B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model

Abstract

Aims/hypothesis: Type 1 diabetes is a T cell-mediated autoimmune disease characterised by the destruction of beta cells in the islets of Langerhans, resulting in deficient insulin production. B cell depletion therapy has proved successful in preventing diabetes and restoring euglycaemia in animal models of diabetes, as well as in preserving beta cell function in clinical trials in the short term. We aimed to report a full characterisation of B cell kinetics post B cell depletion, with a focus on pancreatic islets.

Methods: Transgenic NOD mice with a human CD20 transgene expressed on B cells were injected with an anti-CD20 depleting antibody. B cells were analysed using multivariable flow cytometry.

Results: There was a 10 week delay in the onset of diabetes when comparing control and experimental groups, although the final difference in the diabetes incidence, following prolonged observation, was not statistically significant (p = 0.07). The co-stimulatory molecules CD80 and CD86 were reduced on stimulation of B cells during B cell depletion and repopulation. IL-10-producing regulatory B cells were not induced in repopulated B cells in the periphery, post anti-CD20 depletion. However, the early depletion of B cells had a marked effect on T cells in the local islet infiltrate. We demonstrated a lack of T cell activation, specifically with reduced CD44 expression and effector function, including IFN-γ production from both CD4⁺ and CD8⁺ T cells. These CD8⁺ T cells remained altered in the pancreatic islets long after B cell depletion and repopulation.

Conclusions/interpretation: Our findings suggest that B cell depletion can have an impact on T cell regulation, inducing a durable effect that is present long after repopulation. We suggest that this local effect of reducing autoimmune T cell activity contributes to delay in the onset of autoimmune diabetes.

Keywords: B cell depletion; B cells; Insulitis; NOD mice; Type 1 diabetes.

Fig. 1

Characterisation of anti-CD20 depletion treatment. hCD20/NOD mice aged 6–8 (b–d, h–j) or 12–15 weeks (e–g, k–m) were injected with 2H7 anti-CD20 antibody (grey lines/squares in b–g) or IgG control antibody (black lines/circles in b–g). Diabetes progression was monitored and lymphocyte populations were analysed by flow cytometry at different time points after antibody depletion. (a) Schematic representation of injection regimen. (b–g) Cell numbers for splenic CD19⁺ B cells (b, e), CD8⁺ T cells (c, f) and CD4⁺ T cells (d, g). Data are expressed as mean ± SEM. Each time point includes a minimum of six mice from at least two independent experiments. (h–m) Percentage of B cells depleted or repopulated (calculated as individual numbers from each 2H7-treated mouse/mean number from all control antibody-treated mice) at various time points for different lymphoid organs: spleen (h, k), pancreatic lymph node (i, l), bone marrow (j, m). Horizontal lines indicate medians. **p < 0.01 and ***p < 0.001 (Mann–Whitney U test, control vs 2H7)

Fig. 2

Kinetics of B cell regulatory markers after anti-CD20 antibody treatment. hCD20/NOD mice aged 6–8 weeks (b–d, h–j) or 12–15 weeks (e–g, k–m) were injected with 2H7 anti-CD20 antibody (grey lines/squares in b–g) or IgG control antibody (black lines/circles in b–g) and total splenocytes were analysed. CD19⁺ B cell populations were identified by flow cytometry at different time points after depletion. (a) Representative flow plots (24 h) of spleen compartments marked by CD21 and CD23 (marginal zone [MZ: CD21^hiCD23^low], T2 [CD21^hiCD23^hi]) and follicular zone [FO: CD21^lowCD23^hi], showing flow cytometric gating of control IgG- and 2H7-treated mice (aged 6–8 weeks). (b–g) Number of B cells from MZ (b, e), T2 (c, f) and FO (d, g) spleen compartments. (h–m) Percentage of B cells depleted or repopulated for MZ (h, k), T2 (i, l) and FO (j, m) spleen compartments (calculated as individual numbers from each 2H7-treated mouse/mean number from all control antibody-treated mice). Horizontal lines indicate medians. All surface markers are shown for cells that were gated on viable CD3⁻CD19⁺. Data are expressed as mean ± SEM. Each time point includes a minimum of six mice from at least two independent experiments. **p < 0.01 and ***p < 0.001 (Mann–Whitney U test, control vs 2H7)

Fig. 3

Repopulated B cells are not enriched for IL-10. B cells from the spleen were analysed for intracytoplasmic cytokines 8 and 12 weeks post treatment with control IgG (black circles) or 2H7 anti-CD20 depleting antibody (white squares) in mice aged 6–8 (b, c) or 12–15 weeks (d, e). B cells were either unstimulated or stimulated with 5 μg/ml LPS or anti-CD40. (a) Representative flow plots from 12 weeks post depletion. (b–e) Frequency of IL-10-producing B cells (b, d) and TGF-β-producing B cells (c, e) 8 and 12 weeks post depletion. Horizontal lines represent the medians. Each time point includes a minimum of seven mice, from at least two independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Mann–Whitney U test, control vs 2H7)

Fig. 4

Fewer repopulated B cells express co-stimulatory molecules. Splenic B cells, from mice aged 6–8 (b, c) or 12–15 weeks (d, e) at the time of depletion, were analysed for co-stimulatory molecules 8 and 12 weeks post treatment with control IgG (black circles) or 2H7 anti-CD20 depleting antibody (white squares). The B cells were either unstimulated or were stimulated with 5 μg/ml LPS or anti-CD40 for 24 h. (a) Representative flow plots (12 weeks after depletion) of CD80 and CD86 expression on unstimulated or stimulated B cells. (b–e) Frequency of cells expressing CD80 (b, d) and CD86 (c, e) at 8 and 12 weeks after depletion. Horizontal lines represent the medians. Each time point includes a minimum of seven mice from at least two independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Mann–Whitney U test, control vs 2H7)

Fig. 5

Islet-infiltrating B cells show no enrichment of regulatory cytokines. Pancreatic islets were isolated from mice aged 6–8 weeks (a–c) and 12–15 weeks (d–f) treated with control IgG (black lines/circles) or 2H7 anti-CD20 depleting antibody (grey lines/circles). (a, d) Number of CD19⁺ B cells. Data are expressed as mean ± SEM and numbers represent all islets recovered from individual pancreases. (b, c, e, f) Frequency of islet B cells stimulated with PMA/ionomycin expressing the intracellular cytokines IL-10 (b, e) and TGF-β (c, f). Horizontal lines represent medians. Black circles, control IgG; white squares, 2H7. (g–l) Multivariable analysis of B cells performed by SPICE software at 12 weeks (g, h) and 30 weeks (i, j) post depletion; (g, i) pie charts for controls; (h, j) pie charts for 2H7. Pie charts indicate different heterogeneous subsets; the coloured arcs correspond to the fraction of cells that express specific markers shown in the key. p = 0.2566 at 12 weeks and p = 0.1486 at 30 weeks (permutation test to compare the pie charts, performed by SPICE software). Graphical representations of heterogeneous subsets in pie slices are shown for 12 weeks (k) and 30 weeks (l) post depletion. Horizontal lines represent medians. Black circles, control IgG; white squares, 2H7. Cells were gated on CD8⁻CD4⁻CD19⁺. B cell combinations where the frequency did not exceed 1% are not included in SPICE analysis. Data are representative of a minimum of four mice in each group, from at least two independent experiments. *p < 0.05 and **p < 0.01 (Mann–Whitney U test, control vs 2H7)

Fig. 6

B cell depletion influences islet T cells. Pancreatic islets were isolated from mice aged 6–8 weeks (a–f) and 12–15 weeks (g–l) treated with control IgG or 2H7 anti-CD20 depleting antibody. (a, g) Number of CD4⁺ T cells and (d, j) number of CD8⁺ T cells. Each point represents cells isolated from all islets recovered from individual pancreases. Black lines/circles, control IgG; grey lines/squares, 2H7. (b, e, h, k) Cytokines in islet CD4 T cells: percentage of IFN-γ⁺ (b, h) and IL-10⁺ cells (e, k). (c, f, i, l) Cytokine and cytotoxic markers on pancreatic islet CD8 T cells: percentage of cells positive for cytokine IFN-γ (c, i) and cytotoxic CD107a (f, l). Black circles, control IgG; white squares, 2H7. Data are representative of a minimum of four mice in each group, from at least two independent experiments. Horizontal lines represent medians. *p < 0.05 (Mann–Whitney U test, control vs 2H7)

Fig. 7

Islet CD4⁺ T cells are altered after anti-CD20 treatment. Pancreatic islets were isolated from mice aged 6–8 weeks treated with control IgG or 2H7 anti-CD20 depleting antibody. CD4⁺ T cells were analysed by flow cytometry after 12 or 27–30 weeks. Multivariable analysis of CD4⁺ T cell markers at 12 weeks (a, c) and 30 weeks post depletion (b, d) performed by SPICE software for both control IgG (a, b) and 2H7 treatment (c, d). The pie charts indicate different heterogeneous subsets, with the coloured arcs corresponding to the fraction of cells that expressed specific markers shown in the key. p = 0.0487 at 12 weeks; p = 0.4021 at 30 weeks (permutation test performed by SPICE to compare pies). Graphs illustrate the percentage of islet CD4⁺ T cell populations that were significantly changed at 12 weeks (e) and 30 weeks post depletion (f). Black circles, control IgG; white squares, 2H7. CD4⁺ T cell combinations, where the frequency did not exceed 1%, are not included in SPICE analysis. Mann–Whitney U test (control vs 2H7) was used to determine significance between each population. Data are representative of a minimum of six (12 weeks) or five mice (30 weeks) in each group, from at least two independent experiments. *p < 0.05, **p < 0.01

Fig. 8

B cell depletion affects CD8⁺ T cells long after repopulation. Pancreatic islets were isolated from mice aged 6–8 weeks treated with control IgG or 2H7 anti-CD20 depleting antibody and the CD8⁺ T cells were analysed by flow cytometry 12 or 27–30 weeks later. Multivariable analysis of CD8⁺ T cell markers at 12 weeks (a, c) and 30 weeks post depletion (b, d) was performed by SPICE software for control (a, b) and 2H7 treatment (c, d). The pie charts indicate the proportion of co-expressed markers, with the coloured arcs corresponding to the fraction of cells that expressed specific markers, shown in the key. p = 0.0408 at 12 weeks; p = 0.5680 at 30 weeks (permutation test performed by SPICE to compare pie slices). Graphs illustrate the percentage of individual population of CD8⁺ T cells, expressing different surface markers and cytokines, that were significantly changed at 12 weeks post depletion (e) and the percentage of the individual population of CD69⁺CD103⁺CD8⁺ T cells significantly changed at 30 weeks post depletion (f). Black circles, control IgG; white squares, 2H7. CD8⁺ T cell combinations where the frequency did not exceed 1% are not included in the SPICE analysis. Mann–Whitney U test (control vs 2H7) was used to determine significance between each population. Data are representative of a minimum of six (12 weeks) or five (30 weeks) in each group, from at least two independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001