Biomedical subjects

R Offringa

Publications and source records attributed to R Offringa.

At least 19 recordsLinked to original sources

PubMed2006

BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes.

For immunotherapy of residual disease in patients with Philadelphia-positive leukemias, the BCR-ABL fusion regions are attractive disease-specific T-cell targets. We analyzed these regions for the prevalence of cytotoxic T lymphocyte (CTL) epitopes by an advanced reverse immunology procedure. Seventeen novel BCR-ABL fusion peptides were identified to bind efficiently to the human lymphocyte antigen (HLA)-A68, HLA-B51, HLA-B61 or HLA-Cw4 HLA class I molecules. Comprehensive enzymatic digestion analysis showed that 10 out of the 28 HLA class I binding fusion peptides were efficiently excised after their C-terminus by the proteasome, which is an essential requirement for efficient cell surface expression. Therefore, these peptides are prime vaccine candidates. The other peptides either completely lacked C-terminal liberation or were only inefficiently excised by the proteasome, rendering them inappropriate or less suitable for inclusion in a vaccine. CTL raised against the properly processed HLA-B61 epitope AEALQRPVA from the BCR-ABL e1a2 fusion region, expressed in acute lymphoblastic leukemia (ALL), specifically recognized ALL tumor cells, proving cell surface presentation of this epitope, its applicability for immunotherapy and underlining the accuracy of our epitope identification strategy. Our study provides a reliable basis for the selection of optimal peptides to be included in immunotherapeutic BCR-ABL vaccines against leukemia.

Amino Acid Sequence↗

PubMed2006

Improved peptide vaccine strategies, creating synthetic artificial infections to maximize immune efficacy.

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.

Animals↗

PubMed2004

Dendritic cells in colorectal cancer correlate with other tumor-infiltrating immune cells.

Dendritic cells (DCs) are the most efficient antigen-presenting cells and play a key role in a cellular antitumor immune response. In this study we investigated the exact localization of DCs within colorectal tumors and their relationship to tumor-infiltrating lymphocytes as well as clinical outcome of the patients. Primary tumor specimens of 104 patients with a diagnosis of colorectal cancer were identified retrospectively and analyzed with the dendritic cell markers S-100 protein and human leukocyte antigens (HLA) class II. The markers were individually combined with laminin as a second marker to facilitate the observation of the different tumor localizations. S-100 or HLA class II positive cells were found in the three different compartments of colorectal tumors: tumor epithelium, tumor stroma, and advancing tumor margin, but mainly present in tumor stroma and advancing tumor margin. S-100-positive tumor-infiltrating DCs in direct contact with tumor cells, i.e., in tumor epithelium, significantly correlated to the intraepithelial infiltration of CD4+ (p=0.02) and CD8+ (p=0.01) lymphocytes. High HLA class II+ cell infiltration in the tumor stroma correlated to a lower intraepithelial infiltration of CD8+ (p=0.02) lymphocytes. High intraepithelial infiltration of S-100-positive DCs suggested increased disease-free survival, but was not statistically significant, while high amounts of HLA class II+ cells in the tumor stroma correlated with an adverse survival outcome. Our results show that the infiltration of DCs in colorectal cancer, depending on both location and type of marker, is correlated with local immune interactions and patient prognosis, suggesting a central role for DCs in controlling local tumor immunity.

Adult↗

PubMed2003

Interplay between human papillomaviruses and dendritic cells.

The design of the human papillomavirus (HPV) infection cycle is tightly fitted to the differentiation program of its natural host, the keratinocyte. This has important consequences for the role of antigen-presenting cells in the priming of antiviral immunity. The confinement of HPV infection to epithelia puts the epithelial dendritic cell, the Langerhans cell (LC), in charge of the induction of T cell-dependent immunity. Because HPV-infected keratinocytes cannot reach the regional lymphoid organs, and HPV-infection of LCs does not result in viral gene expression, priming of antiviral T cells exclusively depends on cross-presentation of viral antigens by the LC. Sensitization of the immune system in the regional lymphoid organs elicits systemic anti-HPV immunity as well as intraepithelial immune surveillance by memory-type intraepithelial T cells and locally produced antibodies. The high rate of spontaneous rejections of high-risk HPV-infections and HPV-positive premalignant lesions indicates that in general the LC-driven antigen presentation machinery is capable of raising an effective immune defense against HPV. Epidemiological studies also reveal that a decrease in the vigilance of the immune system is readily exploited by HPV to escape immune destruction, resulting in persistent infections and development of HPV-positive cancers. In view of the inherent antigenicity of HPV, immune intervention strategies constitute a promising approach for both the prevention and the therapeutic treatment of HPV-induced diseases. Importantly, the mechanisms that govern the induction and effector phases of the intraepithelial immune surveillance against HPV must be taken into account when designing such strategies.

Antigen-Presenting Cells↗

PubMed2003

Creation of immune 'stealth' genes for gene therapy through fusion with the Gly-Ala repeat of EBNA-1.

A major obstacle in gene-therapy protocols is T-cell-mediated destruction of transgene-expressing cells. Therefore new approaches are needed to prevent rapid clearance of transduced cells. We exploited the Gly-Ala repeat (GAr) domain of the Epstein-Barr virus nuclear antigen-1, since the GAr prevents cytotoxic T-lymphocyte-epitope generation. Here we show that three different enzymes (viz. the E. coli LacZ gene encoded beta-galactosidase, firefly luciferase, and HSV1 thymidine kinase) fused with the GAr retained their function. Moreover, linking GAr with beta-galactosidase successfully prevented recognition of GAr-LacZ-expressing cells by beta-galactosidase-specific CTL. Nonetheless, vaccination with a GAr-LacZ adenovirus or with an allogeneic cell line expressing GAr-LacZ resulted in the induction of beta-gal-specific CTL. This demonstrates that the GAr domain does not inhibit cross presentation of antigens, but only affects breakdown of endogenously synthesized proteins. These data demonstrate how the GAr domain can be exploited to create immuno'stealth' genes by hiding transgene products from CTL-mediated immune attack.

Adenoviridae↗

PubMed2002

Enhancement of human papillomavirus (HPV) type 16 E6 and E7-specific T-cell immunity in healthy volunteers through vaccination with TA-CIN, an HPV16 L2E7E6 fusion protein vaccine.

TA-CIN is a vaccine that comprises the human papillomavirus (HPV) type 16 L2, E6 and E7 as a single fusion protein. In a mouse model, TA-CIN effectively prevented outgrowth of HPV16-positive tumour cells. To assess the safety and immunogenicity of TA-CIN, a dose escalating (26, 128, 533 micro g), double blind and placebo-controlled phase I study was conducted in 40 healthy volunteers. TA-CIN was administered without adjuvant by intramuscular injection on weeks 0, 4 and 8. No serious adverse events of the vaccination were reported during the study. Both IgG antibodies and proliferative responses against TA-CIN were elicited at all three doses. More importantly, T-cell immunity against the HPV16 E6 and E7 oncoproteins was detected by IFN gamma ELISPOT in 8/11 evaluable subjects vaccinated with the 533 micro g dose.

Adult↗

PubMed2001

Seed development: Early paternal gene activity in Arabidopsis.

Both parental genomes are expressed during embryogenesis, although the time of activation of the paternally inherited genes varies between organisms. Results reported by Vielle-Calzada et al. have suggested that delayed activation of the paternal genome seems to be the rule in plant development. We find, however, that during early embryogenesis in Arabidopsis, paternal genes are expressed and are sufficient for normal development. Our findings indicate that there is no overall maternal control of early embryogenesis, and that the contribution of the parental alleles needs to be assessed for each gene individually.

Arabidopsis↗

PubMed2001

Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9/SPI-6 constitutes a mechanism for immune escape by tumors.

The concept for cellular immunotherapy of solid tumors relies heavily on the capacity of class I MHC-restricted cytotoxic T lymphocytes (CTLs) to eliminate tumor cells. However, tumors often have managed to escape from the cytolytic machinery of these effector cells. Therefore, it is very important to chart the mechanisms through which this escape can occur. Target-cell killing by CTLs involves the induction of apoptosis by two major mechanisms: through death receptors and the perforin/granzyme B (GrB) pathway. Whereas tumors previously were shown to exhibit mechanisms for blocking the death receptor pathway, we now demonstrate that they also can resist CTL-mediated killing through interference with the perforin/GrB pathway. This escape mechanism involves expression of the serine protease inhibitor PI-9/SPI-6, which inactivates the apoptotic effector molecule GrB. Expression of PI-9 was observed in a variety of human and murine tumors. Moreover, we show that, indeed, expression results in the resistance of tumor cells to CTL-mediated killing both in vitro and in vivo. Our data reveal that PI-9/SPI-6 is an important parameter determining the success of T cell-based immunotherapeutic modalities against cancer.

Animals↗

PubMed2001

Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25(+) regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses.

Therapeutic efficacy of a tumor cell-based vaccine against experimental B16 melanoma requires the disruption of either of two immunoregulatory mechanisms that control autoreactive T cell responses: the cytotoxic T lymphocyte-associated antigen (CTLA)-4 pathway or the CD25(+) regulatory T (Treg) cells. Combination of CTLA-4 blockade and depletion of CD25(+) Treg cells results in maximal tumor rejection. Efficacy of the antitumor therapy correlates with the extent of autoimmune skin depigmentation as well as with the frequency of tyrosinase-related protein 2(180-188)-specific CTLs detected in the periphery. Furthermore, tumor rejection is dependent on the CD8(+) T cell subset. Our data demonstrate that the CTL response against melanoma antigens is an important component of the therapeutic antitumor response and that the reactivity of these CTLs can be augmented through interference with immunoregulatory mechanisms. The synergism in the effects of CTLA-4 blockade and depletion of CD25(+) Treg cells indicates that CD25(+) Treg cells and CTLA-4 signaling represent two alternative pathways for suppression of autoreactive T cell immunity. Simultaneous intervention with both regulatory mechanisms is therefore a promising concept for the induction of therapeutic antitumor immunity.

Abatacept↗

PubMed2001

Expression of the serpin serine protease inhibitor 6 protects dendritic cells from cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1 and type 2 cells.

Dendritic cells (DCs) play a central role in the immune system as they drive activation of T lymphocytes by cognate interactions. However, as DCs express high levels of major histocompatibility complex class I, this intimate contact may also result in elimination of DCs by activated cytotoxic T lymphocytes (CTLs) and thereby limit induction of immunity. We show here that immature DCs are indeed susceptible to CTL-induced killing, but become resistant upon maturation with anti-CD40 or lipopolysaccharide. Protection is achieved by expression of serine protease inhibitor (SPI)-6, a member of the serpin family that specifically inactivates granzyme B and thereby blocks CTL-induced apoptosis. Anti-CD40 and LPS-induced SPI-6 expression is sustained for long periods of time, suggesting a role for SPI-6 in the longevity of DCs. Importantly, T helper 1 cells, which mature DCs and boost CTL immunity, induce SPI-6 expression and subsequent DC resistance. In contrast, T helper 2 cells neither induce SPI-6 nor convey protection, despite the fact that they trigger DC maturation with comparable efficiency. Our data identify SPI-6 as a novel marker for DC function, which protects DCs against CTL-induced apoptosis.

Animals↗

PubMed2001

Longevity of antigen presentation and activation status of APC are decisive factors in the balance between CTL immunity versus tolerance.

Encounter of Ag by naive T cells can lead to T cell priming as well as tolerance. The balance between immunity and tolerance is controlled by the conditions of Ag encounter and the activation status of the APC. We have investigated the rules that govern this balance in case an environment that normally induces tolerance is reverted into a milieu that promotes T cell priming, using a minimal CTL epitope derived from human adenovirus type 5 E1A. Vaccination of mice s.c. with E1A peptide in IFA readily induces CTL tolerance, resulting in the inability to control E1A-expressing tumors. The present study shows that efficient CTL priming is achieved when this peptide vaccine is combined with systemic administration of APC-activating compounds like agonistic anti-CD40 mAb or polyriboinosinate-polyribocytidylate. Surprisingly, this CTL response is not long-lasting and therefore fails to protect against tumor outgrowth. Disappearance of CTL reactivity was strongly associated with systemic persistence of the peptide for >200 days. In contrast, peptide administered in PBS does not persist and generates long term CTL immunity capable of rejecting Ad5E1A-positive tumors, when combined with CD40 triggering. Thus, presentation of CTL epitopes in an appropriate costimulatory setting by activated APC, although being essential and sufficient for CTL priming, eventually results in tolerance when the Ag persists systemically for prolonged times. These observations are important for the development of immune intervention schemes in autoimmunity and cancer.

Adenovirus E1A Proteins↗

PubMed2001

Elucidating the autoimmune and antitumor effector mechanisms of a treatment based on cytotoxic T lymphocyte antigen-4 blockade in combination with a B16 melanoma vaccine: comparison of prophylaxis and therapy.

We have previously shown that small B16 melanomas can be successfully treated using a combination of anti-cytotoxic T lymphocyte antigen (CTLA)-4 monoclonal antibody with a granulocyte/macrophage colony-stimulating factor (GM-CSF) producing irradiated tumor cell vaccine. Regression of tumors results in long-lasting immunity and is frequently accompanied by autoimmune depigmentation. Here we examine the cellular and molecular mechanisms of this combined treatment. Histological examination of depigmented lesions revealed infiltration of polymorphonuclear cells and deposition of antibody. The combination therapy also induced tumor rejection and skin depigmentation in B cell-deficient and in CD4(+) T cell-depleted mice. Both effects of the treatment absolutely required CD8(+) T cells. Analysis of the response in successfully treated mice revealed elevated levels of CD8(+) T cells specific for a nonameric peptide consisting of residues 180-188 of the melanocyte differentiation antigen tyrosinase-related protein (TRP)2. There was no evidence of reactivity to the melanocyte antigens gp100, tyrosinase, Mart1/MelanA, or TRP1. Fas-FasL interactions and perforin played a role in mounting the effector response, whereas the tumor necrosis factor pathway was not required. The cellular requirements for tumor rejection in this therapeutic setting were strikingly different from those in a prophylactic setting. In particular, if mice received a prophylactic vaccine consisting of anti-CTLA-4 and B16-GM-CSF before tumor challenge, full protection was obtained even in the absence of CD8(+) T cells. Our data demonstrate that therapeutic autoreactive CD8(+) T cell responses can effectively be generated in tumor-bearing mice and stresses the value of studying tumor immunity in a therapeutic rather than a prophylactic setting.

Abatacept↗

PubMed2001

Pre-clinical safety and efficacy of TA-CIN, a recombinant HPV16 L2E6E7 fusion protein vaccine, in homologous and heterologous prime-boost regimens.

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical intraepithelial neoplasia (CIN) and cancer. A newly designed vaccine, comprising the HPV16 L2, E6 and E7 as a single fusion protein (TA-CIN), was shown to elicit HPV16-specific CTL, T-helper cells and antibodies in a pre-clinical mouse model. These immune responses effectively prevented outgrowth of HPV16-positive tumour cells in a prophylactic setting as well as in a minimal residual disease setting. CTL immunity was optimally induced when TA-CIN was employed in heterologous prime-boost regimens in combination with TA-HPV, a clinical grade vaccinia-based vaccine. These data provide a scientific basis for the use of TA-CIN, alone or in combination with TA-HPV in future human trials.

Animals↗

PubMed2001

Highly efficient transduction of human monocyte-derived dendritic cells with subgroup B fiber-modified adenovirus vectors enhances transgene-encoded antigen presentation to cytotoxic T cells.

The efficiency of dendritic cells (DC) as immunotherapeutic vaccines critically depends on optimal delivery of target Ags. Although DC modified by subgroup C type 5 recombinant adenoviruses (rAd5) provide encouraging results, their clinical application is hampered by the need for high viral titers to achieve sufficient gene transfer, due to the lack of the Ad5 fiber receptor. We now demonstrate that rAd5 carrying subgroup B Ad fibers are up to 100-fold more potent than classical rAd5 for gene transfer and expression in human DC, rAd5 with a type 35 fiber (rAd5F35) being the most efficient vector. This improvement relates to a greater and faster virus entry and to an increased transgene expression especially following DC maturation. Furthermore, these new vectors possess enhanced synergistic effects with other activation signals to trigger DC maturation. Consequently, rAd5F35-infected DC engineered to express the gp100 melanoma-associated Ag largely exceed rAd5-infected DC in activating gp100-specific CTL. Finally, the DC infection pattern of rAd5F35 is fully conserved when DC are in the vicinity of primary skin-derived fibroblasts, suggesting this vector as a candidate for in vivo targeting of DC. Thus, subgroup B fiber-modified rAd5 constitute a major breakthrough in the exploitation of ex vivo rAd-targeted DC as clinically relevant vaccines and may also be suitable for in vivo genetic modification of DC.

Adenoviridae↗

PubMed2001

Natural T-helper immunity against human papillomavirus type 16 (HPV16) E7-derived peptide epitopes in patients with HPV16-positive cervical lesions: identification of 3 human leukocyte antigen class II-restricted epitopes.

Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine-induced immune defense against tumors. Since the majority of cervical cancers express human papillomavirus type 16 (HPV16) E7 oncoprotein, it is important to investigate the Th response against this target antigen in detail. By means of PBMC cultures from HLA-typed healthy donors, we identified the central part of HPV16 E7 (E7(41-72)) as the major immunogenic region within this antigen. Furthermore, we mapped 3 distinct Th epitopes within this region (DR15/E7(50-62), DR3/E7(43-77), DQ2/E7(35-50)). In a parallel approach, employing IFN-gamma ELISPOT analysis, we detected Th immunity against HPV16 E7 in subjects with HPV16+ lesions. Several of these responses matched with the 3 Th epitopes defined in our study. A number of other HPV16+ subjects did not display any E7-specific type 1 cytokine-producing T-cell immunity, indicating failure of the immune response. Our combined data argue for more extensive as well as longitudinal analysis of HPV16-specific T-cell immunity using the ELISPOT assay described, as well as for HPV-specific vaccination of individuals with HPV+ lesions.

Cancer Vaccines↗

PubMed2001

Identification of three non-VNTR MUC1-derived HLA-A*0201-restricted T-cell epitopes that induce protective anti-tumor immunity in HLA-A2/K(b)-transgenic mice.

The human epithelial mucin MUC1 is over-expressed in more than 90% of carcinomas of the breast, ovary, and pancreas as well as in some other tumours, making it a potential target for tumour immunotherapy. We have identified several MUC1-derived peptides mapping outside the variable number tandem repeat region that comply with the peptide-binding motif for HLA-A*0201 and that become processed into stable major histocompatibility complex-peptide complexes as assessed by in vitro assays. In A2/K(b) transgenic mice, 3 peptides, namely MUC(79-87) (TLAPATEPA), MUC(167-175) (ALGSTAPPV) and MUC(264-272) (FLSFHISNL) elicit peptide-specific cytotoxic T lymphocyte (CTL) immunity, which protects these mice against a challenge with MUC1, A2/K(b)-expressing tumour cells. These peptides therefore represent naturally processed MUC1-derived CTL epitopes that could be used as components in peptide-based vaccines and for the analysis of anti-MUC1 CTL responses in A*0201-positive patients with MUC1-expressing tumours.

Animals↗

PubMed2001

Efficient identification of novel HLA-A(*)0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis.

We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A(*)0201-presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved "reverse immunology" strategy. Next to motif-based HLA-A(*)0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A(*)0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA(100-108); SLYSFPEPEA, PRA(142-151); ALYVDSLFFL, PRA(300-309); and SLLQHLIGL, PRA(425-433)) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A(*)0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer.

Amino Acid Sequence↗

PubMed2001

Long lasting p53-specific T cell memory responses in the absence of anti-p53 antibodies in patients with resected primary colorectal cancer.

Colorectal carcinoma is commonly associated with mutation and overexpression of p53, making this antigen a potential target for immune intervention. We analyzed humoral and proliferative immunity against p53 in the blood of patients with resected primary colorectal cancer. The majority of these patients displayed anti-p53 T helper (Th) immunity in the absence of measurable p53 specific antibody levels. The Th responses were long-lasting since they could be detected up to several years after resection of the primary tumor. In a number of cases the Th responses were highly sensitive, reflected by the recognition of naturally processed p53 protein. Our data argue that boosting of these responses in patients with minimal residual disease through p53-specific vaccination, may be employed for improving the chance of disease-free survival of these patients.

Aged↗