Inhibition of c-Kit Is Not Required for Reversal of Hyperglycemia by Imatinib in NOD Mice

<div><p>(1) Aim/Hypothesis</p><p>Recent studies indicate that tyrosine kinase inhibitors, including imatinib, can reverse hyperglycemia in non-obese diabetic (NOD) mice, a model of type 1 diabetes (T1D). Imatinib inhibits c-Abl, c-Kit, and PDGFRs. Next-generation tyrosine kinase inhibitors for T1D treatment should maintain activities required for efficacy while sparing inhibition of targets that might otherwise lead to adverse events. In this study, we investigated the contribution of c-Kit inhibition by imatinib in reversal of hyperglycemia in NOD mice.</p><p>(2) Methods</p><p>The T670I mutation in c-Kit, which confers imatinib resistance, was engineered into the mouse genome and bred onto the NOD background. Hematopoietic stem cells (HSCs) from NOD.c-Kit^T670I mice and NOD.c-Kit^wt littermates were expanded in the presence or absence of imatinib to verify imatinib resistance of the c-Kit^T670I allele. Diabetic mice were treated with imatinib at the onset of hyperglycemia for three weeks, and blood glucose was monitored.</p><p>(3 )Results</p><p><i>In vitro</i> expansion of HSCs from NOD.c-Kit^wt mice was sensitive to imatinib, while expansion of HSCs from NOD.c-Kit^T670I mice was insensitive to imatinib. However, <i>in vivo</i> treatment with imatinib lowered blood glucose levels in both strains of mice.</p><p>(4) Conclusions/Interpretation</p><p>The HSC experiment confirmed that, in NOD.c-Kit^T670I mice, c-Kit is resistant to imatinib. As both NOD.c-Kit^T670I and NOD.c-Kit^wt mice responded comparably to imatinib, c-Kit inhibition does not substantially contribute to the efficacy of imatinib in T1D. Thus, we conclude that inhibition of c-Kit is not required in next-generation tyrosine kinase inhibitors for T1D treatment, and may be selected against to improve the safety profile.</p></div

Diabetic NOD.c-Kit^T670I mice are sensitive to imatinib treatment.

<p>Blood glucose values for individual diabetic NOD.c-Kit^wt mice treated with either PBS (<b>A</b>) or imatinib (<b>B</b>), in comparison to diabetic NOD.c-Kit^T670I mice treated with either PBS (<b>C</b>) or imatinib (<b>D</b>) for 21 consecutive days. <b>E.</b> Average blood glucose values in diabetic NOD.c-Kit^wt or NOD.c-Kit^T670I mice treated with either PBS ((thick, solid line) NOD.c-Kit^wt; (thin, solid line) NOD.c-Kit^T670I) or imatinib ((thick, dashed line) NOD.c-Kit^wt; (thin, dashed line) NOD.c-Kit^T670I) for 21 consecutive days (n = 5–10 mice/group; *** p<0.0001 between PBS vs. imatinib within each mouse group for entire data set, as determined by one-way ANOVA). Note there was no statistically significant difference between NOD.c-Kit^wt and NOD.c-Kit^T670I within imatinib treatment groups. <b>F.</b> Area under curve for diabetes progression over 21 days comparing PBS ((open, white square) NOD.c-Kitwt; (closed, black square) NOD.c-Kit^T670I) or imatinib ((grey, slashed square) NOD.c-Kit^wt; (grey, closed square) NOD.c-Kit^T670I) treated mice. (* p≤0.01 between PBS vs. imatinib within each mouse group, as determined by one-way ANOVA).</p

NOD.c-Kit^T670I mice are imatinib resistant and develop diabetes.

<p>A. Targeting strategy for generation of NOD.c-Kit^T670I mice. PCR genotyping (<b>B</b>) and sequence traces (<b>C</b>) of wild-type (wt) and mutant (mut) mice using primers F and R to generate fragments of 795 bp and 967 bp for wt and mut alleles, respectively. The T670I codon change and accompanying introduction of <i>Bgl-II</i> restriction site are highlighted. <b>D.</b> Expansion of c-Kit⁺/Sca-1⁺ murine HSCs from either NOD.c-Kit^T670I (black bar) or NOD.c-Kit^wt (white bar) littermates in the presence or absence of 5 µM imatinib. (*** p = 0.0002, as determined by two-way ANOVA). <b>E.</b> NOD.c-Kit^T670I (black square) mice develop diabetes comparably to NOD.c-Kit^wt (white circle) littermates (n = 41–45 mice/group; average age of diabetes onset = 14.5 weeks in NOD.c-Kit^wt mice and 15.5 weeks in NOD.c-Kit^T670I mice).</p

IL-2 Immunotherapy Reveals Potential for Innate Beta Cell Regeneration in the Non-Obese Diabetic Mouse Model of Autoimmune Diabetes

<div><p>Type-1 diabetes (T1D) is an autoimmune disease targeting insulin-producing beta cells, resulting in dependence on exogenous insulin. To date, significant efforts have been invested to develop immune-modulatory therapies for T1D treatment. Previously, IL-2 immunotherapy was demonstrated to prevent and reverse T1D at onset in the non-obese diabetic (NOD) mouse model, revealing potential as a therapy in early disease stage in humans. In the NOD model, IL-2 deficiency contributes to a loss of regulatory T cell function. This deficiency can be augmented with IL-2 or antibody bound to IL-2 (Ab/IL-2) therapy, resulting in regulatory T cell expansion and potentiation. However, an understanding of the mechanism by which reconstituted regulatory T cell function allows for reversal of diabetes after onset is not clearly understood. Here, we describe that Ab/IL-2 immunotherapy treatment, given at the time of diabetes onset in NOD mice, not only correlated with reversal of diabetes and expansion of Treg cells, but also demonstrated the ability to significantly increase beta cell proliferation. Proliferation appeared specific to Ab/IL-2 immunotherapy, as anti-CD3 therapy did not have a similar effect. Furthermore, to assess the effect of Ab/IL-2 immunotherapy well after the development of diabetes, we tested the effect of delaying treatment for 4 weeks after diabetes onset, when beta cells were virtually absent. At this late stage after diabetes onset, Ab/IL-2 treatment was not sufficient to reverse hyperglycemia. However, it did promote survival in the absence of exogenous insulin. Proliferation of beta cells could not account for this improvement as few beta cells remained. Rather, abnormal insulin and glucagon dual-expressing cells were the only insulin-expressing cells observed in islets from mice with established disease. Thus, these data suggest that in diabetic NOD mice, beta cells have an innate capacity for regeneration both early and late in disease, which is revealed through IL-2 immunotherapy. </p> </div

Ab/IL-2 immunotherapy augments regeneration through beta cell proliferation.

<p><b>A</b>-<b>D</b>. Recently diabetic NOD mice were treated with Ab/IL-2 or control isotype Ab for one to two weeks, and pancreata were processed for immunofluorescence staining for insulin (green), Ki67 (red), and DAPI (blue). <b>E</b>. Mean percent of proliferating Ki67+/insulin+ beta cells of total insulin+ beta cells was calculated from mouse pancreata samples collected during the first week of treatment by counting beta cells (blinded to treatment group) from n=4-8 mice per group, from each of two representative non-consecutive sections per pancreas, with a minimum of 1800 insulin+ cells per group counted <u>+</u> standard error of the mean. (*P<0.013, *** P<0.0008).</p

Ab/IL-2 immunotherapy enhances survival and preserves beta cells in diabetic NOD mice.

<p>NOD mice were diagnosed with recent-onset diabetes after two consecutive days with measurements of blood glucose between 200-400mg/dl. Diabetic mice were treated daily with intra-peritoneal injections of either control (PBS or isotype Ab) or Ab/IL-2 for 21 days (grey shaded area). Blood glucose (<b>A</b>), and survival (<b>B</b>) were monitored. (n=12 for isotype control; n=13 for Ab/IL-2). <b>C</b>-<b>H</b>. Pancreata from mice of Ab/IL-2 and control groups were processed for immunofluorescence staining for insulin (red), glucagon (green), and DAPI (blue) at the indicated number of days post-onset. <b>I</b>. Islets from Ab/IL2 treated mice retained a higher absolute number of insulin+ cells/islet compared with controls. Insulin+ beta cells in recently diabetic NOD pancreata were counted from n=3-4 mice per treatment group, from each of two representative non-consecutive sections per pancreas, with a minimum of 30 islets and 900 beta cells/group analyzed. Mean insulin+ beta cell number per islet ± standard deviation between individual mice is presented. (**P<0.009, ***P<0.0001) .</p

Ab/IL-2 immunotherapy enhances a rare dual-expressing insulin+/glucagon+ cell population in diabetic NOD mice.

<p>Established or recently diabetic NOD mice were treated with Ab/IL-2 or isotype control Ab for 3 weeks, as described. Pancreata were harvested and processed for immunofluorescence staining for insulin (red), glucagon (green), and DAPI (blue). Insulin+/glucagon+ cell numbers appeared significantly increased by Ab/IL-2 treatment in established diabetic (<b>A</b>-<b>G</b>) and recent onset diabetic (<b>H</b>-<b>N</b>) NOD pancreata. Notably, in established diabetic NOD mice, insulin+/glucagon+ cells were the only detectable source of insulin. (<b>G</b>, <b>N</b>) Percentage of insulin+, glucagon+, and dual-expressing insulin+/glucagon+ (yellow) cells were quantitated by counting all islet cells from each of two representative, non-consecutive sections per pancreas per group. <b>G</b>. In established diabetic mice, a total of n=2898 Ab/IL-2 islet cells and n=1498 control islet cells (n=4-8 mice per group) were counted; (<b>N</b>) in recent onset diabetic mice, n=2264 Ab/IL-2 islet cells and n=961 control islet cells (n=3-4 mice per group) were counted. Due to the rarity of islets themselves and insulin+/glucagon+ cells within the islet, cells from all pancreata within each group were pooled in order to determine the overall percentage. Thus, statistical comparison between individual animals could not be performed. </p

Insulin+/glucagon+ cells do not reflect mature endocrine cells.

<p>Recent onset diabetic NOD pancreata treated with Ab/IL-2 were processed and stained for insulin (red), glucagon (green), DAPI (blue) and mature endocrine markers, Brn4, Pdx1, or Nkx6.1 (white). (<b>A</b>) Staining for Brn4, a transcription factor expressed in mature alpha cells, showed expression in glucagon+ (arrowhead) and insulin+/glucagon+ (arrow) cells. (<b>B</b>) Staining for Pdx1, a beta cell specific transcription factor, showed weak expression in insulin+/glucagon+ (arrow) cells, as well some hormone negative cells (arrowhead). (<b>C</b>) Staining for Nkx6.1 showed only weak or absent nuclear staining (arrows) in insulin+/glucagon+ cells, as well as abnormal cytoplasmic staining (arrowhead). (<b>D</b>) Graph represents quantitation of the percent of insulin+/glucagon+ cells that also express nuclear Brn4, Pdx1 or Nkx6.1 (n=97+ insulin+/glucagon+ cells analyzed per transcription factor, from n≥4 individual samples).</p

Ab/IL-2 immunotherapy promotes survival and C-peptide production in established diabetic NOD mice.

<p>Mice diagnosed with fulminant diabetes at onset (>400mg/dl) were treated with insulin pellets to sustain survival. Starting 4 weeks after diabetes onset, mice were treated with Ab/IL-2 or control isotype Ab for 21 days (from week 4-7 post-onset, grey shaded areas), and further insulin therapy was withheld. Survival (<b>A</b>) and blood glucose (<b>B</b>) were monitored (n=14 isotype control, n=17 Ab/IL-2 treated). <b>C</b>. C-peptide levels were measured in established diabetic mice treated with Ab/IL-2 or control Ab from week 4-7 after onset. Levels after Ab/IL-2 treatment improved and were similar to age-matched control non-diabetic NOD.SCID mice. <b>D</b>. Established diabetic mice were treated for 7 days with Ab/IL-2 or isotype control Ab. Splenocytes, pancreatic lymph node (LN) and pancreatic lymphocytes from n=3 pooled treated mice for each group were analyzed by flow cytometry as described in methods. Plots shown are gated on CD4+ T cells. Treg cells were selectively expanded by Ab/IL-2 treatment in established diabetic NOD mice, as evidenced by increased % of CD4+CD25+FoxP3+ cells. (***P<0.0003, <b>#</b>-Mice sacrificed for histology analysis.) .</p