Role of a selecting ligand in shaping the murine γδ-TCR repertoire.

Unlike αß-T lineage cells, where the role of ligand in intrathymic selection is well established, the role of ligand in the development of γδ-T cells remains controversial. Here we provide evidence for the role of a bona fide selecting ligand in shaping the γδ-T cell-receptor (TCR) repertoire. Reactivity of the γδ-TCR with the major histocompatibility complex (MHC) Class Ib ligands, H2-T10/22, is critically dependent upon the EGYEL motif in the complementarity determining region 3 (CDR3) of TCRδ. In the absence of H2-T10/22 ligand, the commitment of H2-T10/22 reactive γδ-T cells to the γδ fate is diminished, and the specification of those γδ committed cells to the IFN-γ or interleukin-17 effector fate is altered. Furthermore, those cells that do adopt the γδ fate and mature exhibit a profound alteration in the γδTCR repertoire, including depletion of the EGYEL motif and reductions in both CDR3δ length and charge. Taken together, these data suggest that ligand plays an important role in shaping the TCR repertoire of γδ-T cells.

Appl1 and Appl2 are Expendable for Mouse Development But Are Essential for HGF-Induced Akt Activation and Migration in Mouse Embryonic Fibroblasts.

Although Appl1 and Appl2 have been implicated in multiple cellular activities, we and others have found that Appl1 is dispensable for mouse embryonic development, suggesting that Appl2 can substitute for Appl1 during development. To address this possibility, we generated conditionally targeted Appl2 mice. We found that ubiquitous Appl2 knockout (Appl2-/-) mice, much like Appl1-/- mice, are viable and grow normally to adulthood. Intriguingly, when Appl1-/- mice were crossed with Appl2-/- mice, we found that homozygous Appl1;Appl2 double knockout (DKO) animals are also viable and grossly normal with regard to reproductive potential and postnatal growth. Appl2-null and DKO mice were found to exhibit altered red blood cell physiology, with erythrocytes from these mice generally being larger and having a more irregular shape than erythrocytes from wild type mice. Although Appl1/2 proteins have been previously shown to have a very strong interaction with phosphatidylinositol-3 kinase (Pi3k) in thymic T cells, Pi3k-Akt signaling and cellular differentiation was unaltered in thymocytes from Appl1;Appl2 (DKO) mice. However, Appl1/2-null mouse embryonic fibroblasts exhibited defects in HGF-induced Akt activation, migration, and invasion. Taken together, these data suggest that Appl1 and Appl2 are required for robust HGF cell signaling but are dispensable for embryonic development and reproduction.

Rpl22 Loss Impairs the Development of B Lymphocytes by Activating a p53-Dependent Checkpoint.

Although ribosomal proteins facilitate the ribosome's core function of translation, emerging evidence suggests that some ribosomal proteins are also capable of performing tissue-restricted functions either from within specialized ribosomes or from outside of the ribosome. In particular, we have previously demonstrated that germline ablation of the gene encoding ribosomal protein Rpl22 causes a selective and p53-dependent arrest of ab T cell progenitors at the b-selection checkpoint. We have now identified a crucial role for Rpl22 during early B cell development. Germline ablation of Rpl22 results in a reduction in the absolute number of B-lineage progenitors in the bone marrow beginning at the pro­B cell stage. Although Rpl22-deficient pro­B cells are hyporesponsive to IL-7, a key cytokine required for early B cell development, the arrest of B cell development does not result from disrupted IL-7 signaling. Instead, p53 induction appears to be responsible for the developmental defects, as Rpl22 deficiency causes increased expression of p53 and activation of downstream p53 target genes, and p53 deficiency rescues the defect in B cell development in Rpl22-deficient mice. Interestingly, the requirement for Rpl22 in the B cell lineage appears to be developmentally restricted, because Rpl22-deficient splenic B cells proliferate normally in response to Ag receptor and Toll receptor stimuli and undergo normal class-switch recombination. These results indicate that Rpl22 performs a critical, developmentally restricted role in supporting early B cell development by preventing p53 induction.

Noncanonical mode of ERK action controls alternative αß and γδ T cell lineage fates.

Gradations in extracellular regulated kinase (ERK) signaling have been implicated in essentially every developmental checkpoint or differentiation process encountered by lymphocytes. Yet, despite intensive effort, the molecular basis by which differences in ERK activation specify alternative cell fates remains poorly understood. We report here that differential ERK signaling controls lymphoid-fate specification through an alternative mode of action. While ERK phosphorylates most substrates, such as RSK, by targeting them through its D-domain, this well-studied mode of ERK action was dispensable for development of γδ T cells. Instead, development of γδ T cells was dependent upon an alternative mode of action mediated by the DEF-binding pocket (DBP) of ERK. This domain enabled ERK to bind a distinct and select set of proteins required for specification of the γδ fate. These data provide the first in vivo demonstration for the role of DBP-mediated interactions in orchestrating alternate ERK-dependent developmental outcomes.

Origins of γδ T cell effector subsets: a riddle wrapped in an enigma.

αß and γδ T cells are thought to arise from a common precursor in the thymus but play distinct roles in pathogen resistance. Although conventional αß T cells exit the thymus in a naive state and acquire effector function in the periphery, the effector fate of many γδ T cells is specified in the thymus and exhibits limited plasticity thereafter. This review describes the current models that have been proposed to explain the acquisition of effector fate by γδ T cells, as well as the apparent linkage to Vγ gene usage. The two predominant models are the predetermination model, which suggests that effector fate is determined prior to TCR expression, perhaps in association with the developmental timing of Vγ rearrangement, and the TCR-dependence model, which proposes that the nature of the TCR signal, particularly its intensity or duration, plays an important role in influencing effector fate.

The TCR ligand-inducible expression of CD73 marks γδ lineage commitment and a metastable intermediate in effector specification.

Numerous studies indicate that γδ T cell receptor (γδTCR) expression alone does not reliably mark commitment of early thymic progenitors to the γδ fate. This raises the possibility that the γδTCR is unable to intrinsically specify fate and instead requires additional environmental factors, including TCR-ligand engagement. We use single cell progenitor assays to reveal that ligand acts instructionally to direct adoption of the γδ fate. Moreover, we identify CD73 as a TCR ligand-induced cell surface protein that distinguishes γδTCR-expressing CD4(-)CD8(-) progenitors that have committed to the γδ fate from those that have not yet done so. Indeed, unlike CD73(-) γδTCR(+) progenitors, which largely adopt the αß fate upon separation from the intrathymic selecting environment, those that express CD73 remain CD4(-)CD8(-) and committed to the γδ fate. CD73 is expressed by >90% of peripheral γδ cells, suggesting this is a common occurrence during development. Moreover, CD73 induction appears to mark a metastable intermediate stage before acquisition of effector function, suggesting that γδ lineage and effector fate are specified sequentially. These findings have important implications for the role of ligand in γδ lineage commitment and its relationship to the specification of effector fate.

Appl1 is dispensable for Akt signaling in vivo and mouse T-cell development.

Appl1 (Adaptor protein containing pleckstrin homology [PH], phosphotyrosine binding [PTB], and Leucine zipper motifs) is an adaptor that participates in cell signaling by interacting with various signaling molecules including Akt, PI3-kinase (PI3K), Rab5, adiponectin receptor, and TrkA. By using RNA knockdown technology, Appl1 has been implicated in zebrafish development and murine glucose metabolism. To investigate the unambiguous role of Appl1 in vivo, we generated a knockout mouse in which exon1 of the Appl1 gene was disrupted using gene trap methodology. Homozygous Appl1 knockout mice with ubiquitous loss of Appl1 protein expression were viable, grossly normal, and born at expected Mendelian ratios. Moreover, activation of Akt and the downstream effecter Gsk3ß was unaffected in vivo. We next performed glucose and insulin tolerance tests and found that glucose metabolism is normal in Appl1-null mice. We also tested the effect of Appl1 loss on Akt signaling in T cells, because we discovered that Appl1 strongly interacts with the p110ß subunit of PI3K in T lymphocytes. However, such interaction was found to be dispensable for Akt signaling in thymic T cells and T-cell development. Moreover, Appl1 loss did not affect DNA synthesis in cultured thymocytes, although loss of Appl1 was associated with a slight increase in ConA-stimulated splenic T-cell viability/proliferation. Collectively, our findings indicate that Appl1 is dispensable for Akt signaling in vivo and T-cell differentiation.

Expression of cellular FLIP by B cells is required for their participation in an immune response.

High levels of the Fas-signaling antagonist cellular FLIP (cFLIP) in germinal center (GC) B cells suggests an important role for this factor during this stage of the T cell-dependent B cell immune response. To test this idea, we used mice with B cell-specific deletion of a floxed cFLIP allele. Although deletion of cFLIP did not alter their primary development, participation of cFLIP-deficient B cells in the immune response was severely perturbed. Using previously characterized IgH locus-targeted BCR transgenic mice, we showed that adoptively transferred cFLIP-deficient follicular B cells do not effectively participate in the GC response in wild-type hosts. However, this failure was accompanied by severe defects in the initial activation and proliferation of these B cells in vivo. In addition, immunization of mice with B cell-specific cFLIP deletion resulted in selective recruitment into GCs and Ab-forming cell responses of B cells that had not deleted the floxed cFLIP allele. Together, these findings demonstrate that expression of cFLIP is a prerequisite for participation of B cells in all stages of Ag-driven immune responses.

Initial clonal expansion of germinal center B cells takes place at the perimeter of follicles.

Current models of the germinal center (GC) response propose that after stimulation at the edges of T cell zones, pre-GC B cells directly migrate to the center of follicles and proliferate to form GCs. We followed the interrelationship of proliferation, differentiation, and microenvironmental locale in populations of pre-GC B cells responding to antigen. In contrast to the predictions of current models, after accumulation at the T-B interface, these cells appeared at the perimeter of follicles adjacent to the marginal zone. There, they rapidly proliferated for several days but underwent no V gene hypermutation and little heavy-chain class switching. Their chemokine receptor expression pattern indicated that these cells were sessile, yet they had begun to acquire many phenotypic characteristics of GC B cells. The expanded clones were subsequently observed in the center of follicles, suggesting that GCs are created by coalescence of B cells from this follicular perimeter response.

A role for cFLIP in B cell proliferation and stress MAPK regulation.

Fas/Apo-1 signals through the FADD (Fas-associated death domain) adaptor protein, which recruits and activates the apical caspase 8 and leads to apoptosis. Cellular FLIP (cFLIP) is a homolog of caspase 8 and is also capable of binding to FADD. Previous studies suggest that cFLIP could either enhance or inhibit apoptosis and lead to NF-kappaB and Erk1/2 activation. Like FADD or caspase 8 deficiency, a lack of cFLIP disrupts embryogenesis and T cell proliferation. It has been demonstrated that B cells lacking either FADD or caspase 8 were defective in both Fas-induced apoptosis and TLR-induced proliferation, which indicates that these death-inducing proteins have an additional role in regulating innate immunity. To analyze the function of cFLIP in B cells, conditional deletion of cFLIP was induced by using CD19(Cre). The resulting B cell-specific cFLIP-deficient mice were found to have reduced numbers of peripheral B cells that were hypersensitive to Fas-induced apoptosis and impaired in proliferation induced by TLRs and the BCR. Furthermore, there was aberrant expression of costimulatory proteins and activation markers in cFLIP-deficient B cells. Whereas LPS-induced activation of NF-kappaB and Erk1/2 appears to be unaffected, p38 and Jnk were spontaneously activated and hyperinduced in cFLIP-deficient B cells. Therefore, these data revealed novel functions of cFLIP in B cells.

Primary development and participation in a foreign antigen-driven immune response of a chromatin-reactive B cell clonotype are not influenced by TLR9 or other MyD88-dependent TLRs.

Recent findings support a central role for TLRs in both foreign Ag-driven immune responses and systemic autoimmune diseases mediated by B lymphocytes. In vitro studies have shown that the Ag receptors (BCRs) on B cells specific for nuclear autoantigens can facilitate the delivery of these autoantigens to the endocytic compartment, resulting in activation of the nucleic acid-specific TLRs present in this subcellular locale. If this pathway is operative in vivo it might promote the development, survival, or activation of such autoreactive B cells. To test this idea, we evaluated the influence of a deficiency in the CpG DNA-specific TLR, TLR9, or all MyD88-dependent TLRs on the primary development and foreign Ag-driven immune response of B cells in a line of V(H) knockin mice that contains a high frequency of "dual reactive" B cells specific for DNA-based autoantigens such as chromatin, as well as the hapten arsonate. We found that although development and activation of these B cells in vitro are clearly influenced by DNA-based autoantigens, TLR9 or MyD88 deficiencies had no apparent effect on the primary development and participation in the anti-arsonate response of these B cells in vivo. We discuss these results in the context of previous models for the role of TLR9 and other TLRs in the regulation of antinuclear Ag B cell development and activity.