This study examines the possible mechanisms by which radiation and the bacterial toxin, Tetanus toxoid (TT), suppress the murine splenic mononuclear cell (SMNC) response to mitogen. This study demonstrates that the lymphocyte proliferation response of SMNC to the mitogenic lectin PHA can be suppressed by TT in a dose-dependant manner in vitro, without affecting the viability of the cells, in the anti-proliferative concentrations used (0.5-5 $\mu$g/ml). SMNC pre-incubated with TT could suppress the pHA blastogenic response of fresh autologous cells during co-incubation suggestion the involvement of activated suppressor cells. Flow cytometric analysis demonstrated that TT does not produce an alteration in the cellular balance, indicating that the suppression would appear to be dependant upon a change in T cell function. TT down-regulated the expression of class II MHC antigens on antigen-presenting cells which may represent an inappropriate costimulatory signal required for T cell activation. Whole body irradiation has been reported to induce active immune suppression. In the present study, ionizing radiation (0-700 cGy) produced decreased spleen cellularity and decreased ability of surviving SMNC to respond to mitogen. There was no evidence, however, to indicate that irradiation (100 cGy) activated suppressor cells during the first 7 days post-irradiation. Similarly, radiation did not seem to interact with TT to increase the amount of TT-induced suppression. (Abstract shortened by UMI.)

HIV infection is associated with characteristic quantitative changes in T-lymphocyte subsets: progressive depletion of the CD4$\sp{+}$ T-lymphocytes and an increased number of the CD8$\sp{+}$ T-lymphocyte. In this study, I report the results of a flow cytometric analysis of the expression of CD3, CD4, CD8, TCR$\alpha$/$\beta$, and TCR$\gamma$/$\delta$ antigens. I observed that CD8$\sp{+}$TCR$\alpha$/$\beta\sp{+}$ cells increased early in HIV disease (p 0.01) whereas the frequency of CD4$\sp{+}$TCR$\alpha$/$\beta\sp{+}$ cells was relatively unchanged. The frequency of TCR$\gamma$/$\delta\sp{+}$ cells remained unchanged. However, the mean fluorescence intensity (MFI), reflecting surface antigenic density, varied and allowed a clear distinction among different stages of infection. The expression of three activation markers (HLA-DR, CD38, CD57) was clearly increased in HIV infected individuals. The TCR$\alpha$/$\beta$ subset showed more substantial variation for activation markers. In the TCR$\gamma$/$\delta$ subset, the CD57 antigen seemed to be the most affected by the state of the disease and showed the greatest increase (p 0.01). (Abstract shortened by UMI.)

The model pathogen used in the development of the anti-idiotypic antibodies produced in this project was enterovirus-70. This virus is the causative agent of acute hemorrhagic conjunctivitis. In the past twenty-five years, this virus has been responsible for two worldwide pandemics of acute hemorrhagic conjunctivitis. Monoclonal antibodies (MAbs), directed against the prototype enterovirus-70 strain, J670/71, were generated and characterized in order to produce a monoclonal anti-idiotypic antibodies (MAb2s) for use as surrogate immunogens. Radio-immunoprecipitation and western immunoblot assays suggested that all the monoclonal antibodies recognize conformational epitopes on the virion surface. A neutralizing monoclonal antibody, MAb/ev-12, was selected for the production of MAb2s. Five MAb2s were selected for their capacity to inhibit the interaction of MAb/ev-12 with EV-70 in dot immunobinding inhibition and immunofluorescence assays. These five MAb2s also inhibited virus neutralization mediated by MAb/ev-12 suggesting that each recognizes a paratope associated idiotope. In competition enzyme immunosorbent assays, none of the five MAb2s recognized other neutralizing and non-neutralizing enterovirus-70 specific MAbs, thus demonstrating that the MAb2s were specific for private idiotopes. Immunization with each of the MAb2s was carried out for the production of anti-anti-idiotypic antibodies (Ab3). All five MAb2s induced an immune response. Moreover, results suggested that they share idiotopes since MAb2:MAb/ev-12 binding could be inhibited by homologous as well as heterologous Ab3. Ab3 sera were shown to possess antibodies capable of immunoprecipitating $\sp{35}$S-labelled viral proteins in the same manner as MAb/ev-12. Nine of fifteen mice immunized with MAb2s demonstrated Ab3 neutralizing activity specific for the prototype EV-70 strain, J670/71. The potential application of MAb2s to serve as surrogate immunogens for conformational epitopes is substantiated by the results presented in this report.

To determine whether HLA-A,B antigens on platelets are integral membrane constituents or simply represent adsorbed plasma proteins, the degree to which they are adsorbed, and the relative ease with which they elute off platelet membranes was studied using various treatments known to elute passively adsorbed membrane proteins. In addition, this question was investigated at the RNA level using phytohemagglutinin stimulation to determine whether platelets have the capacity to respond with de-novo synthesis of HLA antigens and by enzymatic amplification of platelet derived mRNA to attempt to demonstrate the presence of nascent message encoding for these antigens. HLA antigen present on platelet membranes did not elute when platelets were incubated in autologous plasma or in plasma from homologous, antigen negative donors. When HLA-A2 negative platelets were incubated in HLA-A2 positive plasma a small amount of HLA-A2 antigen was detectable indicating that platelets possess the ability, to a limited extent, to absorb HLA antigen from the plasma, in vitro. The results indicate that the majority of HLA antigen present on platelet membranes can be selectively eluted without a concomitant loss of known integral membrane proteins such as GPIIIa. These findings argue in favor of the existent hypothesis that HLA antigens are absorbed platelet membrane proteins. (Abstract shortened by UMI.)

My objective was to establish the conditions necessary to measure cytotoxic T lymphocyte (CTL) activity. The approach was to clone peripheral blood mononuclear cells (PBMC) from HIV-1$\sp+$ asymptomatic patients to obtain CD8$\sp+$ clones and test these cells against autologous CD4$\sp+$ T lymphoblasts. I also tested the following enhancers of viral infectivity: DEAE-Dextran (DD), Polybrene and Tumor Necrosis Factor-Alpha (TNF$\alpha$) in normal T cells and HUT-78 cells and found that DD greatly increases infection when used as pretreatment and during infection at 10 $\mu$g/ml. Polybrene (2.5-5 $\mu$g/ml) also increases infection when used as pretreatment and during infection but not as much as DD. It proved to be less toxic than DD and would be useful when a slow, less acute infection is desired. The effect of TNF $\alpha$ at 5 ng/ml was not noticeable in normal T cells the first few days after the infection but the infection increased six or seven days later. TNF$\alpha$ did not have any significant effect on the HUT-78 cell line. The improved infection protocol with the use of enhancers will be useful in the production and maintenance of high titer virus stocks in the laboratory and in the infection of sensitive target cells for CTL assays. (Abstract shortened by UMI.)

To determine if blood monocytes from HIV-1 seropositive patients contain HIV-1 antigen and genome, we separated monocytes and T cell subsets using monoclonal antibodies (mAbs) conjugated to magnetic beads and by monocyte adherence to glass. We found: (1) Monocytes cultured without depletion of CD4$\sp+$ T cells (11 of 11 patients) were HIV-1 antigen positive and showed dramatically increased spontaneous formation of MGCs. (2) Monocytes cultured after depletion of CD4$\sp+$ T cells (3 experiments) were HIV-1 antigen negative and MGC formation was markedly decreased. (3) In 14 subsequent patients analyzed by PCR, all patients were positive for HIV-1 proviral DNA in cells enriched for CD4$\sp+$ T cells. In 11 of 14 patients (79%), the monocyte fractions were HIV-1 proviral DNA negative, while in the remaining 3 patients, the monocytes were positive for HIV-1 proviral DNA. In conclusion, the major reservoir for HIV-1 infection in human peripheral blood is CD4$\sp+$ T cells (14 of 14 cases). Fresh blood monocytes from HIV-1 seropositive patients were HIV-1 proviral DNA negative in 11 of 14 cases (79%). Blood monocyte-derived macrophages from HIV-1 seropositive patients may acquire HIV-1 infection in vitro from contaminating infected CD4$\sp+$ T cells. The pathogenic and clinical significance of HIV-1 infected monocytes (21% of patients) remains to be determined. (Abstract shortened by UMI.)

Most conventional vaccines are administered parenterally (e.g., by intramuscular (IM) or intradermal (ID) injection) and induce systemic but rarely mucosal immunity. Novel vaccination strategies capable of inducing both systemic and mucosal immune responses could greatly reduce infection and morbidity. One such strategy is DNA vaccination whereby the antigen is synthesized in vivo after direct introduction of its encoding sequences. In this thesis, we show that the route of administration of plasmid DNA encoding the hepatitis B surface antigen (HBsAg) influences the strength and nature of immune responses in mice and non-human primates. Mucosal immunization using plasmid DNA in saline results in no or weak immune responses. Formulation of DNA with lipid increases levels of reporter gene expression, but apparently not sufficiently to raise immune responses against expressed antigen indicating that other factors are involved. The strong immune responses induced after parenteral administration of DNA appears to be partly due to the adjuvant effect of unmethylated immunostimulatory CpG motifs present in the DNA backbone. Synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG) are potent adjuvants for induction of Th1-like systemic immune responses against parenterally-delivered proteins. Herein, the adjuvanticity of CpG ODN relative to or in combination with cholera toxin (CT), Escherichia coli heat-labile enterotoxin (LT), the B subunit of CT (CTB) and a non-toxic derivative of LT (LTK63) was evaluated with intranasal delivery. We also evaluate the potential of administering immunostimulatory complexes (comprised of HBsAg complexed with antibodies against HBsAg) by a mucosal route and determine whether immune responses are modulated by using CT or CpG as adjuvants. We demonstrate that CpG ODN, CT and LT augment anti-HBs titers equally, and this is more than with CTB or LTK63. CpG ODN acts synergistically with CT and LT but not CTB or LTK63, however for all combinations, CpG induces a more Th1-like response. The mucosal immune response induced is not restricted to the site of application but is also present at distant mucosal surfaces. These studies may lead to the development of novel human vaccines that protect at the level of entry for mucosal pathogens and may thereby prevent many infectious diseases.

The interaction of CD44 with its ligand hyaluronan may play a vital role in many biological processes including leukocyte homing, activation and effector function, hematopoiesis as well as tumor formation and metastasis. This phenomenon may be influenced by the extracellular matrix molecules and cytokines present in the microenvironment. In this study, I investigated the regulation of CD44-HA interactions in normal human B cells and in a panel of B cell lines including: Epstein-Barr virus (EBV)-positive (+) and EBV-negative (-) Burkitt's lymphoma (BL) and lymphoblastoid B cell lines (B-LCL) generated by in vitro EBV-transformation of normal human B cells. Activation of purified B cells with bacterial lipopolysaccharide (LPS), pokeweed mitogen (PWM) or anti-IgM antibodies in the presence or absence of interleukin (IL)-2 or IL-4 failed to induce HA adhesiveness. Stimulation of B cells with PMA however, induced strong HA recognition. Amongst a variety of cytokines that influence B cell activation, proliferation and differentiation, only IFN-gamma and to some extent IL-4 inhibited PMA-induced CD44-HA interactions. IL-13, which shares components of the IL-4 receptor complex and exhibits many biological effects similar to that of IL-4, failed to inhibit HA recognition. EBV infection/transformation of B cells, an alternative method of B cell activation, enhanced the expression of CD44 H, and induced a number CD44 V isofoms but abrogated their ability to bind HA in response to PMA. Stimulation with PMA induced strong HA recognition in the EBV-positive BL cell line BL-30/B95-8, whereas LPS, Staphylococcus aureus Cowan strain I (SAC) or PWM failed to induce HA adhesion. Investigation of the effect of a similar panel of cytokines revealed that in contrast to its inhibitory effect in normal human B cells, IL-4 was capable of inducing HA recognition in BL30/B95-8 cells. IL-13, again, failed to influence HA recognition. In contrast, the ability to recognize HA following PMA or IL-4 stimulation was not observed in B-LCL cells, highlighting the stark contrast between BL30/B95-8 and B-LCL cells. To understand the effect of EBV on CD44 expression and HA recognition, we investigated a panel of EBV- and EBV+ B cell lines. EBV- BL cells failed to express CD44 and hence did not adhere to HA. A remarkable heterogeneity was revealed by the study of various EBV+ B cell lines with respect to CD44 isoform expression and their ability to recognize HA. BL-30/B95-8, Jijoye, IM and all the B-LCLs tested expressed CD44 H and CD44 V isoforms; yet, Jijoye and B-LCLs failed to recognize HA in response to PMA. In conclusion, these results establish that HA recognition and CD44 isoform expression are modulated as a consequence of the mode of human B cell activation, differentiation and/or state of transformation. (Abstract shortened by UMI.)

Multiple sclerosis (MS) is believed to be an autoimmune, inflammatory, demyelinating disease of the central nervous system (CNS), resulting in myelin degradation, loss of the myelin forming cell, the oligodendrocyte (ODC), and axonal degeneration. The hypothesis underlying this work is that gammadelta T cells play a distinct role in MS pathogenesis by initiating, perpetuating, or regulating the immune response directed against the myelin/ODC unit. Initial comparative experiments utilising short term gammadelta T cell lines from peripheral blood (PB) and cerebrospinal fluid (CSF) of MS and other neurological disease (OND) patients, indicated no significant gammadelta subtype or cytotoxicity differences between cells derived from PB and CSF compartments or between MS and OND patients. During the course of these studies, it became apparent that the variably short in vitro lifespans of PB and CSF gammadelta T cells represented a major limitation that hampered further comprehensive studies. Efforts to increase their culture lifespans through restimulation regimens were only marginally successful, so an alternate immortalisation strategy using Herpesvirus saimiri (HVS) was employed. This study reports for the first time, the successful HVS growth transformation of human gammadelta T cells derived from both MS PB and CSF. Multiple HVS transformed PB and CSF gammadelta T cell (t -gammadelta T cell) lines and clones were generated, and have existed in IL-2 dependent culture for periods in excess of 2.5 years without the need for additional stimulation. Comparative analysis of a series of t-gammadelta T cell lines and their personal non-transformed lines indicated the transformation process did not alter their surface molecule expression, cytokine, or cytotoxicity responses. Cell surface characterisation of the t-gammadelta cell lines and clones demonstrated HVS was capable of immortalising a wide spectrum of gammadelta TcR subtypes, along with identifying t -gammadelta T cell clones displaying rare gammadelta T cell phenotypes, not commonly studied. MS t-gammadelta T cells uniformly expressed proinflammatory cytokine profiles (IFN-gamma, TNF-alpha), but only minimal IL-2 or anti-inflammatory IL-4 and IL-10. All gammadelta TcR subtypes displayed identical cytokine pattern expression, suggesting that cytokine expression is independent of gammadelta T cell subtype. t-gammadelta T cell lines demonstrated a graded cytotoxicity towards a panel of tumour cell targets, ranging from high (U937, Jurkat) to moderate (KG-1) to poor (Colo-205). Similar killing patterns of tumour targets were observed for subtype specific t-gammadelta T cell clones, and antigen recognition studies indicated a graded recognition of tumour cell ligands, together suggesting that both cytolytic function and tumour cell recognition, as was seen with cytokine profiles, are independent of gammadelta T cell subtype. In contrast, only Vgamma9Vdelta2 positive t-gammadelta T cells responded to candidate non-peptidic phospholigand and alkylamine antigens. No MS t-gammadelta T cell reactivity was observed to putative MS antigens myelin basic protein, alphaB-crystallin, or Chlamydia pneumoniae. (Abstract shortened by UMI.)

We have examined CD8+ sub-populations to determine whether these subsets are critical to the production of CD8+ T cell nonlytic factors. The production of the beta-chemokines MIP-1alpha, MIP-1beta and RANTES, and the chemoattractant cytokine IL-16 were measured in cells derived from 24 HIV-infected and 25 uninfected subjects. Asymptomatic HIV+ subjects (CD4 > 200/ul) produced significantly higher levels of MIP-1alpha and MIP-1beta from CD8+ T cells and some sub-phenotypes. Higher RANTES levels were produced by CD28-, CD38- and HLA-DR+/- sub-phenotypes. However, IL-16 was only modulated in the CD38+ subset in comparison to total CD8+ T cells. Infection of CD8+ T cells and sub-populations resulted in generally increased levels of chemokine and IL-16 production, which dissipated over a 15 day time course. Moreover, CD8+ antiviral factor (CAF) activity, another major component of CD8+ T cell nonlytic suppression factors, was not associated with chemokine production. However, significantly higher levels of CAF were produced by CD38+ and HLA-DR+ sub-populations. In addition, we also showed that in CD8+ T cell populations, the production of MIP-1alpha, MIP-1beta and IL-16 was inversely correlated with virus copy number. These findings shed light on the noncytotoxic responses of CD8+ T cells in controlling the natural course of HIV infection.