[Long-term expansion of T cell progenitors by JAK3 gene transduced primitive hematopoietic cells in vitro].
OBJECTIVE
To explore whether activation of the JAK3-signaling pathway can stimulate long-term expansion of the earliest T cell progenitors from transduced primitive hematopoietic cells and evaluate their potential ability of committed differentiation.


METHODS
A retrovirus vector (RV) containing JAK3 gene, two binding sites for chemical inducers of dimerization (AP20187), and green fluorescence protein (GFP), MGI-F(2)JAK3, was constructed. The RV vector MGI-F(2)JAK3 was then transduced into murine bone marrow hematopoietic cells. The transduced murine bone hematopoietic marrow cells were divided equally into four groups blank control group (No drug group), AP20187 group (added with AP20187 only), SCF group (added with stem cell factor only), and AP20187 + SCF group. Cytometry was used to detect the GFP marker and observe the survival of cells. The murine bone hematopoietic marrow cells expanded for 50 days were divided into 2 groups: one group was washed to remove the cytokines to observe their survival, and AP20187 + SCF was added into the culture fluid of other group. Cytometry and CELLQuest v3.1 software analysis were used to analyze the phenotype of the cells of AP20187 + SCF after 50 days' expansion. The C-kit(hi) CD44(+)CD25(-)TN cells after 50 days' expansion were further cultured under the condition of SCF + IL7 + IL3 for 5 days. The differentiation rate of Thy1.2 positive cells was observed by cytometry. Five Ly5.2 mice underwent radiation of the dose of 600 cGy, and then injected with the expanded cells into the thymus. Three weeks later the mice were killed and their thymus glands were taken out to prepare suspension of single cells to undergo cytometry to observe the proportions of GFP positive CD3 and CD4 mature T lymphocytes.


RESULTS
One week later the cells of the No Drug group all died, the cells of the AP20187 group and SCF group died 2 - 4 weeks later, however, the cells of the AP20187 + SCF continued to grow and expanded up to 10(12)-fold after 50 days' culture. The cells of the AP20187 + SCF group with the cytokines washed died 2 more weeks later, and those with the cytokines washed and added with AP20187 + SCF continued to grow. The phenotype of the expanded proportion was identified as the earliest T cell progenitors expressing C-kit(hi) CD44(+) CD25(-)TN (triple negative). These progenitors could differentiate into Thy1.2 + T cells in the presence of SCF + IL-7 + IL-3 culture condition. 32% - 96% of the mice thymus cell were GFP positive, 5% +/- 0.8% of the thymus cells were GFP + CD(3) double positive, and 11.0% +/- 2.1% of the T lymphocytes were GFP + CD4(+) double positive.


CONCLUSIONS
AP20187 combined with SCF mediating the activation of JAK3 signaling can dramatically expand the earliest T cell progenitors subpopulation, and acquires the capacity to induce the differentiation into T mature cells. This system may help understand the T cell biology and provide a fundamental basement for gene therapy to immunodeficiency disease in the future.
Zhao
Sheng-ming
SM
Graduate School of Peking Union Medical College, Department of Hematology, Beijing Hospital, Beijing 100730, China.
Gu
Xi-chun
XC
Chang
Nai-bai
NB
Xu
Xiao-dong
XD
Peng
Ming-ting
MT
D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D005260:Female; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018380:Hematopoietic Stem Cell Transplantation; D016130:Immunophenotyping; D007377:Interleukin-3 / Q000494:pharmacology; D015851:Interleukin-7 / Q000494:pharmacology; D053616:Janus Kinase 3 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D013950:Thymus Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D015703:Antigens, CD; D007377:Interleukin-3; D015851:Interleukin-7; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C507935:Jak3 protein, mouse; D053616:Janus Kinase 3