Projects
Project 1: Innate and Adaptive Immune Responses to Lymph-Borne Infections
Principle Investigator : Ulrich H. von Andrian, M.D., Ph.D.
Abstract:
Viruses and antiviral vaccines that enter the body through the skin or mucous membranes gain access to nearby lymph vessels and are transported to regional draining lymph nodes (LNs). When a virus arrives in a LN it must be prevented from continuing further along the lymph conduits, which would channel the pathogen into the systemic circulation causing viremia with potentially fatal consequences. LNs are believed to retain and neutralize lymph-borne micro-organisms, but the mechanisms are poorly understood. LNs also initiate adaptive immune responses to viral infections and vaccines because they recruit and harbor large numbers of lymphocytes and specialized antigen-presenting cells (APCs), which elicit protective effector responses, especially neutralizing antibodies. Our understanding of how lymph-borne viruses are presented to B cells is still very sketchy. Here, we prepose to use multi-photon intravital microscopy (MP-IVM) for time-and space-resolved visualization of the handling of lymph-borne fluorescent virions in LNs. We hope to obtain mechanistic insights into the cellular and molecular mechanisms by which LNs prevent pathogen dissemination and initiate adaptive humoral immunity. Based on preliminary work, our central hypothesis states that intranodal lymph conduits contain specialized cells that are highly adept at capturing viral particles and at presenting these particles to follicular B cells. This hypothesis will be tested in two specific aims:
Aim 1. will explore the cellular mechanisms that determine the fate of lymph-borne viruses in LNs draining a subcutaneous injection site.
Aim 2. will analyze where and how follicular B cells are exposed and respond to lymph-borne viruses.
Project 2: Visualizing Effects of Costimulation on Antiviral Immunity
Project Leaders: Arlene H. Sharpe, M.D., Ph.D.; E. John Wherry, Ph.D.; Gordon Freeman, Ph.D.
Abstracts:
Anti-microbial T cell responses play a major role in determining the outcome of infection. Many microorganisms cause acute infections and are rapidly cleared, while others progress to chronic or persistent infections. The regulation of T cell responses to infection reflects a delicate balance between effector functions needed to eliminate the microbe and the potential to cause immunopathology. An overaggressive anti-microbial T cell response can cause damage to tissues, especially those with delicate mucosal surfaces. There is a dynamic interplay between the pathogenic microbes and the host defenses aimed at eradicating them. For example, pathways in the B7:CD28 family have critical roles in controlling the balance between the stimulatory and inhibitory signals needed for effective immune responses to microbes and maintenance of self-tolerance. Our studies indicate that one of the newer pathways in the B7:CD28 family, the PD-1: PD-1/PD-L2 pathway, delivers inhibitory signals that play a critical role in regulating the balance between T cell activation and tolerance. We and others have found that PD-1 and its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC) play a central role in the interplay between host defenses and microbial strategies that evolved to resist immune responses. Blockade of this pathway can reinvigorate exhausted T cells during chronic viral infection, as well as enhance anti-viral responses during acute viral infection. A better understanding of the immunoregulatory roles of this pathway is needed to determine how it can be effectively modulated to activate anti-viral T cells, while minimizing the risk of autoimmunity and immunopathology.
Our recent work indicates that the function of PD-L1 extends beyond interactions with PD-1. We have identified PD-L1 and B7-1 as binding partners and demonstrated a functionally significant bidirectional interaction between B7-1 and PD-L1 that inhibits T cell responses. The identification of the PD-L1:B7-1 interaction raises a number of important questions that are key to understanding how interactions among PD-1, PD-L1 and their other binding partners regulate anti-microbial immunity: What is the role of the PD-L1:B7-1 pathway in regulating T cell responses? Do PD-L1:PD-1 interactions and PD-L1:B7-1 interactions have unique or overlapping roles in controlling activation of naïve, effector and memory T cells and limiting immunopathology? What are the functions of PD-L1 and B7-1 on T cells? What are the relative roles of these pathways in controlling anti-microbial T cell vs. B cell responses? The answers to these questions are central to the overall goal of the PPG, in achieving a detailed understanding of immune responses to viruses and transcutaneously applied vaccines, particularly the cellular and molecular interactions that regulate the development of protective immune responses.
The main goal of this project is define the functions of the newly discovered PD-L1:B7-1 pathway and compare the relative roles of PD-L1:PD-1 and PD-L1:B7-1 interactions in controlling virus-specific T cell responses and anti-viral immunity. We will test the hypothesis that both PD-L1:PD-1 and PD-L1:B7-1 interactions lead to significant bidirectional inhibitory interactions that inhibit T cell responses, and that PD-L1 may trigger more profound inhibitory effects than PD-1 because PD-L1 has the potential to trigger 2 inhibitory interactions. We will dissect the roles of PD-L1:PD-1 and PD-L1:B7-1 interactions in regulating naïve, effector and memory CD8 T cell responses to viral antigen and influenza virus infection. We have a number of unique tools including KO mice and blocking antibodies that will enable us to dissect PD-L1:PD-1 and PD-L1:B7-1 interactions. We will use engineered influenza virus expressing the gp33 epitope of LCMV ((PR/8-GP33 and X31-GP33), making possible the use of P14 TCR Tg mice and gp33 tetramers to visualize the effects of PD-L1:PD-1 and PD-L1:B7-1 interactions on virus-specific T cells. The use of a TCR Tg system facilitates studies of early events in T cell activation and differentiation as well as migration and homing that are not possible with an endogenous (e.g. non-TCR Tg) response. However, we will also examine the non-TCR Tg T cell response to nature influenza virus epitopes (e.g. Db/NP366) whenever possible. Insights from these studies may lead to improved prophylactic and/or therapeutic vaccination strategies
Aim 1: To analyze the functional significance of PD-L1:B7-1 and PD-L1:PD-1 interactions in controlling the CD8 T cell activation, differentiation and effector responses to model antigens and influenza virus
We are only beginning to understand functions of the newly identified PD-L1:B7-1 interaction. Because our data indicate that B7-1:PD-L1 interactions exert bidirectional inhibitory effects on T cells, we will focus on the roles of B7-1 and PD-L1 on T cells. Here we will investigate the roles of PD-L1:B7-1 interactions, PD-L1:PD-1 interactions individually and together in controlling the CD8 T activation, differentiation, potential to form memory T cells, and effector functions. We will compare the relative roles of PD-L1:PD-1 and PD-L1:B7-1 pathways in controlling 1) responses of naïve and effector CD8 T cells, 2) the functions of PD-L1, PD-1 and B7-1 on CD8 T cells, and 3) CD8 T cell trafficking to lymph nodes and recruitment to inflamed tissue. We will first use a reductionist approach and activate TCR Tg T cells by transfer of peptide-pulsed DCs in the presence or absence of pro-inflammatory stimuli. We then will infect mice with GP33-influenza virus and track CD8 T cell activation, differentiation and effector responses using P14 TCR Tg and gp33 tetramer.
Aim 2: To analyze the roles of the PD-1:PD-L and PD-L1:B7-1 pathways in controlling protective immunity to influenza virus
We will evaluate how PD-L1:PD-1 and PD-L1:B7-1 interactions control memory T cell responses and protective immunity. We will interrogate the roles of these interactions in controlling central and effector memory T cells, their homing to tissue sites, and their fates. We will prime with PR8 gp33 s.c. (a non-pathogenic route of infection) as described in Aim 1. Two different challenge models will be used. First, virus-infected DC will be given s.c. and the dynamics of the secondary T cell response examined in the draining LN. The advantage of this approach is that the early events in secondary T cell activation, differentiation, recruitment and migration can be interrogated using IVM/2P approaches and the roles of the PD-L1:PD-1 and PD-L1:B7-1 interactions can be examined. We will also use a second challenge model of i.n. influenza virus infection (using the x31/GP33 strain). Since influenza virus replicates efficiently in the respiratory tract, this challenge model allows us to investigate the influence of the PD-L1:PD-1 and PD-L1:B7-1 interactions on protective immunity, viral pathogenesis and immunopathology.
Project 3: Compement Receptors in Humoral Immunity to Influenza
Project Leader: Michael Carroll, Ph.D.
Abstract:
Influenza virus represents a major worldwide health problem. Of the 3 known strains, influenza A is probably the best studied as it infects not only humans but other mammals and can be adapted to animal models. The major antigenic targets-hemagglutinin and neuraminidase- undergo structural changes such that antibodies formed against one strain generally are not protective with a related strain. Because of this “antigenic variation”, neutralizing antibodies which are normally protective are not long lasting and necessitate annual vaccination. In addition, B cell memory responses are generally targeted to a few dominant epitopes. Vaccines that enhance humoral immunity to a broader range of epitopes and especially to the conserved regions of the outer coat proteins are less likely to be affected by antigenic variation. The complement system participates in both the innate and adaptive response to influenza. A major role for complement in humoral immunity is mediated via its receptors CD21 and CD35 that are expressed on both B cells and follicular dendritic cells. Understanding how complement receptors enhance persistent antibody to influenza and the B cell memory response could provide insight into design of novel vaccines that would help overcome susceptibility to antigenic variation of the virus. In order to further our understanding of the role of complement receptors in humoral immunity to influenza and to test the efficacy of complement C3d as a molecular adjuvant two aims are proposed:
Aim 1. Test the hypothesis that complement receptors CD21/CD35 are critical for an effective humoral response to influenza.
Aim 2. Identify the mechanism for uptake and transport of influenza virus into peripheral lymph node follicles.
Project 4: Cellular Dynamics and Viral Dissemination in HIV-Infected Lymph Nodes of Humanized Mice
Project Leaders: Andrew Luster, M.D., Ph.D.; Andrew Tager, M.D.; Thorsten Mempel, M.D., Ph.D.
Abstract:
Much has been learned about the pathogenesis of Human Immunodeficiency Virus (HIV)-1 infection over the past two and one half decades since the disease was recognized. Many of these insights have come from careful correlative studies of HIV infected persons and ex vivo studies of their peripheral blood immune cells. While these studies have been very informative, fundamental questions in HIV pathogenesis, immunity and vaccine design remain unanswered. One major limitation of human studies has been the difficulty in interrogating lymphoid tissues. The lymphoid compartment is particularly important in HIV, as it is the primary site of viral replication and CD4+ T cell depletion as well as the site of immune response generation. To investigate host-pathogen interactions in tissue compartments, investigators have often turned to mouse models. These models have been extremely informative in gaining a deeper understanding of viral pathogenesis and the host immune response at sites of infections. In fact, imaging technology, such as multiphoton intravital microscopy (MP-IVM), has advanced to the point where the immune response, and even viral dissemination itself, can now be visualized at the single cell or single particle level in the living mouse. The power of these new technologies have not been able to be brought to bear on pathogens that are restricted in their host range to humans, such as HIV. For these and other reasons there has been much interest in developing a mouse model of HIV that can be readily used to investigate pathogenesis and test vaccine development. We and others have developed an improved humanized mouse model of HIV infection following the transplantation of human CD34+ stem cells and autologous human thymic grafts into NOD-scid mice (NOD-scid-Hu Thy/Liv/HSC mice), which have been referred to as BLT mice (for Bone marrow, Liver and Thymus transplants). Of specific relevance to the focus of this Program Project Grant, we have achieved robust repopulation of mouse lymph nodes with human immune cells, and generated robust anti-HIV cellular and humoral immune responses in our humanized mice. In this project, we will take advantage of this improved humanized mouse model to study questions regarding the biology of HIV not readily approachable through human studies, such as: What are the pathways by which HIV gains access to the lymphoid compartment? What are the pathways and kinetics of viral dissemination once HIV reaches the lymphoid compartment? What are the mechanisms by which CD8+ effector T cells inhibit viral replication in vivo? In addition, our improved humanized mouse model of HIV infection will give us the ability to investigate the mechanism of the functional impairment of cellular immune responses that has been demonstrated to exist in chronic HIV infection. Similar to humans infected with HIV, CD4+ and CD8+ T cells in our humanized mice dramatically increase their cell surface expression of PD-1 following HIV infection. Activation of PD-1 by its ligands PD-L1 or PD-L2 inhibits immune responses, and recent work has shown that blockade of this pathway in mouse LCMV infection restored function of CD8+ T cells and decreased viral load. These exciting findings raise the possibility that inhibiting the PD-1 pathway could reinvigorate CD8+ and CD4+ T cell function in humans infected with HIV and lead to better immune control of viral replication. Specifically, we propose:
Aim 1. To characterize the trafficking patterns of human T cells and dendritic cells in the lymph nodes of our mouse model of a human immune system (the NOD-scid-Hu Thy/Liv/HSC “BLT” mouse model). Using MP-IVM we will, for the first time, observe and quantitatively analyze the migratory behavior of human immune cells in a humanized mouse. We will investigate DC migration into the lymph node from the periphery and the positioning of DCs within the lymph node. We will also visualize the positioning and migration of human naïve and effector T cells within the resting and reactive lymph node.
Aim 2. To use our NOD-scid-Hu Thy/Liv/HSC “BLT” mouse model of HIV infection and MP-IVM to: (i) determine the mechanisms by which HIV is transported into lymphoid tissue either as free virus or in association with DCs and CD4+ T cells using fluorescently labeled virus; (ii) determine the mechanisms of HIV dissemination within LN over time using an HIV mutant inducing the expression of GFP in infected cells; and (iii) determine the mechanism of CD8+ effector killing of HIV-infected cells in the LN in vivo.
Aim 3. To use our humanized mouse model of HIV infection to: (i) define the pattern of PD-1, PD-L1, PD-L2, and B7-1 expression on bulk and HIV-specific CD8+ and CD4+ T cells and on cells within the LN during the course of HIV infection; (ii) determine the effects of inhibiting the PD-1 pathway in vivo on the control of viral replication; and (iii) to determine the effects of inhibiting the PD-1 pathway in vivo on reinvigorating cellular and humoral immune responses as measured by the magnitude and breath of HIV-specific T and B cell responses and T cell function, including proliferation, cytokine secretion, and cytotoxicity.