Shiki Takamura

Activation phenotype, rather than central– or effector–memory phenotype, predicts the recall efficacy of memory CD8+ T cells

The contributions of different subsets of memory CD8+ T cells to recall responses at mucosal sites of infection are poorly understood. Here, we analyzed the CD8+ T cell recall responses to respiratory virus infection in mice and demonstrate that activation markers, such as CD27 and CD43, define three distinct subpopulations of memory CD8+ T cells that differ in their capacities to mount recall responses. These subpopulations are distinct from effector– and central–memory subsets, coordinately express other markers associated with activation status, including CXCR3, CD127, and killer cell lectin-like receptor G1, and are superior to CD62L in predicting the capacity of memory T cells to mediate recall responses. Furthermore, the capacity of vaccines to elicit these memory T cell subpopulations predicted the efficacy of the recall response. These findings extend our understanding of how recall responses are generated and suggest that activation and migration markers define distinct, and unrelated, characteristics of memory T cells.

Premature Terminal Exhaustion of Friend Virus-Specific Effector CD8+ T Cells by Rapid Induction of Multiple Inhibitory Receptors

During chronic viral infection, persistent exposure to viral Ags leads to the overexpression of multiple inhibitory cell-surface receptors that cause CD8+ T cell exhaustion. The severity of exhaustion correlates directly with the level of infection and the number and intensity of inhibitory receptors expressed, and it correlates inversely with the ability to respond to the blockade of inhibitory pathways. Friend virus (FV) is a murine retrovirus complex that induces acute high-level viremia, followed by persistent infection and leukemia development, when inoculated into immunocompetent adult mice. In this article, we provide conclusive evidence that FV infection results in the generation of virus-specific effector CD8+ T cells that are terminally exhausted. Acute FV-induced disease is characterized by a rapid increase in the number of virus-infected erythroblasts, leading to massive splenomegaly. Most of the expanded erythroblasts strongly express programmed death ligand-1 and MHC class I, thereby creating a highly tolerogenic environment. Consequently, FV-specific effector CD8+ T cells uniformly express multiple inhibitory receptors, such as programmed cell death 1 (PD-1), T cell Ig domain and mucin domain 3 (Tim-3), lymphocyte activation gene-3, and CTLA-4, rapidly become nonresponsive to restimulation and are no longer reinvigorated by combined in vivo blockade of PD-1 and Tim-3 during the memory phase. However, combined blockade of PD-1 and Tim-3 during the priming/differentiation phase rescued FV-specific CD8+ T cells from becoming terminally exhausted, resulting in improved CD8+ T cell functionality and virus control. These results highlight FV’s unique ability to evade virus-specific CD8+ T cell responses and the importance of an early prophylactic approach for preventing terminal exhaustion of CD8+ T cells.

DNA vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration

Delivery of foreign genes to the digestive tract mucosa by oral administration of nonreplicating gene transfer vectors would be a very useful method for vaccination and gene therapy. However, there have been few reports on suitable vectors. In the present study, we found that plasmid DNA can be packaged in vitro into a virus-like particle (VLP) composed of open reading frame 2 of hepatitis E virus, which is an orally transmissible virus, and that these VLPs can deliver this foreign DNA to the intestinal mucosa in vivo. The delivery of plasmid DNA to the mucosa of the small intestine was confirmed by the results of immunohistochemical analyses using an expression plasmid encoding human immunodeficiency virus env (HIV env) gp120. After oral administration of VLPs loaded with HIV env cDNA, significant levels of specific IgG and IgA to HIV env in fecal extracts and sera were found. Moreover, mice used in this study exhibited cytotoxic T-lymphocyte responses specific to HIV env in the spleen, Payers patches and mesenteric lymph nodes. These findings suggest that VLPs derived from orally transmissible viruses can be used as vectors for delivery of genes to mucosal tissue by oral administration for the purpose of DNA vaccination and gene therapy.

Chemokine receptor CXCR3 facilitates CD8+ T cell differentiation into short-lived effector cells leading to memory degeneration

CXCR3 regulates CD8+ T cell recruitment to sites of inflammation, thus dictating CD8+ T cell contraction and subsequent effector/memory cell fate.

Quintuple Deglycosylation Mutant of Simian Immunodeficiency Virus SIVmac239 in Rhesus Macaques: Robust Primary Replication, Tightly Contained Chronic Infection, and Elicitation of Potent Immunity against the Parental Wild-Type Strain

ABSTRACT We previously generated a mutant of simian immunodeficiency virus (SIV) lacking 5 of a total of 22 N-glycans in its external envelope protein gp120 with no impairment in viral replication capability and infectivity in tissue culture cells. Here, we infected rhesus macaques with this mutant and found that it also replicated robustly in the acute phase but was tightly, though not completely, contained in the chronic phase. Thus, a critical requirement for the N-glycans for the full extent of chronic infection was demonstrated. No evidence indicating reversion to a wild type was obtained during the observation period of more than 40 weeks. Monkeys infected with the mutant were found to tolerate a challenge infection with wild-type SIV very well. Analyses of host responses following challenge revealed no neutralizing antibodies against the challenge virus but strong secondary responses of cytotoxic T lymphocytes against multiple antigens, including Gag-Pol, Nef, and Env. Thus, the quintuple deglycosylation mutant appeared to represent a novel class of SIV live attenuated vaccine.

Detection of Borna disease virus in a pregnant mare and her fetus

A pregnant mare showing pyrexia, reduced appetite, ataxia and paresis was euthanized and examined for the presence of Borna disease virus (BDV). Her brain, showing multiple neuronal degeneration and necrosis with hemorrhage, and the histologically normal brain of the fetus were both positive for BDV RNA. The BDV nucleotide sequences were identical in the mare and fetus in the second open reading frame (ORF). This is the first report of the possible vertical transmission of BDV in a horse.

Specific niches for lung-resident memory CD8+ T cells at the site of tissue regeneration enable CD69-independent maintenance.

Takamura et al. show that most lung CD8+ TRM cells are not maintained in the inducible bronchus-associated lymphoid tissue (iBALT) but are maintained in specific niches created at the site of tissue regeneration, which are termed as repair-associated memory depots (RAMDs).

Persistence of Viremia and Production of Neutralizing Antibodies Differentially Regulated by Polymorphic APOBEC3 and BAFF-R Loci in Friend Virus-Infected Mice

ABSTRACT Several host genes control retroviral replication and pathogenesis through the regulation of immune responses to viral antigens. The Rfv3 gene influences the persistence of viremia and production of virus-neutralizing antibodies in mice infected with Friend mouse retrovirus complex (FV). This locus has been mapped within a narrow segment of mouse chromosome 15 harboring the APOBEC3 and BAFF-R loci, both of which show functional polymorphisms among different strains of mice. The exon 5-lacking product of the APOBEC3 allele expressed in FV-resistant C57BL/6 (B6) mice directly restricts viral replication, and mice lacking the B6-derived APOBEC3 exhibit exaggerated pathology and reduced production of neutralizing antibodies. However, the mechanisms by which the polymorphisms at the APOBEC3 locus affect the production of neutralizing antibodies remain unclear. Here we show that the APOBEC3 genotypes do not directly affect the B-cell repertoire, and mice lacking B6-derived APOBEC3 still produce FV-neutralizing antibodies in the presence of primed T helper cells. Instead, higher viral loads at a very early stage of FV infection caused by either a lack of the B6-derived APOBEC3 or a lack of the wild-type BAFF-R resulted in slower production of neutralizing antibodies. Indeed, B cells were hyperactivated soon after infection in the APOBEC3- or BAFF-R-deficient mice. In contrast to mice deficient in the B6-derived APOBEC3, which cleared viremia by 4 weeks after FV infection, mice lacking the functional BAFF-R allele exhibited sustained viremia, indicating that the polymorphisms at the BAFF-R locus may better explain the Rfv3-defining phenotype of persistent viremia.

Ag85B of mycobacteria elicits effective CTL responses through activation of robust Th1 immunity as a novel adjuvant in DNA vaccine.

CD4+ T cells play a crucial role in CTL generation in a DNA vaccination strategy. Several studies have demonstrated the requirement of CD4+ T cells for the induction of a sufficient immune response by coadministrating DNAs. In the present study we investigated the effectiveness of Ag85B of mycobacteria, which is known to be one of the immunogenic proteins for Th1 development, as an adjuvant of a DNA vaccine. HIV gp120 DNA vaccine mixed with Ag85B DNA as an adjuvant induced HIV gp120-specific Th1 responses, as shown by delayed-type hypersensitivity, cytokine secretion, and increasing HIV-specific CTL responses. Moreover, these responses were enhanced in mice primed with Mycobacterium bovis bacillus Calmette-Guérin before immunization of HIV DNA vaccine mixed with Ag85B DNA. Furthermore, these immunized mice showed substantial reduction of HIV gp120-expressing recombinant vaccinia virus titers compared with the titers in other experimental mice after recombinant vaccinia virus challenge. Because most humans have been sensitized by spontaneous infection or by vaccination with mycobacteria, these findings indicate that Ag85B is a promising adjuvant for enhancing CTL responses in a DNA vaccination strategy.

A single administration of interleukin-4 antagonistic mutant DNA inhibits allergic airway inflammation in a mouse model of asthma.

Interleukin 4 (IL-4) is essential for the switching of B cells to IgE antibody production and for the maturation of T helper (Th) cells toward the Th2 phenotype. These mechanisms are thought to play a crucial role in the pathogenesis of the allergic airway inflammation observed in asthma. In the present study, we examined the anti-inflammatory effects of DNA administration of murine IL-4 mutant Q116D/Y119D (IL-4 double mutant, IL-4DM), which binds to the IL-4 receptor α and is an antagonist for IL-4. Immunization of BALB/c mice with alum-adsorbed ovalbumin (OVA) followed by aspiration with aerosolized OVA resulted in the development of allergic airway inflammation. A single administration of IL-4DM DNA before the aerosolized OVA challenge protected the mice from the subsequent induction of allergic airway inflammation. Serum IgE level and extent of eosinophil infiltration in bronchoalveolar lavage (BAL) from IL-4DM DNA-administered mice were significantly lower than those in BAL from control plasmid-immunized mice. In our study, IL-4 or IL-4 mutants were not detected in sera from mice that had received a single administration of IL-4DM DNA. The results of this study provide evidence for the potential utility of IL-4 mutant antagonist DNA inoculation as an approach to gene therapy for asthma.