The work reported in this thesis involved the purification, biochemical analysis and sequencing of a novel suppressor factor secreted by a T cell hybridoma, A10. The factor, A10F, isolated from spent medium of A10 cells, was found to consist of two forms with molecular weights at 140 - 160 and 80 kD as suggested by NH₂-terminal sequencing, Western blotting and tryptic peptide mapping experiments. Both forms of A10F were found to be capable of suppressing the in vitro generation of cytotoxic T lymphocyte (CTL) specific for P815 cells by syngeneic (DBA/2) splenocytes. In vitro ³⁵S methionine labeling experiments clearly demonstrated that the 80 kD protein was a secretory product of the A10 cells. The protein, which was specific to the monoclonal antibody (B16G), was absent in the control NS1 and BW5147 cells. Biochemical analysis indicated that the 80 kD molecule, was either a degradation product or a monomer of the 140 - 160 kD molecule. Further degradation products such as the 32 kD molecules were also found. This peptide, however, did not seem to cause substantial suppression in the in vitro CTL assay. When the 140 - 160, 80 and 32 kD proteins were sequenced at the NH₂ terminus, both 140 - 160 and 80 kD proteins were found to possess the same NH₂ terminus sequence. The 32 kD protein, on the other hand, was found to have an NH₂-terminus quite different from that of the 80 kD protein. These findings suggested that the 32 kD fragment was probably located at the distal end of the 140 - 160 kD molecule. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The adaptive immune system is a complex network of cells that protect the body from invasion by foreign pathogens. Crucial to the function of the adaptive immune system is the activation, proliferation and differentiation of T cells in response to foreign pathogen presentation by antigen presenting cells. T cell activation is driven through different signaling pathways that are dependent on phosphorylation of substrates by kinases. In the PLC pathway that activates the il2 gene program, Protein Kinase C-q (PKCq) and Notch1 localize to the immunological synapse and help drive the signaling cascade that leads to robust T cell activation. It has been previously shown that PKCq and Notch1, both interact with the CBM complex at the immunological synapse. Additionally, PKCq and Notch1 both have specific cytoplasmic and nuclear functions that help drive the il2 gene program. Here, we demonstrate the localization of PKCq and Notch1 constructs transfected into HEK 293 cells. The use of deletion constructs of Notch1 was intended to inform us of what functional domain of Notch1 was responsible for the interaction with PKCq, however no direct interaction was demonstrated with the PKCq and Notch1 constructs used in these experiments. We hypothesize that this is likely due to the inactive form of PKCq found in our construct, or a result of the cell type used in these experiments.

archives@tulane.edu / Immune signatures are patterns of gene and protein expression in immune cells that characterize states of activation and response. As such, signatures indicative of viral control during natural infection may guide vaccine development efforts to achieve similar patterns of protection. Here, we used nonhuman primate (NHP) models of Zika virus (ZIKV) and simian immunodeficiency virus (SIV, as a model for HIV) to explore outcomes of infection in these important human pathogens. We employed a multifaceted approach including high dimensional flow cytometry and RNA sequencing to understand cellular responses to ZIKV generally and during pregnancy, as well as to identify the impacts of infection in astrocytes, a neuroglial target of ZIKV thought to be important in the development of neurologic disease. We found that CD8 T cells may restrict ZIKV persistence in tissues but ultimately have a minimal role in protection to either primary or secondary challenge. However, we showed that immune manipulation, either naturally through pregnancy or artificially through depletion experiments, can skew metabolic and innate immune pathways in unexpected ways. While cellular immunity appeared to minimally impact ZIKV infection, such responses in SIV are important in controlling viral replication, which we inversely showed by tracking patterns of viral mutation to evade CD8 responses. We also identified transcriptional signatures in ZIKV infection that may underlie the development of neurologic diseases and found that different virus lineages have unique impacts on gene expression. Together, these experiments showcase the utility of profiling approaches in understanding the immune complexity that accompanies viral infection. / 1 / Blake Schouest

Regulatory T cells (Tregs) provide an essential tolerance mechanism to suppress the immune response. Under normal conditions, Tregs reduce reaction to self-antigens, and conversely, lack of Treg function leads to autoimmune diseases. Reengineering of the immune system with regards to Tregs, such as through adoptive immunotherapy, holds great therapeutic promise for treating a range of diseases. These approaches require production of Tregs, which can be induced from conventional, reactive T cells. This thesis is driven by the concept that changing the mechanical stiffness of biomaterials can be used to direct and optimize this induction process. It is known that T cells sense their extracellular environment, and that T cell activation can be modulated by mechanical cues. However, it is still unclear whether or not human Treg induction is sensitive to material stiffness. We studied this phenomenon by replacing the stiff plastic supports commonly used for T cell activation with planar, elastic substrates — specifically polyacrylamide (PA) gels and polydimethylsiloxane (PDMS) elastomer. Treg induction, as measured by expression of FOXP3, a master transcription factor, was sensitive to stiffness for both materials. Substrate stiffness also modulated the suppressive function and epigenetic profiles of these cells, demonstrating that substrate rigidity can direct Treg induction, complementing the use of chemical and genetic tools. Delving deeper into the mechanisms of T cell mechanosensing, single-cell transcriptomic analysis revealed that substrate rigidity modulates the trajectory of Treg induction from conventional T cells, altering a host of functions including metabolic profile. Together, these studies introduce the use of substrate stiffness and T cell mechanosensing towards directing and optimizing regulatory T cell production. Further development of cell culture systems around this discovery is critical for emerging T cell-based therapies, targeting cancer but also a broad range of diseases.

A memory response is established following primary antigen exposure that stays more or less constant. It appears to adopt a set-point in magnitude but upon re-exposure the response is quicker and better and there is an upward shift in memory frequency that varies with individuals based on the exposure pattern to other microbes or its components. Our investigations were designed to test such differences of non-specific stimulation by PAMPs in lowering the threshold of activation. Neonatal mice were pre-exposed to TLR-ligands intermittently and later analyzed for its resilience to challenge with virus during adult-life. Secondly, adult mice with pre-existing memory to virus were exposed to various TLR-ligands and analyzed for their quality of memory response. The TLR-ligands exposed animals were better responders to a new agent exposure compared to the animals kept in sterile surroundings. Moreover, immune memory recall and the viral specific CD8+ T cells response with TLR-ligands were comparable to the recall response with the cognate antigen. The results provide insights into the role of hyper-sanitized environment versus PAMPs mediated signaling in adaptive immunity and long-term immune memory.

No description available.

Superantigens are exotoxins produced by Staphylococcus aureus and induce extensive T cell proliferation and proinflammatory cytokines, leading to toxic shock syndrome at high concentrations. However, the role of superantigens produced at relatively low concentrations during asymptomatic colonization or chronic infection has not been well established. In this dissertation, we demonstrated that stimulation of human PBMCs with staphylococcal enterotoxin C1 (SEC1) at the dose inducing a half maximal T cell proliferation (suboptimal stimulation) induced immunosuppressive CD4+CD25+FOXP3+ and CD8+CD25+FOXP3+ T cells. The suppression of these cells was mainly mediated by the galectin-1. We found that suboptimal stimulation with SEC1 induced differential activation of PI3K-mTOR-Akt pathway, leading to expression of FOXP3 isoforms preferably localized to the nucleus and induction of PTEN that contributes to maintain stability and suppressive activity of regulatory T cells. Taken together, these results demonstrate the important role of superantigen produced at low concentration during asymptomatic colonization that induce immunosuppressive CD4+ and CD8+ regulatory T cells to promote survival, propagation, and colonization for S. aureus in the host.

Multiple myeloma is a plasma cell cancer that progressively evolves to an aggressive, multi-drug resistant disease, which presents an unmet clinical need. In clinical trials, myeloma shows susceptibility to novel immunotherapeutic agents, particularly those targeting B-cell maturation antigen (BCMA). Among different classes of immunotherapies, T cell-based approaches have progressed the most due to their ability to induce durable responses in patients with advanced drug-resistant blood cancers. Most T cell engineering strategies rely on the use of chimeric antigen receptors (CARs), which although effective, can cause serious life-threatening toxicities. We created a new synthetic receptor, T cell antigen coupler (TAC), which recruits the endogenous T cell receptor and allows T cells to autoregulate their activity. Our experience in solid tumor models has shown that TAC-T cells are similarly efficacious and significantly less toxic than CAR-T cells. This thesis describes our optimization of BCMA-specific TAC-T cells and analysis of different anti-BCMA antigen-binding domains. TAC receptor functions by engaging endogenous TCR-CD3 complex and redirecting it to the target of interest. In Chapter 3, we characterize optimization and humanization of the CD3-recruitment domain in the TAC scaffold and provide evidence that TAC-T cells are effective against multiple myeloma, irrespective of receptor surface levels. In Chapter 4, we describe selection of the human BCMA-binding domain and the creation of a fully humanized TAC receptor against BCMA. Chapters 5 and 6 describe how a BCMA-targeting antigen-binding domain that cross-reacts with an unknown antigen in mice augments in vivo efficacy of TAC- and CAR-T cells, respectively. The work described in Chapters 3 and 4 presents an optimized, fully human BCMA-TAC that is being moved into clinical testing. The work in Chapters 5 and 6 improves our understanding of how antigen-targeting domains in synthetic receptors influence the functionality of engineered T cells. / Thesis / Doctor of Science (PhD) / Multiple myeloma is an incurable blood cancer that has a remarkable ability to develop resistance to different types of chemotherapy. In recent years, treatments redirecting immune cells against tumors have shown impressive clinical responses against different types of chemotherapy-resistant blood cancers, including multiple myeloma. Our lab has developed a new technology for redirecting T cells against tumors, called T cell antigen coupler (TAC) receptor. This thesis describes optimization of a fully human TAC receptor specific for a target on the surface of myeloma cells, known as BCMA. Durable remissions induced by TAC-engineered T cells in a preclinical mouse model of myeloma in the absence of toxicity warrant further testing of this therapeutic in a clinical trial.

Indiana University-Purdue University Indianapolis (IUPUI) / Atopic dermatitis (AD) is a pruritic, chronic inflammatory skin disease with a lifetime prevalence of 10-20% in children and 1-3% in adults, worldwide. In the past three decades, prevalence of the disease has increased by two to three-fold in industrialized countries, with higher incidences in urban regions compared to rural regions. Mice with an activating mutation in STAT6, known as STAT6VT, constitutively express STAT6 in T-cells. Our preliminary data suggests significant differences between the STAT6VT transgenic mice from WT littermate controls treated with SLS. These findings correlate with evidence that there are abnormalities in the barrier function between these mice

Using the mixed lymphocyte reaction (MLR) as a correlate of cell-mediated immunity, an examination was made of positive and negative blastogenic immunoregulation by syngeneic peritoneal macrophages (M_Φ) and splenic T cells from tumor-bearing mice (TBM). As analysis of T cell proliferative response progressed an intricate pattern of immunoregulatory checks and balances unfolded involving both M_Φ and TBM cells. In their capacity as regulators, M_Φ acted, not only to enhance MLR reactivity, but to inhibit it. Results indicated that: i) M_Φ regulation was a concentration dependent phenomena -- high concentrations of M_Φ (or their supernatants) inhibited MLR reactivity, while low doses enhanced MLR reactivity; ii) inhibition occurred via a non-toxic, heat stable, nondialyzable (and therefore non-thymidine) factor; iii) enhancement occurred via a heat labile nondialyzable factor. Since normal M_Φ possessed a factor which, in high concentrations, inhibited T cell blastogenesis, tests were run to determine if the MLR hyporeactivity of T cells from TBM could be attributed to a unique tumor-induced inhibitory M_Φ. Contrary to expectations, TBM M_Φ supernatants, when compared to their normal counterparts on a volume-to-volume basis. showed an increased (not decreased) ability to enhance MLR reactivity. In light of results showing TBM M_Φ enhancement of MLR reactivity, T cell hyporeactivity in TBM was explained after observation of the following dual regulatory mechanism of suppression: i) on a purely quantitative basis, the high in vivo concentration of M_Φ in spleens of TBM inhibits spleen cell response to alloantigen; ii) there also exists a population of mildly nylon wool adherent tumor-induced splenic T cells which elaborate a soluble factor capable of overriding any M_Φ enhancing effect. In their capacity as regulators, tumor-induced splenic T-cells act, both to enhance and inhibit MLR reactivity. Whereas M_Φ regulation is concentration dependent, in the case of the T cell regulator, regulation is based, not upon the relative concentration of the regulator cell, but upon the level of responder cell activity, i.e. M_Φ-depleted MLR cultures (showing minimal proliferation) were enhanced by regulator TEM T cell addition, while M_Φ augmented MLR cultures or PHA stimulated cultures (with a high rate of blastogenesis) were inhibited by the same concentration of TBM regulator cells. Centering around more stringent biophysical and biochemical characterization of M_Φ supernatants, the latest work has resulted in the biochemical separation of M_Φ supernatants into inhibitor and enhancing components. Using anion exchange chromotography and slab gel electrophoresis, inhibitor and enhancing factors have been separated by charge. Treatment of M_Φ with indomethacin did not abrogate release of inhibitor factor, suggesting that it was not prostaglandin. Enhancing factor, obtained from M_Φ sonicates as well as supernatants, could be distinguished from lymphocyte activating factor by its inability to induce a thymocyte PHA response. Thus, analysis of regulation in the cell-mediated immune system has resulted in the elucidation of a most elaborate scheme of immunoregulation involving both M_Φ and T cells. / Ph. D.