Inducible IL-7 Hyperexpression Influences Lymphocyte Homeostasis and Function and Increases Allograft Rejection

The IL-7/IL-7R pathway is essential for lymphocyte development and disturbances in the pathway can lead to immune deficiency or T cell mediated destruction. Here, the effect of transient hyperexpression of IL-7 was investigated on immune regulation and allograft rejection under immunosuppression. An experimental in vivo immunosuppressive mouse model of IL-7 hyperexpression was developed using transgenic mice (C57BL/6 background) carrying a tetracycline inducible IL-7 expression cassette, which allowed the temporally controlled induction of IL-7 hyperexpression by Dexamethasone and Doxycycline treatment. Upon induction of IL-7, the B220+ c-kit+ Pro/Pre-B I compartment in the bone marrow increased as compared to control mice in a serum IL-7 concentration-correlated manner. IL-7 hyperexpression also preferentially increased the population size of memory CD8+ T cells in secondary lymphoid organs, and reduced the proportion of CD4+Foxp3+ T regulatory cells. Of relevance to disease, conventional CD4+ T cells from an IL-7-rich milieu escaped T regulatory cell-mediated suppression in vitro and in a model of autoimmune diabetes in vivo. These findings were validated using an IL-7/anti-IL7 complex treatment mouse model to create an IL-7 rich environment. To study the effect of IL-7 on islet graft survival in a mismatched allograft model, BALB/c mice were rendered diabetic by streptozotocin und transplanted with IL-7-inducible or control islets from C57BL/6 mice. As expected, Dexamethasone and Doxycycline treatment prolonged graft median survival as compared to the untreated control group in this transplantation mouse model. However, upon induction of local IL-7 hyperexpression in the transplanted islets, graft survival time was decreased and this was accompanied by an increased CD4+ and CD8+ T cell infiltration in the islets. Altogether, the findings show that transient elevations of IL-7 can impair immune regulation and lead to graft loss also under immune suppression.

The IL-7/IL-7R pathway is essential for lymphocyte development and disturbances in the pathway can lead to immune deficiency or T cell mediated destruction. Here, the effect of transient hyperexpression of IL-7 was investigated on immune regulation and allograft rejection under immunosuppression. An experimental in vivo immunosuppressive mouse model of IL-7 hyperexpression was developed using transgenic mice (C57BL/6 background) carrying a tetracycline inducible IL-7 expression cassette, which allowed the temporally controlled induction of IL-7 hyperexpression by Dexamethasone and Doxycycline treatment. Upon induction of IL-7, the B220 + c-kit + Pro/Pre-B I compartment in the bone marrow increased as compared to control mice in a serum IL-7 concentration-correlated manner. IL-7 hyperexpression also preferentially increased the population size of memory CD8 + T cells in secondary lymphoid organs, and reduced the proportion of CD4 + Foxp3 + T regulatory cells. Of relevance to disease, conventional CD4 + T cells from an IL-7-rich milieu escaped T regulatory cell-mediated suppression in vitro and in a model of autoimmune diabetes in vivo. These findings were validated using an IL-7/anti-IL7 complex treatment mouse model to create an IL-7 rich environment. To study the effect of IL-7 on islet graft survival in a mismatched allograft model, BALB/c mice were rendered diabetic by streptozotocin und transplanted with IL-7-inducible or control islets from C57BL/6 mice. As expected, Dexamethasone and Doxycycline treatment prolonged graft median survival as compared to the untreated control group in this transplantation mouse model. However, upon induction of local IL-7 hyperexpression in the transplanted islets, graft survival time was decreased and this was accompanied by an increased CD4 + and CD8 + T cell infiltration in the islets. Altogether, the findings show that transient elevations of IL-7 can impair immune regulation and lead to graft loss also under immune suppression.

INTRODUCTION
The IL-7/IL-7 receptor (IL-7R) pathway is indispensable for B and T cell development. IL-7 deficiency results in a severe block in B cell development at the transition from Pro-B to Pre-B cells in mice (1), and studies suggest that IL-7 is also critical for human B cell development (2). IL-7 is also an essential modulator of T cell homeostasis. In the steady state, the immune system relies on low concentrations of IL-7 to regulate T cell homeostasis and preserve T cell repertoire diversity (3,4). During lymphopenia, an IL-7-rich environment provides a milieu for the proliferative expansion of T cells (3,4). IL-7-associated homeostatic expansion is also linked to inflammatory diseases, including graft-vs.-host-disease (5), rheumatoid arthritis (6), and multiple sclerosis (7). In murine models of autoimmune diabetes, IL-7 accelerates disease onset (8) and interference with IL-7 signaling prevents or even reverses disease (9)(10)(11). An increased concentration of IL-7 and homeostatic expansion of T cells, including autoreactive T cells, is observed in patients with Type-1-Diabetes (T1D) who receive immunosuppression as part of transplantation therapy (12). An increased IL-7 concentration also abrogates the ability of human FOXP3 + regulatory T (Treg) cells to suppress autoreactive effector T cell activation in vitro (13). IL-7 is, therefore, suggested to play a pivotal role in the development and recurrence of autoimmunity and graft failure.
A number of pathologies associated with increased IL-7 are associated with the concomitant treatment with immunosuppression, in particular after immune-depletion. Although animal models of increased IL-7 action exist (14)(15)(16)(17)(18)(19), none of these includes hyper-IL-7 concentrations in an immunosuppressed environment. We, therefore, sought to develop such an in vivo mouse model and have used it to study IL-7 driven immune deviations under immunosuppressive conditions. In our model, IL-7 expression can be systemically induced at high levels resulting in bioactive IL-7 to drive population expansion. These findings with the model were validated using IL-7/anti-IL-7 mAb immune complexes, and altogether demonstrate that transient increases in IL-7 can impair immune regulation and decrease allograft survival.

Induction of an IL-7-Rich Environment in vivo
Male and female mice (dTG or Ctrl) were injected i.p. with 2 mg doxycycline (Dox, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and 0.5 mg dexamethasone (Dex, Sigma-Aldrich Chemie GmbH) per 25 g body weight on five consecutive days. Serum IL-7 concentration was measured before and after Dex/Dox administration, using the Mouse IL-7 Quantikine ELISA Kit according to the manufacturer's protocol (R&D Systems, Minneapolis, MN, USA).

Adoptive Transfer Model of Autoimmune Diabetes
Autoimmune diabetes was induced in recipient mice by adoptive transfer of CD4 + T cells with transgenic expression of a diabetogenic T cell receptor. Conventional BDC2.5 + T cells with a naïve surface marker phenotype (CD4 + BDC2.5 + CD62L high CD25 − ) were isolated from pooled LNs and SPL of NOD.BDC2.5 mice by enrichment for CD4 + cells using MACS technology followed by FACS. 5 × 10 5 diabetogenic cells were injected i.v. into NOD.Rag1 −/− mice. The in vivo suppressive capacity of Foxp3 + BDC2.5 + Treg cells was assessed by co-injecting 1 × 10 5 CD4 + BCD2.5 + CD25 + Foxp3 RFP+ cells that had been FACS-purified from pooled LNs and SPL of NOD.BDC2.5 × Foxp3 RFP/GFP mice. Blood glucose concentration of NOD.Rag1 −/− recipient mice were monitored for up to 30 days or until diabetes manifestation (blood glucose levels above 300 mg/dl on two consecutive measurements).

Culture of Pancreatic Islets
To determine the IL-7 release, islets of donor mice of C57BL/6.tet-on-IL-7-irtTA-GBD founders were freshly isolated. One Hundred islets were hand-picked underneath a microscope into open 1.5 ml Eppendorf tubes containing 200 µl RPMI-1640 medium. Islets were cultured in the absence or presence of Dex/Dox for 48 h with at 37 • C and 5% CO 2 . The supernatant was harvested and stored at −80 • C. IL-7 protein was detected in supernatants using a commercial mouse IL-7 ELISA kit (R&D Systems) according to the manufacturer's protocol.

Allograft Model for Islet Transplantation
Eight-week-old BALB/c recipient mice (Charles River, Suelzfeld, Germany) were injected with streptozotocin (STZ, 225 mg/kg) (Sigma-Aldrich Chemie GmbH) to induce diabetes 5 to 7 days prior to islet transplantation. Mice that had a non-fasting blood glucose concentration above 400 mg/dl on two consecutive days were transplanted with 600 islets (dTG or littermate Ctrl) under the kidney capsule. Transplantation was performed using a 1001 TPLT Hamilton syringe (CS-Chromatographie Service, Langerwehe, Germany). Blood glucose was measured at 12 h intervals in the first 3 days post-transplantation. Normoglycemia was defined as non-fasting blood glucose levels <200 mg/dl on at least two consecutive days. At day 7, normoglycemic transplanted recipients were i.p. injected with Dex/Dox for five consecutive days to transiently induce transgenic IL-7 expression in transplanted tet-on-IL-7-irtTA-GBD transgenic islets. Graft rejection was diagnosed when blood glucose increased to >200 mg/dl.

Statistical Analysis
For statistical analysis, Prism 6.07 software (GraphPad Software, San Diego, CA, USA) was used. For statistical analysis of two groups, student's t-test was applied. For comparison of three or more groups, two-way ANOVA in combination with Bonferroni's multiple comparison post-test was used. For survival analysis, Log-rank (Mantel-Cox) test was used.

Generation of Mice With Temporally Controlled IL-7 Hyperexpression
Three lines (founder number: 615, 625, and 644) had stable ins-Hyg-tet-on-IL-7 construct integration after breeding with C57BL/6 wild-type mice ( Figure S1B). The C57BL/6.tet-on-IL-7 founder lines were crossed to C57BL/6.irtTA-GBD mice, giving rise to C57BL/6.tet-on-IL-7-irtTA-GBD mice (dTG) as well as genotype control mice (Ctrl) lacking either the tet-on-IL-7 or the irtTA-GBD transgene. Following Dex/Dox treatment of dTG and Ctrl mice for five consecutive days, all three lines exhibited induced expression ( Figure S1C) of il7 mRNA in multiple lymphoid organs ( Figure 1A) and accumulation of IL-7 protein in serum ( Figure 1B). The highest expression of mRNA and protein was observed in founder line 615 (Figures 1A,B). In time course studies (Figure 1C), serum IL-7 concentrations in founder line 615 peaked on the last day of Dex/Dox treatment (day 5) and declined to concentrations similar to baseline levels within 5 days after discontinuation of Dex/Dox treatment.

IL-7 Expression Levels Correlate With the Population Size of BM Pro/Pre-B I Cells
To assess the bioactivity of induced IL-7 hyperexpression in vivo, B lymphopoietic activity was analyzed (Figure 2). BM from young (4-week-old) and adult (16-week-old) mice was harvested at different time points following the initiation of IL-7 by Dex/Dox treatment. On day 6, the percentage of the B220 + ckit + Pro/Pre-B I cell compartment [nomenclature according to (26)] increased by approximately 3-fold upon IL-7 induction in On day 10, the frequencies of Pro/Pre-B I cells in the BM of the dTG line 615 were similar to those observed in Ctrl mice, while the compartment size of B220 + CD25 + Pre-B II cells and immature B220 + IgM low B cells increased in dTG mice ( Figure S2B, P = 0.0012; P = 0.0005), suggesting developmental progression toward subsequent developmental stages after initial proliferative expansion of Pro/Pre-B I cells (27,28). No increase in mature CD19 + splenocytes was observed (Figure S2C). FACSisolated BM-derived B220 + CD19 + c-kit + Pro/Pre-B I cells of dTG mice cultured in the absence of added IL-7efficiently differentiated into sIgM + cells, albeit with delayed upregulation of sIgM, as compared to their counterparts from Ctrl mice ( Figure S2D; P < 0.0001).
We conclude that the induced hyperexpression of IL-7 in dTG mice appears suitable to modulate IL-7-dependent biological processes in vivo.
Similar changes in Treg frequency and p/tTreg ratio were observed in scLNs, meLNs and SPL of both young and adult mice (Figures S7A,B). These findings were confirmed using rhIL-7/anti-IL-7 mAb (clone: M25) immunocomplex (IL-7/M25) treatment in C57BL/6.Foxp3 RFP/GFP mice (Figure S7C), indicating that the modulation of Treg cell homeostasis by elevated IL-7 bioactivity in vivo is consistent and independent of an immunosuppressive environment.
To determine whether increased Treg suppression or Tresp resistance was dominant, suppression assays were performed with Tresp and Treg cells isolated from Dex/Dox-treated dTG or Dex/Dox-treated Ctrl mice (Figure 4G). When both Tresp and Treg cells were isolated from dTG mice, the IL-7 effect of enhanced Tresp cell resistance to Treg cell-mediated suppression in vitro could not be overcome by the observed IL-7-mediated increase in Treg cell suppressor function (e.g., 1:1 ratio: 9.5 ± 0.5% undivided dTG Tresp cells, P < 0.0001).
These findings were validated by isolating Treg and Tresp cells from an IL-7-rich milieu without additional immunosuppression (i.e., from mice treated with IL-7/M25 immune complexes;

Increased IL-7 Promotes Resistance to Treg Cell-Mediated Suppression of Diabetes Induction
Next, we determined the suppressor function of Treg cells in an IL-7-rich environment in a model of autoimmune diabetes, in which co-transfer of CD4 + Foxp3 + BDC2.5 + Treg cells suppress diabetogenic CD4 + BDC2.5 + T effector cell-mediated destruction of pancreatic beta cells in NOD.Rag1 −/− recipients (30). For this purpose, recipient mice were either left untreated (Ctrl group) or received IL-7/M25 immune complexes following adoptive T cell transfer (post i.v. group) (Figure 5A). Injection of 5 × 10 5 diabetogenic CD4 + BDC2.5 + T cells with a naïve surface marker phenotype resulted in overt diabetes at day 14.5 ± 1.0 in recipient mice of the otherwise untreated Ctrl group (Figure 5B, left), while co-transfer of as few as 1 × 10 5 BDC2.5 + Foxp3 + Treg cells were sufficient to suppress diabetes development. IL-7/M25 treatment of recipient mice starting as early as day 2 after cell transfer shortened the lapse of time until diabetes development to day 11.5 ± 1.0 if only diabetogenic CD4 + BDC2.5 + T cells were transferred (Figure 5B, right; P < 0.05). Interestingly, cotransfer of Foxp3 + BDC2.5 + Treg cells did not prevent diabetes induction in recipient mice as observed for the control group. Here, overt diabetes was detected at day 13.0 ± 0.0 (P < 0.05). Hence, increasing IL-7 was able to increase the pathogenicity of antigen-specific T cells, enabling them to escape Treg cellmediated suppression in an autoimmune diabetes model.

Local IL-7 Hyperexpression in
Transplanted Insulin-Producing Islets Is Sufficient to Promote Allograft Rejection C57BL/6.tet-on-IL-7-irtTA-GBD mice offer an opportunity to study the role of IL-7 in modulating immunity in vivo in FIGURE 4 | Differential impact of increased IL-7 in vivo on CD25 expression, p/tTreg cell abundance, and Foxp3 + Treg cell-mediated suppression in vitro. To track viable populations of Foxp3 + Treg cells, we employed C57BL/6.tet-on-IL-7-irtTA-GBD × Foxp3 RFP/GFP mice, in which all Foxp3 + Treg cells express RFP and developmental sublineages can be distinguished based on differential GFP expression (tTreg, RFP + GFP + ; pTreg, RFP + GFP − ) (21). IL-7 hyperexpression was induced by 5 day Dex/Dox treatment, and scLNs were isolated on day 6. (A-D) Impact of IL-7 hyperexpression in vivo on Foxp3 + Treg cells. CD25 expression levels among (A) total CD4 + T cells and (B) CD4 + -gated total Foxp3.RFP + Treg cells from Dex/Dox-treated Ctrl (for separate results in Ctrl irtTA-GBD and Ctrl tet-on-IL-7 single TG mice see Figure S6) and dTG mice, as revealed by the flow cytometric assessment of median fluorescent intensity (MFI). various experimental settings with clinical relevance. Here, we used the model to assess the impact of IL-7 hyperexpression in the microenvironment of insulin-producing islet transplants in the setting of allograft rejection (C57BL/6 islets BALB/c recipients). Islets isolated from dTG mice released detectable amounts of IL-7 when cultured in the presence of Dex/Dox ( Figure S8A). In vivo, BALB/c mice with STZ-induced diabetes were transplanted with C57BL/6 islets isolated from either dTG or Ctrl donor mice. Mice transplanted with dTG-islets and subsequently treated with Dex/Dox had increased blood glucose concentrations as compared with mice that had received Ctrlislets (247 ± 102 mg/dl after IL-7 induction vs. 151 ± 75 mg/dl control islets; P < 0.05) (Figure 6A). Under immunosuppressive conditions (i.e., after Dex/Dox administration) the rejection of the transplanted islets defined as blood glucose levels > 200 mg/dl was more frequent in mice that received IL-7 releasing islets (63.6% [95% CI 37.6%, 81.1%] at day 20) than in recipients of islets from control mice (18.2% [95% CI 0.1%, 56.4%]; P < 0.05) (Figure 6B). Reduced graft survival was associated with increased infiltration of CD4 + (Figure 6C, upper) and CD8 + (Figure 6C, lower) T cells in transplanted dTG islets with transgenic IL-7 hyperexpression compared to Ctrl-islets. Thus, the selective induction of IL-7 hyperexpression in transplanted dTG islets was sufficient to accelerate allograft rejection, abrogating the immunosuppressive effect of Dex/Dox on graft survival. Foxp3 staining was not performed to determine whether this was due to a reduction in infiltrating Tregs.

DISCUSSION
Cell replacement therapy in T1D must consider strategies to control immune-mediated loss of graft tissue due to reoccurring autoimmunity and graft rejection. Using an immunosuppressive pre-clinical model with transiently inducible IL-7 hyperexpression, we verified inducibility and bioactivity of the transgenic IL-7 protein, demonstrated irregularities in immunoregulation following IL-7 hyperexpression, and showed that local islet IL-7 hyperexpression promoted CD4 + and CD8 + T cell infiltration leading to enhanced graft rejection.
The mouse model, which features both inducible on-off expression and immune-suppression, showed typical responses to increased IL-7 seen in other models and systems (14)(15)(16)(17)(18)(19). Immunophenotyping of our dTG mice revealed a profound increase in Pro/Pre-B-I cells in the BM by IL-7 hyperexpression, while retaining their developmental potential. Constitutively or transiently elevated IL-7 expression without additional immunosuppression was also previously shown to increase this B cell progenitor cell population (14,31,32). In secondary lymphoid organs, CD8 + T cells with a memory surface phenotype were preferentially expanded in an IL-7-rich environment. A predominance of CD8 + to CD4 + T cells was also detected in mice with constitutive transgenic IL-7 expression under the control of the MHC class II promoter, which showed a CD44 + memory phenotype (17,33), independent of IL-15 signaling (33). While we did not address effector functions of the expanded memory-like CD8 + T cells, others have previously shown that in vivo exposure to high levels of transgenically expressed IL-7 led to a higher proportion of IFN-γ-producing cells among the memory CD8 + T cells (33). This is also in line with observations from lymphopenia-derived memory T cells, which upregulated CD44 and elicited effector functions, including rapid IFN-γ secretion (34)(35)(36). In parallel, IL-7 hyperexpression in an immunosuppressive environment reduced the proportion of Treg cells among total CD4 + T cells, confirming previously published findings in IL-7/anti-IL-7 mAb immunocomplex treated mice (37). Hence, we predict that our model, which also include immunosuppression, recapitulates many of the previously reported effects of increased IL-7 activity on B and T lymphocytes. A limitation of our study was that we did not include control experiments in which the activity of transgene derived IL-7 was blocked by antibodies or other means and it, therefore, may be possible that some of the observed effects are not strictly due to IL-7. We also mainly used a mix of two single transgenic animals that had no excess IL-7. These single transgenic animals appeared to be similar in their response to Dex/Dox treatment, but large numbers of each of these were not tested.
Crossing of our inducible mouse model with the Foxp3 RFP/GFP reporter mouse model (21) provided further evidence that IL-7 promotes Foxp3 + Treg cell homeostasis by increasing the pTreg contribution to the overall Treg cell pool. In contrast to our own studies of human Treg cells in the presence of IL-7 in vitro (13), an in vivo exposure to an IL-7-rich environment improved Treg suppressive capacity in vitro. Whether this discrepancy is due to in vitro or in vivo IL-7 exposure or a due to differences between humans and mice needs to be further elucidated. However, this improved Treg suppressive function was outcompeted by the enhanced resistance of Tresp cells to Treg cell-mediated suppression in an IL-7 rich environment. Several studies have described the phenomenon of reduced Tresp cell sensitivity to the suppressive function of Treg cells in various autoimmune diseases, including T1D (38), systemic lupus erythematosus (39), rheumatoid arthritis (40), and juvenile idiopathic arthritis (41), which also have been shown to be associated with lymphopenia and elevated IL-7 levels (42). The mechanism of the observed Tresp cell resistance is unclear. The PI3K/AKT signaling pathway as a downstream target of IL-7/IL-7R signaling and SHP-1 are shown to affect Tresp resistance to Treg (43)(44)(45). Bockade of the STAT5-dependent co-inhibitory receptor LAG3 leads to enhanced homeostatic proliferation of adoptively transferred CD4 + T cells in a lymphopenic host, increased CD25 expression and Tresp cell resistance to Treg-mediated suppression in vitro and in vivo (46). Moreover, Vazquez-Mateo and colleagues showed that blockade of IL-7Rα increased LAG3 as well as PD-1 and Tim-3 expression on CD4 + T cells rendering them more susceptible for co-inhibitory signals (11). Further studies should address whether IL-7 hyperexpression in our model affects PI3K/Akt and SHP-1 pathways, as well as LAG3 and/or other co-inhibitory receptor expression on Tresp cells.
We previously showed that IL-7 levels are elevated post islet transplantation in humans leading to the proliferation of memory T cell clones despite immunosuppression (12). Using isolated islets from our dTG mice in a genetically mismatched alloimmune model (C57BL/6 islet transplantation in diabetic BALB/c mice), we showed that induction of locally restricted IL-7 hyperexpression leads to enhanced CD4 + and CD8 + T cell infiltration resulting in augmented graft rejection. These results indicate that IL-7 can directly contribute to allograft rejection. Age is of potential relevance for the magnitude of the effect. Our experiments showed stronger effects of IL-7 hyperexpression on T cells in 4-week-old mice than 16-week-old mice. The transplant experiments were performed on 8-week-old mice.
Islet transplantation is generally performed in adult patients and an increase in IL-7 may not be of major consequence in adults. On the other hand, increases in IL-7 may be relevant to early autoimmunity in type 1 diabetes, which occurs in young children (47), and where T cells are highly sensitive to IL-7 (48).
In conclusion, we successfully established a pre-clinical model with inducible bioactive IL-7 hyperexpression. We showed that the induced IL-7 protein affects lymphocyte development, homeostasis and function at multiple levels in the model and can aggravate allograft rejection. This model can be used to pre-clinically test therapies in transplantation relevant settings.

ETHICS STATEMENT
This study was carried out in accordance with the recommendations of Landesdirektion Dresden, Ethikkommission an der TU Dresden. The protocol was approved by the Ethikkommission an der TU Dresden.

AUTHOR CONTRIBUTIONS
MS, EB, AH, KA, and KK designed the study, contributed to the conduct of the study, the acquisition, analysis, and interpretation of data, and drafted, reviewed, and approved the manuscript. MW, AK, SS, and CP contributed to the acquisition, analysis and interpretation of data, and approved the manuscript. AH, EB, and KK are the guarantors of this work.