In vivo kinetics and characterization of IFN-gamma-producing cells during a thymus-independent immune response.

Using immunohistochemical techniques, we studied IFN-gamma-producing cells (IFN-gamma-PC) in vivo during immune responses to thymus-independent type-2 (TI-2) Ag. Detection of IFN-gamma-PC in cryostat sections of spleen-tissue was performed with an enzyme labeled mAb directed against IFN-gamma. After TNP-Ficoll immunization, IFN-gamma-PC and TNP-specific antibody-forming cells (TNP-AFC) displayed similar kinetics reaching a maximum number at day 5 to 7. The IFN-gamma-PC were localized in the same compartment as TNP-AFC and a part of them in juxtaposition to TNP-AFC. Immunization with other TI-2 Ag resulted also in a significant increase of the number of IFN-gamma-PC. In a parallel experiment we found both in vivo and in an ELISA-spot assay a significant increase of the number of IFN-gamma-PC and IFN-gamma-spot-forming-cells, respectively, in spleens of mice 6 to 7 days after TNP-Ficoll immunization. Double staining of spleen sections for IFN-gamma and surface Ag revealed that 5 to 7 days after TNP-Ficoll immunization, +/- 40% of the IFN-gamma-PC expressed the MT4 Ag (CD4), +/- 50% the Lyt-2+ Ag (CD8) and +/- 10% the asialo-GM1 Ag (NK cell). This study represents the first description of the in vivo activity and characterization of IFN-gamma-PC during a TI-2 immune response. Moreover, the presented data confirm suggestions from in vitro investigations that IFN-gamma and T cells may play a direct role in the in vivo regulation of a primary immune response against a TI-2 Ag.

Immunocytochemical determination of antigen and epitope specificity of HIV-1-specific B cells in lymph-node biopsies from HIV-1-infected individuals.

Knowledge about B-cell dysfunction and HIV-specific antibody production is necessary for the understanding of both HIV-1-related immunopathology and the (vaccine-induced) humoral immunity involved in protection against AIDS. This paper describes the application of recently developed methods to detect epitope specificity of B cells in lymph-node biopsies with antigen-enzyme conjugates. Cryosections of five lymph-node biopsies from HIV-1-infected individuals and four control tissues were stained with a panel of HIV-1 antigen-enzyme conjugates: recombinant HIV-1 proteins (gp 160, gp 120 and p24), labelled with peroxidase, and synthetic peptides representing neutralizing epitopes from gp120 and gp41, labelled with alkaline phosphatase. Antibody-forming cells (AFCs) were detected in all the HIV-1-infected biopsies with gp160, gp120 and/or p24, in numbers up to 350 per section. AFCs producing specific antibodies against peptide 101 (SP 101), representing the neutralizing epitope 586-608 of gp41, were detected in one patient. These techniques allow correlation of in vivo function of B cells with lymph-node pathology, clinical stage of the disease and serological data. Their potential for the elucidation of HIV-related immunopathogenesis and the development of vaccines is discussed.

Synthesis of fusion proteins with multiple copies of an antigenic determinant of foot-and-mouth disease virus.

A series of four expression plasmids coding for fusion proteins containing foot-and-mouth disease virus (FMDV) sequences was constructed. The fusion proteins contain a large part of beta-galactosidase from Escherichia coli preceded (N-terminal) by 1, 2, 4 or 8 repeats of the antigenic determinant of FMDV consisting of amino acids 137-162 of the capsid polypeptide VP1. All four fusion proteins were efficiently produced in E. coli host bacteria. Immunization of rabbits resulted in FMDV-specific, neutralizing antibodies, the response being dependent on the number of repeats. With enzyme-linked immunosorbent-assay techniques it was shown that the FMDV antigenic determinants are exposed on the surface of the fusion proteins under non-denaturing conditions.