Enhanced selection of FoxP3+ T-regulatory cells protects CTLA-4-deficient mice from CNS autoimmune disease.

It is generally acknowledged that cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4/CD152) plays a pivotal role in the regulation of T-cell activation and the establishment of self-tolerance in the periphery. CTLA-4-deficient (CTLA-4KO) mice develop a lymphoproliferative disorder and die within 4 weeks of birth, suggesting a role for CTLA-4 in T-cell homeostasis or the development and activity of T-regulatory (Treg) cells. To study the role of CTLA-4 in the control of experimental autoimmune encephalomyelitis (EAE), we have generated a CTLA-4KO mouse in which >90% of all CD4(+) T cells bear a Vbeta8.2 transgenic T-cell receptor that is specific for myelin basic protein peptide Ac1-9 (ASQKRPSQR). These mice do not develop spontaneous lymphoproliferative disease or EAE and are resistant to disease induction. This correlates with a higher frequency of functional FoxP3(+) Treg cells in the spleen and thymus of CTLA-4KO mice. The absence of CTLA-4-mediated suppression of CD28 signaling resulted in the early expression of FoxP3 on double-positive cells in the thymic cortex. We conclude that CTLA-4 is not essential for the peripheral function of FoxP3(+) Treg cells but plays a pivotal role in their thymic selection.

Persistent antigenic stimulation alters the transcription program in T cells, resulting in antigen-specific tolerance.

Repetitive antigen stimulation induces peripheral T cell tolerance in vivo. It is not known, however, whether multiple stimulations merely suppress T cell activation or, alternatively, change the transcriptional program to a distinct, tolerant state. In this study, we have discovered that STAT3 and STAT5 were activated in response to antigen stimulation in vivo, in marked contrast to the suppression of AP-1, NF-kappaB and NFAT. In addition, a number of transcription factors were induced in tolerant T cells following antigen challenge in vivo, including T-bet, Irf-1 and Egr-2. The altered transcription program in tolerant cells associates closely with the suppression of cell cycle progression and IL-2 production, as well as with the induction of IL-10. Studies of T-bet and Egr-2 show that the function of T-bet in peptide treatment-induced regulatory T cells is not associated with Th1 differentiation, but correlates with the suppression of IL-2, whereas expression of Egr-2 led to an up-regulation of the cell cycle inhibitors p21(cip1) and p27(kip). Our results demonstrate a balanced transcription program regulated by different transcription factors for T cell activation and/or tolerance during antigen-induced T cell responses. Persistent antigen stimulation can induce T cell tolerance by changing the balance of transcription factors.

Antigen-induced IL-10+ regulatory T cells are independent of CD25+ regulatory cells for their growth, differentiation, and function.

Recent studies have emphasized the importance of T cells with regulatory/suppressor properties in controlling autoimmune diseases. A number of different types of regulatory T cells have been described with the best characterized being the CD25(+) population. In addition, it has been shown that regulatory T cells can be induced by specific Ag administration. In this study, we investigate the relationship between peptide-induced, CD4(+) regulatory T cells and naturally occurring CD4(+)CD25(+) cells derived from the Tg4 TCR-transgenic mouse. Peptide-induced cells were FoxP3(-) and responded to Ag by secreting IL-10, whereas CD25(+) cells failed to secrete this cytokine. Both cell types were able to suppress the proliferation of naive lymphocytes in vitro although with distinct activation sensitivities. Depletion of CD25(+) cells did not affect the suppressive properties of peptide-induced regulators. Furthermore, peptide-induced regulatory/suppressor T cells could be generated in RAG(-/-), TCR-transgenic mice that do not spontaneously generate CD25(+) regulatory cells. These results demonstrate that these natural and induced regulatory cells fall into distinct subsets.

IL-2 overcomes the unresponsiveness but fails to reverse the regulatory function of antigen-induced T regulatory cells.

Intranasal administration of peptide Ac1-9[4Y], based on the N-terminal epitope of myelin basic protein, can induce CD4(+) T cell tolerance, and suppress experimental autoimmune encephalomyelitis induction. The peptide-induced regulatory T (PI-T(Reg)) cells failed to produce IL-2, but expressed IL-10 in response to Ag and could suppress naive T cell responses in vitro. Analysis of Jak-STAT signaling pathways revealed that the activation of Jak1, STAT3, and STAT5 were induced in tolerant T cells after Ag stimulation in vivo. In addition, the expression of suppressor of cytokine signaling 3 was induced in tolerant T cells, suggesting that cytokines regulate the tolerant state of the PI-T(Reg) cells. Stimulation of PI-T(Reg) cells in vitro with IL-10 induced Jak1 and STAT3 activation, but not STAT5, suggesting that IL-10 is important, but not the only cytokine involved in the development of T cell tolerance. Although IL-2 expression was deficient, stimulation with IL-2 in vitro induced Jak1 and STAT5 activation in PI-T(Reg) cells, restored their proliferative response to antigenic stimulation, and abrogated PI-T(Reg)-mediated suppression in vitro. However, the addition of IL-2 could not suppress IL-10 expression, and the IL-2 gene remained inactive. After withdrawal of IL-2, the PI-T(Reg) cells regained their nonproliferative state and suppressive ability. These results underline the ability of the immune system to maintain tolerance to autoantigens, but at the same time having the ability to overcome the suppressive phenotype of tolerant T cells by cytokines, such as IL-2, during the protective immune response to infection.

Severe prekallikrein deficiency associated with homozygosity for an Arg94Stop nonsense mutation.

An elderly patient with no abnormal bleeding presented with prolongation of the activated partial thromboplastin time (aPTT). Preincubation of plasma with aPTT reagent caused shortening of the abnormal clotting time. Plasma prekallikrein (PK) activity and antigen were <5 u/dL. Molecular analysis showed a homozygous Arg94Stop substitution in the PK gene, predicted to prevent expression of the mutant allele. The five heterozygous offspring of the proband each showed a normal aPTT but reduced PK activity and antigen. This is the first description of a kindred in which absence of expression of one or both PK alleles has been confirmed by genotype.

IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4+CD25+ regulatory T cells.

Regulatory T cells (T(Reg)) control immune responses to self and nonself Ags. The relationship between Ag-driven IL-10-secreting T(Reg) (IL-10-T(Reg)) and naturally occurring CD4(+)CD25(+) T(Reg) is as yet unclear. We show that mouse IL-10-T(Reg) obtained using either in vitro or in vivo regimens of antigenic stimulation did not express the CD4(+)CD25(+) T(Reg)-associated transcription factor Foxp3. However, despite the absence of Foxp3 expression, homogeneous populations of IL-10-T(Reg) inhibited the in vitro proliferation of CD4(+)CD25(-) T cells with a similar efficiency to that of CD4(+)CD25(+) T(Reg). This inhibition of T cell proliferation by IL-10-T(Reg) was achieved through an IL-10-independent mechanism as seen for CD4(+)CD25(+) T(Reg) and was overcome by exogenous IL-2. Both IL-10-T(Reg) and CD4(+)CD25(+) T(Reg) were similar in that they produced little to no IL-2. These data show that Foxp3 expression is not a prerequisite for IL-10-T(Reg) activity in vitro or in vivo, and suggest that IL-10-T(Reg) and naturally occurring CD4(+)CD25(+) T(Reg) may have distinct origins.