Steven M. Varga

Attrition of T Cell Memory: Selective Loss of LCMV Epitope–Specific Memory CD8 T Cells following Infections with Heterologous Viruses

Using a variety of techniques, including limiting dilution assays (LDA), intracellular IFNgamma assays, and Db-IgG1 MHC dimer staining to measure viral peptide-specific T cell number and function, we show here that heterologous virus infections quantitatively delete and qualitatively alter the memory pool of T cells specific to a previously encountered virus. We also show that a prior history of a virus infection can alter the hierarchy of the immunodominant peptide response to a second virus and that virus infections selectively reactivate memory T cells with distinct specificities to earlier viruses. These results are consistent with a model for the immune system that accommodates memory T cell populations for multiple pathogens over the course of a lifetime.

Foxp3+ CD4 Regulatory T Cells Limit Pulmonary Immunopathology by Modulating the CD8 T Cell Response during Respiratory Syncytial Virus Infection

Regulatory Foxp3+ CD4 T cells (Tregs) prevent spontaneous inflammation in the lungs, inhibit allergic and asthmatic responses, and contribute to tolerance to inhaled allergens. Additionally, Tregs have previously been shown to suppress the CD8 T cell response during persistent virus infections. However, little is known concerning the role that Tregs play in modulating the adaptive immune response during acute respiratory virus infections. We show following acute respiratory syncytial virus (RSV) infection that Foxp3+ CD4 Tregs rapidly accumulate in the lung-draining mediastinal lymph nodes and lungs. BrdU incorporation studies indicate that Tregs undergo proliferation that contributes to their accumulation in the lymph nodes and lungs. Following an acute RSV infection, pulmonary Tregs modulate CD25 expression and acquire an activated phenotype characterized as CD11ahigh, CD44high, CD43glyco+, ICOS+, and CTLA-4+. Surprisingly, in vivo depletion of Tregs prior to RSV infection results in delayed virus clearance concomitant with an early lag in the recruitment of RSV-specific CD8 T cells into the lungs. Additionally, Treg depletion results in exacerbated disease severity, including increased weight loss, morbidity, and enhanced airway restriction. In Treg-depleted mice there is an increase in the frequency of RSV-specific CD8 T cells that coproduce IFN-γ and TNF-α, which may contribute to enhanced disease severity. These results indicate that pulmonary Tregs play a critical role in limiting immunopathology during an acute pulmonary virus infection by influencing the trafficking and effector function of virus-specific CD8 T cells in the lungs and draining lymph nodes.

Immunopathology in RSV Infection Is Mediated by a Discrete Oligoclonal Subset of Antigen-Specific CD4+ T Cells

Vaccination with the respiratory syncytial virus (RSV) attachment (G) protein results in immune-mediated lung injury after natural RSV infection with pathogenic features characteristic of an exaggerated Th2 response. Here we demonstrate that approximately half of the CD4(+) T cells infiltrating the lungs of G-primed mice utilize a single V beta gene (V beta 14) with remarkably limited CDR3 diversity. Furthermore, elimination of these V beta 14-bearing CD4(+) T cells in vivo abolishes the type 2-like pulmonary injury. These results suggest that a novel subset of CD4(+) T cells may be crucial in the development of pathology during human RSV infection and that genetic or environmental factors prior to or at the time of G antigen exposure may affect the commitment of this discrete antigen-specific T cell subset to Th2 differentiation.

The Attachment (G) Glycoprotein of Respiratory Syncytial Virus Contains a Single Immunodominant Epitope That Elicits Both Th1 and Th2 CD4+ T Cell Responses

BALB/c mice immunized with a vaccinia virus expressing the attachment (G) glycoprotein of respiratory syncytial virus (RSV) develop a virus-specific CD4+ T cell response that consists of a mixture of Th1 and Th2 CD4+ T cells following intranasal infection with live RSV. Recent work has shown that both Th1 and Th2 CD4+ T cells are elicited to a single region comprising aa 183–197 of the G protein. To more precisely define the CD4+ T cell epitope(s) contained within this region, we created a panel of amino- and carboxyl-terminal truncated as well as single alanine-substituted peptides spanning aa 183–197. These peptides were used to examine the ex vivo cytokine response of memory effector CD4+ T cells infiltrating the lungs of G-primed RSV-infected mice. Analysis of lung-derived memory effector CD4+ T cells using intracellular cytokine staining and/or ELISA of effector T cell culture supernatants revealed a single I-Ed-restricted CD4+ T cell epitope with a core sequence mapping to aa 185–193. In addition, we examined the T cell repertoire of the RSV G peptide-specific CD4+ T cells and show that the CD4+ T cells directed to this single immunodominant G epitope use a restricted range of TCR Vβ genes and predominantly express Vβ14 TCR.

Transepithelial migration of neutrophils into the lung requires TREM-1

Acute respiratory infections are responsible for more than 4 million deaths each year. Neutrophils play an essential role in the innate immune response to lung infection. These cells have an armamentarium of pattern recognition molecules and antimicrobial agents that identify and eliminate pathogens. In the setting of infection, neutrophil triggering receptor expressed on myeloid cells 1 (TREM-1) amplifies inflammatory signaling. Here we demonstrate for the first time that TREM-1 also plays an important role in transepithelial migration of neutrophils into the airspace. We developed a TREM-1/3-deficient mouse model of pneumonia and found that absence of TREM-1/3 markedly increased mortality following Pseudomonas aeruginosa challenge. Unexpectedly, TREM-1/3 deficiency resulted in increased local and systemic cytokine production. TREM-1/3-deficient neutrophils demonstrated intact bacterial killing, phagocytosis, and chemotaxis; however, histologic examination of TREM-1/3-deficient lungs revealed decreased neutrophil infiltration of the airways. TREM-1/3-deficient neutrophils effectively migrated across primary endothelial cell monolayers but failed to migrate across primary airway epithelia grown at the air-liquid interface. These data define a new function for TREM-1 in neutrophil migration across airway epithelial cells and suggest that it amplifies inflammation through targeted neutrophil migration into the lung.

CD8 T Cells Inhibit Respiratory Syncytial Virus (RSV) Vaccine-Enhanced Disease

Vaccination of children with a formalin-inactivated (FI) respiratory syncytial virus (RSV) vaccine led to exacerbated disease including pulmonary eosinophilia following a natural RSV infection. Immunization of BALB/c mice with FI-RSV or a recombinant vaccinia virus (vv) expressing the RSV attachment (G) protein (vvG) results in a pulmonary Th2 response and eosinophilia after RSV challenge that closely mimics the RSV vaccine-enhanced disease observed in humans. The underlying causes of RSV vaccine-enhanced disease remain poorly understood. We demonstrate here that RSV M2-specific CD8 T cells reduce the Th2-mediated pathology induced by vvG-immunization and RSV challenge in an IFN-γ-independent manner. We also demonstrate that FI-RSV immunization does not induce a measurable RSV-specific CD8 T cell response and that priming FI-RSV-immunized mice for a potent memory RSV-specific CD8 T cell response abrogates pulmonary eosinophilia after subsequent RSV challenge. Our results suggest that the failure of the FI-RSV vaccine to induce a CD8 T cell response may have contributed to the development of pulmonary eosinophilia and augmented disease that occurred in vaccinated individuals.

Alpha beta and gamma delta T-cell networks and their roles in natural resistance to viral infections

Both αβ and γδ T‐cell populations and natural killer (NK) cells include cytotoxic, interferon (IFN)‐γ‐producing lymphocytes that actively respond to viral infections. We show here that all three populations can provide “natural resistance“ of viruses very early in infection and describe how the T‐tell populations are modulated TO provide this function. γδ T cells were shown to play a role in controlling vaccinia virus (VV) infections, as VV grew to much higher titers in γδ T‐cell knockout mice than in normal mice 3–4 days post‐infection. Our studies of the of T‐cell responses to viruses revealed an interactive network of T cells that is modulated substantially during systemic infections. There is an induction phase associated with a massive virus‐specific CD8 T‐cell response, an apoptosis phase during which the T cells become sensitized to activation‐induced ceil death (AICD). a silencing phase, during which the T‐cell number and activation state is reduced, and. finally, a memory phase associated with the very stable preservation of virus‐specific memory cytotoxic T‐lymphocyte precursors (pCTL). Infection of mice immune to one‐H US with a heterologous virus leads to a selective expansion of memory CTL cross‐reacting between the two viruses, but, after homeostasis is again established, there is a quantitative reduction and quantitative alteration of memory to the first virus. Our results suggest that memory of T cells cross‐reactive between heterologous viruses mediate both immunopathology and protective immunity at early stages of the second viruses infection. Thus, memory αβ T cells can, like γδ T cells and NK cells, provide natural immunity to viral infections.

Multiple CD4+ T Cell Subsets Produce Immunomodulatory IL-10 During Respiratory Syncytial Virus Infection

The host immune response is believed to contribute to the severity of pulmonary disease induced by acute respiratory syncytial virus (RSV) infection. Because RSV-induced pulmonary disease is associated with immunopathology, we evaluated the role of IL-10 in modulating the RSV-specific immune response. We found that IL-10 protein levels in the lung were increased following acute RSV infection, with maximum production corresponding to the peak of the virus-specific T cell response. The majority of IL-10–producing cells in the lung during acute RSV infection were CD4+ T cells. The IL-10–producing CD4+ T cells included Foxp3+ regulatory T cells, Foxp3− CD4+ T cells that coproduce IFN-γ, and Foxp3− CD4+ T cells that do not coproduce IFN-γ. RSV infection of IL-10–deficient mice resulted in more severe disease, as measured by increased weight loss and airway resistance, as compared with control mice. We also observed an increase in the magnitude of the RSV-induced CD8+ and CD4+ T cell response that correlated with increased disease severity in the absence of IL-10 or following IL-10R blockade. Interestingly, IL-10R blockade during acute RSV infection altered CD4+ T cell subset distribution, resulting in a significant increase in IL-17A–producing CD4+ T cells and a concomitant decrease in Foxp3+ regulatory T cells. These results demonstrate that IL-10 plays a critical role in modulating the adaptive immune response to RSV by limiting T-cell–mediated pulmonary inflammation and injury.

Comparison of Histochemical Methods for Murine Eosinophil Detection in an RSV Vaccine-enhanced Inflammation Model:

A comparative study of histochemical detection of eosinophils in fixed murine tissue is lacking. Five histochemical methods previously reported for eosinophil detection were quantitatively and qualitatively compared in an established murine RSV vaccine–enhanced inflammation model. Nonspecific neutrophil staining was evaluated in tissue sections of neutrophilic soft tissue lesions and bone marrow from respective animals. Eosinophils had granular red to orange-red cytoplasmic staining, depending on the method, whereas neutrophils had, when stained, a more homogenous cytoplasmic pattern. Nonspecific background staining of similar coloration was variably seen in vascular walls and erythrocytes. Astra Blue/Vital New Red, Congo Red, Luna, Modified Hematoxylin and Eosin, and Sirius Red techniques were all effective in detecting increased eosinophil recruitment compared to controls; however, differences in eosinophil quantification varied significantly between techniques. Astra Blue/Vital New Red had the best specificity for differentiating eosinophils and neutrophils but had a reduced ability to enumerate eosinophils and was the most time intensive. The Luna stain had excessive nonspecific staining of tissues and a reduced enumeration of infiltrating eosinophils, which made it suboptimal. For multiple parameters such as eosinophil detection, specificity, and contrast with background tissues, the Sirius Red followed by Congo Red and Modified Hematoxylin and Eosin methods were useful, each with their own staining qualities.

IL-13 Is Required for Eosinophil Entry into the Lung during Respiratory Syncytial Virus Vaccine-Enhanced Disease

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in children. Children previously vaccinated with a formalin-inactivated RSV vaccine experienced enhanced morbidity and mortality upon natural RSV infection. Histological analysis revealed the presence of eosinophils in the pulmonary infiltrate of the vaccinated children. Eosinophils are characteristic of Th2 responses, and Th2 cells are known to be necessary to induce pulmonary eosinophilia in RSV-infected BALB/c mice previously immunized with a recombinant vaccinia virus (vv) expressing the RSV G protein (vvG). Using IL-13-deficient mice, we find that IL-13 is necessary for eosinophils to reach the lung parenchyma and airways of vvG-immunized mice undergoing RSV challenge infection. IL-13 acts specifically on eosinophils as the magnitude of pulmonary inflammation, RSV G protein-specific CD4 T cell responses, and virus clearance were not altered in IL-13-deficient mice. After RSV challenge, eosinophils were readily detectable in the blood and bone marrow of vvG-immunized IL-13-deficient mice, suggesting that IL-13 is required for eosinophils to transit from the blood into the lung. Pulmonary levels of CCL11 and CCL22 protein were significantly reduced in IL-13-deficient mice indicating that IL-13 mediates the recruitment of eosinophils into the lungs by inducing the production of chemokines important in Th2 cell and eosinophil chemotaxis.