Identification of a dominant CD4 T cell epitope in the membrane lipoprotein Tul4 from Francisella tularensis LVS

Francisella tularensis is a Gram-negative intracellular bacterium that is the causative agent of tularemia. Small mammals such as rodents and rabbits, as well as some biting arthropods, serve as the main vectors for environmental reservoirs of F. tularensis. The low infectious dose, ability to aerosolize the organism, and the possibility of generating antibiotic resistant strains make F. tularensis a prime organism for use in bioterrorism. As a result, some strains of F. tularensis have been placed on the CDC category A select agent list. T cell immune responses are thought to be a critical component in protective immunity to this organism. However, investigation into the immune responses to F. tularensis has been hampered by the lack of molecularly defined epitopes. Here we report the identification of a major CD4+ T cell epitope in C57Bl/6 (B6) mice. The murine model of F. tularensis infection is relevant as mice are a natural host for F. tularensis LVS and exhibit many of the same features of tularemia seen in humans. Using T cell hybridomas derived from B6 mice that had either been inoculated with F. tularensis and allowed to clear the infection or which had been immunized by conventional means using purified recombinant protein in adjuvant, we have identified amino acids 86–99 of the lipoprotein Tul4 (RLQWQAPEGSKCHD) as an immunodominant CD4 T cell epitope in B6 mice. This epitope is a major component of both the acute and memory responses to F. tularensis infection and can constitute as much as 20% of the responding CD4 T cells in an acute infection. Reactive T cells can also effectively enter the long-term memory T cell pool. The identification of this epitope will greatly aid in monitoring the course of F. tularensis infection and will also aid in the development of effective vaccine strategies for F. tularensis

A broadly applicable approach to T cell epitope identification: Application to improving tumor associated epitopes and identifying epitopes in complex pathogens

Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens

Development and Characterization of Protease Activated Cytokine Fusion Proteins

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2015.Cytokines have long been known to be key immune mediators and are involved in almost all aspects of immune responses. As a result, many have been tested in a variety of cancers with some notable successes. For example, systemic IL-2 delivery is now FDA approved for the treatment of melanoma and kidney cancer. However, side effects of cytokines have greatly limited their utility as well as their efficacy. This may not be surprising given that most cytokines have evolved to act locally over short distances in an autocrine or paracrine fashion. This thesis describes a fundamentally different approach for cytokine use that more closely mimics the normal biology of cytokines but allows for their systemic delivery that could affect all metastatic tumor sites. The overall hypothesis is that by changing the balance of key cytokines at tumor sites, it is possible to alter the character of the anti-tumor immune response and ultimately affect tumor growth. These studies have focused on IL-2 and IL-12. The work in this thesis shows that these cytokines have potent effects individually but when used together in vitro exhibit even more robust effects, preferentially stimulating more cytotoxic effectors and comparatively fewer regulatory T cells. The work presented here, as well as studies in animal models and patients reveal the potential of these cytokines. Nevertheless, other studies reveal this potent activation comes with a price and side effects resulting from widespread stimulation of immune cells. To address many of these issues, this thesis describes the generation and characterization of a series of activatable fusion proteins containing the key cytokines IL-2 and IL-12. The cytokine activated fusion protein (FP) strategy consists of a cytokine linked to an inhibitory component separated by a specific protease cleavage sequence. At the tumor site, the FP can be cleaved by proteases such as Matrix Metalloproteinases (MMPs) that are over-expressed. This cleavage allows the release of the cytokine from the inhibitor and its binding to higher affinity receptors on immune cells. The data presented show that the FPs are greatly inhibited in their native form compared to the same amount of the free cytokine. Moreover, cleavage by MMPs dramatically increases their functional activity. Further, delivery of these fusion proteins engenders little inflammatory responses in vivo compared to the free cytokine. Even long-term expression of the FPs achieved using an AAV gene therapy vector results in little to no inflammatory cytokine responses as measured by luminex assays. These results indicate that the fusion proteins may be used in vivo with greatly reduced toxicity. Further, the activatable fusion protein approach represents a fundamentally different means of delivering cytokines and could serve as a platform technology to deliver any immune modulator

Expanded CD23(+)/CD21(hi) B cells in inflamed lymph nodes are associated with the onset of inflammatory-erosive arthritis in TNF-transgenic mice and are targets of anti-CD20 therapy.

Anti-CD20 B cell depletion therapy (BCDT) is very effective for some patients with rheumatoid arthritis (RA); however the pathogenic role of B lymphocytes in RA and the primary targets of BCDT are unknown. The human TNF transgenic (hTNF-Tg) mouse model of RA displays a chronic, progressive disease that spreads from distal to proximal joints and is generally considered to be adaptive immune system independent. We have previously reported that knee arthritis in hTNF-Tg mice is accompanied by structural and functional changes of the adjoining popliteal lymph node (PLN), detectable by contrast-enhanced magnetic resonance imaging. To better understand these changes, in this paper we show that onset of knee synovitis and focal erosions are paralleled by PLN contraction and accumulation of large numbers of B cells in the lymphatic sinus spaces within the node. Flow cytometry from TNF-Tg mice 2, 4-5, and 8-12 mo old demonstrated that B cell accumulation in the PLN follows ankle arthritis, but commences before knee disease, and involves early expansion of CD21(hi), CD23(+), IgM(hi), CD1d(+), activation marker-negative, polyclonal B cells that are found to be specifically restricted to lymph nodes draining inflamed, arthritic joints. The same B cell population also accumulates in PLNs of K/BxN mice with autoantigen-dependent arthritis. Strikingly, we show that BCDT ameliorates hTNF-Tg disease and clears follicular and CD21(hi), CD23(+) B cells from the PLNs. On the basis of these findings, we propose a model whereby B cells contribute to arthritis in mice, and possibly RA, by directly affecting the structure, composition, and function of joint-draining lymph nodes

A broadly applicable approach to T cell epitope identification: Application to improving tumor associated epitopes and identifying epitopes in complex pathogens

Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens