Viral induction of autoimmunity is thought to occur by either bystander T-cell activation or molecular mimicry. Coxsackie B4 virus is strongly associated with the development of insulin-dependent diabetes mellitus in humans and shares sequence similarity with the islet autoantigen glutamic acid decarboxylase. We infected different strains of mice with Coxsackie B4 virus to discriminate between the two possible induction mechanisms, and found that mice with susceptible MHC alleles had no viral acceleration of diabetes, but mice with a T cell receptor transgene specific for a different islet autoantigen rapidly developed diabetes. These results show that diabetes induced by Coxsackie virus infection is a direct result of local infection leading to inflammation, tissue damage, and the release of sequestered islet antigen resulting in the re-stimulation of resting autoreactive T cells, further indicating that the islet antigen sensitization is an indirect consequence of the viral infection.

Cytokines, particularly those of the common γ chain receptor family, provide extrinsic signals that regulate naive CD4 cell survival. Whether these cytokines are required for the maintenance of memory CD4 cells has not been rigorously assessed. In this paper, we examined the contribution of interleukin (IL) 7, a constitutively produced common γ chain receptor cytokine, to the survival of resting T cell receptor transgenic memory CD4 cells that were generated in vivo. IL-7 mediated the survival and up-regulation of Bcl-2 by resting memory CD4 cells in vitro in the absence of proliferation. Memory CD4 cells persisted for extended periods upon adoptive transfer into intact or lymphopenic recipients, but not in IL-7− mice or in recipients that were rendered deficient in IL-7 by antibody blocking. Both central (CD62L+) and effector (CD62L−) memory phenotype CD4 cells required IL-7 for survival and, in vivo, memory cells were comparable to naive CD4 cells in this regard. Although the generation of primary effector cells from naive CD4 cells and their dissemination to nonlymphoid tissues were not affected by IL-7 deficiency, memory cells failed to subsequently develop in either the lymphoid or nonlymphoid compartments. The results demonstrate that IL-7 can have previously unrecognized roles in the maintenance of memory in the CD4 cell population and in the survival of CD4 cells with a capacity to become memory cells.

The development of effector and memory CD4 cell populations depends upon both T cell receptor (TCR) engagement of peptide/major histocompatibility complex (MHC) class II complexes and ligation of costimulatory molecules with counter receptors on antigen-presenting cells (APCs). We showed previously that sustained interactions with APCs could be crucial for optimal expansion of CD4 cells and for development of effectors that secrete cytokines associated with Th2 cells. Using an adoptive transfer model with TCR transgenic CD4 cells, we now show that responses of CD4 cells primed in B cell–deficient mice become aborted, but are fully restored upon the transfer of activated B cells. Although B cells have the capacity to secrete multiple cytokines that could affect CD4 priming, including IL-4, we were unable to distinguish a role for cytokines that are secreted by B cells. However, B cell costimulation via the OX40L/OX40 pathway that has been implicated in CD4 cell expansion, survival, and Th2 development was required. Th2 but not Th1 responses were impaired in OX40L-deficient recipients and normal responses were restored with OX40L sufficient B cells. The results suggest that without engagement of OX40L on B cells, CD4 cell responses to many protein Ag would be dominated by Th1 cytokines. These data have important implications for strategies to achieve optimal priming of CD4 subsets.

Activated B cells express high levels of class II MHC and costimulatory molecules and are nearly as effective as dendritic cells in their APC ability. Yet, their importance as APC in vivo is controversial and their role, if any, in the development of CD4 memory is unknown. We compared responses of CD4 cells from normal and B cell-deficient mice to keyhole limpet hemocyanin over 6 mo and observed diminished IL-2 production by cells primed in the absence of B cells. This was due to lower frequencies of Ag-responsive cells and not to decreased levels of IL-2 secretion per cell. The absence of B cells did not affect the survival of memory CD4 cells since frequencies remained stable. Despite normal dendritic cell function, multiple immunizations of B cell-deficient mice did not restore frequencies of memory cells. However, the transfer of B cells restored memory cell development. Ag presentation was not essential since B cells activated in vitro with irrelevant Ag also restored frequencies of memory cells. The results provide unequivocal evidence that B cells play a critical role in regulating clonal expansion of CD4 cells and, as such, are requisite for the optimal priming of memory in the CD4 population.

Adaptive regulatory T cells that develop from naive CD4 cells in response to exposure to Ag can act as immunotherapeutic agents to control immune responses. We show that effectors generated from murine islet-specific CD4 cells by TCR stimulation with IL-2 and TGF-β1 have potent suppressive activity. They prevent spontaneous development of type 1 diabetes in NOD mice and inhibit development of pancreatic infiltrates and disease onset orchestrated by Th1 effectors. These regulatory T cells do not require innate CD25+ regulatory cells for generation or function, nor do they share some characteristics typically associated with them, including expression of CD25. However, the adaptive population does acquire the X-linked forkhead/winged helix transcription factor, FoxP3, which is associated with regulatory T cell function and maintains expression in vivo. One mechanism by which they may inhibit Th1 cells is via FasL-dependent cytotoxicity, which occurs in vitro. In vivo, they eliminate Th1 cells in lymphoid tissues, where Fas/FasL interactions potentially play a role because Th1 cells persist when this pathway is blocked. The results suggest that adaptive regulatory CD4 cells may control diabetes in part by impairing the survival of islet-specific Th1 cells, and thereby inhibiting the localization and response of autoaggressive T cells in the pancreatic islets.

The initial steps that determine development of memory in CD4 cells are unknown. To distinguish an intrinsic capacity of effectors to become memory cells from contributions of as yet undefined survival factors, we analyzed the effects of withdrawal of signals via TCR, costimulation, and cytokines from Th1 or Th2 primary effectors induced in vitro from TCR-transgenic CD4 cells. Withdrawal of stimulation caused the transition of effectors to resting populations with a memory phenotype that did not undergo division following transfer to normal syngeneic recipients. The return of effectors to rest was accompanied by acquisition of the capacity to function as memory cells in vivo as defined by extended persistence and a more rapid response to Ag in vivo than naive cells in adoptive hosts. Upon challenge with Ag, these in vitro-rested Th1 and Th2 cells were similar to long-term in vivo-rested memory cells, but distinct from in vitro-generated primary effectors and in vivo-restimulated memory effectors by their ability to resist apoptosis. Cessation of stimulation may occur when activated CD4 cells exit lymphoid tissues after priming and transition to memory may be initiated if effectors either fail to gain access to Ag in peripheral tissues where restimulation can lead to activation-induced cell death or do not receive sufficient stimuli to continue a response. Our results suggest that the first stage leading to stable CD4 memory could occur stochastically and independently of instructional processes and as such, the development of memory may be a default pathway when signals that direct responses are not received.

No abstract

Like dendritic cells (DC), activated B cells are effective antigen‐presenting cells (APC) for naïve CD4 cells due to their expression of MHC class II and multiple costimulatory molecules. We showed previously that CD4 cells primed in B cell‐deficient (μMT) mice undergo more limited expansion than in normal animals after immunization with keyhole limpet hemocyanin. Here we report that in the absence of B cells, priming of effectors with the capacity to produce the Th2 cytokines, IL‐4, IL‐5 and IL‐13, was profoundly reduced whereas the development of effectors that secrete the Th1 cytokine IFN‐γ was much less affected. A blockade of IL‐12 reduced priming of IFN‐γ‐secreting effectors but did not reverse the IL‐4, IL‐5, or IL‐13 deficiency of the response. CD4 cell expansion and priming for Th2 cytokines in μMT mice was reconstituted by adoptive transfer of activated splenic B cells, which were present throughout the primary response. However, transfer of splenic DC from either control or μMT mice also supported development of Th2 cytokine responses, indicating that an APC deficit rather than a unique contribution of B cells accounted for diminished effector priming. We conclude that CD4 cell expansion must be sustained via APC for the development of Th2 cytokine‐secreting effectors in vivo and that in responses to protein antigen, B cells can be a crucial population to serve in this role. The results suggest that the level of APC engagement can not only determine the extent of effector expansion, but also the overall Th1/Th2 cytokine balance.