TAP is responsible for transferring cytosolic peptides into the ER, where they can be loaded onto MHC molecules. Deletion of TAP results in a drastic reduction of MHC class I surface expression and alters the presented peptide pattern. This key molecule in antigen processing is tackled by several viruses and lost in some tumors, rendering the altered cells less vulnerable to T cell-based immune surveillance. Using the TAP-deficient cell line LCL721.174 and its TAP-expressing progenitor cell line LCL721.45, we identified and quantified more than 160 HLA ligands, 50 of which were presented TAP-independently. Peptides which were predominantly presented on the TAP-deficient LCL721.174 cell line had a decreased MHC binding affinity according to their SYFPEITHI and BIMAS score. About half of the identified TAP-independently presented peptides were not derived from signal sequences and may partly be generated by the proteasome. Furthermore, we have excluded the possibility that differences in HLA ligand presentation between LCL721.45 and LCL721.174 were due to varying expression of the source proteins or due to changes in the antigen loading complex. Features of peptides presented independently of TAP as well as proteasomal contribution to their generation provide an insight into basic immunological mechanisms.

The in vitro generation of cytotoxic T lymphocytes (CTLs) for anticancer immunotherapy is a promising approach to take patient-specific therapy from the bench to the bedside. Two criteria must be met by protocols for the expansion of CTLs: high yield of functional cells and suitability for good manufacturing practice (GMP). The antigen presenting cells (APCs) used to expand the CTLs are the key to achieving both targets but they pose a challenge: Unspecific stimulation is not feasible because only memory T cells are expanded and not rare naïve CTL precursors; in addition, antigen-specific stimulation by cell-based APCs is cumbersome and problematic in a clinical setting. However, synthetic artificial APCs which can be loaded reproducibly with MHC-peptide monomers and antibodies specific for costimulatory molecules could resolve these problems. The purpose of this study was to investigate the potential of complex synthetic artificial APCs in triggering the costimulatory molecules CD28 and 4-1BB on the T cell. Anti-4-1BB antibodies were added to an established system of microbeads coated with MHC-peptide monomers and anti-CD28. Triggering via CD28 and 4-1BB resulted in strong costimulatory synergy. The quantitative ratio between these signals determined the outcome of the stimulation with optimal results when anti-4-1BB and anti-CD28 were applied in a 3:1 ratio. Functional CTLs of an effector memory subtype (CD45RA(-) CCR7(-)) were generated in high numbers. We present a highly defined APC platform using off-the-shelf reagents for the convenient generation of large numbers of antigen-specific CTLs.

Acute enteroviral infections ranging from meningitis, pancreatitis to myocarditis are common and normally well controlled by the host immune system comprising virus-specific CD8 + cytotoxic T lymphocytes (CTL). However, in some patients enteroviruses and especially coxsackieviruses of group B are capable of inducing severe chronic forms of diseases such as chronic myocarditis. Currently, it is not known whether divergences in the CTL-related immune response may contribute to the different outcome and course of enterovirus myocarditis. A pre-requisite for the study of CTL reactions in patients with acute and chronic myocarditis is the identification of CTL epitopes. In order to define dominant enterovirus CTL epitopes, we have screened, by using gamma interferon (IFN-c) ELISPOT, 62 HLA-A*01-and 59 HLA-A*02-positive healthy blood donors for pre-existing CTL reactions against 12 HLA-A*01 and 20 HLA-A*02 predicted CTL epitopes derived from coxsackieviruses of group B. Positive CTL reactions were verified by FACS analysis in a combined major histocompatibility complex-tetramer IFN-c staining. A total of 14.8 % of all donors reacted against one of the three identified epitopes MLDGHLIAFDY, YGDDVIASY or GIIYIIYKL. The HLA-A*02-restricted epitope ILMNDQEVGV was recognized by 25 % of all tested blood donors. For this peptide, we could demonstrate specific granzyme B secretion, a strong cytolytic potential and endogenous processing. All four epitopes were homologous in 36-92 % of group B enteroviruses, providing a strong basis for monitoring the divergence of T-cellbased immune responses in enterovirus-induced acute and chronic diseases.

In the development of cell-based medicinal products, it is crucial to guarantee that the application of such an advanced therapy medicinal product (ATMP) is safe for the patients. The consensus of the European regulatory authorities is: "In conclusion, on the basis of the state of art, conventional karyotyping can be considered a valuable and useful technique to analyse chromosomal stability during preclinical studies". 408 chondrocyte samples (84 monolayers and 324 spheroids) from six patients were analysed using trypsin-Giemsa staining, spectral karyotyping and fluorescence in situ hybridisation, to evaluate the genetic stability of chondrocyte samples from non-clinical studies. Single nucleotide polymorphism (SNP) array analysis was performed on chondrocyte spheroids from five of the six donors. Applying this combination of techniques, the genetic analyses performed revealed no significant genetic instability until passage 3 in monolayer cells and interphase cells from spheroid cultures at different time points. Clonal occurrence of polyploid metaphases and endoreduplications were identified associated with prolonged cultivation time. Also, gonosomal losses were observed in chondrocyte spheroids, with increasing passage and duration of the differentiation phase. Interestingly, in one of the donors, chromosomal aberrations that are also described in extraskeletal myxoid chondrosarcoma were identified. The SNP array analysis exhibited chromosomal aberrations in two donors and copy neutral losses of heterozygosity regions in four donors. This study showed the necessity of combined genetic analyses at defined cultivation time points in quality studies within the field of cell therapy.

Immunotherapy utilizing CAMPATH-1H for patients with chemotherapy-refractory chronic lymphocytic leukemia has yielded encouraging results with many reports of complete remission. Here we report the outcome of two patients with CD4-positive T cell prolymphocytic leukemia treated with CAM-PATH-1H. Both patients responded rapidly to treatment and subsequently developed CD4 lymphopenia. One patient remained in complete remission after 14 weeks of treatment. Serial peripheral blood flow cytometry revealed that the CD52 antigen was present throughout treatment. The other patient who was initially CD52-positive, became CD52-negative after 6 weeks of treatment, and developed progressive symptoms of T cell prolymphocytic leukemia. Immunotherapy was stopped, chemotherapy proved futile, and the patient died. This change in phenotype from CD52-positive to -negative during CAM-PATH-1H therapy points out a need to develop strategies for maintaining antigenic expression during monoclonal antibody therapy. Leukemia (2002) 16, 861-864.