Hans Peter Raué

Regulation of innate CD8+ T-cell activation mediated by cytokines

Virus-specific CD8+ T cells develop the ability to function in an “innate” capacity by responding to a remarkable array of cytokines in a TCR-independent manner. Although several cytokines such as IL-12 and IL-18 have been identified as key regulators of CD8+ T-cell activation, the role of other cytokines and the ways in which they interact with each other remain unclear. Here, we have used an unbiased, systematic approach to examine the effects of 1,849 cytokine combinations on virus-specific CD8+ T-cell activation. This study identifies several unexpected cytokine combinations that synergize to induce antigen-independent IFNγ production and CD69 up-regulation by CD8+ T cells in addition to cytokines that exhibit differential regulatory functions, with the ability to either enhance or inhibit T-cell IFNγ production, depending on which cytokine partner is present. These findings underscore the complexity of cytokine interactions while also providing insight into the multifaceted regulatory network controlling virus-specific CD8+ T-cell functions.

Activation of Virus-Specific CD8+ T Cells by Lipopolysaccharide-Induced IL-12 and IL-18

Virus-specific T cells represent a hallmark of Ag-specific, adaptive immunity. However, some T cells also demonstrate innate functions, including non-Ag-specific IFN-γ production in response to microbial products such as LPS or exposure to IL-12 and/or IL-18. In these studies we examined LPS-induced cytokine responses of CD8+ T cells directly ex vivo. Following acute viral infection, 70–80% of virus-specific T cells will produce IFN-γ after exposure to LPS-induced cytokines, and neutralization experiments indicate that this is mediated almost entirely through production of IL-12 and IL-18. Different combinations of these cytokines revealed that IL-12 decreases the threshold of T cell activation by IL-18, presenting a new perspective on IL-12/IL-18 synergy. Moreover, memory T cells demonstrate high IL-18R expression and respond effectively to the combination of IL-12 and IL-18, but cannot respond to IL-18 alone, even at high cytokine concentrations. This demonstrates that the synergy between IL-12 and IL-18 in triggering IFN-γ production by memory T cells is not simply due to up-regulation of the surface receptor for IL-18, as shown previously with naive T cells. Together, these studies indicate how virus-specific T cells are able to bridge the gap between innate and adaptive immunity during unrelated microbial infections, while attempting to protect the host from cytokine-induced immunopathology and endotoxic shock.

Development of a new hydrogen peroxide-based vaccine platform

Safe and effective vaccines are crucial for maintaining public health and reducing the global burden of infectious disease. Here we introduce a new vaccine platform that uses hydrogen peroxide (H2O2) to inactivate viruses for vaccine production. H2O2 rapidly inactivates both RNA and DNA viruses with minimal damage to antigenic structure or immunogenicity and is a highly effective method when compared with conventional vaccine inactivation approaches such as formaldehyde or β-propiolactone. Mice immunized with H2O2-inactivated lymphocytic choriomeningitis virus (LCMV) generated cytolytic, multifunctional virus-specific CD8+ T cells that conferred protection against chronic LCMV infection. Likewise, mice vaccinated with H2O2-inactivated vaccinia virus or H2O2-inactivated West Nile virus showed high virus-specific neutralizing antibody titers and were fully protected against lethal challenge. Together, these studies demonstrate that H2O2-based vaccines are highly immunogenic, provide protection against a range of viral pathogens in mice and represent a promising new approach to future vaccine development.

A Cell-Intrinsic Requirement for NF-κB–Inducing Kinase in CD4 and CD8 T Cell Memory

NF-κB–inducing kinase [(NIK), MAP3K14] is an essential kinase linking a subset of TNFR family members to the noncanonical NF-κB pathway. To assess the cell-intrinsic role of NIK in murine T cell function, we generated mixed bone marrow chimeras using bone marrow from NIK knockout (KO) and wild-type (WT) donor mice and infected the chimeras with lymphocytic choriomeningitis virus (LCMV). The chimeras possess an apparently normal immune system, including a mixture of NIK KO and WT T cells, and the virus was cleared normally. Comparison of the NIK KO and WT CD4 and CD8 T cell responses at 8 d post infection revealed modest but significant differences in the acute response. In both CD4 and CD8 compartments, relatively fewer activated (CD44hi) NIK KO T cells were present, but within the CD44hi population, a comparable percentage of the activated cells produced IFN-γ in response to ex vivo stimulation with antigenic LCMV peptides, although IL-7R expression was reduced in the NIK KO CD8 T cells. Assessment of the LCMV-specific memory at 65 d post infection revealed many more LCMV-specific WT memory T cells than NIK KO memory T cells in both the CD4 and the CD8 compartments, although the small number of surviving NIK KO memory T cells responded to secondary challenge with virus. These results demonstrate a cell-intrinsic requirement for NIK in the generation and/or maintenance of memory T cells in response to acute viral infection.

Transcutaneous yellow fever vaccination of subjects with or without atopic dermatitis

BACKGROUND Atopic dermatitis (AD) is a common inflammatory skin disease with a global prevalence ranging from 3% to 20%. Patients with AD have an increased risk for complications after viral infection (eg, herpes simplex virus), and vaccination of patients with AD with live vaccinia virus is contraindicated because of a heightened risk of eczema vaccinatum, a rare but potentially lethal complication associated with smallpox vaccination. OBJECTIVE We sought to develop a better understanding of immunity to cutaneous viral infection in patients with AD. METHODS In a double-blind randomized study we investigated the safety and immunogenicity of live attenuated yellow fever virus (YFV) vaccination of nonatopic subjects and patients with AD after standard subcutaneous inoculation or transcutaneous vaccination administered with a bifurcated needle. Viremia, neutralizing antibody, and antiviral T-cell responses were analyzed for up to 30 days after vaccination. RESULTS YFV vaccination administered through either route was well tolerated. Subcutaneous vaccination resulted in higher seroconversion rates than transcutaneous vaccination but elicited similar antiviral antibody levels and T-cell responses in both the nonatopic and AD groups. After transcutaneous vaccination, both groups mounted similar neutralizing antibody responses, but patients with AD demonstrated lower antiviral T-cell responses by 30 days after vaccination. Among transcutaneously vaccinated subjects, a significant inverse correlation between baseline IgE levels and the magnitude of antiviral antibody and CD4(+) T-cell responses was observed. CONCLUSIONS YFV vaccination of patients with AD through the transcutaneous route revealed that high baseline IgE levels provide a potential biomarker for predicting reduced virus-specific immune memory after transcutaneous infection with a live virus.

CD8+ T cell immunodominance shifts during the early stages of acute LCMV infection independently from functional avidity maturation

Virus-specific T cell responses are often directed to a small subset of possible epitopes and their relative magnitude defines their hierarchy. We determined the size and functional avidity of 4 representative peptide-specific CD8(+) T cell populations in C57BL/6 mice at different time points after lymphocytic choriomeningitis virus (LCMV) infection. We found that the frequency of different peptide-specific T cell populations in the spleen changed independently over the first 8 days after infection. These changes were not associated with a larger or more rapid increase in functional avidity and yet still resulted in a shift in the final immunodominance hierarchy. Thus, the immunodominance observed at the peak of an antiviral T cell response is not necessarily determined by the initial size or rate of functional avidity maturation of peptide-specific T cell populations.

Cross-Neutralizing and Protective Human Antibody Specificities to Poxvirus Infections

Monkeypox (MPXV) and cowpox (CPXV) are emerging agents that cause severe human infections on an intermittent basis, and variola virus (VARV) has potential for use as an agent of bioterror. Vaccinia immune globulin (VIG) has been used therapeutically to treat severe orthopoxvirus infections but is in short supply. We generated a large panel of orthopoxvirus-specific human monoclonal antibodies (Abs) from immune subjects to investigate the molecular basis of broadly neutralizing antibody responses for diverse orthopoxviruses. Detailed analysis revealed the principal neutralizing antibody specificities that are cross-reactive for VACV, CPXV, MPXV, and VARV and that are determinants of protection in murine challenge models. Optimal protection following respiratory or systemic infection required a mixture of Abs that targeted several membrane proteins, including proteins on enveloped and mature virion forms of virus. This work reveals orthopoxvirus targets for human Abs that mediate cross-protective immunity and identifies new candidate Ab therapeutic mixtures to replace VIG.

Pivotal Advance: CTLA-4+ T cells exhibit normal antiviral functions during acute viral infection

Previous studies have shown that T cells, which are genetically deficient in CTLA‐4/CD152 expression, will proliferate uncontrollably, resulting in lethal autoimmune disease. This and other evidence indicate that CTLA‐4 plays a critical role in the negative regulation of effector T cell function. In contrast to expectations, BrdU incorporation experiments demonstrated that CTLA‐4 expression was associated with normal or even enhanced in vivo proliferation of virus‐specific CD4+ and CD8+ T cells following acute lymphocytic choriomeningitis virus or vaccinia virus infection. When compared with CTLA‐4– T cells directly ex vivo, CTLA‐4+ T cells also exhibited normal antiviral effector functions following stimulation with peptide‐coated cells, virus‐infected cells, plate‐bound anti‐CD3/anti‐CTLA‐4, or the cytokines IL‐12 and IL‐18. Together, this indicates that CTLA‐4 does not directly inhibit antivral T cell expansion or T cell effector functions, at least not under the normal physiological conditions associated with either of these two acute viral infections.

Cytokine-Mediated Activation of NK Cells during Viral Infection

ABSTRACT Natural killer (NK) cells provide a first line of defense against infection via the production of antiviral cytokines and direct lysis of target cells. Cytokines such as interleukin 12 (IL-12) and IL-18 are critical regulators of NK cell activation, but much remains to be learned about how cytokines interact to regulate NK cell function. Here, we have examined cytokine-mediated activation of NK cells during infection with two natural mouse pathogens, lymphocytic choriomeningitis virus (LCMV) and murine cytomegalovirus (MCMV). Using a systematic screen of 1,849 cytokine pairs, we identified the most potent combinations capable of eliciting gamma interferon (IFN-γ) production in NK cells. We observed that NK cell responses to cytokine stimulation were reduced 8 days after acute LCMV infection but recovered to preinfection levels by 60 days postinfection. In contrast, during MCMV infection, NK cell responses to cytokines remained robust at all time points examined. Ly49H-positive (Ly49H+) NK cells recognizing viral ligand m157 showed preferential proliferation during early MCMV infection. A population of these cells was still detected beyond 60 days postinfection, but these divided cells did not demonstrate enhanced IFN-γ production in response to innate cytokine stimulation. Instead, the maturation state of the NK cells (as determined by CD11b or CD27 surface phenotype) was predictive of responsiveness to cytokines, regardless of Ly49H expression. These results help define cytokine interactions that regulate NK cell activation and highlight variations in NK cell function during two unrelated viral infections. IMPORTANCE Natural killer cells play an important role in immunity to many viral infections. From an initial screen of 1,849 cytokine pairs, we identified the most stimulatory cytokine combinations capable of inducing IFN-γ production by NK cells. Ly49H+ NK cells, which can be directly activated by MCMV protein m157, preferentially proliferated during MCMV infection but did not show enhanced IFN-γ production following direct ex vivo cytokine stimulation. Instead, mature CD11b+ and/or CD27+ NK cells responded similarly to innate cytokine stimulation regardless of Ly49H expression. Collectively, our data provide a better foundation for understanding cytokine-mediated NK cell activation during viral infection.

Characterization of CD8+ T Cell Function and Immunodominance Generated with an H2O2-Inactivated Whole-Virus Vaccine

ABSTRACT CD8+ T cells play an important role in protection against both acute and persistent viral infections, and new vaccines that induce CD8+ T cell immunity are currently needed. Here, we show that lymphocytic choriomeningitis virus (LCMV)-specific CD8+ T cells can be generated in response to a nonreplicating H2O2-inactivated whole-virus vaccine (H2O2-LCMV). Vaccine-induced CD8+ T cell responses exhibited an increased ability to produce multiple cytokines at early time points following immunization compared to infection-induced responses. Vaccination with H2O2-LCMV induced the expansion of a narrow subset of the antigen-specific CD8+ T cells induced by LCMV strain Arm infection, resulting in a distinct immunodominance hierarchy. Acute LCMV infection stimulated immunodominance patterns that shifted over time or after secondary infection, whereas vaccine-generated immunodominance profiles remained remarkably stable even following subsequent viral infection. Vaccine-induced CD8+ T cell populations expanded sharply in response to challenge and were then maintained at high levels, with responses to individual epitopes occupying up to 40% of the CD8+ T cell compartment at 35 days after challenge. H2O2-LCMV vaccination protected animals against challenge with chronic LCMV clone 13, and protection was mediated by CD8+ T cells. These results indicate that vaccination with an H2O2-inactivated whole-virus vaccine induces LCMV-specific CD8+ T cells with unique functional characteristics and provides a useful model for studying CD8+ T cells elicited in the absence of active viral infection.