Fig 1.
Autoreactive T cell-mediated inflammation induces CXCR3 expression by ICOS+ Treg cells prior to T1D onset.
Cell suspensions of pancreas and draining LN from 4 week old (A, B) WT and (B) ICOS-/- BDC2.5 mice were assessed for the frequency of CXCR3+ cells and levels of CXCR3 expression (MFI) between the ICOS+ and ICOS- subsets of Treg cells. (C and D) NOD.TCRα-/- mice received MACS sorted BDC2.5 CD4+CD25+ (Treg, 0.75X105) or CD4+CD25- (Teff, 7.5X105) cells alone or at the indicated Treg/Teff cell ratios. When the Teff cell recipient mice displayed hyperglycemia (>33mmol/L), mice were sacrificed and expression of IFN-γ by Teff cells (C) and CXCR3+ percent cells among the ICOS+ Treg cell subset were assessed. (E) NOD.TCRα-/- mice received the indicated ratios of FACS-sorted Thy1.2+ Treg cells to 7.5X105 BDC2.5 Thy1.1+ CD4+Foxp3- Teff cells. After 14 days, the Thy1.2 (tTreg) and Thy1.1+ (pTreg) subsets of ICOS+ Treg cells were compared for percent CXCR3+ cells. (n = 4, peri LN = pooled axial, brachial and inguinal peripheral lymph nodes).
Fig 2.
CXCR3 expression delineates a functionally fit subpopulation of ICOS+ Treg cells.
(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+ and CXCR3- subsets of ICOS+ Treg cells. (B) NOD.TCRα-/- mice received Teff (7.5X105) cells and either CXCR3+ or CXCR3- ICOS+ Treg cells (7.5X104) cells isolated from pooled LN and spleen of BDC2.5 mice. After 14 days suppression was assessed via percent IFN-γ+ cells among Teff cells in the pancreas and draining LN. (n = 4).
Fig 3.
CXCR3-expressing ICOS+ Treg cells adopt a Th1-like phenotype in draining LN.
T-bet and CXCR3 co-expression was assessed in ICOS+ Treg cells from 4 week old WT BDC2.5 mice (A). T-bet expression (MFI) between ICOS+ and ICOS- subsets within Treg cells (B). NOD.TCRα-/- mice received BDC2.5 CD4+CD25+ Treg or CD4+CD25- Teff cells (0.75X105) at the indicated Treg/Teff cell ratios. When mice receiving Teff cells alone became hyperglycemic (>33mmol/L), mice were sacrificed and the frequency of (C) T-bet+ and (D) IFN-γ+ cells within the ICOS+ Treg subset was assessed. (E) NOD.TCRα-/- mice received WT Teff (7.5X105) cells and ICOS-/+ Treg (7.5X105) 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+ and ICOS- Treg cells. (3B-3D n = 4, 3E n = 5)
Fig 4.
Resident leukocyte subsets in the pancreas express CXCR3-activating chemokines.
NOD.TCRα-/- mice initially received BDC2.5 CD4+ T cell (7.5X105) 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+ (A) and CXCL10 in CD11c+ (B) cells was assessed by flow cytometry. (C) NOD.TCRα-/- mice were transferred with CD4+ 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+ cells from diabetic or non-diabetic recipients, as well as un-transferred (without T cell transfer) NOD.TCRα-/- mice. (D) NOD.TCRα-/- mice received Treg or Teff cells either alone or at the indicated Treg/Teff cell ratios. When mice receiving Teff cells alone became hyperglycemic (>33mmol/L), CXCL10 levels were assessed by ELISA in supernatants from pancreatic suspensions. (E), F4/80+ and CD11c+ (1X106/well) cells were seeded in triplicate in lower chambers, and CD4+ (1X106/well) cells in the upper chambers of 24 well Transwell plate. Following a 3 hour incubation period, the percent migrated CXCR3+ cells among ICOS+ Treg cells was compared between wells containing APCs and media alone (control). 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 Treg cells and Teff cells (G), between Trec cells at sites indicated, and between ICOS+ and ICOS+ Treg cells within pancreas (H). (4A-4C n = 6, 4D n = 4, 4E n = 2, 4F-4H n = 5).
Fig 5.
Intra-islet Treg cells reverse Teff cell-mediated abrogation of chemokine secretion by ß-islet cells.
β-islet cells were isolated from 4-week-old BDC2.5 mice. (A) Expression of CXCR3 chemokines was compared between insulin+ cells and glucagon+ cells, and unstained controls. (B), NOD.TCRα-/- mice received Treg or Teff cells either alone or at the indicated Treg/Teff cell ratios. When mice receiving Teff 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+), α (glucagon+) and δ (somatostatin+) cells isolated from 4-week-old BDC2.5 mice. (5B, 5C n = 5).
Fig 6.
IFN-γ regulates CXCR3 expression by ICOS+ Treg cells and their homing to islets.
NOD.TCRα-/- mice received BDC2.5 CD4+ T cells (7.5X105) and, (A) and the percent CXCR3+ and IFN-γR+ cells among total Treg cells in pancreas and draining LNs was assessed at the indicated times post-transfer. (B) Correlation between percent CXCR3+ and IFN-γR+ in pancreas and draining LN at all points examined. (C) BDC2.5 CD4+ T cells were stimulated with various concentrations of recombinant IFN-γ, and STAT1 phosphorylation was assessed by flow cytometry and compared between ICOS+ and ICOS- subsets of Treg cells. (D) NOD.TCRα-/- mice received Teff (7.5X105) 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+ among pTreg cells was compared between groups. (6A, 6B, 6D n = 5. 6C n = 2).