Th1-Like ICOS⁺ Foxp3⁺ T_reg Cells Preferentially Express CXCR3 and Home to β-Islets during Pre-Diabetes in BDC2.5 NOD Mice

<div><p>Type 1 diabetes (T1D) occurs through a breakdown of self-tolerance resulting in the autoimmune destruction of the insulin producing β-islets of the pancreas. A numerical and functional waning of CD4⁺Foxp3⁺ regulatory T (T_reg) cells, prompted by a pancreatic IL-2 deficiency, accompanies Th1 autoimmunity and T1D progression in non-obese diabetic (NOD) mice. Recently, we identified a dominant subset of intra-islet T_reg cells that expresses the ICOS costimulatory receptor and promotes self-tolerance delaying the onset of T1D. ICOS co-stimulation potently enhances IL-2 induced survival and proliferation, and suppressive activity of T_reg cells <i>in situ</i>. Here, we propose an ICOS-dependent mechanism of T_reg cell homing to the β-islets during pre-diabetes in the NOD model via upregulation of the CXCR3 chemokine receptor. The islet-specific ICOS⁺ T_reg cell subset preferentially expresses CXCR3 in the pancreatic lymph nodes (pLN) in response to T_eff cell-mediated pancreatic inflammation, an expression correlating with the onset and magnitude of IFN-γ production by T_eff cells in pancreatic sites. We also reveal that intra-pancreatic APC populations and insulin-producing β, but not α nor δ, islet cells secrete the CXCR3 chemokines, CXCL9, 10 and 11, and selectively promote ICOS⁺CXCR3⁺ T_reg cell chemotaxis <i>in vitro</i>. Strikingly, islet-derived T_reg cells also produce these chemokines suggesting an auto-regulation of homing by this subset. Unlike ICOS^- cells, ICOS⁺ T_reg cells adopt a Th1-like T_reg phenotype while maintaining their suppressive capacity, characterized by expression of T-bet and CXCR3 and production of IFN-γ in the draining pLNs. Finally, <i>in vivo</i> neutralization of IFN-γ blocked T_reg cell CXCR3 upregulation evincing its role in regulating expression of this chemokine receptor by T_reg cells. Thus, CXCR3-mediated trafficking of T_reg cells could represent a mechanism of homeostatic immunoregulation during diabetogeneesis.</p></div

Resident leukocyte subsets in the pancreas express CXCR3-activating chemokines.

<p>NOD.TCRα^<b>-/-</b> mice initially received BDC2.5 CD4^<b>+</b> T cell (7.5X10^<b>5</b>) cells, and then cell suspensions from pancreas, draining pLN and peripheral LN of diabetic mice were examined for expression of CXCL9, CXCL10 and CXCL11 in F4/80^<b>+</b> (A) and CXCL10 in CD11c^<b>+</b> (B) cells was assessed by flow cytometry. (C) NOD.TCRα^<b>-/-</b> mice were transferred with CD4^<b>+</b> T cells in order to induce T1D. Following adoptive transfer, glucose levels were measured daily in order to assess diabetes onset. Pancreatic cell suspensions from recipients were obtained and CXCL10 expression (MFI) was compared among CD11c^<b>+</b> cells from diabetic or non-diabetic recipients, as well as un-transferred (without T cell transfer) NOD.TCRα^<b>-/-</b> mice. (D) NOD.TCRα^<b>-/-</b> mice received T_<b>reg</b> or T_<b>eff</b> cells either alone or at the indicated T_<b>reg/</b>T_<b>eff</b> cell ratios. When mice receiving T_<b>eff</b> cells alone became hyperglycemic (>33mmol/L), CXCL10 levels were assessed by ELISA in supernatants from pancreatic suspensions. (E), F4/80^<b>+</b> and CD11c^<b>+</b> (1X10^<b>6</b>/well) cells were seeded in triplicate in lower chambers, and CD4^<b>+</b> (1X10^<b>6</b>/well) cells in the upper chambers of 24 well Transwell plate. Following a 3 hour incubation period, the percent migrated CXCR3^<b>+</b> cells among ICOS^<b>+</b> T_<b>reg</b> cells was compared between wells containing APCs and media alone (<i>control</i>). Cell suspensions of pancreas and draining LN of 4-week-old BDC2.5 mice were obtained and CXCL10 expression (MFI) was compared (F) between pancreatic T_<b>reg</b> cells and T_<b>eff</b> cells (G), between T_<b>rec</b> cells at sites indicated, and between ICOS^<b>+</b> and ICOS^<b>+</b> T_<b>reg</b> cells within pancreas (H). (4A-4C n = 6, 4D n = 4, 4E n = 2, 4F-4H n = 5).</p

CXCR3-expressing ICOS⁺ T_reg cells adopt a Th1-like phenotype in draining LN.

<p>T-bet and CXCR3 co-expression was assessed in ICOS^<b>+</b> T_<b>reg</b> cells from 4 week old WT BDC2.5 mice (A). T-bet expression (MFI) between ICOS^<b>+</b> and ICOS^<b>-</b> subsets within T_<b>reg</b> cells (B). NOD.TCRα^<b>-/-</b> mice received BDC2.5 CD4^<b>+</b>CD25^<b>+</b> T_<b>reg</b> or CD4^<b>+</b>CD25^<b>-</b> T_<b>eff</b> cells (0.75X10^<b>5</b>) at the indicated T_<b>reg/</b>T_<b>eff</b> cell ratios. When mice receiving T_<b>eff</b> cells alone became hyperglycemic (>33mmol/L), mice were sacrificed and the frequency of (C) T-bet^<b>+</b> and (D) IFN-γ^<b>+</b> cells within the ICOS^<b>+</b> T_<b>reg</b> subset was assessed. (E) NOD.TCRα^<b>-/-</b> mice received WT T_<b>eff</b> (7.5X10^<b>5</b>) cells and ICOS^<b>-/+</b> T_<b>reg</b> (7.5X10^<b>5</b>) cells isolated from pooled LN and spleen. 10 days post-transfer, cell suspensions were obtained from the pancreas and draining pLN and IFN-γR expression (MFI) was compared between ICOS^<b>+</b> and ICOS^<b>-</b> T_<b>reg</b> cells. (3B-3D n = 4, 3E n = 5)</p

IFN-γ regulates CXCR3 expression by ICOS⁺ T_reg cells and their homing to islets.

<p>NOD.TCRα^<b>-/-</b> mice received BDC2.5 CD4^<b>+</b> T cells (7.5X10^<b>5</b>) and, (A) and the percent CXCR3^<b>+</b> and IFN-γR^<b>+</b> cells among total T_<b>reg</b> cells in pancreas and draining LNs was assessed at the indicated times post-transfer. (B) Correlation between percent CXCR3^<b>+</b> and IFN-γR^<b>+</b> in pancreas and draining LN at all points examined. (C) BDC2.5 CD4^<b>+</b> T cells were stimulated with various concentrations of recombinant IFN-γ, and STAT1 phosphorylation was assessed by flow cytometry and compared between ICOS^<b>+</b> and ICOS^<b>-</b> subsets of T_<b>reg</b> cells. (D) NOD.TCRα^<b>-/-</b> mice received T_<b>eff</b> (7.5X10^<b>5</b>) cells and were injected i.p. with either PBS or anti-IFNγ Ab (XMG1.2) on days -1, 1, 3, 5 and 7 post transfer. Mice were sacrificed when the PBS group displayed hyperglycemia (>33mmol/L). Cell suspensions of the pancreatic draining LN were obtained and the percent CXCR3^<b>+</b> among pT_<b>reg</b> cells was compared between groups. (6A, 6B, 6D n = 5. 6C n = 2).</p

Intra-islet T_reg cells reverse T_eff cell-mediated abrogation of chemokine secretion by ß-islet cells.

<p>β-islet cells were isolated from 4-week-old BDC2.5 mice. (A) Expression of CXCR3 chemokines was compared between insulin^<b>+</b> cells and glucagon^<b>+</b> cells, and unstained controls. (B), NOD.TCRα^<b>-/-</b> mice received T_<b>reg</b> or T_<b>eff</b> cells either alone or at the indicated T_<b>reg/</b>T_<b>eff</b> cell ratios. When mice receiving T_<b>eff</b> cells alone became hyperglycemic (>33mmol/L), CXCL10 expression (MFI) in β-islet cells was compared between groups. (C) IFN-γR ΔMFI (ΔMFI was calculated by subtracting the isotype control MFI from IFN-γR antibody MFI) and was compared between β (insulin^<b>+</b>), α (glucagon^<b>+</b>) and δ (somatostatin^<b>+</b>) cells isolated from 4-week-old BDC2.5 mice. (5B, 5C n = 5).</p

Autoreactive T cell-mediated inflammation induces CXCR3 expression by ICOS⁺ T_reg cells prior to T1D onset.

<p>Cell suspensions of pancreas and draining LN from 4 week old (A, B) WT and (B) ICOS^<b>-/-</b> BDC2.5 mice were assessed for the frequency of CXCR3^<b>+</b> cells and levels of CXCR3 expression (MFI) between the ICOS^<b>+</b> and ICOS^<b>-</b> subsets of T_<b>reg</b> cells. (C and D) NOD.TCRα^<b>-/-</b> mice received MACS sorted BDC2.5 CD4^<b>+</b>CD25^<b>+</b> (T_<b>reg</b>, 0.75X10^<b>5</b>) or CD4^<b>+</b>CD25^<b>-</b> (T_<b>eff</b>, 7.5X10^<b>5</b>) cells alone or at the indicated T_<b>reg/</b>T_<b>eff</b> cell ratios. When the T_<b>eff</b> cell recipient mice displayed hyperglycemia (>33mmol/L), mice were sacrificed and expression of IFN-γ by T_<b>eff</b> cells (C) and CXCR3^<b>+</b> percent cells among the ICOS^<b>+</b> T_<b>reg</b> cell subset were assessed. (E) NOD.TCRα^<b>-/-</b> mice received the indicated ratios of FACS-sorted Thy1.2^<b>+</b> T_<b>reg</b> cells to 7.5X10^<b>5</b> BDC2.5 Thy1.1^<b>+</b> CD4^<b>+</b>Foxp3^<b>-</b> T_<b>eff</b> cells. After 14 days, the Thy1.2 (tT_<b>reg</b>) and Thy1.1^<b>+</b> (pT_<b>reg</b>) subsets of ICOS^<b>+</b> T_<b>reg</b> cells were compared for percent CXCR3^<b>+</b> cells. (n = 4, peri LN = pooled axial, brachial and inguinal peripheral lymph nodes).</p

CXCR3 expression delineates a functionally fit subpopulation of ICOS⁺ T_reg cells.

<p>(A) Cell suspensions of pancreatic draining LN from 4-week-old mice were isolated and percent cycle cells, determined by Ki-67 expression, were compared between the CXCR3^<b>+</b> and CXCR3^<b>-</b> subsets of ICOS^<b>+</b> T_<b>reg</b> cells. (B) NOD.TCRα^<b>-/-</b> mice received T_<b>eff</b> (7.5X10^<b>5</b>) cells and either CXCR3^<b>+</b> or CXCR3^<b>-</b> ICOS^<b>+</b> T_<b>reg</b> cells (7.5X10^<b>4</b>) cells isolated from pooled LN and spleen of BDC2.5 mice. After 14 days suppression was assessed via percent IFN-γ^<b>+</b> cells among T_<b>eff</b> cells in the pancreas and draining LN. (n = 4).</p

Relative gene expression of key inflammatory cytokines and growth factors involved in the pathogenesis of Sjögren's syndrome.

<p>TNF-α and TGF-β mRNAs were downregulated in CFA-treated and MSC+CFA versus control NOD mice (upper panel; * <i>P</i><0.05). NOD mice transplanted with only MSCs had the lowest expression of TNF-α (* <i>P</i><0.05). EGF, IGF-IR and FGF-2 mRNAs (middle and lower panels) were upregulated (* <i>P</i><0.05) in MSC and MSC+CFA groups. The combined therapy (MSC+CFA) had the highest upregulation for regenerative genes (EGF, FGF-2). n = 4 to 8 mice per group.</p

Flow cytometry analysis of CD45⁻/Ter119⁻ cells.

<p>After 3 passages, CD45⁻/Ter119⁻ cells were stained with the following surface markers: CD45, Ter119, CD11B, Sca-1, CD106, CD105, CD73, CD29 and CD44. Data are representative of at least three separate experiments. This experiment shows 97.8% CD45⁻, 99.7% TER119⁻, 99.6% CD11b⁻ and 84.4% Sca1⁺, 86.5% CD106⁺, 64.1% CD105⁺, 19.3% CD73⁺, 86.4% CD29⁺, 81.2% CD44⁺ cells.</p

Functional assays for multipotent mesenchymal stromal cell (MSCs).

<p>Photomicrographs of CD45⁻/TER119⁻ cells that undergone chondrocytic, osteogenic and adipocytic differentiation when cultured in differentiation media. Lower right photomicrograph represents CD45⁻/TER119⁻ cells cultured in non-differentiating media (negative control). Collagen II (red) and DAPI (blue nuclei) are used to identify chondrocytes. Von kossa (black) and oil red stainings were used to identify osteoblasts and adipocytes, respectively. Graphs represent the number, size, and frequency of colony-forming unit fibroblasts (CFU-F) of CD45⁻/TER119⁻ cells versus whole bone marrow cells. All three graphs show that CD45⁻/TER119⁻ cells had higher CFU-F than whole bone marrow cells (* <i>P</i><0.05).</p