No abstract

Genetic engineering of T lymphocytes is an attractive strategy to specifically redirect T-cell immunity toward viral infections and malignancies. We previously demonstrated redirected antileukemic reactivity of cytomegalovirus (CMV)-specific T cells by transfer of minor histocompatibility antigen HA-2-specific T-cell receptors (TCRs). HA-2-TCR-transferred CMV-specific T cells were potent effectors against HA-2-expressing leukemic cells, as well as CMV-expressing cells.Functional activity of these T cells correlated with TCR cell-surface expression. In the present study we analyzed which properties of transferred and endogenous TCRs are crucial for efficient cellsurface expression. We demonstrate that expression of the introduced TCR is not a random process but is determined by characteristics of both the introduced and the endogenously expressed TCR. The efficiency of TCR cell-surface expression is controlled by the intrinsic quality of the TCR complex. In addition, we demonstrate that chimeric TCRs can be formed and that efficiency of TCR expression is independent of whether TCRs are retrovirally introduced or naturally expressed. In conclusion, introduced, endogenous, and chimeric TCRs compete for cell-surface expression in favor of the TCR-CD3 complex with best-pairing properties. (Blood. 2007;109: [235][236][237][238][239][240][241][242][243]

Retroviral transfer of T-cell receptors (TCR) to peripheral blood-derived T cells generates large numbers of T cells with the same antigen specificity, potentially useful for adoptive immunotherapy. One drawback of this procedure is the formation of mixed TCR dimers with unknown specificities due to pairing of endogenous and introduced TCR chains. We investigated whether ;D T cells can be an alternative effector population for TCR gene transfer because the ;DTCR is not able to form dimers with the ABTCR. Peripheral bloodderived ;D T cells were transduced with human leukocyte antigen (HLA) class I-or HLA class II-restricted minor histocompatibility antigen (mHag) or virus-specific TCRs. Because most ;D T cells do not express CD4 and CD8, we subsequently transferred these coreceptors. The TCR-transduced ;D T cells exerted high levels of antigen-specific cytotoxicity and produced IFN-; and IL-4, particularly in the presence of the relevant coreceptor. ;D T cells transferred with a TCR specific for the hematopoiesis-specific mHag HA-2 in combination with CD8 displayed high antileukemic reactivity against HA-2-expressing leukemic cells. These data show that transfer of ABTCRs to ;D T cells generated potent effector cells for immunotherapy of leukemia, without the expression of potentially hazardous mixed TCR dimers. (Cancer Res 2006; 66(6): 3331-7) IntroductionCellular immunotherapy is a promising strategy for the treatment of cancer (1). However, adoptive transfer of sufficient numbers of antigen-specific T cells requires complex isolation methods and laborious and time-consuming tissue culture procedures. An alternative method to obtain large numbers of T cells with a defined antigen specificity is the retroviral transfer of a T-cell receptor (TCR). Because T-cell specificity is exclusively determined by the TCR, T-cell specificity can be functionally transferred to other T lymphocytes by retroviral TCR gene transfer. We and others have shown that transfer of human leukocyte antigen (HLA) class I-and HLA class II-restricted TCRs to CD8 + and CD4 + T cells, respectively, generated T cells with converted antigen-specific cytolytic activity and cytokine production (2-10).The potential in vivo efficacy of TCR-transferred T cells was shown in mouse models (8,9). The TCR-transferred T cells were activated in vivo, homed to effector sites, and contributed to tumor clearance.A potential disadvantage of TCR gene transfer to other ah T cells is the formation of mixed TCR dimers. Chains of the introduced TCR can pair with the endogenous TCR chains naturally expressed by the TCR-transferred T cells. The specificity of these mixed TCR dimers is unknown and, therefore, autoreactivity cannot be excluded. To limit the number of T cells with different TCR chains and thus the chance to generate autoreactive T cells, T cells with defined antigen specificity and, therefore, with a limited TCR repertoire can be selected as host cells for TCR gene transfer. We previously showed the reprogramming of cytomegalovirus (CMV)-specifi...

Chromothripsis represents an extreme class of complex chromosome rearrangements (CCRs) with major effects on chromosomal architecture. Although recent studies have associated chromothripsis with congenital abnormalities, the incidence and pathogenic effects of this phenomenon require further investigation. Here, we analyzed the genomes of three families in which chromothripsis rearrangements were transmitted from a mother to her child. The chromothripsis in the mothers resulted in completely balanced rearrangements involving 8-23 breakpoint junctions across three to five chromosomes. Two mothers did not show any phenotypic abnormalities, although 3-13 protein-coding genes were affected by breakpoints. Unbalanced but stable transmission of a subset of the derivative chromosomes caused apparently de novo complex copy-number changes in two children. This resulted in gene-dosage changes, which are probably responsible for the severe congenital phenotypes of these two children. In contrast, the third child, who has a severe congenital disease, harbored all three chromothripsis chromosomes from his healthy mother, but one of the chromosomes acquired de novo rearrangements leading to copy-number changes. These results show that the human genome can tolerate extreme reshuffling of chromosomal architecture, including breakage of multiple protein-coding genes, without noticeable phenotypic effects. The presence of chromothripsis in healthy individuals affects reproduction and is expected to substantially increase the risk of miscarriages, abortions, and severe congenital disease.