Phosphatidylinositol 3-Kinase Regulates the CD4/CD8 T Cell Differentiation Ratio¹

T cell maturation begins in the thymic cortex, where most immature progenitors—cells that lack CD4 and CD8 molecule expression (CD4⁻CD8⁻ double negative (DN)⁴)—are found. These progenitors can be further subdivided into different stages based on CD44 and CD25 expression. The earliest progenitors are CD44⁺CD25⁻ (DN stage I), followed by CD44⁺CD25⁺, CD44⁻CD25⁺, and CD44⁻CD25⁻ populations (stages II–IV, respectively). At the CD44⁻CD25⁺ stage (DN III), cells pass the first T cell differentiation checkpoint. Cells that successfully reorganize the TCR β locus and express a functional receptor complex (known as pre-TCR) proliferate, down-regulate CD25, and differentiate into CD4⁺CD8⁺ double-positive (DP) cells. Assembly of the TCRα-chain in these cells leads to expression of a functional TCRαβ, which permits T cells to pass to the second checkpoint. Cells expressing a TCR that recognizes self Ags are eliminated (negative selection), whereas those whose TCR recognizes the appropriate MHC molecules survive and differentiate into mature single-positive CD4⁺ or CD8⁺ cells (positive selection; reviewed in Refs. 1 and 2).

The intracellular signaling cascades that govern T cell differentiation have begun to be elucidated. In early stages of DN cell differentiation, IL-7R-derived signals as well as Rho GTPase (3, 4) appear essential for driving T cell development. The contribution of the Src-Tyr kinase Lck at the pre-TCR-dependent stage of DN cell differentiation is essential for cells to proceed to the DP differentiation stage (5). Positive selection involves TCR stimulation and requires Lck, Zap 70, mitogen-activated protein kinase (MAPK), and Vav activation (1, 6, 7, 8, 9, 10, 11). How signals that induce positive selection trigger differentiation into the CD4⁺ or CD8⁺ mature cell type is not fully understood. Nonetheless, quantitative differences in MAPK and Lck activation appear to alter the CD4⁺/CD8⁺ differentiation ratio (12, 13).

The class I phosphatidylinositol (PI) 3-kinases (PI3Ks) are heterodimeric proteins composed of a regulatory and a catalytic subunit that induce rapid, transient formation of PI-3,4-P₂ and PI-3,4,5-P₃. Class IB PI3K is composed of the p101 regulatory and the p110γ catalytic subunits and is activated after G protein-coupled receptor (GPCR) stimulation. Class IA PI3K comprises a regulatory (p85α, β, or p55γ) and a p110 catalytic (α, β, or δ) subunit and is activated by Tyr kinase receptor stimulation (reviewed in Ref. 14). PI3K is essential for B cell differentiation (15, 16), but although it is activated by the TCR (17), the role of PI3K in T cell differentiation remains unclear. In fact, p110α^−/− and p110β^−/− mice exhibit embryonic lethality (18, 19), and p85α^−/− mice have no obvious defects in T cell populations, but they still express p85β and p55γ (15, 16). The p110δ isoform was recently shown to be essential for mature B and T cell activation, but it does not seem to affect T cell development (20). Finally, p110γ^−/− mice show a defect in thymus size (21), although the details of how this isoform controls thymic development are presently unknown.

We performed a detailed phenotypic analysis of T cell development in mice deficient in class IB PI3K (p110γ). In addition, to examine the role of the p85/p110 PI3K isoforms (class IA) in T cell development, we analyzed the phenotype of mice expressing an activating mutation of the p85 regulatory subunit, p65^PI3K, in T cells. This mutant regulatory subunit associates with the p110 catalytic subunit, moderately increasing basal PI-3,4,5-P₃ levels and significantly enhancing the transient PI3K activation that follows receptor activation (22, 23). p65^PI3K potentially acts on all three class IA catalytic subunits, constituting a useful tool for the study of the role of class IA p85/p110 PI3K isoforms in T cell development.

We found that p110γ deficiency partially impaired DN-to-DP transition and diminished the proliferative expansion that accompanies development from the DN to the DP stage (thymus growth). In support of a role for PI3K at this stage, p65^PI3K expression increased the pre-TCR-dependent DN-to-DP transition, although it did not affect thymus growth. At the positive selection stage, p110γ deficiency reduced the CD4⁺/CD8⁺ T cell differentiation ratio, whereas p65^PI3K expression augmented this ratio. Moreover, p65^PI3K expression corrected p110γ^−/− mouse differentiation defects, except for thymus growth. These results illustrate that PI3K modulates T cell differentiation at several stages. The p110γ isoform regulates thymic growth. In addition, p110γ influences the pre-TCR-dependent DN-to-DP cell transition and affects the CD4/CD8 T cell differentiation ratio. Enhanced class IA p85/p110 PI3K activation affects the same differentiation steps, except for thymus growth, suggesting that class IA PI3K activation may also contribute to regulation of T cell development. The results presented are the first demonstration of the role of p110γ in the signaling pathways that regulate pre-TCR-dependent differentiation and in CD4⁺/CD8⁺ lineage commitment during positive selection.

p65^PI3K transgenic (Tg) mice were generated as described (24). The Tg colony was maintained by crosses with C57BL/6 females, and offspring were analyzed by PCR 30 days after birth. The p110γ null mice were previously described and maintained in heterozygosis (25). The 5CC7 TCR (Vβ3Vα11) and F5 TCR (Vβ11Vα4) Tg mice were previously described (26, 27) and were kindly provided by Drs. M. Davis (Howard Hughes Medical Institute, Stanford, CA) and D. Kioussis (Medical Research Council, London, U.K.), respectively. 5CC7 TCR Tg mice were crossed with p110γ null mice (on the C57BL/6 background) and with p65^PI3K Tg mice. F5 TCR Tg mice were also crossed with p110γ null mice. Offspring were analyzed by PCR as described (24) and by flow cytometry to verify the appropriate TCR and MHC. All mice were bred and maintained under specific pathogen-free conditions at the National Center for Biotechnology animal facility.

Thymus, spleen, and lymph node cell suspensions were prepared by grinding tissue through sterile wire mesh. Cells were counted by trypan blue exclusion and erythrocytes were lysed with a hypotonic ammonium chloride solution. For cell surface staining, all Abs used were FITC-, PE-, or biotin-conjugated, and cells were stained with saturating Ab concentrations at 4°C. Biotinylated Abs were developed with streptavidin-spectral red (Southern Biotechnology, Birmingham, AL). The Abs used were heat-stable Ag (HSA; CD24, M1/69), CD8 (Ly-2, 53-6, 7), CD4 (L3T4, H129.19), CD3 (145-2C11), CD25 (IL-2R α-chain), CD44 (pgp1, IM7), Vβ3 (KJ25), Vβ11(RR3-15), Vα11 (RR8-1), I-Ek (17-3-3), and H-2b (AF6-88.5), all from BD PharMingen (San Diego, CA). TCRαβ Ab (H57-597) was from Southern Biotechnology, and anti-BrdU Ab was from BD Biosciences (San Jose, CA). BrdU labeling and analysis were performed as described (28). Briefly, BrdU was administrated for 8 days in drinking water to wild type (Wt) and p65^PI3K Tg mice, and then the presence of spleen T cells incorporating BrdU^low was examined by flow cytometry. Intrathymic FITC injections and analysis of recent thymic emigrants were as described (29, 30). Cells were analyzed on an EPICS XL using System II software (Coulter, Miami, FL).

We previously reported that young mice expressing the p65^PI3K transgene in T cells exhibited a 30% increase in the proportion of CD4⁺ peripheral T cells. Mice remain healthy for 1 year; however, at ∼12–15 mo of age, p65^PI3K induces development of a lymphoproliferative disease caused by the enhanced memory cell survival (24). This enhanced memory cell survival in adult mice does not explain the 30% increase in CD4⁺ T cells in young mice. We postulated that p65^PI3K may affect thymic development. Here we examine T cell differentiation in young (5- to 8-wk-old) p65^PI3K Tg mice.

Thymic cellularity was similar in Wt and Tg littermates (data not shown). Nonetheless, the percentage of DN cells was reduced in Tg compared with Wt mice (Fig. 1,A; Student’s t test; p = 0.0001). This represented a systematic reduction in absolute DN cell numbers from a mean of 7.4 ± 2.9 × 10⁶ (Wt thymus) to 4.4 ± 2.2 × 10⁶ (Tg thymus). Examination of DN differentiation steps (31) showed a significant decrease in the percentage of CD44⁻CD25⁺ cells (DN stage III) in p65^PI3K Tg compared with Wt mice (Fig. 1,B; p = 0.002) and no significant changes in the remaining populations. The percentage of DP cells was moderately increased in Tg mice (Fig. 1 C; p = 0.0008). The p65^PI3K Tg mice thus exhibit reduced DN cell numbers, a lower proportion of cells at the CD44⁻CD25⁺ stage, and a moderately increased percentage of DP cells.

We next examined mature thymic T cell populations. The p65^PI3K Tg mouse thymuses had lower percentages of CD4⁺ cells compared with Wt animals (Fig. 2,A; p = 0.007), with no significant differences in the percentage of CD8⁺ thymocytes (Fig. 2,A). We also observed a decrease in the percentage of mature CD3^high cells in Tg thymus (data not shown), indicating that the reduction in CD4⁺ cells is not simply due to down-regulation of the CD4 receptor. The decrease in mature CD4⁺ thymocytes in 5- to 8-wk-old p65^PI3K Tg mice may be caused by a defect in CD4⁺ cell differentiation or may reflect more efficient CD4⁺ cell exit to periphery. To analyze T cell development independently of thymic emigration, we examined neonatal mice (2–2.5 days after birth), when thymocytes have just begun to populate the periphery (2). Neonatal p65^PI3K Tg mice showed increased percentages of mature CD3^high (Fig. 2,B) and mature CD4⁺ thymocytes (Fig. 2, C and D; p < 0.001). The distribution of CD44/CD25-based DN cell subpopulations was similar to that in 5- to 8-wk-old Tg mice (data not shown). The increase in the CD4⁺ cell subpopulation was from a mean of 6.9 ± 1.6% in controls to 11.4 ± 2.8% in p65^PI3K Tg mice. The proportion of thymic CD4⁺ cells subsequently decreased and, between postnatal days 7 and 10, reached values similar to those of 1-mo-old Tg mice. These results indicate that p65^PI3K increases the magnitude of positive selection, promoting generation of CD4⁺ cells, as revealed in neonatal mice.

As the proportion of CD4⁺ cells decreased in the days after birth, we postulated that p65^PI3K expression could enhance CD4⁺ thymocyte emigration. To analyze migration efficiency, we performed in vivo BrdU labeling, which results in BrdU^low incorporation in most developing thymocytes (28, 32). The appearance of BrdU^low-labeled cells in the periphery allows quantitation of thymic migration (28). Although both types of mice incorporated BrdU similarly (60–70% of thymocytes labeled), the percentage of BrdU^low CD4⁺ cells in periphery was higher in p65^PI3K Tg than in Wt mice (Fig. 2,E; p = 0.007), indicating that CD4⁺ thymocytes exit the thymus more efficiently in Tg mice. As an alternative approach, FITC was injected into the thymus of Wt and p65^PI3K Tg mice; 48 h postinjection, we examined the appearance of FITC-labeled recent thymic emigrants in the periphery (29, 30, 33). The number of FITC-labeled CD4⁺ thymic emigrants was consistently higher in p65^PI3K Tg than in Wt littermates (Fig. 2 F; p = 0.006). The p65^PI3K transgene thus enhances generation of CD4⁺ thymocytes and increases their exit to the periphery.

p65^PI3K Tg expression affected DN cell differentiation and increased CD4⁺ cell generation (Figs. 1 and 2). Because p110γ^−/− mice have a smaller thymus and showed a decreased peripheral CD4⁺ population (21), we asked whether the phenotype of these mice might also result from a defect in DN cell differentiation and CD4⁺ cell generation. Total thymocyte numbers were reduced in p110γ^−/− compared with p110γ^+/− littermates (Fig. 3,A; p = 0.001), yielding mean values of 60.2 ± 12.2 × 10⁶ cells in p110γ^+/− mice and 40.0 ± 9.7 × 10⁶ cells in p110γ^−/− mice. The p65^PI3K expression in p110γ^−/− mice did not restore thymus size (Fig. 3 A; p = 0.05; mean, 43.5 ± 9.8 × 10⁶ cells).

The p110γ^−/− mouse thymuses showed an increased percentage of DN cells (Fig. 3,B; p = 0.04). Because the p110γ^−/− thymus is smaller, we calculated absolute cell numbers in these animals to evaluate the status of the different T cell populations more accurately. DN cell numbers were also increased in p110γ^−/− compared with p110γ^+/− mice (Fig. 3,B; p = 0.02). Moreover, the proportion of DN cells at the CD44⁻CD25⁺ stage (DN III) was higher in p110γ^−/− compared with p110γ^+/− mice (Fig. 3,C; p = 0.001), whereas the remaining populations were not significantly affected. This result suggests that p110γ^−/− mice have a partial defect at DN differentiation stage III. In agreement with a role for p110γ at the pre-TCR checkpoint, p110γ^−/− mice had decreased proportions of large CD44⁻CD25⁺ cells (Fig. 3,D; p = 0.002), cells that emerge after pre-TCR ligation (34). Introduction of the p65^PI3K transgene in p110γ^−/− mice complemented the pre-TCR differentiation defect, in that it reduced accumulation of DN cells and of CD44⁻CD25⁺ cells (Fig. 3, B and C), increasing the appearance of large CD44⁻CD25⁺ cells (Fig. 3 D).

The p110γ^−/− mice showed reduced percentages (Fig. 3,E; p = 0.0005) and absolute numbers of DP cells (Fig. 3,E; mean values, 48.0 ± 11.0 × 10⁶ and 30.9 ± 8.2 × 10⁶ in p110γ^+/− and p110γ^−/− mice, respectively; p = 0.008). p65^PI3K expression in p110γ^−/− mice induced a moderate increase in the percentage of DP cells (Fig. 3,E), but DP cell numbers and thymus size (Fig. 3, A and E) remained lower than in p110γ^+/− mice. These observations suggest that p110γ^−/− mice have a partial defect at the pre-TCR checkpoint of DN cell differentiation, which is complemented by p65^PI3K transgene expression. Expansion of the DP pool nonetheless requires p110γ expression.

p110γ regulates macrophage and neutrophil migration (25). To examine CD4⁺ and CD8⁺ cell differentiation independently of thymic emigration defects, we studied neonatal mice. No significant changes were detected in the percentage of CD4⁺ cells (Fig. 4,A), but the lower cellularity of p110γ^−/− thymuses resulted in a moderate reduction in absolute CD4⁺ cell numbers (Fig. 4,A; p = 0.005). In contrast, the percentages and absolute numbers of CD8⁺ cells were significantly increased in neonatal p110γ^−/− compared with p110γ^+/− mice (Fig. 4,B; p = 0.001 and p = 0.05, respectively). As a consequence, p110γ^−/− mice yielded a CD4⁺/CD8⁺ differentiation ratio near 1 (Fig. 4, A and B), rather than the normal CD4⁺/CD8⁺ cell differentiation ratio (more than 2-fold) observed in p110γ^+/− mice. The p65^PI3K transgene expression in p110γ^−/− mice increased generation of CD4⁺ cells and decreased that of CD8⁺ cells (data not shown). Fig. 4 C shows a schematic representation of the proportion of CD4⁺ and CD8⁺ cells in the distinct neonatal mice. Whereas p65^PI3K expression enhanced CD4⁺ cell differentiation, p110γ deficiency enhanced CD8⁺ cell generation.

In contrast with neonatal mice, 5- to 8-wk-old p110γ^−/− mice showed increased percentages of both CD4⁺ and CD8⁺ thymocytes compared with p110γ^+/− mice (Fig. 4,D; p = 0.005 and p = 0.009, respectively). We postulated that p110γ deficiency might impair mature thymocyte exit to the periphery, causing accumulation of mature thymic residents. We examined CD4⁺ and CD8⁺ thymocyte migration. Compared with p110γ^+/− mice, a significant reduction was found in the proportion of CD4⁺ and CD8⁺ BrdU^low thymic emigrants in the periphery of p110γ^−/− mice (Fig. 4,E; p = 0.01 and p = 0.006, respectively). We also examined thymocyte migration by intrathymic FITC injection. The proportion of FITC-labeled CD4⁺ and CD8⁺ recent thymic emigrants was lower in p110γ^−/− than in p110γ^+/− mice (Fig. 4 F; p = 0.006 and p = 0.004, respectively). These results demonstrate p110γ involvement in the regulation of thymic emigration.

Introduction of the p65^PI3K transgene in p110γ^−/− mice nonetheless increased CD4⁺ and CD8⁺ cell migration (Fig. 4, E and F), leading to a reduction in the percentage of mature CD4⁺ and CD8⁺ thymocytes in 5- to 8-wk-old p65^PI3K Tg/p110γ^−/− mice (Fig. 4 D). Examination of neonatal mice showed that p110γ deficiency decreased CD4⁺ cell differentiation and enhanced CD8⁺ cell generation, altering the CD4⁺/CD8⁺ cell differentiation ratio. In addition, p110γ regulated mature thymocyte migration to the periphery.

To confirm the role of PI3K in determining the CD4⁺/CD8⁺ outcome of positive selection, we crossed p110γ^−/− mice and p65^PI3K Tg with TCR Tg mice. The p110γ^−/− mice were crossed with mice expressing an MHC class II-restricted Tg TCR (5CC7 Tg mice) (26) that triggers CD4⁺ cell development in the appropriate MHC context. 5CC7/p110γ^+/− (IE^k/b) mice were obtained and compared with 5CC7/p110γ^−/− (IE^k/b) littermates. Differentiation was examined in the first week of life, to avoid interference with thymic emigration defects in p110γ^−/− mice. Compared with 5CC7/p110γ^+/− mice, 5CC7/p110γ^−/− mice showed a significant decrease in the percentage of differentiated CD4⁺ cells and a significant increase in the percentage of differentiated CD8⁺ cells (Fig. 5,A). This shift in the CD4⁺/CD8⁺ differentiation ratio was also detected within the subpopulation of thymocytes expressing high levels of transgenic TCR Vβ- and Vα-chains (Fig. 5,A). This suggests that the decrease in the CD4⁺/CD8⁺ differentiation ratio occurs within the Tg TCR⁺ cell population, although this MHC class II-restricted TCR normally induces CD4⁺ cell differentiation. The increase in CD8⁺ cell differentiation was also observed in the HSA-negative (HSA⁻) subpopulation, which represents the most mature thymus cell population (12) (Fig. 5 B). The increase in CD8⁺ cells that express an MHC class II-restricted TCR and the concomitant decrease in transgenic TCR⁺CD4⁺ cell differentiation suggest that p110γ deficiency influences CD4⁺/CD8⁺ differentiation fate.

CD4⁺ cell differentiation is increased in p65^PI3K Tg mice (Fig. 2), indicating that enhancement of class IA PI3K activation may also modulate CD4⁺/CD8⁺ cell differentiation. To examine this possibility, p65^PI3K Tg mice were crossed with F5 Tg mice expressing an MHC class I-restricted transgenic TCR (27) that drives CD8⁺ thymocyte development in the appropriate MHC context. We observed that T cell differentiation begins at a slower rate in F5 Tg mice and that the proportion of CD3⁺ mature thymocytes (mostly CD8⁺ cells) continues to increase during the first week of life (data not shown). We thus examined 5- to 8-wk-old F5 Tg (H-2^b) and F5/p65^PI3K double-Tg (H-2^b) mice. p65^PI3K transgene expression in F5 mice reduced CD8⁺ T cell differentiation (Fig. 6,A). Because no appropriate Ab is available to examine expression of the transgenic Vα-chain (Vα4), in this case we only analyzed CD4⁺/CD8⁺ cell differentiation in the subpopulation of cells expressing the transgenic Vβ-chain. A decrease in CD8⁺ cell differentiation was detected within the thymic population expressing high levels of the Tg TCR Vβ-chain (Fig. 6 A).

Mature CD4⁺ thymic residents were very low in both F5 Tg and F5/p65^PI3K double-Tg mice (Fig. 6,A). Nonetheless, an increase in CD4⁺ cell differentiation was found systematically in the HSA⁻ population in F5/p65^PI3K mice (Fig. 6,B). p65^PI3K expression increases CD4⁺ thymic export (Fig. 2). Therefore, it was possible that p65^PI3K expression induced Tg TCR⁺CD4⁺ cell exit to the periphery at a higher rate than in F5 Tg mice. In fact, a larger number of CD4⁺ thymic emigrants was found in the periphery of 5- to 8-wk-old F5/p65^PI3K mice (Fig. 6,C). Moreover, consistent with the enhanced CD4⁺ cell generation in F5/p65^PI3K mice, peripheral CD4⁺ cell numbers were increased in these animals compared with F5 Tg mice (CD4⁺ splenocyte mean values, 4.6 ± 1.7 × 10⁶ and 10.7 ± 3.1 × 10⁶, respectively). Because Tg Vα-chains cannot be examined, we analyzed other Vα-chains (Vα3, Vα11, and Vα11.2); the expression levels of these Vα in F5 Tg mice were very low and were not significantly increased in F5/p65^PI3K double-Tg mice (data not shown). Moreover, the proportion of CD4⁺ cells expressing the transgenic TCR Vβ-chain in the periphery of F5/p65^PI3K double-Tg mice was higher than in F5 Tg mice (Fig. 6 D). This suggests that p65^PI3K expression increases differentiation of CD4⁺ cells that express the transgenic MHC class I-restricted TCR.

We examined thymic development in p110γ-deficient mice and in mice expressing an activating mutation of class IA PI3K, p65^PI3K, in T cells. Both genetic alterations had a moderate effect on pre-TCR-induced DN cell differentiation; p110γ deficiency impaired this transition, and enhanced activation of class IA PI3K favored it. p110γ also regulated thymus growth. Nonetheless, the most striking observation in mice with genetic alterations in PI3K was the effect on CD4⁺/CD8⁺ lineage commitment. p110γ deficiency augmented CD8⁺ cell differentiation, whereas enhancement of class IA PI3K activation increased generation of CD4⁺ cells. These observations support the idea that the magnitude of PI3K activation regulates the CD4⁺/CD8⁺ T cell differentiation ratio. In addition, both alterations influenced migration of mature thymocytes to the periphery.

p110γ involvement in pre-TCR-dependent T cell differentiation is supported by the observation that p110γ^−/− mice show an increase in DN cell numbers. These cells accumulated preferentially at DN stage III, the stage at which pre-TCR triggers differentiation (1, 2). The proportion of large cells at DN stage III, generated after pre-TCR ligation (34), is reduced by 50% in these mice. In support of a role for PI3K at this stage, p65^PI3K mice showed reduced numbers of DN cells and a decreased percentage of DN stage III cells. p65^PI3K expression complemented p110γ function, reducing DN cell accumulation at stage III and restoring the normal proportion of CD44⁻CD25⁺ large cells in p110γ^−/− mice. These results show that class IA PI3K complements p110γ function. This complementation may reflect that both isoforms regulate this transition physiologically or that enhanced p65^PI3K-induced PI3K activity artificially compensates for the lack of p110γ. The p65^PI3K expression nonetheless enhances basal 3-polyphosphoinositide levels only moderately and requires stimulation of a tyrosine kinase-coupled receptor to enhance PI3K activity significantly (22, 23). Pre-TCR, a receptor coupled to Lck tyrosine kinase (5, 35), thus may activate class IA PI3K isoforms physiologically, thereby contributing to pre-TCR-mediated differentiation, as observed in p65^PI3K Tg mice. Moreover, the observation that p65^PI3K expression does not restore thymus growth in p110γ^−/− mice argues against a generalized compensation of p110γ function by increased 3-polyphosphoinositide levels in p65^PI3K Tg mice. Nonetheless, whereas the phenotype of p110γ^−/− mice demonstrates a function for p110γ in pre-TCR-dependent differentiation events, further studies in conditional class IA PI3K knockout mice are required to clarify the contribution of these isoforms.

The p110γ^−/− mice show a defect in the proliferative expansion that accompanies development from the DN to the DP stage (21) (Fig. 4). p110γ is essential for this function, because enhanced class IA PI3K activation caused by p65^PI3K expression did not restore normal thymus size in these mice. Thymus growth thus may require signals other than those needed for DN-to-DP differentiation. We postulate that a receptor that specifically regulates p110γ is involved in control of DP cell expansion. Pertussis toxin treatment resulted in a selective decrease in DP cells (36), supporting GPCR involvement in regulating DP cell expansion. Rho, a GPCR effector, also controls thymus growth (37, 38). Because both p110γ and Rho are activated by GPCR (39, 40, 41), receptors of this family are potential candidates for thymus growth regulation.

Mice with genetic modifications in PI3K showed altered CD4⁺/CD8⁺ differentiation ratios. The contribution of p110γ in the control of lineage commitment was evident in neonatal p110γ^−/− mice, which display increased CD8⁺ and decreased CD4⁺ cell numbers. Crossing these animals with mice expressing an MHC class II-restricted Tg TCR, which normally induces CD4⁺ cell differentiation, resulted in an increase in CD8⁺ cells and a reduction in CD4⁺ cells expressing the Tg TCR. This demonstrates that p110γ activation is a component of the signaling pathways that regulate differentiation to CD8⁺ or CD4⁺ lineages. In the case of enhanced class IA PI3K activation, the result is consistent and complementary. In MHC class I-restricted TCR Tg mice that normally induce predominantly CD8⁺ cell differentiation, p65^PI3K expression increased generation of CD4⁺ cells and reduced that of CD8⁺ cells. In addition to this alteration in lineage commitment, p65^PI3K expression mediated a quantitative increase in positive selection of CD4⁺ cells. This is supported by the observation that neonatal p65^PI3K mice have a larger number of mature CD3⁺ T cells. We found no significant decrease in positive selection in p110γ^−/− mice. Nonetheless, when p110γ^−/− mice were crossed with 5CC7 TCR Tg mice, a decrease of ∼25–30% was detected in the number of mature CD3⁺ thymocytes. These data indicate that, in addition to the effect on CD4⁺/CD8⁺ lineage commitment, PI3K may also regulate the magnitude of positive selection.

To understand the mechanism by which enhanced PI3K activation increases positive selection of CD4⁺ cells, we considered a reduction in apoptosis. Expression of the p65^PI3K mutant (22) did not affect spontaneous apoptosis, γ- or UV-irradiation-induced apoptosis, or apoptosis induced by anti-Fas Ab, dexamethasone, staphylococcal enterotoxin B, or TNF-α (data not shown). We also examined Ag-induced apoptosis in p65^PI3K Tg mice crossed with the 5CC7 TCR Tg mice (26). Administration of the specific antigenic peptide resulted in a comparable reduction in DP cells in 5CC7 and 5CC7 × p65^PI3K Tg mice (data not shown). Thus, we find no reduction in apoptosis in p65^PI3K Tg mice. In addition to increased tumor susceptibility, mice lacking Pten, a phosphatase that reduces 3-polyphosphoinositide levels generated by any PI3K isoform, showed decreased negative selection and increased generation of CD4⁺ T cells with an activated phenotype (42). Thus, it is possible that strong induction of the PI3K pathway, such as that originated by Pten deletion, is required to affect negative selection and quantitatively increase positive selection. Even in the case of Pten, the authors report a selective increase in CD4⁺ cells (42). This and the increase in peripheral CD4⁺ T cells in other mouse models showing PI3K protein kinase B (PKB) pathway activation (43, 44) supports the conclusion that this route skews differentiation toward cells of the CD4⁺ phenotype. Conversely, p110γ^−/− mice have a reduced peripheral CD4⁺ population (21).

It was recently proposed that the difference between signals that generate CD4⁺ and CD8⁺ cell differentiation is quantitative (12, 13). Enhanced activation of Lck or MAPK pathways promotes CD4⁺ cell differentiation, whereas defective induction of these kinases increases CD8⁺ cell differentiation (12, 13). PI3K acts according to this model, because p110γ deletion promotes CD8⁺ generation (even when these cells express an MHC class II-restricted TCR), and transient enhanced class IA PI3K activation promotes CD4⁺ generation (even when these cells express an MHC class I-restricted TCR). The magnitude of this shift is similar to that observed when there are genetic alterations in the MAPK pathway (13), and it is lower than those observed when Lck activity is altered (12). This may reflect the hierarchy of the signaling pathways that regulate differentiation. Lck is a Tyr kinase that is induced shortly after TCR ligation (35). Tyrosine kinases activate PI3K and MAPK (14, 23, 25, 45); thus, these two pathways may cooperate to control the CD4⁺/CD8⁺ cell differentiation ratio. In support of this view, MAPK and Lck control pre-TCR-mediated differentiation and CD4/CD8 lineage commitment. Nonetheless, Lck^−/− mice have a more severe T cell differentiation defect (5, 12) than do MAPK^−/− mice (8, 13, 46).

Class IA PI3K is triggered by TCR ligation (17). p110γ involvement in pre-TCR and TCR-regulated differentiation events was nonetheless unexpected, because these receptors trigger Tyr kinase activation (35), and p110γ is normally activated by GPCR (14). This suggests either that a GPCR cooperates with the pre-TCR and TCR to trigger p110γ activation, or that the pre-TCR and TCR activate p110γ. We have compared TCR-triggered PKB activation in thymocytes and T cells obtained from Wt, p65^PI3K Tg, and p110γ^−/− mice. PKB activation shortly after TCR binding was lower in p110γ^−/− than in Wt mouse cells, and it was higher in cells from p65^PI3K Tg or p110γ^−/−/p65^PI3K Tg mice (data not shown). These results suggest that p110γ participates in TCR-triggered early signals, which may aid in elucidating the way in which this PI3K isoform regulates TCR-mediated differentiation. Future experiments will be oriented to define the mechanism by which TCR activates p110γ.

In addition to these effects on thymic differentiation, lack of p110γ reduces migration of mature thymocytes to the periphery. Moreover, the enhanced class IA PI3K activation by p65^PI3K promotes CD4⁺ cell migration and reconstitutes mature CD4⁺ and CD8⁺ T cell migration in p110γ^−/− mice. This suggests that class IA and IB PI3K may both regulate thymocyte migration to the periphery. Because some receptors that trigger cell migration, such as chemokine receptors, induce class IB PI3K activation and subsequent IA PI3K stimulation (47, 48, 49), activation of these two enzymes in cascade may regulate thymocyte emigration.

The results illustrate that PI3K modulates T cell differentiation at several stages. The p110γ isoform regulates thymic growth. In addition, p110γ influences the pre-TCR-dependent DN-to-DP cell transition and affects the CD4/CD8 T cell differentiation ratio. Class IA p85/p110 PI3K-enhanced activation affects the same differentiation steps except for thymus growth, suggesting that class IA PI3K may contribute to regulation of T cell development. The observations presented demonstrate for the first time the contribution of p110γ to the signaling pathways that regulate pre-TCR-dependent differentiation and CD4⁺/CD8⁺ lineage commitment during positive selection.

We thank Drs. D. Balomenos and J. A. Garcia-Sanz for critical reading of the manuscript and helpful suggestions, Dr. D. Kioussis for F5 Tg mice, Dr. M. M. Davis for 5C.C7 Tg mice, and C. Mark for editorial assistance.