Prevention, but not cure, of autoimmune diabetes in a NOD.scid transfer model by FTY720 despite effective modulation of blood T cells

Abstract

FTY720 modulates lymphocyte trafficking through blood (peripheral blood lymphocyte, PBL) and peripheral lymph nodes (PLN). Treatment with FTY720 causes retention of most blood lymphocytes in PLN. Long-term treatment can slow and/or prevent Type 1 diabetes (T1D) in the nonobese diabetic (NOD) mouse model. B and T cells are both affected by FTY720 binding to sphingosine-1-phosphate receptor 1 (S1P₁). However, little has been done to elucidate which T-cell subsets are differentially affected by FTY720 under healthy conditions, and how this affects disease pathogenesis in T1D. In healthy C57BL/6J (B6) mice, total CD4(+) and CD8(+) T-cell subsets were diminished by FTY720, but recently activated and memory subsets were spared and constituted significantly higher percentage of remaining T cells in blood. FTY720 also lowered PBL counts in NOD mice, but less severely than in B6 mice. This is consistent with a different ratio of naïve, activated, and memory cells in NOD mice compared to those in B6 mice, as well as alterations in S1P₁ and sphingosine-1-phosphate (S1P) levels in PBLs and blood of NOD mice, respectively. To address the functional consequences of PBL T-cell depletion, we studied the effects of FTY720 on disease progression in a timed adoptive transfer model of T1D. Continuous treatment with FTY720 eliminated T1D, if treatment was started before splenocyte transfer. FTY20 treatment started after disease onset slowed disease progression. The inability to fully suppress memory and effector T-cell circulation may explain why FTY720 is only partially effective in the NOD adoptive transfer model of T1D.

Figure 1

Eight-color staining of B6 PBLs. Representative FACS plots of the untreated B6 controls (A) and FTY720-treated B6 PBLs (B). Antibodies used for staining are shown above each column, with the stain on the X-axis listed first. Lymphocyte subsets of naïve, activated, and memory CD4, CD8, or γδ T cells, and NK cells (indicated by groupings underneath graphs) from either untreated B6 control PBLs (C) or FTY720-treated B6 mice (D) are shown. (C) and (D) are gated on CD3⁺ and on subset as indicated. Data are representative of n=5 mice per group.

Figure 2

Scaled pie charts of leukocyte and lymphocyte PBL populations in untreated B6 vs. FTY720-treated B6 mice. Control PBLs (left) and PBLs from FTY720-treated mice are broken down into smaller subsets, ending with tables differentiating CD69 expression on recently activated (CD44^hiCD62L^-) and central memory (CD44^hiCD62L⁺). Total T cells are indicated by the yellow pie pieces. T cell subsets are indicated as follows: blue for CD4⁺, green for CD8⁺, red for γδ T cells, and purple for all others. Subsets for CD4, CD8, and γδ T cells are indicated using patterns of the colors described above. Dots are CD44⁺CD62L^- cells, vertical stripes are CD44⁺CD62L⁺, solids are CD44^-CD62L⁺, and horizontal stripes are CD44^-CD62L^-

Figure 3

Eight-color staining of NOD PBLs. Fully stained samples from untreated NOD PBLs (A), and FTY720-treated NOD PBLs (B). Antibodies used for staining are shown above each column, with the stain on the X-axis listed first. Lymphocyte subsets of naïve, activated, and memory CD4, CD8, or γδ T cells (indicated by groupings underneath graphs) from either untreated NOD (representative data of n=4 for 2 experiments) control PBLs (C) or FTY720-treated NOD mice (representative data of n=5 for 2 experiments) (D) are shown.

Figure 4

Scaled pie charts of leukocyte and lymphocyte PBL populations in untreated NOD vs. FTY720-treated NOD mice. Control PBLs (left) and PBLs from FTY720-treated mice are broken down into smaller subsets, ending with tables differentiating CD69 expression on recently activated (CD44^hiCD62L^-) and central memory (CD44^hiCD62L⁺). Total T cells are indicated by the yellow pie pieces. T cell subsets are indicated as follows: blue for CD4⁺, green for CD8⁺, red for γδ T cells, and purple for all others. Subsets for CD4, CD8, and γδ T cells are indicated using patterns of the colors described above. Dots are CD44⁺CD62L^- cells, vertical stripes are CD44⁺CD62L⁺, solids are CD44^-CD62L⁺, and horizontal stripes are CD44^-CD62L^-

Figure 5

S1P/DHS1P/Total S1P (A) and S1P₁ receptor levels (B) in PBLs of 8 week-old female B6 and NOD mice. Data are representative of 2 experiments. For S1P/DHS1P measurements in blood, data are shown for n=5 mice for each group. For real-time RT-PCR, results normalized to actin are representative of 2 experiments in which n=3 for B6 and n=4 for NOD mice. *p≤ 0.01.

Figure 6

Incidence of T1D in adoptively transferred NOD.scid mice. A) Blood glucose levels for untreated mice pooled from all adoptive transfer experiments (n=15). Each line represents one mouse. B) Kaplan-Meier plot of data in (A) shows that all untreated mice (black circles) adoptively transferred with diabetic NOD splenocytes become diabetic within 38 days. FTY720-treatment was started at the time of transfer (n=4) (C), one week post-transfer (n=4)(D), three weeks post-transfer (n=5) (E), and four weeks post-transfer (n=4) (F). In graphs B-F, black circles with dashed lines represent the untreated mice for each group, and open circles with solid lines represent FTY720-treated mice. Bars under the graphs indicate day treatment was started, and continuation of treatment through the end of each experiment. The curves for each graph are representative of 2 additional experiments. *p<0.05.

Figure 7

Withdrawal of FTY720-treatment in the adoptive transfer model. Adoptively transferred NOD.scid mice were treated with FTY720 starting at 2 weeks (n=7) (A) or 3 weeks (n=11) (B). In all graphs, black circles with dashed lines represent the untreated mice for each group, and open circles with solid lines represent FTY720-treated mice. Treatment length and time at which treatment is stopped are indicated by the bars underneath each graph. The curves for each experiment are combined data from at least 2 separate experiments of the same type. *p<0.05.