Rapamycin is an immunosuppressive compound that is currently used to prevent acute graft rejection in humans. In addition, rapamycin has been shown to allow operational tolerance in murine models. However, a direct effect of rapamycin on T regulatory ( IntroductionRapamycin, a macrolide antibiotic produced by Streptomyces hygroscopicus, is a new effective drug used to prevent allograft rejection. 1 Similarly to the immunosuppressants FK506 and cyclosporin A (CsA), rapamycin exerts its effect by binding to the intracellular immunophilin FK506-binding protein (FKBP12). However, unlike FK506 and CsA, rapamycin does not inhibit T-cell receptor (TCR)-induced calcineurin activity. Rather, the rapamycin-FKBP12 complex inhibits the serine/threonine protein kinase called mammalian target of rapamycin (mTOR), the activation of which is required for protein synthesis and cell-cycle progression. Therefore, rapamycin blocks signaling in response to cytokines/ growth factors, whereas FK506 and CsA exert their inhibitory effects by blocking TCR-induced activation (for a review, see Abraham and Wiederrecht 2 ). Consistent with this mechanism of action, it has been shown that rapamycin (1) blocks T-cell-cycle progression from G 1 to S phase after activation, 3 (2) promotes TCR-induced T-cell anergy even in the presence of costimulation, 4 and (3) allows induction of operational tolerance. 5 However, a direct effect of rapamycin on T regulatory (Tr) cells has not been demonstrated so far.Tr cells are well-characterized T-cell subsets that play a key role in inducing and maintaining immunologic tolerance. Among the CD4 ϩ Tr cells, the Tr-cell subset that expresses the interleukin 2 receptor ␣ (IL-2R␣) chain (CD4 ϩ CD25 ϩ ) is one of the most extensively characterized so far (for a review, see Fehervari and Sakaguchi 6 ). CD4 ϩ CD25 ϩ Tr cells are generated in the thymus and are part of the normal peripheral T-cell repertoire. Suppressive CD4 ϩ CD25 ϩ Tr cells can be distinguished from activated T cells based on the high constitutive expression of CD25, cytotoxic T-lymphocyte antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), and the transcription factor forkhead box P3 (FoxP3). Once generated, thymic CD4 ϩ CD25 ϩ Tr cells migrate to peripheral tissues, where they potently suppress proliferation and cytokine production by both CD4 ϩ and CD8 ϩ T cells via a mechanism that requires cell-cell contact. 6 CD4 ϩ CD25 ϩ Tr cells contribute to tolerance induction after solid organ transplantation and protect from graft-versus-host disease lethality in bone marrow transplantation models. 7 Here, we provide new evidence that in vitro long-term exposure of murine CD4 ϩ T cells to rapamycin induces expansion of the naturally occurring CD4 ϩ CD25 ϩ FoxP3 ϩ Tr cells, which retain their suppressive functions in vitro and in vivo. Materials and methods MiceBalb/c, C57BL/6, and DO11.10 (TCR transgenic [tg] specific for ovalbumin [OVA]) female mice were purchased from Charles River Laboratories (Calco, Italy). All mic...

CD4+CD25+FOXP3+ T regulatory cells (Tregs) are pivotal for the induction and maintenance of peripheral tolerance in both mice and humans. Rapamycin has been shown to promote tolerance in experimental models and to favor CD4+CD25+ Treg-dependent suppression. We recently reported that rapamycin allows in vitro expansion of murine CD4+CD25+FoxP3+ Tregs, which preserve their suppressive function. In the current study, we show that activation of human CD4+ T cells from healthy subjects in the presence of rapamycin leads to growth of CD4+CD25+FOXP3+ Tregs and to selective depletion of CD4+CD25− T effector cells, which are highly sensitive to the antiproliferative effect of the compound. The rapamycin-expanded Tregs suppress proliferation of both syngeneic and allogeneic CD4+ and CD8+ T cells. Interestingly, rapamycin promotes expansion of functional CD4+CD25+FOXP3+ Tregs also in type 1 diabetic patients, in whom a defect in freshly isolated CD4+CD25+ Tregs has been reported. The capacity of rapamycin to allow growth of functional CD4+CD25+FOXP3+ Tregs, but also to deplete T effector cells, can be exploited for the design of novel and safe in vitro protocols for cellular immunotherapy in T cell-mediated diseases.

Human type 1 diabetes (T1D) is an autoimmune disease associated with major histocompatibility complex polymorphisms, β-cell autoantibodies, and autoreactive T cells. However, there is increasing evidence that innate cells may also play critical roles in T1D. We aimed to monitor peripheral immune cells in early stages of T1D (i.e., in healthy autoantibody-positive subjects) and in more advanced phases of the disease (i.e., at disease onset and years after diagnosis). We found a mild but significant and reproducible peripheral neutropenia that both precedes and accompanies the onset of T1D. This reduction was not due to peripheral neutrophil cell death, impaired differentiation, or the presence of anti-neutrophil antibodies. Neutrophils were observed by electron microscopy and immunohistochemical analysis in the exocrine pancreas of multiorgan donors with T1D (both at onset and at later stages of the disease) and not in that of multiorgan donors with type 2 diabetes or nondiabetic donors. These pancreas-infiltrating neutrophils mainly localized at the level of very small blood vessels. Our findings suggest the existence of a hitherto unrecognized clinical phenotype that might reflect unexplored pathogenic pathways underlying T1D.

OBJECTIVEAutoimmune diseases, including type 1 diabetes, are thought to have a Th17-cell bias and/or a T-regulatory cell (Treg) defect. Understanding whether this is a hallmark of patients with type 1 diabetes is a crucial question that is still unsolved, largely due to the difficulties of accessing tissues targeted by the disease.RESEARCH DESIGN AND METHODSWe phenotypically and functionally characterized Th17 cells and Tregs residing in the pancreatic-draining lymph nodes (PLNs) of 19 patients with type 1 diabetes and 63 nondiabetic donors and those circulating in the peripheral blood of 14 type 1 diabetic patients and 11 healthy subjects.RESULTSWe found upregulation of Th17 immunity and functional defects in CD4+CD25bright Tregs in the PLNs of type 1 diabetic subjects but not in their peripheral blood. In addition, the proinsulin-specific Treg-mediated control was altered in the PLNs of diabetic patients. The dysfunctional Tregs isolated from diabetic subjects did not contain contaminant effector T cells and were all epigenetically imprinted to be suppressive, as defined by analysis of the Treg-specific demethylated region within the forkhead box P3 (FOXP3) locus.CONCLUSIONSThese data provide evidence for an unbalanced immune status in the PLNs of type 1 diabetic subjects, and treatments restoring the immune homeostasis in the target organ of these patients represent a potential therapeutic strategy.