Signal-transducing adaptor molecules STAM1 and STAM2 are required for T-cell development and survival

Abstract

We previously reported that the STAM family members STAM1 and STAM2 are phosphorylated on tyrosine upon stimulation with cytokines through the gammac-Jak3 signaling pathway, which is essential for T-cell development. Mice with targeted mutations in either STAM1 or STAM2 show no abnormality in T-cell development, and mice with double mutations for STAM1 and STAM2 are embryonically lethal; therefore, here we generated mice with T-cell-specific double mutations for STAM1 and STAM2 using the Cre/loxP system. These STAM1(-/-) STAM2(-/-) mice showed a significant reduction in thymocytes and a profound reduction in peripheral mature T cells. In proliferation assays, thymocytes derived from the double mutant mice showed a defective response to T-cell-receptor (TCR) stimulation by antibodies and/or cytokines, interleukin-2 (IL-2) and IL-7. However, signaling events downstream of receptors for IL-2 and IL-7, such as activations of STAT5, extracellular signal-regulated kinase (ERK), and protein kinase B (PKB)/Akt, and c-myc induction, were normal in the double mutant thymocytes. Upon TCR-mediated stimulation, prolonged activations of p38 mitogen-activated protein kinase and Jun N-terminal protein kinase were seen, but activations of ERK, PKB/Akt, and intracellular calcium flux were normal in the double mutant thymocytes. When the cell viability of cultured thymocytes was assessed, the double mutant thymocytes died more quickly than controls. These results demonstrate that the STAMs are indispensably involved in T-cell development and survival in the thymus through the prevention of apoptosis but are dispensable for the proximal signaling of TCR and cytokine receptors.

FIG. 1.

Generation of T-cell-specific disruption of both STAM1 and STAM2 genes. (A) Schematic representation of the mSTAM1 cDNA, stam1 genomic locus, targeting vector, and mutated stam1 locus. The positions of stam1 axons are shown as boxes. Restriction sites: B, BamHI; H, HindIII; P, PstI; X, XbaI. (B) Southern blot analysis of the stam1 mutation in mice. Arrows indicate the position of the DNA fragments corresponding to the wild-type (5.5-kb) and mutated (3.0-kb) alleles. (C) PCR analysis of genomic DNA from thymus and tail. PCR primers used are shown in panel A. (D) RT-PCR analysis of total RNA from thymocytes and liver. The primers used are primers E1 and E5, shown in panel A. Note that a shorter PCR product (238-bp) was amplified from Lck-Cre (+) STAM1 lox/lox thymocytes. (E) Immunoprecipitation analysis for STAM1 and STAM2. Lysates of thymocytes from Lck-Cre (+) STAM1^+/+ STAM2^+/+, Lck-Cre (+) STAM1^flox/+ STAM2^+/−, and Lck-Cre (+) STAM1^flox/flox STAM2^−/− mice were immunoprecipitated and then immunoblotted with anti-STAM1 Ab or anti-STAM2 Ab. (F) RT-PCR analysis of total RNA from each thymocyte fraction.

FIG. 2.

Comparison of histological analysis of thymus and thymocyte counts. Size of thymus (A and B), hematoxylin-eosin-stained sections of thymus (C and D), and TUNEL-stained sections of thymus (E and F) from 4-week-old Lck-Cre (+) STAM1^flox/+ STAM2^+/− (A, C, and E) and Lck-Cre (+) STAM1^flox/flox STAM2^−/− (B, D, and F) mice. Magnifications: approximately ×5 (A and B), ×20 (C and D), and ×40 (E and F). (G) Thymocyte counts in 4-week-old Lck-Cre (+) STAM1^flox/+ STAM2^+/− and Lck-Cre (+) STAM1^flox/flox STAM2^−/− mice. The bar indicates the average of thymocyte counts.

FIG. 3.

Flow-cytometric analysis. Total thymocytes, DN thymocytes, splenocytes, and lymph node cells derived from 4-week-old Lck-Cre (+) STAM1^flox/+ STAM2^+/− (n = 8) (left) and Lck-Cre (+) STAM1^flox/flox STAM2^−/− (right) (n = 8) mice were stained with Abs for CD4 and CD8, TCRβ, CD25 and CD44, or CD3 and B220. Numbers indicate the average percentages of the gated cellular subpopulations within the lymphocyte population.

FIG. 4.

Proliferative responses of thymocytes. Total thymocytes (2 × 10⁵ per well) (A) and CD4 SP thymocytes (5 × 10⁴ per well) (B) were stimulated with indicated ligands: plate-coated anti-CD3 MAb (10 mg/ml), plate-coated anti-CD28 MAb (1 mg/ml), murine IL-2 (10 nM), murine IL-7 (10 ng/ml), phorbol myristate acetate (10 ng/ml), and ionomycin (1 mg/ml). They were cultured for 42 h and were then pulsed with [³H]thymidine and harvested after 6 h.

FIG. 5.

In vitro cell survival of thymocytes and Bcl-2 expression in thymocytes. (A) In vitro cell survival experiment using DP thymocytes and CD4 SP thymocytes. After the indicated culture time, cells were stained with annexin V. Viable cells were defined as annexin-V-negative cells in each fraction of thymocytes. Error bars, standard deviations. (B) Bcl-2 expression levels in thymocyte fractions. Each fraction of thymocytes was stained with either isotopic control MAb (dotted line) or anti-Bcl-2 mouse MAb (solid line). The results, analyzed by gating CD4⁻/CD8⁻ (DN) cells, CD4⁺/CD8⁺(DP), CD4⁺ (CD4 SP), or CD8⁺ (CD8 SP) cells, are shown. Representative results of two independent experiments are presented.

FIG. 6.

TCR-induced tyrosine phosphorylation, activation of ERK, JNK, and p38MAPKs, and intracellular calcium fluxes in thymocytes. (A and B) Double-positive thymocytes were incubated with anti-CD3 Ab for the indicated times. Induction of tyrosine phosphorylations of total proteins (A), ERK (B) and JNK (B) in whole-cell lysates was analyzed by immunoblotting. p38MAPK activity was measured using an immunocomplex kinase assay (B). (C) CD4 single-positive thymocytes were incubated with anti-CD3 Ab for the indicated times. Induction of ERK, JNK, and p38MAPK phosphorylation was analyzed by immunoblotting. (D) Intracellular calcium fluxes were measured in double-positive thymocytes or CD4 single-positive thymocytes using Fluo-3. Biotinylated anti-CD3 Ab was first added at 0 s, and then streptavidin was added at 20 s (arrow indicated) for cross-linking.

FIG. 7.

Cytokine-mediated signaling in thymocytes. (A) CD4 SP thymocytes were incubated with murine IL-7 for the indicated times and analyzed for phosphorylation of STAT5, ERK, and Akt by immunoblotting. (B) Total thymocytes were incubated with plate-coated anti-CD3 MAb alone (10 mg/ml) or plate-coated anti-CD3 MAb (10 mg/ml) plus murine IL-2 (10 nM) or murine IL-7 (10 ng/ml) for the indicated times. Total RNA prepared from the cells was first hybridized with the c-myc probe and then rehybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe.