Pediatric Thymus Research Articles

Extrathymic human t lymphocyte development and regulation in immune deficient mice

Abstract We previously studied extrathymic human T lymphocyte development from stem/progenitor subsets in the bone marrow of bnx mice. Current studies examined human T cell development from purified CD34 + CD3 − stem cells in a new strain that we developed, nude/NOD/SCID mice. Human T cells isolated from bnx mice contained double CD4 + /CD8 + and single CD3 + /CD4 + or CD3 + /CD8 + cells, but they were anergic to activation by PMA plus ionomycin (P+1) and to CD3 and CD28 crosslinking. In nude/NOD/SCID mice, there was more variability in human T cell development in the marrow. Double positive CD4/CD8 and CD4 + /cytoCD3 + populations, which expressed CD3 mRNA, were found in some of the mice. But no CD8 single positive cells were found in any mice. In some mice, only myeloid progenitor cells and B cells were found, much like the NOD/SCID strain. The differences may be due to different microenvironments or to the types of human cells engrafted in the starting populations. Studying the influence of implanting human pediatric thymus slices into nude/NOD/SCID on the development of human T cells is in progress. Thymus grafts get well vascularized in the peritoneal cavity of mice. Early studies suggest that the presence of the thymus makes the human T cells less hyporesponsive. Future studies will detemine whether the human cells are being selected in the thymus or whether thymic stromal factors are influencing development and/or activation of human T cells.

William H. Crane of Cincinnati and the first irradiation of the pediatric thymus, 1905.

William H. Crane of Cincinnati and the first irradiation of the pediatric thymus, 1905.A E OestreichAudio Available | Share

CD34-expressing human thymocyte precursors proliferate in response to interleukin-7 but have lost myeloid differentiation potential

CD34 is a marker for pluripotent stem cells also present on lineage- committed hematopoietic progenitors from bone marrow and a subpopulation of immature thymocytes. To characterize these early immature thymocytes, we have studied 24 pediatric thymus samples for CD34/7 expression. Three subpopulations could be defined from these T- cell receptor (TcR-) immature thymocytes: CD34+7++ (12.0 +/- 5.8), CD34- 7++ (12.6 +/- 8.6), and CD34-7+ (71.5 +/- 17.0%). CD7++ represents upregulation of this antigen and is expressed by cells of a blast-like morphology. Three-color flow cytometric analysis of these three subsets suggests the following ordered differentiation sequence: CD34+7++1-4-8- 45RA+-->CD34+7++1+ 4+8-45RA+/- -->CD34-7++1+4+8-+45RO+-->CD34- 7+1++4+8+45RO+. Early immature thymocyte cell division is essential in the thymus to generate a large number of precursors before the initiation of the selection process. We observed that both CD2 as well CD28 activation pathways were inefficient to serve as costimulant with phorbol ester 12-O-tetradecanoyl phorbol 13-acetate or interleukin-2 (IL-2) to induce the proliferation of the three CD34/7 subsets isolated by cell sorting. However, whereas IL-1, IL-2, IL-3, IL-4, granulocyte colony-stimulating factor, and granulocyte-macrophage colony- stimulating factor were ineffective, IL-7 was a potent cytokine, alone or in synergy with stem cell factor (SCF) to induce immature thymocyte proliferation. The proliferation induced by IL-7 or IL-7 + SCF is restricted to the CD34+ cells and, after 4 or 8 days of culture with IL- 7, some CD34+7++ acquire the expression of CD4 and/or CD8, but remain CD3/TcR-. We also tested the myeloid differentiation capacity of these CD34 immature thymocytes. Using two different approaches, myeloid colony formation in methylcellulose and limiting dilution analysis in the presence of myeloid growth factors, we were unable to detect myeloid differentiation capacity from CD34+ early thymocytes, whereas CD34+7+ from bone marrow contained about 10% of the clonogenic cells present in the CD34+7- fraction. Together, these data support the concept that thymic CD34+7++ represents the earliest thymic subset of fully committed T-lineage cells, capable of proliferating specifically to IL-7.

CD34-Expressing Human Thymocyte Precursors Proliferate in Response to Interleukin-7 But Have Lost Myeloid Differentiation Potential

AbstractCD34 is a marker for pluripotent stem cells also present on lineage-committed hematopoietic progenitors from bone marrow and a subpopulation of immature thymocytes. To characterize these early immature thymocytes, we have studied 24 pediatric thymus samples for CD34/7 expression. Three subpopulations could be defined from these T-cell receptor (TcR-) immature thymocytes: CD34+7++ (12.0 +/- 5.8), CD34- 7++ (12.6 +/- 8.6), and CD34-7+ (71.5 +/- 17.0%). CD7++ represents upregulation of this antigen and is expressed by cells of a blast-like morphology. Three-color flow cytometric analysis of these three subsets suggests the following ordered differentiation sequence: CD34+7++1-4-8- 45RA+–>CD34+7++1+ 4+8-45RA+/- –>CD34-7++1+4+8-+45RO+–>CD34- 7+1++4+8+45RO+. Early immature thymocyte cell division is essential in the thymus to generate a large number of precursors before the initiation of the selection process. We observed that both CD2 as well CD28 activation pathways were inefficient to serve as costimulant with phorbol ester 12-O-tetradecanoyl phorbol 13-acetate or interleukin-2 (IL-2) to induce the proliferation of the three CD34/7 subsets isolated by cell sorting. However, whereas IL-1, IL-2, IL-3, IL-4, granulocyte colony-stimulating factor, and granulocyte-macrophage colony- stimulating factor were ineffective, IL-7 was a potent cytokine, alone or in synergy with stem cell factor (SCF) to induce immature thymocyte proliferation. The proliferation induced by IL-7 or IL-7 + SCF is restricted to the CD34+ cells and, after 4 or 8 days of culture with IL- 7, some CD34+7++ acquire the expression of CD4 and/or CD8, but remain CD3/TcR-. We also tested the myeloid differentiation capacity of these CD34 immature thymocytes. Using two different approaches, myeloid colony formation in methylcellulose and limiting dilution analysis in the presence of myeloid growth factors, we were unable to detect myeloid differentiation capacity from CD34+ early thymocytes, whereas CD34+7+ from bone marrow contained about 10% of the clonogenic cells present in the CD34+7- fraction. Together, these data support the concept that thymic CD34+7++ represents the earliest thymic subset of fully committed T-lineage cells, capable of proliferating specifically to IL-7.

T-Cell Receptor β-Chain Gene Rearrangement and Expression During Human Thymic Ontogenesis

T-cell receptor (TCR) beta-chain proteins appear early (approximately 15th week of gestation) during human thymic ontogenesis. These beta-chain proteins, which appear before terminal deoxynucleotidyl transferase (TdT), could be an expression of a fully rearranged (V-D-J), incompletely rearranged (D-J), or germline TCR beta-chain gene. The aims of this study, performed from the 15th week onward, were the following: (1) to investigate whether or not TCR beta gene rearranges at an early stage during human thymic ontogenesis; (2) to investigate whether complete presumptive functional (1.3 kb) TCR beta gene transcript is present at these early stages of development, or if incomplete (1 kb) or germ-line (1.1 kb) transcripts are expressed; (3) to examine the phenotype of TCR beta-chain+ cells with two-color fluorescence using monoclonal antibody (MoAb) beta F1 and MoAbs that recognize CD1, CD2, CD3, CD4, CD8, CD5, and CD7 antigens (rabbit anti-calf TdT antiserum was used to detect TdT); and (4) to demonstrate whether or not beta gene N-diversity regions are detectable as early as the 15th week and whether or not N-nucleotide insertions correlate to TdT expression. Fifteen- to 22-week fetal thymuses and pediatric thymuses were investigated. We demonstrated that TCR beta-chain gene rearranged as early as the 15th week in human thymus and that a complete functional TCR beta gene transcript was expressed at these early stages of human development. No other analyses to date have investigated TCR beta gene expression in early human thymus using molecular biology techniques. No significant differences were detectable between phenotypic analysis of fetal and pediatric samples, except for TdT expression, which appeared after the 20th week. Essentially all mCD3+ (OKT3+) cells were beta-chain+ at the different weeks investigated. A significant percentage of CD1+ cells were beta-chain+, and the percentage increased along with the age of development. After the 20th week, we identified three main populations: TdT+, cCD3+, beta F-(early thymic precursors); TdT+, CD1+, beta F1+ (intermediate maturity cortical thymocytes); and TdT-, mCD3+, beta F1++ (mature medullary thymocytes). Given these values, we may consider beta-chain expression an ordered process. beta gene N-nucleotide insertions were correlated to TdT expression, since N-regions increased considerably after the 20th week. A further increase of N-nucleotide insertions was detected from the 22nd week to the 32nd week.

T-cell receptor beta-chain gene rearrangement and expression during human thymic ontogenesis

T-cell receptor (TCR) beta-chain proteins appear early (approximately 15th week of gestation) during human thymic ontogenesis. These beta- chain proteins, which appear before terminal deoxynucleotidyl transferase (TdT), could be an expression of a fully rearranged (V-D- J), incompletely rearranged (D-J), or germline TCR beta-chain gene. The aims of this study, performed from the 15th week onward, were the following: (1) to investigate whether or not TCR beta gene rearranges at an early stage during human thymic ontogenesis; (2) to investigate whether complete presumptive functional (1.3 kb) TCR beta gene transcript is present at these early stages of development, or if incomplete (1 kb) or germ-line (1.1 kb) transcripts are expressed; (3) to examine the phenotype of TCR beta-chain+ cells with two-color fluorescence using monoclonal antibody (MoAb) beta F1 and MoAbs that recognize CD1, CD2, CD3, CD4, CD8, CD5, and CD7 antigens (rabbit anti- calf TdT antiserum was used to detect TdT); and (4) to demonstrate whether or not beta gene N-diversity regions are detectable as early as the 15th week and whether or not N-nucleotide insertions correlate to TdT expression. Fifteen- to 22-week fetal thymuses and pediatric thymuses were investigated. We demonstrated that TCR beta-chain gene rearranged as early as the 15th week in human thymus and that a complete functional TCR beta gene transcript was expressed at these early stages of human development. No other analyses to date have investigated TCR beta gene expression in early human thymus using molecular biology techniques. No significant differences were detectable between phenotypic analysis of fetal and pediatric samples, except for TdT expression, which appeared after the 20th week. Essentially all mCD3+ (OKT3+) cells were beta-chain+ at the different weeks investigated. A significant percentage of CD1+ cells were beta- chain+, and the percentage increased along with the age of development. After the 20th week, we identified three main populations: TdT+, cCD3+, beta F-(early thymic precursors); TdT+, CD1+, beta F1+ (intermediate maturity cortical thymocytes); and TdT-, mCD3+, beta F1++ (mature medullary thymocytes). Given these values, we may consider beta-chain expression an ordered process. beta gene N-nucleotide insertions were correlated to TdT expression, since N-regions increased considerably after the 20th week. A further increase of N-nucleotide insertions was detected from the 22nd week to the 32nd week.