A mutated cyclin-dependent kinase 4 (CDK4) was identified as a tumor-specific antigen recognized by HLA-A2. 1-restricted autologous cytolytic T lymphocytes (CTLs) in a human melanoma. The mutated CDK4 allele was present in autologous cultured melanoma cells and metastasis tissue, but not in the patient's lymphocytes. The mutation, an arginine-to-cysteine exchange at residue 24, was part of the CDK4 peptide recognized by CTLs and prevented binding of the CDK4 inhibitor p16INK4a, but not of p21 or of p27KIP1. The same mutation was found in one additional melanoma among 28 melanomas analyzed. These results suggest that mutation of CDK4 can create a tumor-specific antigen and can disrupt the cell-cycle regulation exerted by the tumor suppressor p16INK4a.

We investigated impaired cellular immune responses of individuals on chronic hemodialysis by using monoclonal antibodies that trigger differential pathways of T cell activation. Reduced cellular reactivity, which exists in a high proportion of such patients, can be attributed to a failure of the monocyte population to support the process of primary T cell activation in vitro. This defect results in a lack of interleukin 2 production, which is critically dependent on a monocyte-derived signal. In contrast, T lymphocyte function was found to be physiologic. Perhaps more important, the degree of monocyte dysfunction in vitro correlated with the same patients' in vivo responses to hepatitis B vaccination. Addition of recombinant human interleukin 2 fully reconstituted their deficient immune response in vitro.

MicroRNAs (miRs) play major roles in normal hematopoietic differentiation and hematopoietic malignancies. In this work, we report that miR-27a, and its coordinately expressed cluster (miR-23a∼miR-27a∼miR-24-2), was down-regulated in acute leukemia cell lines and primary samples compared to hematopoietic stem-progenitor cells (HSPCs). Decreased miR-23a cluster expression in some acute leukemia cell lines was mediated by c-MYC. Replacement of miR-27a in acute leukemia cell lines inhibited cell growth due, at least in part, to increased cellular apoptosis. We identified a member of the anti-apoptotic 14-3-3 family of proteins, which support cell survival by interacting with and negatively regulating pro-apoptotic proteins such as Bax and Bad, as a target of miR-27a. Specifically, miR-27a regulated 14-3-3θ at both the mRNA and protein levels. These data indicate that miR-27a contributes a tumor suppressor-like activity in acute leukemia cells via regulation of apoptosis, and that miR-27a and 14-3-3θ may be potential therapeutic targets.

We have pursued our analysis of potential tumor-rejection antigens recognized on human melanoma by autologous cytolytic T lymphocytes (CTL). We reported previously that 3 distinct antigens (A,B,C) were recognized on melanoma cell line SK29-MEL in association with HLA-A2. Selection for melanoma-cell variants resistant to anti-A CTL revealed that antigen A consists of at least 2 determinants (Aa, Ab) which can be lost separately. Genetic linkage between Aa and Ab was suggested by concomitant loss of Aa and Ab in an immunoselected tumor-cell variant. This variant was also resistant to an autologous CTL clone restricted by HLA-B45, indicating that this CTL may also recognize a determinant of antigen A. Of 11 allogeneic HLA-A2 melanoma cell lines that were tested, 5 expressed both Aa and Ab, 1 expressed only Aa, and 1 only Ab. None of them was lysed by anti-B or anti-C CTL clones. A CTL clone derived from another HLA-A2-melanoma patient was found to have exactly the same lytic pattern as the anti-Ab CTL of the first patient. This suggested that it may be possible to elicit an anti-Ab response in many HLA-A2 patients. We conclude that there are at least 2 distinct antigens presented in association with HLA-A2 that are common to many melanomas and therefore constitute promising targets for specific immunotherapy.

Population genetic structure of the West Nile Virus vector Culex tarsalis was investigated in 5 states in the western United States using 5 microsatellite loci and a fragment of the mitochondrial reduced form of nicotinamide adenine dinucleotide dehydrogenase 4 (ND4) gene. ND4 sequence analysis revealed a lack of isolation by distance, panmixia across all populations, an excess of rare haplotypes, and a star-like phylogeny. Microsatellites revealed moderate genetic differentiation and isolation by distance, with the largest genetic distance occurring between populations in southern California and New Mexico (F(ST) = 0.146). Clustering analysis and analysis of molecular variance on microsatellite data indicated the presence of 3 broad population clusters. Mismatch distributions and site-frequency spectra derived from mitochondrial ND4 sequences displayed pattern's characteristic of population expansion. Fu and Li's D* and F*, Fu's F(S), and Tajima's D statistics performed on ND4 sequences all revealed significant, negative deviations from mutation-drift equilibrium. Microsatellite-based multilocus heterozygosity tests showed evidence of range expansion in the majority of populations. Our results suggest that C. tarsalis underwent a range expansion across the western United States within the last 375,000-560,000 years, which may have been associated with Pleistocene glaciation events that occurred in the midwestern and western United States between 350,000 and 1 MYA.