We have examined processes leading to the spontaneous development of autoimmune inflammatory arthritis in transgenic mice containing CD4+ T cells targeted to a nominal Ag (hemagglutinin (HA)) and coexpressing HA driven by a MHC class II promoter. Despite being subjected to multiple tolerance mechanisms, autoreactive CD4+ T cells accumulate in the periphery of these mice and promote systemic proinflammatory cytokine production. The majority of mice spontaneously develop inflammatory arthritis, which is accompanied by an enhanced regional immune response in lymph nodes draining major joints. Arthritis development is accompanied by systemic B cell activation; however, neither B cells nor Ab is required for arthritis development, since disease develops in a B cell-deficient background. Moreover, arthritis also develops in a recombinase activating gene-deficient background, indicating that the disease process is driven by CD4+ T cells recognizing the neo-self HA Ag. These findings show that autoreactive CD4+ T cells recognizing a single self-Ag, expressed by systemically distributed APCs, can induce arthritis via a mechanism that is independent of their ability to provide help for autoantibody production.

Autoreactive B cells are not completely purged from the primary B cell repertoire, and whether they can be prevented from maturation into memory B cells has been uncertain. We show here that a population of B cells that dominates primary immune responses of BALB/c mice to influenza virus A/PR/8/34 hemagglutinin (HA) are negatively selected in transgenic mice expressing PR8 HA as an abundant membrane-bound Ag (HACII mice). However, a separate population of B cells that contains precursors of memory B cells is activated by PR8 virus immunization and is subsequently negatively selected during the formation of the memory response. Negative selection of PR8 HA-specific B cells altered the specificity of the memory B cell response to a mutant virus containing a single amino acid substitution in a B cell epitope. Strikingly, this skewed reactivity resulted from an increase in the formation of memory B cells directed to non-self-epitopes on the mutant virus, which increased 8-fold in HACII mice relative to nontransgenic mice and precisely compensated for the absence of autoreactive PR8 HA-specific memory B cells. Negative selection of PR8 HA-specific B cells was a dominant process, since B cells from HACII mice could induce negative selection of PR8 HA-specific B cells from BALB/c mice. Lastly, HA-specific memory responses were unaffected by self-tolerance in another lineage of HA-transgenic mice (HA104 mice), indicating that the amount and/or cell type in which self-Ags are expressed can determine their ability to prevent autoreactive memory B cell formation.

Although somatically mutated autoantibodies are characteristic of many autoimmune diseases, the processes that can lead to their development remain poorly understood. We have examined the formation of autoreactive memory B cells in PevHA mice, which express the influenza virus PR8 hemagglutinin (HA) as a transgenic membrane bound neo-self-Ag. Using a virus immunization strategy, we show that PR8 HA-specific memory B cell formation can occur in PevHA mice, even though a major subset of PR8 HA-specific B cells is negatively selected from the primary repertoire. Moreover, PR8 HA-specific memory B cells develop spontaneously in TS1 × PevHA mice, which coexpress a transgenic PR8 HA-specific TCR and contain a high frequency of HA-specific CD4+ T cells. Notably, autoreactive memory B cell formation occurred in TS1 × PevHA mice even though approximately half of the HA-specific CD4+ T cells were CD25+Foxp3+ cells that could significantly attenuate, but did not completely abolish HA-specific autoantibody production in an adoptive transfer setting. The findings provide evidence that a high frequency of autoreactive CD4+ T cells can be sufficient to promote autoreactive memory B cell formation in the absence of signals provided by overt immunization or infection and despite the presence of abundant autoantigen-specific CD4+CD25+Foxp3+ regulatory T cells.

Autoreactive CD4 1 T cells can undergo deletion and/or become CD25 1 Foxp3 1 Treg as they develop intrathymically, but how these alternative developmental fates are specified based on interactions with self-peptide(s) is not understood. We show here that thymocytes expressing an autoreactive TCR can be subjected to varying degrees of deletion that correlate with the amount of self-peptide. Strikingly, among thymocytes that evade deletion, similar proportions acquire Foxp3 expression. These findings provide evidence that Foxp3 1 Treg can develop among members of a cohort of autoreactive thymocytes that have evaded deletion by a self-peptide, and that deletion and Treg formation can act together to bias the Treg repertoire toward low-abundance self-peptide(s).Key words: Thymic selection . Tolerance . Transgenic models IntroductionDuring their development, thymocytes encounter an array of self-peptides that are expressed in different amounts and by distinct cell types. Self-peptides with which the TCR is strongly reactive can induce thymocyte deletion, which eliminates potentially autoagressive clones from the CD4 1 and CD8 1 T-cell repertoires [1]. Thymocytes can also undergo a program of differentiation to become CD4 1 CD25 1 Foxp3 1 Treg, which additionally participate in preventing autoimmunity [2,3]. The processes that instruct thymocytes to undergo deletion or to develop along a pathway to become Treg remain poorly understood.Evidence that interactions with self-peptides can promote Treg formation came from studies showing increased CD4 1 CD25 1 Treg development in TCR Tg mice that co-express their agonist peptide as a self-peptide [4][5][6]. The idea that signaling from self-peptides can promote Treg formation is supported by studies showing that TCR signaling of progenitor CD4 1 CD8 À (CD4SP) thymocytes can promote Foxp3 1 expression [7].However, as noted above, signaling from self-peptides can also promote thymocyte deletion, and the relationship between thymocyte deletion and Treg formation remains to be defined. One possibility is that differentiation along the Treg pathway is induced by cues other than recognition of self-peptides [8,9]. For example, non-peptide-mediated cues might induce differentiation along a Treg pathway and cause a subset of thymocytes to be more resistant to deletion than are the remaining thymocytes, which are fated to develop into conventional CD4 1 Foxp3 À T cells unless they are signaled by self-peptides to undergo deletion [8]. An alternative possibility is that the fate of an autoreactive thymocyte is dictated by the mode of self-peptide presentation, e.g. expression at high or low densities, or by particular thymic cell types, although recent studies argued against a role for specialized thymic cell types in promoting Treg formation [10,11].We have examined these questions by determining the extent of thymocyte deletion and/or Foxp3 1 Treg formation in Tg mice expressing an MHC class II-restricted TCR, and co-expressing the cognate peptide for this TCR under the control of ...