Erosion of Transplantation Tolerance After Infection.

Recent clinical studies suggest that operational allograft tolerance can be persistent, but long-term surviving allografts can be rejected in a subset of patients, sometimes after episodes of infection. In this study, we examined the impact of Listeria monocytogenes (Lm) infection on the quality of tolerance in a mouse model of heart allograft transplantation. Lm infection induced full rejection in 40% of tolerant recipients, with the remaining experiencing a rejection crisis or no palpable change in their allografts. In the surviving allografts on day 8 postinfection, graft-infiltrating cell numbers increased and exhibited a loss in the tolerance gene signature. By day 30 postinfection, the tolerance signature was broadly restored, but with a discernible reduction in the expression of a subset of 234 genes that marked tolerance and was down-regulated at day 8 post-Lm infection. We further demonstrated that the tolerant state after Lm infection was functionally eroded, as rejection of the long-term surviving graft was induced with anti-PD-L1 whereas the same treatment had no effect in noninfected tolerant mice. Collectively, these observations demonstrate that tolerance, even if initially robust, exists as a continuum that can be eroded following bystander immune responses that accompany certain infections.

HTLV-1 bZIP factor impairs cell-mediated immunity by suppressing production of Th1 cytokines.

Adult T-cell leukemia (ATL) patients and human T-cell leukemia virus-1 (HTLV-1) infected individuals succumb to opportunistic infections. Cell mediated immunity is impaired, yet the mechanism of this impairment has remained elusive. The HTLV-1 basic leucine zipper factor (HBZ) gene is encoded in the minus strand of the viral DNA and is constitutively expressed in infected cells and ATL cells. To test the hypothesis that HBZ contributes to HTLV-1-associated immunodeficiency, we challenged transgenic mice that express the HBZ gene in CD4 T cells (HBZ-Tg mice) with herpes simplex virus type 2 or Listeria monocytogenes, and evaluated cellular immunity to these pathogens. HBZ-Tg mice were more vulnerable to both infections than non-Tg mice. The acquired immune response phase was specifically suppressed, indicating that cellular immunity was impaired in HBZ-Tg mice. In particular, production of IFN-γ by CD4 T cells was suppressed in HBZ-Tg mice. HBZ suppressed transcription from the IFN-γ gene promoter in a CD4 T cell-intrinsic manner by inhibiting nuclear factor of activated T cells and the activator protein 1 signaling pathway. This study shows that HBZ inhibits CD4 T-cell responses by directly interfering with the host cell-signaling pathway, resulting in impaired cell-mediated immunity in vivo.

Role of PKR and Type I IFNs in viral control during primary and secondary infection.

Type I interferons (IFNs) are known to mediate viral control, and also promote survival and expansion of virus-specific CD8+ T cells. However, it is unclear whether signaling cascades involved in eliciting these diverse cellular effects are also distinct. One of the best-characterized anti-viral signaling mechanisms of Type I IFNs is mediated by the IFN-inducible dsRNA activated protein kinase, PKR. Here, we have investigated the role of PKR and Type I IFNs in regulating viral clearance and CD8+ T cell response during primary and secondary viral infections. Our studies demonstrate differential requirement for PKR, in viral control versus elicitation of CD8+ T cell responses during primary infection of mice with lymphocytic choriomeningitis virus (LCMV). PKR-deficient mice mounted potent CD8+ T cell responses, but failed to effectively control LCMV. The compromised LCMV control in the absence of PKR was multifactorial, and linked to less effective CD8+ T cell-mediated viral suppression, enhanced viral replication in cells, and lower steady state expression levels of IFN-responsive genes. Moreover, we show that despite normal expansion of memory CD8+ T cells and differentiation into effectors during a secondary response, effective clearance of LCMV but not vaccinia virus required PKR activity in infected cells. In the absence of Type I IFN signaling, secondary effector CD8+ T cells were ineffective in controlling both LCMV and vaccinia virus replication in vivo. These findings provide insight into cellular pathways of Type I IFN actions, and highlight the under-appreciated importance of innate immune mechanisms of viral control during secondary infections, despite the accelerated responses of memory CD8+ T cells. Additionally, the results presented here have furthered our understanding of the immune correlates of anti-viral protective immunity, which have implications in the rational design of vaccines.

Listeriosis associated with upper gastrointestinal endoscopy in an HIV-infected patient.

An HIV-infected man developed listeriosis six days after an upper gastrointestinal (GI) endoscopy. Although listeriosis has been rarely described following lower GI endoscopy, we believe this is the first case related to upper GI endoscopy.

Cytomegalovirus generates long-lived antigen-specific NK cells with diminished bystander activation to heterologous infection.

Natural killer (NK) cells play a key role in the host response to cytomegalovirus (CMV) and can mediate an enhanced response to secondary challenge with CMV. We assessed the ability of mouse CMV (MCMV)-induced memory Ly49H(+) NK cells to respond to challenges with influenza, an acute viral infection localized to the lung, and Listeria monocytogenes, a systemic bacterial infection. MCMV-memory NK cells did not display enhanced activation or proliferation after infection with influenza or Listeria, as compared with naive Ly49H(+) or Ly49H(-) NK cells. Memory NK cells also showed impaired activation compared with naive cells when challenged with a mutant MCMV lacking m157, highlighting their antigen-specific response. Ex vivo, MCMV-memory NK cells displayed reduced phosphorylation of STAT4 and STAT1 in response to stimulation by IL-12 and type I interferon (IFN), respectively, and IFN-γ production was reduced in response to IL-12 + IL-18 compared with naive NK cells. However, costimulation of MCMV-memory NK cells with IL-12 and m157 antigen rescues their impaired response compared with cytokines alone. These findings reveal that MCMV-primed memory NK cells are diminished in their response to cytokine-driven bystander responses to heterologous infections as they become specialized and antigen-specific for the control of MCMV upon rechallenge.

The Tat pathway is prevalent in Listeria monocytogenes lineage II and is not required for infection and spread in host cells.

Listeria monocytogenes, a foodborne pathogenic bacterium, remains a serious public health concern due to its frequent occurrence in food products coupled with a high mortality rate. Bacterial pathogenicity depends greatly on the ability to secrete virulence factors to or beyond the bacterial cell surface. The Tat pathway, one of the secretion systems present in L. monocytogenes, was until now only investigated in silico. In L. monocytogenes strain EGDe two genes constitute this pathway, tatC(lmo0361) and tatA(lmo0362). Here we show that tatC and tatA are cotranscribed in a bicistronic- and growth-phase-dependent manner, being downregulated in the stationary phase. An EGDe tatAC mutant strain (EGDe ΔtatAC) was constructed, confirming that the Tat pathway is not essential for L.monocytogenes survival or biofilm-forming ability. When compared to the wild-type EGDe, deletion of tatAC did not decrease the virulence potential of EGDe ΔtatAC in HT-29 human epithelial cell line and even increased (p < 0.05) the virulence potential for mice. Moreover, we show that tat genes are prevalent in L. monocytogenes strains belonging to genetic lineage II and are generally absent from lineage I, which is more associated with human cases, thus excluding the possibility of using the Tat system as a target for novel antimicrobial compounds targeting L.monocytogenes.

Synthetic toll-like receptor 4 agonists stimulate innate resistance to infectious challenge.

A compound family of synthetic lipid A mimetics (termed the aminoalkyl glucosaminide phosphates [AGPs]) was evaluated in murine infectious disease models of protection against challenge with Listeria monocytogenes and influenza virus. For the Listeria model, intravenous administration of AGPs was followed by intravenous bacterial challenge 24 h later. Spleens were harvested 2 days postchallenge for the enumeration of CFU. For the influenza virus model, mice were challenged with virus via the intranasal/intrapulmonary route 48 h after intranasal/intrapulmonary administration of AGPs. The severity of disease was assessed daily for 3 weeks following challenge. Several types of AGPs provided strong protection against influenza virus or Listeria challenge in wild-type mice, but they were inactive in the C3H/HeJ mouse, demonstrating the dependence of the AGPs on toll-like receptor 4 (TLR4) signaling for the protective effect. Structure-activity relationship studies showed that the activation of innate immune effectors by AGPs depends primarily on the lengths of the secondary acyl chains within the three acyl-oxy-acyl residues and also on the nature of the functional group attached to the aglycon component. We conclude that the administration of synthetic TLR4 agonists provides rapid pharmacologic induction of innate resistance to infectious challenge by two different pathogen classes, that this effect is mediated via TLR4, and that structural differences between AGPs can have dramatic effects on agonist activity in vivo.

Cutting edge: re-evaluating the in vivo cytokine responses of CD8+ T cells during primary and secondary viral infections.

Virus-specific CD8(+) T cells produce IFN-gamma after Ag contact and, in the absence of this cytokine, the host often cannot eradicate infection. However, our ability to identify cells that are actively expressing this critical effector function in vivo is limited, because the protein is rapidly secreted. In this study, we describe a simple approach that circumvents the need for ex vivo Ag stimulation and allows the enumeration of CD8(+) T cells that are actively synthesizing IFN-gamma in vivo during primary and secondary virus infections. The proportion of Ag-specific primary CD8(+) T cells producing IFN-gamma peaks at 5 days postinfection, when the T cell population is still expanding exponentially. In vivo IFN-gamma synthesis by memory cells is explosive, peaking at approximately 12 h after secondary infection and terminating hours thereafter. This technique will be useful when evaluating in vivo immune cell activity in many situations, including a variety of noninfectious (e.g., autoimmune) diseases.

Listeria monocytogenes mutants that fail to compartmentalize listerolysin O activity are cytotoxic, avirulent, and unable to evade host extracellular defenses.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. Escape of the bacterium from the phagosome to the cytosol is mediated by the bacterial pore-forming protein listeriolysin O (LLO). LLO has multiple mechanisms that optimize activity in the phagosome and minimize activity in the host cytosol. Mutants that fail to compartmentalize LLO activity are cytotoxic and have reduced virulence. We sought to determine why cytotoxic bacteria have attenuated virulence in the mouse model of listeriosis. In this study, we constructed a series of strains with mutations in LLO and with various degrees of cytotoxicity. We found that the more cytotoxic the strain in cell culture, the less virulent it was in mice. Induction of neutropenia increased the relative virulence of the cytotoxic strains 100-fold in the spleen and 10-fold in the liver. The virulence defect was partially restored in neutropenic mice by adding gentamicin, an antibiotic that kills extracellular bacteria. Additionally, L. monocytogenes grew more slowly in extracellular fluid (mouse serum) than within tissue culture cells. We concluded that L. monocytogenes controls the cytolytic activity of LLO to maintain its nutritionally rich intracellular niche and avoid extracellular defenses of the host.

Regulation of CD8+ T cells undergoing primary and secondary responses to infection in the same host.

Naive Ag-specific CD8(+) T cells expand, contract, and become memory cells after infection and/or vaccination. Memory CD8(+) T cells provide faster, more effective secondary responses against repeated exposure to the same pathogen. Using an adoptive transfer system with low numbers of trackable nontransgenic memory CD8(+) T cells, we showed that secondary responses can be comprised of both primary (naive) and secondary (memory) CD8(+) T cells after bacterial (Listeria monocytogenes) and/or viral (lymphocytic choriomeningitis virus) infections. The level of memory CD8(+) T cells present at the time of infection inversely correlated with the magnitude of primary CD8(+) T cell responses against the same epitope but directly correlated with the level of protection against infection. However, similar numbers of Ag-specific CD8(+) T cells were found 8 days postinfection no matter how many memory cells were present at the time of infection. Rapid contraction of primary CD8(+) T cell responses was not influenced by the presence of memory CD8(+) T cells. However, contraction of secondary CD8(+) T cell responses was markedly prolonged compared with primary responses in the same host mice. This situation occurred in response to lymphocytic choriomeningitis virus or L. monocytogenes infection and for CD8(+) T cell responses against multiple epitopes. The delayed contraction of secondary CD8(+) T cells was also observed after immunization with peptide-coated dendritic cells. Together, the results show that the level of memory CD8(+) T cells influences protective immunity and activation of naive precursors specific for the same epitope but has little impact on the magnitude or program of the CD8(+) T cell response.