A number of ultraviolet light-induced tumors that do not grow progressively in 3- or 9-month-old animals grow progressively in animals starting at approximately 15 months of age. We investigated the immune response to one such tumor, 6130, in young and old mice to determine in nature of this breakdown in immune protection with age. The 6130 tumor cells that grow progressively in 22-month-old animals still retain sensitivity of cytotoxic T lymphocytes (CTLs) generated against the highly immunogenic "6130-A" antigen and consequently are still immunologically rejected when transplanted into 3- and 9-month-old animals, but not in mice older than 15 months. Investigation of the CD8+ CTL response found that in animals 3-12 months old, primary and secondary tumor-specific CTL responses to 6130 can be generated in vivo and in vitro. On the other hand, the CD8+ CTL responses to 6130 seen in animals 15 months of age or older were significantly depressed or absent. These results indicate that a decline in antigen-specific CD8+ CTL is the major cause of increased susceptibility to 6130 tumor growth in older animals.

Ly-6A is a murine antigen which is implicated in lymphocyte activation and may be involved in activation of hematopoietic stem cells. Antibody cross-linking studies and antisense experiments have suggested that Ly-6A is a lymphocyte coactivation molecule. To better understand the function of Ly-6A, we used gene targeting to produce Ly-6A null mice which are healthy and have normal numbers and percentages of hematopoietic lineages. However, T lymphocytes from Ly-6A–deficient animals proliferate at a significantly higher rate in response to antigens and mitogens than wild-type littermates. In addition, Ly-6A mutant splenocytes generate more cytotoxic T lymphocytes compared to wild-type splenocytes when cocultured with alloantigen. This enhanced proliferation is not due to alterations in kinetics of response, sensitivity to stimulant concentration, or cytokine production by the T cell population, and is manifest in both in vivo and in vitro T cell responses. Moreover, T cells from Ly-6A–deficient animals exhibit a prolonged proliferative response to antigen stimulation, thereby suggesting that Ly-6A acts to downmodulate lymphocyte responses.

Fimbriae from Porphyromonas gingivalis are believed to play an important role in the pathogenesis of periodontal diseases. The aim of the present study was to identify the fimbrial protective T-cell epitopes in CBA/J mice. A truncated protein corresponding to amino acids 1 to 198, PgF1-198, was generated and allowed us to demonstrate that the N terminus of the protein contains T-cell epitopes. With synthetic peptides, an immunodominant sequence was identified between amino acids 103 and 122. The corresponding peptide, PgF-P8, induced T-cell proliferation after in vitro restimulation of in vivo-primed cells, giving a stimulation index comparable to the one obtained with r-fimbrillin, and induced production of both Th1 and Th2 cytokines. Growth supernatant contained significant levels of interleukin 2 (IL-2), gamma interferon, IL-4 (28 pg/ml), and tumor necrosis factor alpha. Immunization of mice with r-fimbrillin, PgF1-198, and PgF-P8 induced production of antibodies specific to r-fimbrillin and PgF-P8. In addition, by using the mouse chamber model we found that mice immunized with PgF-P8 were dramatically protected against a normally lethal injection of P. gingivalis. Animals immunized with PgF-P8 40 days prior to challenge showed a 60% survival rate when challenged with P. gingivalis, compared with just 25% survival in control animals and just 5% survival in mice immunized with PgF-P8 only 21 days prior to challenge. Although the protection depended on the time of immunization before the bacterial challenge, it did not correlate with in vivo local cytokine production (IL-2, IL-4, IL-6, tumor necrosis factor alpha, and gamma interferon), specific antibody levels, or the isotype of anti-PgF-P8 antibodies produced.

Fimbrillin is the major subunit protein of fimbriae from the human periodontal pathogen Porphyromonas (Bacteroides) gingivalis. We describe here the generation and initial characterization of recombinant fimbrillin (r-fimbrillin) isolated from P. gingivalis 381. A fragment of DNA encoding the gene for fimbrillin was generated by polymerase chain reaction and cloned into the expression vector pET11b. Plasmids containing the recombinant gene were transfected into Escherichia coli. Clones were selected on plates for ampicillin resistance and individually screened by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein production after activation with IPTG (isopropyl-beta-D- thiogalactopyranoside). One clone, OW0.2, produced significant amounts of a 42-kDa protein after induction with IPTG. This clone contained the pET11b plasmid with a 1-kb insert that had sequence homology to the gene encoding fimbrillin. The majority of recombinant protein from clone OW0.2 was found in the cytoplasm within inclusion bodies. Protein aggregates were solubilized in 8 M urea, and SDS-PAGE analysis showed two major protein bands, one at 42 kDa and the other at 17 kDa. These two proteins coeluted from a DEAE-Sepharose column at 0.15 M NaCl and were reactive to rabbit antiserum to fimbrillin in a Western blot (immunoblot). A preparation giving a single protein band at 42 kDa in SDS-PAGE was obtained by size fractionation by using continuous-elution electrophoresis. Lymph node cells from animals immunized with either fimbrillin from P. gingivalis or r-fimbrillin showed antigen-specific proliferation to both P. gingivalis fimbrillin and r-fimbrillin in an in vitro recall assay. Therefore, it appears that r-fimbrillin is chemically, antigenically, and serologically identical to fimbrillin isolated from P. gingivalis 381.