The major finding documented here is that injury to β cells mediated by CTLs does not occur in the absence of IFN-γ (Fig. ). Further, IFN-γ is not required for the generation of LCMV-specific CTLs (Table ) and killing of target cells in vitro, and clearance of acute LCMV infection in vivo can occur in the absence of IFN-γ (21
). Thus, CTLs generated in IFN-γ-deficient mice can lyse target cells in vitro, clear virus infections in vivo, and enter the pancreas in vivo (Fig. ). A likely mechanism for the ablation of the autoimmune process and the failure of IDDM to occur in the absence of IFN-γ is the insufficient upregulation of MHC class I molecules on β cells and class II molecules on APCs, but not a defect in CTL generation or function. As a consequence, there is a lack of antigen presentation and functional CTLs are not retained in the islets. These findings provide a clear rationale for suppressing inflammatory cytokine levels like IFN-γ locally in the islets as a treatment of IDDM.
IFN-γ is a key cytokine produced by activated CTLs. It is involved in the upregulation of MHC molecules and in antiviral host defense (21
). Recent data show that IFN-γ knockout mice generate LCMV-specific primary and memory CTLs with equivalent activities as found in non-tg littermates (21
). The generation of LCMV-specific primary CTLs in IFN-γ–deficient mice terminates an acute LCMV viral infection (21
). However, when memory CTLs from IFN-γ knockout mice are adoptively transferred into persistently infected recipients, they are unable to clear the virus showing that, while IFN-γ is not required for CTL activity in vitro or control of acute infection in vivo, it is required for viral clearance of a persistent infection by CTLs in vivo (22
). The role IFN-γ plays in IDDM is likely mediated by the upregulation of MHC molecules on β cells and APCs in the islets. First, upregulation of MHC class I glycoproteins is a frequent marker during the development of IDDM (11
). Second, IDDM does not occur in the absence of MHC class I expression on β cells (25
). For example, expression of the adenoviral E3 gene complex can prevent MHC class I trafficking to the cell surface. When the E3 complex is co-expressed with LCMV proteins under the RIP in β cells, upregulation of MHC class I Db
molecules is suppressed specifically and IDDM is prevented (von Herrath, M., S. Efrat, M. Oldstone, and M. Horwitz, manuscript submitted for publication). However, upregulation of MHC expression itself in the absence of a specific (viral) trigger or in the absence of autoreactive CTLs is not sufficient for the development of IDDM (43
). Further, APCs expressing MHC class II are not found in islets of RIP LCMV IFN-γ (−/−) mice (data not shown) indicating that the lack of IFN-γ also results in a loss of infiltration by APCs that are required to propagate the autoimmune process leading to IDDM.
IFN-γ could also contribute to the autoimmune process in the pancreas by “bystander” activation of autoreactive CTLs and other inflammatory cells (48
). The recovery of lower numbers of antiself (viral) CTLs from the pancreas of IFN-γ–deficient RIP LCMV mice 60 d after infection (Table ) suggests that this may also occur. Hence, both upregulation of MHC molecules and “bystander” activation may be needed for IDDM to develop.
Recently, Kagi et al. reported that IDDM did not occur in perforin–deficient RIP LCMV transgenic mice and concluded that β cell destruction was predominantly a consequence of perforin-mediated lysis by antiself (viral) CTLs (34
). In this model, virus-induced MHC class I restricted CTLs are the key factor for the induction of IDDM. Diabetes does not occur in the absence of MHC class I expression on β cells (25
) or the absence of CD8+
). Both our own (Tishon, T., and M.B.A. Oldstone, unpublished data) and other results (49
) indicate that while perforincompetent CTLs are required to lyse target cells in vitro, they are unable to destroy β cells in vivo in the absence of IFN-γ to cause IDDM (Fig. ). Thus, the cytokine IFN-γ plays an important role in the pathogenesis of IDDM and without it, IDDM does not occur, even after an 8-mo observation period. Kagi et al (34
) reported infiltration and retention of CD8+
lymphocytes in the islets was observed in the absence of perforin although IDDM did not occur over the 2-mo observation period after LCMV infection. Whether IDDM could have occurred at later times in the absence of perforin and in the presence of IFN-γ is unknown.
It has been reported (24
) that IDDM is delayed, but not aborted, in NOD mice in the absence of IFN-γ. A likely reason why IDDM occurs in IFN-γ–deficient NOD mice, but fails to occur in the RIP LCMV tg model, is that NOD mice are usually genetically prone to spontaneously develop IDDM (50
). They express genes that convey susceptibility to IDDM, and it is likely that their β cells are more sensitive to destruction. By contrast the RIP LCMV tg mice do not spontaneously develop diabetes (6
), even after a 2-yr observation period (Tishon, T., and M.B.A. Oldstone, unpublished data).
From our data, it is unlikely that the lack of IDDM in IFN-γ (−/−) and the lower incidence of IDDM in IFN-γ (+/−) mice is due to an IDDM-protective gene linked to the IFN-γ (−/−) mutation for several reasons. First, similar kinetics of IDDM are observed in all groups of mice independent of whether F-1 backcrosses to IFN-γ (−/−) background or F-5 backcrosses to the RIP LCMV background are used (Fig. ). Second, recent work by Krakowski et al. (51
) demonstrated a protective effect of the IFN-γ (+/+) genotype for experimental allergic encephalitis (EAE), whereas the encephalitis was enhanced on the IFN-γ (−/−) background. Such findings were not mirrored in our report. Finally, and most importantly, we find less IFN-γ production by splenocytes from IFN-γ (+/−) compared to IFN-γ (+/+) splenocytes. This finding correlates with the lower incidence of IDDM in IFN-γ (+/−) mice and suggests a quantitative gene-dosage effect in IFN-γ production as the explanation for the different incidence of IDDM observed in RIP LCMV IFN-γ (+/+) and (+/−) mice.
In conclusion, a multifactorial process leads to IDDM and autoimmune destruction of β cells. The development of insulitis involves a cascade of events. Generation of antiself islet-antigen–specific CTLs, a functional perforin pathway, the presence of IFN-γ, upregulation of MHC class I on β cells, and likely the presence of CD4 lymphocytes are required. Identification of the factors involved will suggest the strategies that can be applied to hinder or prevent the autoimmune process from continuing and hopefully prevent IDDM.