ABSTRACT
In this paper we show that the effects of transgenic coreceptor expression on thymocyte development depend on the onset of transgene expression. Thus, a CD8 transgene expressed on CD44+CD25+ (DN2) and CD44-CD25+ (DN3) cells causes a partial block at the stage when TCRbeta selection takes place and diminishes expansion at the subsequent developmental stages, resulting in increased DN3 and markedly reduced double-positive (DP) thymocyte numbers. This effect is evident on a polyclonal TCR repertoire as well as in TCR-transgenic mice (F5). By contrast, a CD8 transgene that leads to the same degree of overexpression on DP thymocytes, but is not expressed on double-negative subsets, has no effect on thymus size or composition. Therefore, the reduction of DP thymocyte numbers in CD8 TCRtg mice can be attributed to interferences at early developmental stages rather than to increased negative selection of DP cells.
Subject(s)
CD8 Antigens/biosynthesis , CD8 Antigens/genetics , Gene Expression Regulation/immunology , Receptors, Antigen, T-Cell, alpha-beta/antagonists & inhibitors , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Thymus Gland/immunology , Thymus Gland/metabolism , Transgenes/immunology , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Division/genetics , Cell Division/immunology , Crosses, Genetic , Flow Cytometry , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor/immunology , Humans , Lymphocyte Count , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Knockout , Mice, Transgenic , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Interleukin-2/biosynthesis , T-Lymphocyte Subsets/cytology , Thymus Gland/cytologyABSTRACT
CD4 and CD8 are crucial for the development and function of T cells. An intergenic deoxyribonuclease I hypersensitive site region (cluster CIII) directs expression in mature CD8 T cells only. Here, we show that two further independent regions from the CD8 gene locus in conjunction with cluster CIII restore transgene expression in appropriate immature thymocytes. Deletion of two of the intergenic cluster CIII DNaseI-HSS in homozygous mutant mice affects expression of CD8alphaalpha homodimers on intraepithelial T cells (IEL), particularly on the gammadeltaTCR+ subset. Surprisingly, none of the thymocyte or peripheral alphabetaTCR T cell subsets are affected by this mutation, indicating hierarchical activation of these elements within the different T cell subsets.
Subject(s)
CD8 Antigens/genetics , T-Lymphocyte Subsets/immunology , Alleles , Animals , Cell Differentiation , Chromosome Mapping , DNA/genetics , Deoxyribonuclease I , Female , Gene Expression Regulation, Developmental , Genes, Reporter , Male , Mice , Mice, Knockout , Mice, Mutant Strains , Mice, Transgenic , Mutation , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, gamma-delta/genetics , Sequence Deletion , T-Lymphocyte Subsets/cytologyABSTRACT
The coreceptors CD4 and CD8 play a crucial role during thymocyte development and T cell effector function, and their expression is developmentally regulated. To determine the underlying molecular mechanisms of CD8 gene regulation we cloned the murine CD8 gene locus from genomic libraries and analyzed this region for deoxyribonuclease (DNase I) hypersensitive sites (HSS). Here we report, using transgenic mice, deletion analysis of one of the identified clusters of DNase I hypersensitivity, consisting of three DNase I-HSS and located in the intergenic region between the CD8alpha and CD8beta genes. Our data show that at least two of the DNase I-HSS constituting this cluster are individually sufficient to direct CD8alpha or heterologous transgene expression to the mature CD8 single-positive T cell subset and that this expression coincides temporally with the appearance of positively selected T cells.
Subject(s)
CD8 Antigens/genetics , CD8-Positive T-Lymphocytes/immunology , Regulatory Sequences, Nucleic Acid , T-Lymphocyte Subsets/immunology , Animals , DNA Footprinting , Deoxyribonuclease I/metabolism , Mice , Mice, Inbred CBA , Mice, Transgenic , Receptors, Antigen, T-Cell, alpha-beta/genetics , Sequence Deletion , Thymus Gland/cytologyABSTRACT
Helper and cytotoxic T cell subsets require the expression of different coreceptors (CD4 and CD8, respectively) for their development and function. We have cloned the CD8 gene locus from genomic cosmid and P1 libraries and analyzed the region around the CD8alpha and CD8beta genes for gene expression regulatory elements. DNase I (DNase I) hypersensitivity analysis of 80 kb in the CD8 locus identified four clusters of putative regulatory regions, three of which are thymocyte specific. Transgenic mice carrying the cloned CD8alphabeta genomic locus and containing the identified DNase I-hypersensitive site clusters express the transgenic CD8 in a developmentally regulated, tissue-specific, and CD8 T cell subset-specific manner.
Subject(s)
CD8 Antigens/genetics , Regulatory Sequences, Nucleic Acid , T-Lymphocyte Subsets/physiology , Animals , Cloning, Molecular , DNA Footprinting , Gene Expression Regulation , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Spleen/physiology , Thymus Gland/physiology , Transcription, GeneticABSTRACT
In a flow model the effective orifice areas (Ae) of 17 mechanical heart valve prostheses were determined. We measured the Ae-values of several sizes of three types of mechanical prostheses (Medtronic-Hall, St. Jude Medical, and Omnicarbon) under quasi-steady flow conditions using the continuity equation: Ae = flow/maximal transprosthetic velocity. The flow through the model could be determined exactly by directly measuring the decreasing fluid level within the feed tank, while the maximal velocities were calculated from CW-Doppler echocardiographic spectra. It was found that 1) over a range of 200-800 cm3/s Ae was constant for all prostheses and 2) in small aortic prostheses the Ae could be determined with only little scattering of the obtained values, while in large mitral prostheses there was a considerable variation within the results of repeated investigations. For example, in the 21- and 31-Omnicarbon-valves mean values of Ae were calculated as 1.41 and 4.03 cm2, respectively, with standard deviations of 0.05 and 0.49 cm2 as a result of about 70 single calculations in each valve. 3) The absolute values of Ae were smaller than those of comparable in vitro studies based on the Gorlin formula. We conclude that the effective orifice areas of prosthetic heart valves can be easily determined in a flow model by the combination of flow and Doppler echocardiographic measurements. As determinations are based on the same principle, the obtained values should clinically be referred to patients where the corresponding continuity equation for pulsatile flow is used as Ae = stroke volume/time integral of the maximal transvalvular velocity.
