Broad induction of immunoregulatory mechanisms after a short course of anti-IL-7Rα antibodies in NOD mice
© The Author(s). 2017
Received: 19 September 2016
Accepted: 22 March 2017
Published: 29 March 2017
Type 1 diabetes is an autoimmune disease caused by T cell-mediated destruction of the insulin-producing β-cells in the pancreas. Therefore, approaches that effectively halt the pathogenic T cell response are predicted to have preventive or therapeutic benefit for type 1 diabetes patients. We previously demonstrated that long-term blocking of IL-7 signaling, which is critical for the survival and function of T cells, prevented and reversed type 1 diabetes in non-obese diabetic mice. However, such persistent inhibition of T cell responses raises concerns about causing immunodeficiency. Here, we asked whether a reduced duration of the treatment with anti-IL-7Rα antibodies retained efficacy in preventing diabetes. Moreover, we sought to identify immunoregulatory mechanisms induced by anti-IL-7Rα administration.
Anti-IL-7Rα antibodies were administered to prediabetic NOD mice for 3 weeks and blood samples were taken at the end of treatment and 2 weeks later to analyze changes in T cell phenotypes in response to IL-7Rα blockade. We found that the co-inhibitory receptors LAG-3, Tim-3 and PD-1 were increased on peripheral blood CD4+ and CD8+ T cells from anti-IL-7Rα-treated mice. Expression of these receptors contributed to reduced T cell cytokine production in response to TCR stimulation. In addition, the frequency of Tregs within the circulating CD4+ T cells was increased at the end of anti-IL-7Rα antibody treatment and these Tregs showed a more activated phenotype. In vitro restimulation assays revealed that effector T cells from anti-IL-7Rα-treated mice were more sensitive to co-inhibitory receptor induction after TCR stimulation. Importantly, these changes were accompanied by delayed type 1 diabetes disease kinetics.
Together, our data show that short-term blockade of IL-7Rα induces detectable changes in co-inhibitory receptor expression and Treg frequencies in peripheral blood of NOD mice. These changes appear to have long-lasting effects by delaying or preventing type 1 diabetes incidence. Hence, our study provides further support for using anti-IL-7Rα antibodies to modulate autoreactive T cell responses.
KeywordsType 1 diabetes Interleukin 7 T cells Autoimmunity Tregs Inhibitory receptors Non-obese diabetic mice
Type 1 diabetes is a progressive autoimmune disease caused by infiltration of autoreactive lymphocytes in the islets of Langerhans which, ultimately, will destroy the insulin-producing β-cells. As a result of the loss of β-cells, blood sugar levels increase leading to a severe risk of secondary organ complications. Despite current advances in the understanding of type 1 diabetes, treatment remains largely limited to insulin replacement therapy and attempts to prevent or cure the disease in humans have so far been unsuccessful [1, 2].
IL-7 is a cytokine with an important role in T cell survival and function and is an emerging target for the treatment of multiple autoimmune diseases . We and others previously demonstrated that blocking IL-7 receptor alpha (IL-7Rα) prevented and reversed diabetes in non-obese diabetic (NOD) mice and hence has potential to be translated as an immunotherapy for human type 1 diabetes [4, 5]. Initial analyses of CD4+ T cells in anti-IL-7Rα-treated mice revealed increased expression of the co-inhibitory receptor Programmed Death-1 (PD-1) in effector/memory CD4+ T cells (TE/M) and an increased frequency of polyclonal regulatory T cells (Tregs) in lymphoid organs . These observations suggested that anti-IL-7Rα antibodies shift the balance in the immune system from active autoreactivity to a more regulated state, impacting disease progression.
Co-inhibitory receptors play critical roles in maintaining self-tolerance to autoantigens and are also associated with “T cell exhaustion”, caused by chronic antigenic stimulation of virus- and tumor-specific TE/M cells [6–8]. Hence, increasing co-inhibitory receptor expression and “exhaustion” in autoreactive T cells are predicted to be desirable outcomes for the treatment of autoimmune diseases such as type 1 diabetes. Loss-of-function studies of the co-inhibitory receptors PD-1 and LAG-3 have demonstrated a critical role for these co-inhibitory receptors in suppressing anti-islet T cell responses in NOD mice, reflected by an accelerated kinetics of disease course [9–12]. Contributions of other co-inhibitory receptors, e.g., Tim-3 and B7x, in regulating type 1 diabetes are emerging as well [13, 14]. The role of Tregs in maintaining islet tolerance is also firmly established and defects in Tregs may underlie susceptibility for type 1 diabetes [15, 16]. Various approaches to increase Treg activity for the treatment of type 1 diabetes are intensively being developed and, in some cases, have entered clinical trials .
The initiation of clinical trials to use anti-IL-7Rα antibodies for the treatment of type 1 diabetes and other autoimmune diseases  underscores the necessity to better understand the treatment modalities and mechanisms underlying protection against type 1 diabetes provided by anti-IL-7Rα administration. Therefore, we treated prediabetic NOD mice with a short course of anti-IL-7Rα antibodies and expanded our analysis of co-inhibitory receptor expression and Tregs. We found that in addition to PD-1, LAG-3 and Tim-3 were also induced on T cells in response to IL-7Rα blockade. Importantly, changes in these receptors could be found not only in lymphoid organs but on peripheral blood T cells as well and may serve as a biomarker of treatment efficacy. Furthermore, we show that IL-7Rα blockade increases the frequency and changes the phenotype of polyclonal Tregs. Together, our data suggest that anti-IL-7Rα antibodies promote two key mechanisms of protection against autoimmunity: increased expression of co-inhibitory receptors and increased Treg activity. Moreover, short-term IL-7Rα blockade retained some capacity to alter the kinetics of the disease.
Prediabetic female NOD mice (9–11 weeks) were purchased from The Jackson Laboratory (US). All animal experiments were approved by the Institutional Animal Care and Use Committee of Boston University Medical Campus.
Diabetes incidence was followed weekly by urine analysis (Diastix, Bayer, US) and measuring of blood glucose levels with a Contour glucose meter (Bayer; US). The percentage of diabetic mice (glucose levels >250 mg/dL) over a 32-weeks time course was calculated by the Survival Curves method using GraphPad Prism.
In vivo antibody treatment
Anti-IL-7Rα (rat IgG2a, clone A7R34) antibodies for in vivo blocking experiments were produced by a hybridoma cell line and purified with Protein G Sepharose 4 Fast Flow (GE Healthcare, US) in our laboratory. Rat IgG (Jackson ImmunoResearch Laboratories, US) was used as a control. For anti-IL-7Rα and rat IgG antibodies, 0.5 mg was administered in PBS intraperitoneally.
In vitro stimulation assays and ELISA
Cells were cultured in RPMI 1640 media (Invitrogen, US) supplemented with 1 mM each of L-glutamine, nonessential amino acids, sodium pyruvate, Hepes, penicillin, streptomycin, 50 μM 2-Mercaptoethanol (Gibco by Life Technologies, US), and 10% FCS (Omega Scientific, US), and incubated at 37 °C in 5% CO2. In vitro assays to measure cytokine production were performed by stimulating 5×105 cells from spleen and pancreatic lymph nodes (PLN) with soluble anti-CD3 (1 μg/ml) (clone 145-2C11; eBioscience, US) and anti-CD28 (2 μg/ml) (clone 37.51; eBioscience, US) antibodies in round-bottom 96-well plates (BD Falcon, US) in the absence or presence of blocking antibodies (10 μg/ml) for PD-L1 (clone MIH5), LAG-3 (clone C9B7W) and Tim-3 (clone RMT3-23) (Bio X Cell, US). Supernatants from the cultures were harvested after 18 h and IFN-γ and IL-2 content determined by ELISA (eBioscience, US), following the manufacturer’s instructions. For assays to measure induction of co-inhibitory receptor expression, PLN cells from mice treated with anti-IL-7Rα or rat IgG antibodies were stimulated in vitro with soluble anti-CD3- (0.1 or 10 μg/ml) and anti-CD28 (1 μg/ml) antibodies. Cell cultures were set up in flat-bottom 96-well plates (BD Falcon, US) and harvested after 3 days for flow cytometric analysis.
Antibodies and staining procedures
Blood samples (50–100 μl) were drawn from mouse tail vein and an equal volume of EDTA (50 mM) (Sigma, US) was added immediately to avoid coagulation. Prior to staining, erythrocytes were lysed for spleen and blood samples. To distinguish live from dead cells, cells were preincubated with a fixable viability dye (eBioscience, US) according to manufacturer’s instructions. Fc receptors were blocked with anti-CD16/CD32 antibodies for 5 min at 4 °C before any antibody staining procedures were started. The following antibodies were used for detection of murine activation and proliferation markers and co-inhibitory receptors: anti-CD4; anti-CD8; anti-PD-1; anti-Tim-3; anti-LAG-3; anti-CD44; anti-Foxp3 and anti-CD25 (eBioscience, Biolegend or BD Pharmingen, US). Extracellular staining was performed by incubating with antibodies for 15–30 min at 4 ° C. For Foxp3 intracellular staining cells were fixed and permeabilized with a Foxp3 staining buffer set (eBioscience, US) following manufacturer’s instructions.
Phenotypic analysis of cell populations was performed by multiparameter flow cytometry. Fluorescence intensities were measured on a LSRII flow cytometer and data were analyzed with FlowJo software.
Statistically significant differences between groups were determined using the Mantel–Cox log-rank test (for diabetes incidence) and one- or two-tailed paired or unpaired t tests (for flow cytometry data) using Graph Pad Prism. P values ≤ 0.05 were considered significant. Horizontal lines in graphs indicate statistical significance (* = p ≤ 0.05, ** = p ≤ 0.005, *** = p ≤ 0.0005, ns = p > 0.05).
A short course of IL-7Rα blocking antibodies delays type 1 diabetes onset in NOD mice
Circulating T lymphocytes from anti-IL-7Rα-treated NOD mice show enhanced expression of multiple co-inhibitory receptors
Peripheral blood Tregs show increased frequency and an activated phenotype after anti-IL-7Rα antibody treatment
Co-inhibitory receptor expression on T cells from anti-IL-7Rα-treated mice impairs cytokine production
Absence of IL-7 signaling sensitizes T cells to express co-inhibitory receptors in response to TCR stimulation
In this study we sought to further characterize the protective mechanisms induced by treatment with anti-IL-7Rα antibodies during an ongoing autoreactive T cell response. We found that, in addition to PD-1, two other co-inhibitory receptors, Tim-3 and LAG-3, show increased expression in T cells from anti-IL-7Rα-treated NOD mice. Moreover, IL-7Rα blockade promoted an expansion of the polyclonal Treg population in NOD mice and, interestingly, increased their activation status, indicating enhanced suppressive potential. Our results indicate that a broad program of immunoregulation underlies the slower disease kinetics afforded by IL-7Rα blockade in type 1 diabetes and, that markers of anti-IL-7Rα antibody activity can be detected in peripheral blood following a short course of treatment.
PD-1, Tim-3 and LAG-3 are co-inhibitory receptors that are critical for controlling autoimmunity: studies with blocking antibodies as well as gene-deficient mice demonstrated that these pathways, individually or synergistically, play important roles in type 1 diabetes and other autoimmune diseases [9, 11–13, 32]. Hence, developing methods to promote activity of these pathways is a promising approach towards novel therapies for type 1 diabetes and other autoimmune diseases. Our data indicate that this desired effect is achieved as a consequence of IL-7Rα blockade. Interestingly, such a broad increase of multiple co-inhibitory receptors in CD4+ Foxp3− and CD8+ TE/M cells resembles the phenotype described in previous studies for exhausted T cells responding to tumors or chronic viral infections [7, 8, 23]. In these settings, continuous antigen exposure results in inhibited, “exhausted” T cells that have lost effector functions (e.g., IFN-γ production) necessary to effectively combat tumors and chronic viral infections [8, 23, 33]. Importantly, IL-7 restored functionality in CD8+ T cells during chronic viral infections and in tumor models [34, 35]. These observations thus support the idea that IL-7 is an environmental factor promoting T cell responses during chronic antigen challenge. As a corollary, inappropriate IL-7 signaling during autoreactive T cell activation may contribute to the development of a pathogenic T cell response. In this respect, murine models of autoimmune diseases treated with inhibitors of IL-7/IL-7Rα signaling show preventive or therapeutic efficacy [4, 5, 36–38]. In many of these models, it remains to be investigated whether increased co-inhibitory receptor expression plays a role as a protective mechanism. Due to the multiple immunoregulatory pathways induced in anti-IL-7Rα-treated NOD mice, it will be a challenging endeavor to unequivocally demonstrate the contribution of each to controlling autoimmunity. In this regard, one interesting finding from our study is that cells expressing PD-1 vs Tim-3 and/or LAG-3 appear with different kinetics in the blood after anti-IL-7Rα mAb administration. This may be due to a slower kinetics of initial induction or because Tim-3 and LAG-3-expressing cells are preferentially retained in the lymphoid organs while PD-1 expressing cells belong to a population that more readily enters the circulation. Besides enhanced co-inhibitory receptor expression on TE/M cells, PD-1, Tim-3 and LAG-3 were also increased on Tregs. Tregs expressing PD-1 , Tim-3  and LAG-3  are found at sites of active immune responses, e.g., in tumors and transplants, and are thought to possess higher suppressive activity.
The molecular mechanisms underlying increase of co-inhibitory receptors in the absence of IL-7 signaling remain to be determined. However, it is reasonable to speculate that a direct effect of IL-7 on T cells during priming is involved. For example, recent data show that IL-7 provides additional early signals (increased ERK, STAT5, Akt) during TCR engagement that promote optimal T cell activation [40, 41]. These IL-7-induced signals are important for expression of the glucose transporter Glut1 in T cells  and, intriguingly, decreased Glut1 and glucose uptake have been associated with increased PD-1 and Tim-3 expression and T cell exhaustion . Hence it is feasible that absence of IL-7 signaling during T cell priming promotes expression of co-inhibitory receptors, perhaps as a consequence of defective metabolic regulation.
Efforts to translate pre-clinical studies showing efficacy of anti-IL-7Rα antibodies for the treatment of type 1 diabetes have been initiated in the clinic . Our study shows that a limited treatment with anti-IL-7Rα antibodies is sufficient to induce detectable changes in the peripheral blood T cell phenotype, increasing expression of several co-inhibitory receptors in CD4+ and CD8+ TE/M cells and promoting Treg presence. Hence, our data support the rationale for clinical trials with anti-IL-7Rα mAbs and suggest that a T cell biomarker in the blood based on co-inhibitory receptor expression may be helpful in following individual patients’ response to IL-7Rα blockade. The shorter, 3-week course of treatment we tested here lost some efficacy to prevent type 1 diabetes compared to persistent treatment , suggesting that it may be ideally suited to combine with another intervention to improve efficacy while maintaining the increased safety profile presumably associated with limited treatment duration.
Pancreatic lymph nodes
The authors thank Dr. Cristina Penaranda (Massachusetts General Hospital) for useful comments on the manuscript. This work was supported by the Boston University Flow Cytometry Core Facility.
This research was supported by American Diabetes Association Basic Science Grant #1-13-BS-038 (to H.D.), National Institutes of Health Grant R01 DK-102911 (to H.D.) and by the Boston University Arthritis Center. The funders had no role in the design of the study, the collection, analysis and interpretation of data, and in writing the manuscript.
Availability of data and materials
The data and data analysis that support the findings of the current study are available from the corresponding author on reasonable request.
CVM designed and performed experiments, analyzed data and wrote the manuscript. JC planned and conducted experiments and contributed to discussion. MF performed experiments and contributed to discussion. HD conceived the study, analyzed data and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All animal experiments were approved by the Institutional Animal Care and Use Committee of Boston University Medical Campus under protocol number AN-15302.
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