Rejection of intraocular tumors by CD4(+) T cells without induction of phthisis.

Immune privilege of the eye protects against sight-threatening inflammatory events, but can also permit outgrowth of otherwise nonlethal immunogenic tumors. Nonetheless, ocular tumor growth can be controlled by cellular immune responses. However, this will normally result in phthisis of the eye, in case tumor rejection is mediated by a delayed-type hypersensitivity response orchestrated by CD4(+) T cells. We now show that intraocular tumors can be eradicated by CD4(+) Th cells without inducing collateral damage of neighboring ocular tissue. Injection of tumor cells transformed by the early region 1 of human adenovirus type 5 in the anterior chamber of the eye leads to intraocular tumor formation. Tumor growth is transient in immunocompetent mice, but lethal in immunodeficient nude mice, indicating that T cell-dependent immunity is responsible for tumor clearance. Tumor rejection has all the characteristics of a CD8(+) T cell-mediated immune response, as the tumor did not express MHC class II and only tumor tissue was the subject of destruction. However, analysis of the molecular and cellular mechanisms involved in tumor clearance revealed that perforin, TNF-alpha, Fas ligand, MHC class I, and CD8(+) T cells did not play a crucial role in tumor eradication. Instead, effective tumor rejection was entirely dependent on CD4(+) Th cells, as CD4-depleted as well as MHC class II-deficient mice were unable to reject their intraocular tumor. Taken together, these observations demonstrate that CD4(+) T cells are able to eradicate MHC class II-negative tumors in an immune-privileged site without affecting surrounding tissues or the induction of phthisis.

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.

The role of CD40 in peripheral T cell tolerance and immunity.

CD40 and CD40 ligand (CD40L) have been implicated as important molecules for the transformation of nonactivated antigen-presenting cells (APC) into cells that are potent inducers of cytotoxic T lymphocyte (CTL) immunity. The onset of a successful immune response lies within the control of the CD4+ T helper cells which, after specific antigen recognition, can up-regulate CD40L and subsequently activate APC through CD40 signaling. Triggering of CD40 with antibodies in vivo can replace the need for CD40L-expressing CD4+ T helper cells for cross-priming of CTL. Blocking of CD40-CD40L interactions can also have profound effects on the generation of T cell immunity. Interestingly, differential involvement of CD40/CD40L in immune responses can be observed between various immunological sites in the body. In most sites of the periphery interruption of CD40-CD40L interactions can lead to the induction of T cell tolerance whereas in mucosal tissues this interruption can lead to abrogation of T cell tolerance. Furthermore, in vivo CD40 activation can convert specific T cell tolerance following peptide vaccination into efficient T cell priming. Thus intervention of CD40-CD40L interactions can result in enhancement or down-modulation of T cell reactivity and therefore modulation of these interactions may form the foundation of new treatment modalities directed against malignancies, allergies, organ rejections and autoimmunity.

Successful immunotherapy of an intraocular tumor in mice.

Immune privilege in the eye is considered essential in the protection against local sight-threatening inflammatory responses. However, the deviant immune responses in the eye may also provide an ideal opportunity to uncontrolled growth of viruses or tumors by inhibiting intraocular immunological attack. To establish to what extent immune privilege interferes with T cell-mediated antitumor immunotherapy, we established a new ocular tumor model in the mouse and tested whether well-defined tumor-specific CTLs can eradicate an immunogenic intraocularly growing tumor. Tumor cells, transformed by human adenovirus type 5 early region 1 (Ad5E1), injected s.c. in a dose of 10(7) cells, did not induce s.c. tumor growth in C57BL/6 mice. However, an injection of 0.3 x 10(6) of these cells into the anterior chamber of the eye led to intraocular tumor growth in 95% of mice (n = 20). Tumor growth in the eye did not induce systemic tumor-specific tolerance, because 70% of the mice were able to eradicate the tumor spontaneously after 5 weeks. Mice vaccinated s.c. with irradiated tumor cells were protected against intraocular tumor challenge, indicating that preactivated memory T cells are able to protect against intraocular tumor growth. Moreover, an i.v. injection of an Ad5E1-specific CTL clone was able to eradicate established intraocular Ad5E1-transformed tumors, whereas the anatomy of the eye remained intact. These results demonstrate that tumor-specific, CTL-mediated immunity can be used successfully for the prevention and eradication of tumors growing in the immune-privileged anterior chamber of the eye, without detectable destruction of the eye.

CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy.

The outcome of antigen recognition by naive CD8+ cytotoxic T lymphocytes (CTLs) in the periphery is orchestrated by CD4+ T-helper cells, and can either lead to priming or tolerization. The presence of T-helper cells favors the induction of CTL immunity, whereas the absence of T-helper cells can result in CTL tolerance. The action of T helper cells in CTL priming is mediated by CD40-CD40 ligand interactions. We demonstrate here that triggering of CD40 in vivo can considerably enhance the efficacy of peptide-based anti-tumor vaccines. The combination of a tolerogenic peptide vaccine containing a minimal essential CTL epitope with an activating antibody against CD40 converts tolerization into strong CTL priming. Moreover, CD40 ligation can provide an already protective tumor-specific peptide vaccine with the capacity to induce therapeutic CTL immunity in tumor-bearing mice. These findings indicate that the CD40-CD40 ligand pair can act as a 'switch', determining whether naive peripheral CTLs are primed or tolerized, and support the clinical use of CD40-stimulating agents as components of anti-cancer vaccines.

Angelman syndrome without detectable chromosome 15q11-13 anomaly: clinical study of familial and isolated cases.

The clinical findings in 12 Angelman syndrome (AS) patients (4 sib pairs and 4 sporadic cases, aged 12-55 years) without a cytogenetic or molecular detectable defect at the AS locus were compared to those of 28 AS patients (aged 11-50 years) with a deletion, in order to determine whether the clinical spectrum differed between the two groups. There were only two minor differences, i.e., mandibular prognathism was always found in the patients with a defect (100% vs. 58%), whereas truncal hypotonia was found less frequently in the group with a detectable genetic defect (54% vs. 91%). All other clinical and physical characteristics were equally represented in the two groups. Epileptic seizures occurred in 93% and 75%, respectively, of patients with and without a detectable chromosome 15 defect. Specific EEG patterns were found in 90% of both groups. The clinical signs and symptoms of our patients closely resemble those in familial AS cases reported in the literature, with the exception of scoliosis, which was present in 55% of the patients in our study. We conclude that the absence of a detectable cytogenetic or molecular defect at the AS locus is not associated with a strikingly different AS phenotype, compared to those with such a defect. Mutation analysis of the UBE3A gene in our patients without a detectable genetic defect, especially in the familial cases, is currently underway.

Angelman syndrome in adulthood.

We studied the clinical and EEG-findings in 28 adult patients (aged 20-53 years) with Angelman syndrome (AS). Twenty-three showed a maternal chromosome 15q11-13 deletion; in 5, the diagnosis was based on a combination of typical clinical findings. Compared to the clinical manifestations present in childhood, "coarsening" of facial traits (100%), thoracic scoliosis (71%), and being wheelchair-bound (39%) were found more frequently. Paroxysms of laughter were still observed in adulthood (79%), but less frequently than in childhood. Most adult patients could feed themselves, but needed help with many daily activities. The majority (82%) had epileptic seizures. Abnormal EEG-activity consisting of 2-3/s rhythmic triphasic waves of high amplitude with a maximum over the frontal regions, which has been identified in many AS children, was found in 67% of these adult patients.