No abstract

Cytotoxic chemotherapies may expose the immune system to high levels of tumor antigens and expand the CD8(+) T-cell response to include weak or subdominant antigens. Here, we evaluated the in vivo CTL response to tumor antigens using a murine mesothelioma tumor cell line transfected with a neotumor antigen, ovalbumin, that contains a known hierarchy of epitopes for MHC class I molecules. We show that as tumors progress, effector CTLs are generated in vivo that focus on the dominant epitope SIINFEKL, although a weak response was seen to one (KVVRFDKL) subdominant epitope. These CTLs did not prevent tumor growth. Cisplatin treatment slowed tumor growth, slightly improved in vivo SIINFEKL presentation to T cells and reduced SIINFEKL-CTL activity. However, the CTL response to KVVRFDKL was amplified, and a response to another subdominant epitope, NAIVFKGL, was revealed. Similarly, gemcitabine cured most mice, slightly enhanced SIINFEKL presentation, reduced SIINFEKL-CTL activity yet drove a significant CTL response to NAIVFKGL, but not KVVRFDKL. These NAIVFKGL-specific CTLs secreted IFNγ and proliferated in response to in vitro NAIVFKGL stimulation. IL-2 treatment during chemotherapy refocused the response to SIINFEKL and simultaneously degraded the cisplatin-driven subdominant CTL response. These data show that chemotherapy reveals weaker tumor antigens to the immune system, a response that could be rationally targeted. Furthermore, while integrating IL-2 into the chemotherapy regimen interfered with the hierarchy of the response, IL-2 or other strategies that support CTL activity could be considered upon completion of chemotherapy.

The purpose of this study was to examine the ability of gamma(delta) T cells to cause graft-vs.-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) and to determine whether these cells offered any therapeutic advantages relative to alphabeta T cells. Due to the paucity of naive gamma(delta) T cells in mice and humans, gamma(delta), T cells (obtained from alpha(beta) T cell-deficient murine donors) were ex vivo activated and expanded in interleukin (IL)-2 so as to achieve sufficient cell numbers and to serve as a more clinically feasible strategy. After transplantation into lethally irradiated hosts, donor gamma(delta) T cells were detected in target organs of GVHD such as the spleen and intestines 2 weeks after BMT and constituted the primary T cell subpopulation. Large doses (150 x 10(6)) of activated gamma(delta) T cells, which we have previously shown capable of facilitating engraftment in MHC-disparate recipients, failed to cause fatal GVHD in lethally irradiated recipients of MHC-incompatible donor marrow grafts (C57BL/6 [H-2b]-->B10.BR [H-2k] and C57BL/6 [H-2b]-B6D2F1[H-2b/d]). The absence of GVHD was confirmed by histologic analysis of target organs, splenic B cell reconstitution, and appropriate negative selection in the thymus, that were all comparable to those observed in mice transplanted with T cell-depleted BM only. While early splenic reconstitution was attributable to donor gamma(delta) T cells, analysis of durably engrafted chimeras 2 months posttransplant revealed that the vast majority of donor splenic T cells expressed the alpha(beta) T cell receptor. The results of secondary adoptive transfer assays showed that these cells were tolerant of recipient alloantigens in vivo, demonstrating that gamma(delta) T cells did not prevent the subsequent development of donor anti-host tolerance in BM-derived alpha(beta) T cells. When comparatively evaluated, the minimal number of naive alpha(beta) T cells necessary for donor engraftment caused significantly more fatal GVHD than the corresponding minimal dose of activated gamma(delta) T cells and thus had a superior therapeutic index. These studies indicate that doses of activated gamma(delta) T cells that are able to promote alloengraftment do not cause lethal GVHD in mice transplanted with MHC-incompatible marrow grafts.

No abstract

The TRUGENE HCV 5NC genotyping kit (GeneLibrarian modules 3.1.1 and 3.1.2) and VERSANT HCV genotyping assay were compared by using 96 hepatitis C virus (HCV) RNA-positive patient specimens, including HCV genotypes 1, 2, 3, 4, 5, 6, and 10. The TRUGENE HCV 5NC genotyping kit (GeneLibrarian module 3.1.2) yielded the most accurate genotyping results.Hepatitis C virus (HCV) has been classified into at least six distinct genotypes that can be further differentiated into multiple subtypes (12, 13). Recent anti-HCV treatment algorithms suggest that tailoring antiviral therapy based on HCV genotype as well as HCV RNA titer can optimize therapeutic outcome (2, 7). While sequence variability is found throughout the HCV genome, the 5Ј noncoding (5ЈNC) region remains highly conserved and is the target of choice for detection by reverse transcription-PCR (RT-PCR) (5,14,15). Despite the limited sequence diversity found within the HCV 5ЈNC region compared to that of a highly diversified genomic target such as the nonstructural (NS) 5B region, practical considerations have made the 5ЈNC region the preferred target for HCV genotyping in most diagnostic laboratories (3,6,8). Several HCV genotyping assays are currently commercially available, including the TRUGENE HCV 5ЈNC genotyping kit (TRUGENE 5ЈNC; Bayer HealthCare LLC, Berkeley, Calif.) and the VERSANT HCV genotype assay (LiPA; Bayer HealthCare LLC). Recently, there have been several published comparisons of these two assays (1,8,10), in addition to several other evaluations of the TRUGENE 5ЈNC (4, 11). To date, none of these comparisons has evaluated the TRUGENE 5ЈNC in conjunction with the new HCV 5ЈNC GeneLibrarian module 3.1.2 (GL 3.1.2; Bayer HealthCare LLC). The GL 3.1.2 is a new 5ЈNC sequence database validated by phylogenetic analysis of the 5ЈNC and corresponding NS5B sequences from over 250 HCV strains collected worldwide and is designed for use with the TRUGENE 5ЈNC