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In our previous studies we have shown that bacterial enterotoxin B subunits are effective vehicles to deliver antigen into the MHC class I processing route. Here we have used the non-toxic Escherichia coli heat labile enterotoxin B subunit (EtxB) conjugated to OVA peptide (EtxB-peptide) to address the impact on induction of specific CD8(+) T cells in vivo. Although incubation of DCs with these EtxB-peptide conjugates as such did not induce DC maturation in vitro MHC class I antigen presentation was much more efficient as compared to peptide alone. Antigen presentation was further enhanced upon DC maturation with the TLR-4 ligand LPS. Injection of matured DCs incubated with EtxB-peptide conjugates lead to strong induction of OVA-specific CD8(+) T lymphocytes and fully prevented the outgrowth of lethal B16 melanoma in wild type mice. Our data demonstrate that bacterial non-toxic B subunit-peptide conjugates are potent vaccine vehicles for induction of protective CD8(+) T cell responses.

Covalent conjugation of synthetic Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides provides well-defined constructs that have significantly improved capacity to induce efficient priming of CD8(+) T lymphocytes in vivo. We have recently explored the cellular mechanisms underlying the efficient induction of a CD8(+) cytotoxic T lymphocyte response by such synthetic model vaccines [Khan, S., Bijker, M.S., Weterings, J.J., Tanke, H.J., Adema, G.J., van, H.T., Drijfhout, J.W., Melief, C.J., Overkleeft, H.S., van der Marel, G.A., Filippov, D.V., van der Burg, S.H., Ossendorp, F., 2007. Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells. J. Biol. Chem. 282, 21145-21159.]. In the current study we have investigated the behaviour of two diastereomers of the TLR-2 ligand Pam(3)CSK(4) (Pam) derivatives, namely the R- and S-epimers at C-2 of the glycerol moiety. Other studies have shown that the Pam(3)Cys based lipopeptides of R-configuration (Pam(R)) in the glycerol moiety enhanced macrophage and B-cell activation compared to those with S-configuration (Pam(S)). Here we report that Pam(R)-conjugates lead to better activation of dendritic cells than the Pam(S)-conjugates as judged by higher IL-12 secretion, upregulation of relevant markers for dendritic cell maturation. In contrast both epimers were internalized equally efficient in a clathrin-dependent manner indicating no qualitative difference in the uptake of the two stereoisomeric Pam-conjugates. We conclude that the enhanced DC activation is due to enhanced TLR-2 triggering by the Pam(R)-conjugate in contrast to the Pam(S)-conjugate. Importantly, induction of specific CD8(+) T-cells was significantly higher in mice injected with the Pam(R)-conjugates compared to mice injected with the Pam(S)-conjugate. In summary we show that the favourable effects of the Pam(R)-configuration of TLR-2 ligand can be attributed to direct effects on dendritic cells resulting in enhancement of CD8(+) T-cell responses.

Cancerous lesions promote tumor growth, motility, invasion, and angiogenesis via oncogene-driven immunosuppressive leukocyte infiltrates, mainly myeloid-derived suppressor cells, tumor-associated macrophages, and immature dendritic cells (DCs). In addition, many tumors express or induce immunosuppressive cytokines such as TGF-beta and IL-10. As a result, tumor-antigen crosspresentation by DCs induces T cell anergy or deletion and regulatory T cells instead of antitumor immunity. Tumoricidal effector cells can be generated after vigorous DC activation by Toll-like receptor ligands or CD40 agonists. However, no single immunotherapeutic modality is effective in established cancer. Rather, chemotherapies, causing DC activation, enhanced crosspresentation, lymphodepletion, and reduction of immunosuppressive leukocytes, act synergistically with vaccines or adoptive T cell transfer. Here, I discuss the considerations for generating promising therapeutic antitumor vaccines that use DCs.

A systematic comparison of the immunostimulatory capacity of TLR 2, 3, 4, 5, 7 and 9 agonists and an agonistic CD40-specific antibody was performed in a single long peptide vaccination model. All adjuvants activated DC in vitro but not all induced a strong functional T-cell response in vivo. Optimal clonal CD8(+) T-cell expansion depended on the capacity of agonists to mature pro-inflammatory DC and the duration of their in vivo stimulatory effect. Strong agonists promoted the induction of both antigen-specific IFNgamma-producing CD4(+) T-helper cells and high numbers of IFNgamma producing CD8(+) effector T-cells that killed target cells in vivo. Importantly, the capacity of an agonist to function as an adjuvant depended on the vaccine strategy used. Collectively, the multi-parameter system presented here can be used as a general road map to develop therapeutic vaccines.

The full-length E7 oncoprotein of HPV16 was prepared from two peptide fragments using native chemical ligation and purified by a single gel-filtration step under denaturing conditions.

Soon after it was realized that T-cells recognize their target antigens as small protein fragments or peptides presented by MHC molecules at the cell surface, these peptide epitopes have been tried as vaccines. Human testing of such vaccines, although protective in mouse models, has produced mixed results. Since these initial trials, there has been an tremendous increase in our understanding of how infectious organisms can induce potent immune responses. In this article we review the key changes in the design, formulation and delivery of synthetic peptide vaccines that are applied to improve peptide vaccine strategies.

We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membranebound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian pop ulation. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptidebinding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.

Efficacious vaccines against cancers and infectious diseases will, in general, need to elicit comprehensive immune responses, including cytotoxic T lymphocyte activity. Because of their unique T cell stimulatory capacities, dendritic cells (DC) have emerged as the most potent antigen-presenting cell. Vaccination strategies should therefore aim at the acquisition and display of the antigen(s) of choice by DC. Results from vaccination studies, in animal models and in humans, stress the need for optimized antigen delivery systems to DC, to increase vaccination efficacy as well as to improve control on the immunological outcome. Here, we discuss the advantages and limitations of several recently described methodologies for antigen delivery into DC.

The synthesis of an array of 2-azidoalkoxy substituted 7-hydro-8-oxoadenines is described. The relation of the structure of these compounds and their ability to induce maturation of dendritic cells is evaluated.

A single injection of HPV16 L1 virus-like particles induced potent CD8-mediated protection from tumor challenge by C3 cells, a line derived from embryonic mouse cells transfected with the HPV16 genome. L1 RNA, but not protein, was detected biochemically in C3 cells. These results indicate that low-level expression of HPV16 L1 can occur in proliferating cells and serve as a tumor vaccine target. Although L1 expression is generally thought to be restricted to terminally differentiated epithelial cells, these results suggest that additional analysis for low-level L1 expression in proliferating cells of HPV-induced lesions is warranted and might help in predicting the clinical potential of HPV L1 virus-like particle-based vaccines.

T cell-mediated immunity plays an important role in the defense against human papillomavirus (HPV) associated cervical cancer as evident from data of the etiology of the virus, several animal models and clinical data. This review addresses the most recent observations in this field which have led to the first clinical trials that are aimed at an immunotherapeutic intervention in this disease via induction of HPV specific T-cell responses.

Functional polymorphism in the rat peptide transporter associated with antigen processing (TAP) changes the peptide pool available for binding and presentation by a class I MHC allele, RT1.Aa. The peptide binding motif for RT1.Aa, determined by stabilization with synthetic peptides, included a strong preference for arginine at the peptide C terminus. Analysis of natural peptides bound to RT1.Aa by both pool sequencing and anhydrotrypsin chromatography revealed that TAP polymorphism determined the presence or absence of arginine as the peptide C-terminal residue. This result highlights the in vivo impact of TAP–peptide selectivity, and provides evidence against a high rate of generation of new C termini by protease activity in the endoplasmic reticulum.

Extensive sets of in total about 2000 synthetic peptides were investigated for their binding affinities to HLA-A ∗ 0201. Comparisons of the amino acid compositions of binding to nonbinding sets of peptides provided new information concerning the rules for 9-, 10-, and 11-mer peptide binding at the amino acid level. Preferred primary anchors were shown to depend on peptide length, longer peptides being more demanding in this respect. A clear preference exists for certain amino acids at several nonanchor positions. In addition, the presence of particular amino acids at those positions almost completely precludes peptide binding. We found no evidence for preferred anchor pairs. From these results new and detailed HLA-A ∗ 0201 peptide-binding motifs for 9-, 10-, and 11-mer peptide binding were deduced. The motifs are in accordance with earlier reports but include new findings, including C as a C-terminal anchor, the importance of D at position 4 for binding, and the deleterious effect of R at position 5 (in 9-mers). The motifs are presented in such a way that they can be used to predict peptide binding to HLA-A ∗ 0201 by computer analysis (see accompanying paper [56]). Human Immunology 43, 1–12 (1995)

Vaccination with peptides recognized by antigen-specific CTLs can prevent lethal virus infections and tumor growth. In order to avoid the synthesis and testing of the numerous overlapping peptide of long AA sequences of proteins of interest, we developed a computer program which utilizes the rules, "motifs" which govern how peptides bind to HLA class I molecules, to derive a predicted binding score for each overlapping peptide. Correlations between the predicted and actual binding results to HLA-A*0201 for 100 peptides selected from six early and two late protein sequences of human papillomavirus type 1a revealed an acceptable level (61%) of concordance. The program is very flexible with regard to the input of protein sequences and motif definitions and is able to handle various motif and peptide lengths.

Cell lineage-specific cellular proteins, oncogenes from viral or cellular origin and tumor suppressor genes encode tumor-specific/associated antigens. Such antigens can elicit an major compatibility complex (MHC) class I-restricted cytotoxic T lymphocyte (CTL) response, either naturally in cancer patients or following appropriate immunostimulation (in vitro or in vivo). The reported immune responses in humans to the melanoma-associated MAGE gene products, GP100 and tyrosinase, all self-proteins, support the idea to use wild-type p53 products as targets for T cells. An important step towards this goal is identification of potential p53 CTL epitopes. We identified the wild-type p53 peptides with the highest affinity to the HLA-A∗0201 molecule using two assays: the previously described MHC peptide-binding assay and the peptide competition assay. We obtained CTL against four p53 peptides with a high affinity for the HLA-A∗0201 molecule. These findings are discussed next to a short review concerning the p53 literature.

The immunogenicity of viral oncoproteins has been established beyond doubt. Cytotoxic T lymphocytes directed against viral oncogene products can eradicate large established tumor masses. This stage has not yet been reached for cellular oncogene and tumor suppressor gene products, but T cells have been raised against MHC-binding peptides encoded by both mutant and wild-type alleles of the ras oncogene and the p53 tumor suppressor gene. In addition, T cells specific for joining region peptides of abnormal fusion proteins resulting from chromosome translocation in tumor cells have been generated. Some of these peptides are processed in cells infected with, for example, vaccinia-ras, but direct anti-tumor effects of peptide specific T lymphocytes remain to be demonstrated.

We have developed an in vitro immunization system for the production of B-cell lines that secrete HLA-specific human mAbs. For this purpose, peripheral blood lymphocytes of parous women were stimulated with pools of allogeneic lymphocytes. Preferential outgrowth of B-lymphocytes was effected by inclusion of rIL-2 and a B-cell specific nucleoside analogue. Stimulated B cells were immortalized by EBV transformation, and specific antibody-producing transformants were fused to heteromyeloma or mouse myeloma cell lines, yielding stable hybridomas. This approach has led to the successful development of two human heterohybridomas producing HLA-specific mAbs reactive by complement-mediated cytotoxicity. The specificities of these human mAbs, reactive with HLA-B12(44 + 45) and HLA-B60, respectively, are fully concordant with those of HLA-typing sera.

The in vivo efficacy of virus- and tumor-specific cytotoxic T lymphocytes (CTL) is discussed as well as ways to activate these cells in vivo with lymphokines, monoclonal antibodies and immunization. In vivo and in vitro peptide immunization can induce such virus- and tumor-specific CTL but several questions have to be answered before peptide vaccination can be implemented as a novel immunotherapeutic or preventive approach in man.

A 49-year-old male with an 8 year history of lowered Hb level, granulocytopenia and fatigue presented in 1986 with progressive fatigue, a dramatically reduced Hb level (45 g/l) and an increased lymphocyte count (6.6 x 10(9)/l). Clinical picture and laboratory studies led to the diagnosis of chronic T lymphocytosis with expansion of CD8+ T cells expressing CD16 IgG Fc receptors (Fc gamma RIII). DNA analyzed with T-cell receptor (TcR) gamma and beta probes revealed extra rearranged bands representing a clonal expansion of T lymphocytes. These T lymphocytes expressed T-cell receptor alpha beta as evaluated by staining with monoclonal antibodies. Because of the severe progressive anemia the patient was transfused with packed red cells. He was then treated with cyclophosphamide. After one month of treatment the transfusions could be discontinued and two months later cyclophosphamide treatment was stopped because of normalized Hb level and lymphocyte counts. The patient remained in a hematologically stable condition, though a minor T-cell population representing the clonal expansion, an inverted CD4/CD8 ratio and low immunoglobulin levels persisted. This is the first report of regression of proven monoclonal CD8+ T gamma-cell expansion and the associated anemia following cyclophosphamide therapy. These observations implicate the expanded monoclonal CD8+ lymphocytes in the pathogenesis of the anemia and granulocytopenia.

One way to solve the problem of human donor organ shortage is the use of animal organs. Therefore, it is important to study the T-cell response against xenogeneic major histocompatibility complex (MHC) antigens. In the present study, we have used HLA-B27 transgenic mice in a xenogeneic transplantation model. The results indicate that both transgenic skin transplantation and intravenous (IV) injection of transgenic spleen cells can reverse specific T-cell low responsiveness against the transgenic HLA-B27 antigen into high responsiveness in vivo and in vitro. In contrast, IV injection of spleen cells across an allogeneic H-2 class I disparity results in transplantation tolerance. Thus, despite T-cell low responsiveness against the transgenic HLA-B27 antigen, IV injection of transgenic HLA-B27 disparate lymphocytes does not tolerize, but rather immunizes for the xeno-MHC antigen.

Cytotoxic T lymphocyte (CTL) clones against adenovirus type 5 (Ad5) early region 1 (E1)-transformed cells were generated in C57BL/6 (B6) mice. A defined peptide encoded by Ad5 E1A is the target structure for H-2Db-restricted CTLs. Upon intravenous injection into B6 nude mice bearing Ad5 E1-induced tumors, these CTLs, if combined with recombinant IL-2, destroy subcutaneous tumor masses up to 10 cm3. The in vivo action of CTLs is highly specific, and long-term "memory" persists in treated nude mice months after tumor regression. Our data show an important role for CTLs directed against a viral nuclear oncogene product in tumor eradication.

The antibody response against the envelope proteins of a variety of cloned highly and poorly oncogenic dualtropic mink cell focus-inducing (MCF) murine leukemia viruses (MuLV) was studied and compared with the antibody response against ecotropic isolates. MCF viruses evoke stronger antibody responses than ecotropic MuLV isolates. Although these MCF viruses are highly polymorphic with respect to their gp70 and p15(E) envelope proteins, generally a similar H-2-linked immune response gene control of anti-viral antibody responses is observed. Neonatally infected BALB/c (H-2d) and C57BL/10 (B10,H-2b) mice are high responders and B10.A (H-2a) mice, congenic at the major histocompatibility complex (H-2) with B10, low responders. No correlation was found between the expression of any single gp70 or p15(E) epitope of the infecting MCF virus and the magnitude of the antibody response. This indicates that the level of the H-2 regulated anti-MuLV envelope antibody response is most likely determined by a MuLV antigen shared by all MuLV isolates examined. The magnitude of the antibody response against highly oncogenic and against poorly oncogenic MCF virus does not differ significantly. The combined data indicate that the intrinsic oncogenic potency encoded by the viral genome is a more important feature of oncogenesis than the level of antiviral envelope antibody response evoked by the MCF virus. However, the oncogenic properties of a single murine leukemia virus may vary among H-2 congenic mice, correlated with their H-2-dependent capacity to produce antiviral antibodies.

In antigen-specific cytotoxic T-lymphocyte (CTL) responses H-2 class I mutations usually result in a decreased recognition of the antigen in association with the mutant molecule by CTL from the strain of origin. However, the influence of class I mutations on the magnitude and specificity of CTL responses in the mutants has been studied in only a few instances, in which usually a partial or complete loss of responsiveness was found. We now report that class I mutants extensively use gained (novel) CTL restriction sites, generated by the mutations in the CTL response against the hapten trinitrophenyl (TNP), demonstrated both at the population level and in limiting dilution. TNP-specific CTL clones, restricted by mutant-specific determinants, were detected in all mutants. The percentages mutant-specific CTL clones in limiting dilution experiments were 43, 40, 35, and 13 in the K b mutants bm1, bm8, bm3 and bm5, respectively, and 35 in the D b mutant bm14. It is concluded that H-2 class I mutations led to changes in the TNP-specific CTL repertoire resulting in gain of CTLs uniquely restricted to the mutant molecule.

A review is presented of functional studies performed with the expanded T cells in Sézary syndrome, T-cell chronic lymphocytic leukemia (T-CLL), T-prolymphocytic leukemia (T-PLL), adult T-cell leukemia lymphoma (ATLL), T-cell acute lymphoblastic leukemia (T-ALL) and T gamma lymphocytosis. The immunological and clinical relevance of immunological studies of functional properties of T-cell expansions is discussed.

The prevalence of different host-range classes of murine leukemia virus (MuLV) was studied in C57BL mice with (V+) and without (V-) milk transmission of a naturally occurring B-tropic ecotropic MuLV. Virus isolates were studied with respect to growth properties, XC-plaque formation, antigen profiles of their envelope proteins (gp70 and P15(E)), gp70 tryptic-peptide maps, and their potential to induce lymphomas after inoculation into newborn mice. B-tropic ecotropic MuLV with the capacity to cause plaques in XC cells was isolated from almost all lymphomas of both V+ and V- sublines. The reaction patterns of these ecotropic isolates with monoclonal antibodies reactive with MuLV-env proteins and the tryptic-peptide maps of the gp70 molecule indicate that they are similar to each other and differ only slightly from the ecotropic MuLV in the spleens of young V+ animals, which is identical to the milk-transmitted virus. XC − , B-tropic dualtropic mink cell focus-inducing (MCF) viruses were isolated from the majority of different types of lymphoma (B cell, T cell, or neither B nor T cell derived), but not from the spleens or milk of young V+ or V- animals. The env proteins of the MCF isolates are highly heterogeneous, but most isolates originating from B10.AV+ T-cell lymphomas share MCF-related epitopes in their gp85 envelope precursor with AKR MCF-247 virus. Most MCF viruses isolated from non-T lymphomas do not possess these epitopes. The results indicate that also in this model the generation of dualtropic MCF viruses might be important in lymphoma induction, although only some of the cloned MCF viruses show enhanced oncogenic properties in comparison with ecotropic isolates. A cloned oncogenic MCF virus induced different lymphoma types in C57BL/10 (=B10, H-2 b ) and B10.A (H-2 a ) mice, similar to what was found earlier with the milk-transmitted virus. Hence, the lymphoma-type differences are not due to differences in the B-tropic ecotropic viruses transmitted through the milk in these strains, but reflect an influence of the H-2 complex on the phenotype of the virus-induced lymphomas.