Recipient Organization
UNIV OF PENNSYLVANIA
(N/A)
PHILADELPHIA,PA 19104
Performing Department
School Of Veterinary Medicine
Non Technical Summary
Influenza virus causes dangerous, potentially fatal infections, especially in patients with preexisting conditions such as cardiovascular diseases. The main goal of our study is to analyze if the T cells that enter the lung to fight infection with influenza virus are able to exit the lung and how this exit is regulated. T-cells are part of the immune system which is the body's defense against infections. T cells enter infected tissues to eliminate pathogens. They are distributed throughout the body via the blood to reach different tissues and organs where they migrate from the blood vessel into the surrounding tissue. Subsequently, T cells can as well exit tissues and organs by entering lymphatic vessels, the exit route. The duration of stay of the T cells is a likely mechanism to control the successful elimination of influenza virus. In parallel the presence of T cells within the lung can also cause potential damage to the lung tissue, as T cells are not only harmful to the virus but can also injure lung cells. We are pursuing two specific questions. Our first question is how important a recently discovered exit-factor is for T cells to leave the lung. To address this question we are investigating the behavior of T cells in influenza virus infection in mice using an experimental system to transplant T cells into the lungs of mice infected with influenza virus. We then observe if the T cells can leave the organ. Our first experiments indicate that the exit-factor is important and T cells that lack it do not leave while normal T cells do. We also test mice that have too much of the exit-factor. Our second question is which mice cope best with influenza virus infection, those that get normal T cells, those that get T cells lacking exit factor or those that get T cells with too much exit factor. We believe that during mild infection mice that get T cells with too much exit factor will do best while during severe infection mice that get T cells lacking exit factor will recover the fastest. Our experiments will help us to understand how the duration of stay of T cells in the lung during influenza virus infection does influence the recovery from infection. Knowledge of the mechanisms that control the presence of virus fighting T cells in the infected lung can be used to develop new drugs that would help the body's own immune cells to eliminate influenza virus more efficiently. Patients with heart disease will especially benefit from faster recovery from influenza virus infection and less severe progression of the infection.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Influenza A virus infection is a severe infectious disease causing up to 36,000 death per year in the US. Pre-existing cardiovascular disease is a critical risk factor for the development of severe pneumonia, the most common complication during influenza A virus infection that can lead to fatal respiratory dysfunction. Immune protection against acute influenza A virus infection is dependent on cytotoxic CD8 T cells and requires their direct contact with infected cells of the respiratory epithelium. Entry into the lung as well as retention at the site of infection is essential for such protective cytotoxic T cell responses against respiratory virus infections. Mechanisms of lymphocyte migration from the bloodstream into peripheral tissues such as the lung have been extensively studied; however, mechanisms responsible for lymphocyte exit from and retention within tissues are poorly defined. Lymphocyte egress from peripheral tissue and migration into draining lymph nodes through afferent lymph is a key determinant of lymphocyte accumulation and contributes to immunosurveillance and inflammation. In contrast to previous assumptions, recent studies in our lab showed that lymphocyte egress from peripheral tissue is indeed regulated and that T cells require the expression of the chemokine receptor CCR7 for egress under steady-state (non-inflammatory) conditions. Here, I will test the role of CCR7 in T cell exit from different lung compartments during the course of influenza virus infection. Most notably, I will determine the importance of T cell retention in versus egress from the lung for pathogen clearance and host survival during influenza A virus infection. I hypothesize that upon recognition of viral antigens, antigen-specific protective effector/memory T cells transiently down-regulate CCR7 expression to prevent CCR7-driven tissue exit, ensuring the most efficient viral clearance. Conversely, I propose, that T cells that lack the exit receptor CCR7 will accumulate in the infected lung and provide superior viral clearance. Identifying the control mechanisms responsible for lymphocyte exit from the lung via the afferent lymph will likely reveal new targets on which to base therapeutics designed to regulate tissue inflammation or improve viral clearance.
Project Methods
To test the role of CCR7 in effector T cell exit from the lung at different stages of influenza A virus infection, I have established a mouse model that allows visualization of lymphocyte exit from this organ and to determine the molecular requirements for this process. CD8 T cells are activated in vitro under conditions that induce IFN-γ producing effector T cells (Tc1 cells), labeled with fluorochromes (CFSE or PKH26) and intranasally transferred into the airways of infected recipient mice. Subsequently, the ability of Tc1 cells to migrate to the draining mediastinal lymph node passing through the lung parenchyma and entering the afferent lymph can be determined by flow cytometry (FACS). The number of labeled cells that appear in the node can be reliably quantified 20h after transfer, a time point when transferred cells can not be found in non-draining lymph nodes or spleens, confirming that they have not yet reentered the circulation. Using this system with CCR7-deficient (CCR7-/-) and WT cells from sex and age-matched control animals, this method can be applied to competitive migration studies that address the role of CCR7 in Tc1 exit from the lung at various time points after influenza A virus infection. Expression of exit receptors such as CCR7 likely affects the retention of T cells within the lung and the immune response in consequence. Future experiments are designed to test the impact of effector T cell egress from the lung on virus clearance during influenza. In order to address this question I modified an established influenza model in mice (Cerwenka et al. 1999). Briefly, mice lethally infected with A/PR/8/34 receive CL-4 Tc1 cells within one hour of infection. Mice that received Tc1 cells by intranasal application (i.n.) can recover from the infection in contrast to animals that not receive cells. Future experiments will test the consequences of dysregulated versus lack of CCR7 expression on transferred Tc1 cells for virus clearance and rescue. Experiments will be analyzed by monitoring survival and body weight, lung pathology via histology and by virus titer detection. To analyze progression of CCR7 expression on virus specific effector T cells throughout influenza virus infection I will examine CCR7 expressionon T cell receptor transgenic CD8 T cells at different time points after infection with the influenza virus strain A/PR/8/34. I employ CL-4 mice, which express a transgenic (tg) T cell receptor for a hemagglutinin peptide from A/PR/8/34. Total CL-4 lymphocytes are transferred into Balb/c recipients 2 days prior to sublethal infection with A/PR/8/34. FACS Staining for CCR7 on CD8 T cells recovered from BAL, lung, dLN, non-draining popliteal lymph nodes and blood on day 5 and 8 post infection (p.i.) shows the percentage of CCR7+ CL-4 CD8 T cells at these sites in comparison to polyclonal endogenous CD8 T cells. In addition, I will examine how Ag-recognition alters CCR7 expression by direct transfer of CD8 T cells into the airways of mice infected with different strains of influenza A virus.