Katherine A. Forbush

Notch1 Signaling Promotes the Maturation of CD4 and CD8 SP Thymocytes

Notch proteins regulate many developmental processes. Notch1 is highly expressed on thymocytes, but its role in regulating their development is not known. We show that activation of Notch1 signaling in CD4+CD8+ double positive thymocytes promotes the maturation of both CD4+ and CD8+ single positive thymocytes and that this occurs in the absence of interactions between the T cell receptor and MHC molecules expressed on thymic epithelial cells. We have also identified several genes that are transcriptionally regulated by Notch1 in T cells and show that they are upregulated during maturation into both single positive lineages. These observations suggest that Notch1 signaling plays a role in promoting maturation into both the CD4 and CD8 T cell lineages.

Regulation of T cell receptor signaling by a src family protein-tyrosine kinase (p59fyn)

Engagement of the clonotypic antigen receptor (TCR) on T lymphocytes provokes an activation response leading to cell proliferation and lymphokine secretion. To examine the molecular basis of T cell signaling, we generated transgenic animals in which a lymphocyte-specific nonreceptor protein-tyrosine kinase p59fyn(T) is 20-fold overexpressed in developing T lineage cells. Thymocytes from these mice, analyzed using both cellular and biochemical assays, were remarkably hyperstimulable. Moreover, the responsiveness of normal thymocytes to TCR-derived signals correlated well with the extent to which p59fyn was expressed in these cells. Overexpression of a catalytically inactive form of p59fyn substantially inhibited TCR-mediated activation in otherwise normal thymocytes. These effects are unique to p59fyn; overexpression of a closely related T cell-specific tyrosine kinase, p56lck, elicits dramatically different phenotypes. Our results suggest that p59fyn is a critically important component of the TCR signal transduction apparatus.

A dominant-negative transgene defines a role for p56lck in thymopoiesis.

The lymphocyte‐specific protein tyrosine kinase p56lck participates in T cell signaling through functional interactions with components of the T cell antigen receptor complex and the interleukin‐2 receptor. Additional insight into the function of p56lck has now been obtained through the generation of transgenic animals expressing high levels of a catalytically inactive form of this kinase (p56lckR273). Mice bearing the lckR273 transgene manifested a severe defect in the production of virtually all T lymphocytes. Those exceptional CD3+ cells that escaped the effects of the lckR273 transgene were confined primarily to the T cell subset that expresses gamma/delta T cell receptors. Remarkably, construction of a dose‐response curve for the effects of the lckR273 transgene revealed that developmental arrest of thymocytes occurred at a discrete stage in the normal T cell maturation pathway, corresponding to a point at which thymoblasts ordinarily begin a series of mitotic divisions that result in expansion and maturation. These results suggest that p56lck normally regulates T cell production by metering the replicative potential of immature thymoblasts.

Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase

Stimulation of the T lymphocyte antigen receptor‐CD3 complex (TCR‐CD3) causes T cell activation by a process associated with increased phosphatidylinositol‐specific phospholipase C (PI‐PLC) activity. Evidence exists suggesting that GTP‐binding (G) proteins, particularly the pertussis toxin (PT)‐sensitive Gi proteins, participate in this signal transduction pathway. To clarify the role of Gi proteins in TCR‐CD3 signaling, and to investigate other possible functions of Gi molecules in T cells, we expressed the S1 subunit of PT in the thymocytes of transgenic mice using the lymphocyte‐specific lck promoter. Transgenic thymocytes contained S1 activity and exhibited profound depletion of Gi protein PT substrates in a manner suggesting their inactivation by S1 in vivo. Nevertheless, treatment of transgenic thymocytes with mitogenic stimuli provoked normal increases in intracellular free Ca2+ concentrations and IL‐2 secretion, indicating that Gi proteins are not required for T cell activation. These normal signaling responses notwithstanding, mature thymocytes accumulated in lck‐PT mice and did not appear in secondary lymphoid organs or in the circulation. Viewed in the context of the known features of Bordetella pertussis infection, our results suggest that a PT‐sensitive signaling process, probably involving Gi proteins, regulates thymocyte emigration.

Fyn can partially substitute for Lck in T lymphocyte development.

Lck, a Src family tyrosine kinase, transduces signals important for the development of alphabeta and gammadelta T cells. However, T cell development is only partially compromised in Lck-deficient mice, suggesting that other kinases may also transduce pre-TCR or TCR signals. One candidate is Fyn, a Src kinase coexpressed with Lck in immature and mature T cells. Here we show that T cell development is completely compromised in lck(-/-)fyn(-/-) mice. In addition, we demonstrate that expression of a gain-of-function mutant fyn(T) transgene completely restores production of immature CD4/CD8 double positive thymocytes and gammadelta T cells and improves the representation of CD4 or CD8 single positive thymocytes. These observations reveal that Fyn can subserve some Lck-like functions in T cell development.

Control of B cell development by Ras-mediated activation of Raf

Cell fate commitment in a variety of lineages requires signals conveyed via p21ras. To examine the role of p21ras in the development of B lymphocytes, we generated transgenic mice expressing a dominant‐negative form of Ras in B lymphocyte progenitors, using a novel transcriptional element consisting of the Eμ enhancer and the lck proximal promoter. Expression of dominant‐negative Ras arrests B cell development at a very early stage, prior to formation of the pre‐B cell receptor. Furthermore, an activated form of Raf expressed in the same experimental system could both drive the maturation of normal pro‐B cells and rescue development of progenitors expressing dominant‐negative Ras. Hence p21ras normally regulates early development of B lymphocytes by a mechanism that involves activation of the serine/threonine kinase Raf.

The extracellular matrix protein mindin is a pattern-recognition molecule for microbial pathogens

Microbial pathogens use a variety of their surface molecules to bind to host extracellular matrix (ECM) components to establish an effective infection. However, ECM components can also serve as an integral part of the innate immunity. Mice lacking expression of mindin (spondin 2), a highly conserved ECM protein, have an impaired ability to clear bacterial infection, and mindin-deficient macrophages show defective responses to a broad spectrum of microbial stimuli. Moreover, mindin binds directly to bacteria and their components and functions as an opsonin for macrophage phagocytosis of bacteria. Thus, mindin is essential in the initiation of the innate immune response and represents a unique pattern-recognition molecule in the ECM for microbial pathogens.

Thymocyte expression of cathepsin L is essential for NKT cell development

CD1d antigen presentation to natural killer T (NKT) cells expressing the semi-invariant T cell receptor Vα14Jα18 requires CD1d trafficking through endosomal compartments; however, the endosomal events remain undefined. We show that mice lacking the endosomal protease cathepsin L (catL) have greatly reduced numbers of Vα14+NK1.1+ T cells. In addition, catL expression in thymocytes is critical not only for selection of these cells in vivo but also for stimulation of Vα14+NK1.1+ T cells in vitro. CD1d cell-surface expression and intracellular localization appear normal in catL-deficient thymocytes, as does the lysosomal morphology; this implies a specific role for catL in regulating presentation of natural CD1d ligands mediating Vα14+NK1.1+ T cell selection. These data implicate lysosomal proteases as key regulators of not only classical major histocompatibility complex class II antigen presentation but also nonclassical CD1d presentation.

B7h is required for T cell activation, differentiation, and effector function

T helper (Th) cell activation, differentiation, and immune function are regulated by costimulatory molecules. Inducible costimulator (ICOS) is a recently identified costimulatory receptor expressed on activated T cells. A ligand for ICOS, B7h, is expressed on B cells and other types of antigen-presenting cells (APC). Although ICOS has been shown to be essential in T cell activation and differentiation, the regulatory roles of B7h at different stages of T cell immune responses have not been examined genetically. In this study, we generated and analyzed B7h-deficient mice. We present evidence that B7h is the only ligand for ICOS, and ICOS, its only corresponding receptor. Th cells, when activated with B7h-deficient APC, exhibited reduced proliferation and IL-2 production. In addition, Th cells produced significantly reduced amounts of IL-4 and -13 after differentiation at the presence of B7h–/– APC. This cytokine defect was associated with a deficiency in c-Maf expression and could be rescued completely by c-Maf overexpression in T cells. Furthermore, we showed that effector T cells, when restimulated in the presence of B7h-deficient APC, exhibited reduced Th2 cytokine production. Therefore, B7h is required for proper Th cell activation, differentiation, and effector cytokine expression.

Differential Regulation of Cathepsin S and Cathepsin L in Interferon γ–treated Macrophages

Cathepsin S (catS) and cathepsin L (catL) mediate late stages of invariant chain (Ii) degradation in discrete antigen-presenting cell types. Macrophages (Mφs) are unique in that they express both proteases and here we sought to determine the relative contribution of each enzyme. We observe that catL plays no significant role in Ii cleavage in interferon (IFN)-γ–stimulated Mφs. In addition, our studies show that the level of catL activity is significantly decreased in Mφs cultured in the presence of IFN-γ whereas catS activity increases. The decrease in catL activity upon cytokine treatment occurs despite the persistence of high levels of mature catL protein, suggesting that a specific inhibitor of the enzyme is up-regulated in IFN-γ–stimulated peritoneal Mφs. Similar inhibition of activity is observed in dendritic cells engineered to overexpress catL. Such enzymatic inhibition in Mφs exhibits only partial dependence upon Ii and therefore, other mechanisms of catL inhibition are regulated by IFN-γ. Thus, during a T helper cell type 1 immune response catL inhibition in Mφs results in preferential usage of catS, such that major histocompatibility complex class II presentation by all bone marrow–derived antigen-presenting cell is regulated by catS.