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The progressive maturation of T cells is accompanied by their migration through the thymus, with each selection stage occurring in distinct microenvironments. Many specialized receptor-ligand pairs have been defined that drive T cell differentiation, but our understanding of the complex relationship between T cells and the thymic stroma is incomplete. Recent reports have identified a role for the chemokine stromal cell-derived factor 1α and its receptor CXC chemokine receptor 4 in β-selection. This review explores these findings in detail.

Perforin and the serine protease granzymes are key effectors of CD8+ T cell granule-mediated cytotoxicity, but the requirements for their expression remain largely undefined. We show in this study that IL-2 increased the expression of perforin and granzyme A, B, and C mRNA; intracellular granzyme B protein levels; and cytolytic function in a dose-dependent manner during primary activation of murine CD8+ T cells in vitro. Two approaches showed that these responses were not a consequence of the effects of IL-2 on cell survival and proliferation. First, IL-2 enhancement of perforin and granzyme expression was equivalent in CD8+ T cells from wild-type and bcl-2 transgenic mice, although only the latter cells survived in low concentrations or the absence of added IL-2. This property of bcl-2 transgenic T cells also allowed the demonstration that induction of granzyme A, B, and C mRNA and granzyme B protein required exogenous IL-2, whereas induction of perforin and IFN-γ expression did not. Second, analysis of perforin and granzyme mRNA levels in cells separated according to division number using the dye CFSE showed that the effects of IL-2 were unrelated to division number. Together, these findings indicate that IL-2 can directly regulate perforin and granzyme gene expression in CD8+ T cells independently of its effects on cell survival and proliferation.

Thymocytes are tested for productive rearrangement of the tcrb locus by expression of a pre-TCR in a process termed β-selection, which requires both Notch1 and CXCR4 signaling. It has been shown that activation of the GTPase Ras allows thymocytes to proliferate and differentiate in the absence of a Pre-TCR; the direct targets of Ras at this checkpoint have not been identified, however. Mice with a mutant allele of p110γ unable to bind active Ras revealed that CXCR4-mediated PI3K activation is Ras dependent. The Ras–p110γ interaction was necessary for efficient β-selection–promoted proliferation but was dispensable for the survival or differentiation of thymocytes. Uncoupling Ras from p110γ provides unambiguous identification of a Ras interaction required for thymic β-selection.

The generation of high-affinity Abs is essential for immunity and requires collaboration between B and T cells within germinal centers (GCs). By using novel mouse models with a conditional deletion of the p110δ catalytic subunit of the PI3K pathway, we established that p110δ is required in T cells, but not in B cells, for the GC reaction. We found the formation of T follicular helper (TFH) cells to be critically dependent on p110δ in T cells. Furthermore, by deleting phosphatase and tensin homolog deleted on chromosome 10, which opposes p110δ in activated T cells, we found a positive correlation between increased numbers of TFH cells and GC B cells. These results are consistent with the hypothesis that T cell help is the limiting factor in the GC reaction. P110δ was not required for the expression of B cell lymphoma 6, the downregulation of CCR7, or T cell entry into primary follicles. Instead, p110δ was the critical catalytic subunit for ICOS downstream signaling and the production of key TFH cytokines and effector molecules. Our findings support a model in which the magnitude of the GC reaction is controlled by the activity of the PI3K pathway in TFH cells.

T cell development requires phosphatidylinositol 3-kinase (PI3K) signaling with contributions from both the class IA, p110delta, and class IB, p110gamma catalytic subunits. However, the receptors on immature T cells by which each of these PI3Ks are activated have not been identified, nor has the mechanism behind their functional redundancy in the thymus. Here, we show that PI3K signaling from the preTCR requires p110delta, but not p110gamma. Mice deficient for the class IB regulatory subunit p101 demonstrated the requirement for p101 in T cell development, implicating G protein-coupled receptor signaling in beta-selection. We found evidence of a role for CXCR4 using small molecule antagonists in an in vitro model of beta-selection and demonstrated a requirement for CXCR4 during thymic development in CXCR4-deficient embryos. Finally, we demonstrate that CXCL12, the ligand for CXCR4, allows for Notch-dependent differentiation of DN3 thymocytes in the absence of supporting stromal cells. These findings establish a role for CXCR4-mediated PI3K signaling that, together with signals from Notch and the preTCR, contributes to continued T cell development beyond beta-selection.

Inflammatory activation of monocytes is an essential part of both innate immune responses and the pathogenesis of conditions such as atherosclerosis. However, the mechanisms which modulate the response of monocytes to inflammatory stimuli are still poorly understood. Here, we report that tribbles-2 (trb-2) is a novel regulator of inflammatory activation of monocytes. Down-regulation of trb-2 levels potentiates LPS-induced IL-8 production via enhanced activation of the extracellular signal-regulated kinase and jun kinase mitogen-activated protein kinase (MAPK) pathways. In keeping with this, the endogenous level of trb-2 expression in human primary monocytes is inversely correlated to the cell’s ability to produce IL-8. We show that trb-2 is a binding partner and a negative regulator of selected MAPKs. The potential in vivo relevance of these findings is highlighted by the observation that modified low-density lipoprotein profoundly down-regulates trb-2 expression, which may, in turn, significantly contribute to the inflammatory processes in the development of vascular disease. Taken together, our results define trb-2 as a potent novel regulator of monocyte biology, controlling the activation of these cells.\ud \ud

Exposure to IL-4 during activation of naive murine CD8+ T cells leads to generation of IL-4-producing effector cells with reduced surface CD8, low perforin, granzyme B and granzyme C mRNA, and poor cytolytic function. We show in this study that maximal development of these cells depended on exposure to IL-4 for the first 5 days of activation. Although IL-4 was not required at later times, CD8 T cell clones continued to lose surface CD8 expression with prolonged culture, suggesting commitment to the CD8low phenotype. This state was reversible in early differentiation. When single CD8low cells from 4-day cultures were cultured without IL-4, 65% gave rise to clones that partly or wholly comprised CD8high cells; the proportion of reverted clones was reduced or increased when the cells were cloned in the presence of IL-4 or anti-IL-4 Ab, respectively. CD8 expression positively correlated with perforin and granzyme A, B, and C mRNA, and negatively correlated with IL-4 mRNA levels among these clones. By contrast, most CD8low cells isolated at later time points maintained their phenotype, produced IL-4, and exhibited poor cytolytic function after many weeks in the absence of exogenous IL-4. We conclude that IL-4-dependent down-regulation of CD8 is associated with progressive differentiation and commitment to yield IL-4-producing cells with little cytolytic activity. These data suggest that the CD4−CD8− cells identified in some disease states may be the product of a previously unrecognized pathway of effector differentiation from conventional CD8+ T cells.

Control of the intracellular levels of phosphatidylinositol-(3, 4, 5)-trisphosphate by PI3K and phosphatase and tensin homolog (PTEN) is essential for B cell development and differentiation. Deletion of the PI3K catalytic subunit p110δ leads to a severe reduction in B1 and marginal zone (MZ) B cells, whereas deletion of PTEN results in their expansion. We have examined the relationship between these two molecules by generating mice with a B cell-specific deletion of PTEN (PTENB) and a concurrent germline deletion of p110δ. The expanded B1 cell population of PTENB mice was reduced to normal levels in PTENB/p110δ mutant mice, indicating a critical role for the p110δ isoform in the expansion of B1 cells. However, numbers of MZ B cells in the PTENB/p110δ mutants was intermediate between wild-type and PTENB-deficient mice, suggesting an additional role for other PI3K catalytic isoforms in MZ differentiation. Furthermore, the defective class switch recombination in PTENB B cells was only partially reversed in PTENB/p110δ double mutant B cells. These results demonstrate an epistatic relationship between p110δ and PTEN. In addition, they also suggest that additional PI3K catalytic subunits contribute to B cell development and function.