Enrico Arpaia

The development of inflammatory T(H)-17 cells requires interferon-regulatory factor 4

Interferon-regulatory factor 4 (IRF4) is essential for the development of T helper type 2 cells. Here we show that IRF4 is also critical for the generation of interleukin 17–producing T helper cells (TH-17 cells), which are associated with experimental autoimmune encephalomyelitis. IRF4-deficient (Irf4−/−) mice did not develop experimental autoimmune encephalomyelitis, and T helper cells from such mice failed to differentiate into TH-17 cells. Transfer of wild-type T helper cells into Irf4−/− mice rendered the mice susceptible to experimental autoimmune encephalomyelitis. Irf4−/− T helper cells had less expression of RORγt and more expression of Foxp3, transcription factors important for the differentiation of TH-17 and regulatory T cells, respectively. Altered regulation of both transcription factors contributed to the phenotype of Irf4−/− T helper cells. Our data position IRF4 at the center of T helper cell development, influencing not only T helper type 2 but also TH-17 differentiation.

Defective T cell receptor signaling and CD8+ thymic selection in humans lacking Zap-70 kinase

We have previously described a type of selective T cell deficiency (STD) characterized by persistent infections reminiscent of severe combined immunodeficiency. We show here that STD patients carry a mutation of zap-70, resulting in loss of the activity of this kinase. The thymi of zap-70-/- patients show the presence of CD4+CD8+ cells in the cortex; however, only CD4, not CD8, single-positive cells are present in the medulla. Peripheral CD4+ T cells from the zap-70-/- patients exhibit markedly reduced tyrosine phosphorylation, fail to produce interleukin-2, and do not proliferate in response to T cell receptor stimulation by mitogens or antigens. Thus, Zap-70 kinase appears to be indispensable for the development of CD8 single-positive T cells as well as for signal transduction and function of single-positive CD4 T cells.

The interaction between caveolin-1 and Rho-GTPases promotes metastasis by controlling the expression of alpha5-integrin and the activation of Src, Ras and Erk.

Proteins containing a caveolin-binding domain (CBD), such as the Rho-GTPases, can interact with caveolin-1 (Cav1) through its caveolin scaffold domain. Rho-GTPases are important regulators of p130Cas, which is crucial for both normal cell migration and Src kinase-mediated metastasis of cancer cells. However, although Rho-GTPases (particularly RhoC) and Cav1 have been linked to cancer progression and metastasis, the underlying molecular mechanisms are largely unknown. To investigate the function of Cav1–Rho-GTPase interaction in metastasis, we disrupted Cav1–Rho-GTPase binding in melanoma and mammary epithelial tumor cells by overexpressing CBD, and examined the loss-of-function of RhoC in metastatic cancer cells. Cancer cells overexpressing CBD or lacking RhoC had reduced p130Cas phosphorylation and Rac1 activation, resulting in an inhibition of migration and invasion in vitro. The activity of Src and the activation of its downstream targets FAK, Pyk2, Ras and extracellular signal-regulated kinase (Erk)1/2 were also impaired. A reduction in α5-integrin expression, which is required for binding to fibronectin and thus cell migration and survival, was observed in CBD-expressing cells and cells lacking RhoC. As a result of these defects, CBD-expressing melanoma cells had a reduced ability to metastasize in recipient mice, and impaired extravasation and survival in secondary sites in chicken embryos. Our data indicate that interaction between Cav1 and Rho-GTPases (most likely RhoC but not RhoA) promotes metastasis by stimulating α5-integrin expression and regulating the Src-dependent activation of p130Cas/Rac1, FAK/Pyk2 and Ras/Erk1/2 signaling cascades.

HUNK suppresses metastasis of basal type breast cancers by disrupting the interaction between PP2A and cofilin-1

Metastasis leads to the death of most cancer patients, and basal breast cancer is the most aggressive breast tumor type. Metastasis involves a complex cell migration process dependent on cytoskeletal remodeling such that targeting such remodeling in tumor cells could be clinically beneficial. Here we show that Hormonally Up-regulated Neu-associated Kinase (HUNK) is dramatically down-regulated in tumor samples and cell lines derived from basal breast cancers. Reconstitution of HUNK expression in basal breast cancer cell lines blocked actin polymerization and reduced cell motility, resulting in decreased metastases in two in vivo murine cancer models. Mechanistically, HUNK overexpression sustained the constitutive phosphorylation and inactivation of cofilin-1 (CFL-1), thereby blocking the incorporation of new actin monomers into actin filaments. HUNK reconstitution in basal breast cancer cell lines prevented protein phosphatase 2-A (PP2A), a phosphatase putatively acting on CFL-1, from binding to CFL-1. Our investigation of HUNK suggests that the interaction between PP2A and CFL-1 may be a target for antimetastasis therapy, particularly for basal breast cancers.

ICA69null Nonobese Diabetic Mice Develop Diabetes, but Resist Disease Acceleration by Cyclophosphamide

ICA69 (islet cell Ag 69 kDa) is a diabetes-associated autoantigen with high expression levels in β cells and brain. Its function is unknown, but knockout of its Caenorhabditis elegans homologue, ric-19, compromised neurotransmission. We disrupted the murine gene, ica-1, in 129-strain mice. These animals aged normally, but speed-congenic ICA69null nonobese diabetic (NOD) mice developed mid-life lethality, reminiscent of NOD-specific, late lethal seizures in glutamic acid decarboxylase 65-deficient mice. In contrast to wild-type and heterozygous animals, ICA69null NOD congenics fail to generate, even after immunization, cross-reactive T cells that recognize the dominant Tep69 epitope in ICA69, and its environmental mimicry Ag, the ABBOS epitope in BSA. This antigenic mimicry is thus driven by the endogenous self Ag, and not initiated by the environmental mimic. Insulitis, spontaneous, and adoptively transferred diabetes develop normally in ICA69null NOD congenics. Like glutamic acid decarboxylase 65, ICA69 is not an obligate autoantigen in diabetes. Unexpectedly, ICA69null NOD mice were resistant to cyclophosphamide (CY)-accelerated diabetes. Transplantation experiments with hemopoietic and islet tissue linked CY resistance to ICA69 deficiency in islets. CY-accelerated diabetes involves not only ablation of lymphoid cells, but ICA69-dependent drug toxicity in β cells that boosts autoreactivity in the regenerating lymphoid system.

IMMUNODEFICIENCY CAUSED BY PURINE NUCLEOSIDE PHOSPHORYLASE DEFICIENCY

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway with adenosine deaminase (ADA). Inherited deficiency of either one of two purine salvage enzymes results in severe combined immunodeficiency (SCID) in humans. 32,33,50 One of the striking features of ADA and PNP deficiencies is the selective effect on the immune system, although the activities of these enzymes are missing in every cell in the body. 50 The biochemical mechanism(s) explaining the pathology of PNP deficiency attempts to address the specificity of this disease toward the immune system. 50,68 Enzyme deficiencies can cause deleterious phenotypes as a result of lack of product of the enzymatic reaction or as a consequence of the cytotoxicity of the accumulating substrates. The lack of products in PNP deficiency is unlikely to contribute to the immune deficiency because deficiency of the next enzyme in the purine salvage pathway hypoxanthine-guanine phosphoribosyl transferase (HGPRT) causes Lesch-Nyhan syndrome with normal immune function. 62 Of the four purine nucleoside substrates of the PNP reaction, inosine, deoxyinosine, guanosine, and deoxyguanosine, only deoxyguanosine has an alternative metabolic fate (i.e., phosphorylation by deoxyguanosine kinase) (Fig. 1). 28,90 Deoxyguanosine is the only logical candidate to mediate the lymphoid abnormalities observed in PNP deficiency. The challenge remains to explain the metabolic, biochemical, and cellular mechanisms that lead to the unique lymphoid specificity of PNP deficiency despite its ubiquitous expression.

Micromanagement of lymphomas.

B cell receptor signaling participates in the genesis of lymphoma and influences the characteristics of the tumor cells.

CD8 LYMPHOCYTOPENIA CAUSED BY ZAP-70 DEFICIENCY

ZAP-70 is a member of a distinct family of cytoplasmic tyrosine kinases involved in signal transduction from the antigen receptors. The family currently consists of ZAP-70, which is found in T lymphocytes and natural killer (NK) cells and Syk, which is most highly expressed in B lymphocytes and platelets. 12 ZAP-70 was isolated initially as an ATP-binding phosphoprotein associating with a component of the T-cell receptor (TCR) signaling complex, CD3ζ, after TCR stimulation. 9,87,90 ZAP-70 has a single protein tyrosine kinase catalytic domain and tandem SH2 domains responsible for binding to the CD3ζ receptor chain. This structure is shared by the closely related Syk kinase. 12 Both kinases also have multiple tyrosine residues predicted to form recognition sites for SH2 domains. ZAP-70 is expressed from an extremely early point in T-cell differentiation, and is present in all the major thymocyte subpopulations. 11 Expression does not seem to be inducible, but remains constant throughout the developmental process. A single gene on chromosome 2q12 encodes the human ZAP-70 kinase. 38 Although playing a critical role in T-cell antigen receptor signaling, the activation of ZAP-70 is not the primary event following TCR ligation. Rather, ZAP-70 activation seems to lie downstream of members of the src tyrosine kinase family, and, as a result of their activity, ZAP-70 is activated. 82,89 This is, in part, because of phosphorylation of CD3ζ by the src kinases, primarily p56lck, and, in part, because of phosphorylation of ZAP-70 itself. On TCR stimulation, ZAP-70 rapidly moves from the cytoplasm to the plasma membrane, binding to the CD3ζ chain of the TCR complex. It then is tyrosine phosphorylated rapidly and its kinase domain is activated. Various signal transduction proteins become associated with ZAP-70, some of which are substrates for its kinase domain and others that modify and regulate its activity. Localization to the plasma membrane plays an important role in ZAP-70 activation, as artificial targeting to the membrane results in ZAP-70 phosphorylation and activation of the downstream ras/MAPK and Ca 2+ /calcineurin pathways. 25

Isolation of mutants of CHO cells resistant to 6 (p-hydroxyphenylazo)-uracil II. Mutants auxotrophic for deoxypyrimidines

Two classes of CHO mutants resistant to the drug 6(p-hydroxyphenylazo)-uracil have been characterized. Both classes exhibited a nutritional requirement that could be satisfied by deoxypyrimidines and uridine but not other ribopyrimidines. A biochemical investigation of these mutants revealed a structural defect in ribonucleotide reductase resulting in a two- to fourfold increase in the Kmfor UDP and CDP. As a consequence of this lesion, the cells had imbalanced deoxypyrimidine pools and showed an increase in the rate of spontaneous mutation to 6-thioguanine resistance but not emetine resistance.

Isolation of mutants of CHO cells resistant to 6(p-hydroxyphenylazo)-uracil I. A novel BrdU cross-resistant phenotype

Three classes of mutants resistant to the drug 6(p-hydroxyphenylazo)-uracil have been isolated from mutagenized cultures of CHO cells. One class of these mutants designated HPURA exhibits a unique form of cross-resistance to bromodeoxyuridine in that it is resistant to this drug only in the presence of thymidine. The molecular basis of the BrdU resistance is unknown but does not appear to involve the known targets of the drug. An interesting feature of these mutants is that they give rise, at a high frequency, to a subpopulation of cells which are much more resistant to BrdU.