Sarah L. Londrigan

BH3 mimetics antagonizing restricted prosurvival Bcl-2 proteins represent another class of selective immune modulatory drugs

Death by apoptosis shapes tissue homeostasis. Apoptotic mechanisms are so universal that harnessing them for tailored immune intervention would seem challenging; however, the range and different expression levels of pro- and anti-apoptotic molecules among tissues offer hope that targeting only a subset of such molecules may be therapeutically useful. We examined the effects of the drug ABT-737, a mimetic of the killer BH3 domain of the Bcl-2 family of proteins that induces apoptosis by antagonizing Bcl-2, Bcl-XL, and Bcl-W (but not Mcl-1 and A1), on the mouse immune system. Treatment with ABT-737 reduced the numbers of selected lymphocyte and dendritic cell subpopulations, most markedly in lymph nodes. It inhibited the persistence of memory B cells, the establishment of newly arising bone marrow plasma cells, and the induction of a cytotoxic T cell response. Preexisting plasma cells and germinal centers were unaffected. Notably, ABT-737 was sufficiently immunomodulatory to allow long-term survival of pancreatic allografts, reversing established diabetes in this model. These results provide an insight into the selective mechanisms of immune cell survival and how this selectivity avails a different strategy for immune modulation.

N-Linked Glycosylation Facilitates Sialic Acid-Independent Attachment and Entry of Influenza A Viruses into Cells Expressing DC-SIGN or L-SIGN

ABSTRACT It is widely recognized that sialic acid (SA) can mediate attachment of influenza virus to the cell surface, and yet the specific receptors that mediate virus entry are not known. For many viruses, a definitive demonstration of receptor function has been achieved when nonpermissive cells are rendered susceptible to infection following transfection of the gene encoding a putative receptor. For influenza virus, such approaches have been confounded by the abundance of SA on mammalian cells so that it has been difficult to identify cell lines that are not susceptible to infection. We examined influenza virus infection of Lec2 Chinese hamster ovary (CHO) cells, a mutant cell line deficient in SA. Lec2 CHO cells were resistant to influenza virus infection, and stable cell lines expressing either DC-SIGN or L-SIGN were generated to assess the potential of each molecule to function as SA-independent receptors for influenza A viruses. Virus strain BJx109 (H3N2) bound to Lec2 CHO cells expressing DC-SIGN or L-SIGN in a Ca2+-dependent manner, and transfected cells were susceptible to virus infection. Treatment of Lec2-DC-SIGN and Lec2-L-SIGN cells with mannan, but not bacterial neuraminidase, blocked infection, a finding consistent with SA-independent virus attachment and entry. Moreover, virus strain PR8 (H1N1) bears low levels of mannose-rich glycans and was inefficient at infecting Lec2 CHO cells expressing either DC-SIGN or L-SIGN, whereas other glycosylated H1N1 subtype viruses could infect cells efficiently. Together, these data indicate that human C-type lectins (DC-SIGN and L-SIGN) can mediate attachment and entry of influenza viruses independently of cell surface SA.

Growth of rotaviruses in continuous human and monkey cell lines that vary in their expression of integrins

Rotavirus replication occurs in vivo in intestinal epithelial cells. Cell lines fully permissive to rotavirus include kidney epithelial (MA104), colonic (Caco-2) and hepatic (HepG2) types. Previously, it has been shown that cellular integrins alpha 2 beta 1, alpha 4 beta 1 and alpha X beta 2 are involved in rotavirus cell entry. As receptor usage is a major determinant of virus tropism, the levels of cell surface expression of these integrins have now been investigated by flow cytometry on cell lines of human (Caco-2, HepG2, RD, K562) and monkey (MA104, COS-7) origin in relation to cellular susceptibility to infection with monkey and human rotaviruses. Cells supporting any replication of human rotaviruses (RD, HepG2, Caco-2, COS-7 and MA104) expressed alpha 2 beta 1 and (when tested) alpha X beta 2, whereas the non-permissive K562 cells did not express alpha 2 beta 1, alpha 4 beta 1 or alpha X beta 2. Only RD cells expressed alpha 4 beta 1. Although SA11 grew to higher titres in RD, HepG2, Caco-2, COS-7 and MA104 cells, this virus still replicated at a low level in K562 cells. In all cell lines tested, SA11 replicated to higher titres than did human strains, consistent with the ability of SA11 to use sialic acids as alternative receptors. Levels of cell surface alpha 2 integrin correlated with levels of rotavirus growth. The alpha 2 integrin relative linear median fluorescence intensity on K562, RD, COS-7, MA104 and Caco-2 cells correlated linearly with the titre of SA11 produced in these cells at 20 h after infection at a multiplicity of 0.1, and the data best fitted a sigmoidal dose-response curve (r(2)=1.00, P=0.005). Thus, growth of rotaviruses in these cell lines correlates with their surface expression of alpha 2 beta 1 integrin and is consistent with their expression of alpha X beta 2 and alpha 4 beta 1 integrins.

The fate of influenza A virus after infection of human macrophages and dendritic cells

Airway macrophages (MΦ) and dendritic cells (DC) are important components of the innate host defence. Historically, these immune cells have been considered to play a critical role in controlling the severity of influenza A virus (IAV) infection by limiting virus release, initiating local inflammatory responses and by priming subsequent adaptive immune responses. However, some IAV strains have been reported to replicate productively in human immune cells. Potential amplification and dissemination of IAV from immune cells may therefore be an important virulence determinant. Herein, we will review findings in relation to the fate of IAV following infection of MΦ and DC. Insights regarding the consequences and outcomes of IAV infection of airway MΦ and DC are discussed in order to gain a better understanding of the pathogenesis of influenza virus.

Monkey Rotavirus Binding to α2β1 Integrin Requires the α2 I Domain and Is Facilitated by the Homologous β1 Subunit

ABSTRACT Rotaviruses utilize integrins during virus-cell interactions that lead to infection. Cell binding and infection by simian rotavirus SA11 were inhibited by antibodies (Abs) to the inserted (I) domain of the α2 integrin subunit. To determine directly which integrins or other proteins bind rotaviruses, cell surface proteins precipitated by rotaviruses were compared with those precipitated by anti-α2β1 Abs. Two proteins precipitated by SA11 and rhesus rotavirus RRV from MA104 and Caco-2 cells migrated indistinguishably from α2β1 integrin, and SA11 precipitated β1 from α2β1-transfected CHO cells. These viruses specifically precipitated two MA104 cell proteins only, but an additional 160- to 165-kDa protein was precipitated by SA11 from Caco-2 cells. The role of the α2 I domain in rotavirus binding, infection, and growth was examined using CHO cell lines expressing wild-type or mutated human α2 or α2β1. Infectious SA11 and RRV, but not human rotavirus Wa, specifically bound CHO cell-expressed human α2β1 and, to a lesser extent, human α2 combined with hamster β1. Binding was inhibited by anti-α2 I domain monoclonal Abs (MAbs), but not by non-I domain MAbs to α2, and required the presence of the α2 I domain. Amino acid residues 151, 221, and 254 in the metal ion-dependent adhesion site of the α2 I domain that are necessary for type I collagen binding to α2β1 were not essential for rotavirus binding. Rotavirus-α2β1 binding led to increased virus infection and RRV growth. SA11 and RRV require the α2 I domain for binding to α2β1, and their binding to this integrin is distinguishable from that of collagen.

Cell-surface receptors on macrophages and dendritic cells for attachment and entry of influenza virus

Airway MΦ and DCs are important components of innate host defense and can play a critical role in limiting the severity of influenza virus infection. Although it has been well established that cell‐surface SA acts as a primary attachment receptor for IAV, the particular receptor(s) or coreceptor(s) that mediate IAV entry into any cell, including MΦ and DC, have not been clearly defined. Identifying which receptors are involved in attachment and entry of IAV into immune cells may have important implications in regard to understanding IAV tropism and pathogenesis. Recent evidence suggests that specialized receptors on MΦ and DCs, namely CLRs, can act as capture and/or entry receptors for many viral pathogens, including IAV. Herein, we review the early stages of infection of MΦ and DC by IAV. Specifically, we examine the potential role of CLRs expressed on MΦ and DC to act as attachment and/or entry receptors for IAV.

Versatile co-expression of graft-protective proteins using 2A-linked cassettes.

Fisicaro N, Londrigan SL, Brady JL, Salvaris E, Nottle MB, O’Connell PJ, Robson SC, d’Apice AJF, Lew AM, Cowan PJ. Versatile co‐expression of graft‐protective proteins using 2A‐linked cassettes. Xenotransplantation 2011; 18: 121–130.

In situ protection against islet allograft rejection by CTLA4Ig transduction.

Background. Immunosuppression focused at or near the graft site would reduce the need for systemic immunosuppression thus educing fewer side effects. We investigated whether locally produced CTLA4Ig, mediated by adenovirus (Adv) transduction of mouse islets, would protect allografts and whether such immunosuppression would remain localized. Methods. Adv-CTLA4Ig- or Adv-control-transduced islets were grafted under the kidney capsule of fully allogeneic diabetic mice. CTLA4Ig secreted from the grafted islets was detected by enzyme immunoassay of blood or immunohistochemistry of graft sections. Graft survival was monitored by blood glucose measurement. Histologic scores of graft sections stained with Gomori aldehyde fuchsin to detect insulin granules or hematoxylin-eosin to detect inflammation were used to compare grafts placed at different sites within the same mouse. Results. Adv-CTLA4Ig-transduced islet grafts secreted CTLA4Ig that was detected transiently in the circulation but persistently at the graft site. Survival of these grafts was significantly enhanced compared with control Adv-transduced and untransduced grafts. The kidney graft site availed elucidation of the site of action of CTLA4Ig. Histologic scores indicated that CTLA4Ig-producing grafts were protected by comparison with control grafts on the contralateral kidney. Hence, graft protection was not attributable to general systemic immunosuppression. Indeed, survival of CTLA4Ig-producing grafts was enhanced over control grafts placed at the opposite pole of the same kidney, indicating that graft protection was not solely due to inhibition of priming in the common draining lymph nodes. Conclusions. Islet allografts are protected by locally produced CTLA4Ig-disrupting immune interactions at the effector site.

Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets

Abstract:  There is considerable interest in the viral modification of insulin‐producing islets, including porcine islets, in the context of islet xenotransplantation to treat type 1 diabetes. Adenovirus (Adv) gene delivery offers the potential to modify pre‐transplant islets for enhanced survival. Modifications include transfer of cytoprotective molecules to ensure islet survival immediately post‐transplant, and molecules to dampen the immune system and prevent chronic islet graft rejection. In this study, we compared different promoters (three promiscuous and two tissue‐specific promoters) for their efficiency in driving gene expression in neonatal pig islet tissue after Adv delivery. We also compared the efficiency of these promoters in adult islets from mouse and human pancreata. We observed that the promiscuous cytomegalovirus promoter was the most potent, eliciting high luciferase expression in neonatal pig islets, as well as in human and mouse islets. In contrast, the mammalian EF1‐α promoter educed comparatively intermediate gene expression. The mouse major histocompatibility complex class I promoter H‐2Kb and the pancreatic‐specific promoters insulin and human pdx‐1 (area II) performed poorly in islets from all three species. This has important implications for the generation of modified neonatal pig islets for transplantation into humans.

Prolonged local expression of anti-CD4 antibody by adenovirally transduced allografts can promote long-term graft survival

Currently, successful transplantation of allografts requires the systemic use of immunosuppressive drugs. These can cause serious morbidity due to toxicity and increased susceptibility to cancer and infections. Local production of immunosuppressive molecules limited to the graft site would reduce the need for conventional, generalized immunosuppressive therapies and thus educe fewer side effects. This is particularly salient in a disease like type 1 diabetes, which is not immediately life‐threatening yet islet allografts can effect a cure.