Vaccines are one of the most cost effective and extraordinarily successful medical interventions. Most of those vaccines depend on CD4 T+ cells and their help to B cells. Our understanding of in vivo, specific human CD4+ T cell responses to pathogens remains hazy, due to the complexity of the biology, the rarity of the cells, relatively inaccessible tissue localization, and technical challenges of identifying specific CD4+ T cells. Therefore, our approach to this serious problem has been to develop multiple new techniques to study human CD4+ T cells over the past several years. While CD4+ T cell-dependent antibody responses have been the source of protection for most licensed vaccines (Project 1), there is a very good argument to be made that many of the diseases for which we do not have successful vaccines require adaptive immune responses beyond antibodies for protection (Projects 2 and 3). The three Projects proposed here vigorously pursue an understanding of the mechanisms regulating human anti-pathogen CD4+ T cells, linked by new experimental approaches.
Our U19 Center consists of three Cores and three Projects:
CROTTY, SHANE P, Core Lead
The purpose of Core A (Administrative Core) is to provide administrative, fiscal, and information technology (IT) support to all projects within the CCHI. The Core will be based at LJI, the CCHI’s home institution and will be utilized equally by all projects. The Administrative Core provides a central focus for the program. The Administrative Core of the CCHI will facilitate interactions between the projects and with the National Institutes of Health. The goal of this core is to provide programmatic direction and promote interaction between the investigators. It will be the responsibility of the Administrative Core to assure access to core facilities by all Projects funded by this application.
CLINICAL STUDIES AND LN FNA CORE
SETTE, ALESSANDRO, Core Lead
The success of the entire program is contingent upon successful accrual of human samples. We are confident in our ability to amass the necessary samples that will enable the most robust and compelling studies. The Clinical Studies and LN FNA Core will provide a centralized resource for accrual of human lymph node (LN), lung, peripheral blood mononuclear cells (PBMC) and plasma samples, and ensure the quality and uniformity of handling of the samples received. These functions are crucial to provide a strong foundation for the studies described in the three proposed projects. The Clinical Studies and LN FNA Core will work closely with the individual Projects, the Administrative Core, the Sequencing and Epigenetics Core, and the clinical subcontractors to enable success of all objectives in this program proposal. This Core will be responsible for the preparation and maintenance of IRB protocols, oversight of subject recruitment, quality control of coded clinical information and donor samples. The cohorts to be used for this program include samples from well- regarded and successful collaborators and clinicians. We will work closely with Drs. F. Hasteh (UCSD) and S. Yekezare (UCSD) and the Altman Clinical and Translational Research Institute (ACTRI) at UCSD to enable acquisition of lymph node fine needle aspirate (LN FNA) samples for Projects 1, 2 and 3. We have a long- standing collaboration with Dr. M.T. Brigger (Rady Children’s, UCSD) to obtain tonsil samples to be used for Projects 1 and 3. We also will collaborate with Dr. Ottensmeier (Southampton, UK) to obtain lung and lung- draining lymph node tissue samples for Projects 1 and 3. LJI’s in-house Clinical Core will manage the banked samples from vaccinated individuals for Project 2 and will provide all new samples from pertussis- and yellow fever virus-vaccinated donors needed for Projects 1, 2 and 3.
SEQUENCING AND EPIGENETICS CORE
SEUMOIS, GREGORY, Core Lead
The proposed projects in this CCHI program will use genetic approaches to define comprehensive molecular properties of specific immune cells in different aspects of immune responses in different diseases and after vaccination from blood and human clinical tissues (lymph nodes and lung tissue). Facing the inherent challenge, of cell abundance and heterogeneity when working with human samples, the CCHI Sequencing and Epigenetics Core, unlike any other external facility, will provide a unique and comprehensive set of procedures especially validated for isolating and processing small numbers of immune cell populations and support downstream single- cell and bulk transcriptomic as well as epigenetic studies. First, the centralized team of the Sequencing Core will provide assistance to all CCHI project users for careful collection of small number of immune cells, adequate processing and storage of samples for all transcriptomic and epigenetic assays listed in the proposal. Second, the Sequencing Core will perform all micro-scaled bulk RNA-seq as well as single-cell RNA sequencing and will provide support for the whole transcriptome analysis, TCR and BCR highly variable sequence extraction from bulk and single-cell RNA-seq datasets. Finally, the Sequencing Core will perform micro-scaled library preparation for epigenetic assays (ATAC- and ChIP-Seq).
T FOLLICULAR HELPER (TFH) CD4+ T CELL, GERMINAL CENTER, AND ANTIBODY RESPONSE DYSFUNCTION IN HUMAN RECURRENT TONSILLITIS
CROTTY, SHANE P, Project Lead
T follicular helper CD4 T cells (Tfh cells) are required for germinal centers, and thus the majority of high affinity antibody responses. Tfh cells have important roles in protection from infectious diseases. Much remains to be learned about Tfh cells. We have developed a novel method for quantifying pathogen-specific Tfh cells, the activation immune marker (AIM) assay. The goal of this proposal is to ascertain the immunologic mechanisms tied to recurrent strep throat, and the GAS virulence factors that cause recurrent tonsillitis disease. It has remained a longstanding mystery why some children are susceptible to recurrent tonsillitis. Tonsillectomies are among the most common pediatric surgery in America, with recurrent tonsillitis associated with S. pyogenes (group A streptococcus, GAS) being a primary cause. We have preliminary data that there is an immunological basis for recurrent streptococcal tonsillitis. Antibody responses are a central part of the immune system. Completion of this study could help identify children who are likely to be susceptible to recurrent strep throat, and mechanistic studies proposed herein could allow for rational design of countermeasures to prevent the disease in the future.
MECHANISMS OF DIFFERENTIAL RESPONSES TO WHOLE CELL AND ACELLULAR PERTUSSIS VACCINATION
SETTE, ALESSANDRO, Project Lead
In the mid-1990s, vaccine-related side effects prompted the replacement of the whole Pertussis (wP) vaccine by a new acellular Pertussis vaccine (aP). Unexpectedly, whooping cough cases have recently increased, particularly in teenagers. In the preliminary data, we compared individuals born before 1995 and vaccinated in infancy with wP, with individuals born in 1996 or later and vaccinated with aP in infancy. Activation Induced Marker (AIM) assays allowed examination of responses directly ex vivo, highlighting a Th2 vs Th1/Th17 polarization of pertussis (PT)-specific CD4+ T cells. Remarkably, we detected differences in response to a contemporary aP booster, even though the first aP or wP priming occurred more than 18 years previously. aP priming was associated with 1) increased IL-9 and TGF-β and decreased IL-17 production in response to PT epitopes ex vivo, and 2) defective ex vivo capacity to expand memory cells one to two months following a booster aP immunization and in vitro proliferation in response to PT epitopes. However, the biological significance of these observations is limited by the fact that only responses in blood are measured and whether the same phenotypic differences are observed in situ, in the lymphatic tissues where memory T cell reside and immune responses are orchestrated. Accordingly, we propose to define the molecular mechanisms underlying the differential IL-9/IL-17 polarization of donors originally primed with aP vs wP (Aim 1). We will define the cellular characteristics of IL-9+ PT-specific CD4+ T cells after aP vaccination and molecular mechanisms underlying their development. We will study the IL-17 signature seen in wP vaccine-primed individuals, by targeted flow cytometry and AIM studies, and perform single-cell RNA-seq with the PT-specific CD4+ T cells. In Aim 2, the mechanisms involved in differential proliferation will be addressed by blocking in vitro TGF-b related pathways, and downregulating the ANAPC2 and WDR1 genes, which were identified in the preliminary data. The cellular basis of the differential proliferation of donors originally primed with aP vs wP will be investigated by cell depletion experiments. Aim 3 will use Lymph Node (LN) Fine Needle Aspirates (FNA) to interrogate PT responses in individuals originally primed with either wP or aP to assess differential germinal center (GC) Tfh cell and GC B cell responses in LNs of vaccine recipients, define whether differential antibody isotype responses of wP- vs aP-primed individuals is due to biases in GC Tfh helper functions to B cells and determine if circulating CD4+ T cells correlate with the vaccine immune response in the draining LN, the primary immune response site. In conclusion, we believe that results from this study will illuminate molecular reasons for lesser aP efficacy and illustrate human CD4+ T cell biological principles generally applicable to vaccinology and immunology.
BIOLOGY OF HUMAN CYTOTOXIC CD4 T CELLS (CD4-CTLS) IN VIRAL INFECTIONS
VIJAYANAND, PANDURANGAN, Project Lead
The existence of MHC class II-restricted CD4+ helper T cells with cytotoxic potential (CD4-CTLs) has been reported in humans with several viral infections. Importantly, the CD4-CTLs have been reported to play a protective role in several of these viral infections. However, little is known in humans about the biology of CD4- CTL generation and their functional properties. We recently performed single-cell RNA-seq in over 9000 cells to unravel CD4-CTL heterogeneity and functional properties. Our analysis led to the discovery of a distinct subset of long-lived CD4-CTL memory precursors. Understanding the biology of such long-lived CD4-CTL precursors will pave the way for developing strategies to boost durable CD4-CTL immune responses following vaccination against infections. In Project 3, specific aim 1, we will determine the phenotype and functional properties of pathogen-specific and tissue-specific CD4-CTLs in humans. (A) Pathogen-specific features of CD4-CTLs: We will compare the phenotype and molecular features of circulating DENV-, hCMV-, EBV-reactive CD4-CTL precursors and effector cells. (B) Tissue-specific features of CD4-CTLs: Here, we will compare hCMV-, EBV- and pertussis vaccine-reactive CD4-CTLs present in human lungs to those circulating in the blood. In addition, we will compare pathogen-specific CD4-CTLs present in tonsil (lymphoid) tissue vs. blood (collaboration with Crotty, Project 1). (C) Vaccine-induced CD4-CTLs: In collaboration with Crotty (Project 1), we will determine whether yellow fever vaccination induces the generation of CD4-CTL precursors in the blood and lymph nodes. In specific aim 2, (A) we will identify molecular transcription factors driving CD4-CTL differentiation and (B) test their function in in vivo models and in human cells. In summary, our work will fundamentally advance our understanding of the molecular basis of CD4-CTL immunity in humans and will benefit from the synergistic interactions with other Projects.