MaterialA fter exposure to Ag via immunization or infection, B cells have the capacity to generate plasma cells and develop an extrafollicular response or, together with follicular dendritic cells, initiate the germinal center (GC) reaction (1). The GC is a microanatomical structure formed in B cell follicles in secondary lymphoid organs where Ag-specific B cells undergo division, isotype switching, somatic hypermutation, and differentiation into memory B cells or plasma cells. Cognate interaction between B and T cells in the GC is essential for the selection of high-affinity Ag-specific B cells, and access to T cell help is thought to be a limiting factor for the positive selection of GC B cells (2).The PI3K pathway has been implicated in lymphocyte development and activation. It transduces extracellular signals into the production of the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) by phosphorylation of phosphatidylinositol 4,5-bisphosphate. In lymphocytes, PIP 3 is generated by the class I PI3K catalytic subunits of which there are four isoforms named a, b, d, and g. The a, b, and d enzymes form heterodimers with one of five Src homology 2 domain-containing regulatory subunits termed p85a, p85b, p55g, p50a, and p55a, which mediate recruitment to phosphotyrosine-containing signalosomes (3). The levels of cellular PIP 3 are also regulated by phosphatases: phosphatase and tensin homolog deleted on chromosome 10 (PTEN) directly opposes PI3K by removing the 39 phosphate from PIP 3 ; the SHIP enzymes generate the second messenger, phosphatidylinositol 3,4-bisphosphate.Mice with germline mutations of the p110d catalytic subunit have demonstrated its importance for the GC reaction. However, it remains unclear whether this is a reflection of the requirement for p110d in B cells, T cells, dendritic cells (DCs), or other cell types required for the GC response (4, 5). B cells from p110d-deficient mice display in vitro defects in survival and proliferation that correlate with defective signal transduction following stimulation with Abs via the BCR or CD19 (6-8). Whether these in vitro defects are relevant in vivo is unclear since B cell-specific deletion of PTEN impaired class-switch recombination but not the magnitude of the GC response (9). T cell-specific deletion of the class IA regulatory subunits showed that the GC reaction and Ag-specific Ab titers were reduced, implicating a T cell-intrinsic requirement for class IA PI3K but leaving unresolved the nature of the relevant catalytic subunits (10). Using the p110d D910A mouse model, which carries a point mutation that renders p110d catalytically inactive, adoptive transfer experiments revealed impaired Th1/Th2 cytokine production and a 2-fold reduction in clonal expansion (5, 11). In contrast, T cell-specific deletion of PTEN removed the requirement for CD28 costimulation (12) and allowed enhanced IL-4 production (13). Gaining a better understanding of the cell intrinsic role for p110d in Ab responses should prove helpful in determining the mech...

ZFP36L1 and ZFP36L2 are RNA-binding proteins (RBPs) which interact with AU-rich elements in the 3'UTR of mRNA, leading to mRNA degradation and translational repression. Mice lacking ZFP36L1 and ZFP36L2 during thymopoiesis develop a Notch1-dependent T cell acute lymphoblastic leukaemia (T-ALL). Prior to the onset of T-ALL, thymic development is perturbed with accumulation of cells which have passed through the β-selection checkpoint without first expressing T cell receptor β (TCR-β). Notch1 expression is increased in non-transformed thymocytes in the absence of ZFP36L1 and ZFP36L2. Both RBPs interact with evolutionarily conserved AU-rich elements within the 3' untranslated region of Notch1 and suppress its expression. These data establish a role for ZFP36L1 and ZFP36L2 during thymocyte development and in the prevention of malignant transformation.The development of T cells in the thymus proceeds through a series of developmental stages characterised by progressive rearrangement of the T cell receptor (TCR) genes and regulated by a series of developmental checkpoints. This ordered process is orchestrated by transcription factor networks which integrate environmental cues to initiate gene expression programs appropriate to the developmental stage of the thymocyte1 -3. However there is increasing recognition that gene expression during lymphocyte development is also subject to regulation by post-transcriptional mechanisms. These affect the half-life of mRNA though promotion or

T cell development requires phosphatidylinositol 3-kinase (PI3K) signaling with contributions from both the class IA, p110δ, and class IB, p110γ 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 p110δ, but not p110γ. 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 β-selection. We found evidence of a role for CXCR4 using small molecule antagonists in an in vitro model of β-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 β-selection.

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.

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.

The guanosine triphosphatases (GTPases) of the immunity-associated protein (GIMAP) family of putative GTPases has been implicated in the regulation of T-lymphocyte development and survival. A mouse conditional knockout allele was generated for the immune GTPase gene

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.

An interleukin (IL)-4-containing tumor environment is reported to be beneficial for immune clearance of tumor cells in vivo; however, the effect of IL-4 on the effector CD8 + T cells contributing to tumor clearance is not well defined. We have used the immunogenic HLA-CW3-expressing P815 (P.CW3) mastocytoma and investigated whether IL-4 expression by the tumor affects tumor clearance and, if so, whether it alters the tumor-induced VB10 + CD8 + T-cell response. P.CW3 were stably transfected with IL-4 or the empty control vector, and independent cell lines were injected i.p. into syngeneic DBA/2 mice. After apparent clearance of primary tumors over 12 to 15 days, secondary tumors arose that lacked surface expression and H-2-restricted antigen presentation of CW3 in part due to the loss of the HLA-CW3 expression cassette. Surprisingly, mice that received IL-4-producing tumor cells showed delayed primary tumor clearance and were significantly more prone to develop secondary tumors compared with mice receiving control tumor cells. Tumor clearance was dependent on CD8 + T cells. The IL-4-secreting P.CW3 tumor cells led to markedly higher mRNA expression of IL-4 and granzyme A and B but no differences in IFN-; and IL-2 production, cell proliferation, or ex vivo CTL activity in primary VB10 + CD8 + T cells when compared with the control tumor cells. We concluded that tumor-derived IL-4 selectively changed the quality of the tumor-induced CD8 + T-cell response and resulted in unexpected negative effects on tumor clearance. These data bring into question the delivery of IL-4 to the tumor environment for improving tumor immunotherapy. (Cancer Res 2006; 66(1): 571-80)