induction-scoring-graft-versus-host-disease-xenogeneic-murine-model

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

University of Kansas Medical Center

The Ikaros family of transcription factors is essential for normal T-cell development, but their expression pattern in human thymocytes remains poorly defined. Our goal is to determine how protein levels of Ikaros, Helios and Aiolos change as human thymocytes progress through the positive selection and lineage commitment stages. To accomplish this goal, we used multi-parameter flow cytometry to define the populations in which positive selection and lineage commitment are most likely to occur. After human thymocytes express CD3 and receive positive selection signals, the cells down-regulate expression of CD4 to become transitional single-positive (TSP) CD8 thymocytes. At this stage, there was a transient increase in the Ikaros, Helios and Aiolos protein levels. After the TSP CD8 developmental stage, some thymocytes re-express CD4 and become CD3 double-positive thymocytes before down-regulating CD8 to become mature single-positive CD4 thymocytes. Except for regulatory T cells, Helios protein levels declined and Aiolos protein levels transiently increased during CD4 T-cell maturation. For thymocytes progressing toward the CD8 T-cell lineage, TSP CD8 thymocytes increase their expression of CD3 and maintain high levels of Aiolos protein as the cells complete their maturation. In summary, we defined the TSP CD8 developmental stage in human T-cell development and propose that this stage is where CD4/CD8 lineage commitment occurs. Ikaros, Helios and Aiolos each undergo a transient increase in protein levels at the TSP stage before diverging in their expression patterns at later stages.

Expression patterns of Ikaros family members during positive selection and lineage commitment of human thymocytes.

The Ikaros family of transcription factors includes five highly homologous members that can homodimerize or heterodimerize in any combination. Dimerization is essential for their ability to bind DNA and function as transcription factors. Previous studies showed that eliminating the function of the entire family blocks lymphocyte development while deletion of individual family members has relatively minor defects. These data indicate that multiple family members function during T cell development, so we examined the changes in expression of each family member as thymocytes progressed from the CD4CD8 double negative (DN) to the CD4CD8 double positive (DP) developmental stage. Further, we compared the expression of each family member in murine and human thymocytes. In both species, Ikaros and Aiolos mRNA levels increased as thymocytes progressed through the DN to DP transition, but the corresponding increases in protein levels were only observed in mice. Further, Ikaros and Aiolos underwent extensive alternative splicing in mice, whereas only Ikaros was extensively spliced in humans. Helios mRNA and protein levels decreased during murine T cell development, but increased during human T cell development. These differences in the expression and splicing of Ikaros family members between human and murine thymocytes strongly suggest that the Ikaros family of transcription factors regulates murine and human T cell development differently, although the similarities across Ikaros family members may allow different proteins to fulfill similar functions.

Expression and splicing of Ikaros family members in murine and human thymocytes.

Regulatory T (Treg) cells are a subset of immune cells that maintain homeostasis by promoting immune tolerance and suppressing the immune response via a variety of mechanisms such as secreting cytokines, killing reactive immune cells, and inducing anergy. Dysfunction of Treg cells has been implicated in inflammatory diseases such as autoimmunity and transplant rejection. Conversely, too many or hyperresponsive Treg cells has been observed in cancer and chronic infections. Treg cells have proven to be difficult to study as there are no definitive Treg surface markers. Additionally, Tregs can gain pro-inflammatory phenotype depending on stimuli. In this commentary, we discuss the expression and function of members of the Ikaros family of transcription factors during Treg cell development and activation.

Amara Seng

Department of Microbiology,
Molecular Genetics,
and Immunology,
Department of Microbiology, Molecular Genetics, and Immunology

University of Kansas Medical Center

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Amara Seng

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microbiology,Molecular Genetics,and Immunology,Department of Microbiology, Molecular Genetics, and Immunology

University of Kansas Medical Center

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Department of Microbiology,
Molecular Genetics,
and Immunology,
Department of Microbiology, Molecular Genetics, and Immunology