Janice S. Blum - US grants

Antigens are processed by acidic proteases prior to presentation in the context of class II histocompatibility proteins. The formation of a trimolecular complex of processed antigen, class II proteins, and T-cell receptor is essential for immune recognition and responses. This study will investigate the role of acidic endosomal proteases in antigen processing and invariant chain release from HLA class II proteins. The coordinate regulation of these proteolytic events is critical to antigen presentation, as invariant chain release must occur prior to peptide antigens binding to class II proteins. In humans, the precise enzymes which function in antigen and invariant chain processing have not been identified nor have the factors which regulate protease activity been defined. Immune responsiveness may be linked to protease expression, as differences in antigen processing have been detected among individuals. Proteolytic processing is a prerequisite for the presentation and recognition of both foreign and self antigens. The generation of self peptides which bind specific class II alleles, may be a key event initiating autoimmune reactivity. Thus, protease expression may represent a previously unrecognized genetic factor in susceptibility to autoimmune disorders. The long-term goals of this research program are to define the critical intracellular events which regulate antigen presentation and determine how protease expression controls responses to foreign and self antigens in humans. For these purposes, antigen processing and presentation will be studied in human B-cell lines which display high levels of class II proteins. In B-cells both invariant chain release from class II proteins and antigen processing may occur in endosomes. Specifically, this proposal will: 1) Determine if antigenic peptides are generated by endosomal proteases and how changes in endosomal protease activity modulate antigen presentation; 2) Evaluate if polymorphic class II antigens found in endosomes influence processing and the formation of antigenic determinants; 3) Investigate the requirement for aspartyl proteases, such as cathepsin D in the processing of antigens and invariant chain through the generation of mutant cell lines. A combination of cell biological as well as molecular and cellular immunological approaches will be employed for each of these specific aims.

Macrophages. dendritic cells and B-lymphocytes are sequentially recruited into the islets. serving as potent antigen presenting cells during the development of IDDM. This project will investigate the role of macrophages in the initiation of autoreactivity and islet cell damage. The efficient processing and presentation of autoantigens by macrophages is potentially a critical factor triggering pathogenesis. Engulfment of islet cell antigens by macrophages may lead to the generation of unique peptides which bind class II antigens and stimulate autoreactive T-cells. Studies of autoantigen presentation in humans have focussed on B- lymphocytes, yet little is known concerning the molecular steps which regulate antigen processing and presentation in macrophages. Antigen presenting cells may also regulate the progression of inflammatory responses, thus the influx of dendritic cells and later, B-lymphocytes into the pancreas may facilitate the presentation of new antigenic determinants. In support of this hypothesis, early T-lymphocyte responses are directed against a limited number of peptides derived from islet cell antigens followed by a broad spreading in T-cell reactivity with progression to IDDM. The overall goal of this project is to understand the molecular events which trigger immunological responses to islet cell antigens and the development of IDDM. For this purpose, the processing and presentation of an islet cell antigen, glutamic acid decarboxylase (GAD) will be studied in human macrophages, dendritic cells and B-lymphocytes. The initiation and amplification of autoimmune responses in IDDM, may be dependent upon the recruitment of specialized antigen presenting cells. Specifically, this proposal will test: 1) if macrophages, dendritic cells and B-lymphocytes differentially process the islet cell antigen GMD such that a distinct spectrum of antigenic peptides is produced within each of these APC; 2) if macrophages selectively display a unique series of GAD peptides bound to HLA class II antigens genetically linked with susceptibility to IDDM; 3) if macrophages preferentially activate early T-cell responses in prediabetes, while dendritic cells and B-lymphocytes are responsible for the spread of immunoreactivity with progression to IDDM. Insights into the relevant antigen presenting cells as well as the peptides derived from islet cell antigens, should prove useful in devising novel therapeutic interventions for IDDM.

DESCRIPTION (Provided by the Applicant): MHC class II molecules present peptides derived from self and foreign antigens to CD4+ T-cells. Peptides derived from exogenous antigens and proteins localized in endosomes, are captured by class II dimers as these MHC molecules transit to the cell surface. Remarkably, epitopes from select cytoplasmic antigens also gain access to class II proteins. Cytoplasmic antigens have long been viewed as the primary source of peptides for MHC class molecules. Yet mechanisms appear to have evolved to allow class II proteins to sample select peptides within the cytoplasm, potentiating helper T-cell responses to intracellular pathogens, tumors and the maintenance of self tolerance. The purpose of this application is to elucidate the pathway(s) required for processing and delivery of cytoplasmic antigens to MHC class II proteins. We hypothesized that proteases in the cytoplasm regulate epitope abundance, with a subset of the resulting peptides being directly translocated into membrane organelles for binding to class II molecules. Indeed, neutral proteases within the cytoplasm can modulate epitope production for class II restricted presentation. In aim 1, studies are proposed to further identify these proteases and to define their regulation in APC. Class II-restricted presentation of exogenous antigens is controlled by co-factors such as the Invariant chain, DM and DO. In aim 2, the importance of these accessory molecules in cytoplasmic antigen presentation will be examined. As these co-factors associate with class II proteins in distinct membrane organelles, elucidating their role in cytoplasmic antigen presentation will help dissect the relative importance of the endoplasmic reticulum vs. endosomes in this novel class II pathway. Studies also suggest that translocation of cytoplasmic epitopes into distinct membrane organelles must occur to deliver these ligands to class II molecules. Yet, the essential features guiding cytoplasmic epitope translocation to receptive class II proteins, remain undefined. In aim 3, experiments to elucidate the role of peptide transporters and heat shock proteins in cytoplasmic antigen presentation via MHC class II molecules are therefore proposed. These studies will provide key insights into this novel pathway for class II presentation with implications for vaccine development.

DESCRIPTION (provided by applicant): This is a new application for a 5 year program for pre- and postdoctoral training in Immunology and Infectious Disease. The program's goal is to develop a well-educated, diverse workforce of scientists capable of responding to the nation's health needs. The emergence of new infectious agents, the potential for misuse of pathogenic microbes and their products, coupled with the increasing incidence of disease linked to autoimmunity and allergy, demonstrate the growing need for dedicated, creative scientists with expertise in Immunology and Infectious Disease research. The Immunology and Infectious Disease Program at Indiana University School of Medicine offers students and fellows formal and applied training to meet this need in an interactive, multi-disciplinary medical center environment. The program's fourteen preceptors and resource faculty are drawn from both basic and clinical sciences, offering trainees exposure to advanced molecular and cellular technologies as well as the chance to gain knowledge from patient samples and in vivo models of disease. Strengths of the program include the diversity of research problems addressed by the faculty, and preceptors' track record of relevant, quality publications and external funding. Trainees will benefit from active, collaborative research including NIH-funded program projects and centers, as well as opportunities for co-mentoring. Comprehensive training is offered through didactic courses including classes in bioethics, research seminars, an established journal club, a grant writing workshop, and hands-on faculty mentoring. Support is requested for 3 predoctoral trainees, 3 postdoctoral fellows, and 3 short-term graduate students to study in this program at Indiana University School of Medicine. Trainees will work with experienced faculty from the Departments of Microbiology and Immunology, Biochemistry and Molecular Biology, Medicine (Infectious Disease, Pulmonary) and Pediatrics. Short-term trainees are part of an initiative to transition minority students into pre- and postdoctoral training, and ultimately careers in academic research.

Description: The potential for widespread dissemination of lethal poxviruses such as smallpox during bioterrorism has led to renewed scientific efforts to understand the pathogenesis of this virus and related orthopoxviruses. Poxviruses have evolved many strategies to modulate and evade host immunity, compromising viral recognition and clearance by the host as well as the development of long-lasting immune responses. Vaccinia virus has been widely used as an infectious vaccine agent to induce immunity against smallpox. However, concerns related to the effectiveness of vaccinia-induced immunity, as well as complications associated with vaccinia virus immunization in the current populace, have lead to a call for development of improved poxvirus vaccines. In this resubmitted Program Project application, vaccinia virus will be used as a model for orthopoxvirus infection and to study the induction of host immunity. Immune recognition of viruses is in part, regulated by host antigen presenting cells. The overall goal of the program is to elucidate how poxviruses specifically alter the function of these antigen presenting cells, and thus subvert host immunity. A central hypothesis for the program therefore, is that vaccinia virus regulates the function of host antigen presenting cells to diminish the development of adaptive immunity and enhance viral pathogenesis. To address this hypothesis, the program application links three related projects involving a team of highly interactive investigators using complementary, interdisciplinary approaches. Projects within the program address: the specific mechanisms employed by vaccinia virus to modulate class II antigen presentation (Project 1);interplay between host cell signaling pathways and vaccinia virus leading to disruption of CDId-mediated antigen presentation (Project 2);and the importance of lung dendritic cells in regulating respiratory vaccinia virus infection in the context both normal and atopic lung microenvironments (Project 3). These research projects will be supported by an administrative core, and two research facilities including a virus core and a pulmonary biology core, each designed to facilitate studies of viral pathogenesis and host immunity.

[unreadable] Description: This application will evaluate the role of T cell secreting the pro-inflammatory cytokine IL-17 in virus infections. The IL-17-producing T cells may function to protect from infection or may mediate inflammation and immunopathology. IFN is known to promote the development of Th1/Tc1 cells while it inhibits the formation of IL-17 producing T cells. [unreadable] [unreadable] The vaccinia virus model will be investigated. The hypothesis is that vaccinia virus may promote the generation of IL-17-producing T cells because: (1) vaccinia virus drives the production of cytokines important for the differentiation of IL-17-producing T cells (IL-6, TGFbeta1, TNFalpha); and (2) vaccinia virus encodes immunomodulatory proteins that block IFN action (which inhibits the generation of IL-17-producing T cells). Two specific aims are proposed: [unreadable] [unreadable] (1) Define the transcription factors that contribute to the differentiation of IL-17-producting T cells and the production of specific cytokines by these cells. Combination of cells from mice deficient in signal transducer and activator of transcription (STAT3), STAT4 or T-bet and the treatment in culture with different cytokines will be employed. [unreadable] [unreadable] (2) To investigate the function of IL-17-producing T cells in mice infected with vaccinia virus, characterizing deficient mice with altered development of IL-17-producing T cells, the effect of adoptive transfer of IL-17-producing cells and the neutralization of IL-17 with antiserum. [unreadable] [unreadable] These studies may help the development of improved vaccinia virus vaccines which may regulate the formation of IL-17-producing T cells and reduce the damaging inflammation caused by the smallpox vaccine. These investigations will define the role of IL-17-producing T cells in virus infections. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Peptides derived from foreign and self antigens are presented in the context of MHC class II molecules for recognition by CD4+ T cells. Class II molecules transit the endosomal network of cells, acquiring peptides from exogenous antigens as well as endogenous cell surface and endosomal proteins. Surprisingly, epitopes from cytoplasmic antigens also are presented by class II molecules. The overall goal of this proposal is to understand the mechanisms regulating class II cytoplasmic Ag presentation, and to identify new means to modulate immunity to self and tumor Ags. With subversion of class I presentation by tumors and viruses, exploiting novel pathways for cytoplasmic Ag presentation remains important. Targeting specific cytoplasmic Ags for class II presentation offers a unique approach to promote immune recognition and overcome such immune evasion. The current proposal focuses on a selective pathway, chaperone mediated autophagy (CMA) which facilitates class II cytoplasmic antigen presentation. We propose that cytoplasmic antigens are selectively targeted by cytosolic chaperones for proteolysis during CMA. The resulting peptides may then be chaperoned to a membrane transporter, LAMP-2A for translocation into the endosomal network for class II presentation. Studies are proposed to define the importance of chaperones, HSC70 and HSP90 in guiding cytoplasmic antigens for processing and translocation into organelles for class II presentation. Multiple isoforms of the LAMP-2 gene are expressed in B cells and melanomas, potentially regulating class II cytoplasmic antigen presentation by autophagy. Studies are proposed to elucidate the role of individual LAMP- 2 isoforms in class II presentation pathways. This project will offer critical insights into the molecular events and essential co-factors for CMA which faciliate class II access to cytoplasmic antigens for immune recognition. Notably, autophagy pathways are often up-regulated in tumor cells to promote tumor growth and survivial. Such changes in cellular autophagy pathways may enhance tumor antigen recognition. Studies in this project will examine the importance of distinct autophagy pathways in cytoplasmic antigen presentation by melanomas. This work also has relevance to our understanding of autoimmunity to self antigens and tolerance induction. Exploiting the selectivity of the CMA pathway may also offer opportunities for novel approaches to vaccine development. PUBLIC HEALTH RELEVANCE: This work has relevance to our general understanding of how the immune system distinguishes self and foreign molecules. The project has implications for cancer as autophagy is often up-regulated in tumors, and this could be exploited to promote immune recognition of tumor cells. These studies may also be important in understanding autoimmunty, and lead to new approaches for vaccine development.

DESCRIPTION (provided by applicant): This is a new application for an institutional NRSA sponsored training program to provide hypothesis-driven biomedical research experience to students during the early stages of their medical education at Indiana University School of Medicine (IUSM). Our purpose is to provide a structured research environment that engages medical students' interest in biomedical research, creating opportunities for basic and translational research experience, and education in research ethics. The main objective of the program is to serve as a portal to train and recruit physician-scientists. The proposed program is based on a highly successful mentorship and training program at IUSM, the Student Research Program in Academic Medicine (SRPAM). Medical students with strong academic credentials are selected for SRPAM, which pairs students with highly qualified faculty mentors for 12 week summer research internships with an accompanying lecture series focused on research communication and writing, ethics, and translational investigations. The current application seeks support to enroll 24 students/year in the program to conduct basic and translational research within the basic and clinical departments and centers at IUSM. The program is designed: a) to increase student awareness of the value of doing biomedical research by challenging students to take on independent projects during the internship; and b) to strongly support students interested in a career in academic medicine by providing access to opportunities in the MD/PhD program and assistance with research fellowship applications to HHMI and NIH. Trainees will engage in mentored research experience in areas that reflect the school's strength and international reputation in hematopoiesis, host defense and pulmonary biology, cancer biology, cardiovascular, diabetes and medical informatics. The strengths of our program are: i) a cadre of highly successful mentors including physician-scientists with extramural funding, outstanding training records and solid experience with short-term trainees; ii) an outstanding scientific environment characterized by an intense interdisciplinary spirit and access to cutting-edge technologies and excellent resources; iii) a supportive community and a solid network providing students with opportunities for leadership and mentoring; iv) integration with the medical school curriculum with student credit for level III competency in biomedical knowledge; and v) an unequivocal commitment from the leadership at the Indiana University School of Medicine. The long term goal of the program is to increase the number of physician scientists nationally by exposing students in their early years of medical education to hypothesis-driven research focused on the molecular and cellular basis of disease and the potential for clinical translation.

Project Summary/Abstract This supplement project addresses the need to create safer and more inclusive research training environments for NIH-supported projects through a novel curriculum that includes modules on bias/harassment/discrimination based on gender, sexual orientation, gender identity, ability, citizenship, and race/ethnicity. The curriculum has the following learning objectives: (1) increase trainee skills to become (self-) advocates when navigating unsafe and unwelcoming environments; and (2) improve the institutional training environment by enhancing mentor skills for recognizing and preventing bias, harassment, and discriminatory events. Curriculum modules, presented separately to trainees and faculty/staff, consist initially of a podcast using narrative to describe an unwelcomed event to help trainees and mentors understand the impact of bias and discrimination in the training environment and ways to mitigate it. After listening to the podcast, participants will engage in in-class activities, which include a case study, discussion, and role play. Through these participatory sessions, participants will develop the tools necessary to confidently communicate about bias and discrimination. The curriculum will be evaluated using a mixed-method design to obtain qualitative and quantitative formative and summative evidence that demonstrates how well the proposed strategies and interventions promote safety and inclusion in the training environment. The most effective components will be institutionalized for use throughout the IUPUI community and made available on the IPREP website for external use.

IPREP Project Summary The IUPUI Postbaccalaureate Research Education Program (IPREP) will provide academic enhancement and extensive research experiences to prepare talented college graduates from diverse backgrounds for success in rigorous Ph.D. and M.D./Ph.D. programs in the biomedical and behavioral sciences. Six students will be recruited annually from historically black colleges and universities, predominantly Hispanic serving institutions and tribal colleges as well as from a pool of high performing underrepresented minority students from existing IUPUI programs. IPREP Fellows will begin the pro- gram on June 1st each year with a one-week orientation where their skills and needs will be assessed, and from which a detailed personal development plan will be established. Weeks 2-7 will be two-week rotations in three labs chosen by the Fellows. Every IPREP Fellow will then be paired with an experienced research mentor with active sponsored research, who will incorporate the student as a paid employee into their research enterprise. IPREP Fellows will receive additional ongoing support from the IUPUI Center for Research and Learning that will include individualized advising, assistance in preparing graduate school applications, planning for graduate school visits and interviews, as well as monthly monitoring and reporting of progress to the Program Director. IPREP Fellows will leave the program with enhanced laboratory and research sills, including improved abilities to ask and solve research questions (critical thinking), increased understanding of expectations of graduate programs, awareness of process for seeking external funding for graduate education, improved self-confidence and ability to succeed in a competitive academic environment. At least five of the six IPREP Fellows will be successful in obtaining admission to rigorous Ph.D. and Ph.D./M.D. programs and will successfully complete their graduate/professional programs.

The National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) is a highly competitive, federal fellowship program. GRFP helps ensure the vitality and diversity of the scientific and engineering workforce of the United States. The program recognizes and supports outstanding graduate students who are pursuing research-based master's and doctoral degrees in science, technology, engineering, and mathematics (STEM) and in STEM education. The GRFP provides three years of financial support for the graduate education of individuals who have demonstrated their potential for significant research achievements in STEM and STEM education. This award supports the NSF Graduate Fellows pursuing graduate education at this GRFP institution.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary/Abstract The IUPUI Postbaccalaureate Research Education Program (IPREP) provides academic enhancement and extensive research experiences to prepare talented college graduates from diverse backgrounds for success in rigorous Ph.D. and MD./Ph.D. programs in biomedical and behavioral research. IPREP trainees from underrepresented minority groups at predominantly white institutions may face marginalizing experiences such as discrimination that may lead to both mental health problems and poor academic performance. Research demonstrates that engaging trainees in extracurricular activities, such as physical activity participation, is a best practice. Physical activity (PA) has been shown to improve assertiveness, confidence, memory, attention, depression, anxiety, resiliency, and work errors. Despite PA?s contribution to cognitive and mental health outcomes, nationally representative accelerometry data show that less than 10% of all adults meet the American College of Sports Medicine (ACSM) recommended guidelines of 150 minutes of moderate to vigorous PA per week. A key barrier to PA participation is a lack of understanding of how to participate in PA while navigating obstacles such as cost, time, transportation, and lack of social support. The goal of the proposed project is to provide a guided program in PA education that serves as a life-long tool to promote IPREP trainee well-being, resiliency, and persistence in challenging careers focused on biomedical and behavioral research. Trainees will complete a baseline mental health assessment, exercise self-efficacy scale, and assessment of resilience. Additionally, their cardiorespiratory fitness, muscular strength and endurance, and body composition will be measured. All trainees will be given a Fitbit, and their activity and sleep will be monitored via Fitabase. We will use the ACSM Exercise is Medicine® evidence-based curriculum for self- paced, didactic education over an eight-week period. We will monitor trainee progress on the didactic modules via the Canvas learning management software. After eight weeks, IPREP trainees will be assigned to an Exercise Science student who is experienced in PA programming and health coaching and will serve as the IPREP trainee?s personal trainer. IPREP trainees will select from a variety of exercise programs, sports, or other self-identified physical activities that the personal trainer will support. We will re-evaluate the baseline measures at weeks 8, 16, 32, and 64. The evaluators will collect, analyze, and report data to assist the leadership team in deciding which elements of the intervention should be institutionalized and incorporated into the IPREP and other training programs. Clinically meaningful levels of depressive symptoms (i.e. PHQ score > 10) will be reported to the trainee and referral to their healthcare provider for further evaluation and treatment. Research results will be presented at relevant professional meetings and submitted for peer-reviewed publication as an education research manuscript and a program guide shared nationally for implementation in PREP and graduate training programs.