EFFICACY AND MECHANISM OF â-DEFENSIN2 FUSED ANTIGEN PROTEIN VACCINES

Vaccines which use the strategy of fusing adjuvant murine â-defensin2 (mBD2) to an antigen in order to elicit stronger anti-antigen immune responses are referred to as murine â-defensin2 (mBD2) vaccines. Previous studies have validated the potential of mBD2 vaccines, thus in this study we focus on increasing vaccine efficacy as well as mechanism elucidation. Initially, we demonstrate superior IFN-ã release levels by antigen specific effector T cells when antigen is crosspresented by dendritic cells (DC) which absorbed mBD2 vaccine (mBD2 fused antigen protein) over antigen alone. We move unto an in vivo model and note significant increases in the expansion of antigen specific class I T cells but not class II T cells when receiving mBD2 vaccine over antigen alone. Further, knowing mBD2’s link with CC chemokine receptor 6 (CCR6) and Toll-like receptor 4 (TLR4) we note that this enhanced class I T cell expansion is CCR6 independent but TLR4 dependent. With anti-tumor responses desired, we demonstrate in tumor protection experiments with mice, compelling tumor protection when combining adoptive T cell therapy and mBD2 vaccine immunization. We further note that mBD2 vaccines are not limited by the antigen and characterize a viable strategy for enhancing tumor antigen immunogenicity.

Role of janus tyrosine kinase -3 (JAK3) and signal transducer and activator of transcription (STAT5) pathway in T lymphocyte activation

T cell activation and expansion is essential for immune response against foreign antigens. However, uncontrolled T cell activity can be manifested as a number of lymphoid derived diseases such as autoimmunity, graft versus host disease, and lymphoma. The purpose of this research was to test the central hypothesis that the Jak3/Stat5 pathway is critical for T cell function. To accomplish this objective, two novel Jak3 inhibitors, AG490 and PNU156804, were identified and their effects characterized on Jak3/Stat5 activation and T cell growth. Inhibition of Jak3 selectively disrupted primary human T lymphocyte growth in response to Interleukin-2 (IL-2), as well as other γ c cytokine family members including IL-4, IL-7, IL-9, and IL-15. Inhibition of Jak3 ablated IL-2 induced Stat5 but not TNF-α mediated NF-κβ DNA binding. Loss of Jak3 activity did not affect T cell receptor mediated signals including activation of p56Lck and Zap70, or IL-2 receptor a chain expression. To examine the effects of Jak3/Stat5 inhibition within a mature immune system, we employed a rat heart allograft model of Lewis (RT1 1) to ACI (RT1a). Heart allograft survival was significantly prolonged following Jak3/Stat5 inhibition when rats were treated with AG490 (20mg/kg) or PNU156804 (80mg/kg) compared to non-treated control animals. This effect was synergistically potentiated when Jak3 inhibitors were used in combination with a signal 1/2 disrupter, cyclosporine, but only additively potentiated with another signal 3 inhibitor, rapamycin. This suggested that sequential inhibition of T cell function is more effective. To specifically address the role of Stat5 in maintaining T cell activity, novel Stat5 antisense oligonucleotides were synthesized and characterized in vitro. Primary human T cells and T-cell tumor lines treated with Stat5 antisense oligonucleotide (7.5 μM) rapidly underwent apoptosis, while no changes in cell cycle were observed as measured by FACS analysis utilizing Annexin-V-Fluorescein and Propidium iodide staining. Evidence is provided to suggest that caspase 8 and 9 pathways mediate this event. Thus, Stat5 may act rather as a negative regulator of apoptotic signals and not as a positive regulator of cell cycle as previously proposed. We conclude that the Jak3/Stat5 pathway is critical for γc cytokine mediated gene expression necessary for T cell expansion and normal immune function and represents an therapeutically relevant effector pathway to combat T cell derived disease. ^

Characterization and optimization of antigen-specific T cell responses during ex vivo expansion of melanoma tumor-infiltrating lymphocytes

Treatment of metastatic melanoma with tumor reactive T cells (adoptive T cell therapy, ACT) is a promising approach associated with a high clinical response rate. However, further optimization of this treatment modality is required to increase the clinical response after this therapy. ACT in melanoma involves an initial phase (pre-REP) of tumor-infiltrating lymphocyte (TIL) expansion ex vivo from tumor isolates followed by a second phase, “rapid expansion protocol” (REP) generating the billions of cells used as the TIL infusion product. The main question addressed in this thesis was how the currently used REP affected the responsiveness of the CD8+ T cells to defined melanoma antigens. We hypothesized that the REP drives the TIL to further differentiate and become hyporesponsive to antigen restimulation, therefore, proper cytokine treatment or other ways to expand TIL is required to improve upon this outcome. We evaluated the response of CD8+ TIL to melanoma antigen restimulation using MART-1 peptide-pulsed mature DC in vitro. Post-REP TILs were mostly hypo-responsive with poor proliferation and higher apoptosis. Phenotypic analysis revealed that the expression of CD28 was significantly reduced in post-REP TILs. By sorting experiment and microarray analysis, we confirmed that the few CD28+ post-REP TILs had superior survival capacity and proliferated after restimulation. We then went on to investigate methods to maintain CD28 expression during the REP and improve TIL responsiveness. Firstly, IL-15 and IL-21 were found to synergize in maintaining TIL CD28 expression and antigenic responsiveness during REP. Secondly, we found IL-15 was superior as compared to IL-2 in supporting the long-term expansion of antigen-specific CD8+ TIL after restimulation. These results suggest that current expansion protocols used for adoptive T-cell therapy in melanoma yield largely hyporesponsive products containing CD8+ T cells unable to respond in vivo to re-stimulation with antigen. A modification of our current approaches by using IL-15+IL-21 as supporting cytokines in the REP, or/and administration of IL-15 instead of IL-2 after TIL infusion, may enhance the anti-tumor efficacy and long-term persistence of infused T cells in vivo.

Proteomic identification and functional characterization of prohibitin 1 and 2 in T cell activation, expansion, survival and disease

Several immune pathologies are the result of aberrant regulation of T lymphocytes. Pronounced T cell proliferation can result in autoimmunity or hematologic malignancy, whereas loss of T cell activity can manifest as immunodeficiency. Thus, there is a critical need to characterize the signal transduction pathways that mediate T cell activation so that novel and rational strategies to detect and effectively control T cell mediated disease can be achieved. ^ The first objective of this dissertation was to identify and characterize novel T cell regulatory proteins that are differentially expressed upon antigen induced activation. Using a functional proteomics approach, two members of the prohibitin (Phb) family of proteins, Phb1 and Phb2, were determined to be upregulated upon activation of primary human T cells. Furthermore, their regulated expression was dependent upon CD3 and CD28 signaling pathways which synergistically increased their expression. In contrast to previous reports of Phb nuclear localization, both proteins were determined to localize to the mitochondrial inner membrane of human T cells. Additionally, novel Phb phosphorylation sites were identified and characterized using mass spectrometry, phosphospecific antibodies and site directed mutagenesis. ^ Prohibitins have been proposed to play important roles in cancer development however the mechanism of action has not been elucidated. The second objective of this dissertation was to define the functional role of Phbs in T cell activity, survival and disease. Compared to levels in normal human T cells, Phb expression was higher in the human tumor T cell line Kit225 and subcellularly localized to the mitochondrion. Ablation of Phb expression by siRNA treatment of Kit225 cells resulted in disruption of mitochondrial membrane potential and significantly enhanced their sensitivity to cell death, suggesting they serve a protective function in T cells. Furthermore, Q-RT-PCR analysis of human oncology cDNA expression libraries indicated the Phbs may represent hematological cancer biomarkers. Indeed, Phb1 and Phb2 protein levels were 6-10 fold higher in peripheral blood mononuclear cells isolated from malignant lymphoma and multiple myeloma patients compared to healthy individuals. ^ Taken together, Phb1 and Phb2 are novel phosphoproteins upregulated during T cell activation and transformation to function in the maintenance of mitochondrial integrity and perhaps energy metabolism, thus representing previously unrecognized intracellular biomarkers and therapeutic targets for regulating T cell activation and hematologic malignancies. ^

Motile response patterns and ultrastructural observations of the isolated outer hair cell

The tonotopic organization of the mammalian cochlea is accompanied by structural gradients which include the somatic lengths of outer hair cells (OHCs). These receptors rest upon the vibrating portion of the basilar membrane and have been reported to exhibit motile responses following chemical and electrical stimulation. These movements were examined in detail in this dissertation. It was found that isolated OHCs cultured in vitro respond to chemical depolarization with slow tonic movements, and to electrical waveforms with bi-directional, frequency following movements extending from DC to at least 10 kHz.^ Slow contractions were also elicited following electrical stimulation, bath incubation in carbachol (a cholinergic agonist), and increases in extracellular K+ concentration as little as 50 mM.^ Isolated OHCs display anatomical features which are remarkable when contrasted with those prepared from intact receptor organs. A complex structure located between the cuticular plate and the nuclear membrane was consistently observed and was examined by serial cross-sections which revealed a network of non-membrane bound densities. This corresponded to a granular complex seen at the light microscope level. The complex was composed of dense regions of organelles, striated structures embedded within the core, and a circumferential network of microtubules residing in the peri-nuclear portion of the cell. In cells which had lost their nuclear attachment to the terminal synaptic body, the granular complex could be made to contract without effecting any change in cellular length, implying that the complex may be the driving force behind certain aspects of the motile response.^ Most cells displayed movements which revealed asymmetries analogous to those reported for OHC receptor potentials in vivo. The contraction phase (for longer cells) was shown to have a small time constant (approximately 400 microseconds) and saturated with limited displacements. The expansion phase had time constants as large as 1.3 milliseconds but yielded displacements as much as 60 percent larger than those seen for contractions.^ Additional waveform characteristics seen in the in vivo response could be emulated either by biasing the cell's resting length with either direct current, triggering contractions via large electrical displacements, or incubation with depolarizing compounds.^ Alternatively, short (20-30 um) cells revealed more linear response characteristics to the probe stimulus. Partial saturation was achieved and revealed a DC component which was opposite in polarity to that seen in longer cells. (Abstract shortened with permission of author.) ^