Irina Apostolou

Inducing and expanding regulatory T cell populations by foreign antigen

Evidence suggests that regulatory T cells expressing the transcription factor Foxp3 develop extrathymically and intrathymically. Mechanisms of extrathymic induction require further scrutiny, especially as proliferation and/or phenotypic changes of preexisting suppressor cells must be distinguished from true de novo generation. Here we report the conversion of truly naive CD4+ T cells into suppressor cells expressing Foxp3 by targeting of peptide-agonist ligands to dendritic cells and by analysis of Foxp3 expression at the level of single cells. We show that conversion was achieved by minute antigen doses with suboptimal dendritic cell activation. The addition of transforming growth factor-β or the absence of interleukin 2 production, which reduces proliferation, enhanced the conversion rate. In addition, regulatory T cell populations induced in subimmunogenic conditions could subsequently be expanded by delivery of antigen in immunogenic conditions. The extrathymic generation and proliferation of regulatory T cells may contribute to self-tolerance as well as the poor immunogenicity of tumors and may be exploited clinically to prevent or reverse unwanted immunity.

Origin of regulatory T cells with known specificity for antigen

T cell receptor agonists can induce the differentiation of regulatory T (TR) cells. We report here that the immunoglobulin κ–controlled expression of an agonist in different cell types correlated with the phenotype of the generated TR cells. We found that aberrant expression on thymic stroma yielded predominantly CD4+CD25+ TR cells, which—under physiological conditions—may be induced by ectopically expressed organ-specific antigens and thus prevent organ-specific autoimmunity. Expression of the agonist antigen by nonactivated hematopoietic cells produced mostly CD4+CD25− TR cells. This subset can be derived from mature monospecific T cells without “tutoring” by other T cells and can be generated in the absence of a functioning thymus. Suppression of CD4+ T cell proliferative responses by both CD25+ and CD25− subsets was interleukin 10 (IL-10)–independent and was overcome by IL-2. These data suggest that distinct pathways can be exploited to interfere with unwanted immune responses.

In Vivo Instruction of Suppressor Commitment in Naive T Cells

The induction of antigen-specific tolerance in the mature immune system of the intact organism has met with limited success. Therefore, nonspecific immunosuppression has been the treatment of choice to prevent unwanted immunity. Here, it is shown that prolonged subcutaneous infusion of low doses of peptide by means of osmotic pumps transforms mature T cells into CD4+25+ suppressor cells that can persist for long periods of time in the absence of antigen and confer specific immunologic tolerance upon challenge with antigen. The described procedure resembles approaches of tolerance induction used decades ago, induces tolerance in the absence of immunity, and holds the promise to become an effective means of inducing antigen-specific tolerance prospectively, whereas its power to suppress already ongoing immune responses remains to be determined.

Thymic selection revisited: how essential is it?

Intrathymic T cell development represents one of the best studied paradigms of mammalian development. Lymphoid committed precursors enter the thymus and the Notch1 receptor plays an essential role in committing them to the T cell lineages. The pre‐T cell receptor (TCR), as an autonomous cell signaling receptor, commits cells to the αβ lineage while its rival, the γδTCR, is involved in generating the γδ lineage of T cells. Positive and negative selection of immature αβTCR‐expressing cells are essential mechanisms for generating mature T cells, committing them to the CD4 and CD8 lineages and avoiding autoimmunity. Additional lineages of αβT cells, such as the natural killer T cell lineage and the CD25+ regulatory T cell lineage, are formed when the αβTCR encounters specific ligands in suitable microenvironments. Thus, positive selection and receptor‐instructed lineage commitment represent a hallmark of the thymus. Ectopically expressed organ‐specific antigens contribute to thymic self–nonself discrimination, which represents an essential feature for the evolutionary fitness of mammalian species.

Making regulatory T cells with defined antigen specificity: role in autoimmunity and cancer

Summary:  There is increasing evidence that agonist ligand presentation either intrathymically or extrathymically plays a crucial if not essential role in the generation of regulatory T cells (Tregs). Thus, it is possible to induce Tregs of any desired specificity in vivo. The same goal can be achieved in vitro by expanding antigen‐specific CD4+ T cells and retrovirally transducing them. In contrast, in vitro expansion of Tregs is limited to antigens that have resulted in Treg generation in vivo. Antigen‐specific Tregs can be used in cellular therapy with the goal to prevent autoimmune disease or even to interfere with established autoimmunity. The latter requires that the Tregs can suppress effector cells that have already caused harm, which is possible because of the antigen‐dependent homing properties of Tregs, i.e. these cells can accumulate in antigen‐draining lymph nodes and exit into inflamed tissue. Generally, the in vivo interference is dependent on cytokines such as transforming growth factor‐β and interleukin‐10 that were dispensable in in vivo analysis of immunosuppression. The precise mechanisms of suppression remain enigmatic, however, but may be further elucidated by the molecular analysis of suppressed versus non‐suppressed T cells.

Induction of antigen-specific regulatory T cells in wild-type mice: Visualization and targets of suppression

Antigen-specific transplantation tolerance in the absence of immunosuppressive drugs is a rarely achieved goal. Immune responses to Y chromosome-encoded transplantation antigens (HY) can have life-threatening consequences in the clinic. Here, we have adopted a procedure developed in T cell antigen receptor (TCR)-transgenic mice to convert naïve T cells into male-specific Foxp3+ regulatory T cells (Tregs) in WT female mice. For this purpose, female mice were infused by osmotic minipumps with a single class II MHC-presented HY peptide and Tregs visualized by tetramer staining. As a result, animals developed Treg-mediated long-term tolerance to all HY transplantation antigens, irrespective of whether they were recognized by CD4 or CD8 T cells, on skin or hematopoietic grafts from male donors.

Effective Destruction of Fas-deficient Insulin-producing β Cells in Type 1 Diabetes

In type 1 diabetes, autoimmune T cells cause destruction of pancreatic β cells by largely unknown mechanism. Previous analyses have shown that β cell destruction is delayed but can occur in perforin-deficient nonobese diabetic (NOD) mice and that Fas-deficient NOD mice do not develop diabetes. However, because of possible pleiotropic functions of Fas, it was not clear whether the Fas receptor was an essential mediator of β cell death in type 1 diabetes. To directly test this hypothesis, we have generated a β cell–specific knockout of the Fas gene in a transgenic model of type 1 autoimmune diabetes in which CD4+ T cells with a transgenic TCR specific for influenza hemagglutinin (HA) are causing diabetes in mice that express HA under control of the rat insulin promoter. Here we show that the Fas-deficient mice develop autoimmune diabetes with slightly accelerated kinetics indicating that Fas-dependent apoptosis of β cells is a dispensable mode of cell death in this disease.

Kruppel-like Factor KLF10 Targets Transforming Growth Factor-β1 to Regulate CD4+CD25− T Cells and T Regulatory Cells

CD4+CD25+ regulatory T cells (T regs) play a major role in the maintenance of self-tolerance and immune suppression, although the mechanisms controlling T reg development and suppressor function remain incompletely understood. Herein, we provide evidence that Kruppel-like factor 10 (KLF10/TIEG1) constitutes an important regulator of T regulatory cell suppressor function and CD4+CD25− T cell activation through distinct mechanisms involving transforming growth factor (TGF)-β1 and Foxp3. KLF10 overexpressing CD4+CD25− T cells induced both TGF-β1 and Foxp3 expression, an effect associated with reduced T-Bet (Th1 marker) and Gata3 (Th2 marker) mRNA expression. Consistently, KLF10−/− CD4+CD25− T cells have enhanced differentiation along both Th1 and Th2 pathways and elaborate higher levels of Th1 and Th2 cytokines. Furthermore, KLF10−/− CD4+CD25− T cell effectors cannot be appropriately suppressed by wild-type T regs. Surprisingly, KLF10−/− T reg cells have reduced suppressor function, independent of Foxp3 expression, with decreased expression and elaboration of TGF-β1, an effect completely rescued by exogenous treatment with TGF-β1. Mechanistic studies demonstrate that in response to TGF-β1, KLF10 can transactivate both TGF-β1 and Foxp3 promoters, implicating KLF10 in a positive feedback loop that may promote cell-intrinsic control of T cell activation. Finally, KLF10−/− CD4+CD25− T cells promoted atherosclerosis by ∼2-fold in ApoE−/−/scid/scid mice with increased leukocyte accumulation and peripheral pro-inflammatory cytokines. Thus, KLF10 is a critical regulator in the transcriptional network controlling TGF-β1 in both CD4+CD25− T cells and T regs and plays an important role in regulating atherosclerotic lesion formation in mice.

Peripherally Induced Treg: Mode, Stability, and Role in Specific Tolerance

Foxp3-expressing regulatory T cells (Treg) have an essential function of preventing autoimmune disease in man and mouse. Foxp3 binds to forkhead motifs of about 1,100 genes and the strength of binding increases upon phorbol 12-myristate 13-acetate/ionomycin stimulation. In Foxp3-expressing T cell hybridomas, Foxp3 promoter binding does not lead to activation or suppression of genes which becomes only visible after T cell activation. These findings are in line with observations by others that Foxp3 exerts important functions in collaboration with T cell receptor (TCR)-dependent transcription factors in a DNA-binding complex. Tregs can be generated when developing T cells encounter TCR agonist ligands in the thymus. This process apparently depends on costimulatory signals. In contrast, extrathymic conversion of naïve T cells into Tregs appears to depend on transforming growth factor (TGF)-β and is inhibited by costimulation. In fact, dendritic cell-derived retinoic acid helps the conversion process by counteracting the negative impact of costimulation. Tregs induced by subimmunogenic antigen delivery in vivo are much more stable than Tregs induced by antigenic stimulation in the presence of TGF-β in vitro which correlates with the extent of demethylation of the Foxp3 locus. Tregs can be induced by conversion of antigen-specific T cells that occur with a very low frequency in wt mice. Conversion of naïve cluster of differentiation (CD)4 T cells into Tregs by a single peptide of HY antigens results in complete antigen-specific tolerance to an entire set of HY epitopes recognized by CD4 as well as CD8 T cells when presented with male skin or hemopoietic grafts.

Targeting of CD25 and Glucocorticoid-Induced TNF Receptor Family-Related Gene-Expressing T Cells Differentially Modulates Asthma Risk in Offspring of Asthmatic and Normal Mother Mice

Immunological mechanisms leading to increased asthma susceptibility in early life remain obscure. In this study, we examined the effects of neonatal Ab treatments targeting T cell populations on the development of an asthma syndrome. We used a model of increased asthma susceptibility where offspring of asthmatic BALB/c mother mice are more prone (than normal pups) to develop the disease. Neonatal pretreatment of naive pups with mAb directed against the IL-2Rα chain (CD25), the costimulatory molecule glucocorticoid-induced TNFR family related gene, and the inhibitory molecule CTLA-4 elicited contrasting effects in offspring depending on the mother’s asthma status. Specifically, neonatal CD25high T cell depletion stimulated asthma susceptibility in normal offspring whereas it ameliorated the condition of pups born of asthmatic mothers. Conversely, glucocorticoid-induced TNFR family related gene ligation as a primary signal reduced the spleen cellularity and largely abrogated asthma susceptibility in asthma-prone offspring, without inducing disease in normal pups. Striking changes in Th1/Th2 cytokine levels, especially IL-4, followed mAb pretreatment and were consistent with the impact on asthma susceptibility. These results point to major differences in neonatal T cell population and responsiveness related to maternal asthma history. Interventions that temporarily remove and/or inactivate specific T cell subsets may therefore prove useful to attenuate early life asthma susceptibility and prevent the development of Th2-driven allergic airway disease.