Cytotoxic T lymphocyte antigen 4 is induced in the thymus upon in vivo activation and its blockade prevents anti-CD3-mediated depletion of thymocytes.

The development of a normal T cell repertoire in the thymus is dependent on the interplay between signals mediating cell survival (positive selection) and cell death (negative selection or death by neglect). Although the CD28 costimulatory molecule has been implicated in this process, it has been difficult to establish a role for the other major costimulatory molecule, cytotoxic T lymphocyte antigen (CTLA)-4. Here we report that in vivo stimulation through the T cell receptor (TCR)-CD3 complex induces expression of CTLA-4 in thymocytes and leads to the association of CTLA-4 with the SH2 domain-containing phosphatase (SHP)-2 tyrosine phosphatase. Moreover, intrathymic CTLA-4 blockade dramatically inhibits anti-CD3-mediated depletion of CD4+CD8+ double positive immature thymocytes. Similarly, anti-CD3-mediated depletion of CD4+CD8+ double positive cells in fetal thymic organ cultures could also be inhibited by anti-CTLA-4 antibodies. Thus, our data provide evidence for a role of CTLA-4 in thymic selection and suggest a novel mechanism contributing to the regulation of TCR-mediated selection of T cell repertoires.

Paradoxical effects of overexpression of the type I insulin-like growth factor (IGF) receptor on the responsiveness of human breast cancer cells to IGFs and estradiol.

Estrogens increase the proliferative response of estrogen-responsive breast cancer cells to insulin-like growth factors (IGFs). The mechanisms involved are unclear, but the observation that estradiol increases type I IGF receptor levels in MCF-7 breast cancer cells has suggested that the increased response may be due to increased expression of type I IGF receptor. The purpose of this study was to investigate this hypothesis by using a retroviral expression vector to constitutively over-express the type I IGF receptor in estrogen-responsive breast cancer cells. We isolated clones of infected MCF-7 cells that expressed up to 4.5-fold more receptor than the estradiol-induced level in cells infected with a control vector. Hybridization of a type I IGF receptor complementary DNA probe to RNA extracted from these clones showed that most of the receptor RNA was transcribed from the retroviral provirus. Estrogen receptor continued to be expressed in clones overexpressing type I IGF receptor, and overexpression had little effect on the induction of an estrogen-regulated gene by estradiol and the proliferative response to IGFs alone or estradiol alone. Overexpression did, however, alter the proliferative response to IGFs in the presence of estradiol. The three clones analyzed showed an increased sensitivity to low IGF-I concentrations and a paradoxical attenuation of the synergistic effect between estradiol and IGF-I at high IGF-I concentrations. Collectively, these experiments show that the level of expression of the type I IGF receptor is an important determinant in the responsiveness of breast cancer cells to estrogen, but the observation that the response of cells to estradiol alone is not affected by constitutive overexpression of the type I IGF receptor suggests that estrogens stimulate the proliferation of breast cancer cells by regulating the expression of genes in addition to the type I IGF receptor.

Interactions between the oestrogen and insulin-like growth factor signalling pathways in the control of breast epithelial cell proliferation.

There is increasing evidence for interactions between steroid and growth factor signalling pathways. Oestrogens modulate the responsiveness of breast epithelial cells to insulin-like growth factors (IGFs), and this may be the mechanism by which oestrogens modulate cell proliferation. Oestrogens appear to act at several points in the IGF signal transduction pathway. Despite earlier studies suggesting that breast epithelial cells do not synthesize IGF-I, we have shown by PCR that IGF-I is expressed and that its expression is regulated by oestrogen. IGF-II is expressed at markedly higher levels than IGF-I and is also regulated by oestrogen, consistent with it being an oestrogen-regulated autocrine growth factor. Oestrogens regulate the expression of IGF binding proteins and the type I IGF receptor. The biological significance of oestrogen regulation of IGF binding protein expression is not clear. Experiments in which the type I IGF receptor has been constitutively overexpressed have suggested that oestrogen regulation of the receptor is not involved in mediating the effects of oestrogen on cell proliferation. Recent studies have started to assess the effects of oestrogen on the expression of components of the IGF signal transduction pathway, and have shown that the expression of insulin receptor substrate-1, the principal substrate for the tyrosine kinase of the type I IGF receptor, is regulated by oestradiol.

Location-specific regulation of transgenic Ly49A receptors by major histocompatibility complex class I molecules.

Inhibitory receptors expressed on natural killer (NK) cells and T cells specific for major histocompatibility complex (MHC) class I are believed to prevent these cells from responding to normal self tissues. To understand the regulation and function of Ly49 receptor molecules in vivo, we used the CD2 promoter to target Ly49A expression to all thymocytes, T cells, and NK cells. In animals expressing its MHC class I ligand, H-2Dd or H-2Dk, there was a large decrease in the expression of Ly49A on thymocytes, peripheral T cells, and NK1.1+ cells. The extent of the down-regulation of Ly49A was dependent on the expression of the MHC ligand for Ly49A and on the site where the cells were located. The level of expression of endogenous Ly49A was similarly found to be dependent upon the organ where the cells resided. Data from bone marrow chimeras indicated that most cell types may regulate Ly49A expression, but the efficacy to regulate receptor expression may vary depending on the cell type.

H-2Dd engagement of Ly49A leads directly to Ly49A phosphorylation and recruitment of SHP1.

We have used a number of in vitro and in vivo techniques to identify the molecules that can bind to the cytoplasmic tail of the Ly49A receptor. Affinity chromatography using peptides corresponding to the N-terminal 18 amino acids of Ly49A allowed the recovery of a number of proteins that bound preferentially to the tyrosine-phosphorylated peptide, including SH2-containing phosphatase-1 (SHP1) and the SH2-containing inositol 5' phosphatase (SHIP). In another approach, using the entire cytoplasmic domain of the Ly49A receptor, we found that SHP2 also interacted with the tyrosine-phosphorylated form of the Ly49A cytoplasmic tail. Using BIACORE(R)2000 analysis, we determined that both SHP1 and SHP2 bound to the tyrosine-phosphorylated cytoplasmic tail of Ly49A with affinities in the nanomolar range, whilst SHIP showed no binding. Mutation of tyrosine-36 to phenylalanine did not significantly affect the affinities of these proteins for the tyrosine-phosphorylated cytoplasmic tail of Ly49A. In addition, using a whole-cell system with T-cell lymphoma cell lines that expressed the Ly49A receptor or its H-2Dd ligand, we determined that engagement of Ly49A by its major histocompatibility complex (MHC) ligand leads to tyrosine-phosphorylation events and recruitment of SHP1. Recruitment of SHP1 was rapid and transient, reaching a maximum after 5 min. These data suggest that mechanisms for the inhibitory signal are generated following receptor engagement. They also provide direct evidence that ligand engagement of the Ly49A receptor is responsible for recruitment of downstream signalling molecules.

Cloning and characterization of a novel mouse myeloid DAP12-associated receptor family.

The presence of a negatively charged residue in the transmembrane domain of DAP12 precludes its cell surface expression in the absence of a partner receptor containing a positive charge in its transmembrane domain. We utilized this property of DAP12 to screen a BALB / c macrophage cDNA library for novel molecules that induce cell surface expression of DAP12. By this method, we cloned a cell surface receptor with a single Ig (V) domain, a transmembrane lysine residue, and a short cytoplasmic domain. By homology screening of BALB / c macrophage libraries, we identified a second cDNA for a highly homologous receptor. These receptors appear to be the mouse orthologues of a recently identified human cDNA, TREM-2, so we have designated the receptors as mouse TREM-2a and TREM-2b. By Northern blotting, transcripts for TREM-2 were found in each of three macrophage cell lines but not in a variety of other hematopoietic cell lines. We further demonstrate that TREM-2a is associated with endogenous DAP12 in macrophage cells, and cross-linking of TREM-2a on the surface of macrophages leads to the release of nitric oxide. Our studies define TREM-2 as a receptor family in mouse macrophages and demonstrate the capacity of these receptors to activate macrophage function through DAP12.

Immune rejection of a large sarcoma following cyclophosphamide and IL-12 treatment requires both NK and NK T cells and is associated with the induction of a novel NK T cell population.

Combined immunotherapy with cyclophosphamide (Cy) and IL-12, but not IL-12 alone, stimulates eradication of a large established solid tumor (20 mm), MCA207, a methylcholanthrene-induced murine sarcoma. In these studies we demonstrate that NK1.1(+) cells and CD1d-dependent NK T cells each play important yet distinct roles in regression of a large tumor in response to Cy and IL-12, and we define a novel NK T cell subset, selectively increased by this treatment. Mice depleted of NK1.1(+) cells demonstrated more rapid initial tumor growth and prolonged tumor regression following treatment, but tumors were eventually eradicated. In contrast, initial tumor regression following therapy was unimpaired in CD1d(-/-) mice, which are deficient in most NK T cells, but tumors recurred. No tumor regression occurred following Cy and IL-12 therapy in CD1d(-/-) mice that were depleted of NK1.1(+) cells. We found that Cy and IL-12 induced the selective increase in liver and spleen lymphocytes of a unique NK T subpopulation (DX5(+)NK1.1(-)CD3(+)). These cells were not induced by treatment in CD1d(-/-) mice. Our studies demonstrate a contribution of both NK and NK T cells to the Cy- and IL-12-stimulated anti-tumor response. We describe the selective induction of a distinct NK T cell subset by Cy and IL-12 therapy, not seen following IL-12 therapy alone, which we suggest may contribute to the successful anti-tumor response induced by this immunotherapeutic regimen.

Physiologic functions of activating natural killer (NK) complex-encoded receptors on NK cells.

Natural killer (NK) cells express a superfamily of surface proteins that share common structural features: dimeric type II integral membrane proteins with extracellular domains resembling C-type lectins. These receptors are encoded by a single genetic region called the NK complex (NKC). The NKC encompasses several families of genes including NKR-PI, Ly-49, CD94/NKG2, and NKG2D. Different NKC-encoded receptors have been shown to activate or to inhibit NK-cell function, and different receptors within the same family can have opposing functions. Within an individual NK cell, inhibitory receptors typically predominate over stimulatory receptors, calling into question the teleologic requirement or physiologic significance of lectin-like activating receptors in NK cells. Despite the widespread expression of inhibitory receptors, however, subtle features of activating receptor biology enable them to stimulate effector functions in vivo and in vitro. Activating receptors and inhibitory receptors differ in their subset expression, in their structural constraints for binding to common ligands, in their ligand repertoires, and in that divergent families of activating receptors utilize different signaling pathways. These subset, binding, repertoire, and signaling diversities may allow activating receptors to manifest their effects in spite of inhibitory receptor functions during pathologic conditions in vivo. In this review, we will present a detailed analysis of the data supporting this hypothesis with particular relevance toward physiologic NK-cell functions.