Erin J. Adams - US grants

DESCRIPTION (provided by applicant): ?d T cells constitute an important component of the immune response against infectious agents and cancerous transformations, yet the biochemical mechanisms by which they detect antigen through their somatically recombined T cell receptor (TCR) remain unclear. Unlike a[unreadable]TCRs, which are restricted to recognizing antigens in the context of Major Histocompatibility Complex (MHC) molecules, ?d TCRs can recognize a diversity of ligands ranging from self MHC to intact, unprocessed, viral glycoproteins. While the ?d TCR is structurally similar to the a[unreadable]TCR, and both use components of the CD3 signaling complex for signal transduction, it is clear ?d TCRs bind antigen in a distinct and divergent way. The lack of concrete biochemical characterization of these receptors and their interactions with ligands has hindered a true understanding of the role of these receptors in ?d T cell activation. Thus, the long-term goal of this proposal is to understand the biochemical and structural mechanisms of ?d TCR engagement and how this binding event discriminates between healthy and unhealthy tissue to initiate T cell activation. We plan to study these questions using two parallel strategies that are directly complementary, yet unique. The first is a structure/function-based approach combining biochemistry, structural biology and cell-based functional assays to determine the specific molecular details that govern ?d TCR recognition of defined ligands. We will focus our biochemical, biophysical and structural studies on two receptor/ligand interactions: ?d TCRs specific for 1) the murine MHC T107T22 molecules and 2) the human MHC CD1c molecules. The second approach involves using a cross-species sequence-based analysis to understand the selective, evolutionary mechanisms that shape the ?d TCR V, D and J gene repertoire. This approach will define what types of selective pressures (diversifying, neutral or purifying) have governed the evolution of these genes in humans and non-human primates, indicating the nature of the ligands to which they bind. These approaches, together, will help to elucidate the molecular recognition principles of ?d TCR interactions, and determine if ?d TCRs recognize ligands in a convergent manner, such as seen in a[unreadable] TCR/MHCp interactions, or whether there are a diversity of recognition solutions, unique to each ?d T cell population and particular ligand.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Tech R&D Core Support for AIDS Research

DESCRIPTION (provided by applicant): CD1c is a member of the CD1 family of proteins that are similar in structure to Major Histocompatibility Complex class I molecules. CD1 molecules have evolved unique lipid binding pockets (called A' and F') ideally suited for binding diverse lipid antigens via their hydrophobic hydrocarbon tails. CD1c falls into the Group 1 family of CD1 molecules found in humans; homologues are lacking in the mouse. Because we lack a tractable model by which to probe the function of Group 1 CD1 molecules, little is known about the immunological function of CD1c and the ¿¿ T cells that recognize it. Our recent crystal structure of CD1c shed light on this topic, revealing several unique features of this molecule that endows it with the capacity to bind diverse antigens. This structure also showed how CD1c presents a cell-wall lipid of Mycobacterium tuberculosis (MPM), a key potential target for vaccine development, and provides clues as to how a dodecameric N- terminally acylated lipopeptide (lipo12) can also be presented. However, it is still unknown how CD1c presents other lipids, how this complex is recognized by ¿¿ T cells, and what characteristics dictate the ¿¿ T cell repertoire specific for CD1c (and whether this changes with different lipid antigens). In order to address these questions and more fully understand CD1c and its role in presenting diverse lipids to ¿¿ T cells, we will use the following strategies: our first aim, To characterize the biophysical and structural details of CD1c lipid presentation. is focused on understanding the structural features of CD1c presentation and how intrinsic (lipid structure) and extrinsic (pH and chaperones) factors modulate CD1c's lipid repertoire. Our second aim, To define how ¿¿ TCRs recognize CD1c/lipid ligands at the molecular level. is focused on using structural, biophysical and functional approaches to reveal how ¿¿ TCRs recognize CD1c/lipid at the molecular level. This is a key question relevant to globally understanding T cell recognition: How do diverse ¿¿ T cells see CD1 molecules? Is this recognition like classical ¿¿ TCR/MHC-like? Or are there features reminiscent of invariant Natural Killer T cell recognition of CD1d? Currently we have no information on this process. Finally, our third aim, To characterize the repertoire of ¿¿ T cells that recognize CD1c. will focus on identifying the ¿¿ T cell repertoire that is CD1c responsive and to characterize these T cells for cell-surface markers (co-receptors, memory markers), effector functions (cytokine secretion and/or cytotoxicity) and the sequence of their TCRs, specifically to determine whether there are motifs in the CDR loops that are used in this recognition process. We will explore the human repertoire from health and diseased individuals and will make use of a humanized CD1c murine model to explore the T cell repertoire there. Currently our understanding of CD1c's contribution to human immunity is in its infancy, yet hints at its importance in pathogen recognition (such as M. tuberculosis) are already evident. Our proposal will expand our understanding of CD1c lipid presentation and the T cells that are reactive to it.

? DESCRIPTION (provided by applicant): The University of Chicago Biological Sciences Division (BSD) has a traditional culture of academic research with training and exposure focused predominantly on research career paths in the academy. Yet ten years post-graduation, less than one-quarter (~21%) of our trainees become faculty members in a research-intensive institution. When surveyed, 86% of our trainee respondents supported a more concerted effort to prepare them for jobs outside academia and over 90% of faculty respondents believed their mentees should be provided exposure to a range of career options that use their training. A few enterprising pre- and postdocs have begun to capitalize on expanding translational and education-training programming outside the BSD. Major collaborators in these programs include our Booth School of Business (regularly ranked #1 in the world), its Polsky Center for Entrepreneurship, and our Center for Technology Development & Ventures (UChicagoTech). The University's new Center for Teaching Excellence has also attracted growing participation. These efforts reflect a culture shifting significantly towards broader engagement with innovation and interdisciplinary collaboration, fueled by trainee demand and the reality of fewer faculty job opportunities. With solid support of the President, Provost, and Deans of the University, we propose a program, Chicago Options-In-Careers Empowerment (my- CHOICE), to systematically provide a BSD on-ramp to link to, legitimize, leverage, and help grow these pro- grams, expanding career exposure offerings to our pre- and postdoctoral trainees. We are partnering with UChicagoTech, Polsky and a vast network of internal and external individuals we call Mentors from career areas including biotech, entrepreneurship, medicine, science policy and law, science communication, teaching, and administration to develop and implement this program. We will greatly expand existing Postdoc and Bio- tech Association seminar series to provide broad career path EXPOSURE, create a range of mini-courses for more in-depth EDUCATION in career domains, and guide trainees to a growing set of opportunities for deeper EXPERIENCE in areas of focus. We will use existing infrastructure, and add no extension to training time. We will also hold a portion of the myCHOICE programming in a new facility, the Chicago Innovation Exchange, an accelerator/incubator/event space, to locate myCHOICE events alongside many exemplars of PhDs pursuing non-academic careers. We have developed an innovative evaluation plan to test the hypotheses that more extensive participation in myCHOICE predicts greater trainee career choice empowerment, satisfaction with chosen career, and improved correlation between the FASEB myIDPCareer Fit assessment and career selection. We seek also to quantify the change in academic cost (the salary/lifestyles trainees would sacrifice to be an academic) as they progress through myCHOICE. We will establish strong bidirectional dissemination mecha- nisms, within and outside the University, to share and learn from other BEST programs. Our proposal enjoys extremely strong institutional support and commitment to sustainability beyond the funding period.

? DESCRIPTION (provided by applicant): Human V?9V?2 T cells are the major subset of ?? T cells that are found in human blood, comprising up to 5% of the T cells in healthy individuals and expanding to 20-50% during infection or disease. These cells play important roles in mediating immunity against microbial pathogens, including Mycobacterium tuberculosis and Mycobacterium leprae (the causative agents of tuberculosis and leprosy, respectively), and can respond potently against certain types of tumor cells. V?9V?2 T cells respond to these threats through recognition of structurally related non-peptidic phosphorylated antigens (pAgs). In tumor cells these are intermediate metabolites that accumulate due to over-production of the mevalonate pathway and in microbes they are generated during isoprenoid biosynthesis. It is largely unknown how V?9V?2 T cells recognize these pAgs; this proposal seeks to improve our overall understanding of the mechanisms behind V?9V?2 T cell activation and improve our ability to modulate this population in clinical settings. These cells also represent a prime opportunity to study alternative recognition strategies by ?? T cells, as classical and most no-classical MHC molecules do not appear to be involved in the recognition process. Our collaborators were the first to define the role of the BTN3A molecules in pAg-mediated V?9V?2 T cell stimulation and we have since then demonstrated the intracellular domain of one on them, BTN3A1, is the molecular sensor for pAg accumulation. We seek to understand the events that occur after pAg binding in our Aim1: To determine the direct molecular consequences of pAg binding to the BTN3A1 B30.2 intracellular domain. by using structural and dynamic studies including crystallography, NMR and fluorescence based measurements (FRET). In our Aim 2. To determine the supra-molecular organization of BTN3A1 molecules in the plasma membrane upon pAg or 20.1 mAb binding and its role in V?9V?2 T cell activation, we will use FRET based approaches, microscopy and protein engineering to study the factors that mediate cell surface assembly of BTN3A molecules. Finally, we will employ cross-linking and pull-downs combined with a SILAC-based mass spectrometry approach, complemented with a high-throughput genome-wide knockdown screen to pursue our Aim 3. From BTN3A1 to the V?9V?2 TCR: discovery of the relevant molecular players linking BTN3A1 to V?9V?2 T cell activation, which will define the other molecular players that are involved in pAg recognition in inducing the signals that directly lead to V?9V?2 T cell activation.

PROJECT ABSTRACT Foxp3+ regulatory T (Treg) cells are critical for the regulation of immune responses to self, foreign, and tumor- associated antigens, prompting considerable interest in the therapeutic manipulation of Treg cells in the context of cancer, autoimmunity, and transplantation. Over the past decade, a paradigm has emerged holding that thymic-derived Treg (tTreg) cells recognize self antigens. Furthermore, substantial evidence indicates that antigen recognition is critical for many aspects of Treg biology, including development, activation, anatomical distribution, and suppressor function. However, the natural self antigens recognized by tTreg cells remained unidentified. Without this knowledge, it was not possible to determine the biochemical nature of antigen recognition by Treg cells, to elucidate the role of TCR signal strength in promoting Treg development and clonal deletion, and to directly characterize endogenous antigen-specific Treg populations using peptide/MHC multimers. In recent work, we have identified a self antigen recognized by an endogenous population of naturally occurring Treg cells. The antigen, referred to as ?C4? in this proposal, is an unmodified I-Ab-restricted peptide derived from a prostate-specific protein, and is recognized by a canonical Treg clone named ?MJ23?. The studies outlined in this proposal will utilize the MJ23 TCR:C4/I-Ab system to address long-standing critical gaps in knowledge regarding Treg biology and the mechanisms by which dominant and recessive tolerance are enforced. The objectives of this proposal are to elucidate the biochemical nature of pMHC recognition by tTreg TCRs, to identify the mechanisms driving the thymic development of C4-specific Treg cells, and to elucidate the processes coordinating the peripheral homeostasis of C4-specific Tregs. It is our central hypothesis that high-affinity recognition of C4/I-Ab drives the thymic development of C4-specific Treg cells and the further selection of optimal C4-specific Treg clones in the periphery. We will test our central hypothesis and accomplish the objectives of this application by pursuing the following specific aims. In Aim 1, we will elucidate the molecular basis of Treg TCR recognition of self pMHC. In Aim 2, we will identify the signals driving the thymic development of C4-specific Treg cells. In Aim 3, we will determine the impact of peripheral selection on C4-specific Treg cells. The work outlined in this proposal is expected to reveal the biochemical nature of self antigen recognition by naturally occurring tTreg specificities. Moreover, the studies are expected to elucidate whether TCR signal strength is a primary determinant of whether self-reactive thymocytes will differentiate into the Treg lineage or undergo clonal deletion. Finally, these studies are expected to reveal the extent to which antigen encounter in the periphery shapes the repertoire of antigen-specific tTreg cells throughout life. In all, our work is expected to yield new insights into Treg cell biology and the mechanisms by which host tissues are protected from autoimmune attack.

Project Summary/Abstract Mucosal associated invariant T (MAIT) cells are an innate-like T cell subset prevalent in humans and enriched in the airway. Human MAIT cells have been defined by the expression of the semi-invariant TCR? chain TRAV1- 2/TRAJ12/20/33 and their restriction by the non-polymorphic MHC class I-like molecule, MHC-related protein 1 (MR1). MAIT cells recognize Mtb and can be activated by small organic molecules, derived from the riboflavin biosynthesis pathway. We have shown that MR1-restricted T cells can use TCRs that are not TRAV1-2, and can recognize organisms (S. pyogenes) that cannot produce riboflavin. Consequently, we define MAIT cells as a subset of MR1-restricted T cells (MR1Ts). Furthermore, we find that not all MR1Ts can be defined based on MR1 tetramer bound to the known MAIT agonist / MR1 ligand 5-(2-oxopropylideneamino)- 6-D- ribitylaminouracil (5-OP-RU), in that they can be defined based on their MR1-dependent response to microbial infection and binding to alternate MR1 tetramers. We have generated a pipeline approach for identifying new, microbially-derived MR1 antigens, and demonstrate that MR1Ts in the lung are characterized by oligoclonal enrichments, possibly driven by these antigens. Together, these data support the specific aims of this grant which are to 1) define the repertoire of ligands presented by MR1 from M. smeg/Mtb and define the structural basis of their presentation by MR1. We focus on Mtb for its disease relevance to human health but also from our preliminary data demonstrating migration of MR1 reactive T cells to the lung during Mtb infection. Our Aim 2 is to define the T cell repertoire of MR1Ts recognizing antigens presented by MR1 from M. smeg/Mtb and define the structural basis of their recognition of the MR1- antigen complex. This is an obvious extension from preliminary data from us and others demonstrating that the MR1T population contains diversity previously unappreciated. We seek to know whether this diversity in the TCR repertoire drives antigen selectivity. Directly related to Aims 1 & 2 is our Aim 3 which will determine the biological significance of MR1-ligand/MR1T cell selectivity in human health and disease. We hypothesize that MR1T cells with a diverse TCR repertoire selectively expand at infected tissue sites in response to microbe/ligand recognition via MR1. Here our focus will be on Mtb, and we capitalize on the expertise and patient accessibility of Dr. Waltz (Capetown) to derive lung (BAL) and PBMC samples from infected and control individuals. Ultimately, the work from this project would support MR1T cell targeted vaccines and immune-therapies as a means to improve resistance to disease following exposure to Mtb.

Project Summary/Abstract HLA-F is a nonclassical class I MHC (Ib) molecule that has been found expressed on a variety of cancers, shown to play a role in HIV infection and the neurological autoimmune disease ALS, and is expressed throughout pregnancy. Despite the potential importance of this protein in these conditions, little is known about this molecule in terms of its function or even in which state it is expressed. We have recently shown that, in addition to being expressed as a heavy chain only state, or open conformer (HLA-FOC), HLA-F can also be expressed as a bona fide peptide presenting molecule, associated with the ?2m subunit (pHLA-F). Peptides are presented in an unconventional way, with the N-terminus not anchored within the groove and the potential for post-translational modifications featuring in peptide anchoring. Despite these advances, there remains much unknown about the role for presented peptide in HLA-F function and how HLA-F engages its various receptors in each of these states. Thus, the aims of this proposal focus on addressing these questions and are: Aim 1: Develop and validate nanobodies towards peptide-loaded (pHLA-F) and open conformer (HLA- FOC) states of HLA-F and determine their expression profile in cancer cell lines. As HLA-F has been described to function in both the peptide-bound and free states, we will develop and validate novel nanobodies that specifically bind to these different forms of HLA-F. We will validate them through the use of HLA-F over expressing and knock out cell lines by flow cytometry, immunoprecipitation, and western blot. Finally, validated nanobody-binders will be used to determine the dominant form of HLA-F (HLA-FOC, pHLA-F, or both) expressed in and on the surface of 1) standard cell lines and 2) from gynecologic, neuroblastoma, and hepatocellular carcinoma cancer cell lines. Aim 2: Determine the peptide-antigen repertoire of HLA-F from selected cancer cell lines and determine their effect on NK-cell receptor engagement. As we have previously published, we have the ability to produce recombinant pHLA-F from HEK293T cells using an engineered baculovirus. We also showed that we can use this purified protein to determine the peptide repertoire of HLA-F from this cell line using MS/MS. Using this approach, we will produce pHLA-F from the cancer cell lines mentioned in SA1 and determine the peptide repertoire from these cancer specific lineages. In addition, we will pull out endogenous protein from these lines and confirm our results or act as an alternative approach should recombinant protein prove difficult to produce. Finally, we will determine the role of these peptide-antigens in NK-cell receptor engagement using in vitro binding experiments.

?? T cells constitute an important component of the immune response against infectious agents and cancerous transformations, yet the biochemical mechanisms by which they detect antigen through their somatically recombined T cell receptor (TCR) remain unclear. Unlike ??TCRs, which are restricted to recognizing antigens in the context of Major Histocompatibility Complex (MHC) molecules, ??TCRs can recognize a diversity of ligands ranging from self MHC to intact, unprocessed, viral glycoproteins. Our recent work has established CD1 molecules as ligands for a subpopulation of human V?1 ?? T cells, producing robust functional, biochemical and structural evidence. We seek to extend our studies to the human gut, where ?? T cells, and in particular, V?1+ T cells, predominate. Our preliminary data suggests that CD1 recognition is robust and present in all individuals examined, and that there exist important functional differences between CD1 reactive ?? T cells in tumors versus healthy adjoining tissue. Thus, the long-term goal of this proposal is to fully characterize this CD1 reactive population in tumors versus healthy tissue, examining their functional effector phenotypes, TCR repertoire and immunomodulatory signals, in addition to the TCR, that shape the recruitment, activation and potential expansion of these cells in the context of a highly relevant human disease, colorectal cancer. Our first aim, ?Characterization of CD1-specific ?? T cells in normal and diseased tissue.?, seeks to use classical cellular expansions complemented by direct ex vivo functional and transcript analysis to profile CD1 reactive and non- reactive T cell populations derived from tumor and adjoining healthy tissue. These data will provide insight into the signals that regulate ?? T cells within the tumor microenvironment compared to healthy tissue. Our second aim, ?Elucidation of the molecular mechanisms by which ?? TCRs bind to CD1/lipid complexes.?, will focus on characterizing the interaction between the ?? TCRs expressed by these cells and CD1/lipid antigen. We will use protein biochemistry, biophysics and x-ray crystallography to elucide the molecular mechanisms by which the ?? TCR recognizes CD1/lipid. Our effort will significantly expand our understanding of the specific signals that regulate ?? T cell activity in human health and disease. Our third aim, ?Determine the presence and role of ligand, co-stimulatory and/or co-receptor molecules in CD1 specific ?? T cell activation and phenotype in the colon? will characterize the ligand and immunomodulatory signals that may regulate the activity of CD1 reactive ?? T cells in the context of human colorectal cancer. We will combine RNAseq and differentiation assays using cord blood derived, naïve V?1 cells to test the relevance of candidate signals. This will be complemented by in vitro derived native V?1 T cells through the OP9/DL1 system. ?? T cells can be either pro-inflammatory or regulatory, therefore we seek to understand which role these cells play, if any, in this disease state. Together, these aims will begin to unravel the mystery of ?? T cells in human immunobiology, both at the cellular and molecular levels.