Scott A. Brown

TNF/iNOS-producing dendritic cells are the necessary evil of lethal influenza virus infection

Respiratory infection with highly pathogenic influenza A viruses is characterized by the exuberant production of cytokines and chemokines and the enhanced recruitment of innate inflammatory cells. Here, we show that challenging mice with virulent influenza A viruses, including currently circulating H5N1 strains, causes the increased selective accumulation of a particular dendritic cell subset, the tipDCs, in the pneumonic airways. These tipDCs are required for the further proliferation of influenza-specific CD8+ T cells in the infected lung, because blocking their recruitment in CCR2−/− mice decreases the numbers of CD8+ effectors and ultimately compromises virus clearance. However, diminution rather than total elimination of tipDC trafficking by treatment with the peroxisome proliferator-activated receptor-γ agonist pioglitazone moderates the potentially lethal consequences of excessive tipDC recruitment without abrogating CD8+ T cell expansion or compromising virus control. Targeting the tipDCs in this way thus offers possibilities for therapeutic intervention in the face of a catastrophic pandemic.

Distinct Contributions of Vaccine-Induced Immunoglobulin G1 (IgG1) and IgG2a Antibodies to Protective Immunity against Influenza

ABSTRACT Vaccination represents the most effective form of protection against influenza infection. While neutralizing antibodies are typically measured as a correlate of vaccine-induced protective immunity against influenza, nonneutralizing antibodies may contribute to protection or amelioration of disease. The goal of this study was to dissect the individual contributions of the immunoglobulin G1 (IgG1) and IgG2a antibody isotypes to vaccine-induced immunity against influenza virus. To accomplish this, we utilized an influenza vaccine regimen that selectively enhanced IgG1 or IgG2a antibodies by using either DNA or viral replicon particle (VRP) vectors expressing influenza virus hemagglutinin (HA) (HA-DNA or HA-VRP, respectively). After HA-DNA vaccination, neutralizing antibodies were detected by both in vitro (microneutralization) and in vivo (lung viral titer) methods and were associated with increased IgG1 expression by enzyme-linked immunosorbent assay (ELISA). Vaccination with HA-VRP did not strongly stimulate either neutralizing or IgG1 antibodies but did induce IgG2a antibodies. Expression of IgG2a antibodies in this context correlated with clearance of virus and increased protection against lethal influenza challenge. Increased induction of both antibody isotypes as measured by ELISA was a better correlate for vaccine efficacy than neutralization alone. This study details separate but important roles for both IgG1 and IgG2a expression in vaccination against influenza and argues for the development of vaccine regimens that stimulate and measure expression of both antibody isotypes.

Paired analysis of TCRα and TCRβ chains at the single-cell level in mice

Characterizing the TCRα and TCRβ chains expressed by T cells responding to a given pathogen or underlying autoimmunity helps in the development of vaccines and immunotherapies, respectively. However, our understanding of complementary TCRα and TCRβ chain utilization is very limited for pathogen- and autoantigen-induced immunity. To address this problem, we have developed a multiplex nested RT-PCR method for the simultaneous amplification of transcripts encoding the TCRα and TCRβ chains from single cells. This multiplex method circumvented the lack of antibodies specific for variable regions of mouse TCRα chains and the need for prior knowledge of variable region usage in the TCRβ chain, resulting in a comprehensive, unbiased TCR repertoire analysis with paired coexpression of TCRα and TCRβ chains with single-cell resolution. Using CD8+ CTLs specific for an influenza epitope recovered directly from the pneumonic lungs of mice, this technique determined that 25% of such effectors expressed a dominant, nonproductively rearranged Tcra transcript. T cells with these out-of-frame Tcra mRNAs also expressed an alternate, in-frame Tcra, whereas approximately 10% of T cells had 2 productive Tcra transcripts. The proportion of cells with biallelic transcription increased over the course of a response, a finding that has implications for immune memory and autoimmunity. This technique may have broad applications in mouse models of human disease.

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin

Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell lysates by Western blotting and in cells by immunofluorescence, and K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a strategy for the generation of similar reagents against other forms of polyUb.

BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation

The mitochondrial pathway of apoptosis is initiated by mitochondrial outer membrane permeabilization (MOMP). The BCL-2 family effectors BAX and BAK are thought to be absolutely required for this process. Here, we report that BCL-2 ovarian killer (BOK) is a bona fide yet unconventional effector of MOMP that can trigger apoptosis in the absence of both BAX and BAK. However, unlike the canonical effectors, BOK appears to be constitutively active and unresponsive to antagonistic effects of the antiapoptotic BCL-2 proteins. Rather, BOK is controlled at the level of protein stability by components of the endoplasmic reticulum (ER)-associated degradation pathway. BOK is ubiquitylated by the AMFR/gp78 E3 ubiquitin ligase complex and targeted for proteasomal degradation in a VCP/p97-dependent manner, which allows survival of the cell. When proteasome function, VCP, or gp78 activity is compromised, BOK is stabilized to induce MOMP and apoptosis independently of other BCL-2 proteins.

Hidden Epitopes Emerge in Secondary Influenza Virus-Specific CD8+ T Cell Reponses

Influenza A virus-specific CD8+ T cell responses in H2b mice are characterized by reproducible hierarchies. Compensation by the DbPB1-F262 epitope is apparent following infection with a variant H3N2 virus engineered to disrupt the prominent DbNP366 and DbPA224 epitopes (a double knockout or DKO). Analysis with a “triple” knockout (TKO) virus, which also compromises DbPB1-F262, did not reveal further compensation to the known residual, minor, and predicted epitopes. However, infection with this deletion mutant apparently switched protective immunity to an alternative Ab-mediated pathway. As expected, TKO virus clearance was significantly delayed in Ab-deficient MHC class II−/− and Ig−/− mice, which were much more susceptible following primary, intranasal infection with the TKO, but not DKO, virus. CD8+ T cell compensation was detected in DKO, but not TKO, infection of Ig-deficient mice, suggestive of cooperation among CD8+ T cell responses. However, after priming with a TKO H1N1 mutant, MHC II−/− mice survived secondary intranasal exposure to the comparable H3N2 TKO virus. Such prime/challenge experiments with the DKO and TKO viruses allowed the emergence of two previously unknown epitopes. The contrast between the absence of compensatory effect following primary exposure and the substantial clonal expansion after secondary challenge suggests that the key factor limiting the visibility of these “hidden” epitopes may be very low naive T cell precursor frequencies. Overall, these findings suggest that vaccine approaches using virus vectors to deliver an Ag may be optimized by disrupting key peptides in the normal CD8+ T cell response associated with common HLA types.

Clustering of Th cell epitopes on exposed regions of HIV envelope despite defects in antibody activity

A long-standing question in the field of immunology concerns the factors that contribute to Th cell epitope immunodominance. For a number of viral membrane proteins, Th cell epitopes are localized to exposed protein surfaces, often overlapping with Ab binding sites. It has therefore been proposed that Abs on B cell surfaces selectively bind and protect exposed protein fragments during Ag processing, and that this interaction helps to shape the Th cell repertoire. While attractive in concept, this hypothesis has not been thoroughly tested. To test this hypothesis, we have compared Th cell peptide immunodominance in normal C57BL/6 mice with that in C57BL/6μMT/μMT mice (lacking normal B cell activity). Animals were first vaccinated with DNA constructs expressing one of three different HIV envelope proteins, after which the CD4+ T cell response profiles were characterized toward overlapping peptides using an IFN-γ ELISPOT assay. We found a striking similarity between the peptide response profiles in the two mouse strains. Profiles also matched those of previous experiments in which different envelope vaccination regimens were used. Our results clearly demonstrate that normal Ab activity is not required for the establishment or maintenance of Th peptide immunodominance in the HIV envelope response. To explain the clustering of Th cell epitopes, we propose that localization of peptide on exposed envelope surfaces facilitates proteolytic activity and preferential peptide shuttling through the Ag processing pathway.

Differential subcellular localization of the regulatory T-cell protein LAG-3 and the coreceptor CD4

CD4 binds to MHC class II molecules and enhances T‐cell activation. The CD4‐related transmembrane protein LAG‐3 (lymphocyte activation gene‐3, CD223) binds to the same ligand but inhibits T‐cell proliferation. We have previously shown that LAG‐3 cell surface expression is tightly regulated by extracellular cleavage in order to regulate its potent inhibitory activity. Given this observation and the contrasting functions of CD4 and LAG‐3, we investigated the cell distribution, location and transport of these related cell surface molecules. As expected, the vast majority of CD4 is expressed at the cell surface with minimal intracellular localization, as determined by flow cytometry, immunoblotting and confocal microscopy. In contrast, nearly half the cellular content of LAG‐3 is retained in intracellular compartments. This significant intracellular storage of LAG‐3 appears to facilitate its rapid translocation to the cell surface following T‐cell activation, which was much faster for LAG‐3 than CD4. Increased vesicular pH inhibited translocation of both CD4 and LAG‐3 to the plasma membrane. While some colocalization of the microtubule organizing center, early/recycling endosomes and secretory lysosomes was observed with CD4, significantly greater colocalization was observed with LAG‐3. Analysis of CD4:LAG‐3 chimeras suggested that multiple domains may contribute to intracellular retention of LAG‐3. Thus, in contrast with CD4, the substantial intracellular storage of LAG‐3 and its close association with the microtubule organizing center and recycling endosomes may facilitate its rapid translocation to the cell surface during T‐cell activation and help to mitigate T‐cell activation.

Molecular Approaches to Differentiate Subpopulations or Formae speciales of the Fungal Phytopathogen Microbotryum violaceum

Microbotryum violaceum infects more than 200 species of Caryophyllaceae, although individual isolates of the fungus are limited in host range to one or a few species. The goal of the present study was to establish a molecular profile of individual haploid M. violaceum strains from different host species and to evaluate different methods for producing such profiles. Eighteen different sporidial strains from seven different host species were examined for electrophoretic karyotype, for random amplification of polymorphic DNA (RAPD) profile, by phylogenetic analysis of intron sequences in the γ-tubulin gene, and by parsimony analysis of melting curves of total genomic DNA (DNA thermal profiles). While no one method could resolve all isolates from closely related host species, together the results obtained with these methods were able to group isolates based on their host species of origin. Taken together, host range/host species of origin and the genetic differences indicated by the molecular methods in this study would appear to define pathovars or formae speciales of this fungus.

Apoptosis-Inducing-Factor-Dependent Mitochondrial Function Is Required for T Cell but Not B Cell Function

The role of apoptosis inducing factor (AIF) in promoting cell death versus survival remains controversial. We report that the loss of AIF in fibroblasts led to mitochondrial electron transport chain defects and loss of proliferation that could be restored by ectopic expression of the yeast NADH dehydrogenase Ndi1. Aif-deficiency in T cells led to decreased peripheral T cell numbers and defective homeostatic proliferation, but thymic T cell development was unaffected. In contrast, Aif-deficient B cells developed and functioned normally. The difference in the dependency of T cells versus B cells on AIF for function and survival correlated with their metabolic requirements. Ectopic Ndi1 expression rescued homeostatic proliferation of Aif-deficient T cells. Despite its reported roles in cell death, fibroblasts, thymocytes and B cells lacking AIF underwent normal death. These studies suggest that the primary role of AIF relates to complex I function, with differential effects on T and B cells.