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

Dendritic Cell-Targeted Pancreatic β-Cell Antigen Leads to Conversion of Self-Reactive CD4+ T Cells Into Regulatory T Cells and Promotes Immunotolerance in NOD Mice

Studies employing T cell receptor transgenic T cells have convincingly shown that selective delivery of non-self model antigens to DEC-205+ dendritic cells (DCs) in the steady-state can induce Foxp3-expressing CD4+CD25+ regulatory T (Treg) cells from conventional CD4+CD25-Foxp3- T cells. Although of considerable clinical interest, the concept of DC-targeted de novo generation of antigen-specific Treg cells has not yet been evaluated for self-antigens and self-reactive CD4+ T cells in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). Here, we show in proof-of-principle experiments that targeting a mimotope peptide to the endocytic receptor DEC-205 on DCs in NOD mice induces efficient conversion of pancreatic β-cell-reactive BDC2.5 CD4+ T cells into long-lived Foxp3+ Treg cells. Of note, conversion efficiency in normoglycemic and hyperglycemic mice with early diabetes onset was indistinguishable. While de novo generation of BDC2.5 Treg cells did not interfere with disease progression, anti-DEC-205-mediated targeting of whole proinsulin in prediabetic NOD mice substantially reduced the incidence of diabetes. These results suggest that promoting antigen-specific Treg cells in vivo might be a feasible approach towards cellular therapy in T1D

Vagaries of fluorochrome reporter gene expression in Foxp3+ regulatory T cells.

CD4(+)CD25(+) regulatory T (Treg) cell lineage commitment and expression of the transcription factor Foxp3 can be induced at the CD4(+)CD8(+) double-positive (DP) and CD4(+)CD8(?) single-positive stages of thymic development, as well as in postthymic CD4(+) T cells in peripheral lymphoid tissues. The availability of transgenic mice with Foxp3-dependent fluorochrome reporter gene expression has greatly facilitated studies on the intra- and extrathymic generation of murine Foxp3(+) Treg cells. Here, we performed a comparative analysis of thymic Treg cell development and peripheral compartments of mature Treg cells in various transgenic strains with gene targeted and bacterial artificial chromosome (BAC)-driven Foxp3-fluorochrome expression. These studies revealed a relative deficiency of Foxp3(+) DP thymocytes selectively in mice with targeted insertion of the fluorochrome reporter gene coding sequence into the endogenous Foxp3 gene. While Foxp3 BAC-driven fluorochrome expression in ex vivo CD4(+) T cells was found to faithfully reflect Foxp3 protein expression, we provide evidence that Foxp3 BAC transgenesis can result in sizable populations of Foxp3(+) Treg cells that lack fluorochrome reporter expression. This could be attributed to both timely delayed up-regulation of BAC expression in developing Treg cells and the accumulation of peripheral Foxp3(+) Treg cells with continuous transcriptional inactivity of the Foxp3 BAC transgene

Foxp3+ Regulatory T Cells in Mouse Models of Type 1 Diabetes

Studies on human type 1 diabetes (T1D) are facilitated by the availability of animal models such as nonobese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, as well as a variety of genetically engineered mouse models with reduced genetic and pathogenic complexity, as compared to the spontaneous NOD model. In recent years, increasing evidence has implicated CD4+CD25+ regulatory T (Treg) cells expressing the transcription factor Foxp3 in both the breakdown of self-tolerance and the restoration of immune homeostasis in T1D. In this paper, we provide an overview of currently available mouse models to study the role of Foxp3+ Treg cells in the control of destructive β cell autoimmunity, including a novel NOD model that allows specific and temporally controlled deletion of Foxp3+ Treg cells

Cell spheroids are as effective as single cells suspensions in the treatment of critical-sized bone defects

Background!#!Due to their multilineage potential and high proliferation rate, mesenchymal stem cells (MSC) indicate a sufficient alternative in regenerative medicine. In comparison to the commonly used 2-dimensional culturing method, culturing cells as spheroids stimulates the cell-cell communication and mimics the in vivo milieu more accurately, resulting in an enhanced regenerative potential. To investigate the osteoregenerative potential of MSC spheroids in comparison to MSC suspensions, cell-loaded fibrin gels were implanted into murine critical-sized femoral bone defects.!##!Methods!#!After harvesting MSCs from 4 healthy human donors and preculturing and immobilizing them in fibrin gel, cells were implanted into 2 mm murine femoral defects and stabilized with an external fixator. Therefore, 26 14- to 15-week-old nu/nu NOD/SCID nude mice were randomized into 2 groups (MSC spheroids, MSC suspensions) and observed for 6 weeks. Subsequently, micro-computed tomography scans were performed to analyze regenerated bone volume and bone mineral density. Additionally, histological analysis, evaluating the number of osteoblasts, osteoclasts and vessels at the defect side, were performed. Statistical analyzation was performed by using the Student's t-test and, the Mann-Whitney test. The level of significance was set at p = 0.05.!##!Results!#!μCT-analysis revealed a significantly higher bone mineral density of the MSC spheroid group compared to the MSC suspension group. However, regenerated bone volume of the defect side was comparable between both groups. Furthermore, no significant differences in histological analysis between both groups could be shown.!##!Conclusion!#!Our in vivo results reveal that the osteo-regenerative potential of MSC spheroids is similar to MSC suspensions

Comparative quantification of GFP⁺ DP cells.

<p>Flow cytometric isolation of GFP⁺ DP thymocytes from (<b>A</b>) Foxp3^GFP, BAC-Foxp3^Cre-GFP and (<b>B</b>) Foxp3^IRES-GFP mice. Representative dot plots (from left to right) show presort analysis of CD4/CD8 expression among total thymocytes and CD25/GFP expression among CD25-enriched populations of gated DP cells, as well as postsort analysis of CD25/GFP and CD4/CD8 expression after flow cytometric isolation according to sort gates for CD25-enriched CD25⁺GFP⁺ cells, as indicated. Lines with arrowheads illustrate the gating scheme. Numbers in dot plots indicate percentages of cells in the respective quadrant or gate. (<b>C</b>) Quantification of GFP⁺ thymocytes. Percentages (left) and numbers (right) of GFP⁺ DP cells (top) and GFP⁺ CD4SP cells (bottom) from indicated Foxp3 reporter strains, revealed after postsort analysis as depicted in (<b>A,B</b>). All mice were six weeks old. (<b>D</b>) Numbers of GFP⁺ DP thymocytes from eleven-week-old Foxp3^GFP mice on the C57BL/6 (left) and BALB/c (middle) genetic background, as compared to age-matched BAC-Foxp3^Cre-GFP mice (right). Dots and horizontal lines represent individual mice and mean values, respectively. * p < 0.05, *** p < 0.001, ns, non-significant (one-way ANOVA with Bonferroni’s multiple comparison post test).</p

Genetic lineage tracing of Foxp3⁺ Treg cells in BAC-Foxp3^Cre-GFP × R26Y mice.

<p>Analysis of Foxp3 expression by Foxp3 ICS, in FACS purified GFP⁻YFP⁻ and GFP⁻/GFP⁺YFP⁺ populations among (<b>A</b>) CD25⁺ CD4SP thymocytes and (<b>B</b>) CD4⁺CD25⁺ T cells from LNs of BAC-Foxp3^Cre-GFP × R26Y mice. Representative presort analysis of GFP and YFP expression among gated CD4⁺CD25⁺ cells and postsort analysis are shown as dot plots. Histograms depict Foxp3 expression (ICS) in sorted GFP⁻YFP⁻ (left) and GFP⁻/GFP⁺YFP⁺ (right) cells. Numbers in dot plots and histograms indicate percentages of cells in the respective quadrant or gate. Data are representative of three independent experiments including at least three mice. (<b>C</b>) Percentages of GFP⁺ cells among <i>ex vivo</i> populations of YFP⁻CD25⁺ CD4SP thymocytes (left) and peripheral YFP⁻CD4⁺CD25⁺ T cells (right). (<b>D</b>) mRNA expression of GFP, YFP and Foxp3 was determined by real-time RT-PCR in sorted GFP⁻YFP⁻ and GFP⁻/GFP⁺YFP⁺ cells presented in (<b>A and B</b>).</p

Infidelity of BAC-Foxp3^Cre-GFP-dependent GFP expression.

<p>(<b>A-C</b>) Tracking Foxp3⁺ cells that lack GFP expression during thymic Treg cell lineage commitment. (<b>A</b>) Representative dot plots depict presort analysis of CD25 and GFP expression among gated DP and CD4SP thymocyte subsets from six-week-old BAC-Foxp3^Cre-GFP mice before and after magnetic bead enrichment of CD25⁺ cells, as indicated. Histograms show postsort analysis of GFP expression (left) and Foxp3 expression (right), as revealed by Foxp3 ICS, among indicated postsort populations. (<b>B</b>) Percentages and (<b>C</b>) numbers of Foxp3⁺ cells (ICS) among GFP⁻ and GFP⁺ cells that had been sorted from DP and CD4SP thymocyte compartments. (<b>D-F</b>) Tracking Foxp3⁺ Treg cells that lack GFP expression in peripheral lymphoid tissues. (<b>D</b>) Representative dot plots depict presort analysis of CD25 and GFP expression among gated CD4⁺ T cells from LNs before and after magnetic bead enrichment of CD25⁺ cells. Histograms show postsort analysis of GFP expression (left) and Foxp3 expression, as revealed by Foxp3 ICS (right), among indicated postsort populations. (<b>E</b>) Percentages and (<b>F</b>) numbers of Foxp3-expressing (ICS) CD4⁺CD25⁺ T cells among GFP⁻ and GFP⁺ cells that had been isolated by flow cytometry from pooled scLNs of BAC-Foxp3^Cre-GFP mice. All mice were six weeks old. Lines with arrowheads in dot plots illustrate the gating scheme. Numbers in dot plots and histograms indicate percentages of cells in the respective quadrant or gate. Dots and horizontal lines in graphs indicate individual mice and mean values, respectively.</p