The linker for activation of T cells (LAT) is an adaptor protein that couples TCR engagement to downstream signaling cascades. LAT is important in early thymocyte development as LAT-deficient mice have a complete block at the double-negative (DN) 3 stage. To study the role of LAT beyond the DN3 stage, we generated mice in which the lat gene could be deleted by the Cre recombinase. Analysis of these mice showed that deletion of LAT after the DN3 stage allowed thymocytes to develop past the DN3 to DN4 checkpoint and to generate double-positive thymocytes. However, LAT-deficient DP thymocytes were severely defective in responding to stimulation via the TCR and failed to differentiate into single-positive thymocytes efficiently. Consequently, few LAT-deficient mature T cells could be found in the periphery. These T cells had undergone extensive homeostatic proliferation and expressed low levels of the TCR on their surface. Collectively, our data indicate that in addition to its role in pre-TCR signaling, LAT also plays an essential role in thymocyte development during transition from the double-positive to single-positive stage.

Linker for activation of T cells (LAT)3 is a membrane-associated adaptor protein that couples the engagement of TCR to Ras-MAPK activation and Ca2+ mobilization. Upon TCR engagement, LAT is phosphorylated by ZAP-70 tyrosine kinase on its membrane-distal tyrosine residues and directly interacts with Grb2, Gads, and PLCγ1 (1, 2, 3, 4, 5). LAT binding of Gads recruits an important cytosolic adaptor protein, SLP-76, to the membrane, which can further interact with PLCγ1, Vav1, and other signaling molecules (6, 7). Together, LAT and SLP-76 bring PLCγ1 to the plasma membrane to be phosphorylated and activated. Activated PLCγ1 then hydrolyzes PIP2 into IP3 and DAG. IP3 interacts with the IP3 receptor and induces intracellular Ca2+ mobilization, whereas DAG activates PKCs and binds to RasGRP1 to activate the Ras-MAPK pathway (8, 9). LAT also contributes to Ras-MAPK activation through the recruitment of Grb2, which in turn brings Sos to the plasma membrane to activate Ras (10).

The essential role of LAT in T cell development has been clearly demonstrated in LAT-deficient mice. Thymocyte development in LAT−/− mice is completely blocked at the CD25+CD44 double-negative (DN) 3 stage, indicating an absolute requirement for LAT in pre-TCR-mediated signal transduction. As a result, LAT−/− mice completely lack double-positive (DP) and single-positive (SP) thymocytes in the thymus and mature T cells in the periphery (11). Similar phenotypes were seen in mice with the four membrane-distal tyrosines (Y136, Y175, Y195, and Y235) mutated simultaneously, indicating the importance of these tyrosines in LAT function (12). Interestingly, mice with a mutation at the PLCγ1-binding site of LAT (Y136) have a partial block in thymocyte development; however, they develop a polyclonal lymphoproliferative disease due to hyperactivation and expansion of CD4+ T cells (13, 14, 15). While both positive and negative thymocyte selection are impaired, the development of the naturally arising CD4+CD25+ regulatory T (Treg) cells is severely impaired in the LAT Y136F mice (16). Introduction of CD4+CD25+ Treg cells into these mice is able to prevent the development of the autoimmune disease (16). Interestingly, mice with mutations at the other three membrane-distal tyrosines (Y175, Y195, and Y235) also develop a similar disease (17). These data suggest that LAT-mediated signaling is important in T cell activation and the control of autoimmunity.

The early block of thymocyte development in LAT−/− mice makes it impossible to study the function of LAT beyond the DN3 stage. Thus, the role of LAT in the transition from DN3 to DP and, further, from DP to SP stages remains unclear. Additionally, the function of LAT in TCR-mediated signaling events, such as Ras-MAPK activation and calcium mobilization, has mostly been demonstrated in Jurkat cell lines (2, 3). Therefore, its role in primary T cells remains to be determined. To address these questions, we generated LAT knock-in mice in which the lat gene could be deleted upon expression of the Cre recombinase. By using CD4Cre transgenic mice, we successfully deleted LAT between the DN3 and DP stages. Our data showed that deletion of LAT after the DN3 stage allowed for the development of DP thymocytes. However, these LAT-deficient DP thymocytes were not able to respond to stimulation from the TCR and failed to further differentiate into mature T cells. Our data indicate that LAT plays an essential role in thymocyte development during transition from the DP to SP stage.

The targeted embryonic stem cells were injected into 129/Sv blastocysts to generate chimeric mice, which were subsequently crossed with Flp transgenic mice to generate LATf/+ mice. LATf/+ mice were backcrossed with C57BL/6 mice for at least six generations before analysis. Flp, CD4Cre, and β-actin-Cre mice were purchased from The Jackson Laboratory. LATf/f mice were crossed with CD4Cre+LAT−/− mice to generate CD4Cre+LATf/− and LATf/− mice. All mice were used in accordance with the National Institutes of Health guidelines. The experiments described in this study were reviewed and approved by the Duke University Institutional Animal Care Committee. Mice were housed in specific pathogen-free conditions.

Fluorescence-conjugated Abs used in flow cytometry, such as anti-CD3, CD4, CD8, CD25, CD44, CD62L, CD5, CD69, HSA, TCRβ, TCRγδ, and Foxp3, were all purchased from eBioscience. For cell surface marker staining, single-cell suspensions were prepared from mouse thymi, lymph nodes, or spleens and were incubated with the 2.4G2 Ab (anti-Fcγ receptor) before staining with different Ab mixtures. Further intracellular staining of Foxp3 was performed using the Foxp3 staining buffer set (eBioscience) according to the instructions from the manufacturer. FACS data were acquired on FACSDiva (BD Biosciences) and analyzed with the FlowJo software. DP thymocytes were sorted on FACSDiva after staining with Abs against CD4 and CD8.

FACS-sorted DP cells (5 × 106) were either left untreated or stimulated by crosslinking CD3 for 2 min, and they were subsequently lysed in RIPA buffer. The postnuclear lysates were resolved on SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were then blotted with different primary Abs. The anti-pErk, pPLCγ1, and PLCγ1 Abs were purchased from Cell Signaling Technology. Anti-Erk2 was purchased from Santa Cruz Biotechnology. The anti-LAT (11B12) and anti-pY (4G10) Abs were from Upstate Biotechnology. For secondary Abs, Alexa Fluor 680 anti-mouse-IgG (Molecular Probes) or IRDye 800 anti-rabbit-IgG (Rockland) were used accordingly. The membranes were scanned by the LI-COR Odyssey infrared imaging system.

Thymocytes were first loaded with Indo-1 (Molecular Probes) in loading buffer (1× HBSS with 10 mM HEPES and 1% FBS) for 30 min and then stained with PE-anti-CD4 and PECy5-anti-CD8 Abs. Calcium flux was initiated by the addition of biotinylated anti-CD3 (5 μg/ml) and anti-CD4 (1 μg/ml) or anti-CD8 (1 μg/ml) followed by crosslinking with streptavidin (25 μg/ml final concentration; Sigma-Aldrich). Ionomycin (2 μg/ml; Sigma-Aldrich) was used to induce TCR-independent calcium flux to ensure equal loading of Indo-1 dye. Calcium flux was assayed by monitoring the fluorescence emission ratio at 405/510 nm with a BD FACStar flow cytometer (BD Biosciences) and analyzed using the FlowJo software.

LAT-deficient mice have an early block in thymocyte development in the DN3 stage. To study the function of LAT in T cells beyond DN3, we generated mice in which the lat gene can be deleted conditionally upon expression of the Cre recombinase. As shown in Fig. 1 a, the lat gene consists of 12 exons with a stop codon at exon 11 and the 3′ untranslated region at exon 12. We replaced exons 7–11 with an artificial exon containing the corresponding cDNA fragment of these exons. These exons encode the C-terminal region of LAT, which has six membrane-distal tyrosine residues. These tyrosine residues are important for LAT function in T cell activation and development; mutation of these residues renders LAT nonfunctional (12, 18). To monitor deletion of the lat gene, we inserted an artificial exon containing a modified gfp gene that lacks its own start codon. Without deletion of the lat gene, GFP should not be expressed from the floxed allele, because the lat stop codon precedes it. However, upon expression of the Cre recombinase, the floxed lat sequence, including the stop codon, will be deleted, and a LAT-GFP fusion will be expressed from the deleted allele. Therefore, T cells should be GFP+ after deletion of LAT.

FIGURE 1.

Generation of LAT knock-in mice. a, The LAT knock-in targeting strategy. Twelve lat exons are indicated. The initiation codon (*) is at the exon 1. The stop codon (•) is at the exon 11. Exon 7–11 in the targeting construct was made from the corresponding lat cDNA fragment. The gray triangles represent the FRT sites. The black triangles represent the LoxP sites. b, T cell development in LATf/−, LAT+/−, LATd/−, and LAT−/− mice. The thymi and spleens from 5-wk-old mice were analyzed by FACS. Top, CD4 vs CD8 profile of thymocytes. Middle, CD25 vs CD44 profile of the CD4CD8 (DN) thymocytes. Bottom, CD4 vs CD8 profile of splenocytes. c, The GFP expression profile in different subsets of thymocytes from the age-matched LAT+/− and LATd/+ mice.

FIGURE 1.

Generation of LAT knock-in mice. a, The LAT knock-in targeting strategy. Twelve lat exons are indicated. The initiation codon (*) is at the exon 1. The stop codon (•) is at the exon 11. Exon 7–11 in the targeting construct was made from the corresponding lat cDNA fragment. The gray triangles represent the FRT sites. The black triangles represent the LoxP sites. b, T cell development in LATf/−, LAT+/−, LATd/−, and LAT−/− mice. The thymi and spleens from 5-wk-old mice were analyzed by FACS. Top, CD4 vs CD8 profile of thymocytes. Middle, CD25 vs CD44 profile of the CD4CD8 (DN) thymocytes. Bottom, CD4 vs CD8 profile of splenocytes. c, The GFP expression profile in different subsets of thymocytes from the age-matched LAT+/− and LATd/+ mice.

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Targeted embryonic stem cells were used to generate mice with the floxed lat allele (LATf/+). To examine whether the floxed allele is functional, we crossed LATf/+ mice with LAT knockout mice (LAT−/−). Analysis of thymocyte development in LATf/− mice showed that T cells developed normally. The size of thymus from LATf/− mice was similar to that from LAT+/− mice. Normal percentages of DP and SP thymocytes were seen (Fig. 1,b). Mature T cells were also present in the periphery of LATf/− mice, although at a lower percentage, likely due to a reduced level of LAT expression (Fig. 1 b). T cells from LATf/− mice expressed normal TCR levels on their cell surface (data not shown). These data indicated that the floxed lat allele is functional during thymocyte development.

To show the loss of function after deletion, we first crossed LATf/+ mice with β-actin Cre transgenic mice, in which the Cre recombinase is ubiquitously expressed, to generate LATd/+ mice (d, deleted), and then crossed LATd/+ mice with LAT−/− mice to obtain LATd/− mice. Similar to LAT−/− mice, these LATd/− mice showed a profound block in thymocyte development at the DN3 stage and contained no mature T cells in the periphery (Fig. 1 b). These data indicated that the floxed lat allele can be successfully deleted upon Cre expression and that such deletion renders LAT nonfunctional during thymocyte development.

Additionally, since GFP was inserted into the lat locus, GFP expression upon deletion of LAT should be able to mark cells that normally express LAT. To examine LAT expression during thymocyte development, we monitored GFP expression in different subsets of thymocytes in LATd/+ mice. Corresponding thymocyte subsets from LAT+/− mice were used as negative controls in FACS analysis. As shown in Fig. 1 c, the GFP fluorescence signal was detected in as early as the DN1 stage, although the expression level was low when compared with other subsets of thymocytes. GFP expression was seen throughout the later stages of thymocyte development from DN2 to SP. Mature T cells in the periphery of LATd/+ mice expressed GFP as well (data not shown). In contrast, mature B cells were GFP (data not shown). We did not observe any T cell development defects in LATd/+ as compared with LAT+/− mice (data not shown), suggesting that the LAT-GFP fusion protein does not exert a dominant negative role during thymocyte development. These data showed that GFP can be expressed upon deletion of the lat gene and can be used as a marker to monitor the lat gene deletion on a single-cell basis.

To study LAT function during thymocyte development beyond DN3, we crossed LAT knock-in mice (LATf/f) with CD4Cre transgenic mice that had been bred onto the LAT−/− background (CD4Cre+LAT−/−) to generate CD4Cre+LATf/− mice. In these mice, expression of the Cre recombinase is driven by the CD4 proximal promoter and is initiated at the late DN3 stage (19, 20). Thymocyte development in CD4Cre+LATf/− mice should progress through the DN3 checkpoint and possibly beyond.

Thymi from 4-wk-old CD4Cre+LATf/− mice were slightly smaller than those from their littermate controls (LATf/−), and their total cellularity was relatively normal (Fig. 2,b). FACS analysis of thymocytes showed that, in contrast to LAT−/− mice, which lack both DP and SP thymocytes, DP thymocytes were clearly present in CD4Cre+LATf/− mice (Fig. 2,a). The percentage of DP thymocytes in these mice was slightly increased compared with LATf/− controls (Fig. 2,a); the absolute number of DP thymocytes was similar (Fig. 2,b). The percentage of DN thymocytes in CD4Cre+LATf/− mice was slightly increased (Fig. 2,a), although the number of DN thymocytes was similar (Fig. 2,b). Despite a large number of DP thymocytes, very few SP thymocytes were present in the CD4Cre+LATf/− mice. Both the percentages and the numbers of SP thymocytes were dramatically reduced compared with LATf/− controls (Fig. 2, a and b). To confirm Cre-mediated deletion, we analyzed GFP expression in different thymocyte subsets of CD4Cre+LATf/− mice. As shown in Fig. 2,c, GFP+ cells appeared as early as the DN3 stage, although the percentage of GFP+ DN3 cells was very low. This is consistent with previous publications showing that Cre activity is initiated in the late DN3 stage in this CD4-Cre transgenic line (20). A small number of GFP+ DN4 or immature single-positive (ISP) cells were detected as well. Most of the DP thymocytes clearly expressed GFP. To confirm the absence of LAT protein after deletion, GFP+ DP cells from CD4Cre+LATf/− mice were sorted by FACS, with DP cells from LATf/− mice also sorted as a control. Lysates of these cells were subjected to Western blotting by an anti-LAT Ab. As shown in Fig. 2 d, LAT protein was not detected in GFP+ DP cells from CD4Cre+LATf/− mice. These data indicated that, although Cre-mediated deletion of LAT started in as early as the DN3 stage, such deletion mainly occurred in, and was nearly completed by, the DP stage.

FIGURE 2.

Thymocyte development in the CD4Cre+LATf/− mice. a, The CD4 vs CD8 expression profile of total thymocytes from 4-wk-old LATf/− and CD4Cre+LATf/− littermates. The figure shown is one representative of five mice analyzed. b, The total numbers of different thymocyte populations from 4-wk-old LATf/− and CD4Cre+LATf/− littermates. Five mice for each genotype were analyzed. ∗∗, p < 0.01 (Student’s t test). c, GFP expression in different subsets of thymocytes. d, Western blot analysis of LAT expression in GFP+ DP thymocytes. GFP+ DP thymocytes were sorted by FACS and lysed. Postnuclear lysates were analyzed by Western blotting. An anti-Erk2 blot was used as a loading control. e, The CD25 vs CD44 expression in the DN thymocytes from the LATf/− and CD4Cre+LATf/− mice. f, The development of γδ T cells in CD4Cre+LATf/− mice. The numbers in each panel represent the percentages of the gated population.

FIGURE 2.

Thymocyte development in the CD4Cre+LATf/− mice. a, The CD4 vs CD8 expression profile of total thymocytes from 4-wk-old LATf/− and CD4Cre+LATf/− littermates. The figure shown is one representative of five mice analyzed. b, The total numbers of different thymocyte populations from 4-wk-old LATf/− and CD4Cre+LATf/− littermates. Five mice for each genotype were analyzed. ∗∗, p < 0.01 (Student’s t test). c, GFP expression in different subsets of thymocytes. d, Western blot analysis of LAT expression in GFP+ DP thymocytes. GFP+ DP thymocytes were sorted by FACS and lysed. Postnuclear lysates were analyzed by Western blotting. An anti-Erk2 blot was used as a loading control. e, The CD25 vs CD44 expression in the DN thymocytes from the LATf/− and CD4Cre+LATf/− mice. f, The development of γδ T cells in CD4Cre+LATf/− mice. The numbers in each panel represent the percentages of the gated population.

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We further examined thymocyte development before the DP stage. Analysis of DN thymocytes from CD4Cre+LATf/− and LATf/− mice by expression of CD44 and CD25 revealed that DN thymocyte development in CD4Cre+LATf/− mice was relatively normal, as no obvious differences in the percentages of DN subsets were seen (Fig. 2,e). We also assessed the development of ISP thymocytes (CD4CD8+TCRlow-intHSAhigh), an intermediate stage between DN and DP (21, 22). Although very few CD8+ SP thymocytes were present in CD4Cre+LATf/− mice (Fig. 2,a), most of them were TCRlow-intHSAhigh, a phenotype typical of ISP cells (Fig. 3,a). The number of ISP thymocytes in CD4Cre+LATf/− mice was comparable to those in control mice (Fig. 2,b). Normal development of thymocytes from DN to ISP was expected since the lat gene was only deleted in a small fraction of these two populations (Fig. 2 c).

FIGURE 3.

The defective positive selection in the CD4Cre+LATf/− mice. a, TCRβ vs HSA expression on CD4CD8+ thymocytes from 4-wk-old LATf/− and CD4Cre+LATf/− littermates. b, Expression of CD69, CD5, and TCRβ on DP thymocytes from the LATf/− and CD4Cre+LATf/− mice. c, GFP expression in the CD4+ SP and CD8+ SP thymocytes (gated on HSAneg-low population) in the LATf/− and CD4Cre+LATf/− mice. The shadowed area represents cells from LATf/− mice. d, GFP vs surface CD69 expression in CD4+ SP thymocytes. e, GFP vs surface TCRβ expression in the CD4+ SP and CD8+ SP thymocytes (gated on HSAneg-low population).

FIGURE 3.

The defective positive selection in the CD4Cre+LATf/− mice. a, TCRβ vs HSA expression on CD4CD8+ thymocytes from 4-wk-old LATf/− and CD4Cre+LATf/− littermates. b, Expression of CD69, CD5, and TCRβ on DP thymocytes from the LATf/− and CD4Cre+LATf/− mice. c, GFP expression in the CD4+ SP and CD8+ SP thymocytes (gated on HSAneg-low population) in the LATf/− and CD4Cre+LATf/− mice. The shadowed area represents cells from LATf/− mice. d, GFP vs surface CD69 expression in CD4+ SP thymocytes. e, GFP vs surface TCRβ expression in the CD4+ SP and CD8+ SP thymocytes (gated on HSAneg-low population).

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Additionally, we examined the development of γδ T cells, which are absent in LAT-deficient mice (11). The divergence of γδ and αβ T cells is first evident in the DN2 stage and is largely complete by the DN3 stage (23, 24, 25). Thus, CD4Cre-mediated LAT deletion is not expected to significantly affect the development of γδ T cells. As predicted, the number of CD3+TCRγδ+ thymocytes in CD4Cre+LATf/− mice was similar to those in control mice (Fig. 2,b), although the percentage of γδ T cells was increased slightly due to the decreased number of total thymocytes in these mice (Fig. 2,f). While the majority of the γδ-TCR+ thymocytes remained GFP (Fig. 2 c), there was a small population of GFP+γδTCR+ thymocytes (∼7.5%). It is possible that Cre is also expressed in γδ thymocytes, even though it is driven by the CD4 proximal promoter. Another possibility is that these GFP+γδTCR+ thymocytes were derived from DN3 cells in which there was some residual LAT protein after deletion of the lat gene.

Collectively, the data above showed that deletion of LAT by CD4-Cre was able to drive thymocyte development past the DN3 checkpoint and resulted in relatively normal development of DP thymocytes. Deletion of the lat gene in these DP cells was nearly complete, as indicated by both the expression of GFP and the absence of LAT protein. CD4Cre+LATf/− mice can be used to study the role of LAT in DP thymocyte development and signaling.

The signals initiated by the interaction between αβTCRs and MHC-peptide complexes drive DP thymocytes through both positive and negative selection to develop into SP thymocytes (26, 27). The existence of LAT-deficient DP thymocytes in the CD4Cre+LATf/− mice enabled us to investigate whether LAT is required during the transition from DP to SP. To assess this, we next examined the development of SP thymocytes in CD4Cre+LATf/− mice. Unlike DP cells, very few SP thymocytes were generated in these mice (Fig. 2,a). The number of CD4+ SP cells was reduced >10-fold (Fig. 2,b). Among the CD4CD8+ thymocytes, >85% were TCRlow-intHSAhigh ISP cells (Fig. 3,a), and the number of mature CD8+SP (CD4CD8+TCRhigh HSAlow-int) cells decreased by ∼20-fold (Fig. 2 b).

The absence of LAT completely abrogates TCR-mediated Erk activation and calcium mobilization in Jurkat cells (2, 3). If LAT plays a similar role in DP thymocytes to that observed in Jurkat cells, LAT deficiency should severely impair positive selection. To examine positive selection of thymocytes in CD4Cre+LATf/− mice, we analyzed the surface expression of CD5, CD69, and TCRβ on DP thymocytes. Up-regulation of these markers is considered to be one of the characteristics of positive selection (28, 29, 30, 31, 32). While a small percentage of DP thymocytes from LATf/− mice up-regulated CD5, CD69, or TCRβ, these cells were greatly reduced in the GFP+ DP thymocytes from CD4Cre+LATf/− mice (Fig. 3 b). Taken together, these data clearly demonstrated that deletion of LAT in DP thymocytes impaired positive selection and blocked the development of SP thymocytes. Therefore, in addition to its essential role in pre-TCR-mediated signaling in DN thymocytes, LAT is also indispensable for TCR-dependent maturation of DP thymocytes.

Interestingly, although the vast majority of DP thymocytes expressed GFP, ∼46% of mature CD4+ and 70% of mature CD8+ SP thymocytes were GFP (Fig. 3,c). These GFP SP thymocytes were likely derived from a few DP thymocytes that had escaped lat deletion. Although our data strongly suggested that LAT played an important role during the transition from DP to SP, we did detect some GFP+CD4+ or GFP+CD8+ SP thymocytes in these mice (Fig. 3,c). Moreover, similar to their GFP counterparts, a significant percentage of these GFP+ SP thymocytes also expressed surface CD69, indicating that they had undergone recent positive selection (Fig. 3,d). Interestingly, surface expression of the TCR on these GFP+ SP thymocytes was considerably lower than that on their GFP counterparts (Fig. 3 e).

It is possible that GFP+ (LAT-deficient) DP cells are still capable of developing into SP cells or that they are likely derived from GFP DP or SP cells with continued Cre-mediated deletion. To examine whether GFP+ DP cells can develop into SP cells, DP thymocytes (CD45.2+) from LATf/− mice or GFP+ DP thymocytes from CD4Cre+LATf/− mice were sorted by FACS and cultured on a thymic stromal layer from CD45.1+ mice. While distinct populations of CD4 and CD8 SP thymocytes were derived from LATf/− DP thymocytes, GFP+CD4Cre+LATf/− DP thymocytes failed to develop into SP thymocytes (data not shown). These results indicated that LAT is essential in thymocyte development during the transition from DP to SP.

The developmental block during the DP to SP transition in CD4Cre+LATf/− mice is likely caused by the inability of LAT-deficient DP thymocytes to respond to TCR engagement. In LAT-deficient Jurkat cells, TCR-mediated Erk activation and calcium flux are defective. To investigate the function of LAT in thymocytes, we first examined TCR-mediated calcium flux in DP thymocytes. As shown in Fig. 4,a, GFP+ DP thymocytes from CD4Cre+LATf/− mice failed to mobilize calcium upon crosslinking of CD3. These thymocytes were able to respond normally to ionomycin treatment, suggesting that their calcium release apparatus was intact. Next, we performed biochemical analysis of TCR-mediated signaling events. GFP+ DP thymocytes were sorted from CD4Cre+LATf/− mice. DP thymocytes from LATf/− mice were also sorted as a control. The postsort purity was >99% (data not shown). These thymocytes were stimulated with anti-CD3 before lysis. As shown in Fig. 4 b, while the total tyrosine phosphorylation of proteins in the GFP+CD4Cre+LATf/− DP thymocytes was relatively normal, phosphorylated LAT was absent. Phosphorylation of PLCγ1 in these cells was diminished, which was consistent with the calcium data. Erk activation was also diminished as evidenced by its dramatically reduced phosphorylation. These data indicated that TCR-mediated Erk activation and calcium mobilization were both defective in LAT-deficient DP thymocytes. The lack of proper activation necessary for positive selection following engagement of the TCR in CD4Cre+LATf/− DP thymocytes likely prevented them from further differentiating into SP thymocytes.

FIGURE 4.

Defective TCR-mediated signaling in LAT-deficient DP thymocytes. a, Calcium flux. Thymocytes from the LATf/− and CD4Cre+LATf/− mice were loaded with Indo-1 and then stimulated by crosslinking CD3. Ionomycin was also added at the indicated time point. Calcium in GFP+ DP thymocytes was monitored by flow cytometry and represented as the ratio of fluorescence at 405 and 510 nm. The figure shown is a representative of four experiments performed. b, Tyrosine phosphorylation of proteins. GFPLATf/− and GFP+CD4Cre+LATf/− DP thymocytes were sorted by FACS and stimulated for 2 min by anti-CD3 crosslinking. Postnuclear lysates were analyzed by Western blotting with anti-pTyr, pPLCγ1, and pErk. PLCγ1 and Erk2 blots were shown as loading controls.

FIGURE 4.

Defective TCR-mediated signaling in LAT-deficient DP thymocytes. a, Calcium flux. Thymocytes from the LATf/− and CD4Cre+LATf/− mice were loaded with Indo-1 and then stimulated by crosslinking CD3. Ionomycin was also added at the indicated time point. Calcium in GFP+ DP thymocytes was monitored by flow cytometry and represented as the ratio of fluorescence at 405 and 510 nm. The figure shown is a representative of four experiments performed. b, Tyrosine phosphorylation of proteins. GFPLATf/− and GFP+CD4Cre+LATf/− DP thymocytes were sorted by FACS and stimulated for 2 min by anti-CD3 crosslinking. Postnuclear lysates were analyzed by Western blotting with anti-pTyr, pPLCγ1, and pErk. PLCγ1 and Erk2 blots were shown as loading controls.

Close modal

We next examined the T cell compartment in the periphery of CD4Cre+LATf/− mice. As expected, the percentage of T cells was greatly reduced in both spleens (Fig. 5,a) and lymph nodes (data not shown). The numbers of CD4+ and CD8+ T cells were also significantly decreased (Fig. 5,b). Interestingly, there were more CD8+ T cells than CD4+ T cells in the periphery of CD4Cre+LATf/− mice compared with LATf/− mice (Fig. 5,c). Most of these CD4+ and CD8+ T cells appeared to be GFP (Fig. 5,d), an indication that these cells escaped Cre-mediated deletion of LAT. Interestingly, compared with the GFP T cell population, GFP+ T cells had a lower surface expression of TCRβ (Fig. 5 e).

FIGURE 5.

Abnormal T cells in the periphery of CD4Cre+LATf/− mice. a, Reduced number of mature T cells in the CD4Cre+LATf/− mice. b, The total numbers of CD4+ and CD8+ T cells in spleen and lymph nodes. ∗, p < 0.05 and ∗∗, p < 0.01 (Student’s t test). c, The CD4 vs CD8 expression profile of TCRβ+ splenocytes. d, GFP expression in splenic CD4+ and CD8+ T cells from the LATf/− (gray) and CD4Cre+LATf/− (solid line) mice. e, TCRβ expression in the splenic CD8+ T cells. f, The CD44 vs CD62L expression profile of the splenic CD8+ T cells from the LATf/− and CD4Cre+LATf/− mice.

FIGURE 5.

Abnormal T cells in the periphery of CD4Cre+LATf/− mice. a, Reduced number of mature T cells in the CD4Cre+LATf/− mice. b, The total numbers of CD4+ and CD8+ T cells in spleen and lymph nodes. ∗, p < 0.05 and ∗∗, p < 0.01 (Student’s t test). c, The CD4 vs CD8 expression profile of TCRβ+ splenocytes. d, GFP expression in splenic CD4+ and CD8+ T cells from the LATf/− (gray) and CD4Cre+LATf/− (solid line) mice. e, TCRβ expression in the splenic CD8+ T cells. f, The CD44 vs CD62L expression profile of the splenic CD8+ T cells from the LATf/− and CD4Cre+LATf/− mice.

Close modal

Even though the number of peripheral T cells was significantly reduced in CD4Cre+LATf/− mice, it was higher than expected considering the severe block in thymocyte development from the DP to SP stage. Since most of the T cells in the periphery were GFP, it is possible that they had undergone a tremendous homeostatic expansion driven by the lymphopenic environment in CD4Cre+LATf/− mice. To test this hypothesis, we analyzed the surface expression of CD44 and CD62L on these peripheral T cells. As shown in Fig. 5 f, most CD8+ T cells in the CD4Cre+LATf/− spleen were CD44highCD62Llow, a phenotype of T cells that had undergone homeostatic expansion. A similar phenotype was also observed in CD4+ T cells from these mice (data not shown). These results suggested that the mature T cells in CD4Cre+LATf/− mice likely developed from the few thymocytes that escaped LAT deletion and greatly expanded in the lymphopenic periphery.

LAT Y136F mice, in which the PLCγ1-binding site of LAT was mutated, lack naturally arising CD4+CD25+Foxp3+ Treg cells (16), suggesting that the LAT-PLCγ1 interaction plays an important role in Treg cell development. To examine the role of LAT in the development of Treg cells, we examined the expression of Foxp3 in the LAT knock-in mice. While a small but significant population of CD4+Foxp3+ thymocytes existed in the LATf/− mice, few, if any, could be found in age-matched CD4Cre+LATf/− mice (Fig. 6,a). This result was consistent with the severe defect seen in thymocyte development from the DP to SP stage. Interestingly, despite lacking CD4+Foxp3+ thymocytes, the CD4Cre+LATf/− mice contained a high percentage of Foxp3+ cells (∼25%) in their peripheral CD4+ T cell compartment when compared with the LATf/− controls (Fig. 6,a). Upon closer examination, we found that such peripheral Foxp3+ Treg cells were exclusively GFP, indicating that they had escaped LAT deletion (Fig. 6,b). The same observation was made using CD25 as a marker for the naturally arising Treg cells (Fig. 6 b). The complete lack of LAT-deficient Treg cells in the periphery further demonstrated that the expression of LAT in DP thymocytes is critical for Treg cell development.

FIGURE 6.

Development of Treg cells in the CD4Cre+LATf/− mice. a, Foxp3 expression in total thymocytes and splenocytes of LATf/− and CD4Cre+LATf/− mice (10 wk old). b, Expression of GFP and Foxp3 (top) or CD25 (bottom) in CD4+ splenic T cells.

FIGURE 6.

Development of Treg cells in the CD4Cre+LATf/− mice. a, Foxp3 expression in total thymocytes and splenocytes of LATf/− and CD4Cre+LATf/− mice (10 wk old). b, Expression of GFP and Foxp3 (top) or CD25 (bottom) in CD4+ splenic T cells.

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In this study, we generated LAT knock-in mice and used them to study the role of LAT during thymocyte development and TCR-mediated signaling. In these knock-in mice, the GFP sequence was inserted into the lat locus to monitor deletion of LAT on a single-cell basis. This approach allowed us to identify T cells with LAT deletion and to study the effect of this deletion on thymocyte development and TCR-mediated signaling. By using the CD4Cre transgenic mice, we successfully deleted LAT after the DN3 stage. Because LAT−/− mice have a severe block of thymocyte development at the DN3 stage, this strategy allowed us to study the role of LAT beyond DN3. Our data showed that deletion of LAT after the DN3 stage effectively rescued the development of DP thymocytes but severely blocked the generation of SP thymocytes, eventually resulting in a significant loss of mature T cells in the periphery. Our data indicate that, in addition to its essential role in pre-TCR-mediated signaling, LAT also plays an important role in TCR signaling in DP thymocytes and is required during thymocyte differentiation from DP to SP.

In CD4Cre+LATf/− mice, there was a severe block in thymocyte development from DP to SP; however, some GFP+ SP thymocytes still remained. As shown in Fig. 3,c, ∼50% of the CD4+ and 30% of the CD8+ SP thymocytes in CD4Cre+LATf/− mice were GFP+. Moreover, a significant percentage of such GFP+ SP thymocytes expressed surface CD69, an indicator of recent positive selection (Fig. 3,d). Consistent with the thymocyte data, GFP+ mature T cells could be found in the periphery as well. These data led to the question as to whether GFP+ LAT-deficient DP thymocytes have completely lost their capability of further differentiation. There are three possibilities for the existence of these GFP+ SP thymocytes. First, these cells might have developed from GFP DP thymocytes, in which the lat gene was deleted after differentiation into SP cells. Even though our GFP reporter system allows us to identify cells that have deleted LAT, it is impossible to pinpoint exactly when such deletion occurs. This possibility is supported by the fact that we consistently observed a higher percentage of GFP+ cells in CD4+ SP than in CD8+ SP thymocytes, because the CD4 promoter that controls Cre expression should remain active in CD4+ SP thymocytes. The second possibility is that the LAT protein might have a long half-life. Even after deletion of the lat gene, there might be a sufficient amount of LAT protein left to drive further thymocyte development. The third possibility is that the LAT-deficient DP thymocytes might still have some ability, although limited, to differentiate into SP thymocytes. TCR-mediated signaling, such as Erk activation and Ca2+ flux, might not be totally dependent on LAT. As shown in Fig. 4,a, TCR-dependent calcium flux in GFP+ DP thymocytes was extremely weak; however, it was not completely abolished. Similarly, weak activation of TCR-dependent Erk was also observed in these cells (Fig. 4 b). Such residual signals might enable a few GFP+ DP thymocytes to further differentiate. Our data showed that GFP+ DP thymocytes from CD4Cre+LATf/− mice failed to differentiate into SP cells in vitro, further supporting the essential role of LAT during the transition from DP to SP.

What caused the aforementioned residual TCR-dependent signals in GFP+ DP thymocytes remains to be determined. In the LAT-deficient Jurkat cell line (JCaM2), TCR-dependent calcium mobilization and Erk are completely abolished (2, 3). We are more inclined to argue that a low level of LAT expression, which might be difficult to detect by Western blotting, may be present in these GFP+ DP thymocytes. This possibility is supported by our unpublished data using ERCre+LATf/− mice in which LAT can be deleted effectively within 4 days after tamoxifen injection. No TCR-mediated calcium flux was observed in GFP+ peripheral T cells from tamoxifen-treated ERCre+LATf/− mice, indicating the requirement of LAT in TCR-mediated calcium mobilization. However, it remains possible that other TCR-mediated LAT-independent signals play a role in DP thymocytes (33).

Mature T cells found in the periphery of the CD4Cre+LATf/− mice were CD44highCD62Llow, a phenotype that is likely a consequence of lymphopenia-driven homeostatic expansion. Interestingly, while there were about the same number of GFP+ as GFP SP cells in the thymus, GFP mature T cells clearly dominated in the periphery. Thus, the GFP mature T cells in the CD4Cre+LATf/− mice may have expanded much more vigorously than did their GFP+ counterparts. On the other hand, it is also possible that GFP+ mature T cells might be defective in cell survival upon LAT deletion. An adoptive transfer approach will be taken to further address this possibility in the future.

We noticed that GFP+ SP thymocytes in CD4Cre+LATf/− mice expressed a considerably lower level of surface TCR than did their GFP counterparts. A similar phenomenon was observed in peripheral mature T cells as well. Previous studies have also linked the down-regulation of TCR expression with a defect in LAT-mediated signaling. In knock-in mice that express LAT with a point mutation at Y136, TCR and CD3 expression on the peripheral T cells is also much lower than on normal T cells (13, 14). Moreover, deficiency of SLP-76, which also plays an essential role in mediating TCR-dependent signaling, similarly causes down-regulation of TCR expression on the surface of peripheral T cells (34). Thus, normal signaling downstream of TCR engagement appears to positively regulate the expression of the TCR through mechanisms that are yet to be explored.

Given that LAT plays an essential role in SP thymocyte development, it was not surprising that the CD4Cre+LATf/− mice lacked CD4+Foxp3+ thymocytes. However, it is intriguing that, while LAT-deficient GFP+ CD4 T cells could be found in the periphery, albeit few, GFP+ Treg cells were completely missing. Studies using TCR transgenic models have demonstrated that, compared with conventional T cells, Treg cells likely require higher TCR/MHC-peptide affinity for positive selection (35, 36, 37, 38), although this conclusion is being debated. The Treg cell development may be more strictly dependent on LAT. Further supporting this hypothesis, CD4+CD25+ Treg cells are absent in both the thymus and the periphery in LAT Y136F mice, while the positive selection for conventional T cells is only partially defective (15, 16). Therefore, the residual TCR signaling in the GFP+ DP thymocytes, whether due to LAT-independent signaling pathways or to the incomplete deletion of LAT protein, might have enabled them to develop into conventional T cells, but not Treg cells.

The phenotype of our CD4Cre+LATf/− mice carried a striking resemblance to the SLP-76 conditional knockout mice that have recently been described (34). While SLP-76−/− mice suffer a severe block in thymocyte development at the DN3 stage and fail to generate any DP and SP thymocytes, just like the LAT−/− mice (39, 40, 41), depletion of SLP-76 after the DN3 stage in CD4CreSLP-76f/f mice also results in the reappearance of DP thymocytes. SLP-76-deficient DP thymocytes fail to respond to TCR stimulation and are defective in both positive and negative selection. As a result, few SP thymocytes and mature T cells develop in the CD4CreSLP-76F/F mice. Collectively, these data strongly suggest that the LAT-GADS-SLP-76 complex is absolutely required to transduce signals from the TCR that would lead to positive and negative selection of DP thymocytes.

Although our studies clearly demonstrated the important role of LAT during thymocyte development after the DN3 stage, its role in mature T cells still remains to be determined. There were only a small number of GFP+ T cells present in the periphery of CD4Cre+LATf/− mice, which makes it extremely difficult to study the role of LAT in mature T cells. Moreover, such T cells, which were CD44highCD62LlowTCRlow and had undergone homeostatic proliferation, are not likely to be ideal cells for future study. Additional work is being done to effectively delete LAT in normally developed T cells, and studies on these cells will shed more light on the role of LAT in mature T cell activation, survival, and homeostasis.

The authors have no financial conflicts of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Heath Grants AI048674 and AI056156. W.Z. is a scholar of Leukemia and Lymphoma Society. J.L. is supported by a National Science Scholarship from A*STAR, Singapore.

3

Abbreviations used in this paper: LAT, linker for activation of T cells; DN, double negative; DP, double positive; ISP, immature single positive; SP, single positive; Treg cell, regulatory T cell.

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