Hua Gu

Grb2 (growth-factor receptor-bound protein-2) is a signaling adaptor that interacts with numerous receptors and intracellular signaling molecules. However, its role in B-cell development and function remains unknown. Here we show that ablation of Grb2 in B cells results in enhanced B-cell receptor signaling; however, mutant B cells do not form germinal centers in the spleen after antigen stimulation. Furthermore, mutant mice exhibit defects in splenic architecture resembling that observed in B-cell–specific lymphotoxin-β–deficient mice, including disruption of marginal zone and follicular dendritic cell networks. We find thatgrb2^−/−B cells are defective in lymphotoxin-β expression. Although lymphotoxin can be up-regulated by chemokine CXCL13 and CD40 ligand stimulation in wild-type B cells, elevation of lymphotoxin expression ingrb2^−/−B cells is only induced by anti-CD40 but not by CXCL13. Our results thus define Grb2 as a nonredundant regulator that controls lymphoid follicle organization and germinal center reaction. Loss of Grb2 has no effect on B-cell chemotaxis to CXCL13, indicating that Grb2 executes this function by connecting the CXCR5 signaling pathway to lymphotoxin expression but not to chemotaxis.

Grb2 is an adaptor molecule that mediates Ras-MAPK activation induced by various receptors. Here we show that conditional ablation of Grb2 in thymocytes severely impairs both thymic positive and negative selections. Strikingly, the mutation attenuates T-cell antigen receptor (TCR) proximal signaling, including tyrosine phosphorylation of multiple signaling proteins and Ca²⁺ influx. The defective TCR signaling can be attributed to a marked impairment in Lck activation. Ectopic expression of a mutant Grb2 composed of the central SH2 and the C-terminal SH3 domains in Grb2^−/− thymocytes fully restores thymocyte development. Thus, Grb2 plays a pivotal role in both thymic positive and negative selection. It amplifies TCR signaling at the top end of the tyrosine phosphorylation cascade via a scaffolding function.

The function of microRNAs (miRNAs) in hematopoietic stem cells (HSCs), committed progenitors, and leukemia stem cells (LSCs) is poorly understood. We show that miR-29a is highly expressed in HSC and down-regulated in hematopoietic progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors results in acquisition of self-renewal capacity by myeloid progenitors, biased myeloid differentiation, and the development of a myeloproliferative disorder that progresses to acute myeloid leukemia (AML). miR-29a promotes progenitor proliferation by expediting G1 to S/G2 cell cycle transitions. miR-29a is overexpressed in human AML and, like human LSC, miR-29a-expressing myeloid progenitors serially transplant AML. Our data indicate that miR-29a regulates early hematopoiesis and suggest that miR-29a initiates AML by converting myeloid progenitors into self-renewing LSC.

Summary:  Lymphocyte development, activation, and tolerance depend on antigen receptor signaling transduced via multiple intracellular signalosomes. These signalosomes are assembled by different adapters. Given that signaling molecules can be either positive or negative regulators for a biochemical target, the complex of a target with different regulator may dictate the final signaling outcome. Grb2 is a simple adapter known to be involved in a variety of growth factor receptor signaling. However, its role in antigen receptor signaling as well as lymphocyte development and function has emerged only recently. Despite its simple molecular structure, recent experiments show that Grb2 may play a complex role in T and B‐cell antigen receptor signaling. In this article, we review recent findings about the physiological role of Grb2 in T and B‐cell development and activation and summarize the current mechanistic understanding of how Grb2 exerts its function following T and B‐cell antigen receptor stimulation.

Summary: Negative regulation of intracellular signaling delivered by the antigen receptors and coreceptors plays an important role in lymphocyte development and activation. Recent data from our laboratory and others have identified the Cbl family of ubiquitin ligases as important negative regulators in both T‐cell and B‐cell antigen receptor and coreceptor signaling. We show that c‐Cbl and Cbl‐b, two members of the Cbl family of proteins, play a redundant role in establishing the major histocompatibility complex‐dependent development of thymocytes and in thymic selection. They also control the activation threshold and CD28 costimulatory signaling in peripheral T cells. In B cells, c‐Cbl and Cbl‐b set the B‐cell receptor signaling threshold critical for proper B‐cell maturation and anergy induction. Biochemical studies indicate that the immune regulation by Cbl proteins correlate with their ubiquitin ligase function. Inactivation of Cbl‐b also renders mice resistant to both transplanted and spontaneous tumors due to an enhanced anti‐tumor immunity of CD8⁺ T cells. These findings thus place Cbl proteins at the center of a complex immune network regulation and suggest that modulation of this signaling pathway may be beneficiary to the treatment of autoimmunity and cancer.

It has been shown that the transgene insertional mutations TM1 and TM2 constitute a genetic trait controlling thymocyte development. Here we conducted a detailed analysis of the impact of TM1 and TM2 double mutation on thymocyte development. We found that the hemizygous TM1 and TM2 double transgenic mice possessed much smaller thymi. Flow cytometric analysis revealed a severe blockage of T‐cell development at the transition from DN3 to DN4 stage and pre‐T‐cell receptor (pre‐TCR)/TCR signaling appeared to be impaired. We could not identify any known gene that was implicated in a similar function in the chromosomal regions 7E–F1 and 11B5–C, where TM1 and TM2 mutations were mapped to respectively. Thus, TM1 and TM2 mutations represent two novel alleles that define a genetic trait controlling DN3 thymocyte development, possibly through modulating the signals downstream of the pre‐TCR.

Transgene insertion is instrumental to identifying genes with defined physiological functions. In this paper, we show that mice homozygous for either TM1 or TM2, two mutant alleles with distinct transgene insertions, exhibited embryo lethality, suggesting that these two alleles play essential roles in embryogenesis. Interestingly, although hemizygous TM1 or TM2 alone did not have obvious alteration in thymocyte development, together, they exhibited a compound effect on thymocyte development, blocking the development from CD4 and CD8 double-negative to double-positive stage of T cells. TM1 and TM2 mutations were mapped to chromosome regions 7E-F1 and 11B5-C, respectively, where we could not identify any known gene that was implicated in a similar function. Thus, TM1 and TM2 represent two novel alleles that define a genetic trait controlling thymocyte development.

A significant challenge to efforts aimed at inducing effective antitumor immune responses is that CD8⁺ T cells, which play a prominent role in these responses, may be unable to respond to tumors that lack costimulatory signals and that are protected by an immune suppressive environment such as that mediated by TGF-β produced by tumor cells themselves or by infiltrating Tregs, often resulting in tolerance or anergy of tumor-specific T cells. Here we show that the in vitro activation of Cblb^–/– CD8⁺ T cells does not depend on CD28 costimulation and is resistant to TGF-β suppression. In vivo studies further demonstrated that Cblb^–/– mice, but not WT controls, efficiently rejected inoculated E.G7 and EL4 lymphomas that did not express B7 ligands and that introduction of the Cblb^–/– mutation into tumor-prone ataxia telangiectasia mutated–deficient mice markedly reduced the incidence of spontaneous thymic lymphomas. Immunohistological study showed that E.G7 tumors from Cblb^–/– mice contained massively infiltrating CD8⁺ T cells. Adoptive transfer of purified Cblb^–/– CD8⁺ T cells into E.G7 tumor-bearing mice led to efficient eradication of established tumors. Thus, our data indicate that ablation of Cbl-b can be an efficient strategy for eliciting immune responses against both inoculated and spontaneous tumors.