Regulatory T cells essential to prevent the loss of self-tolerance in murine models of erythrocyte-specific autoantibody responses.

The spontaneous appearance of anti-erythrocyte autoantibodies resulting in autoimmune hemolytic anemia described in NZB mice more than 40 years ago provided a model for the study of mechanisms behind the loss of self-tolerance. We developed an in vitro model of this anti-MRBC response in which CD8(+) suppressor T cells were shown to be a controlling element. CD8(+) T cells from young NZB mice co-cultured with spleen cells from old, actively autoimmune NZB mice suppressed the anti-MRBC responses of the old mice. Eliminating the CD8(+) cells from young NZB spleen cells or even from non-autoimmune BALB/c spleen cells prior to culture removed the controlling influence of these CD8(+) cells and allowed the development of anti-MRBC-secreting cells. This review will consider the role of the CD8(+) suppressive cells in the anti-self-erythrocyte model in light of insights provided by current 'regulatory T cell' literature.

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-gamma) 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-gamma 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-gamma 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-gamma plays in protection against viral infections.

Different response requirements for IFNgamma production in ELISPOT assays by CD4+ T cells from mice early and late after immunization.

Antigen specific immune responses that occur early after antigen exposure differ from those that are present late in the response. The present study focused on detecting changes in production of IFNgamma by CD4+ T cells over time during chronic antigen exposure. (C3HxCB17)F1 mice were primed with recombinant hepatitis B core antigen (rHBcAg) in incomplete Freund's adjuvant to allow persistent antigen exposure. To assay the CD4+ T cell response to HBcAg, splenocytes from immunized mice were restimulated with rHBcAg for 24 or 48 h in vitro and tested for IFNgamma and IL-5 secreting cells by ELISPOT. Results showed that early after antigen exposure (7 days for primary and 3 days for secondary exposures), the maximal number of IFNgamma secreting cells was detected in the ELISPOT after 24 h of restimulation. However, late after antigen exposure (28 days for primary and 14 days for secondary exposures), the maximum number of IFNgamma secreting cells was not detected until 48 h of restimulation in this assay. This delay in IFNgamma production was related to the availability of IL-2, since addition of IL-2 allowed the delayed cells from late responses to develop peak IFNgamma production in vitro by 24 h, equivalent to that of cells from early responses. This IL-2 dependent delay occurred in Th1-type IFNgamma responses but not in Th2-type IL-5 responses. These observations indicate that, when detecting IFNgamma secreting cells it is important to screen responses at different times of restimulation or in the presence and absence of IL-2 to ensure optimal detection. This approach should prove critical, particularly when evaluating patients with chronic infections and in determining the effectiveness of vaccines since these deal with both early and late responses.