Michael R. Betts - US grants

DESCRIPTION (provided by applicant): Human CD8+ T cells play an important role in controlling HIV and SIV disease progression, but the precise mechanisms involved in control remain unclear. CD8+ T cells are capable of multiple effector functions, including cytolysis and production of various cytokines and chemokines. It is now possible, through polychromatic flow cytometry, to measure at least five CD8+ T cell functions simultaneously. The functional profile of HIV-specific CD8+ T cells using this technique is highly complex, and variable between different HIV antigens and disease progression states. We hypothesize that the functional nature of the HIV-specific CD8+ T cell response can vary between individual HIV epitopes, and therefore place differential selective pressure upon HIV epitopes to influence escape mutation. The ability of a particular CD8+ T cell functional profile to induce escape would indicate that it has an adverse effect on viral replication. Specific Aim #1 will determine if, within an HIV-infected individual, the functional nature of the CD8+ T cell response to distinct HIV epitopes is variable. We will perform epitope-mapping studies employing polychromatic flow cytometry to identify CD8+ T cell functional profiles to multiple HIV epitopes, and whether differential thresholds exist for multiple CD8+ T cell functions. Finally, we will examine CMV, EBV, and influenza virus-specific CD8+ T cell functionality to identify differential functional ability within and between these viruses and HIV. In Specific Aim 2, we will determine if particular CD8+ T cell functional profiles place differential selective pressure upon HIV epitopes. We will examine epitope escape mutations for recognition by subjects with appropriate epitope-specific CD8+ T cells for altered functionality in response to escape epitopes. The long term goal of this project is to determine if escape mutation occurs as a result of particular CD8+ T cell functional patterns. Defining the role of different CD8+ T cell functional patterns at the epitope level will lead to insights into disease pathogenesis and immune correlates of protection for HIV vaccine modalities. (Lay language description): The human immune system is able to control HIV disease progression, but the underlying mechanisms are unclear. This proposal seeks to determine what aspects of the HIV-specific immune response are important in controlling HIV. Our findings will further our understanding of HIV disease, and assist in the development of an HIV vaccine.

A critical issue in the development of an effective HIV vaccine is the need to understand the immune correlates of protection in HIV and SIV infection and to develop and understand vaccine approaches that drive effective mucosal immune responses. Without such knowledge, it is difficult to ascertain the potential effectiveness of a vaccine. Indeed, despite promising immunogenicity, few viable vaccine candidates designed to stimulate T cell responses have proven particularly effective in controlling pathogenic SIV challenge, which may in part be due to the lack of vaccine-induced mucosal responses. Additionally, a major challenge has been the difficulty in identifying the qualitative aspects of T cell functionality most correlated with protection. Recent work in our laboratory using polychromatic flow cytometry that can simultaneously assess multiple T cell functions (degranulation, IFNg, TNFa, IL2,and MIP1b) has revealed dramatic differences in the functional profile of the T cell response to HIV in progressive and non-progressive HIV disease. Through these studies, we have found that the functional profile of the HIV-specific CD8+ T cell response itself represents a correlate of immune protection. In this program we combine our knowledge-based understanding of T cell functional characterization with novel vaccines that induce mucosal immune responses. Here, we will extend our techniques into murine and primate HIV DNAvaccine models to better understand the nature of the immune responses these vaccine candidates elicit. Our preliminary data indicates that through systemic administration of DNA vaccines and chemokine adjuvants, we can expand the mucosal component of the vaccine-induced response. Thus, we will therefore be able to define the functional, phenotypic, and protective qualities of a systemic and mucosal vaccine-induced immune response. Ultimately, such knowledge will be pivotal in the design and implementation of effective HIV vaccines designed to stimulate protective mucosal immune responses. There are three hypotheses: (1) Systemic immunization will elicit T cell responses with distinct qualitative profiles at mucosal and systemic compartments, based on multiple functions assessed simultaneously. (2) The mucosal chemokines TECK, MEC, and CTACK can be utilized as adjuvants to manipulate the functional quality and phenotype of systemic and mucosal DNA vaccine-induced T cells to enhance vaccine efficacy. (3) Chemokine adjuvants can influence mucosal trafficking of DNA vaccine-induced T cells through recruitment of new responses or alteration of the functional profiles of cells responding to the DNA vaccine itself.

[unreadable] DESCRIPTION (provided by applicant): Considerable evidence indicates that CD8+ cells play a critical role in controlling HIV/SIV disease progression, but neither the quantity nor breadth of the HIV-specific CD8+ T cell response as measured using standard approaches (MHC class I tetramer and single function analysis) conclusively correlate with control of replication or disease progression. Recently, using a novel polyfunctional analytical approach, we have identified patterns of CD8+ T cell function that appear to provide discriminate HIV disease progression status. HIV-infected subjects during chronic uncontrolled infection possess functionally limited HIV-specific CD8+ T cell responses, whereas long-term nonprogressors maintain polyfunctional responses. We believe that these polyfunctional patterns may enable us to identify correlates of natural immune protection and, furthermore, that they may have implications for vaccine development strategies. It is pertinent to define HIV-specific CD8+ T cell responses during acute infection, as the events during this period define future disease progression. Furthermore, an effective HIV vaccine will need to stimulate responses protective during this particular phase of infection. Our goals are to define the polyfunctional and phenotypic characteristics of HIV-specific CD8+ T cells that arise during primary infection, determine how they are modulated, and define their relationship to viral control and disease course. We will also determine if these responses can be altered by vaccination, using as a model a DNA/Adenovirus approach and exploiting the unique resource of a small number of vaccinated individuals who became HIV infected. Together, these studies will provide critical information regarding immune correlates of protection against which HIV vaccine candidates can be measured for potential protective effects. Lay Description: Development of a vaccine against HIV requires the understanding of the natural immune responses against HIV after infection occurs. In this proposal, we will determine how the immune system fights HIV during early infection and apply this knowledge to the development and testing of HIV vaccines. [unreadable] [unreadable] [unreadable]

Gene therapy holds great promise for the treatment of various genetic diseases. However, successes in expressing transgenes in vitro are often not reproduced when transferred in vivo. It became clear in several studies that immune responses to the vector and/or transgene product heavily influence in vivo gene therapy oerformance. The Immunology Core has focused its efforts on studying the natural existing T and B cell mmunity to Adenovirus (AdV) and Adeno-associated virus (AAV) in human and non human primates (NHPs). We have also .centered our studies on vector and transgene specific T and B cell responses after systemic or local administration of various rAAV and rAdV serotypes carrying different transgenes in NHP models. This will help us to determine if rAAV and rAdV administration can re-activate existing or induce new T cell responses to the vector or the transgene product, and whether these have any effect on the outcome of the gene therapy and the health of the patient. Moreover we studied the activation of hCFTR-specific T cells in cystic fibrosis mice following gene transfer. The mission of the Immunology Core is to assist Center Participants in developing and conducting assays to evaluate cell mediated and humoral immune responses after gene therapy treatment in small and large animal models including mice, dogs, non human primates and humans. In all these studies we isolated lymphocytes from blood, liver, intestine, primary and secondary lymphoid organs and characterized the T cell response by ex vivo and cultured IFNy ELISPOT, lymphoproliferation assay, in vivo CTL assay, and polychromatic flow cytometric analysis (5-11 colors) of antigen specific T cells. B cell responses were characterized measuring total and neutralizing antibodies in serum and mucosal secretions.

DESCRIPTION (provided by applicant): In this pilot proposal, we will address the hypothesis that CD200 and CD200R are dysregulated in HIV infected individuals. CD200 and CD200R form a critical pathway by which monocyte, macrophage, and dendritic cell function can be regulated by T cells. Triggering of this pathway has been shown to directly suppress monocyte and macrophage activation, which in turn reduces T cell activation. In Aim 1 of this proposal we will perform the initial studies to characterize the expression of CD200 on T cells and its relatio to chronic immune activation in HIV infected individuals from various stages of infection. In Aim 2 we will examine expression of the receptor for CD200, CD200R, on cells of the myeloid lineage in HIV infected individuals. We will also examine the relationship between CD200R expression on myeloid lineage cells and chronic activation markers on T cells, as well as lipopolysaccharide levels in the plasma. Altered regulation of either CD200 or CD200R (or both) in HIV infected individuals could directly influence chronic immune activation, so it is important to assess each part of the pathway. If successful, these results will pave the way to future studies designed to directly study the mechanistic effects of the CD200/CD200R pathway on chronic immune activation, as well as a means to either abrogate or promote CD200R signaling as a therapeutic strategy. PUBLIC HEALTH RELEVANCE: Immune activation is a primary driver of HIV disease progression. This study will examine one potential pathway that controls activation of monocytes, macrophages, and dendritic cells, the CD200-CD200R pathway, in the context of HIV infection. We will determine whether there are defects in the expression of CD200 and CD200R that could lead to heightened immune activation in HIV disease.

DESCRIPTION (provided by applicant): Recently we demonstrated that T-bet expression in human CD8+ T cells is directly correlated to CD8+ T cell effector function. In chronic HIV infection, low expression of T-bet within HIV-specific CD8+ T cells is associated with poor effector CD8+ T cell responses and a failure to control HIV viremia. Furthermore, our preliminary data demonstrates that despite a massive (40-75% of T cells) acute effector CD8+ T cell response during acute HIV infection, these responses cannot be maintained and ultimately result in failure to durably control HIV viremia perhaps due dysregulation of T-bet. HIV specific CD4+ T cells during acute HIV infection also upregulate T-bet (as is required for Th1 function), but lose this expression within weeks of infection. This loss likely has severe ramifications on the proper programming, maturation, and maintenance of HIV-specific CD8+ T cell effector function that is critical for long-term control of HIV viral load. Here, we will address these concepts in the rhesus macaque model in order to address two hypotheses: 1) we hypothesize that high T- bet expression by SIV/HIV-1 specific CD8+ T cells enables functionally protective responses against viral replication and subsequent disease progression, and that the failure to express adequate T-bet leads to loss of effector function and functional exhaustion, and 2) that rapid loss of T-bet mediated CD4+ T cell Th1 responses will fundamentally alter subsequent T cell help necessary for proper HIV-1-specific CD8+ T cell development and maintenance of effector and memory function. In Aim 1, we will determine the kinetics of T-bet expression within SIV-specific CD4+ and CD8+ T cells during acute SIV infection, and the relationship between these functional properties and the propensity to induce immune escape. Animals will be serially necropsied during acute SIV infection in order to assess these factors within multiple tissue compartments. In Aim 2, we will then explore the role of CD4+ T cell help in promoting optimal T-bet expressing CD8+ T cells. We will approach this in two ways~ first we will preserve CD4+ T cell function through early antiretroviral therapy, and second we will deplete CD4+ T cells prior to SIV challenge. In both approaches we will monitor the resultant effects upon the CD8+ T cell compartment with a particular focus on T-bet expression. We will also characterize the propensity to induce immune escape in these situations, and more broadly assess CD8+ T cell function using microarray analysis. Finally, in Aim 3, we will conduct an SIV challenge study to define the role of T-bet expression in SIV-specific CD4+ T and CD8+ T cells in predicting elite control or disease progression in the Mamu B*08/B*17 model of HIV elite control. Together, these complementary Aims will define the importance of T-bet in protective CD4+ and CD8+ T cell responses in HIV/SIV infection, and provide key information on protective T cell functional programming for HIV vaccine development.

DESCRIPTION (provided by applicant): In this R01 renewal application, we propose to continue our work examining the role of T cell function in the control of acute and chronic HIV infection, for the purpose of developing meaningful correlates of protection in HIV infection that can be applied to HIV vaccine development. Here we will focus on the regulation of CD8+ T effector cell and CD4+ T helper type-1 cell function,by the transcription factors T-bet and eomesodermin (eomes), in order to understand the failings of the HIV-specific T cell immune response during acute and chronic HIV infection. We will further study the ability of different vaccine and adjuvant strategies to stimulate the expression of these key transcription factors. Previously we have shown that in chronic HIV infection, low expression of T-bet within HIV-specific CD8+ T cells is associated with poor effector CD8+ T cell responses and a failure to control HIV viremia. Our preliminary data demonstrates that although a massive (40-75% of T cells) acute effector CD8+ T cell response is raised during acute HIV infection, these responses cannot be maintained, perhaps due to their failure to upregulate and properly localize T-bet or eomes within responding cells, ultimately resulting in failure to control viremia. During this sam period, T-bet expressing HIV-specific CD4+ T cells are lost within weeks of infection, suggesting a rapid loss of T helper type 1 responses critical for the generation and maintenance of effective CD8+ T cell responses and antibody responses. Importantly however, T-bet expression is maintained in both HIV-specific CD4+ and CD8+ T cells in HIV elite controllers, suggesting that these responses play a key role in effective HIV-specific immune responses. Based upon these premises, we hypothesize a successful HIV vaccine will require stimulation of T-bet/eomes dependent HIV-specific CD8 effector and CD4 Th1 responses in order to mediate clearance or control of initial viremia. In this application we will therefore focus on defining effector CD8+ ad CD4 Th1 T cell responses in the control of HIV viremia, the transcriptional regulation of these responses, and whether these types of responses can be induced by different vaccine/adjuvant strategies. In Aim 1 we will define the relationship between CD8+ T cell effector function and T-bet/eomes expression and localization in HIV acute, chronic, and nonprogressive infection and how they relate to long-term control of viremia. In Aim 2, we will define the induction and maintenance of T-bet/eomes expressing CD4+ Th1 T cells in acute, chronic, and nonprogressive infection, and how these directly relate to the quality of the HIV-specific CD8+ T cell response and maintenance of CD8+ T cell effector function. Finally, in Aim 3, we will define the potential for T-bet and eomes to be modulated within human and rhesus macaque CD4+ and CD8+ T cells by current HIV vaccine and adjuvant strategies for application to both prophylactic and therapeutic (cure-based) HIV vaccine development.

DESCRIPTION (provided by applicant): According to the CDC, there are over 20,000 deaths and 200,000 hospitalizations due to influenza each year, 90% of which were in people over age 65. The administration of the seasonal trivalent inactivated influenza vaccine remains the primary public health measure recommended by the CDC; however, this vaccine has significantly reduced efficacy, often below 50%, in this older population that needs protection most. Thus, a better understanding of immunologic and vaccination failure is urgently needed to develop better strategies to reduce influenza-related morbidity and mortality in older adults. At the tissue level, the underlying mechanisms of vaccine failure--immunologic non-responsiveness to influenza vaccination--in older adults remain to be defined. Most key immunologic responses to vaccination occur within regional draining lymph nodes (LN), not the peripheral blood. In light of this, the central novelty and innovation of this proposal is to defin the underlying B and T cell properties within the regional draining LN that can account for immunologic non- responsiveness and vaccination failure in older adults. Here we propose direct examination of influenza vaccine-induced CD4+ T and B cells within proximal LN biopsies obtained after vaccination. Recent studies in mice have identified T follicular helper (Tfh) cells key role in the development of the germinal center in the LN after vaccination to provide optimal stimulation of B lymphocytes for the development of long lasting humoral immunity with high affinity antibodies. Our preliminary data suggest that Tfh cells in blood decline with aging, but more-so for the subset of older adults who fail to respond to influenza vaccine. As such, we hypothesize that defects in T follicular helper cell (Tfh) and T helper type 1 (Th1) transcription programming, levels, phenotype, and function result in reduced antibody quantity and quality after influenza vaccination in older adults, clinically translating to non- responsiveness and vaccine failure. Moreover, we hypothesize that these defects will be most pronounced in older vaccine non-responders and contribute to the immunosenescent phenotype observed in older persons. We will address these hypotheses in the following Specific Aims: Aim 1: To determine if baseline differences in total and flu-specific CD4+ Th1/Tfh subsets and B cells in LN and blood prior to vaccination predict old influenza vaccine failures/non-responders (NR) and old vaccine-responders (R). Aim 2: To determine the specific defects in the induction of CD4+ Th1/Tfh cell and B cell responses in regional LN and blood acutely after influenza vaccination that result in the old influenza vaccine NR and R phenotypes.

? DESCRIPTION (provided by applicant: Lymphoid tissue is the key site for the initial seeding, dissemination, and long-term maintenance of the HIV reservoir. As such, understanding those T cell properties and mechanisms required for control and elimination of HIV directly within lymphoid tissue are of critical importance to the HIV cure, eradication, and vaccine agenda. The central question underlying this proposal is whether HIV-specific CD8+ and CD4+ T cells in the lymph node (LN) have or can acquire the functional capabilities necessary to control or eliminate HIV infected cells. These studies are predicated on the concept that cytotoxic CD8+ (and CD4+) T cells are normally absent from the lymph node in HIV- subjects or those without underlying inflammatory conditions. Intuitively, this makes biological sense: why would cytotoxic T cells be wanted in a LN? Cytotoxic T cells (either CD8+ or CD4+) in the LN would function in part to eliminate antigen presenting cells, thereby dampening immune responses. However, in HIV infection, HIV vaccines, and especially HIV cure-based strategies, cytotoxic HIV- specific CD8+ T cells within the LN would be needed to control or eliminate virally infected CD4+ T cells. Our preliminary data show the presence of dysregulated perforin+ HIV-specific CD8+ T cells in the LN from chronically infected subjects, with very low expression of CXCR5, a marker required for entry into the lymphoid follicle, where HIV-infected CD4+ T cells are concentrated. Unexpectedly, antiretroviral therapy appears to reverse this, with heightened CXCR5 expression on LN CD8+ T cells, but lower levels of perforin. This is opposite of what one would want as the kill component of an HIV shock and kill cure strategy. Our findings overall indicate that LN CD8+ T cells have fundamentally different functional abilities compared to the conventional wisdom driving the HIV cure and vaccine field. These considerations suggest that were an effective anti-HIV cytolytic T cell response present, it would not target the LN HIV reservoir in the majority of HIV-infected people. To address these issues, we will examine LN T cell responses against HIV in subjects with progressive HIV infection, ART-treated HIV infection, and elite control of HIV infection. Our studies will provide critical information for therapeutic manipulation, engineering, or vaccine-mediated strategies designed to induce HIV-specific CD8+ T cells capable of homing to appropriate regions of the LN in order to eliminate HIV reservoirs. In Aim 1 we will define the effect of antiretroviral therapy on LN total and HIV-specifc CD8+ and CD4+ T cells compared to HIV infected chronic progressors and elite controllers. In Aim 2, we will determine whether LN- and PBMC-derived CD8+ and CD4+ T cells from ART-treated HIV-infected individuals have effective cytolytic activity. Finally, in Aim 3, we will determine the effect of ART on LN CD8+ and CD4+ HIV-specific T cell transcriptional regulation, activation, cytotoxic properties, and LN retention/egress markers after activation.

Penn Human Pancreas Procurement and Analysis Program Abstract Utilizing our existing infrastructure and scientific collaborations, we have assembled 6 cores with expertise ranging from pancreas procurement and islet isolation to data integration for a comprehensive and integrated Human Pancreas Procurement and Analysis Program based at the University of Pennsylvania. Core A will procure a spectrum of human pancreata and detailed donor medical history; perform high resolution HLA typing by next generation sequencing; isolate islets; and distribute islets and tissues to the other Cores for further analysis or processing. Core B will perform physiological phenotyping on the isolated islets. Core C will quantify and characterize memory T cell subsets by flow cytometry and single cell qPCR analysis; characterize suppressive activity of Tregs and the ability of related effector cells to be suppressed; B cell phenotyping; and generate chromatin accessibility maps of enhancers in pathogenic cell types. Core D will perform multiple advanced modalities for the molecular profiling of isolated islets including RNAseq and microRNAseq of sorted islet cell populations; mass cytometry for single cell quantification of more than 20 cell surface and intracellular markers; and single cell RNAseq. Core E will process tissues using multiple modalities that will allow for analysis using advanced technologies such as multiplexed immunoflourescent staining, combinatorial barcoded FISH (combFISH), whole slide imaging, and quantitative image analysis of protein markers and immune cell infiltrates. This Core will adapt 2-dimensional mass cytometry to pancreatic sections utilizing multiplexed ion beam imaging (MIBI) technology. This Core will also archive tissues as well as DNA and blood, and facilitate sample distribution to HPPAP approved researchers. Finally, Core F will assemble, annotate and maintain an open access database for the Program and its member-researchers, and collaborate with the HIRN in the sharing of data from both programs. The entire Program will be executed by an Administrative Core consisting of the PIs, with assistance from an Executive Committee consisting of the core leaders. The Administrative Core will interface with an external committee to review applications for HPPAP biosample use, and will collaborate with the HIRN. The Program will also interact with the HPPAP member/PANC DB user community to provide a richly annotated source of physiologic, genomic and immunologic data on the tissue-based landscape governing T1D.

Scientific and administrative leadership and effective program project management of ?Immunologic strategies to modulate SIV rebound from ART interruption? is the goal of Core A. The core will oversee scientific progress relative to project aims and milestones and lead scientific efforts in conjunction with the Executive Committee and Scientific Advisory Board. It will coordinate financial management, research program management, regulatory compliance, sample shipments, and reporting functions. In addition to coordinating these activities, the Administrative Core will be responsible for promoting and documenting all aspects of scientific, IACUC and financial regulatory compliance at Penn and collaborating institutions. Staff on this core are located within the Penn CFAR laboratory suites on the 4th and 5th floor of Johnson Pavilion, within the laboratories and administrative offices of Drs. Bar, Betts and Shaw, thus taking advantage of a highly experienced and integrated management team. The University of Pennsylvania provides infrastructure support for administration, information technology, and human resources, and also provides dedicated pre- and post-award units to expedite the work. Penn will provide this support for this Program. A highly functional administrative core will contribute to and facilitate the successful advancement of the proposed science. Specific aims of Core A are to: (i) provide scientific and administrative leadership and program coordination; (ii) ensure timely financial accounting and reporting; (iii) ensure regulatory compliance with all institutional and federal research guidelines; (iv) coordinate timely and appropriate shipments of samples, and (v) develop and implement a communications and data sharing plan.

Abstract. The majority of antiretroviral (ART) treated HIV-infected individuals experience detectable rebound in HIV plasma viremia within weeks following interruption of ART. The kinetics of rebound virus reactivation are a correlate of the size of the archived reservoir and a key measure of reductions therein. Recent mathematical estimates suggest that virus reactivation events occur every 5-8 days following ART removal. Immunologic strategies to prevent or extend the time to virus reactivation and HIV rebound are critically needed to enable durable control or eradication of HIV within infected individuals. Rebounding plasma virus following ART interruption is thought to primarily originate from long-lived resting CD4+ T cell reservoirs, largely PD-1+ T follicular helper cells, within secondary lymphoid tissue (LT) such as lymph nodes, spleen, tonsil, bone marrow, and lymphoid aggregates within gut tissue. This P01 proposal will explore and model two immunologic strategies to prevent, limit, or delay viral rebound by (1) directly targeting rebounding virus through passive nAb therapy in transmitted founder-SHIV infected rhesus macaques (K. Bar/G. Shaw, Project 1) and (2) inhibiting lymphocyte egress from LT to both prevent infected CD4+ T cell redistribution and enable the interaction between reactivated infected CD4+ T cells and protective CD8+ T cells in lymphoid tissue after ART interruption in SIV infected rhesus macaques (M. Betts, Project 2). Importantly, we will employ bar-coded SIV and SHIV viruses in both projects (B. Keele), allowing us to precisely track reactivation rate at a clonal level, and to discern potential tissue and cell types of viral reactivation. These projects will be supported by experts in mathematical modeling (M. Davenport, Analysis and Modeling Core) and nonhuman primate studies (M. Paiardini/G. Silvestri Non-Human Primate Core). Together these studies will provide novel insights into immunological strategies to delay or prevent viral rebound after ART interruption. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page