1. Overexpression of suppressor of cytokine signaling-1 impairs pre-T-cell receptor–induced proliferation but not differentiation of immature thymocytes
by Sébastien Trop, Paulo De Sepulveda, Juan Carlos Zúñiga-Pflücker, and Robert Rottapel
Blood
Volume 97(8):
April 15, 2001
©2001 by American Society of Hematology

2. SOCS-1 is expressed at all stages of T-cell development
SOCS-1 is expressed at all stages of T-cell development.Thymocytes from CD1 fetal thymic lobes or adult thymus were fractionated according to surface expression of CD25 and CD117 (DNI-DNIV) or CD4 and CD8 (DP and SP).
SOCS-1 is expressed at all stages of T-cell development.Thymocytes from CD1 fetal thymic lobes or adult thymus were fractionated according to surface expression of CD25 and CD117 (DNI-DNIV) or CD4 and CD8 (DP and SP). The cDNAs were prepared from total RNA, and serial 3-fold dilutions of cDNAs were analyzed for expression of β-actin and SOCS-1 mRNA by RT-PCR. As control (H2O), RT-PCR reactions lacking template were amplified simultaneously.
Sébastien Trop et al. Blood 2001;97:
©2001 by American Society of Hematology

3. Experimental strategy
Experimental strategy.(A) Retrovirus constructs were generated by subcloning cDNA for wild-type SOCS-1 or mutated SOCS-1 lacking a functional SH2 domain (SOCS-1:SH2*) into the MIEV retroviral vector.
Experimental strategy.(A) Retrovirus constructs were generated by subcloning cDNA for wild-type SOCS-1 or mutated SOCS-1 lacking a functional SH2 domain (SOCS-1:SH2*) into the MIEV retroviral vector. The presence of an internal ribosomal entry site (IRES) upstream of a GFPreporter gene leads to the production of bicistronic mRNA under the control of the 5′ long terminal repeat (LTR). As control, a retroviral vector was generated containing only the Pcmvpromoter derived from the pcDNA3.1 plasmid. (B) Experimental procedure for retroviral gene transfer into fetal liver–derived hematopoietic precursors (top) or fetal thymic lobes (bottom). Hematopoietic precursors were enriched from fetal liver by complement-mediated depletion of CD24+ cells. Precursors were then cocultured on a monolayer of GP+E 86 retroviral packaging cells. The following day, infected cells were sorted based on the coexpression of GFP and CD117 and subsequently introduced into dGuo-treated fetal thymic lobes to allow T-cell differentiation to occur. Alternatively, fetal thymic lobes were cocultured with GP+E 86 cells and subsequently cultured under normal FTOC conditions.
Sébastien Trop et al. Blood 2001;97:
©2001 by American Society of Hematology

4. Overexpression of SOCS-1 abrogates T-cell differentiation from hematopoietic progenitors.Fetal liver–derived hematopoietic precursors were infected with the indicated retrovirus constructs and introduced into dGuo-treated FTOC. (A) Single-cell suspensions w...
Overexpression of SOCS-1 abrogates T-cell differentiation from hematopoietic progenitors.Fetal liver–derived hematopoietic precursors were infected with the indicated retrovirus constructs and introduced into dGuo-treated FTOC. (A) Single-cell suspensions were obtained from fetal thymic lobes at day 6 or 14 of culture and analyzed for surface expression of CD4 and CD8 or of CD25 and CD117; percentages of cells in each quadrant are shown. The analysis was confined to infected donor cells by gating on GFP expression, as indicated by the rectangular gate in the GFP versus forward light scatter (FSC) dot plots. (B) Single-cell suspensions were obtained from fetal thymic lobes at day 4 of culture and analyzed for the presence of GFP-expressing cells. As control in each set of experiments, fetal thymic lobes receiving no donor cells were also analyzed.
Sébastien Trop et al. Blood 2001;97:
©2001 by American Society of Hematology

5. Overexpression of SOCS-1 impairs T-cell differentiation from committed thymic precursors.Fetal thymic lobes were infected with the indicated retrovirus constructs for 3 days and subsequently cultured for a further 1, 3, or 6 days.
Overexpression of SOCS-1 impairs T-cell differentiation from committed thymic precursors.Fetal thymic lobes were infected with the indicated retrovirus constructs for 3 days and subsequently cultured for a further 1, 3, or 6 days. Single-cell suspensions were obtained from fetal thymic lobes at the indicated time and analyzed for surface expression of CD4 and CD8; percentages of cells in each quadrant are shown. The analysis was confined to infected donor cells by gating on GFP expression, as indicated by the rectangular gate in the GFP versus forward light scatter (FSC) dot plots; cell counts for the absolute number of GFP+ cells per lobe are shown in parentheses. Data obtained with uninfected (GFP−) cells are provided as a control for the ability of the thymic lobes to sustain normal T-cell development.
Sébastien Trop et al. Blood 2001;97:
©2001 by American Society of Hematology

6. Expression of SOCS-1 is suppressed following pre-TCR signaling
Expression of SOCS-1 is suppressed following pre-TCR signaling.Neonatal RAG2 −/− mice were injected intraperitoneally with anti-CD3ε mAb.
Expression of SOCS-1 is suppressed following pre-TCR signaling.Neonatal RAG2 −/− mice were injected intraperitoneally with anti-CD3ε mAb. Injections were given 36 or 72 hours prior to harvest to obtain CD25Low or ISP and DP thymocytes, respectively. CD25+ thymocytes were obtained from untreated RAG2 −/− mice. The cDNAs were prepared from total RNA and analyzed by RT-PCR for expression of β-actin, SOCS-1, TCRα (germline transcripts), and CD45 mRNA. As control (H2O), RT-PCR reactions lacking template were amplified simultaneously.
Sébastien Trop et al. Blood 2001;97:
©2001 by American Society of Hematology

7. Overexpression of SOCS-1 hinders pre-TCR–driven expansion of immature thymocytes.Fetal thymic lobes from RAG2 −/− mice were infected with the indicated retrovirus constructs for 2 days and subsequently cultured for a further 6 days in the presence of anti-C...
Overexpression of SOCS-1 hinders pre-TCR–driven expansion of immature thymocytes.Fetal thymic lobes from RAG2 −/− mice were infected with the indicated retrovirus constructs for 2 days and subsequently cultured for a further 6 days in the presence of anti-CD3ε mAb. Single-cell suspensions were obtained from fetal thymic lobes and analyzed for surface expression of CD4 and CD8; percentages of cells in each quadrant are shown. Results obtained for uninfected (GFP−) and infected (GFP+) donor cells are presented; cell counts for the absolute number of GFP+ cells per lobe are shown in parentheses. The ratio—calculated from the total thymic cellularity—of infected cells (GFP+) to uninfected cells (GFP−) expressing CD4/CD8 coreceptors is provided.
Sébastien Trop et al. Blood 2001;97:
©2001 by American Society of Hematology

8. The γc family of cytokines is not required for transition to the CD4+CD8+ stage.(A) The IL-7–dependent cell line B23 was infected with the MIEV–SOCS-1 retroviral vector or with the empty MIEV vector as control.
The γc family of cytokines is not required for transition to the CD4+CD8+ stage.(A) The IL-7–dependent cell line B23 was infected with the MIEV–SOCS-1 retroviral vector or with the empty MIEV vector as control. Stably infected cells were obtained by sorting for GFP expression. Cells were then cultured for the indicated time and pulsed with [3H]thymidine for 6 hours to measure cell proliferation. Mean values ± SD for triplicate samples are given. (B) Newborn RAG2 −/− andRAG2 −/− × γc−/− mice were injected with anti-CD3ε mAb or with control Ab (Armenian hamster [Ahm] IgG). After 3 days, single-cell suspensions were prepared from the thymus and analyzed for surface expression of CD4 and CD8 by flow cytometry; percentages of cells in each quadrant are shown. Absolute cell numbers were calculated (mean ± SD) from 3 to 6 mice of each genotype. (C) Role of SOCS-1 during early T-cell development. A simple scheme of early T-cell development is shown that is adapted from Di Santo et al36; solid and dashed lines indicate where overexpression of SOCS-1 affects a complete or partial block in T-cell development, respectively. Bold circles represent cycling cells.
Sébastien Trop et al. Blood 2001;97:
©2001 by American Society of Hematology