Transfusion of nonobese diabetic mice with allogeneic newborn blood ameliorates autoimmune diabetes and modifies the expression of selected immune response genes

Abstract

Although allogeneic bone marrow transplantation has been shown to prevent autoimmune diabetes in heavily irradiated nonobese diabetic (NOD) mice, a similar procedure is not suitable for the treatment of patients with type 1 diabetes because of associated severe side effects. Therefore, we evaluated whether mouse newborn blood (NBB), equivalent to human umbilical cord blood, could be used for diabetes prevention without recipient preconditioning. To test this hypothesis, unconditioned, prediabetic female NOD mice were given a single injection of whole NBB derived from the allogeneic diabetes-resistant mouse strain C57BL/6. Transfusion of allogeneic NBB but not adult blood prevented diabetes incidence in a majority of treated mice for a prolonged period of time. This was accompanied by the release of insulin in response to a challenge with glucose. Invasive cellular infiltration of islets was also substantially reduced in these mice. Although NBB transfusion induced a low level of hematopoietic microchimerism, it did not strictly correlate with amelioration of diabetes. Induction of genes implicated in diabetes, such as Il18, Tnfa, and Inos but not Il4, Il17 or Ifng, was repressed in splenocytes derived from protected mice. Notably, expression of the transcription factor Tbet/Tbx21 but not Gata3 or Rorgt was upregulated in protected mice. These data indicate that allogeneic NBB transfusion can prevent diabetes in NOD mice associated with modulation of selected cytokine genes implicated in diabetes manifestation. The data presented in this study provide the proof of principle for the utility of allogeneic umbilical cord blood transfusion to treat patients with autoimmune diabetes.

Figure 1. Phenotype of NBB derived from C57BL/6 mice

Peripheral blood was obtained from newborn mice up to 5 days of age in 5 different experiments (n=50). After RBC lysis, cells were stained with various antibodies and analyzed by flow cytometry. Cells were electronically gated based on forward angle light and side scatter properties. The expression of CD44 was detected in cells found in Gate B, C and D in various proportions (see Table I). Only cells in Gate D expressed all of the determinants examined.

Figure 2. Amelioration of diabetes in NOD mice by allogeneic NBB transfusion

Incidence of diabetes (>250 mg/dL) in untreated NOD mice and those treated with allogeneic NBB or adult blood is shown. Arrow indicates the time of injection of cells. Statistical significance: untreated vs. newborn blood transfused, P=0.006, and untreated vs. adult blood transfused, P=0.54, NS.

Figure 3. Allogeneic NBB transfusion preserved the beta cell function

A. Intraperitoneal glucose tolerance test was performed at 28 to 32 wk of age. Blood glucose levels in various groups of mice challenged with glucose are shown. Values indicate mean +/- SD per group. Numbers of mice tested are indicated in parentheses. B. Corresponding insulin levels of these mice are shown. Mean values +/- SD per group are indicated.

Figure 4. Reduction in invasive infiltration of islets in protected mice

Top row shows H & E stained sections of pancreata displaying islets with no/minimal (Grade 1, A), moderate (Grade 2, B) and severe cellular infiltration (Grade 3, C) (original magnification × 200). Bottom row shows confocal images of islets displaying insulin-producing beta cells (red) in corresponding sections. Nuclei were stained with Hoechst (blue). Bar indicates 20 μM (original magnification × 630). D. Histological scores of pancreata in indicated sets of mice are shown.

Figure 5. Relationship between microchimerism and protection against diabetes

A. The presence of H-2K^b-expressing cells was determined by flow cytometry in the peripheral blood of untreated NOD mice and in those transfused with indicated numbers of mononuclear cells containing NBB at various time points. Statistical significance between groups is shown (n=8-10 mice per group). B. Microchimerism was analyzed in splenocytes after 14 to 18 wk of NBB transfusion by flow cytometry. Data are shown for NBB transfused but remained diabetic and those protected from diabetes after NBB transfusion (n= 10 per group). C. RT-PCR analysis of splenocytes obtained after 18 wk of NBB transfusion. B6: spleen cells from C57BL/6 positive control; N-1 through N-7: spleen cells from NOD mice transfused with C57BL/6 NBB; NOD: spleen cells from NOD mice. Amplicon sizes of K^b and β-2 microglobulin are shown.

Figure 6. Modulation of gene expression in NBB transfused and protected mice

A. Spleens were harvested from NOD mice protected from diabetes after transfusion with NBB derived from C57BL/6 mice and from untreated-diabetic mice at 32 wk of age and stimulated with PMA and ionomycin. Unstimulated splenocytes cultured over night in complete media served as controls. Real-time RT-PCR analysis was performed and the relative levels of gene expression in stimulated vs. unstimulated cells were analyzed using Gapdh as the normalizer. Data shown are from a single experiment (mean+/-SD of triplicates) of a total of 5 experiments, each containing 3 to 5 mice per group. P values between protected and diabetic groups are indicated. B. Prediabetic NOD mice were killed at 12 wk of age and their splenocytes were activated as described above. Relative gene expression in stimulated vs unstimulated cells was assessed using Gapdh as the normalizer. Data shown are from a single experiment (mean+/-SD of triplicates) in a total of 3 experiments, each containing 3 to 5 mice per group. The data shown are from an experiment analyzed simultaneously with that shown in A. C. Splenocytes were obtained from NBB transfused-non-diabetic and those untreated-diabetic mice and stimulated with PMA and ionomycin or left untreated as described in A. After overnight culture, supernatants were obtained and tested for various cytokines by ELISA. Shown is mean +/- SE of duplicate samples from a representative of 3 independent experiments with similar results. * P= 0.05.