Antigen Loading (e.g., Glutamic Acid Decarboxylase 65) of Tolerogenic DCs (tolDCs) Reduces Their Capacity to Prevent Diabetes in the Non-Obese Diabetes (NOD)-Severe Combined Immunodeficiency Model of Adoptive Cotransfer of Diabetes As Well As in NOD Mice

Abstract

Tolerogenic DCs (tolDCs) are being researched as a promising intervention strategy also in autoimmune diseases including type 1 diabetes (T1D). T1D is a T-cell-mediated, organ-specific disease with several well-defined and rather specific autoantigens, i.e., proinsulin, insulin, glutamic acid decarboxylase 65 (GAD65), that have been used in animal as well as human intervention trials in attempts to achieve a more efficient, specific immunotherapy. In this study, we have tested tolerogenic DCs for their effectiveness to prevent adoptive transfer of diabetes by diabetogenic splenocytes into non-obese diabetes (NOD)-severe combined immunodeficiency (NOD-SCID) recipients. While i.p. application of tolDCs prepared from bone marrow of prediabetic NOD mice by vitamin D2 and dexamethasone significantly reduced diabetes transfer into the NOD-SCID females, this effect was completely abolished when tolDCs were loaded with the mouse recombinant GAD65, but also with a control protein-ovalbumin (OVA). The effect was not dependent on the presence of serum in the tolDC culture. Similar results were observed in NOD mice. Removal of possible bystander antigen-presenting cells within the diabetogenic splenocytes by negative magnetic sorting of T cells did not alter this surprising effect. Tolerogenic DCs loaded with an immunodominant mouse GAD65 peptide also displayed diminished diabetes-preventive effect. Tolerogenic DCs were characterized by surface maturation markers (CD40, CD80, CD86, MHC II) and the lipopolysaccharide stability test. Data from alloreactive T cell proliferation and cytokine induction assays (IFN-γ) did not reveal the differences observed in the diabetes incidence. Migration of tolDCs, tolDCs-GAD65 and tolDCs-OVA to spleen, mesenteric- and pancreatic lymph nodes displayed similar, mucosal pattern with highest accumulation in pancreatic lymph nodes present up to 9 days after the i.p.

Application: These data document that mechanisms by which tolDCs operate in vivo require much better understanding for improving efficacy of this promising cell therapy, especially in the presence of an antigen, e.g., GAD65.

Keywords: autoantigen; cell therapy; dendritic cells; glutamic acid decarboxylase 65; non-obese diabetes mice; non-obese diabetes-severe combined immunodeficiency mouse; tolerogenic; type 1 diabetes.

Figure 1

Tolerogenic DCs but not tolDCs-GAD65 prevent diabetes in the adoptive transfer model of non-obese diabetes (NOD)-severe combined immunodeficiency (NOD-SCID) mice. Tolerogenic DCs (tolDCs) or glutamic acid decarboxylase 65 (GAD65) (2 µg/mL) loaded tolDCs (tolDCs-GAD65) were generated from bone marrows of 8- to 10-week-old NOD females by cultivation in the presence of GM-CSF and IL-4 followed by additions of dexamethasone/vitamin D2 and monophosphoryl lipid A. Dendritic cells (3 × 10⁶) were resuspended in phosphate bovine saline (PBS) together with 5 × 10⁶ diabetogenic splenocytes from 13-week-old prediabetic NOD females (n = 8). Cells were then injected i.p. (left side of the belly) in a volume of 300 µL PBS to 8-week-old NOD-SCID female recipients (n = 12). Diabetogenic splenocytes in PBS were used as the Control group. Data are presented as cumulative diabetes incidence in NOD-SCID recipients, p-values were compensated for multiple comparisons, tolDC vs. tolDC-GAD65: *p = 0.0159.

Figure 2

Antigen-loaded tolDCs fail to lower induction of diabetes in non-obese diabetes (NOD)-severe combined immunodeficiency (NOD-SCID) mice by transfer of diabetogenic NOD splenocytes but also by transfer of splenic T cells. Diabetogenic splenocytes (5 × 10⁶ per mouse) were isolated from 13-week-old prediabetic NOD females (n = 11). T cells were enriched (cell purity >92%) by negative selection (EasySep T cell Enrichment kit, Stemcell Tech.), and equivalent of 33% of splenocytes, i.e., 1.65 × 10⁶ T cells per mouse were used for diabetes induction in NOD-SCID recipients. Tolerogenic DCs, glutamic acid decarboxylase 65 (GAD65)- (1 µg/mL) or OVA- (1 µg/mL) loaded tolDCs were generated from bone marrows of 8- to 10-week-old NOD females by cultivation in the presence of GM-CSF and IL-4 followed by additions of dexamethasone/vitamin D2 and stabilized by monophosphoryl lipid A (MPLA). Diabetogenic splenocytes or enriched T cells (groups marked as T cell⁺) were resuspended in phosphate bovine saline (PBS) together 3 × 10⁶ tolDCs and injected i.p. (left side of the belly) in a volume of 300 µL PBS to 8-week-old NOD-SCID female recipients (n = 8). Diabetogenic splenocytes in PBS were used as the Control group. Data are presented as cumulative diabetes incidence in NOD-SCID recipients, p-values were compensated for multiple comparisons.

Figure 3

Serum-free cultured tolDCs and GAD65-loaded tolDCs in diabetes prevention using the non-obese diabetes (NOD)-severe combined immunodeficiency (NOD-SCID) model of adoptive transfer of diabetes. Dendritic cells were generated from bone marrows of 8- to 10-week-old NOD females by cultivation in the presence of GM-CSF and IL-4 followed by additions of dexamethasone/vitamin D2 and final maturation with monophosphoryl lipid A (MPLA). (A) Tolerogenic DCs loaded with 1 µg/mL of glutamic acid decarboxylase 65 (GAD65) (tolDC-GAD65) or OVA (tolDC-OVA) were prepared in SF medium, whereas tolDCs were cultured in both serum-supplemented (10% fetal bovine serum RPMI-1640) and SF media (tolDC SF). (B) In another experiment, unloaded tolDCs were compared to tolDCs loaded with 1 µg/mL of the GAD65-immunodominant peptide no. 35 (tolDC-pept) and prepared in both serum-supplemented and SF media. Diabetogenic splenocytes (5 × 10⁶ per mouse) from 12-week-old prediabetic NOD females (n = 10) and above listed groups of tolDCs (3 × 10⁶) were mixed and applied i.p. (left side of the belly) in a volume of 300 µL phosphate bovine saline (PBS) to 7-week-old NOD-SCID female recipients (n = 8). Diabetogenic splenocytes in PBS were used as the Control group in both experiments. Data are presented as cumulative diabetes incidence in NOD-SCID recipients and p- values were compensated for multiple comparisons (A) Control PBS vs. tolDC SF: *p = 0.026.

Figure 4

Diabetes-preventive effect of tolDCs but not antigen-loaded tolDCs in the spontaneous model of type 1 diabetes—the non-obese diabetes (NOD) mice. Tolerogenic DCs, GAD65- (1 µg/mL) or OVA- (1 µg/mL) loaded tolDCs were generated from bone marrows of 8- to 10-week-old NOD females by cultivation in serum-supplemented (10% fetal bovine serum RPMI-1640) medium in the presence of GM-CSF and IL-4 followed by additions of dexamethasone/vitamin D2 and stabilized by MPLA. Tolerogenic DCs were also prepared in SF media (tolDC SF). TolDCs (3 × 10⁶) were resuspended in phosphate bovine saline (PBS) and injected i.p. (left side of the belly) in a volume of 200 µL to 4-week-old NOD females (n = 16). i.p. application of 200 µL PBS alone was used for the Control group. Diabetes incidence was observed weekly (from week 12) until the age of 310 days. Data are presented as cumulative diabetes incidence, p-values were compensated for multiple comparisons.

Figure 5

Phenotypic characteristics, anti-inflammatory cytokine profile, and stability of tolDCs and antigen-loaded tolDCs. (A) Expression of maturation markers CD40, CD80, CD86, and MHC II on immature bone marrow-derived dendritic cells (iDCs), control matured bone marrow-derived dendritic cells (cDCs), tolDCs, tolDCs-GAD65, tolDCs-OVA, and DCs loaded with 1 µg/mL of glutamic acid decarboxylase 65 (GAD65)-immunodominant peptide no. 35 (tolDCs-pept), cultured in serum-supplemented (RPMI-1640 with 10% fetal bovine serum) and SF conditions, was determined by surface staining of live cells and flow cytometry. Data are expressed as mean fluorescence intensities (MFI) from 4 to 5 (serum-supplemented conditions) or 2 to 3 (SF conditions) experiments ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. (B) Example dot plots from flow cytometry analyses of additional surface markers, i.e., CD103, CCR7, and IL-7Ra (CD127) on tolDCs and autoantigen-loaded tolDCs-GAD65. (C) Stability test of above listed types of DCs was carried out by additional 24 h culture (Control) or restimulation with 1 µg/mL lipopolysaccharide (LPS). Changes in expression of maturation markers CD40, CD80, CD86, and MHC II were assessed by flow cytometry and are displayed as MFI. (D) Interleukin 10 (IL-10) release after MPLA activation or 24 h LPS restimulation (stability test) by iDCs, cDCs, tolDCs, tolDCs-GAD65, and tolDCs-OVA. Data are expressed as means from two to four parallel cell cultures.

Figure 6

Allogeneic T cell proliferation and IFN-γ production by stimulation with tolDC vs. antigen-loaded tolDCs. (A) Allogeneic proliferative responses of immature bone marrow-derived dendritic cells (iDCs), control matured bone marrow-derived dendritic cells (cDCs), tolDCs, tolDCs-GAD65, tolDCs-OVA, and tolDC-pept were assessed by coculture of CFSE-labeled splenocytes (6- to 8-week-old C57BL/6 females) with DCs (8-week-old non-obese diabetes females) at 10:1 ratio for 3 and 5 days. Proliferation was measured as CFSE dilution in live CD3⁺ cells by flow cytometry. Splenocytes cultured alone were used as a control. All experiments were carried out in the serum-supplemented RPMI-1640 medium. Data are expressed as mean percentage of CFSElowCD3⁺ cells ± SEM of four experiments, **p < 0.01, ***p < 0.001. (B) Example of proliferation analysis by the flow cytometry of CFSE-labeled CD3⁺ splenocytes. (C) Induction of INF-γ in allogeneic CD4⁺CD3⁺ T cells was measured after 5 days of coculture with iDCs, cDCs, tolDCs, tolDCs-GAD65, tolDCs-OVA, and tolDCs-pept, following 4-h restimulation with phorbo-12-myristate-13-acetate/ionomycin by intracellular staining and flow cytometry analysis. Data are expressed as mean percentage of CD4⁺CD3⁺ cells ± SEM of four experiments, **p < 0.01, ***p < 0.001. (D) Example flow cytometry data of allogeneic induction of IFN-γ by DCs within CD3⁺CD4⁺ splenocytes.

Figure 7

CD8⁺ and CD4⁺ T-cell-mediated in vitro killing of tolDCs vs. antigen-loaded tolDCs. (A) Equal number (5 × 10⁶) of immature bone marrow-derived dendritic cells (iDCs), tolDCs, GAD65-, or OVA-loaded tolDCs was mixed 1:1 with CD4⁺ or CD8⁺ splenic T cells (enriched by negative magnetic selection) and cocultured for 4, 8, 12, and 24 h. Dendritic cells cultured without splenic T cells were used as controls. Percentage of live nonapotopic cells was measured by flow cytometry as DCs (gated according to the FSC, SSC and CD3⁻CD11c⁺ parameters) stained double negative for Hoechst33342⁻ and AnnexinV⁻. Data are expressed as mean ± SEM of two to three experiments, *p < 0.05, **p < 0.01, ***p < 0.001. (B) Example of CD4⁺ and CD8⁺ T cell enrichment by negative magnetic selection. (C) Example of flow cytometry analyses of CD3⁻CD11c⁺AnnexinV⁻Hoechst33342⁻ cells at the 4 h timepoint.

Figure 8

In vivo migration of PKH26-labeled tolDCs and tolDCs-GAD65. Bone marrow-derived dendritic cells were prepared from 8-week-old non-obese diabetes (NOD) mice. Tolerogenic DCs and tolDCs-GAD65 (1 µg/mL) were labeled with fluorescent PKH26 dye and 5 × 10⁶ cells were applied i.p. (left side of the belly) to 6-week-old NOD females. Unlabeled tolDCs were used as a negative control. FACS detection of PKH26⁺ cells was carried out on cell suspensions from spleen, mesenteric lymph nodes (MLNs), pancreatic lymph nodes (PLNs), and systemic inguinal lymph nodes (ILNs) after 3, 5, 7, 9, and 12 days (three mice per group for day 12, five mice per group for all other timepoints). Following doublets exclusion cells were gated according to the FSC-A and SSC-A parameters and dead cells were excluded by Hoechst 33258. PKH26⁺ tolDCs and tolDCs-GAD65 are displayed as percentage of live CD11c⁺ cells (1–2 × 10⁶ events per sample). Example of a larger (no. of time points) of two independent experiments.