AUTOIMMUNITY

Research ArticleImmunology Free access | 10.1172/jci.insight.126471 Stem cell–derived tissue-associated regulatory T cells suppress the activity of pathogenic cells in autoimmune diabetes Mohammad Haque,1 Fengyang Lei,2 Xiaofang Xiong,1 Jugal Kishore Das,1 Xingcong Ren,3 Deyu Fang,4 Shahram Salek-Ardakani,5 Jin-Ming Yang,3 and Jianxun Song1 First published February 19, 2019 - More info Abstract The autoantigen-specific Tregs from pluripotent stem cells (PSCs), i.e., PSC-Tregs, have the ability to suppress autoimmunity. PSC-Tregs can be programmed to be tissue associated and to infiltrate into local inflamed tissues to suppress autoimmune responses after adoptive transfer. Nevertheless, the mechanisms by which the autoantigen-specific PSC-Tregs suppress the autoimmune response remain to be fully elucidated. In this study, we generated functional autoantigen-specific Tregs from the induced PSC (iPSCs), i.e., iPSC-Tregs, and investigated the underlying mechanisms of autoimmunity suppression by these Tregs in a type 1 diabetes (T1D) murine model. A double-Tg mouse model of T1D was established in F1 mice, in which the first generation of RIP-mOVA Tg mice that were crossed with OT-I T cell receptor (TCR) Tg mice was challenged with vaccinia viruses expressing OVA (VACV-OVA). We show that adoptive transfer of OVA-specific iPSC-Tregs greatly suppressed autoimmunity in the animal model and prevented the insulin-secreting pancreatic β cells from destruction. Further, we demonstrate that the adoptive transfer significantly reduced the expression of ICAM-1 in the diabetic pancreas and inhibited the migration of pathogenic CD8+ T cells and the production of the proinflammatory IFN-γ in the pancreas. These results indicate that the stem cell–derived tissue-associated Tregs can robustly accumulate in the diabetic pancreas, and, through downregulating the expression of ICAM-1 in the local inflamed tissues and inhibiting the production of proinflammatory cytokine IFN-γ, suppress the migration and activity of the pathogenic immune cells that cause T1D. Graphical Abstract Introduction Type 1 diabetes (T1D) develops due to autoimmune self-destruction of pancreatic β cells that produce insulin, and life-time administration of insulin is required for treatment of this disease (1). Although accumulating knowledge has contributed greatly to our understanding of the autoimmune pathogenesis of T1D, the precise causes remain unclear. It has been generally appreciated that autoimmune diseases arise from the breakdown of immune tolerance (2, 3). A number of studies found the massive infiltration of CD8+ T cells into the pancreases of newly diagnosed T1D patients whereas the number of CD4+ T cells was greatly reduced (4, 5). Infiltrated CD8+ T cells are pathogenic to the islets, can cause the destruction of pancreatic β cells, and ultimately reduce the secretion of insulin. Currently, there is no definitive treatment for controlling blood glucose level in T1D except durable insulin therapy. As a result, generation and transplantation of exogenous β cells to replace dead or dysfunctional endogenous β cells are considered as a promising strategy for controlling blood glucose level in patients with T1D. However, since the autoimmune disease is a continuous process, after β cell transplantation, diabetes may develop and progress again through destruction of the pancreatic islets by pathogenic T cells. Therefore, β cell transplantation might not be a long-lasting solution for the control of blood glucose level in T1D. Over the past several years, there has been an increasing interest in Tregs, which play a fundamental role in controlling various autoimmune responses. Numerous preclinical studies suggest that adoptive transfer of Tregs can prevent or cure T cell–mediated autoimmune diseases, such as T1D and arthritis (6, 7). There are several advantages of the Treg adoptive transfer over conventional treatments. These advantages include (a) the potential of antigen (Ag) specificity without general immunosuppression; (b) the option of inducing “physiological” long-lasting regulation in vivo; and (c) the possibility of Treg-based immunotherapy as a customized or personally designed agent for each patient, with reduced side effects (8, 9). It is now generally believed that adoptive transfer of in vitro–generated Tregs can reduce the hazards of complicated surgical events throughout the life. However, the use of Tregs has been complicated due to difficulties in expanding and characterizing this minor subset of T cells. Here, we report the development of a robust technique for producing a large amount of autoantigen-specific Tregs from induced pluripotent stem cells (iPSCs), i.e., iPSC-Tregs that retain all the quintessential characteristics of this T cell subset, including expressions of CD25, CTLA-4, and FoxP3 and production of IL-10. We show that adoptive transfer of these autoantigen-specific iPSC-Tregs significantly reduces the high ratio of CD8+ to CD4+ T cells in the pancreases of diabetic mice and markedly decreases the expression of intracellular adhesion molecule-1 (ICAM-1) in the pancreases of prediabetic and diabetic mice. These results demonstrate the great potential of stem cell–derived autoantigen-specific Tregs in T1D immunotherapy. Moreover, we show that the stem cell–derived tissue-associated Tregs control autoimmune diabetes via preventing the ICAM-1–mediated migration of pathogenic CD8+ T cells in the pancreas and suppressing the production of proinflammatory cytokine IFN-γ. Results Characterization of autoantigen-specific naturally occurring Treg–like iPSC-Tregs. Although FoxP3 is a master regulator and a specific molecular marker for naturally occurring Tregs (nTregs), there is evidence that FoxP3 expression is not a distinct and reliable marker or a sole regulator of functionally stable Tregs. In addition, recent evidence showed that various transcription factors are critical for the development of FoxP3+ Tregs, including Nr4a1, Ikzf4, Tnfrsf18, and Tbx21 (10). These genes, which are substantially expressed in nTregs, are essential for regulating Treg transcriptional programs and maintaining the lineage stability in Tregs. We previously showed the generation of functional Ag-specific iPSC-Tregs, which had the ability to suppress the development of autoimmune arthritis after adoptive transfer in murine models (11, 12). Because the iPSC-Tregs are retrovirally transduced with FoxP3 and TCR genes and have a similar phenotype to iPSC-Tregs in expression of IL-10 and TGF-β, we examined the Treg signature genes in the autoantigen-specific iPSC-Tregs. We adoptively transferred Rag 1–/– mice with pre-iPSC-Tregs that had been cocultured with OP9-DL1/DL4/I-Ab cells for 7 days. Six weeks later, mice were sacrificed and their spleens and lymph nodes (LNs) were removed for the isolation of CD4+CD25+ cells. Conversely, CD4+CD25+ cells were sorted from the spleens and LNs of normal C57BL/6 mice. RT-PCR analysis of the Treg signature genes showed that there were no significant differences between the mice in gene expression of the transcription factors (Nr4a1, Il2ra, Ikzf4, Tnfrsf18, and Bcl6) involved in the regulation of Treg functions (all P > 0.05, Figure 1). Collectively, these results and suggest that the autoantigen-specific iPSC-Tregs are nTreg-like suppressive cells. Figure 1 Characterization of autoantigen-specific nTreg-like iPSC-Tregs. PCR analysis was performed by TaqMan real-time PCR. Primers for sequences were used as follows: (A) Nr4a1, forward 5′-TGTGAGGGCTGCAAGGGCTTC-3′, reverse 5′-AAGCGGCAGAACTGGCAGCGG-3′; (B) Il2ra, forward 5′-AACTGCCAGTGCACCAGCAAC-3′, reverse 5′ GAGGTGGCTCCCTGCAGTGAC-3′; (C) Ikzf4, forward 5′-CAATCTGCTTCGCCACATCAAG-3′, reverse 5′-GCCACAGTAGTTGCACTTGTAG-3′; (D) Tbx21, forward 5′-GGAGCCCACTGGATGCGCCAG-3′, reverse 5′ AGGCAGCCTCTGGCTCTCCATC-3′, Tnfrsf18, forward 5′-CCTGCCAACCAGGCCAGAGGG-3′, reverse 5′-GTCCAAAGTCTGCAGTGACCG-3′; and (E) Bcl6, forward 5′-CACACCCGTCCATCATTGAA-3′, reverse 5′-TGTCCTCACGGTGCCTTTTT-3′. Data shown are representative of 3 identical experiments. The values represent mean ± SEM (n = 3). ns, P > 0.05, Student’s 1-tailed t test. Accumulation of activated CD8+ T cells in autoantigen-expressing pancreases of diabetic mice. To show the expression of autoantigen in the pancreases of double-Tg mice (B6-mOVA with OT-I), we performed immunofluorescence examination. The results showed the expression of OVA autoantigen in the double-Tg mice but not in the B6 or OT-I control mice (Figure 2A). Because the OVA-specific CD8+ T cells have the ability to migrate into the pancreases of the F1 generation that results from crossing B6 mOVA with OT-I TCR Tg mice and to cause destruction of the islets, we examined the fate of OVA-specific CD8+ T cells in vivo (Figure 2B). The migrations of CD8+ T cells in diabetic mice were further confirmed by flow cytometry in which more numbers of both CD8+ and TCRVβ5+ cells were present in diabetic mice (Figure 2C). In F1 mice after vaccinia virus (VACV) infection (Supplemental Figure 3; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.126471DS1), the draining LNs and spleens of the F1 mice were analyzed for the presence of OT-I CD8+ T cells. The proportions of OVA-specific CD8+ cells among the total CD8+ cells in the pancreatic LNs were significantly higher than in the pyloric, mesenteric, inguinal, cervical, or splenic LNs. No apparent accumulation or homing was observed in the non-Tg control B6 mice (Figure 2D). Additionally, all F1 mice after VACV infection developed autoimmune diabetes at age of 9 weeks, which was confirmed by the measurement of blood sugar (Figure 2E). These results verify that autoantigen-specific CD8+ T cells are the main pathogenic immune cells that induce autoimmune diabetes in the murine model. Figure 2 Accumulation of activated CD8+ T cells in autoantigen-expressing pancreases in diabetic mice. Pancreases were isolated from B6-mOVA × OT-I TCR double-Tg mice and B6 mice at 9 weeks of age, including 1 week in which mice were challenged with vaccinia viruses expressing OVA (VACV-OVA). (A) Detection of OVA expression by immunohistochemically staining. OVA expression (arrow) is indicated (original magnification, ×200). Data are representative of 5 mice per group in 3 independent experiments. Scale bar: 20.1 μm. (B) CD8+ T cell infiltration in the pancreas. CD8+ T cells (arrow) are indicated. Data are representative of 5 mice per group in 3 independent experiments. Scale bar: 20.1 μm. (C) OVA-specific CD8+ T cells in the pancreas. OVA-specific TCRVβ5 was analyzed by flow cytometry, after gating on CD8+ populations from the pancreas. Data shown are representative of 3 independent experiments (P < 0.001, Student’s 1-tailed t test). (D) Summarized analyses of OVA-specific CD8+ T cells in various locations. Data shown are representative of 5 mice per group in 3 independent experiments. Data shown are representative of 3 individual experiments. The values represent mean ± SD. **P < 0.01; ns, P > 0.05, multiple Student’s 1-tailed t test. (E) Blood glucose measurement. Data shown are representative of 3 individual experiments (n = 5). The values represent mean ± SD. **P < 0.01, Student’s 1-tailed t test. In vivo specificity of nTreg-like autoantigen-specific iPSC-Tregs. To exert their suppressive effects, Tregs need to migrate to specific tissues or organs, and this requires Ag specificity (8). We and others have previously reported that Tregs are detected within inflamed tissues and transplanted grafts, suggesting that these Tregs could control effector T cells in the peripheral tissues at sites of ongoing immune responses (12–14). Tregs may block the pathogenic cells from filling up their appropriate niche by taking up the space. The Treg population was reduced in autoimmune prone animals and patients, and Tregs were defective in NOD mice (15). In this study, we observed that mice receiving autoantigen OVA-specific iPSC-Tregs maintained a high population of both CD4+ and FoxP3+ cells in the pancreas, as compared with diabetic mice receiving control cells (iPSC-derived cells with empty vector) or nontissue-associated SM1-specific iPSC-Tregs by immunofluorescence examination (Figure 3A) and by flow cytometric analysis (90.1% vs. 12.5% or 16.8% at week 13, Figure 3B). These results suggest that the autoantigen-specific iPSC-Tregs are organ/tissue-associated nTreg-like suppressive cells. Figure 3 Accumulation of autoantigen-specific iPSC-Tregs in the pancreases of diabetic mice following adoptive transfer. B6-mOVA Tg × OT-I TCR double-Tg mice were immunized with VACV-OVA. At week 10, control cells or OVA- or SM1-specific pre-iPSC-Tregs were transferred into diabetic mice. Before or after the cell transfer, mice were sacrificed and their pancreases were isolated for analysis of CD4 and FoxP3. (A) Immunohistology at week 13 (original magnification, ×200). Scale bar: 20.1 μm. (B) Flow cytometric analysis at week 10 (before the cell transfer), 13, and 16. Data are representative of 5 mice per group in 3 independent experiments. Autoantigen-specific iPSC-Tregs can prevent destruction of pancreaticβcells and reduce autoimmune diabetes. Next, we injected diabetic mice with autoantigen-specific iPSC-Tregs. Two weeks after the cell transfer, blood glucose levels were measured. This cell transfer with autoantigen OVA-specific iPSC-Tregs but not control cells or nontissue-associated SM1-specific iPSC-Tregs significantly reduced blood glucose level with urine discharge in all of the diabetic mice (P < 0.0001, Figure 4A and Supplemental Figure 3). T1D develops due to the destructive autoreactive immune response in which CD8+ T cells play a critical role. CD8+ T cells infiltrated into the pancreatic islets of the T1D patients at both initial and final destructive phases of autoimmune β cells attack (16). The blood glucose level was increased because of the destruction of β cells in the islets by pathogenic CD8+ T cells in the diabetic pancreas, which was evidenced in the above mouse diabetic model. More CD8+ T cells were accumulated in the pancreases of mice receiving control cells or nontissue-associated Ag-specific iPSC-Tregs than autoantigen-specific iPSC-Tregs by immunofluorescence examination (Figure 4B) and by flow cytometric analysis (49.8% or 46.1% vs. 6.5%, Figure 4C). Only mice receiving autoantigen-specific iPSC-Tregs had a significantly reduced percentage of incidence of diabetes (P < 0.001, Figure 4D). A large number of inflammatory cells had infiltrated into the pancreases of mice receiving control cells, whereas the infiltration was substantially reduced in mice receiving autoantigen-specific iPSC-Tregs, as shown by H&E staining (Figure 4E). The total number of the islets was also reduced in mice receiving control cells as compared with autoantigen-specific iPSC-Tregs (P < 0.01 or P < 0.001, Figure 4F). To detect the destruction of β cells in the islets of diabetic mice, we stained pancreases with insulin. The islets were reduced in size and number in mice receiving control cells, whereas mice receiving autoantigen-specific iPSC-Tregs were protected from the destruction, by immunofluorescence examination (Figure 4G). The islets were partially destructed by pathogenic CD8+ T cells, which caused the increase of blood sugar and developed autoimmune diabetes, but some insulin+ staining in diabetic mice was still present. Collectively, these results indicate that autoantigen-specific iPSC-Tregs are specific and effective in protecting the hosts from islet destruction and in promoting insulin secretion to prevent the mice from diabetes mellitus. Figure 4 Prevention of destruction of pancreatic β cells and suppression of autoimmune diabetes by autoantigen-specific iPSCs-Tregs. Control cells or pre-iPSC-Tregs were adoptively transferred into diabetic mice, as described in Figure 3. (A) Blood sugar measurement at various weeks. Data shown are representative of 5 mice per group in 3 independent experiments. **P < 0.001; ***P < 0.0001, 2-way ANOVA analysis. (B) Immunofluorescence detection of pathogenic immune cells. Mice were sacrificed and pancreases were isolated for immunohistochemistry staining with CD8+ T cells (original magnification, ×200). Scale bar: 20.1 μm. Data are representative of 5 mice per group in 3 independent experiments. (C) Flow cytometric analysis of pathogenic immune cells. Pancreatic lymph nodes (LNs) were isolated, and single-cell suspensions were prepared for OVA-specific CD8+ TCRVβ5+ staining and analyzed by flow cytometry. Data shown are representative of 3 identical experiments (P < 0.001, Student’s 1-tailed t test). (D) Percentage of incidence of diabetes at week 22. Data shown are representative of 5 mice per group in 3 independent experiments. The values represent mean ± SD. **P < 0.001; ***P < 0.001, 1-way ANOVA analysis. (E) Representative photomicrographs (H&E staining) of the islet inflammation (original magnification, ×200). Scale bar: 20.4 μm. Cellular infiltrations (arrow) are indicated. Data are representative of 5 mice per group in 3 independent experiments. (F) Islet count from sections of 5 individual pancreases in each group. Data are represented as the mean ± SD of 3 independent experiments (***P < 0.001, 1-way ANOVA analysis). (G) Representative photomicrographs (immunofluorescence staining) of islet destruction (original magnification, ×200). Scale bar: 20.1 μm. Insulin-producing cells (arrow) are indicated. Data are representative of 5 mice per group in 3 independent experiments. Autoantigen-specific iPSC-Tregs can decrease the expression of ICAM-1. A number of mononuclear cells, including macrophages and CD4+ T cells, were found to be initially predominant in the autoimmune process taking place in T1D (17, 18). In autoimmune diabetes, these cells played an important role in the early infiltrating process and were shown to express ICAM-1, which served to bind to lymphocytes and possibly to monocytes and polymorphonuclear leukocytes (19). The infiltrated ICAM-1 can promote the release of several cytokines that modulate the expression of the adhesion molecules, thus increasing the adhesion of leukocytes and other autoreactive T cells (20). These autoreactive T cells, after migrating into the pancreas, start to destroy β cells. We thus examined the expression of ICAM-1 in the pancreases. The high expression of ICAM-1 in the pancreases of the diabetic mice, which was undetectable in the normal pancreases, was significantly reduced in diabetic mice receiving autoantigen-specific iPSC-Tregs but not other control cells (Figure 5, A and B). In addition, the high expression of ICAM-1 on the effector CD4+ T cells in the pancreases of the diabetic mice receiving control cells (84.4% or 78.9%), which was undetectable in the normal pancreases (0.35%), was markedly reduced in those of mice receiving autoantigen-specific iPSC-Tregs (18.4%) (Figure 5C). In addition, the expression of ICAM-1 on CD11b+ cells in the pancreases of diabetic mice receiving control cells (15.64% or 12.83%), which was at a low level in the normal pancreases (4.89%), was considerably reduced in those of mice receiving autoantigen-specific iPSC-Tregs (5.68%) (Figure 5D). Because CD11b is expressed on the surface of various leukocytes, including monocytes/macrophages, granulocytes, neutrophils, NK cells, and dendritic cells, the reduction of CD11b+ICAM-1+ cells indicated the inhibition of leukocyte adhesion and migration to mediate the inflammatory response. Taken together, these results suggest that the autoantigen-specific iPSC-Tregs can reduce the expression of ICAM-1 in diabetic mice, inhibit the migration of autoreactive CD8+ T cells into the pancreas, and, ultimately, protect the pancreatic β cells from destruction. Figure 5 Reduction of ICAM-1 expression in the diabetic pancreas by autoantigen-specific iPSC-Tregs. Mice were sacrificed 4 weeks after adoptive cell transfer, and the pancreases were isolated from nondiabetic, diabetic (cell transfer control), and diabetic (Ag-specific iPSC-Treg transfer) mice. Samples were prepared for immunohistochemistry staining. (A) ICAM-1+ zones (arrow) are indicated (original magnification, ×200). Data are representative of 5 mice per group in 3 independent experiments. (B) Quantification of ICAM-1 expression from sections of 5 individual pancreases in each group. Data are represented as the mean ± SD from 3 independent experiments (**P < 0.01; ***P < 0.001, 1-way ANOVA). (C and D) Flow cytometry analysis of ICAM-1 expression in pancreatic CD4+ T or CD11b+ cells. The pancreatic lymph nodes (LNs) were isolated, and single-cell suspensions were prepared for ICAM-1 staining on CD4+ or CD11b+ cells and analyzed by flow cytometry. Data shown are representative of 3 identical experiments. Autoantigen-specific iPSC-Tregs can migrate to the pancreas and produce a large amount of suppressive cytokines. In BDC2.5/NOD mice, FoxP3 was highly expressed in insulitic lesions where the function was impaired due to alterations in their gene expression profile (21, 22). We previously showed that the iPSC-Tregs exhibited a similar expression of Treg signature genes and produced IL-10 and TGF-β (12). In the current study, we observed very few numbers of FoxP3-expressing T cells in the pancreases of diabetic mice, and these cells did not secret IL-10 and TGF-β, which are the two key cytokines that are the hallmark for functional Tregs and secreted by activated and functional Tregs. We further showed that the pancreatic CD4+ T cells from mice receiving autoantigen-specific iPSC-Tregs produced substantially more IL-10 (66.1% vs. 12.9% or 15.1%) and TGF-β (5.61% vs. 0.62% or 1.68%), as compared with those from mice receiving control cells (Figure 6). These results support the assumption that autoantigen-specific iPSC-Tregs accumulating in the pancreas can secrete large amounts of suppressive cytokines (IL-10 and TGF-β), reduce the expression of ICAM-1, and prevent the migration of pathogenic CD8+ T cells into the pancreas, thus protecting the pancreas from destruction. Figure 6 Induction of IL-10 and TGF-β by autoantigen-specific iPSC-Tregs. Mice were sacrificed 4 weeks after adoptive cell transfer, and single-cell suspension was prepared from the pancreatic lymph nodes (LNs). Cells were stimulated with plate-coated CD3 and soluble CD28 antibodies and then stained with CD4, TGF-β, and IL-10. The CD4 population was gated, and the production of TGF-β and IL-10 was analyzed. Data shown are representative of 3 identical experiments. Autoantigen-specific iPSC-Tregs can decrease the production of IFN-γby pathogenic immune cells in the diabetic pancreatic islets. Treatment of NOD mice with an anti-IFN-γ monoclonal antibody could block diabetes (23); conversely, overproduction of IFN-γ in pancreatic islets provoked the disease (24). We next determined the secretion of IFN-γ in the diabetic islet. We found more IFN-γ–producing pancreatic CD8 and CD4 T cells in mice receiving control cells (31.4% and 30.4% or 29.7% and 28.6%); by contrast, both of the IFN-γ–producing pancreatic CD8 and CD4 T cells were significantly reduced in mice receiving autoantigen-specific iPSC-Tregs (14.6% and 10.9%) (Figure 7). These results indicate that the autoantigen-specific iPSC-Tregs can protect the islets from destruction by suppressing the secretion of IFN-γ produced by the pathogenic immune cells. Figure 7 Downregulation of IFN-γ by autoantigen-specific iPSC-Tregs in the diabetic pancreatic islets. Mice were sacrificed 4 weeks after adoptive cell transfer, and single cell suspension was prepared from the pancreatic lymph nodes (LNs). Cells were stimulated with plate-coated CD3 and soluble CD28 antibodies, and then stained with CD4, CD8, and IFN-γ for flow cytometric analysis. (A) IFN-γ production in CD4+ or CD8+ population. Data shown are the representative of 3 identical experiments. (B) Quantification of IFN-γ production. Data shown are representative of 3 identical experiments (**P < 0.01; ***P < 0.001, 1-way ANOVA). Discussion We and others have previously reported that adoptive transfer of stem cells has or stem cell–derived Tregs has the ability to suppress the development of autoimmunity in various animal models (11, 12, 25, 26). However, the suppressive mechanisms behind this suppression remain to be fully defined. By investigating the adoptive transfer of autoantigen-specific iPSC-Tregs in a murine model of T1D, we demonstrate that adoptive transfer significantly reduced the high ratio of CD8+ to CD4+ T cells in the pancreases of diabetic mice. We also show a critical role of this transfer in reducing the expression of ICAM-1 in inflamed pancreatic tissues and preventing the accumulation of pathogenic CD8+ T cells in the pancreases and diabetic pathogenesis. We further demonstrate that autoantigen-specific iPSC-Tregs associated with inflamed tissue and protected the islets from destruction through suppressing the production of proinflammatory cytokine IFN-γ. These findings may help us better understand the pathogenesis of autoimmunity in T1D and provide a foundation for therapeutic use of the stem cell–derived tissue-associated Tregs in the treatment of T1D (27). Among animal models of autoimmune diabetes, the NOD mouse is a widely used animal of autoimmune T cell–mediated T1D (spontaneously nonobese diabetic). The NOD mouse model clearly shows the leukocytic infiltration into the pancreatic islets (insulitis) and autoimmune destruction of the pancreatic β cell in female mice (2–4 weeks), which occurs later in male mice (5–7 weeks). Insulitis in NOD mice shows a combination of T cells (both CD4+ and CD8+), B cells (28), and inconstant numbers of macrophages/dendritic cells. In addition, a dominant-negative mutation in the mouse insulin 2 gene (Ins2Akita) produces a severe insulin deficiency syndrome exclusive of autoimmune participation and various transgenes overexpressed in β cells. Moreover, pharmacologically induced T1D (without autoimmunity) by alloxan or streptozotocin produces hyperglycemia in most strains of mice. Several low doses of streptozotocin combing direct β cell toxicity with local inflammation also elicit T1D in a male sex-specific fashion (29). To determine the suppressive mechanisms of stem cell–derived tissue-associated Tregs, we employed a mouse model of T1D by crossing B6-mOVA Tg mice with OT-I TCR Tg mice. Around 8 weeks of age, blood glucose levels were measured in F1 mice. Approximately 20%–40% of the mice were found to be diabetic; one potential explanation for this occurrence is that the OT-I CD8+ T cells may be tolerated during T cell development in the thymuses of F1 mice. As we propose in our model in which mice will develop autoimmune diabetes due to a large amount and the autoreactivity of CD8+ T cells, we activated tissue-associated CD8+ T cells by inoculating VACV-OVA into the mice in which the viruses-induced CD8+ T cells became highly polyfunctional (30). All mice developed diabetes with high blood glucose levels (Supplemental Figure 3) and more urine discharge, for which mice needed additional animal care. In this mouse model of T1D, OVA protein serves as inflamed tissue–associated autoantigen and is highly expressed in the pancreas. OVA-specific CD8+ T cells act as central pathogenic immune cells that accumulate in the pancreas and cause pancreatic β cell destruction and T1D (Figure 2). In addition, in this mouse model of T1D, we observed a non-OVA-specific CD4+ population existing in the pancreas (Figure 3), and these CD4+ T cells produced IFN-γ (Figure 7), which might also involve to the disease pathogenesis. These FoxP3–CD4+ T cells in the pancreas were TCRVα2–/Vβ5–, indicating that T cell activation was not through OVA-specific TCR. Of note, the number of these effector CD4+ T cells was dramatically reduced in mice receiving OVA-specific but not SM1-specific iPSC-Tregs (Supplemental Figure 4). These results also indicate that tissue-associated (OVA-specific) iPSC-Tregs are more effective than nontissue-associated (SM1-specific) iPSC-Tregs. Because of the challenge of VACV-OVA, these non-OVA-specific CD4+ T cells mainly originating from the RIP-mOVA descent may undergo bystander activation (31, 32). However, addition studies are needed on the pathways leading to bystander T cell activation under the condition. Tregs are an important component of self-tolerance, and numerous studies have demonstrated the effects of Tregs on natural and induced autoimmune diseases in various mouse models (33, 34). Targeting Tregs as a treatment for autoimmune disorders is an attractive approach, as there is an emerging consensus that many patients suffering from autoimmune disease have dysfunctional Tregs (35, 36). Given the manageability of murine models, the evidence for Tregs in control of autoimmune diseases has recently become clearer, particularly as a therapeutic intervention. In well-established models of rheumatoid arthritis (37), multiple sclerosis (38), and systemic lupus erythematosus (39), adoptive transfer of polyclonal Tregs could prevent or slow disease progression when administered prior to disease occurrence. In multiple sclerosis, Ag-specific Tregs targeting the disease-associated Ags were highly efficacious in reverting the ongoing disease (8, 40). Specifically, the nonobese T1D NOD.Cd28–/– mouse model deficient in Tregs developed diabetes at an accelerated rate as compared with NOD mice. Injection of NOD Tregs into NOD.Cd28–/– mice could delay and, in some cases, prevent the development of diabetes (41). In the current study, we have confirmed that adoptive transfer of stem cell–derived autoantigen-specific Tregs but not control cells, nonspecific iPSC-Tregs, or nontissue-associated Ag-specific iPSC-Tregs caused a high level of accumulation of these cells in the pancreas and reversed the disease process of autoimmune diabetes in the mouse model of T1D that was caused by pathogenic CD8+ T cells (Figure 4 and Supplemental Figure 3). A small number of Ag-specific iPSC-Tregs was observed in LNs and spleen (Supplemental Figure 5). A possible working mechanism of stem cell–derived autoantigen-specific Tregs is mediated through the secretion of large amounts of suppressive cytokines (e.g., IL-10 and TGF-β) that were identified (Figure 6). Of note, Ag-specific iPSC-Tregs have a functional stability for which we had previously demonstrated in an autoimmune condition (12), and FoxP3 expression in these cells persisted for 6 weeks after adoptive transfer in the diabetic mice (Figure 3). We further show that ICAM-1 (also known as CD54), a member of the Ig-like superfamily of adhesion proteins, plays a critical role in the accumulation of the pathogenic CD8+ T cells in the pancreases of the mice with autoimmune diabetes. ICAM-1 is a cell surface glycoprotein expressed in endothelial cells and some immune cells. ICAM-1 expressed in diabetic pancreatic tissues can bind to integrins of type CD11a/CD18 or CD11b/CD18 on pathogenic CD8+ T cells, and this binding facilitates the accumulation of pathogenic CD8+ T cells in the panaceas. The expression of ICAM-1 in the pancreases of diabetic mice is much higher than that in normal mice. In contrast, in the pancreases of diabetic mice, the adoptive transfer of autoantigen-specific iPSC-Tregs can dramatically reduce the expression of ICAM-1. This cell transfer can also reduce the accumulation of autoreactive CD8+ T cells in the pancreases and eventually protect the pancreatic β cells from further devastation (Figure 5). This conclusion is in line with those of previous studies showing that the expression of ICAM-1 is critical for the development of experimental autoimmune encephalomyelitis (42, 43). The role of IFN-γ, a prototypical Th1 cytokine, has been appreciated in the development of T1D, because the treatment of NOD mice with an anti-IFN-γ monoclonal antibody prevented the development of the autoimmune diabetes (23, 24). We detected an elevated amount of IFN-γ in both effector CD8 and CD4 T cells of the diabetic islets and showed that adoptive transfer of stem cell–derived autoantigen-specific Tregs markedly decreased the production of the proinflammatory cytokine in the pathogenic T cells. We demonstrated that the autoantigen-specific iPSC-Tregs protected the islets from mass destruction via suppressing the production of pathogenic IFN-γ (Figure 7). As other proinflammatory cytokines, such as IL-21 (44), IL-17 (45), TNF-α (46), and IL-1β (47) also play roles in the pathogenesis of T1D, studies on these proinflammatory cytokines would be needed to better exploit the stem cell–derived tissue-associated Tregs as a therapeutic approach against autoimmune disorders. The Tg model used in this study highly expressed OVA as surrogate autoantigen and received approximately 2 million of OVA-specific iPSC-Tregs for the treatment, which may be much higher than in the clinical setting with diverse low-abundance target peptides. However, the understanding of islet Ags has previously resulted in translation into new strategies by targeting tissue-specific immune interventions to prevent disease progression as well as to reverse T1D. A big advantage of our approach is the development of large numbers of autoantigen-specific nTreg-like suppressive cells (Figure 1 and Supplemental Figures 1 and 2), which can accumulate in the inflamed tissues and suppress local pathogenic immune cells. In addition, the stem cell–derived Tregs maintain stability for up to 3 months (11, 12), because the overexpression of FoxP3 has the ability to suppress the potential switch of Tregs to Th17 cells. Of note, the selection of optimal autoantigens for the development of tissue-associated Tregs is critical for the success of the Treg-based T1D immunotherapy. Taken together, the current study provides insight into the mechanisms by which stem cell–derived tissue-associated Tregs suppress autoimmunity in T1D and suggests that stem cell–derived autoantigen-specific Tregs have a great potential to be adapted as an immunotherapy for T1D. Methods Cell lines and mice. The mouse iPS-MEF-Ng-20D-17 cell line was obtained from the RIKEN Cell Bank (48). The OP9-DL1/DL4/I-Ab cell line was generated by a retroviral transduction of the OP9 cells (12). The SNL76/7 cell line (ATCC SCRC-1049) was purchased from ATCC. C57BL/6 (B6), Rag1–/–, B6 mOVA RIP (RIP-mOVA) Tg, and OT-I TCR Tg mice were purchased from The Jackson Laboratory. Cell culture. iPSCs were maintained on feeder layers of irradiated SNL76/7 cells in 6-well culture plates (Nunc) and were passaged every 3 days (48). Retroviral transduction and generation of OVA-specific iPSC-Tregs. cDNA for FoxP3 with OVA 322–339 (ISQAVHAAHAEINEAGR)-specific I-Ab-restricted TCR genes (Vα2 and Vβ5; obtained from Dario A. Vignali, University of Pittsburgh, Pittsburgh, Pennsylvania, USA) or LCMV (SMARTA1; SM1) gp61 (GLKGPDIYKGVYQFKSVEFD)-specific I-Ab-restricted TCR genes (Vα2 and Vβ8; obtained from Matthew A. Williams, University of Utah, Salt Lake City, Utah, USA) was used for retroviral transduction of mouse iPSCs and the generation of OVA- or SM1-specific iPSC-Tregs (12). Antibodies. PE-, PE/Cy7-, Alexa Fluor 647–, APC-, or APC/Cy7-conjugated anti-mouse TCRVβ5 (MR9-4), CD4 (GK1.5), CD11b (M1/70), TGF-β1 (TW7-16B4), and FoxP3 (MF-14) were obtained from Biolegend. FITC- or PE-conjugated anti-mouse CD8 (6A242) was obtained from Santa Cruz Biotechnology. APC-conjugated IL-10 (JES5-16E3) was purchased from BD Biosciences. Rabbit insulin antibody (C27C9; 3014) was purchased from Cell Signaling and anti-ICAM1 (YN1/1.7.4; ab25375) antibody was obtained from Abcam. Flow cytometric analysis. T cells from the pancreatic LNs were collected and intracellular IL-10 and TGF-β were analyzed by flow cytometry after gating on live CD4+ or CD8+ cells. RT-PCR. MiDR OTII 2A FoxP3-transduced mouse iPSCs were cocultured with OP9-DL1-DL4-I-Ab in the presence of mFlt-3L for 7 days. Differentiated iPSCs were separated and adoptively transferred into Rag1–/– mice. Mice were housed for 6 weeks for in vivo maturation of the iPSC-Tregs. Six weeks later, spleens and LNs were collected from normal B6 mice and cell transferred Rag1–/– mice. CD4+CD25+ cells were sorted and total RNA was extracted from sorted cells using QIAgen RNeasy mini kits. Samples were subjected to reverse transcription using a high-capacity cDNA synthesis kit (Applied Biosystems). PCR analysis was performed by TaqMan real-time PCR (Thermo Fisher Scientific). Ex vivo stimulation assay. Mice were sacrificed 4 weeks after adoptive cell transfer and single-cell suspension was then prepared from the pancreatic LNs. Cells were stimulated with plate-coated CD3 plus soluble CD28 antibodies. Productions of IL-10 and TGF-β were determined by intracellular cytokine staining (12). Murine autoimmune diabetes model. Autoimmune diabetes was induced in F1 mice that were crossed between RIP-mOVA Tg with OT-I TCR Tg mice by i.p. injection with VACV-OVA (2 × 106 PFU/mouse) (49). All of the mice developed diabetes after viral injection. Blood glucose was measured 1 week after the VACV-OVA injection. Blood glucose measurement. Blood glucose levels were determined using a Glucometer Ascensia Elite XL (Bayer). Six hundred milligrams per deciliter is the maximum measurable glucose reading. Mice were typically considered diabetic with readings of >250 mg/dl. Adoptive cell transfer. B6-mOVA Tg × OT-I TCR double-Tg mice (F1) were immunized with VACV-OVA for 1 week. At week 10, OVA-specific pre-iPSC-Tregs or iPSC-derived control cells (3 × 106) were i.v. transferred into diabetic mice. Four to six weeks later, mice were euthanized, and the pancreatic tissues were removed for histopathological examination. Histology and immunohistochemistry. For H&E staining, pancreatic tissues were fixed with 10% neutral formalin solution (VWR), and the fixed samples were prepared and stained as described previously (48). For immunofluorescent microscopy, the pancreatic tissues were frozen in cryovials on dry ice immediately after resection. Cryosectioning and immunofluorescent staining were performed as described previously (48). Statistics. Multiple Student’s 1-tailed t test or 1-way ANOVA or 2-way ANOVA analysis was performed to analyze the differences between the groups, using GraphPad Prism. A P value of less than 0.05 was considered significant. Study approval. The present studies in mice were reviewed and approved by The Texas A&M University Animal Care Committee and were in accordance with the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care. Author contributions JS and JMY designed the experiments, analyzed data, and contributed to the writing of the paper. MH, FL, and JKD performed the experiments. DF and SSA provided reagents in the animal model. XX and XR analyzed data. Supplemental material View Supplemental data Acknowledgments JS is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. This work was supported by National Institutes of Health grants R01AI121180, R01CA221867, and R21AI109239 and the American Diabetes Association (1-16-IBS-281 to JS). Footnotes Conflict of interest: The authors have declared that no conflict of interest exists. Copyright: © 2019 American Society for Clinical Investigation Reference information: JCI Insight. 2019;4(7):e126471. https://doi.org/10.1172/jci.insight.126471. References Delovitch TL, Singh B. The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD. Immunity. 1997;7(6):727–738. 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View this article via: PubMed CrossRef Google Scholar Version history Version 1 (February 19, 2019): In-Press Preview Version 2 (April 4, 2019): Electronic publication

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Abstract Epidemiological data suggest that previous infections can alter an individual's susceptibility to unrelated diseases. Nevertheless, the underlying mechanisms are not completely understood. Substantial research efforts have expanded the classical concept of immune memory to also include long‐lasting changes in innate immunity and antigen‐independent reactivation of adaptive immunity. Collectively, these processes provide possible explanations on how acute infections might induce long‐term changes that also affect immunity to unrelated diseases. Here, we review lasting changes the immune compartment undergoes upon infection and how infection experience alters the responsiveness of immune cells towards universal signals. This heightened state of alert enhances the ability of the immune system to combat even unrelated infections but may also increase susceptibility to autoimmunity. At the same time, infection‐induced changes in the regulatory compartment may dampen subsequent immune responses and promote pathogen persistence. The concepts presented here outline how infection‐induced changes in the immune system may affect human health. 1 Introduction People react differently to immunological challenges, such as infections or cancer, and show large variance in their susceptibility to inflammatory diseases, such as allergies or autoimmunity. The responsiveness of the immune system is naturally to a large degree determined by genetic factors. However, a series of recent studies have revealed that genetic factors can only explain about 50–75% of the immune trait variance and the immune profile of an individual is thus to an astonishingly large degree determined by environmental factors.1, 2 There is robust epidemiologic evidence that the decreasing frequency of infections in developed countries correlates with a rising prevalence of allergies and other inflammatory disorders, as already put forward in the hygiene hypothesis almost 30 years ago.3 The hypothesis has since been expanded and adapted to account also for the rise in autoimmunity in developed countries.4 Indeed, reduced incidence of infections and lower microbiotic diversity in developed countries correlate with an increased prevalence of clinical conditions that are caused by inappropriate immune responses, such as allergy and autoimmunity—and possibly even cancer.5 These data indicate that both pathogenic and nonpathogenic microorganisms play a fundamental role in educating the immune system.6 While the specific mechanisms regulating how the immune response to one pathogen alters the response to a later infection with another pathogen or the susceptibility to autoimmunity, allergy, or cancer have been studied in some individual combinations, a global picture of how infection history affects disease susceptibility is still lacking. Twin studies have been used to determine heritable versus nonheritable influences in immune responses elicited by vaccinations and the development of autoimmune diseases and found that both aspects strongly affect the immunological status of an individual.7 The microbes (commensal and pathogenic) an individual encounters throughout his or her life are most likely a major determinant for the nonheritable factors. Indeed monozygous twin pairs, from which only one twin acquired cytomegalovirus (CMV) infection, show greatly enhanced variation for immune parameters after the infection.1 Chronic infections, like CMV, and continuous interactions with commensal microbiota thus shape the immune system. Intriguingly, accumulating evidence suggests that transient challenges, such as acute infections and vaccinations, also have nonspecific effects on the ability of the immune system to react to other diseases.8, 9 Several adaptations of the immune system that could contribute to this altered reactivity have been reported, which include changes in the innate and adaptive arm of the immune system as well as alterations in its regulatory mechanisms. The following sections illustrate the long‐term changes that pathogen encounters elicit in these different parts of the immune system (Figure 1), how the sum of these changes contributes to shaping the immune system over time and how this may affect susceptibility to unrelated diseases. 2 Infection‐induced Changes in the Immune System 2.1 Infections Generate Pathogen‐Specific and Heterologous Immunity The capability to memorize previous pathogenic encounters and thereby confer superior protection if the same pathogen is re‐encountered represents a well‐established core feature of the vertebrate adaptive immune system. Memory T cells can rapidly re‐expand and are activated upon engagement of their cognate antigen with their T‐cell receptor (TCR). Antibody production by memory B cells furthermore contributes to rapid and more robust responses upon re‐encounter with a specific pathogen. Although these mechanisms constitute the basis for vaccinations, early observations have noted that certain vaccines such as Bacillus Calmette‐Guérin (BCG), which protects against tuberculosis, can improve overall childhood survival.10 These observations have hinted that the immune system cannot only learn to defend its host against re‐encounter with the same pathogens but might also become superior in fighting other, unrelated pathogens under certain circumstances. To date, a number of mechanisms have been described that are believed to contribute to heterologous immunity (Table 1). These include cross‐reactivity and bystander activation of T cells as well as trained immunity of innate immune cells. A general feature of innate and adaptive immune cells that have participated in an immune response is an altered epigenetic landscape.28, 29 These alterations could put them in a “poised state” and add an additional layer of responsiveness towards inflammatory cues, such as cytokines and the engagement of germline‐encoded receptors, during heterologous immune challenges. To date, a number of studies have shown how mechanisms of heterologous immunity can, on the one hand, contribute to protection against newly encountered infections of their host but, on the other hand, pose a potential risk for immunopathology or the establishment of autoimmune disorders. Process Protection against heterologous infection Reference Memory Cross‐reactivity 42-44, 46 or [36], 45 Bystander activation 56, 57 Trained immunity 11, 14, 57 Microbiota/chronic infection 27, 61 or 72 Physical remodeling 9, 64 Tregs 73, 75 2.2 How Selective Are Adaptive Immune Cells? The most striking feature of T cells and antibodies produced by B cells is their unique specificity for their cognate antigen. This enables the formation of immunological memory, directed responses, and protects from the emergence of autoreactive T cells. But just how specific are T cells and antibodies really? About two decades ago, Mason30 argued that cross‐reactivity of the TCR is required for robust immunological protection. He supported this hypothesis with a simple but conclusive model, in which the number of monospecific naïve T cells required to cover all possible foreign peptides would be impossible to generate and maintain in a physiological context. Indeed, cross‐reactivity has emerged as a common, or even necessary, feature of T cells and antibodies with potentially beneficial and adverse consequences for the host.31 Whether a T cell or an antibody cross‐reacts with different antigens is determined by the sequence and structural similarities of these antigens. Such similarities, also known as molecular mimicry, can occur between self‐ and foreign antigens.32 Importantly, molecular mimicry can result in autoimmunity through inappropriate responses of cross‐reactive T cells that were primed during microbial encounters and are subsequently activated by recognition of self‐antigens.32, 33 Conversely, T‐cell clones with a strong affinity towards a specific self‐antigen will be deleted during their maturation process and thereby the T‐cell pool responsive towards a similar microbial or tumor antigen might be reduced.34 In addition to this, cross‐reactivity towards different foreign antigens was also frequently observed in subsequent infections with related but also unrelated viruses. Some studies have shown that cross‐reactive memory T‐cell clones can be favored over non‐cross‐reactive clones during heterologous infections, thereby altering the relative contributions of different T‐cell clonotypes, also known as immunodominance (Figure 2).35 In extreme cases this could skew the immune response towards cross‐reactive epitopes and thereby facilitate viral escape by mutation of the respective epitope.36 Although cross‐reactive memory T cells could help to confer protective immune responses towards unrelated pathogens, they also pose a risk for immunopathology.37, 38 This is showcased by a study, in which lymphocytic choriomeningitis virus (LCMV)‐immune mice were challenged with influenza A virus (IAV) infection and the degree of IAV‐specific T cells cross‐reacting with LCMV‐peptides correlated with the degree of lung injury.39 Several studies have also found evidence for the significance of cross‐reactive T cells in human infectious diseases. Epstein–Barr virus (EBV) is a common infection in humans and well known as the causative agent of infectious mononucleosis (IM). IM involves pronounced activation and proliferation of CD8+ T cells and can vary considerably in severity. T‐cell clones recognizing IAV epitopes were identified among the T cells activated during IM.40 Moreover, the frequency of IAV and EBV cross‐reactive CD8+ T cells was reported to correlate with disease severity during IM.41 Conversely, a more recent study suggested that some IAV‐specific T‐cell clones might be protective against infection with EBV.42 These contrasting results highlight that the relationship between cross‐reactive T cells and the outcome of heterologous infections may be very complex and not only depend on the respective pathogens but also on the specific cross‐reactive antigens or T‐cell clones. Antibodies and T cells are also commonly cross‐reactive to different species of flavivirus, including the Zika virus (ZIKV) and the dengue virus (DENV). CD8+ T cells from DENV‐immune mice can contribute to immunity against experimental ZIKV infection.43 In line with these reports, a stronger T‐cell response and altered immunodominance pattern were also observed in DENV‐pre‐exposed patients upon ZIKV infection.44 In contrast to the potentially protective role of cross‐reactive CD8+ T cells, several publications have indicated that cross‐reactive antibodies from DENV‐exposed humans or mice can enhance ZIKV infection.45 Thus, it is exceedingly difficult to dissect and determine the overall contribution of previous DENV exposure to infection with ZIKV or disease pathogenesis. Nevertheless, a recent epidemiological study of local residents in Brazil found that previous exposure to DENV is associated with a lower risk of ZIKV infection.46 Overall, the evidence for protective functions of cross‐reactive T cells or antibodies in humans is still rare. This does not seem surprising, as it would likely not result in any clinical symptoms. However, memory‐phenotype CD4+ T cells with specificities for human immunodeficiency virus (HIV), CMV, and herpes simplex virus (HSV) have been found in the blood of donors who were never infected with these viruses.16 The same study also showed that IAV‐specific T cells that expanded in individuals following flu vaccination were able to recognize other microbial peptides, thus indicating that vaccinations could induce long‐lived T cells that cross‐react with peptides derived from unrelated pathogens. 2.3 Training Shapes Innate Immunity The long‐standing dogma that memory features can only be acquired by adaptive immune cells has been overthrown by a number of studies within the last decade. Innate immune cells such as natural killer cells (NK cells) and myeloid cells can also form a type of memory that has been termed “trained immunity.”47 For instance, NK cells that were preactivated had a higher cytokine response upon reactivation.48 Furthermore, preactivated NK cells showed higher proliferative capacity than nonactivated controls and could thereby maintain enhanced function.49 Importantly, it was demonstrated that preactivation of NK cells can enable them to respond more potently towards tumor cells.50 In contrast to other innate cells, NK cells are also able to form antigen‐dependent memory by expression of the germline‐encoded receptor Ly49H, which specifically recognizes a mouse cytomegalovirus (MCMV)‐encoded glycoprotein.51 Through this mechanism, MCMV‐specific NK cells can generate a pool of long‐lived memory cells. Myeloid cells are also able to acquire a “trained” phenotype, which enables them to respond more efficiently to inflammatory stimuli. Trained monocytes were able to confer protection from reinfection with the opportunistic fungal pathogen Candida albicans in mice lacking adaptive immune cells.11 This study also showed that β‐glucans, a component of the fungal cell wall, could train the monocytes and induced changes in their histone methylation. Indeed, epigenetic changes have been shown to be a hallmark of trained monocytes.28 Training of monocytes could also contribute to a potential heterologous protection through BCG vaccinations, which has been frequently discussed.12 In a study addressing this hypothesis, monocytes from BCG‐vaccinated donors acquired epigenetic modifications and exhibited higher functional responsiveness to heterologous inflammatory stimuli when compared to monocytes that were collected before vaccination.13 Furthermore, a BCG vaccination trial in humans showed that epigenetic remodulation of monocytes correlated with cross‐protection against experimental yellow fever virus (YFV) challenge.14 Different functional states in immune cells are accompanied and supported by changes in cell metabolism.17 In accordance with this observation, metabolic and epigenetic changes associated with trained immunity are closely interlinked. BCG‐induced trained immunity in monocytes is dependent on alterations of cellular metabolism, most notably the induction of glycolysis.18 More evidence for the connection between trained immunity and metabolism was provided by a recent study, which showed that the metabolite mevalonate can induce trained immunity in monocytes.19 Because monocytes are rather short‐lived cells, the question arose whether their precursor cells in the bone marrow might acquire a different phenotype through immune training and thereby influence myelopoiesis. A combination of three studies approaching the subject with different murine models of immune training—β‐glucan administration, BCG vaccination, and sterile inflammation induced by a high‐fat western diet—all found that the hematopoetic precursors of myeloid cells were modified and thereby constitute an important component of trained immunity.20 However, trained immune cells may also be detrimental to host fitness in certain settings and contribute to disease manifestation. Monocytes isolated from patients with symptomatic atherosclerosis showed epigenetic modifications and expression levels of glycolytic enzymes that could be attributed to trained immunity.15 Innate immune cells in the brain can also acquire a trained state and thereby affect central nervous system inflammation. In mice, inflammation‐mediated modulation of brain‐resident macrophages (microglia) affected neuropathology in diseases like an experimental Alzheimer's model.52 Interestingly, the authors of the latter study found pronounced, microglia‐dependent differences in brain cytokine content depending on the administration of the inflammatory stimulus. Repeated injection resulted in a tolerant state, whereas a single administration was connected to a training effect and therefore a lower threshold for activation. This highlights the ability of the innate immune system to adapt to different kinds of stimuli and thereby provides important insights for the design of immunotherapies. 2.4 T Cells Show Unexpected Talents As previously mentioned, T cells express a TCR that is unique for each clonotype and can recognize its cognate antigen in the context of MHC presentation. Two additional signals are required in order to direct the T cell to an appropriate response or differentiation: the engagement of costimulatory receptors and signals provided by cytokines. However, depending on the differentiation status of T cells, they can also be activated independent of TCR stimulation and therefore respond in a nonspecific manner during a heterologous immune challenge. This phenomenon has been termed “bystander” or “innate‐like” activation of T cells. While TCR‐dependent activation of T cells is rather well described, the understanding of T‐cell activation by cytokines or germline‐encoded receptors without recognition of cognate antigen is still quite limited. The first observations that memory‐phenotype CD8+ T cells can be activated by cytokines in the absence of TCR signaling were already made about 20 years ago. In particular, the proliferation of memory‐phenotype CD8+ T cells in mice could be induced with cytokine interleukin‐15 (IL‐15).53 Some groups that observed bystander activation or proliferation of T cells have argued that the biological relevance would be rather minimal.37, 54 Although this may be true for the models used in the respective studies, other groups have argued for a significant contribution of bystander T cells in various settings. It has become clear that innate‐like activation of memory T cells depends on a number of variables like the cytokine environment, tissue homing or residency, and ligand expression of other cells, which are likely not met by all commonly examined virus infections or inflammation models. Importantly, the cytokine profile that is induced by innate cells during a heterologous challenge needs to fit the requirements for innate‐like activation of T cells. Some cytokines, in particular IL‐12, IL‐18, or IL‐15, were shown to potently induce interferon‐γ (IFN‐γ) production or cytolytic activity in memory CD8+ T cells.55 In mouse models, activation of memory CD8+ T cells in an antigen‐independent manner contributed to protection against heterologous infection with Listeria monocytogenes.56 In addition to cytokines, engagement of germline‐encoded receptors can also contribute to TCR‐independent activation of memory CD8+ T cells. Such receptors can be expressed by innate as well as adaptive immune cells and detect a wide range of signals that are associated with infections or malignant cells. Some adaptive immune cells can also be activated in the absence of their cognate antigen through the recognition of these general signals in combination with cytokine stimuli. NK receptors like NKG2D are broadly expressed on memory CD8+ T cells in mice and humans. Antigen‐independent activation of memory T cells through NKG2D engagement could have beneficial effects for early pathogen control and thereby support innate immunity.57 In contrast to this protective role, engagement of NKG2D in bystander memory T cells may also result in immunopathology under certain conditions, as shown in the context of Leishmania major infection in mice.58 In humans, NKG2D engagement on CD8+ T cells is thought to contribute to immunopathology in celiac disease.25 Furthermore, a recent report described how TCR‐independent activation of memory CD8+ T cells by IL‐15 and NK‐receptor engagement might significantly contribute to liver injury in patients during acute hepatitis A virus infection.26 Overall, these findings reveal that innate‐like activation of bystander T cells might have a significant impact on disease outcome depending on the inflammatory context (Figure 3). It has become clear that cytokines released by innate cells and engagement of NK receptors can trigger TCR‐independent activation of memory or effector T cells and thereby add an additional layer to the immune response. Nevertheless, many important questions concerning this mode of T‐cell activation remain to be answered: for instance, which specific T‐cell subsets can be activated independent of antigen recognition. 2.5 Microbe Exposure Shapes the Immune System The human body is not just challenged by infections; it is also colonized by a diverse collection of viruses, as well as the microbiota, which is composed of microorganisms like bacteria, fungi, and protists. The microbiota mostly resides at barrier sites such as the skin or the gut and plays an important role in training and shaping the host immune system, allowing for induction of protective immunity to combat infections but also the establishment of immune tolerance.59 However, changes in the microbiota composition or diversity induced by hygiene conditions, overuse of antibiotics, or diet can prevent the maturation and maintenance of a healthy and balanced immune system and predispose to inflammatory diseases and autoimmunity. Furthermore, depending on the context, commensal microorganisms can become pathogenic and vice versa.60 Microbiota as well as ongoing chronic infections continuously condition cells of the immune system and thereby enable a rapid response to infectious challenges through trained immunity and bystander activation. This is illustrated by the observation that antibiotic treatment, which leads to a transient elimination of the bacterial microbiota, markedly reduces the efficacy of vaccinations as well as parasite‐specific immune responses.27 Similarly, several chronic infections were shown to enhance immune responses to unrelated pathogens.61 At the same time, the heightened state of alert in persistent virus infections can also enhance immune responses that are harmful to the host as, e.g., those causing colitis or other inflammatory disorders and thus exacerbate disease.62 2.6 Infections Can Cause Long‐Lasting Physical Changes In addition to changing the composition and function of immune cells, infections often also induce physical changes in the host that, in some cases, even persist after the infection has been cleared. These changes can alter the microenvironment of the affected organ as outlined here for sepsis or they can induce persisting structural changes as seen in Yersinia pseudotuberculosis (Y. pseudotuberculosis) infection. These changes have a large impact on subsequent immune responses as they alter the ability of the host to counter infections but also influence the development of inflammatory disorders and therefore affect susceptibility to a broad spectrum of diseases that are not related to the initial infectious agent. Severe, life‐threatening infections can trigger massive immune responses that are known as sepsis. Severe sepsis entails multiorgan dysfunction and is often accompanied by sepsis‐induced immunosuppression and a high risk of developing pneumonia. This immunosuppressed state persists for weeks even after patients appear to have “recovered” from sepsis itself.63 A recent study revealed that the primary infection induces lasting changes in the local microenvironment in that it promotes the induction of regulatory T cells (Tregs). These dampen the immune response and compromise effective immune responses to a secondary challenge, leaving the host susceptible to infections.64 This suggests that intervention strategies that interfere with Treg induction or function may be able to revert the generalized immune suppression in sepsis patients and improve their survival. Indeed, blockade of the PD‐1/PD‐L1 pathway, which interferes with Treg function, can restore immune function and improve survival of sepsis patients.65 Overall, this suggests that preceding infections induce long‐lasting changes in the local cytokine milieu, immune cell composition, and function that can alter the susceptibility to subsequent immune challenges. The potent immune responses induced to rapidly eliminate the infectious agents are often also accompanied by a certain degree of collateral tissue damage. Once the infection is cleared, tissue damage is usually repaired and function is restored. However, in some instances, such as in Y. pseudotuberculosis infection, extensive structural changes occur and persist even after the infection is cleared.9 Acute infection with Y. pseudotuberculosis results in the relocation of dendritic cells that persists even beyond the clearance of Yersinia and leads to a markedly reduced efficacy of subsequent oral vaccinations.9 At the same time, the sustained inflammation might set the stage for chronic disorders, such as inflammatory bowel disease or celiac disease, which share many features with the inflammation induced by Yersinia infection.66 Infection‐induced structural changes can thus serve as a direct link between acute infections and the development of chronic inflammatory disorders. In both examples listed here, sepsis and Yersinia challenge, the infection leads to long‐lasting changes in the local cytokine milieu that can be immune suppressive or stimulating and persists beyond the clearance of the infection. Given their constant exposure to external challenges as well as constant stimulation through commensals, barrier tissues might be at particularly high risk of accumulating these kinds of immunological scars, predisposing for inflammatory disorders such as inflammatory bowel or celiac disease, psoriasis, allergies, or asthma but also infectious diseases such as pneumonia or gastrointestinal infections. 2.7 Pathogen Encounter Shapes the Regulatory Immune Response Pathogen encounter not only initiates a proinflammatory immune response to eliminate the infection, but also induces a regulatory response that balances these effects to limit immunopathology resulting from excessive activity. At the same time, these regulatory responses can favor pathogen persistence and chronic infections. Myeloid suppressor cells, regulatory B cells, and Tregs are the mediators of this regulatory response. Tregs act through diverse mechanisms,67 and like all T cells, are activated through the engagement of their TCR by antigen together with costimulatory signals.68 However, in contrast to conventional T cells, the TCR repertoire of Tregs is shifted towards self‐antigens.69 This implies that Tregs can be activated independently of foreign antigen in all settings that enhance costimulatory signals. Once activated, their suppressive mechanisms allow Tregs to inhibit responder T cells irrespective of their antigen specificity, a mechanism known as bystander suppression. Furthermore, cytokines released upon tissue damage, such as IL‐33 and IL‐18, have been shown to be able to activate Tregs even in the absence of a TCR signal,70 allowing Tregs to rapidly respond to tissue damage in a bystander fashion and limit immune pathology by dampening the immune response. Pathogen‐ or microbiota‐induced changes in Treg frequency or function could therefore have a strong impact on immune responses to heterologous challenges. The composition of the microbiota has been shown to be a major determinant for the induction of pro‐ versus anti‐inflammatory T‐cell responses. While segmented filamentous bacteria promote proinflammatory T‐cell responses and systemic autoimmunity,71 Clostridia and their metabolic by‐products induce Tregs that have systemic suppressive effects and contribute to maintaining immune homeostasis.72 Similarly, persistent pathogens often induce an increase in Tregs and thereby limit the pathogen‐specific immune response, allowing for pathogen persistence (Figure 4). This extends to all classes of persistent pathogens, including chronic viral, bacterial, and parasitic infections, such as tuberculosis, leprosy, or malaria, where patients with poor anti‐pathogen responses and persistent disease show an increase in Tregs.73 An extensive body of work also outlines the central role of Tregs in parasite persistence in helminth infections and the extensive interplay between the parasites and the immune system.74 Importantly, the generalized suppression of immunity by helminth‐induced Tregs also extends to modulation of unrelated immune responses, as helminth‐infected patients show reduced immune responses towards childhood vaccines or other parasites.75 Given that coinfections with helminths and malaria and/or tuberculosis are still frequent in many low‐income countries, it will be important to determine whether the immune‐suppressive effect of pathogen‐induced Tregs continues to increase with increasing infectious burden and how this could be counteracted to allow for pathogen clearance and efficient vaccination strategies against prevalent diseases. Interestingly, the high levels of parasitic infections in low‐income countries also seem to have beneficial effects in that inflammatory and autoimmune disorders are much less frequent than in the developed world.74 In a cohort of Argentinian multiple sclerosis patients, individuals who unintentionally acquired a helminth infection displayed elevated Treg frequencies and remission from the symptomatic disease. This was reversed upon anti‐helminth treatment and accompanied by a loss of the regulatory response and clinical relapse.21 These findings could also be recapitulated in experimental animal models where helminth infection protects mice from inflammatory disorders such as celiac disease and allergy and this protection can be transferred to naïve animals via Treg transfer.22 These findings have led to therapeutic approaches in clinical trials that capitalize on the ability of parasite extracts to induce Tregs and thereby dampen inflammatory immune responses. The stimulation of Treg activity has thus emerged as a central concept that explains the beneficial effects of certain microbiota and parasitic infections in ameliorating inflammatory diseases such as allergy and autoimmune disorders as well as improving transplantation tolerance.23 Recent research has also revealed that Tregs themselves represent a mixture of functionally diverse subsets. Furthermore, infectious challenges can transiently alter the composition and function of Tregs to allow for the induction of anti‐pathogen responses but also efficiently shutting down the effector response once the infection has been cleared (Figure 4).24 Most likely, these temporary changes in Treg composition not only affect immune responses against the ongoing infection but also would alter suppression of autoreactive immune cells or cells specific for persistent pathogens. Indeed, epidemiological data revealed that several autoimmune diseases show a correlation with certain pathogens, although these do not induce the disease. In the case of multiple sclerosis, many different viruses were investigated as a possible cause, most recently EBV. However, there is no proof that any of them causes multiple sclerosis.76 Similarly, there is a strong correlation between enterovirus species and type 1 diabetes, but again, there is no indication that the virus can actually cause islet destruction.77 As such, the viral infections that are associated with the diseases likely serve as their environmental triggers without being the causative agent. We propose that in addition to infection‐induced training of innate immunity, bystander activation of memory cells and reactivation of cross‐reactive lymphocytes, changes in Treg composition and function may allow an autoreactive immune response to unfold and thus contribute to the manifestation of autoimmune diseases. In contrast, infection‐induced changes in Treg composition could also result in a more potent suppression of immune responses and thereby promote pathogen or tumor persistence. 2.8 Speaking the Same Language—Educated Immune Cells Respond to Universal Signals As outlined in the preceding sections, infections trigger long‐lasting changes in both the innate and adaptive arm of the immune system that go far beyond classical memory. Infection experience seems to equip both types of immune cells with an altered responsiveness towards two types of universal signals: cytokines and the ligation of germline‐encoded receptors. Innate cells are thus able to respond more quickly and potently as observed in trained immunity. Adaptive immune cells are able to rapidly participate in an immune response even in the absence of their cognate antigen as observed in bystander activation. Following infection, immune cells adopt a state of alertness that persists even beyond pathogen clearance and is maintained by epigenetic modifications.28, 29 This education of the immune system likely re‐enforces a first line of defense in heterologous challenges and thereby reduces the magnitude of the immune response required to control the secondary infection and allows the host to preserve its resources. In addition, this heightened responsiveness might serve as a means to compensate for the loss of clonal diversity that is observed with age and contributes to the higher susceptibility of older people to infections.78 Indeed, a recent study found that the effect of non‐heritable factors on the immune response becomes more dominant with age, most likely due to the accumulating effect of environmental influences.1, 79 Interestingly, the frequency of Tregs was more strongly determined by environmental factors with progressing age,1 suggesting that infection‐induced changes in Treg frequencies and function might be an important factor in shaping the immune response and determining susceptibility to infectious and inflammatory diseases. Trained immunity and bystander activation both show positive effects on pathogen control and maybe even anti‐cancer immunity as outlined above (Table 2).50 At the same time, heterologous immunity can also have negative consequences for the host such as increased tissue damage as seen in hepatitis26 and likely also contributes to disease in the context of autoimmunity. Most of the changes observed in an experienced immune system reported to date result in enhanced inflammatory responses and increased cytotoxicity (through NK and cytotoxic T cells). Whether similar mechanisms also exist in cells contributing to allergic responses or anti‐helminth immunity, such as mast cells, eosinophils, or T helper cells, remains to be determined (see Table 2). Similarly, how cells participating in a heterologous response are regulated is still completely unclear. While Tregs have been shown to have tissue protective effects upon antigen‐independent activation,70 it is unclear which cell type might dampen and limit heterologous immunity to prevent immune pathology. Although many questions still remain open, recent developments have made it clear that immune cells are more versatile than previously appreciated. In addition to their classical functions, infection‐experienced immune cells rapidly respond to stimulation by cytokines or germline‐encoded receptors and initiate potent heterologous immune responses that contribute to pathogen control but might also enhance immune pathology. Branch Cell type Mechanisms Possible disease associations Potential impact on host Innate Macrophages ● Epigenetic changes in progenitor cells ● Heterologous protection27, 60 Beneficial ● Proinflammatory phenotype ● Neuropathology68 Harmful ● Enhanced responsiveness ● Atherosclerosis67 NK cells ● Cytokine‐induced epigenetic changes ● Antitumor responses56-58 Beneficial ● Cytokine production ● Heterologous protection? ● Cytotoxicity ● Tissue damage? (mechanism may be similar to memory bystander CD8+ T cells) Harmful Granulocytes ● Epigenetic changes in progenitor cells? ● Heterologous protection? Beneficial ● Allergic disease? Harmful Mast cells ● Epigenetic changes? ● Heterologous protection? Beneficial ● Allergic disease? Harmful Adaptive Effector and memory CD8+ T cells ● Epigenetic changes ● Heterologous protection21-24 Beneficial ● Cytokine production ● Antitumor responses? ● Cytotoxicity ● Tissue damage76-78 Harmful Effector and memory CD4+ T‐helper cells ● Epigenetic changes ● Heterologous protection? Beneficial ● Cytokine production ● Autoimmunity? Harmful Regulatory ● Epigenetic changes? ● Tissue protection? Beneficial T cells ● Tissue abundance? ● Immune homeostasis? ● Enhanced responsiveness? ● Promoting pathogen persistence? Harmful ● Hampered antitumor responses? 3 Conclusions and Outlook The concepts presented here outline how pathogens and commensals induce long‐term changes in the immune system that affect subsequent immune responses and impact human health. Animal models and clinical studies show that vaccinations, prior infections, or coinfections can modify the immune response to unrelated pathogens. Additionally, a number of inflammatory diseases have been linked to infections but finding direct associations between pathogens and the initiation of these disorders has remained difficult. This might in part be due to the fact that infections can induce long‐term changes in the immune system that may persist even once the infection is cleared. These effects likely accumulate over time and the onset of inflammatory disease does not have to coincide with the infectious trigger(s). Similarly, infection history can also induce changes that enhance the regulatory properties of the immune system and thus favor the establishment of chronic infections and prevent protective immunity against cancer. Future work should be aimed at further unraveling the mechanistic basis for these changes in the immune system and most importantly also at bringing these concepts together to reveal a more comprehensive picture of the mechanisms underlying heterologous immunity. Each new infection or immune trigger an individual is exposed to during a lifetime will educate and potentially alter the dynamics of their immune system and it is important that this is taken into account in preclinical models. Future research should identify the factors that shape the immune system in these processes and determine how heterologous secondary immunity affects vaccine efficiency and immune therapies targeting inflammatory disorders or cancer to be able to optimize preventive and therapeutic interventions in the future. Conflict of Interest The authors declare no conflict of interest. References

Abstract Autologous human islet transplantation to mitigate or prevent surgically induced diabetes after total pancreatectomy for the relief of chronic pancreatitisinduced pain was pioneered in the late 1970s. Islet allotransplantation using islets from human donors to treat type 1 diabetes was first reported several years later. Over the last 40 years, methods for clinical islet transplantation have been methodically standardized to become the methods used today. Human islets are also isolated from donors with pathologies with the aim to conduct a broad range of investigational studies. Human islet isolation undertaken for research can provide the opportunity to further optimize and develop methods that may be helpful in the clinic. The variability of conditions associated with donor and pancreas makes consistent success in isolating islets a challenge, thus, we should re-evaluate the effectiveness of our methods of islet isolation. In order to do this, we must first consider that the intended outcomes for clinical islet transplantation and experimental investigations involving islet isolation are, in fact, quite different and that these differences impact the status and future considerations of islet isolation procedures. To cite this article Bottino R., Bertera S., Knoll C., Knoll M. F., Trucco M. Human islet isolation: status and future considerations* CellR4 2019; 7: e2607 DOI: 10.32113/cellr4_20194_2607 Publication History Submission date: 18 Dec 2018 Revised on: 15 Mar 2019 Accepted on: 01 Apr 2019 Published online: 18 Apr 2019

It is often not the lack of a cure, but the lack of will. Many great strides have been made with diabetes, when people stopped trying to develop medicines, and instead focused on how to enable the body to produce insulin.

Via Krishan Maggon