The transcription factors Runx1 and Runx3 are required for permanent silencing of CD4 in maturing CD8+ thymocytes. Runx binding to consensus sites within the CD4 silencer region is required for CD4 silencing post-positive selection. The Runx nuclear matrix targeting sequence (NMTS) is required for CD4 silencing and is implicated in binding to the histone acetyltransferase (HAT) p300, histone deacetylases (HDAC), and histone methyltransferase (HMT) SUV39H1 proteins. Epigenetic modifications of chromatin or post-translational modifications of Runx itself as a result of Runx association with these enzymes may be important for establishment of long-term CD4 silencing. In this study, we show that treatment of thymocytes with the HDAC inhibitor trichostatin A (TSA) and a resulting increase in histone acetylation in the CD4 silencer region results in an increase in CD4 silencing in a Runx-independent manner. We evaluated the role played by Runx lysines and their potential post-translational modification in CD4 silencing by mutational analysis of nine Runx lysine residues. Disruption of lysines within the Runt DNA binding domain known to reduce Runx DNA-binding activity resulted in CD4 derepression, indicating that Runx DNA-binding is required for CD4 silencing. Mutation of other lysines not involved with DNA-binding and reported to be acetylated or methylated did not affect CD4 silencing by Runx. The transduction of thymocytes with the C-terminally truncated Runx1 or Runx1 lacking the NMTS fused with the p300 HAT domain, HDAC1, or SUV39H1 resulted in CD4 derepression, indicating that enforced association of these individual enzymes with the Runx DNA-binding domain promotes CD4 transactivation rather than CD4 silencing. Profiling chromatin marks present under c-terminally truncated Runx1d.190 treatment conditions revealed H3K9me3/H3K4me3 coenrichment in CD4 promoter and silencer regions suggesting the involvement of a dynamic instruction profile in the establishment of CD4 silencing.

NKG2D is a unique immunoreceptor of key significance to immune surveillance of tumor cells, and it represents an attractive candidate in the development of both molecular and cell-based immunotherapies. NKG2D is a stimulatory receptor expressed by human natural killer (NK) cells, cytokine induced killer/lymphokine activated killer (CIK/LAK) cells, γδ T cells and CD8+ &agr;β T cells. The NKG2D receptor plays a pivotal role in both innate and adaptive immunity, where it stimulates the cytotoxic function of (NK) cells and CD8 + T cells upon recognition of a diverse array of MHC-related ligands. In turn, these ligands are specifically induced under pathological conditions.Current research has established a prominent role for the NKG2D receptor in the modulation of tumor immunity by both NK cells and T cells. We discovered a novel genetic modifier that limits the effectiveness of NK/T cell immunotherapy in human cancer patients. Our objective of this study was to investigate how these genetic modifiers affect NK receptor-ligand interactions and ultimately tumor cell recognition, immunity, and immune evasion. We characterized an alternate splice variant of human NKG2D that encodes a truncated receptor lacking the ligand-binding ectodomain. This truncated NKG2D variant (NKG2DTR) does not activate cytotoxicity in a ligand-dependent manner, and its enforced expression inhibits killing mediated by the full-length NKG2D isoform (NKG2DFL). Enforced expression of NKG2DTR resulted in the retention of NKG2DFL in intracellular compartments. The relative abundance of NKG2DTR transcripts varies among PBMC and activated LAK/CIK cells and NK cells of diverse donors and inversely correlates with the killing capacity of expressing cells. Furthermore, specific shRNA-mediated knockdown of the endogenous NKG2D TR isoform in human CIK cells and NK cells enhances endogenous NKG2D FL-mediated cytotoxicity. Co-immunoprecipitation studies revealed that NKG2DTR pairs with DAP10 and heterodimerizes with NKG2D FL, suggesting that its dominant negative impact on cytotoxicity is due to the formation of heterodimeric NKG2DTR/FL receptor complexes incapable of ligand binding. Thus, competitive interference via an alternatively spliced NKG2D variant constitutes a novel mechanism for regulation of NKG2D-mediated signaling and cytotoxicity in LAK/CIK cells and NK cells.

WC1 proteins specifically expressed on the surface of γδ T cells are members of group B Scavenger Receptor Cysteine Rich (SRCR) superfamily, in which receptors contain several SRCR domains in the extracellular region. WC1+ γδ T cells play a critical role in bridging innate and adaptive immunity, organizing granulomas in response to Mycobacterium and producing IFNγ in response to Leptospira. The serologically-defined WC1.1+ γδ T cells exclusively respond to spirochete Leptospira ; the serologically-defined WC1.2+ subpopulation responds to rickettsias Anaplasma; shRNA silencing three WC1.1+ proteins significantly reduced γδ T cell response to Leptospira antigen. Co-ligation of WC1 with TCR/CD3 potentiates T cell activation, and tyrosine phosphorylation of the WC1 cytoplasmic domain is required for WC1 co-receptor activity. We hypothesized that WC1 receptors encode antigen specificity and contribute to T cell activation in response to leptospirosis. Our data showed SRCR domains from a WC1.1 type receptor, WC1-3, directly interact with vaccines and liquid cultures of leptospires. Vaccine Leptospira interacts with more SRCR domains as compared to liquid cultures, suggesting that vaccine preparation may enhance ligand accessibility to SRCR domains. Importantly, we did not observe any Leptospira binding from WC1-4 SRCR domains, a representative WC1.2 type receptor. The binding assay showed that SRCR a1 domains from WC1.1 type proteins contribute to Leptospira recognition, but none from WC1.2 proteins does. Alkaline phosphatase treatment suggests that a phosphorylation pattern is recognized by SRCR a1 domain, supporting that WC1 functions as a pattern recognition receptor. PMA-induced CD4/WC1-3 endocytosis is mediated by a membrane-proximal dileucine motif, which is essential for the recruitment of AP2 complexes. The disruption of the dileucine motif greatly accumulates the overall levels of CD4/WC1-3 molecules on the cell surface and in the cytoplasm. Co-crosslinking CD4/WC1-3 and TCR/CD3 indicates that the dileucine motif acts as a negative regulator for downstream cytokine production. Moreover, a double serine motif upstream the dileucine motif mediates signaling through WC1-3 for the downstream event. Taken together, the data support that co-ligation of WC1 and the γδ TCR by pathogen-associated molecular patterns (PAMPs) induces specific γδ T cell activation.

The anatomical compartment within which the B-cell develops is different in different animals. This study attempts to trace B-cell ontogeny in cattle by investigating the expression profile of B-cell specific genes in cattle. Bovine fetal spleen and liver are the site of lymphopoeisis as both RAG1 and RAG2 could be detected at 60 days of gestation by RT-PCR. VpreB and heavy chain genes were also detected at 60 days of gestation, but no light chain expression could be found. TdT expression could not be detected and analysis of expressed light chain genes revealed lack of any junctional diversity in the expressed sequences, suggesting that N-nucleotide addition has no role in diversification of the primary repertoire in cattle. Most mammals express two Ig light chain isotypes—lambda and kappa. Cattle has long been known to produce only the lambda light chain. This study attempts to characterize the kappa light chain expression in cattle. While kappa light chain could be detected at both the transcript and protein levels, the number of B-cells expressing kappa light chain in the periphery was limited and varied from animal to animal. The diversity of the expressed kappa light chain was determined by RT-PCR cloning and sequencing. The expressed kappa light chain genes show very limited—about half the diversity as compared to the lambda light chain. The complexity of kappa light chain genes present in the cattle genome is comparable to that of the lambda light chain as determined by southern blot analysis of genomic DNA indicating similar number of genes in both the light chain loci. The cloning of kappa germline genes revealed that the nanomer sequence of the RSS elements consistently have a substitution at the fourth base position and the heptamer sequence is also not always conserved. The kappa J/C intronic region was also cloned and sequenced. The conserved enhancer sequence, found in most mammals, was partly missing as only two of the three enhancer elements could be detected.

Runx1/AML1 transcription factor expression in hematopoietic cell lineages is differentially regulated via usage of two distinct promoters. The 5' UTR and a 19 amino acid encoding sequence transcribed from the distal promoter is inserted via alternative splicing into the 5' end of the mRNA transcript, replacing the 5' UTR and a 5 amino acid encoding sequence usually transcribed from the proximal promoter. Expression of proximal Runx1 in 32Dcl.3 cells delays G-CSF induced neutrophil terminal differentiation by increasing viability compared to distal Runx1. We utilized Runx1 N-terminal deletion and point mutants of three evolutionarily conserved residues to describe dual N-terminal isoform motifs that promote two distinct differentiation phenotypes as regulatory elements in hematopoietic cell differentiation. Runx1 isoforms were evaluated in established hematopoietic in vitro and ex vivo differentiation systems. Deletion of amino acids 3’-14’ (Δ3-14) or 3’-19’ (Δ3-19) of the distal Runx1 N-terminus delayed terminal differentiation of the 32Dcl.3 myeloid cell line, indicating a regulatory motif in distal Runx1 abrogates the delay of terminal differentiation induced by proximal Runx1. Deletion of amino acids 3’-8’ (Δ3-8) or mutation of amino acids serine 3’, serine 5’ and phenylalanine 7’ of the distal Runx1 N-terminus reduce Runx1 expression in the 32Dcl.3 cell line. The N-terminus motif, runt domain and nuclear matrix-targeting sequence of Runx1 modulated Ets1 activity on the KIR3DL1 bidirectional promoter element. The transcription factor YY1 promotes both forward and reverse activation of the KIR3DL1 bidirectional promoter element dominantly in the presence of Runx1, and additively with Ets1. Distinct Runx1 proximal and distal N-termini induced phenotypes were observed in myeloid and thymocyte differentiation, but not with the KIR3DL1 luciferase assay system. This work identifies a previously unknown N-terminal regulatory motif that acts with spatio-temporal and gene target specificity to add another level of control over Runx1 activity during hematopoiesis.

The success of an oral vaccine depends upon the ability of the antigen to target the Peyer's Patches of the small intestine, uptake and presentation of the antigen to local immune effector cells, and the antigenicity of the antigen. Most proteins are poor mucosal immunogens and therefore have been coupled to a carrier molecule to target the Peyer's Patches. However, the carrier molecule must be immunologically inert so that it may be used for successive immunizations. Additionally many pathogenic bacteria are coated with carbohydrates and lipids which are weak immunogens. Therefore it is of value to develop vaccine strategies which employ proteins which mimic these antigens. These experiments investigated poly-D,L-glutamic acid for use as a delivery vehicle for oral immunization with protein antigens, and attempted to induce an idiotypic specific immune response at mucosal surfaces using idiotypic antibodies. Tetanus toxoid mimicking anti-idiotypic antibodies were produced in a horse by immunizing it with anti-tetanus toxoid antibodies isolated from immune guinea pig sera. The anti-idiotypic antibodies were affinity purified using horse anti-tetanus toxoid antibodies. These anti-idiotypic antibodies specifically inhibited the binding of labelled tetanus toxoid to immobilized horse anti-tetanus toxoid antibodies. Poly-D,L-glutamic acid produced by Bacillus licheniformis was isolated by ethanol precipitation and coupled with idiotypic antibodies for both parental and oral immunization. Horse antibodies coupled to the polymer retained antigen binding specificity as determined by binding to labelled antigen, and induced the same anti-xenotypic response as carrier free horse antibodies when injected into guinea pigs and rabbits. Idiotypic antibody-polymer complexes were packaged in enteric coated gelatin capsules, tablets or suspended in NaHCO$\sb3$ and used to orally immunize primates, guinea pigs and mice respectively. No anti-xenotypic or anti-idiotypic antibodies were detected in the serum or secretion samples collected. These studies demonstrate that poly-D,L-glutamic acid does not optimize nonspecific uptake of protein antigen by Peyer's Patches but do not rule out the use of anti-idiotypic antibodies to induce immunity at mucosal surfaces.

Includes bibliographical references / A growing body of evidence indicates that long noncoding RNAs (lncRNAs), the most abundant noncoding RNA (ncRNA) species of the pervasively transcribed mammalain genome, have functional roles in the gene regulation of an array of cellular processes. These observations have since discredited the long standing central dogma formulated by Franscis Crick in 1958, which states that genetic output is entirely conducted by protein. Recent studies collectively indicate that lncRNAs play important functional roles in the transcriptional regulation of a wide array of cellular processes. In the last year alone, a handful of studies have identified lncRNAs linc-Cox2, Lethe, PACER and THRIL as central players in host cell innate immune response against microbial infection. These discoveries and the vast numbers of uncharacterized lncRNAs identified by high-throughput nextgeneration transcriptome sequencing technologies, set a precedence for further investigation and characterization of lncRNAs in infection biology. Importantly, lncRNAs may serve as important diagnostic markers of infection as well as therapeutic targets. These aspect of lncRNAs field although extensively being explored in cancer research, have been neglected in infection biology, particularly in microbial infection. In this study, next-generation technologies were used to identify subtle vairations in transcriptional activity, with particular emphasis to lncRNA differential expression, and uncover their physiological relevance during Listeria monotocytogenes infection. To this end, an RNA-Seq dataset of Listeria-infected HeLa cells was subjected to several variations of data analysis lncRNA discovery pipelines. Potential lncRNA functioning was hypothesized using the Rinn & Chang "guilt by association" approach in which lncRNA functioning was hypothesized based on the known functions of tightly co-expressed protein coding mRNAs. "Guilty" lncRNAs were then knocked down in the HeLa cells using transcription activator-like nucleases (TALENs) to validate their candidacy as infection-regulating lncRNAs. Preliminary investigations conducted in this study have revealed potential Listeria infectionregulating lncRNA candidates. Furthermore, we explored the use of the physiologically relevant cellular model of iPSC-MDMs to validate identified lncRNA candidates. This work provides a framework for lncRNA discovery from RNA-Seq data by iterative and intergrative analysis.

Bibliography: leaves 75-90. / Absence of the TH2 inducing cytokine IL-4 has been shown not to increase the TH1 response and resistance to mycobacterial infection. This study asked whether the combined absence of IL-4 and IL3, as compared to IL-4 deficiency only, would increase resistance to an aerogenic Mycobacterium tuberculosis infection. By using IL-4 gene-deficient mice, it was confirmed that endogenous IL-4 does not reduce host immunity. In contrast, IL-4Rα genedeficient mice, which lack both IL-4 and IL-13 signalling, had reduced bacterial burden in the lungs and other organs, increased survival and cellular immunity with macrophage activation. Therefore IL-4Rα gene-deficient mice have increased resistance to Mycobacterium tuberculosis infection.

abstract: The human papillomavirus (HPV) is a double-stranded DNA virus responsible for causing upwards of 80% of head and neck cancers in the oropharyngeal region. Current treatments, including surgery, chemotherapy, and/or radiation, are aggressive and elicit toxic effects. HPV is a pathogen that expresses viral-specific oncogenic proteins that play a role in cancer progression. These proteins may serve as potential targets for immunotherapeutic applications. Engineered T cell receptor (TCR) therapy may be an advantageous approach for HPV-associated cancers. In TCR therapy, TCRs are modified to express a receptor that is specific to an immunogenic antigen (part of the virus/cancer capable of eliciting an immune response). Since HPV-associated oropharyngeal cancers typically express unique viral proteins, it is important to identify the TCRs capable of recognizing these proteins. Evidence supports that head and neck cancers typically experience high levels of immune cell infiltration and are subsequently associated with increased survival rates. Most of the immune cell infiltrations in HPV+ HNSCC are CD8+ T lymphocytes, drawing attention to their prospective use in cellular immunotherapies. While TCRs are highly specific, the TCR repertoire is extremely diverse; enabling the immune system to fight off numerous pathogens. In project 1, I review approaches to analyzing TCR diversity and explore the use of DNA origami in retrieving paired TCR sequences from a population. The results determine that DNA origami can be used within a monoclonal population but requires further optimization before being applied in a polyclonal setting. In project 2, I investigate HPV-specific T-cell dysfunction; I detect low frequency HPV-specific CD8+ T cells, determine that they are tumor specific, and show that HPV+HNSCC patients exhibit increased epitope-specific levels of CD8+T cell exhaustion. In project 3, I apply methods to expand and isolate TCRαβ sequences derived from donors stimulated with a previously identified HPV epitope. Single-cell analysis provide ten unique TCRαβ pairs with corresponding CDR3 sequences that may serve as therapeutic candidates. This thesis contributes to fundamental immunology by contributing to the knowledge of T cell dysfunction within HPV+HNSCC and further reveals TCR gene usage within an HPV stimulated population, thus identifying potential TCR pairs for adoptive cell therapies. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2020

Synthesis, storage and release of “neuronal” Acetylcholine (ACh) has long been studied in the context of neurons. In addition, the roles of ACh in neurotransmission, regulation of movement, heart rate, digestion and induction of vasodilation has been well-established. However, for decades now multiple studies have shown synthesis of ACh in non-neuronal cells, such as immune cells. Synthesis of ACh catalyzed by cholinesterase (ChAT) enzyme within CD4+ and CD8+ T cells in the context of viral infection has been reported. Furthermore, it has been reported that ChAT expression is induced in CD4+ and CD8+ T cells during chronic viral infection in an interleukin (IL)-21 dependent manner. Lastly, studies have demonstrated that deletion of ChAT within T cells abolishes vasodilation, and that ChAT expression is required to control chronic viral infection. However, ChAT production by CD8+ T cells in the context of acute viral infection is not well-understood. Furthermore, the kinetics of ChAT production and ACh release following acute viral infection, remains unknown. Here we show that ChAT production by CD8+ T cells is only induced following acute viral infection with Lymphocytic choriomeningitis virus (LCMV). Our results also indicate ChAT production peaks and decreases overtime throughout the course of the infection. In addition, if we introduce cognate peptide after viral clearance, production of ChAT by CD8+ T cells remains constant. Our data demonstrates that after rechallenge of CD8+ effector memory T cells the upward and downward trend in ChAT production over time does not change. Previous work from our lab has demonstrated viral infection induces expression of CX3C chemokine receptor 1 (CX3CR1) on CD8+ effector T cells and have identified 3 antiviral CX3CR1 CD8+ effector T cell subsets. We found that CX3CR1high CD8+ effector T cells remained in circulation and discovered expression of ChAT in all 3 subsets. In this study, we show the kinetics of ChAT expression in CX3CR1 CD8 effector T cells subsets following acute viral infection, peptide stimulation, and rechallenge in the memory phase. We found that most antigen-experienced CD8+ T cells producing ChAT are CX3CR1high. In parallel, we analyzed diastolic blood pressure (diastolic BP) in the animals as way of indirectly measuring vasodilation and found that a decrease in blood pressure after stimulating antigen-experienced cells with cognate peptide. Additionally, we attempted to measure vasodilation in vivo by intravascular imaging, however we only found pulsing of arterioles following antigen stimulation. We also measured ACh release in serum, but due technical issues, we were not able to reach conclusive results. Finally, we detected expression of cholinergic/acetylcholine receptor M3 (Chrm3) in arteriolar and post-capillary venular endothelial cells, but more expression of Chrm3 in post-capillary venules. Although our data indicates antigen-experienced CD8+ T cells might have a vasorelaxant role in response to acute infection, more work has to done to prove this hypothesis. / 2024-11-30T00:00:00Z