Steven G. Nadler

Differential expression and sequence-specific interaction of karyopherin α with nuclear localization sequences

The process of nuclear protein transport requires the interaction of several different proteins, either directly or indirectly with nuclear localization or targeting sequences (NLS). Recently, a number of karyopherins α, or NLS-binding proteins, have been identified. We have found that the karyopherins hSRP1 and hSRP1α are differentially expressed in various leukocyte cell lines and could be induced in normal human peripheral blood lymphocytes. We show that the two karyopherins bind with varied specificities in a sequence specific manner to different NLSs and that the sequence specificity is modulated by other cytosolic proteins. There was a correlation between binding of karyopherins α to different NLSs and their ability to be imported into the nucleus. Taken together, these data provide evidence for multiple levels of control of the nuclear import process.

The clinical utility of inhibiting CD28‐mediated costimulation

Summary:  This volume covers many topics in the field of T‐cell costimulation. The need for such a volume is testament to the growth of the field. From its beginning as a concept in the 1980s, we have now progressed to the point where many molecules now have functionally defined roles in T‐cell costimulation. In addition, the field has progressed ‘from bench to bedside’. Abatacept [cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4)‐immunoglobulin (Ig) (CTLA‐4‐Ig)], an inhibitor of CD28‐mediated T‐cell costimulation, was approved for the treatment of moderate‐to‐severe rheumatoid arthritis in 2006 by the Food and Drug Administration and in 2007 by the European Medicines Agency. This chapter first presents a personal historical perspective on the early basic studies on the elucidation of the CD28/B7 T‐cell costimulatory pathway and the discovery of CTLA‐4‐Ig. We next present an overview of studies of CTLA‐4‐Ig in preclinical animal studies. The material discussed in these first two sections is selective rather than exhaustive; their purpose is to provide context for the final section, a summary of human clinical studies performed with abatacept.

Control of Memory CD4 T Cell Recall by the CD28/B7 Costimulatory Pathway

The CD28/B7 costimulatory pathway is generally considered dispensable for memory T cell responses, largely based on in vitro studies demonstrating memory T cell activation in the absence of CD28 engagement by B7 ligands. However, the susceptibility of memory CD4 T cells, including central (CD62Lhigh) and effector memory (TEM; CD62Llow) subsets, to inhibition of CD28-derived costimulation has not been closely examined. In this study, we demonstrate that inhibition of CD28/B7 costimulation with the B7-binding fusion molecule CTLA4Ig has profound and specific effects on secondary responses mediated by memory CD4 T cells generated by priming with Ag or infection with influenza virus. In vitro, CTLA4Ig substantially inhibits IL-2, but not IFN-γ production from heterogeneous memory CD4 T cells specific for influenza hemagglutinin or OVA in response to peptide challenge. Moreover, IL-2 production from polyclonal influenza-specific memory CD4 T cells in response to virus challenge was completely abrogated by CTLA4Ig with IFN-γ production partially inhibited. When administered in vivo, CTLA4Ig significantly blocks Ag-driven memory CD4 T cell proliferation and expansion, without affecting early recall and activation. Importantly, CTLA4Ig treatment in vivo induced a striking shift in the phenotype of the responding population from predominantly TEM in control-treated mice to predominantly central memory T cells in CTLA4Ig-treated mice, suggesting biased effects of CTLA4Ig on TEM responses. Our results identify a novel role for CD28/B7 as a regulator of memory T cell responses, and have important clinical implications for using CTLA4Ig to abrogate the pathologic consequences of TEM cells in autoimmunity and chronic disease.

Co-ligation of the Antigen and Fc Receptors Gives Rise to the Selective Modulation of Intracellular Signaling in B Cells REGULATION OF THE ASSOCIATION OF PHOSPHATIDYLINOSITOL 3-KINASE AND INOSITOL 5′-PHOSPHATASE WITH THE ANTIGEN RECEPTOR COMPLEX

Cross-linking of the Fc receptor (FcR) to surface immunoglobulin (sIg) on B cells inhibits the influx of extracellular calcium and abrogates the proliferative signal. The mechanism by which this occurs is not well understood. In this report we show that co-cross-linking the FcR to the antigen receptor gives rise to very selective modulation of signal transduction in B cells. Co-cross-linking sIg and the FcR enhanced the phosphorylation of the FcR, the adapter protein, Shc, and the inositol 5′-phosphatase Ship. Furthermore, phosphorylation of the FcR induced its association with Ship. Cross-linking of the FcR and sIg decreased the tyrosine phosphorylation of CD19, which led to a reduction in the association of phosphatidylinositol 3-kinase. In addition, the phosphorylation of several other proteins of 73, 39, and 34 kDa was reduced. Activation of the cells with either F(ab′)2 or intact anti-IgG induced very similar changes in levels of tyrosine phosphorylation of most other proteins, and no differences in the activation of several protein kinases were observed. These results indicate that the inhibitory signal that is transmitted through the FcR is not mediated by a global shutdown of tyrosine phosphorylation but is, rather, a selective mechanism involving localized changes in the interactions of adapter proteins and the enzymes Ship and phosphatidylinositol 3-kinase with the antigen receptor complex.

The Amino-terminal Immunoglobulin-like Domain of Activated Leukocyte Cell Adhesion Molecule Binds Specifically to the Membrane-proximal Scavenger Receptor Cysteine-rich Domain of CD6 with a 1:1 Stoichiometry

Activated leukocyte cell adhesion molecule (ALCAM) was recently identified as a ligand for CD6, a signaling receptor expressed on T cells, a subset of B cells, and some cells in the brain. Receptor-ligand binding assays, antibody blocking experiments, and examination of the tissue distribution of these two cell surface proteins suggest that CD6-ALCAM interactions play an important role in mediating the binding of thymocytes to thymic epithelial cells and of T cells to activated leukocytes. Presently, the details of CD6-ALCAM interactions and of signaling through CD6 are unknown. A series of truncated human ALCAM and CD6 immunoglobulin fusion proteins were produced and tested in different binding assays to analyze ALCAM-CD6 interactions in more detail. In this study, we report that the amino-terminal Ig-like domain of human ALCAM specifically binds to the third membrane-proximal scavenger receptor cysteine-rich (SRCR) domain of human CD6. Using thrombin-cleaved Ig fusion proteins containing single or multiple ALCAM or CD6 domains, we were able to determine that the stoichiometry of the interaction between the amino-terminal ALCAM domains and the membrane-proximal CD6 SRCR domain is 1:1. These results provide the first example of an Ig-like domain mediating an interaction with an SRCR domain.

Phosphodiesterase 7A-Deficient Mice Have Functional T Cells

Phosphodiesterases (PDEs) are enzymes which hydrolyze the cyclic nucleotide second messengers, cAMP and cGMP. In leukocytes, PDEs are responsible for depletion of cAMP which broadly suppresses cell functions and cellular responses to many activation stimuli. PDE7A has been proposed to be essential for T lymphocyte activation based on its induction during cell activation and the suppression of proliferation and IL-2 production observed following inhibition of PDE7A expression using a PDE7A antisense oligonucleotide. These observations have led to the suggestion that selective PDE7 inhibitors could be useful in the treatment of T cell-mediated autoimmune diseases. In the present report, we have used targeted gene disruption to examine the role PDE7A plays in T cell activation. In our studies, PDE7A knockout mice (PDE7A−/−) showed no deficiencies in T cell proliferation or Th1- and Th2-cytokine production driven by CD3 and CD28 costimulation. Unexpectedly, the Ab response to the T cell-dependent Ag, keyhole limpet hemocyanin, in the PDE7A−/− mice was found to be significantly elevated. The results from our studies strongly support the notion that PDE7A is not essential for T cell activation.

Partial restoration of cAMP-stimulated CFTR chloride channel activity in ΔF508 cells by deoxyspergualin

Deletion of the codon encoding phenylalanine 508 (ΔF508) is the most common mutation in cystic fibrosis (CF) and results in a trafficking defect. Mutant ΔF508-CF transmembrane conductance regulator (CFTR) protein retains functional activity, but the nascent protein is recognized as abnormal and, in consequence, is retained in the endoplasmic reticulum (ER) and degraded. It has been proposed that this retention in the ER is mediated, at least in part, by the cellular chaperones heat shock protein (HSP) 70 and calnexin. We have investigated the ability of deoxyspergualin (DSG), a compound known to compete effectively for binding with HSP70 and HSP90, to promote trafficking of ΔF508-CFTR to the cell membrane. We show that DSG treatment of immortalized human CF epithelial cells (ΔF508) and cells expressing recombinant ΔF508-CFTR partially restored cAMP-stimulated CFTR Cl-channel activity at the plasma membrane. Although there are several possible explanations for these results, one simple interpretation is that DSG may have altered the interaction between ΔF508-CFTR and its associated chaperones. If this is correct, agents capable of altering the normal functioning of cellular chaperones may provide yet another means of restoring CFTR Cl- channel activity to CF subjects harboring this class of mutations.

Abatacept Limits Breach of Self-Tolerance in a Murine Model of Arthritis via Effects on the Generation of T Follicular Helper Cells

Abatacept modulates CD28-mediated T cell costimulation and is efficacious in the treatment of rheumatoid arthritis (RA). Its mechanism of action has not been fully elucidated but will likely reveal critical pathologic pathways in RA. We show that abatacept substantially modulated Ag-specific T and B cell responses in vivo. Ag-specific T cell proliferation was reduced, and the acquisition of an activated phenotype, characterized by upregulation of CD69, OX40, ICOS, and programmed death-1 and downregulation of CD62L, was suppressed. Furthermore, abatacept suppressed the production of inflammatory cytokines, such as IFN-γ and IL-17. These effects were associated with a failure of Ag-specific T cells to acquire the CXCR5+ICOS+ T follicular helper cell phenotype. This, in turn, led to a failure of these cells to enter B cell follicles, resulting in reduced specific Ab responses, despite normal B cell clonal expansion. To test the pathologic significance of this, we used a novel model of RA associated with breach of self-tolerance to self-Ag and demonstrated that abatacept prevented the emergence of self-reactivity. Thus, CD28-dependent signaling is required for optimal T follicular helper cell maturation and expansion, and its inhibition prevents loss of self-tolerance in a model of articular pathology. Thus, we provide a novel mode of action for abatacept with profound implications for its potential usefulness in early inflammatory arthropathies associated with autoantibody expression.

Therapeutic effect of cytotoxic T lymphocyte antigen 4/immunoglobulin on a murine model of primary biliary cirrhosis

Collectively, the data in both humans and murine models of human primary biliary cirrhosis (PBC) suggest that activated T cells, particularly CD8 T cells, play a critical role in biliary cell destruction. Under physiological conditions, T‐cell activation involves two critical signals that involve the major histocompatibility complex and a set of costimulatory molecules, which include a receptor on T cells termed cytotoxic T lymphocyte antigen 4 (CTLA‐4). Germane to the studies reported herein, signaling by CTLA‐4 has the potential to modulate costimulation and induce inhibitory signals. In this study, we have taken advantage of our well‐defined murine model of PBC, in which mice are immunized with 2‐octynoic acid coupled to bovine serum albumin (2OA‐BSA), leading to the production of high‐titer antimitochondrial autoantibodies (AMAs) and portal cellular infiltrates. To investigate the potential of CTLA‐4‐Ig (immunoglobulin) as an immunotherapeutic agent, we treated mice both before and after induction of autoimmune cholangitis. First, we demonstrate that CTLA‐4‐Ig treatment, begun 1 day before 2OA‐BSA immunization, completely inhibits the manifestations of cholangitis, including AMA production, intrahepatic T‐cell infiltrates, and bile duct damage. However, and more critically, treatment with CTLA‐4‐Ig, initiated after the development of autoimmune cholangitis in previously immunized mice, also resulted in significant therapeutic benefit, including reduced intrahepatic T‐cell infiltrates and biliary cell damage, although AMA levels were not altered. Conclusion: These data suggest that an optimized regimen with CTLA‐4‐Ig has the potential to serve as an investigative therapeutic tool in patients with PBC. (HEPATOLOGY 2013)

Intranuclear targeted delivery of functional NF-κB by 70 kDa heat shock protein

The 70 kDa heat shock protein (Hsp70) is a highly conserved, ubiquitous protein involved in chaperoning proteins to various cellular organelles. Here we show that when added exogenously to cells, Hsp70 is readily imported into both cytoplasmic and nuclear compartments in a cell‐type‐specific fashion. We exploited this ability of Hsp70 to deliver NF‐κB, a key transcriptional regulator of inflammatory responses. We demonstrate that a fusion protein composed of a C‐terminal Hsp70 peptide and the p50 subunit of NF‐κB was directed into the nucleus of cells, could bind DNA specifically, and activated Igκ expression and TNFα production. We therefore propose that Hsp70 can be used as a vehicle for intracytoplasmic and intranuclear delivery of proteins or DNA to modulate gene expression and thereby control immune responses.