Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Regulation of the immune response is mediated, in part, by the action of suppressor T cells (Ts). One intriguing aspect of these cells is the description of T cell suppressor factor (TsF): a soluble analog of the cell that shares many of its properties, such as the ability to bind free antigen (Ag) and suppress an Ag-specific immune response. The exact molecular nature of TsF and the relationship of TsF to Ts are unknown. The immune response to the small, bacterial protein, ferredoxin (Fd), was used as a model system to study TsF. A Fd-specific suppressor cell network has been described in mice that are genetically nonresponsive to this Ag. Previously, a soluble mediator, known as Fd11F, was found in the culture supernatant (SN) of the Ts hybridoma, Fd11. Fd11F possessed both Ag-binding activity and the ability to suppress the anti-Fd Ab response in mice. The TsF-specific monoclonal antibody, B16G, was used for both the recovery of Fd11F-enriched material from SN and its detection by the enzyme-linked immunosorbent assay. ' Further immunochemical, biological, and biochemical characterization of Fd11F was done with emphasis on describing the Ag-binding properties of Fd11F. It was found that Fd11F bound to solid- and liquid-phase Fd, and demonstrated preferential binding to the carrier determinant of the Ag. A spleen cell culture assay was devised which showed that Fd11F suppressed Ab production in a concentration-dependent manner. Additional experiments suggested that the suppressive effect was Ag-specific. The identification of the Ag-binding molecule was attempted by the fractionation of Fd11F-enriched material using high performance gel filtration or preparative SDS-PAGE (run under non-reducing conditions). Using SDS-PAGE, a unique, single polypeptide of about 30k relative molecular mass (Mr) was identified as the Ag-binding moiety of Fd11F. The possible relationship of this moiety to other identified materials is discussed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

T cell surface antigens are thought to play significant roles in immunological functions. They are involved in cellular interactions and T cell activation and proliferation. Characterization of T cell antigens is important in understanding the molecular machanisms underlying immune responses. The subject of this thesis is to characterize a novel murine T cell surface antigen called YE1/48. YE1/48, defined by two rat monoclonal antibodies YE1/48.10.6 and YE1/32.8.5, is a dimeric glycoprotein with molecular size and charge resembling the murine T cell antigen receptor α/β. It was initially detected at high levels on two T cell lymphomas, EL-4 and MBL-2. In my thesis studies, the YE1/48 antigen was characterized biochemically, a cDNA clone was isolated, and its expression in lymphoid cell populations was determined. The YE1/48 antigen was found to be distinct from the T cell receptor based on direct comparisons of their primary sequences as well as immunological analyses. It is likely a homodimer with similar or identical subunits. No homology with any known proteins could be detected, including the human T cell activation antigen CD28 (T44) which also has a similar dimeric structure as YE1/48. No function of the YE1/48 antigen could be derived from its primary sequence or with the use of the two monoclonal antibodies because the antibodies do not appear to bind to the surface of intact normal T lymphocytes. Some intriguing characteristics of the YE1/48 antigen were observed in the current studies. The YE1/48 antigen belongs to a rare group of type II membrane proteins with orientation of the amino-terminus inside the cell and the carboxy-terminus outside. The YE1/48 gene may have two alleles among different mouse strains and may belong to a multigene family. YE1/48 is expressed at low levels on a wide range of T cells with no restriction to their differentiation stages, and on spleen B cells as well as bone marrow cells. Its expression on lymphocytes is not related to activation or proliferation. However, YE1/48 expression appears to be induced at high levels by Abelson Murine Leukemia Virus-transformation of pre-B cells. Moreover, the epitopes defined by the YE1/48.10.6 and YE1.32.8.5 antibodies seem to be exposed only on three T lymphomas but not on normal T cells. It is thus tantalizing to speculate a correlation of the high level expression of YE1/48 antigen and its epitope exposure on transformed lymphocytes with cellular transformation. In summary, YE1/48 was found to be a novel T cell surface antigen which has similar dimeric structure as the murine T cell receptor α/β and human CD28 (T44). It has now been characterized biochemically, molecularly cloned, and its expression on lymphoid cells has been determined. Although the function of YE1/48 antigen remains unknown, a number of intriguing characteristics observed in the current studies have certainly called for further studies on the antigen and the determination of its function. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

DNA immunization, inoculation with an antigen-expressing plasmid DNA, is a new method for generating an antigen-specific immune response. At the time these investigations began, very little was known about the immune response produced by DNA vaccines. Thus, the first aim of our studies was to perform a detailed examination of the antibody response generated by DNA immunization with an influenza hemagglutinin (HA)-expressing DNA in BALB/c mice. Using several different routes and methods of DNA immunization, we observed a number of findings. Although all three forms of DNA immunization elicited strong anti-HA antibody responses, i.m. and i.d. saline DNA immunization required approximately 100 times more DNA than a gene gun DNA immunization to raise an equivalent titer of anti-HA antibody. Indeed, as little as one inoculation and one boost by gene gun of 0.0004 μg of DNA produced a measurable antibody response in 50% of mice. Unexpectedly, we found the isotype of the antibody response differed among groups of mice immunized by different forms of DNA immunization. Intramuscular and i.d. saline DNA immunization produced predominantly an IgG2a anti-HA antibody response, whereas gene gun DNA immunization elicited mostly an IgG1 anti-HA antibody response. Considering that IgG2a and IgG1 antibody isotypes were known to correlate with Th1 and Th2 immune responses, respectively, we analyzed the type of immune responses produced by i.m., i.d., and gene gun DNA immunization. We found that i.m. and i.d. saline DNA immunization produced a Th1 predominant cellular immune response. In contrast, gene gun DNA immunization produced a Th2 cellular immune response. The differences in the type of immune responses were found to be due to the method of DNA immunization, and not due to the route of DNA inoculation. A gene gun DNA immunization of muscle produced the same IgG1, Th2 immune response as a gene gun DNA immunization of skin, while a saline DNA immunization of muscle and skin produced mostly an IgG2a, Th1 immune response. Each method of DNA immunization created good memory Th cell responses. The type of immune response created by an initial DNA immunization remained fixed even after multiple boosts with the identical method of DNA immunization, following a boost with the alternative method of DNA immunization, or after a viral challenge. The differentiation of naive Th cells into Th1 or Th2 cells depends on a variety of factors. We performed many experiments to elucidate which factors played a role in the generation of Th1 or Th2 immune responses following saline DNA immunization and gene gun DNA immunization. DNA dose response studies revealed the use of different doses of DNA between groups of saline DNA and gene gun DNA immunized mice did not account for the differentiation of distinct Th cell subsets. Cytokine production inducible by a number of factors inherently associated with either saline DNA or gene gun DNA immunization did not affect Th differentiation. For instance, contamination of plasmid DNA with lipopolysaccharide did not account for differences in the immune response. Immunostimulatory CpG sequences did not affect Th differentiation following DNA immunization, but they did enhance the IgG2a antibody response to coinoculated HA protein. Finally, cotransfection of IFNγ or IL-4 expressing plasmids with an HA-expressing plasmid by gene gun inoculation or as a saline DNA injection did not shift the type of immune response in a Th1 or Th2 direction, respectively. Thus, it appeared that increased cytokine stimulation was not responsible for selective Th subset differentiation. One factor related to the method of DNA immunization did seem to correlate with Th1 differentiation. Deposition of plasmid DNA extracellulary by saline DNA injections (as opposed to intracellular DNA delivery by gene gun) may have stimulated Th1 immune responses. Manipulating a gene gun DNA immunization to deliver DNA to the dermis (and thus extracellularly) shifted the immune response from that of a Th2 type to a mixed Th1/Th2 type. Furthermore, evidence was gathered demonstrating that pDNA can interact with cell surface molecules and that specific sequences in pDNA can act as a ligand and bind to molecules. Taken together, our data led us to propose a new model for Th1 differentiation following saline DNA immunization. We believe extracellular pDNA binds to an APC cell surface molecule which activates the cell. The activated APC preferentially stimulates naive Th cells to differentiate into Th1 cells. Finally, studies using a variety of mice differing in their genetic backgound and MHC genotype demonstrated the generality of our findings regarding i.m. saline DNA inoculations of an HA-expressing pDNA. Saline DNA immunization produced IgG2a, Th1-predominant immune responses independent of the genetic background and MHC genotype of the mice. In contrast, the type of immune response elicited by a gene gun DNA immunization was dependent on the MHC genotype of mice. Thus the type of immune response produced by gene gun DNA immunization probably depends on the specific antigen (and its effect on MHC-peptide/TcR interaction and signaling) and is less likely due to any inherent feature associated with the process of gene gun DNA delivery.