In chronic viral infections, CD8 + T cells become functionally deficient and display multiple molecular alterations. In contrast, only little is known of self-and tumor-specific CD8 + T cells from mice and humans. Here we determined molecular profiles of tumor-specific CD8 + T cells from melanoma patients. In peripheral blood from patients vaccinated with CpG and the melanoma antigen Melan-A/MART-1 peptide, we found functional effector T cell populations, with only small but nevertheless significant differences in T cells specific for persistent herpesviruses (EBV and CMV). In contrast, Melan-A/MART-1-specific T cells isolated from metastases from patients with melanoma expressed a large variety of genes associated with T cell exhaustion. The identified exhaustion profile revealed extended molecular alterations. Our data demonstrate a remarkable coexistence of effector cells in circulation and exhausted cells in the tumor environment. Functional T cell impairment is mediated by inhibitory receptors and further molecular pathways, which represent potential targets for cancer therapy.

To study telomere length dynamics in hematopoietic cells with age, we analyzed the average length of telomere repeat sequences in diverse populations of nucleated blood cells. More than 500 individuals ranging in age from 0 to 90 yr, including 36 pairs of monozygous and dizygotic twins, were analyzed using quantitative fluorescence in situ hybridization and flow cytometry. Granulocytes and naive T cells showed a parallel biphasic decline in telomere length with age that most likely reflected accumulated cell divisions in the common precursors of both cell types: hematopoietic stem cells. Telomere loss was very rapid in the first year, and continued for more than eight decades at a 30-fold lower rate. Memory T cells also showed an initial rapid decline in telomere length with age. However, in contrast to naive T cells, this decline continued for several years, and in older individuals lymphocytes typically had shorter telomeres than did granulocytes. Our findings point to a dramatic decline in stem cell turnover in early childhood and support the notion that cell divisions in hematopoietic stem cells and T cells result in loss of telomeric DNA.

We adoptively transferred donor-derived cytomegalovirus (CMV)-specific T-cell lines into 8 stem cell transplant recipients lacking CMV-specific T-cell proliferation. All patients, of whom one was infected by a CMV strain that was genotypically ganciclovir resistant, had received unsuccessful antiviral chemotherapy for more than 4 weeks. CMVspecific lines had been prepared by repetitive stimulation with CMV antigen, which increased the percentage of CMV-specific T cells and ablated alloreactivity completely even against patients mismatched for 1 to 3 HLA antigens. After transfer of 10 7 T cells/m 2 at a median of 120 days (range, 79-479 days) after transplantation, no side effects were noticed. Despite cessation of antiviral chemotherapy, the CMV load dropped significantly in all 7 evaluable patients, with a maximal reduction after a median of 20 days (range, 5-31 days). In 2 patients with high virus load, the antiviral effect was only transient. One of these patients received a second T-cell infusion, which cleared the virus completely. At a median of 11 days after transfer, CMV-specific T-cell proliferation was demonstrated in 6 patients, and an increase in CMV-specific CD4 ؉ T cells was demonstrated in 5 patients. In 6 patients, 1.12 to 41 CMV-specific CD8 ؉ T cells/L blood were detected at a median of 13 days after transfer, with an increase in all patients lacking CMV-specific CD8 ؉ T cells prior to transfer. Hence, anti-CMV cellular therapy was successful in 5 of 7 patients, whereas in 2 of 7 patients, who received an intensified immune suppression at the time of or after T-cell therapy, only transient reductions in virus load were obtained. (Blood. 2002;99: 3916-3922)

To measure the average length of telomere repeats at chromosome ends in individual cells we developed a flow cytometry method using fluorescence in situ hybridization (flow FISH) with labeled peptide nucleic acid (PNA) probes. Results of flow FISH measurements correlated with results of conventional telomere length measurements by Southern blot analysis (R = 0.9). Consistent differences in telomere length in CD8+ T-cell subsets were identified. Naive and memory CD4+ T lymphocytes in normal adults differed by around 2.5 kb in telomere length, in agreement with known replicative shortening of telomeres in lymphocytes in vivo. T-cell clones grown in vitro showed stabilization of telomere length after an initial decline and rare clones capable of growing beyond 100 population doublings showed variable telomere length. These results show that flow FISH can be used to measure specific nucleotide repeat sequences in single cells and indicate that the very large replicative potential of lymphocytes is only indirectly related to telomere length.

The paracaspase MALT1 is pivotal in antigen receptor-mediated lymphocyte activation and lymphomagenesis. MALT1 contains a caspase-like domain, but it is unknown whether this domain is proteolytically active. Here we report that MALT1 had arginine-directed proteolytic activity that was activated after T cell stimulation, and we identify the signaling protein Bcl-10 as a MALT1 substrate. Processing of Bcl-10 after Arg228 was required for T cell receptor-induced cell adhesion to fibronectin. In contrast, MALT1 activity but not Bcl-10 cleavage was essential for optimal activation of transcription factor NF-kappaB and production of interleukin 2. Thus, the proteolytic activity of MALT1 is central to T cell activation, which suggests a possible target for the development of immunomodulatory or anticancer drugs.