In a human melanoma model of tumor antigen (TA)-based immunization, we tested the functional status of TA-specific CD8 ؉ cytotoxic T lymphocytes. A "quiescent" phenotype lacking direct ex vivo cytotoxic and proliferative potential was identified that was further characterized by comparing its transcriptional profile to that of TA-specific T cells sensitized in vitro by exposure to the same TA and the T-cell growth factor interleukin 2 (IL-2). Quiescent circulating tumor-specific CD8 ؉ T cells were deficient in expression of genes associated with T-cell activation, proliferation, and effector function. This quiescent status may explain the observed lack of correlation between the presence of circulating immunizationinduced lymphocytes and tumor regression. In addition, the activation of TAspecific T cells by in vitro antigen recall and IL-2 suggests that a complete effector phenotype might be reinstated in vivo to fulfill the potential of anticancer vaccine protocols. (Blood. 2004;104:1970-1978 © 2004 by The American Society of Hematology IntroductionThe coexistence in patients with cancer of tumor and tumorspecific circulating CD8 ϩ T cells remains unexplained, 1,2 particularly in the context of tumor antigen (TA)-specific immunization that consistently induces TA-specific immune responses rarely associated with tumor regression. [3][4][5][6] This discrepancy could be due to a progressive escape of tumor cells from T-cell recognition as a result of immune editing. 7 However, we and others postulated that in humans the primary reason for the lack of tumor immune responsiveness is ineffectual T-cell function, whereas tumor escape mechanisms likely play a role only in adaptation to those rare cases of successful immune rejection. 1,2 Some investigators attributed a status of "unresponsiveness" to circulating TA-specific T cells. 8 Others, however, reported that at least a subset of circulating TA-specific T cells can exert tumorspecific cytolytic activity ex vivo. 9 In addition, total T-cell anergy does not apply to immunization-induced T cells because they express markers of T-cell activation 10 and respond to relevant antigen stimulation with interferon ␥ (IFN-␥) secretion. [11][12][13] Cytokine production, however, may not comprehensively portray the globality of cytotoxic T-cell functions 14 and other parameters could more comprehensively characterize T cells. 10,[15][16][17][18][19][20][21] For instance, Speiser et al 22 noted that immunization-induced circulating T cells lack effector features displayed by virus-specific T cells. In addition, in a previous study we were impressed by the low levels of perforin constitutively expressed. 18 The poor correlation observed in melanoma between frequency of circulating TA-specific T cells and clinical effectiveness is not shared by other diseases. During acute viral infection, expansion of pathogen-specific CD8 ϩ T cells is paralleled by resolution of the infectious process. 23 During chronic viral infections, the CD8 ϩ T-cell phenotype may vary greatly according to ...
Background: Although the sequence of events leading to wound repair has been described at the cellular and, to a limited extent, at the protein level this process has yet to be fully elucidated. Genome wide transcriptional analysis tools promise to further define the global picture of this complex progression of events.
BackgroundEndogenous reference genes are commonly used to normalize expression levels of other genes with the assumption that the expression of the former is constant in different tissues and in different physiopathological conditions. Whether this assumption is correct it is, however, still matter of debate. In this study, we searched for stably expressed genes in 384 cDNA array hybridization experiments encompassing different tissues and cell lines.ResultsSeveral genes were identified whose expression was highly stable across all samples studied. The usefulness of 8 genes among them was tested by normalizing the relative gene expression against test genes whose expression pattern was known. The range of accuracy of individual endogenous reference genes was wide whereas consistent information could be obtained when information pooled from different endogenous reference genes was used.ConclusionsThis study suggests that even when the most stably expressed genes in array experiments are used as endogenous reference, significant variation in test gene expression estimates may occur and the best normalization is achieved when data from several endogenous reference genes are pooled together to minimize minimal but significant variation among samples. We are presently optimizing strategies for the preparation of endogenous reference gene mixtures that could yield information comparable to that of data pooled from individual endogenous reference gene normalizations.
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