Cholera toxin subunit B peptide fusion proteins reveal impaired oral tolerance induction in diabetes‐prone but not in diabetes‐resistant mice

The cholera toxin B subunit (CTB) has been used as adjuvant to improve oral vaccine delivery in type 1 diabetes. The effect of CTB/peptide formulations on Ag‐specific CD4⁺ T cells has remained largely unexplored. Here, using tetramer analysis, we investigated how oral delivery of CTB fused to two CD4⁺ T‐cell epitopes, the BDC‐2.5 T‐cell 2.5mi mimotope and glutamic acid decarboxylase (GAD) 286–300, affected diabetogenic CD4⁺ T cells in nonobese diabetic (NOD) mice. When administered i.p., CTB‐2.5mi activated 2.5mi⁺ T cells and following intragastric delivery generated Ag‐specific Foxp3⁺ Treg and Th2 cells. While 2.5mi⁺ and GAD‐specific T cells were tolerized in diabetes‐resistant NODxB6.Foxp3^EGFP F1 and nonobese resistant (NOR) mice, this did not occur in NOD mice. This indicated that NOD mice had a recessive genetic resistance to induce oral tolerance to both CTB‐fused epitopes. In contrast to NODxB6.Foxp3^EGFP F1 mice, oral treatment in NOD mice lead to strong 2.5mi⁺ T‐cell activation and the sequestration of these cells to the effector‐memory pool. Oral treatment of NOD mice with CTB‐2.5mi failed to prevent diabetes. These findings underline the importance of investigating the effect of oral vaccine formulations on diabetogenic T cells as in selected cases they may have counterproductive consequences in human patients.     

Partial and transient modulation of the CD3–T‐cell receptor complex,elicited by low‐dose regimens of monoclonal anti‐CD3, is sufficient to induce disease remission in non‐obese diabetic mice

It has been established that a total of 250 μg of monoclonal anti‐mouse CD3 F(ab′)₂ fragments, administered daily (50 μg per dose), induces remission of diabetes in the non‐obese diabetic (NOD) mouse model of autoimmune diabetes by preventing β cells from undergoing further autoimmune attack. We evaluated lower‐dose regimens of monoclonal anti‐CD3 F(ab′)₂ in diabetic NOD mice for their efficacy and associated pharmacodynamic (PD) effects, including CD3–T‐cell receptor (TCR) complex modulation, complete blood counts and proportions of circulating CD4⁺, CD8⁺ and CD4⁺ FoxP3⁺ T cells. Four doses of 2 μg (total dose 8 μg) induced 53% remission of diabetes, similarly to the 250 μg dose regimen, whereas four doses of 1 μg induced only 16% remission. While the 250 μg dose regimen produced nearly complete and sustained modulation of the CD3 –TCR complex, lower doses, spaced 3 days apart, which induced similar remission rates, elicited patterns of transient and partial modulation. In treated mice, the proportions of circulating CD4⁺ and CD8⁺ T cells decreased, whereas the proportions of CD4⁺ FoxP3⁺ T cells increased; these effects were transient. Mice with greater residual β‐cell function, estimated using blood glucose and C‐peptide levels at the initiation of treatment, were more likely to enter remission than mice with more advanced disease. Thus, lower doses of monoclonal anti‐CD3 that produced only partial and transient modulation of the CD3–TCR complex induced remission rates comparable to higher doses of monoclonal anti‐CD3. Accordingly, in a clinical setting, lower‐dose regimens may be efficacious and may also improve the safety profile of therapy with monoclonal anti‐CD3, potentially including reductions in cytokine release‐related syndromes and maintenance of pathogen‐specific immunosurveillance during treatment.     

Inhibition of type 1 diabetes in filaria‐infected non‐obese diabetic mice is associated with a T helper type 2 shift and induction of FoxP3+ regulatory T cells

We sought to determine whether Litomosoides sigmodontis, a filarial infection of rodents, protects against type 1 diabetes in non‐obese diabetic (NOD) mice. Six‐week‐old NOD mice were sham‐infected or infected with either L3 larvae, adult male worms, or adult female worms. Whereas 82% of uninfected NOD mice developed diabetes by 25 weeks of age, no L. sigmodontis‐infected mice developed disease. Although all mice had evidence of ongoing islet cell inflammation by histology, L. sigmodontis‐infected mice had greater numbers of total islets and non‐infiltrated islets than control mice. Protection against diabetes was associated with a T helper type 2 (Th2) shift, as interleukin‐4 (IL‐4) and IL‐5 release from α‐CD3/α‐CD28‐stimulated splenocytes was greater in L. sigmodontis‐infected mice than in uninfected mice. Increased circulating levels of insulin‐specific immunoglobulin G1, showed that this Th2 shift occurs in response to one of the main autoantigens in diabetes. Multicolour flow cytometry studies demonstrated that protection against diabetes in L. sigmodontis‐infected NOD mice was associated with significantly increased numbers of splenic CD4⁺ CD25⁺ FoxP3⁺ regulatory T cells. Interestingly, injection of crude worm antigen into NOD mice also resulted in protection against type 1 diabetes, though to a lesser degree than infection with live L. sigmodontis worms. In conclusion, these studies demonstrate that filarial worms can protect against the onset of type 1 diabetes in NOD mice. This protection is associated with a Th2 shift, as demonstrated by cytokine and antibody production, and with an increase in CD4⁺ CD25⁺ FoxP3⁺ regulatory T cells.     

Abrogation of ICOS/ICOS ligand costimulation in NOD mice results in autoimmune deviation toward the neuromuscular system

NOD mice spontaneously develop insulin‐dependent diabetes around 10–40 wk of age. Numerous immune gene variants contribute to the autoimmune process. However, genes that direct the autoimmune response toward β cells remain ill defined. In this study, we provide evidence that the Icos and Icosl genes contribute to the diabetes process. Protection from diabetes in ICOS^?/? and ICOSL^?/? NOD mice was unexpectedly associated with the development of an autoimmune disorder of the neuro‐muscular system, characterized by myositis, sensory ganglionitis and, to a reduced extent, inflammatory infiltrates in the CNS. This syndrome was reproduced upon adoptive transfer of CD4⁺ and CD8⁺ T cells from diseased donors to naïve NOD.scid recipients. Our data further show that protection from diabetes results from defective activation of autoimmune diabetogenic effector T cells in ICOS^?/? NOD mice, whereas acceleration of diabetes in BDC2.5 ICOS^?/? NOD mice is induced by a dominant defect in Treg. Taken together, our findings indicate that costimulation signals play a key role in regulating immune tolerance in peripheral tissues and that the ICOS/ICOSL costimulatory pathway influences the balance between Treg and diabetogenic effector T cells.     

BAFF regulates activation of self‐reactive T cells through B‐cell dependent mechanisms and mediates protection in NOD mice

Targeting the BAFF/APRIL system has shown to be effective in preventing T‐cell dependent autoimmune disease in the NOD mouse, a spontaneous model of type 1 diabetes. In this study we generated BAFF‐deficient NOD mice to examine how BAFF availability would influence T‐cell responses in vivo and the development of spontaneous diabetes. BAFF‐deficient NOD mice which lack mature B cells, were protected from diabetes and showed delayed rejection of an allogeneic islet graft. Diabetes protection correlated with a failure to expand pathogenic IGRP‐reactive CD8⁺ T cells, which were maintained in the periphery at correspondingly low levels. Adoptive transfer of IGRP‐reactive CD8⁺ T cells with B cells into BAFF‐deficient NOD mice enhanced IGRP‐reactive CD8⁺ T‐cell expansion. Furthermore, when provoked with cyclophosphamide, or transferred to a secondary lymphopenic host, the latent pool of self‐reactive T cells resident in BAFF‐deficient NOD mice could elicit beta cell destruction. We conclude that lack of BAFF prevents the procurement of B‐cell‐dependent help necessary for the emergence of destructive diabetes. Indeed, treatment of NOD mice with the BAFF‐blocking compound, BR3‐Fc, resulted in a delayed onset and reduced incidence of diabetes.     

CD40‐mediated signalling influences trafficking,T‐cell receptor expression,and T‐cell pathogenesis,in the NOD model of type 1 diabetes

CD40 plays a critical role in the pathogenesis of type 1 diabetes (T1D). The mechanism of action, however, is undetermined, probably because CD40 expression has been grossly underestimated. CD40 is expressed on numerous cell types that now include T cells and pancreatic β cells. CD40⁺ CD4⁺ cells [T helper type 40 (TH40)] prove highly pathogenic in NOD mice and in translational human T1D studies. We generated BDC2.5.CD40^−/− and re‐derived NOD.CD154^−/− mice to better understand the CD40 mechanism of action. Fully functional CD40 expression is required not only for T1D development but also for insulitis. In NOD mice, TH40 cell expansion in pancreatic lymph nodes occurs before insulitis and demonstrates an activated phenotype compared with conventional CD4⁺ cells, apparently regardless of antigen specificity. TH40 T‐cell receptor (TCR) usage demonstrates increases in several Vα and Vβ species, particularly Vα3.2⁺ that arise early and are sustained throughout disease development. TH40 cells isolated from diabetic pancreas demonstrate a relatively broad TCR repertoire rather than restricted clonal expansions. The expansion of the Vα/Vβ species associated with diabetes depends upon CD40 signalling; NOD.CD154^−/− mice do not expand the same TCR species. Finally, CD40‐mediated signals significantly increase pro‐inflammatory Th1‐ and Th17‐associated cytokines whereas CD28 co‐stimulus alternatively promotes regulatory cytokines.     

Genetic deletion of granzyme B does not confer resistance to the development of spontaneous diabetes in non‐obese diabetic mice

Granzyme B (GzmB) and perforin are proteins, secreted mainly by natural killer cells and cytotoxic T lymphocytes that are largely responsible for the induction of apoptosis in target cells. Because type 1 diabetes results from the selective destruction of β cells and perforin deficiency effectively reduces diabetes in non‐obese diabetic (NOD) mice, it can be deduced that β cell apoptosis involves the GzmB/perforin pathway. However, the relevance of GzmB remains totally unknown in non‐obese diabetic (NOD) mice. In this study we have focused on GzmB and examined the consequence of GzmB deficiency in NOD mice. We found that NOD.GzmB^–/– mice developed diabetes spontaneously with kinetics similar to those of wild‐type NOD (wt‐NOD) mice. Adoptive transfer study with regulatory T cell (T_reg)‐depleted splenocytes (SPCs) into NOD‐SCID mice or in‐vivo T_reg depletion by anti‐CD25 antibody at 4 weeks of age comparably induced the rapid progression of diabetes in the NOD.GzmB^–/– mice and wt‐NOD mice. Expression of GzmA and Fas was enhanced in the islets from pre‐diabetic NOD.GzmB^–/– mice. In contrast to spontaneous diabetes, GzmB deficiency suppressed the development of cyclophosphamide‐promoted diabetes in male NOD mice. Cyclophosphamide treatment led to a significantly lower percentage of apoptotic CD4⁺, CD8⁺ and CD4⁺CD25⁺ T cells in SPCs from NOD.GzmB^–/– mice than those from wt‐NOD mice. In conclusion, GzmB, in contrast to perforin, is not essentially involved in the effector mechanisms for β cell destruction in NOD mice.     

Th17 cells promote pancreatic inflammation but only induce diabetes efficiently in lymphopenic hosts after conversion into Th1 cells

IDDM is characterized by leukocyte invasion to the pancreatic tissues followed by immune destruction of the islets. Despite the important function of Th17 cells in other autoimmune disease models, their function in IDDM is relatively unclear. In this study, we found association of elevated Th17 cytokine expression with diabetes in NOD mice. To understand the function of Th17 cells in IDDM, we differentiated islet‐reactive BDC2.5 TcR transgenic CD4⁺ cells in vitro into Th17 cells and transferred them into NOD.scid and neonate NOD mice. NOD.scid recipient mice developed rapid onset of diabetes with extensive insulitic lesions, whereas in newborn NOD mice, despite extensive insulitis, most recipient mice did not develop diabetes. Surprisingly, BDC2.5⁺ cells recovered from diabetic NOD.scid mice, in comparison with those from neonate NOD mice, showed predominant IFN‐γ over IL‐17 expression, indicating conversion of donor cells into Th1 cells. Moreover, diabetes progression in NOD.scid recipients was dependent on IFN‐γ while anti‐IL‐17 treatment reduced insulitic inflammation. These results indicate that islet‐reactive Th17 cells promote pancreatic inflammation, but only induce IDDM upon conversion into IFN‐γ producers.     

Detection of vasostatin‐1‐specific CD8+ T cells in non‐obese diabetic mice that contribute to diabetes pathogenesis

Chromogranin A (ChgA) is an antigenic target of pathogenic CD4⁺ T cells in a non‐obese diabetic (NOD) mouse model of type 1 diabetes (T1D). Vasostatin‐1 is a naturally processed fragment of ChgA. We have now identified a novel H2‐K^d‐restricted epitope of vasostatin‐1, ChgA 36‐44, which elicits CD8⁺ T cell responses in NOD mice. By using ChgA 36‐44/K^d tetramers we have determined the frequency of vasostatin‐1‐specific CD8⁺ T cells in pancreatic islets and draining lymph nodes of NOD mice. We also demonstrate that vasostatin‐1‐specific CD4⁺ and CD8⁺ T cells constitute a significant fraction of islet‐infiltrating T cells in diabetic NOD mice. Adoptive transfer of T cells from ChgA 36‐44 peptide‐primed NOD mice into NOD/severe combined immunodeficiency (SCID) mice led to T1D development. These findings indicate that vasostatin‐1‐specific CD8⁺ T cells contribute to the pathogenesis of type 1 diabetes in NOD mice.     

Vasostatin‐1 antigenic epitope mapping for induction of cellular and humoral immune responses to chromogranin A autoantigen in NOD mice

The chromogranin A (ChgA) 29–42 sequence is the antigenic epitope for the BDC2.5 CD4⁺ T‐cell receptor in NOD mice (H‐2^g7). We have now characterized the binding register of the ChgA 29–42 peptide for the I‐A^g7 molecule. Truncation of the peptide demonstrated that the KCVLEVISD sequence 34–42 is the binding register and extension of this sequence by flanking residues increased its binding affinity and antigenic capacity. We employed anti‐ChgA peptide antibodies generated against different fragments of ChgA for immunostaining of pancreatic islet sections from NOD mice. A strong immuno‐staining pattern was observed for the ChgA 17–38 peptide antibodies that overlap with the ChgA 29–42 sequence. Moreover, sera from diabetic NOD mice showed elevated titers of autoantibodies to the ChgA 29–42 peptide. These findings indicate that peptides from the N‐terminal region of ChgA are able to induce cellular and humoral immune responses in NOD mice.     

CD40 engagement of CD4+ CD40+ T cells in a neo-self antigen disease model ablates CTLA-4 expression and indirectly impacts tolerance

Biomarkers defining pathogenic effector T (Teff) cells slowly have been forthcoming and towards this we identified CD4⁺ T cells that express CD40 (CD4⁺CD40⁺) as pathogenic in the NOD type 1 diabetes (T1D) model. CD4⁺CD40⁺ T cells rapidly and efficiently transfer T1D to NOD.scid recipients. To study the origin of CD4⁺CD40⁺ T cells and disease pathogenesis, we employed a dual transgenic model expressing OVA_323–339 peptide as a neo‐self antigen on islet β cells and medullary thymic epithelial cells (mTECs) and a transgenic TCR recognizing the OVA_323–339 peptide. CD4⁺CD40⁺ T cells and Treg cells each recognizing the cognate neo‐antigen, rather than being deleted through central tolerance, drastically expanded in the thymus. In pancreatic lymph nodes of DO11.RIPmOVA mice, CD4⁺CD40⁺ T cells and Treg cells are expanded in number compared with DO11 mice and importantly, Treg cells remain functional throughout the disease process. When exposed to neo‐self antigen, CD4⁺CD40⁺ T cells do not express the auto‐regulatory CTLA‐4 molecule while naïve CD4⁺CD40⁺ T cells do. DO11.RIPmOVA mice develop autoimmune‐type diabetes. CD40 engagement has been shown to prevent CTLA‐4 expression and injecting anti‐CD40 in DO11.RIPmOVA mice significantly exacerbates disease. These data suggest a unique means by which CD4⁺CD40⁺ T cells thwart tolerance.     

The microbial product lipopolysaccharide confers diabetogenic potential on the T cell repertoire of BDC2.5/NOD mice: implications for the etiology of autoimmune diabetes

Both genetic predisposition and environmental factors participate in the etiology of Type-1 diabetes. To test the role of the microbial product lipopolysaccharide (LPS) as an environmental trigger of autoimmune diabetes, we employed transgenic (tg) BDC2.5/NOD mice that bear an islet-specific CD4(+) T cell repertoire (>95%), but do not develop the spontaneous diabetes that typifies the NOD (nonobese diabetic) strain. LPS administration provoked diabetes in BDC2.5/NOD mice by their 16th week of age. However, LPS administration in NOD mice did not accelerate their diabetes. This finding indicates that the frequency of islet-specific T cells influences LPS-mediated diabetes. Furthermore, in vitro LPS-cultured splenocytes from BDC2. 5/NOD and BDC2.5-microMT (B-cell-deficient) mice effectively transferred diabetes into immunodeficient NOD-scid/scid mice but not immunosufficient NOD mice. Therefore, B lymphocytes are not required for LPS-provoked autoimmune diabetes. Flow cytometric analysis then revealed that LPS-stimulation in vitro induced the expression of an IL-2 receptor (CD25) on CD4 T cells; this indicates that the activation of islet-specific T cells is a prerequisite to eliciting diabetes in this situation. Overall, these results point to microbial LPS as an etiopathogenic agent of autoimmune diabetes.     

Plasmacytoid dendritic cells and type 1 interferon promote peripheral expansion of forkhead box protein 3+ regulatory T cells specific for the ubiquitous RNA‐binding nuclear antigen La/Sjögren's syndrome (SS)‐B

RNA‐binding nuclear antigens are a major class of self‐antigen to which immune tolerance is lost in rheumatic diseases. Serological tolerance to one such antigen, La/Sjögren's syndrome (SS)‐B (La), is controlled by CD4⁺ T cells. This study investigated peripheral tolerance to human La (hLa) by tracking the fate of hLa‐specific CD4⁺ T cells expressing the transgenic (Tg) 3B5.8 T cell receptor (TCR) after adoptive transfer into lymphocyte‐replete recipient mice expressing hLa as a neo‐self‐antigen. After initial antigen‐specific cell division, hLa‐specific donor CD4⁺ T cells expressed forkhead box protein 3 (FoxP3). Donor cells retrieved from hLa Tg recipients displayed impaired proliferation and secreted interleukin (IL)?10 in vitro in response to antigenic stimulation. Transfer of highly purified FoxP3‐negative donor cells demonstrated that accumulation of hLa‐specific regulatory T cells (T_reg) was due primarily to expansion of small numbers of donor T_reg. Depletion of recipient plasmacytoid dendritic cells (pDC), but not B cells, severely hampered the accumulation of FoxP3⁺ donor T_reg in hLa Tg recipients. Recipient pDC expressed tolerogenic markers and higher levels of co‐stimulatory and co‐inhibitory molecules than B cells. Adoptive transfer of hLa peptide‐loaded pDC into mice lacking expression of hLa recapitulated the accumulation of hLa‐specific T_reg. Blockade of the type 1 interferon (IFN) receptor in hLa Tg recipients of hLa‐specific T cells impaired FoxP3⁺ donor T cell accumulation. Therefore, peripheral expansion of T_reg specific for an RNA‐binding nuclear antigen is mediated by antigen‐presenting pDC in a type 1 IFN‐dependent manner. These results reveal a regulatory function of pDC in controlling autoreactivity to RNA‐binding nuclear antigens.     

The type 1 diabetes resistance locus B10 Idd9.3 mediates impaired B‐cell lymphopoiesis and implicates microRNA‐34a in diabetes protection

NOD.B10 Idd9.3 mice are congenic for the insulin‐dependent diabetes (Idd) Idd9.3 locus, which confers significant type 1 diabetes (T1D) protection and encodes 19 genes, including microRNA (miR)‐34a, from T1D‐resistant C57BL/10 mice. B cells have been shown to play a critical role in the priming of autoantigen‐specific CD4⁺ T cells in T1D pathogenesis in non‐obese diabetic (NOD) mice. We show that early B‐cell development is impaired in NOD.B10 Idd9.3 mice, resulting in the profound reduction of transitional and mature splenic B cells as compared with NOD mice. Molecular analysis revealed that miR‐34a expression was significantly higher in B‐cell progenitors and marginal zone B cells from NOD.B10 Idd9.3 mice than in NOD mice. Furthermore, miR‐34a expression in these cell populations inversely correlated with levels of Foxp1, an essential regulator of B‐cell lymphopoiesis, which is directly repressed by miR‐34a. In addition, we show that islet‐specific CD4⁺ T cells proliferated inefficiently when primed by NOD.B10 Idd9.3 B cells in vitro or in response to endogenous autoantigen in NOD.B10 Idd9.3 mice. Thus, Idd9.3‐encoded miR‐34a is a likely candidate in negatively regulating B‐cell lymphopoiesis, which may contribute to inefficient expansion of islet‐specific CD4⁺ T cells and to T1D protection in NOD.B10 Idd9.3 mice.     

Pathogenic T helper type 17 cells contribute to type 1 diabetes independently of interleukin‐22

We have shown that pathogenic T helper type 17 (Th17) cells differentiated from naive CD4⁺ T cells of BDC2·5 T cell receptor transgenic non‐obese diabetic (NOD) mice by interleukin (IL)‐23 plus IL‐6 produce IL‐17, IL‐22 and induce type 1 diabetes (T1D). Neutralizing interferon (IFN)‐γ during the polarization process leads to a significant increase in IL‐22 production by these Th17 cells. We also isolated IL‐22‐producing Th17 cells from the pancreas of wild‐type diabetic NOD mice. IL‐27 also blocked IL‐22 production from diabetogenic Th17 cells. To determine the functional role of IL‐22 produced by pathogenic Th17 cells in T1D we neutralized IL‐22 in vivo by using anti‐IL‐22 monoclonal antibody. We found that blocking IL‐22 did not alter significantly adoptive transfer of disease by pathogenic Th17 cells. Therefore, IL‐22 is not required for T1D pathogenesis. The IL‐22Rα receptor for IL‐22 however, increased in the pancreas of NOD mice during disease progression and based upon our and other studies we suggest that IL‐22 may have a regenerative and protective role in the pancreatic islets.     

Toll-like receptor 7 deficiency suppresses type 1 diabetes development by modulating B-cell differentiation and function

Innate immunity mediated by Toll-like receptors (TLRs), which can recognize pathogen molecular patterns, plays a critical role in type 1 diabetes development. TLR7 is a pattern recognition receptor that senses single-stranded RNAs from viruses and host tissue cells; however, its role in type 1 diabetes development remains unclear. In our study, we discovered that Tlr7-deficient (Tlr7^−/−) nonobese diabetic (NOD) mice, a model of human type 1 diabetes, exhibited a significantly delayed onset and reduced incidence of type 1 diabetes compared with Tlr7-sufficient (Tlr7^+/+) NOD mice. Mechanistic investigations showed that Tlr7 deficiency significantly altered B-cell differentiation and immunoglobulin production. Moreover, Tlr7^−/− NOD B cells were found to suppress diabetogenic CD4⁺ T-cell responses and protect immunodeficient NOD mice from developing diabetes induced by diabetogenic T cells. In addition, we found that Tlr7 deficiency suppressed the antigen-presenting functions of B cells and inhibited cytotoxic CD8⁺ T-cell activation by downregulating the expression of both nonclassical and classical MHC class I (MHC-I) molecules on B cells. Our data suggest that TLR7 contributes to type 1 diabetes development by regulating B-cell functions and subsequent interactions with T cells. Therefore, therapeutically targeting TLR7 may prove beneficial for disease protection.     

Stable activity of diabetogenic cells with age in NOD mice: dynamics of reconstitution and adoptive diabetes transfer in immunocompromised mice

The non-obese diabetic (NOD) mouse is a prevalent disease model of type 1 diabetes. Immune aberrations that cause and propagate autoimmune insulitis in these mice are being continually debated, with evidence supporting both dominance of effector cells and insufficiency of suppressor mechanisms. In this study we assessed the behaviour of NOD lymphocytes under extreme expansion conditions using adoptive transfer into immunocompromised NOD.SCID (severe combined immunodeficiency) mice. CD4⁺ CD25⁺ T cells do not cause islet inflammation, whereas splenocytes and CD4⁺ CD25⁻ T cells induce pancreatic inflammation and hyperglycaemia in 80–100% of the NOD.SCID recipients. Adoptively transferred effector T cells migrate to the lymphoid organs and pancreas, proliferate, are activated in the target organ in situ and initiate inflammatory insulitis. Reconstitution of all components of the CD4⁺ subset emphasizes the plastic capacity of different cell types to adopt effector and suppressor phenotypes. Furthermore, similar immune profiles of diabetic and euglycaemic NOD.SCID recipients demonstrate dissociation between fractional expression of CD25 and FoxP3 and the severity of insulitis. There were no evident and consistent differences in diabetogenic activity and immune reconstituting activity of T cells from pre-diabetic (11 weeks) and new onset diabetic NOD females. Similarities in immune phenotypes and variable distribution of effector and suppressor subsets in various stages of inflammation commend caution in interpretation of quantitative and qualitative aberrations as markers of disease severity in adoptive transfer experiments.     

Human adipose‐tissue derived mesenchymal stem cells induce functional de‐novo regulatory T cells with methylated FOXP3 gene DNA

Due to their immunomodulatory properties, mesenchymal stem cells (MSC) are interesting candidates for cellular therapy for autoimmune disorders, graft‐versus‐host disease and allograft rejection. MSC inhibit the proliferation of effector T cells and induce T cells with a regulatory phenotype. So far it is unknown whether human MSC‐induced CD4⁺CD25⁺CD127^–forkhead box P3 (FoxP3)⁺ T cells are functional and whether they originate from effector T cells or represent expanded natural regulatory T cells (nT_reg). Perirenal adipose‐tissue derived MSC (ASC) obtained from kidney donors induced a 2·1‐fold increase in the percentage of CD25⁺CD127^–FoxP3⁺ cells within the CD4⁺ T cell population from allostimulated CD25^–/dim cells. Interleukin (IL)‐2 receptor blocking prevented this induction. The ASC‐induced T cells (iT_reg) inhibited effector cell proliferation as effectively as nT_reg. The vast majority of cells within the iTreg fraction had a methylated FOXP3 gene Treg‐specific demethylated region (TSDR) indicating that they were not of nT_reg origin. In conclusion, ASC induce T_reg from effector T cells. These iT_reg have immunosuppressive capacities comparable to those of nT_reg. Their induction is IL‐2 pathway‐dependent. The dual effect of MSC of inhibiting immune cell proliferation while generating de‐novo immunosuppressive cells emphasizes their potential as cellular immunotherapeutic agent.