The age-associated decrease in functionality of the human immune system is thought to have a negative impact on the capacity to provide protection against infection, in turn leading to increased incidence of mortality. In a previous longitudinal study of octogenarians, we identified an immune risk phenotype (IRP) in the very elderly defined by an inverted CD4/CD8 ratio, which was associated with increased mortality and persistent CMV infection. In this study, we analyzed the CD8 clonal composition of nonagenarians and middle-aged individuals. An increased number of CD8 T cell clones was observed in the nonagenarians, and was associated with CMV-seropositivity. Surprisingly, CMV-seropositive nonagenarians with the IRP had a significantly lower number of clones compared with non-IRP individuals. The decrease in clone numbers in IRP individuals was associated with shorter survival time. MHC/peptide multimer staining indicated that the frequency of CMV-specific T cells was higher in nonagenarians than in the middle-aged, but the ratio of functionally intact cells was significantly lower. The lowest ratio of functional CMV-specific T cells was found in an IRP individual. A thorough longitudinal analysis of the CMV-specific T cells in nonagenarians showed a stable pattern with respect to frequency, phenotype, and clonal composition. We hypothesize that the number of different CD8 T cell clonal expansions increases as the individual ages, possibly, as a compensatory mechanism to control latent infections, e.g., CMV, but eventually a point is reached where clonal exhaustion leads to shrinkage of the CD8 clonal repertoire, associated with decreased survival.

Mutations in LKB1/STK11, a gene mapping to chromosome 19p13.3 and encoding a widely expressed serine/threonine kinase, were recently identi®ed as the cause of Peutz-Jeghers syndrome. Despite the hamartomatous polyps and increased cancer risk associated with this syndrome, somatic alterations in LKB1/STK11 have not been identi®ed in human tumours. Prompted by another feature of the syndrome, lentigines of the lips and oral mucosa, we evaluated the status of LKB1/ STK11 expression, deletion, and mutation in cell lines and tumour samples from 35 patients with sporadic malignant melanoma. Two somatic mutations were identi®ed, a nonsense mutation (Glu170Stop) causing exon skipping and intron retention, and a missense mutation (Asp194Tyr) a ecting an invariant residue in the catalytic subunit of LKB1/STK11. Our data suggest that LKB1/STK11 may contribute to tumorigenesis in a small fraction of malignant melanomas.

The TAM family of receptor tyrosine kinases (TYRO3, AXL, and MERTK) is known to be expressed on antigen-presenting cells and function as oncogenic drivers and as inhibitors of inflammatory responses. Both human and mouse CD8 þ T cells are thought to be negative for TAM receptor expression. In this study, we show that T-cell receptor (TCR)-activated human primary CD8 þ T cells expressed MERTK and the ligand PROS1 from day 2 postactivation. PROS1-mediated MERTK signaling served as a late costimulatory signal, increasing proliferation and secretion of effector and memory-associated cytokines. Knockdown and inhibition studies confirmed that this costimulatory effect was mediated through MERTK. Transcriptomic and metabolic analyses of PROS1-blocked CD8 þ T cells demonstrated a role of the PROS1-MERTK axis in differentiation of memory CD8 þ T cells. Finally, using tumor-infiltrating lymphocytes (TIL) from melanoma patients, we show that MERTK signaling on T cells improved TIL expansion and TIL-mediated autologous cancer cell killing. We conclude that MERTK serves as a late costimulatory signal for CD8 þ T cells. Identification of this costimulatory function of MERTK on human CD8 þ T cells suggests caution in the development of MERTK inhibitors for hematologic or solid cancer treatment.

Accumulation of T cells carrying identical T-cell receptors (TCR) is associated with a number of immunological and non-immunological diseases. Therefore, it is of interest to be able to analyze complex T-cell populations for the presence of clonally expanded subpopulations. Here, we describe a simple method combining reverse transcription (RT)-PCR and denaturing gradient gel electrophoresis (DGGE) for rapid detection and characterization of T-cell clonality. The detection of clonally expanded T cells by DGGE relies on the fact that clonal transcripts have no junctional diversity and therefore resolve at a fixed position in the gel, which is determined by their melting properties. For polyclonal populations with a high degree of junctional diversity, the different DNA molecules will resolve at different positions in the gel and together will be revealed as a smear. For each of the TCR beta-variable gene (BV) 1-24 families, cloned transcripts were amplified and shown to resolve as distinct bands in the denaturing gradient gel, whereas the analysis of polyclonal T-cell populations resulted in a smear in the gel. The present method might prove useful to test for clonotypic T-cells in a variety of pathological and physiological conditions and for monitoring T-cell responses in diagnostic and therapeutic settings.

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