Catherine E. Calkins

Rabies virus glycoprotein as a carrier for anthrax protective antigen.

Abstract Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen was also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems.

T Cells Infiltrating the Skin of Tsk2 Scleroderma-Like Mice Exhibit T Cell Receptor Bias

The T cell repertoire expressed by Tsk2 mice, a novel experimental model of systemic sclerosis, was examined to determine whether cells infiltrating the areas of involved skin exhibit a T cell receptor (TCR) bias. Reverse transcription-polymerase chain reactions (RT-PCR) were conducted using RNA extracted from lymph nodes and skin from TSk2 mice and from normal mice, with an oligonucleotide primer library specific for the variable region of the TCR (beta) chain. RT-PCR signals were observed in all lymph node cell (LNC) samples from both Tsk2 mice and control mice, with eighteen of the twenty-one Vbeta types present. In contrast, cDNA extracted from areas of involved skin from Tsk2 mice exhibited a restricted pattern, with positive Vbeta signals corresponding to eight T cell subtypes (Vbeta1, 6, 8.1, 8.2, 10, 11, 16, and 18). Band strength analysis revealed that three Vbeta subtypes dominated within this restricted pattern (Vbeta8.1, 11, and 18). Moreover, this pattern of Vbeta bias was consistent among the four skin samples from different Tsk2 mice. These data suggest that a restricted T cell population participates in the inflammatory cell infiltrate of Tsk2 skin.

Transient inhibition of Th1-type cytokine production by CD4+ T cells in hepatitis B core antigen immunized mice is mediated by regulatory T cells

The non‐cytopathic hepatitis B virus (HBV) can induce chronic infections characterized by weak and limited T cell responses against the virus. The factors contributing to the failure to clear HBV and subsequent development of chronic HBV infections are not clearly understood, but a strong interferon‐gamma (IFN‐γ) response by CD4+ T cells against the nucleocapsid hepatitis B core antigen (HBcAg) of the virus appears to be important for viral clearance. The present study documents depressed numbers of CD4+ T cells secreting IFN‐γ and interleukin‐2 (IL‐2) in enzyme‐linked immunospot assay (ELISPOT) assays restimulated for 24 hr with antigen following both primary and secondary immunizations of mice with recombinant hepatitis B core antigen (rHBcAg). The kinetics of these responses showed that the depression occurred following a peak response and lasted approximately 2 weeks before returning to the previous peak levels. The depression was abrogated by depletion of CD25+ cells prior to culture in the ELISPOT assay, suggesting inhibition by regulatory T cells. This inhibition of IFN‐γ and IL‐2 production was also reversed by in vitro restimulation of the test cells for 48 hr rather than 24 hr in the assay. No such transient, reversible inhibition was detected in the production of IL‐5, a Th2‐type cytokine. The inhibition in cytokine production did not appear to correlate with the number of antibody‐secreting cells or the isotypes produced. This delay by regulatory T cells of Th1‐type cytokine production could contribute to viral persistence in chronic HBV infection by interfering with the critical role IFN‐γ plays in protection against viral infections.

Demonstration of active suppressor cells in spleens of young nzb mice.

Abstract NZB mice, a strain prone to the development of autoimmune disease, have during the first 2 weeks of life suppressor cells in their spleens which can in coculture with adult spleen cells suppress the antibody response to sheep red blood cells (SRBC) generated in culture by the adult cells. The suppressive activity of spleen cells from NZB mice in the first week after birth is similar to that of spleen cells from 4-day-old C57BL/6 mice, a strain which does not spontaneously develop autoimmune disease. As in “normal” strains of mice, suppressor cell activity in NZB mice is diminished at 2 weeks and undetectable at 3 weeks of age. The data indicate that there is no defect inherent in the suppressor cells detected in the spleens of newborn and young NZB mice and suggest that the development of autoimmune responses does not result from a lack of suppressor cells in the young animals.

Effect of antigen priming on T-cell suppression. I: Activity of Ly 1+2+ feedback suppressor T-cell precursors after isolation from competing Ly 2− T cells

Previous experiments have demonstrated that feedback suppression of murine antibody responses occurs in vitro after exposure of unprimed T-cell subsets to suppression-inducing signals from primed cells, resulting in suppression of primary and secondary IgM as well as IgG anti-SRBC responses. However, following priming with antigen when cells appear which are capable of inducing feedback suppression, the ability of unfractionated splenic T-cell populations to mediate detectable feedback suppression in vitro rapidly disappears, suggesting that priming alters the expression of feedback suppression at the same time as providing for its induction. In the present study, we have succeeded in isolating active feedback suppressor T-cell precursors (preTs) in the Ly 1+2+ and L3T4- T-cell populations from SRBC-primed as well as from unprimed mice, demonstrating that preTs are not lost after priming. The preTs isolated from primed mice resemble those isolated from unprimed mice in Ly and L3T4 phenotype, cell dose requirements, kinetics, level of suppression, and requirement for in vitro activation by primed cells. These results imply that antigen priming neither significantly depresses nor enhances the ability of Ly 1+2+ preTs to participate in feedback suppression and that activated suppressor effector cells are not detectable in the Ly 1+2+ splenic T-cell subset. Priming does, however, induce an enhancing activity in Ly 2-, L3T4+ T cells which appears to compete with feedback suppression and thus may account for the absence of detectable feedback suppression when unfractionated T cells from primed mice are the only source of preTs.

Formation of antigen specific foci as a complement independent assay for individual antibody-secreting cells

Antibody-forming cells (AFC) could be demonstrated by the binding of antigen to secreted antibody localized around the AFC. Using sheep red blood cells (SRBC) as antigen, this antigen binding was detected as foci of erythrocytes surrounding individual lymphocytes. Focus formation was antigen specific and involved the active secretion of antibody. AFC specific for antigens resistant to lysis, such as human red blood cells were also demonstrable with this assay. The focus assay provides a complement independent method for enumerating antibody-forming cells.

Detection of antibody-induced feedback suppression in spleen cell populations of primed and unprimed mice.

Feedback suppression has been proposed as a natural mechanism of regulating immune responses. Both activated T and B cells, and their products have been shown to induce feedback regulatory circuits by interacting with T cells from normal, unprimed mice (1–4). If feedback suppression is a regulatory mechanism that occurs in vivo to down-regulate antibody responses, it would require that the antigen-primed host be able to generate feedback suppression in the absence of the exogenous unprimed T cells used to detect suppression in the experimental models. This study reports the results of experiments designed to investigate whether all the cells participating in the feedback suppression circuit are present in the antigen-primed host.