph nodes (TDLNs)] undergo profound alterations due to the presence of the upstream tumor. The antigen-presenting cell population in TDLNs becomes modified such that tumor-derived antigens are cross-presented by host cells in a tolerizing fashion. In addition, the number and suppressor activity of regulatory T cells (Tregs) are increased in the TDLN. Emerging evidence suggests that some of these Tregs may be generated de novo against specific tumor-derived antigens, and thus they arise as a direct consequence of antigen presentation in the TDLN. Others may represent Tregs against self-antigens, which undergo preferential activation in the tolerogenic milieu of the TDLN. The TDLN thus becomes an anatomic context in which presentation of new antigens not only fails to elicit a protective immune response but also actively creates systemic tolerance. In this regard, the TDLN displays features analogous to classical immune privilege. Accumulating evidence thus suggests that the TDLNs, although small in size, may exert a profound tolerizing influence on the rest of the immune system. These mechanisms will need to be interrupted in order for clinical anti-tumor immunotherapy to be successful.

Immunotherapy of Cancer Based on DC-Tumor Fusion Vaccine

Fusions of dendritic cells (DC) and tumor cells are increasingly used in tumor immunotherapy. The strategy for DC-tumor fusion vaccine is based on the fact that DC are the most potent antigen-presenting cells in the body, whereas tumor cells express abundant tumor antigens. The fusion of these two cell types creates a heterokaryon with both DCderived costimulatory molecules, efficient antigen- processing and -presentation machinery, and tumor-derived antigens. In animal and human studies, DC-tumor fusion cells (FC) have been shown to possess the elements essential for processing and presenting tumor antigens to host immune cells and for inducing effective immune response that is able to break T-cell tolerance to tumor-associated antigens; moreover, they have been demonstrated to provide protection against challenge with tumor cells and to regress established tumors. Despite these unique features of DC-tumor fusion cells and the observation of tumor eradication in animal studies, only limited, yet encouraging, success has been seen in clinical trials. This article reviews the methods used for the preparation and selection of DC-tumor fusion cells and analyzes the factors influencing the success or failure of FC-mediated immunotherapy. In addition, it discusses the challenges facing FC vaccine, including preparation, selection, and quality control of DC-tumor fusion cells, and approaches for enhancing antitumor immunity. Keywords: DC-tumor fusion, anti-tumor Immunity, antigen presentation, vaccination, T cell activation

DC-NK cell cross talk as a novel CD4+ T-cell–independent pathway for antitumor CTL induction

It is generally accepted that priming of antitumor CD8+ cytotoxic T lymphocytes (CTLs) needs help that can be provided by CD4+ T cells. We show that interactions between dendritic cells (DCs) and natural killer (NK) cells can bypass the T helper arm in CTL induction. Bone marrow-derived DCs caused rejection of the A20 lymphoma and induced tumor-specific long-term memory, although they were not loaded with tumor-derived antigen. Experiments using CD40(-) knock-out mice and cell depletion showed that this effect did not require CD4+ cells. Both primary rejection and long-term CTL memory were the result of NK cell activation by DCs. NK cytotoxicity, which was necessary for primary rejection, was dependent on expression of natural killer group 2 D (NKG2D) ligands on tumor cells. Blocking of these ligands using NKG2D tetramers abrogated tumor killing in vitro and in vivo. The long-term response was due to CTLs directed against antigen(s) expressed on A20 and in vitro-differentiated DCs. The mechanism leading to CD4+ helper cell-independent CTL responses was elucidated as a cascade that was initiated by NK cell activation. This pathway was dependent on inter-feron-gamma expression and involved priming endogenous DCs for interleukin-12 production. Our data suggest a novel pathway linking innate and adaptive immunity.

Preclinical evaluation of autologous dendritic cells transfected with mRNA or loaded with apoptotic cells for immunotherapy of high-risk neuroblastoma

Children with high-risk neuroblastoma (NB) have a poor clinical outcome. The purpose of the present study was to evaluate different strategies for immunotherapy of high-risk NB based on vaccination with antigen-loaded dendritic cells (DCs). DCs are professional antigen-presenting cells with the ability to induce antitumor T-cell responses. We have compared DCs either loaded with apoptotic tumor cells or transfected with mRNA from the NB cell line HTB11 SK-N-SH, for their capacity to induce T-cell responses in vitro. Monocyte-derived DCs from healthy donors were loaded with tumor-derived antigens in the form of apoptotic cells or mRNA, matured and used to prime autologous T cells in vitro. After 1 week, T-cell responses against antigen-loaded DCs were measured by ELISPOT assay. DCs loaded with apoptotic NB cells or transfected with NB-cell mRNA were both able to efficiently activate autologous T cells. Both T cells of the CD8+ and CD4+ subset were activated. T cells activated by NB mRNA transfected DCs extensively crossreacted with DCs loaded with apoptotic NB cells and vice versa. The results indicate that loading of DCs with apoptotic NB cells or transfection with tumor mRNA represent promising strategies for development of individualized cancer vaccines/cancer gene therapy in treatment of NB.

Activation of Dendritic Cells That Cross-Present Tumor-Derived Antigen Licenses CD8+ CTL to Cause Tumor Eradication

The fate of naive CD8(+) T cells is determined by the environment in which they encounter MHC class I presented peptide Ags. The manner in which tumor Ags are presented is a longstanding matter of debate. Ag presentation might be mediated by tumor cells in tumor draining lymph nodes or via cross-presentation by professional APC. Either pathway is insufficient to elicit protective antitumor immunity. We now demonstrate using a syngeneic mouse tumor model, expressing an Ag derived from the early region 1A of human adenovirus type 5, that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c(+) APC. Although this event results in division of naive CTL in tumor draining lymph nodes, it does not establish a productive immune response. Treatment of tumor-bearing mice with dendritic cell-stimulating agonistic anti-CD40 mAb resulted in systemic efflux of CTL with robust effector function capable to eradicate established tumors. For efficacy of anti-CD40 treatment, CD40 ligation of host APC is required because adoptive transfer of CD40-proficient tumor-specific TCR transgenic CTL into CD40-deficient tumor-bearing mice did not lead to productive antitumor immunity after CD40 triggering in vivo. CpG and detoxified LPS (MPL) acted similarly as agonistic anti-CD40 mAb with respect to CD8(+) CTL efflux and tumor eradication. Together these results indicate that dendritic cells, depending on their activation state, orchestrate the outcome of CTL-mediated immunity against tumors, leading either to an ineffective immune response or potent antitumor immunity.

Tumor Antigen Immunization of Sibling Stem Cell Transplant Donors in Multiple Myeloma.

Strategies to enhance the specific anti-tumor effect of the graft without increasing the risk of graft-versus-host disease (GVHD) are needed to improve the outcome in allotransplant recipients. In this pilot study, we vaccinated sibling donors against a defined, patient tumor-derived antigen prior to bone marrow harvest. The objective of this strategy was to induce tumor antigen-specific immunity in healthy donors and to transfer this immunity to the recipients by bone marrow transplantation (BMT). For this purpose, the unique antigenic determinants (Idiotype [Id]) of the immunoglobulin secreted by the myeloma tumor can serve as a safe, tumor-specific antigen for active immunotherapy. The vaccine formulation consisted of Id isolated from the plasma of myeloma patients chemically conjugated to a carrier molecule keyhole limpet-hemocyanin (KLH) and administered together with GM-CSF as an adjuvant. Immunization of the HLA-matched sibling donors (n = 5) prior to bone marrow harvest, induced specific cellular and/or humoral immune responses against Id and KLH in all five donors. No short-term or long-term toxicities were observed in any of the donors following vaccination. The recipients received priming vaccination pretransplant and booster immunizations starting approximately 3 months after the myeloablative transplantation. Following BMT, two patients died within 30 days due to transplant related complications. Id-specific and KLH-specific T cell responses were detected in all three surviving patients post-, but not pre-BMT. In at least one patient, Id-specific T cell immunity persisted beyond 18 months after completion of the vaccination schedule. All three surviving patients converted from a partial response to a complete response following BMT. Two of the three patients (both recipients of immune BMT from syngeneic twins) remain in complete remission 8 years and 7 years after the transplant, and the third patient developed chronic steroid-dependent GVHD and died of renal failure after 5.5 years while in complete remission from the disease. Our results suggest that Id vaccination induces specific T cell immunity in the donors that is transferable by BMT, is associated with a prolonged disease-free survival in recipients and may represent a novel strategy to enhance the graft versus myeloma effect. These encouraging results have prompted us to initiate a clinical trial to evaluate this approach of donor immunization in the context of non-myeloablative allogeneic peripheral blood stem cell transplantation in myeloma patients.

Reduction of immature myeloid cells by treatment with all-trans-retinoic acid (ATRA) improves the immunotherapeutic effect of the combination of local radiation with CTLA-4 blockade

Accumulated evidence supports the contention that ionizing radiation therapy (RT) effects on tumor-host interactions extend beyond its ability to eliminate the cancer cells within a tumor. The ability of RT to generate inflammatory signals, to enhance the penetration of antigen-presenting cells (APC) and effector T-cells into solid tumors, to free tumor-derived antigens from the dying cancer cells, and to enhance the expression of some surface receptors on the cancer cells that survive, may all be important determinants of the effects of RT on tumor immunogenicity.

Reduction of immature myeloid cells by treatment with all-trans-retinoic acid (ATRA) improves the immunotherapeutic effect of the combination of local radiation with CTLA-4 blockade

Accumulated evidence supports the contention that ionizing radiation therapy (RT) effects on tumor-host interactions extend beyond its ability to eliminate the cancer cells within a tumor. The ability of RT to generate inflammatory signals, to enhance the penetration of antigen-presenting cells (APC) and effector T-cells into solid tumors, to free tumor-derived antigens from the dying cancer cells, and to enhance the expression of some surface receptors on the cancer cells that survive, may all be important determinants of the effects of RT on tumor immunogenicity. We have recently shown that RT can enhance the immunogenicity of the poorly immunogenic, spontaneously metastasizing mouse mammary carcinoma 4T1 (Int J Radiat Oncol Biol Phys. 2003 Oct 1;57(2 Suppl):S259). Blockade of the CTLA4 receptor by the 9H10 antibody on T-cells lowers their activation threshold and has been shown to induce powerful anti-tumor immunity to partially immunogenic tumors. We find that a single dose (12 Gy) of RT can convert the poorly immunogenic 4T1 mammary carcinoma which is otherwise refractory to the treatment with CTLA-4 blocking antibodies into a responsive phenotype. Thus, treatment of 4T1 tumor-bearing mice with local RT followed by 9H10 administration induces a CD8 T cell-mediated anti-tumor immune response that inhibits the metastatic growth in the lungs and extends the survival of the mice. However, mice eventually succumb to their disease. 4T1 tumor growth is associated with dysregulation of APC maturation resulting in accumulation of immature (Gr1+CD11b+) myeloid cells (iMy) with immune suppressive function. The aim of the present study is to test whether depletion of iMy can result in a stronger and longer-lasting anti-tumor immune response following treatment with the combination of RT and CTLA-4 blockade. BALB/C mice were injected on day 0 with the syngeneic mammary carcinoma cells 4T1 in the flank at a single s.c. site considered as the primary tumor and treatment started when the growing tumors were at least 5mm in average diameter. All-trans-retinoic acid (ATRA) or placebo pellets (5 mg, 21-day release) were implanted s.c. on Day 7. Mice were randomly assigned to four groups: 1) non-specific hamster Ig (Ig)+ placebo; 2)Ig+ATRA; 3) RT+9H10 + placebo; 4) RT+9H10 + ATRA. RT was delivered to the s.c. tumor at a single dose of 12 Gy. The monoclonal hamster antibody against mouse CTLA4, 9H10, was given i.p. at 200 ug 1, 4 and 7 days after RT. Mice were followed for tumor growth and survival. Survival differences among treatment groups were analyzed using a Weibull model. Growth of 4T1 tumors was associated with progressive accumulation of Gr1+CD11b+ iMy cells in the spleen (up to 20–40% of total splenocytes). iMy cells also infiltrated the tumor, representing ∼5% of total cells. ATRA implantation on Day 7 dramatically reduced the percentage of iMy in the spleen of tumor-bearing mice compared to placebo and increased the percentage of dendritic cells, as measured on Day 20. Relative to placebo, administration of ATRA resulted in a statistically significant increase in survival in the presence of RT+9H10 (p = 0.0013) but had no significant effect in its absence. Furthermore, in these mice with more advanced tumors, relative to Ig control, the treatment with RT+9H10 significantly improved survival when in combination with ATRA (p = 0.0001) but had no significant effect on survival when administered with placebo. Tumor-induced immunosuppression is a great obstacle to the development of more effective immunotherapy treatments. These results show that, like other tumor vaccination strategies, the effectiveness of local RT combined with CTLA-4 blockade is limited by the accumulation of iMy cells, and their elimination by ATRA may be required to improve the therapeutic effect of this new treatment strategy

Differential immunization identifies PHB1/PHB2 as blood-borne tumor antigens.

Early diagnosis of cancer is crucial for successful treatment. Noninvasive assays for detecting tumor-derived antigens in serum and other bodily fluids have the potential to screen healthy individuals for hitherto undetected cancers. Very few such assays have been successfully developed, in part because identifying potential target antigens remains a challenge. To identify new blood-borne tumor antigens for the purpose of establishing such assays, we have developed a novel technique called differential immunization. Using this method, we have identified PHB1 and PHB2, proteins thought to function as mitochondrial chaperones and transcriptional regulators, as antigens released from colorectal tumors in vivo. Serum from colorectal patients contains significantly higher levels of these antigens compared to serum from healthy volunteers. These data demonstrate that differential immunization is an effective new method for identifying tumor-derived antigens in serum.

Concurrent delivery of tumor antigens and activation signals to dendritic cells by irradiated CD40 ligand-transfected tumor cells resulted in efficient activation of specific CD8+ T cells.

To improve the efficacy of tumor cell-based and dendritic cell (DC)-based cancer vaccines, this study explored the potential of a new cancer vaccine strategy, that is, the use of CD40 ligand-transfected tumor (CD40L-tumor) cells to simultaneously deliver both tumor-derived antigens (Ag) and maturation stimuli to DCs. Materials from frozen/thawed or irradiated human tumor cells, with or without surface CD40L, were internalized efficiently by immature DCs after coincubation. However, during the internalization process, only coculturing with irradiated CD40L-tumor cells resulted in concurrent, optimal DC maturation and production of proinflammatory chemokines and pro-Th1 cytokines, such as IL-6, IL-8, IL-12, IFN-gamma, and TNF-alpha. These activated DCs were the most potent cells to support the growth of CD8+, IFN-gamma-producing T cells, and to process tumor Ag for the generation of specific cytotoxic T cells in vitro. Animals vaccinated with irradiated CD40L-tumor cell-pulsed DCs were better protected against subsequent challenge of a weakly immunogenic tumor cell line than animals vaccinated with irradiated CD40L-tumor cells alone. Thus, our results strongly support the future clinical application of using DCs pulsed with irradiated CD40L-tumor cells as a cancer vaccine.

IDO and tolerance to tumors

Tumors create a pathological state of tolerance towards tumor-associated antigens. The mechanisms by which this occurs are still largely unknown. Recent studies suggest that one mechanism contributing to this phenomenon could be tryptophan catabolism carried out by the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO). IDO can be expressed by tumor cells themselves, and we propose that the recruitment of antigen-presenting cells expressing IDO to tumor-draining lymph nodes might constitute another potent mechanism for inducing tolerance to tumor-derived antigens. Therapeutic strategies based on blocking this pathway might represent a novel class of adjuvants for tumor immunotherapy.

In vivo priming of natural killer T cells by dendritic cells pulsed with hepatoma-derived acid-eluted substances.

Many murine tumor cells express not only individual haplotype-matched class I MHC molecules, but also species-specific CD1d molecules. The former class I MHC molecules generally present internally synthesized tumor-derived peptide antigens to highly specific CD8(+) cytotoxic T lymphocytes (CTLs) in acquired immunity. In contrast, the latter CD1d molecules may present tumor-associated glycolipid antigens to broadly crossreactive natural killer T (NKT) cells, which might correlate with controlling tumor metastasis. Here, we showed that murine hepatoma cell line Hepa1-6-derived acid-eluted substances might contain both D(b) class I MHC-restricted antigens and CD1d-restricted substances, which could sensitize not only syngeneic bone marrow-derived DCs (BM-DCs), but also allogeneic BM-DCs expressing haplotype-mismatched class I MHC and species-specific CD1d molecules. To our surprise, intravenous (i.v.) immunization of C57BL/6 mice with the former syngeneic BM-DCs carrying acid-eluted materials primed both CD4(-)CD8(-) and CD8(+) NKT cells in the spleen, whereas immunization with the latter allogeneic BM-DCs loaded the tumor-derived substances primed CD4(-)CD8(-), but not CD8(+) NKT cells. The findings shown in the present study will open a new area for cancer immunotherapy using allogeneic DCs and tumor-derived acid-eluted substances.

Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8+cells in patients with hepatocellular carcinoma

Background/Aims: Members of the melanoma antigen encoding gene family are expressed in tumors of different histological types but not in normal tissue. For this reason, they are attractive targets for cancer immunotherapy. Methods: In the present study, we analyzed the expression of MAGE-1 and -3 genes in the hepatocellular carcinoma (HCC) tissue as well as frequency, phenotype and function of circulating and tumor infiltrating CD8+ cells specific for HLA-A1 and -A2 restricted epitopes of MAGE-1 and -3. Results: Our study shows for the first time the presence of MAGE/tetramer+ CD8 cells in the tumor tissue of patients with HCC. These cells are able to recognize the MAGE-1 sequence 161–169 and the MAGE-3 sequence 271–279. In a patient with a particularly high frequency of MAGE-1 sequence 161–169-specific T cells, phenotypic and functional analysis was performed showing a phenotype of recently-primed CD8 cells (CD28+CD27+CD45RA−CCR7). Conclusions: The observation of a spontaneous in vivo priming of a MAGE-specific T cell response in patients with HCC and the high frequency of MAGE antigens expression in this tumor, makes this antigen a potential candidate for a MAGE-specific immunotherapy in hepatocellular carcinoma.

Mini-review: Regulation of cytotoxic T lymphocyte responses by dendritic cells: peaceful coexistence of cross-priming and direct priming?

Cross-priming is the process whereby professional APC, mainly dendritic cells (DC), prime T cells by presenting antigens processed from proteins of other cells such as tumor cells or virus-infected cells. It has been argued that the importance of cross-priming of CD8(+) CTL responses has been overemphasized, despite strong evidence that these mechanisms operate when infectious organisms, tumors or self antigens cannot efficiently access the biosynthetic MHC class I processing pathway of DC. Since DC are ideally equipped to capture exogenous antigen and also, particularly in the mature state, express costimulatory molecules, it is difficult to distinguish whether effects are due to cross-presentation, costimulation, or both. Whether cross-priming or cross-tolerance occurs depends on the maturation state of the DC, as well as the levels of MHC class I-bound peptides they present. Cross-presentation of tumor-derived antigens has been demonstrated but, importantly, requires adequate APC activation to prime CTL responses. Similarly, cross-presentation of antigens from infectious organisms appears to be common, and frequently leads to cross-priming. Interactions between cross-presenting DC, CD4(+) cells and CD8(+) T cells in the establishment of immunological memory are still not well defined. Experiments utilizing DC depletion are needed to further examine the role of processes such as cross-priming and costimulation in the immune response.

CD4+ CD25+ T cells responding to serologically defined autoantigens suppress antitumor immune responses.

A variety of tumor-derived antigens have been defined by IgG antibodies in tumor bearers' sera with serological identification of antigens by recombinant expression cloning (SEREX), a serological expression cloning method. The majority of these antigens show no structural abnormality and seem to be wild-type autoantigens. Coimmunization with DNA encoding these autoantigens and tumor-specific cytotoxic T lymphocytes epitopes heightened CD8+ T cell responses and increased resistance to tumor challenge in a CD4+ T cell-dependent manner. In contrast, immunization with these SEREX-defined autoantigens alone leads to heightened susceptibility to tumor challenge. This suppressive effect of immunization is mediated by CD4+ CD25+ T cells. In mice immunized with one of the SEREX-defined autoantigens, Dna J-like 2, the number of alpha-GalCer/CD1d tetramer+ CD3+ T cells [representing natural killer T (NKT) cells] was reduced in the pulmonary compartment, whereas no evident change in the number of other T cell subsets was observed. Experiments with Jalpha281-/- mice lacking most NKT cells indicate that NKT cells are primarily responsible for metastasis suppression and that their activity is inhibited by immunization with Dna J-like 2. We propose that SEREX identifies a pool of autoantigens that maintains and regulates immunological homeostasis via CD4+ CD25+ regulatory T cells.

Autologous lung cancer tissues derived gp96-peptide complex/dendritic cell vaccine induces the cytotoxic T cell response in vitro

To investigate whether autologous lung cancer tissues derived gp96-peptide complex/dendritic cell vaccine could induce peptide specific cytotoxic T lymphocyte (CTL) response in vitro. A patient's tumor-derived antigens including gp96-peptide complexes and tumor cell lysate were co cultured with DCs derived from the same patient's bone marrow blood mononuclear cells. The various antigen/DC vaccines were used to stimulate peripheral lymphocytes. Interferon-γ (IFN-γ) level of activated lymphocytes was detected by ELISA method and the Cr51 release test was performed to evaluate the gp96-peptide specific CTL response in three kinds of target cells including the primary cultured tumor cells, PG cells and K562 cells. IFN-γ could be observed from the supernate collected in all antigen groups after the cognate T lymphocytes were stimulated by various vaccines. The concentration of IFN-γ induced by gp96-peptide complexes/DC vaccine was higher than that of other groups. In addition, the killing effect of the activated T lymphocytes on patient's primary tumor cells was higher than that on PG and K562 cells. Autologous tumor-derived gp96-peptide complexes can induce a peptide complex specific CTL response, and the CTL response is significantly intensified after DCs are pulsed.

Getting peptide vaccines to work: just a matter of quality control?

Optimizing peptide vaccine composition Synthetic peptide–based vaccines, which are designed to elicit T cell immunity, are an attractive approach to the prevention or treatment of infectious diseases and malignant disorders (1–4). CTLs recognize antigens as small protein fragments of eight to ten residues (known as peptide epitopes) that associate with MHC molecules, which are expressed on the surface of antigen-presenting cells (APCs) such as infected or malignant cells (5). As the result of the specific interaction between the T cell receptor for antigen (TCR) and the peptide/MHC complexes, the CTLs are able to kill target cells expressing infectious or tumorderived antigens. In consequence, manufacturing these T cell epitopes by conventional organic peptide synthesis methods is a relatively simple, expedient, and cost-effective way to produce vaccines for any type of disease in which CTLs could play a role in the elimination of the offending agent. Unfortunately, the advantages that peptide vaccines have to offer are to some extent diminished by their inherent lack of immunogenicity, which so far has been reflected by their not-sospectacular results in the clinic. Because the immune system in most species has evolved through time to fight life-threatening infectious agents (and perhaps tumors), it should not be surprising that vaccines consisting of aseptic, endotoxin-free peptides are likely to be ignored and will likely be ineffective at inducing T cell immunity. In addition, peptides that are injected in aqueous solutions will be unsuccessful at stimulating CTL responses, either because of their rapid biodegradation (e.g., by proteases) or, worse, because of the induction of T cell tolerance/anergy, which results from the antigenic stimulation of CTLs by nonprofessional APCs (6, 7). A means for enhancing the immunogenicity of synthetic peptide vaccines has been the use of adjuvants, which aim at mimicking the danger signals produced by pathogens, provoking inflammatory reactions that awaken the immune system (8). In addition, some adjuvants also create a depot effect, preventing access of the immunogen to proteases and allowing their slow release into the extracellular compartments. Thus, it is hoped that peptides that are appropriately formulated in adjuvants can elicit CTL responses that in some instances will have positive effects against infections or tumors. An additional complication resulting from the use of synthetic peptide–derived vaccines is the induction of CTLs that, while capable of killing target cells that are exogenously pulsed with peptide, are not able to recognize target cells that naturally process and present the peptide epitope, such as infected or malignant cells. Obviously, these “low-quality” CTLs would have little effect in fighting and controlling disease. One of the reasons for the generation of such lowquality CTLs by peptide vaccines is the induction of CTLs with low affinity for antigen, which will require a high density of specific peptide/MHC complexes on the target-cell surface to exert their effector function. In vitro, the induction of low-affinity CTLs usually results from the use of high concentrations of peptide, generating a high level of specific peptide/MHC complexes on APCs, which will effectively activate these CTLs (9). The prediction is that high densities of peptide/MHC complexes on APCs in vivo resulting from an excessive peptide dose will also produce low-quality CTLs with low affinity for antigen. Another cause for the induction of low-quality CTLs is the use of vaccines produced with synthetic peptides representing cryptic T cell epitopes, which are not expressed on the surface of the infected or tumor target cells (10). The peptides corresponding to cryptic epitopes are immunogenic, because they bind well to MHC molecules on APCs and are able to activate T cells. However, because the same peptide/MHC complexes are not generated through the natural antigen-processing machinery in the infected or tumor cells, CTLs recognizing cryptic epitopes will be unable to interact with infected or tumor target cells and will also be useless in disease control.