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Keywords:

  • CD25;
  • CD70;
  • CD8;
  • islet infiltration;
  • non-obese diabetic (NOD) mice

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Destruction of pancreatic islets in type 1 diabetes is caused by infiltrating, primed and activated T cells. In a clinical setting this autoimmune process is already in an advanced stage before intervention therapy can be administered. Therefore, an effective intervention needs to reduce islet inflammation and preserve any remaining islet function. In this study we have investigated the role of targeting activated T cells in reversing autoimmune diabetes. A combination therapy consisting of CD25-, CD70- and CD8-specific monoclonal antibodies was administered to non-obese diabetic (NOD) mice with either new-onset diabetes or with advanced diabetes. In NOD mice with new-onset diabetes antibody combination treatment reversed hyperglycaemia and achieved long-term protection from diabetes (blood glucose <13·9 mmol/l) in >50% of mice. In contrast, in the control, untreated group blood glucose levels continued to increase and none of the mice were protected from diabetes (P < 0·0001). Starting therapy early when hyperglycaemia was relatively mild proved critical, as the mice with advanced diabetes showed less efficient control of blood glucose and shorter life span. Histological analysis (insulitis score) showed islet preservation and reduced immune infiltration in all treated groups, compared to their controls. In conclusion, antibody combination therapy that targets CD25, CD70 and CD8 results in decreased islet infiltration and improved blood glucose levels in NOD mice with established diabetes.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Type 1 diabetes (T1D) is an autoimmune disease that results from T cell-mediated destruction of insulin-producing β cells. Disease transfer has been obtained with both CD4+[1,2] and CD8+[1,3] T cell clones after injection into immuno-incompetent recipient mice, which speaks in favour of T cells being the main therapeutic target in T1D. In the clinical setting, T1D is usually diagnosed at an advanced stage of islet destruction when infiltrating T cells are likely to have a primed/memory phenotype. Importantly, current treatments have limited effects on primed/memory T cells [4]. Indeed, immune responses against self and/or allogeneic islets transplanted into non-obese diabetic (NOD) mice with established disease are extremely difficult to control, with only a small number of treatments achieving successful outcomes [5].

We have postulated that targeting activated T cells by using a combination of antibodies specific for CD25, CD70 and CD8 could prevent diabetes onset and restore normoglycaemia in newly hyperglycaemic NOD mice. CD25 and CD70 are expressed on activated T cells and ensure selective targeting of effector T cells, which could be involved in islet destruction. In this strategy, a low dose of CD8-specific antibody is added to the combination therapy to increase total antibody binding to CD8+ T cells as the activated, primed CD8+ T cells are less sensitive to treatment with CD25- and CD70-specific antibodies [6]. We have focused on activated T cells as the probable effector cells critical for the islet destruction. However, CD25 and CD70 are also expressed on activated B cells; thus, it is possible that our antibody combination therapy affects activated B cells, which could contribute to its efficacy.

The objectives of this study were to investigate the impact of the therapeutic antibody combination on the course of hyperglycaemia, insulitis and long-term protection from diabetes of NOD mice. The antibodies used were murine-specific reagents equivalent to those currently licensed for medical use (anti-CD25) [7,8], or in an advanced stage of clinical development (anti-CD70 and anti-CD8) [9,10]. Therefore, our therapeutic approach has considerable potential for transfer from the laboratory to the clinic. The side effects of these therapeutic antibodies are likely to be acceptable due to their specificity of action, as well as their long half-life and prolonged effects (thus requiring only intermittent administration). We have focused on the use of a combination of antibodies, as opposed to a single antibody, although most of the currently developed therapeutics in both research and the clinic are based on the use of the single compound. Our reasoning is that by increasing the quantity of antibody bound to selected target T cells a threshold should be reached that will result in their efficient control or depletion [6,11], as well as the increasing acceptance that a complex disease such as T1D will most probably require a combination approach [12].

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Mice

Female NOD mice >12 weeks old, generally accepted to represent a clinically relevant model for the investigation of the potential efficacy of novel treatments for newly diagnosed diabetes [5,13], were obtained from Taconic Farms, Inc. (Germantown, NY, USA) and cared for in accordance with institutional guidelines. Mice were bred under specific pathogen-free conditions at the Biological Services Unit for Animal Care at NHH, King's College London. All procedures were carried out in accordance with UK Home Office regulations.

Assessment of diabetes

New-onset diabetes was defined as blood glucose levels >250 mg/dl (>13·9 mmol/l) for a minimum of 2 consecutive days. Advanced disease was defined as blood glucose levels >306 mg/dl (>17 mmol/l) for a minimum of 2 consecutive days. Blood was taken from the lateral tail vein (approximately 200 µl per draw) and glucose was measured using a OneTouch Ultra blood glucose meter and matching test strips (LifeScan, Johnson&Johnson, Bracknell, Berkshire, UK) with maximum readable blood glucose of 33·3 mmol/l.

Antibody combination therapy

Anti-mouse CD70-specific monoclonal antibody FR70 and CD8-specific antibody YTS169 (both BioXcell, West Lebanon, NH, USA) were used at 500 µg and 5 µg/dose, respectively. CD25-specific antibody PC615-3 (AbD Serotec, Kidlington, UK) was used at a 200 µg/dose. Immediately before use, antibodies were pooled and administered together intraperitoneally (i.p.) as a single dose. Mice were given five i.p. injections of antibody combination therapy on days 0, 2, 4, 7 and 10 (day 0 marking the time-point when hyperglycaemia was confirmed for the second time in 2 consecutive days). Notably, our treatment regimen was not supported by insulin administration, but relied solely upon the therapeutic effects of the antibody combination.

Histology

Pancreata were harvested at specific time-points and fixed in 10% formalin prior to paraffin-embedding. Staining was performed using 1 µm-thick tissue sections cut using a microtome (Leica, Nussloch, Germany). Once cut to the desired thickness, sections were collected onto standard microscope slides and placed into a 65°C chamber to soften the wax prior to staining. Immediately before staining, slides were briefly soaked in xylene to de-wax, then passed through an ethanol gradient (100, 90, 70%) and finally immersed in water.

Pancreatic sections were stained with haematoxylin (7 min) and with 1% aqueous eosin for 3 min. After staining, they were dehydrated in an ethanol gradient, dipped in xylene and then mounted with DPX Mountant (Sigma-Aldrich, Dorset, UK). Slides were analysed using standard light microscopy.

Islet scoring

A score from 0 to 4 was assigned based on islet infiltration, as described previously [14]. A minimum of 30 islets per group were analysed, pooled from different mice. Insulitis score was graded as follows: grade 0, normal islets; grade 1, mild mononuclear infiltration (<25%) at the periphery; grade 2, 25–50% of the islets infiltrated; grade 3, more than 50% of the islets infiltrated; and grade 4, islets completely infiltrated with no residual parenchyma remaining. Analysis was conducted from islets in different sections at intervals of 200 µm. The scoring was performed by two experienced pathologists blinded for the treatment status and the data were averaged.

Statistical analysis

Data are expressed as mean ± standard error. Protection from diabetes data were analysed using the Kaplan–Meier method, with the log-rank test used to verify the significance of the difference between groups. When groups were compared, the two-sided unpaired Student's t-tests (for parametric data) or Mann–Whitney U-tests (for non-parametric data) were used according to the data distribution. A value of P less than 0·05 was considered statistically significant. Prism software was used for drawing graphs (GraphPad Software, Inc., San Diego, CA, USA). Data were analysed using sas version 8·02 (SAS Institute Inc., Cary, NC, USA).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

A combination therapy consisting of CD25-, CD70- and CD8-specific antibodies reverses hyperglycaemia and achieves long-term protection from diabetes of new-onset diabetic NOD mice

Female NOD mice older than 12 weeks were monitored for their blood glucose levels regularly and considered diabetic after detecting levels of more than 13·9 mmol/l for a minimum of 2 consecutive days. On the day that the new onset of diabetes was determined, NOD mice were treated with the combination of antibodies using the 10-day treatment regimen (as described in the Methods section). Mice in the untreated control group (n = 9, Fig. 1a) had difficulties in maintaining euglycaemia from the day of detection of diabetes, with three mice reaching hyperglycaemic plateau (levels of blood glucose above 33·3 mmol/l) by day 5. Notably, none of the untreated, control group mice managed to restore euglycaemia after being hyperglycaemic for 2 or more days. In contrast, all mice from the treated group showed a reduction of the blood glucose level after treatment was administered. In the treated group (n = 18, including seven mice that were killed for islet histological analysis), one mouse reached tha hyperglycaemic plateau and died on day 21 and six mice reached the hyperglycaemic plateau by day 48. Importantly, the remaining six mice from the treated group maintained a euglycaemic state for >200 days and had their survival prolonged indefinitely, showing no signs of disease (Fig. 1b). Thus, in contrast to untreated controls, a significant proportion of the treatment group (>50%) showed long-term protection from diabetes (blood glucose <13·9 mmol/l) (Fig. 1c, P < 0·0001).

image

Figure 1. Antibody combination therapy restores normoglycaemia in non-obese diabetic (NOD) mice with new-onset diabetes. NOD mice with blood glucose >13·9 mmol/l for 2 consecutive days were treated with the antibody combination comprising anti-CD70 (500 µg/dose), anti-CD8 (5 µg/dose) and anti-CD25 (200 µg/dose) over a period of 10 days (intraperitoneal injections on days 0, +2, +4, +7, +10). (a) None of the untreated control mice (n = 9) restored euglycaemia and survived beyond day 24. (b) In contrast, treated mice showed better control of blood glucose, with six mice restoring normoglycaemia and surviving indefinitely. Randomly selected mice (n = 7, marked with *) were killed at days 21 and 28 and their tissues were analysed. (c) The comparison between treated and control groups showed long-term protection from diabetes in the treated group (P < 0·0001).

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Seven mice from the treated group were selected randomly and killed at various time-points before day 24 and their tissues were taken in order to compare their histology to the control group.

Antibody combination therapy reduces insulitis and improves islets structure when administered to NOD mice with new-onset diabetes

In order to investigate the effect of the combination antibody therapy on insulitis and islet destruction, we examined pancreata of NOD mice killed at various time-points before and after hyperglycaemia arose.

At 16 weeks of age, the vast majority of our NOD mice were still euglycaemic and showed no signs of disease. When these normoglycaemic mice were killed (n = 6) and their tissue harvested for histological analysis and islet scoring, their pancreata showed that although the animals were normoglycaemic, none of their islets was free from infiltration. A representative islet with a relatively low level of infiltration is shown in Fig. 2a. Furthermore, islet scoring in the euglycaemic 16-week-old mice revealed that 45% of the islets showed mild infiltration (less than 25% of the islet infiltrated – light yellow in the graph; Fig. 2g, column 1), whereas approximately 10% of the islets showed moderate (25–50%) infiltration and approximately 20% of islets showed severe (>50%) infiltration. Notably, more than 20% of the islets in this group were completely destroyed (shown dark red in the graph; Fig. 2g).

image

Figure 2. Diabetic non-obese diabetic (NOD) mice treated with antibody combination therapy show decrease in islet infiltration compared to untreated controls. (a) A representative islet from a 16-week-old normoglycaemic NOD mouse shows mild angular infiltration. (b) A typical islet from a 26-week-old NOD mouse displays more islet destruction, with infiltrate erasing the distinctive border between the islet and the surrounding exocrine tissue. (c) A tissue section from the 30-week-old NOD control group, 2 weeks after the onset of diabetes, shows completely destroyed islets, with the loss of the distinctive shape and morphology of the islet, massive perivascular and peri-islet infiltration, with islets replaced with organized ectopic lymphoid structures consisting of inflammatory cells, which stretch between the blood vessels and former islets. (d) A healthy-looking islet with minimal infiltration from a treated 30-week-old NOD mouse, 2 weeks after the onset of diabetes. (e) An islet that appears protected from penetration by inflammatory cells, a feature observed only in the treated group. (f) An islet from a control, untreated 30-week-old NOD mouse shows cell infiltration that appears to have destroyed the islet border and entered the islet. (g) Scoring islet infiltration shows that increased islet destruction correlates with increased age of the mice in the control, untreated group (columns 1–3). In contrast, the treated 30-week-old NOD mice, 2 weeks after the onset of diabetes, show a reduced percentage of destroyed islets (column 4). The data were based on sections from four mice in the treated and control groups, with four slides taken from each mouse (a total of 16 slides for each group), yielding a total of 33 islets for controls and 66 islets for the treated group.

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At 26 weeks of age, a significant proportion of mice were still normoglycaemic. When their pancreata were harvested (n = 7), an expectedly higher grade of infiltration compared to the previous group was evident (a representative islet is shown in Fig. 2b). Indeed, the islet scoring showed that the proportion of completely destroyed islets was higher (approximately 30%) than that observed in the 16-week-old group (Fig. 2g, column 2). This corresponded to the percentage of mildly (<25%) infiltrated islets being decreased from 45% in the 16-week-old group to 30% in the 26-week-old group.

At the age of 28 weeks, the majority of mice were hyperglycaemic, and we administered antibody treatment. Two weeks after the onset of diabetes, at the age of 30 weeks, pancreata from both the control (n = 4) and treated (n = 4) groups were harvested. The 30-week-old hyperglycaemic control group showed a further deterioration in islet score, with >35% of completely destroyed islets (Fig. 2g, column 3). Typically, in this group the cellularity of the islets was compromised severely and it was difficult to determine their borders to distinguish the exocrine pancreatic tissue. Furthermore, the islets were often replaced with organized foci of inflammatory cells connecting the area between the islets and blood vessels (a representative destroyed islet is shown in Fig. 2c). Notably, compared to the treated group of the same age, the number of islets per pancreas was approximately halved: 16 sections from the control group yielded 33 islets, compared to 66 islets from the same number of sections obtained from the treated group.

Strikingly, islets from treated mice in the 30-week-old group were much better-preserved, showing minimum infiltration and healthy-like morphology (a representative, healthy-looking islet from the treated group is shown on Fig. 2d). The most prominent effect of antibody combination therapy was the smaller number of destroyed islets in the treated group, <10% in the treated group compared to >35% in the control, hyperglycaemic 30-week-old group (Fig. 2g, columns 4 and 3). Furthermore, this was less than in the normoglycaemic 26-week-old mice (30% destroyed islets, Fig. 2g, column 2). Notably, the percentage of completely destroyed islets in the 30-week-old treated group was slightly smaller than the percentage of destroyed islets in the normoglycaemic 16-week-old group, before the onset of diabetes (<10% compared to 20% in the 16-week-old group, Fig 2g, columns 4 and 1). This suggests that antibody combination therapy decreases infiltration and preserves islet structure so that pancreas histology resembles that observed before the onset of the disease.

The islets of the treated group showed some unique features that were absent in the untreated control NOD mice of 16, 26 and 30 weeks of age. Namely, some islets in the treated group appeared protected from the penetration by inflammatory cells, despite a large number of cells surrounding the islet (peri-insulities; Fig. 2e). In contrast, the islet from a matched control, untreated 30-week-old NOD mouse showed cellular infiltrates that cross the islet border (Fig. 2f).

Treatment with antibody combination improves the blood glucose profile of NOD mice with advanced diabetes

To test the antibody combination therapy in a more stringent disease model, the effects of the treatment in female NOD mice with blood glucose levels of >17 mmol/l for a minimum of 2 consecutive days (representing advanced disease) was evaluated. Notably, our therapy was not supported by insulin administration.

The control untreated group showed rapid and irreversible changes in blood glucose towards the hyperglycaemic plateau, which was reached in all the mice (n = 14) by day 10. This was followed by the death of all the mice before day 30 (Fig. 3a). The treated group showed an improved blood glucose profile from the initiation of therapy with a drop in blood glucose levels during the first 10 days in most recipients (Fig. 3b). Three mice from the treated group were killed before day 10 and their tissues were harvested for the analysis of islet histology. The remaining six mice in the treated group showed a slow but steady increase in blood glucose levels, resulting in a hyperglycaemic plateau by days 35–65 (Fig. 3b). Therefore, even in this more challenging setting (diabetes treated only after blood glucose levels reached >17 mmol/l – advanced disease stage) the treated group showed improved glucose profile. However, only a small proportion (16%) of treated mice were protected from diabetes (blood glucose <13·9 mmol/l) at any time.

image

Figure 3. Antibody combination therapy improves blood glucose control in non-obese diabetic (NOD) mice with advanced diabetes. NOD mice with blood glucose >17 mmol/l for 2 consecutive days were assigned randomly to the control untreated group or treated with antibody combination. (a) A rapid, irreversible change in blood glucose towards a hyperglycaemic plateau was seen in the control group. (b) An undulating curve with drops in glucose levels corresponding to administered therapy (days 0, 2, 4, 7, 10) can be observed in the treated group.

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Antibody combination therapy reduces infiltration and preserves islet morphology when administered to NOD mice with advanced diabetes

In the group of female NOD mice with blood glucose >17 mmol/l that were treated with the antibody combination therapy, analysis of the pancreata (n = 5) histology showed reduced cellular infiltration and more healthy-looking islets (Fig. 4a), compared to untreated controls (n = 5) (Fig. 4b). Furthermore, islet scoring revealed a substantial decrease in the number of destroyed islets in the treated group (15% compared to 45% in the control group, Fig. 4c). Notably, more than half (55%) the islets in the treated group were healthy or mildly (less than 25%) infiltrated, compared to fewer than 10% of minimally affected islets in the control group (Fig. 4c).

image

Figure 4. Histology of islets in non-obese diabetic (NOD) mice with advanced diabetes. Five NOD mice were analysed for each group, four sections per pancreas, totalling 20 slides for each group. The control group slides contained a total of 57 islets compared to 76 islets in the treated group. (a) An islet from the treated group showing mild infiltration. (b) Histology of a representative islet from the control, untreated group shows peri-islet and perivascular cell infiltration with the destruction of tissue structure between the small vessels. (c) Islet scoring of NOD mice with blood glucose >17 mmol/l, which were treated with antibody combination, thereby showed improved preservation and reduced islet infiltration compared with those from control mice.

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As the control untreated group deteriorated rapidly and survived for only a short period (Fig. 3a) pancreata of the treated group were harvested early, during the 10-day therapy period. In this way we could compare the islet histology of the two groups at a similar stage of the disease.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Recent reviews of the therapeutic strategies used in the NOD model of type 1 diabetes have revealed that more than 190 treatments are able to prevent or delay the onset of diabetes [15–17]. In contrast, a very small number of therapies showed any efficacy in NOD mice that were already hyperglycaemic/diabetic [18–21]. Furthermore, only one therapeutic approach, that of non-depleting CD3-specific antibody, showed promise for translation into clinical use [22]. We have developed a novel antibody combination therapy that is able to reverse hyperglycaemia in >50% of NOD mice with new-onset diabetes. These findings are particularly important regarding the highly stringent and challenging model, namely non-transgenic NOD mice that develop diabetes spontaneously. Taking this into consideration, the efficacy of our combination therapy equals or surpasses any other therapy used in the NOD model published to date [23]. Notably, we have not used insulin as a supportive therapy, although in all likelihood this could have improved further the efficacy of our therapy.

The antibody combination therapy used in this study is based on reagents already available for clinical use (anti-CD25) or in the late stage of clinical development (anti-CD70) [24]. CD70 and CD25 are expressed on activated T and B cells, suggesting that these specific antibodies may target both cell populations. As recent data emphasize the importance of both B cells [25–27] and T cells [28] in autoimmune diabetes, this antibody combination may result in strong, synergistic effects on the key cells involved in disease pathology. There are other reagents with promising therapeutic potential, such as CD154-specific antibody and rapamycin, which could be used as single therapeutics or as part of a combination therapy [29,30]. However, we believe that these reagents could face substantial obstacles for their clinical use due to potentially serious side effects.

Our published work provides evidence that when used as individual reagents antibodies specific for CD25, CD70 and CD8 are not effective in controlling primed/memory T cells [6,31]. As primed T cells play a key role in the later stages of islet pathology in NOD mice with established diabetes [32–34], these antibodies are highly unlikely to show effects when used as individual reagents. Nevertheless, it is possible that individual antibodies might be beneficial when administered earlier, i.e. when NOD mice show mild islet infiltration, before substantial islet damage and hyperglycaemia has occurred.

The mechanisms of action of the combination therapy in NOD mice were not studied in detail. Nevertheless, the use of therapy was based on the finding that it had beneficial effects in another model where primed T cells play a major role in pathology, cardiac transplantation in presensitized recipients [11], and the assumption that this might translate into the NOD model. Indeed, this success has now been replicated in the autoimmune diabetes setting, implying that the mechanisms of action of the therapy may be similar in these two settings. The transplant model offers some advantages in mechanistic studies, therefore most of the insights into the effects of this specific antibody combination therapy are from this model [6,11,30,35].

Our published data have shown that CD70-specific antibody acts primarily via Fc-mediated depletion of activated T cells and possibly B cells [31]. However, at present it remains unclear whether the depletion of T effector cells is responsible for the efficacy of antibody therapy in the NOD model. The synergy between CD70- and CD8-specific antibodies in targeting activated T cells has been described previously [6]. However, the role of CD25-specific antibody within a combination therapy is less clear. Our recent data in a transplant model suggest that treatment with CD25-specific antibody results in down-regulation of CD25 on T cells (data not shown). Furthermore, in the transplant model the therapy with a combination of CD25-, CD70- and CD8-specific antibodies results in a striking decrease in T cells that produce interferon (IFN)-γ on rechallenge with transplant antigens (data not shown). Such strong effects of the combination therapy on effector T cells were difficult to demonstrate in the NOD model, as the use of islet autoantigens for ex-vivo stimulation is less established as a means to examine changes in effector cell function [36].

We have tested islet-reactive T cells from the spleens of NOD mice using enzyme-linked immunospot assays (ELISPOT) to detect interleukin (IL)-2 and IFN-γ production in response to the mimetic peptide NRP-V7 (KYNKANVFL) [37]. This peptide provided a weak stimulus for IL-2 production, with only 10–15 spots per 400 000 splenocytes harvested from diabetic NOD mice (two to five spots in the absence of peptide). The NRP-V7 peptide induced a strong increase in IFN-γ production by T cells from diabetic NOD mice when compared to their normoglycaemic controls (>100 spots versus 30–60 spots per 400 000 splenocytes) or no-peptide controls (two to five spots per 400 000 splenocytes). Interestingly, we found that T cells harvested from the spleens of the treated, diabetic NOD mice produced high levels of cytokines directly ex vivo without peptide stimulation (>100 spots per 400 000 splenocytes). This high background prevented the detection of signals in response to islet-specific peptide stimulation.

Furthermore, we explored the use of tetramer technology to analyse CD8+ T cells specific for NRP-V7 (KYNKANVFL) peptide [38,39]. The percentages of tetramer-positive CD8+ T cells were relatively low in normoglycaemic NOD mice (0·02–0·06%) and were only slightly higher in mice that became diabetic (0·38–0·80%). The effects of therapy were difficult to interpret due to the treatment with anti-CD8 component of the therapy, which resulted in partial depletion of the CD8+ T cell population (data not shown) and thus difficulty in relative quantitation.

In the treated NOD mice with advanced diabetes we observed that the blood glucose levels showed an undulating pattern in the first 20–30 days, which corresponds with the half-life of the antibodies in vivo[7,40], but climb continuously thereafter. This suggests the need for a ‘rescue’ therapy, whereas an additional dose of the antibodies after day 20 could improve the glucose profiles and survival.

Importantly, the level of islet damage must be taken into consideration when discussing the possibility of restoring normoglycaemia. It is estimated that hyperglycaemia occurs when >80% of the islets are destroyed [41]. Therefore, we may be able to slow down islet destruction without reversing hyperglycaemia, due to the simple fact that the number of islets still available is too low to produce sufficient quantities of insulin. In the clinic, this might manifest as a successful therapy which stabilizes inflammation, prevents further loss of β cell function and allows the patient to control glucose homeostasis with minimal doses of exogenous insulin. The Diabetes Care and Complication (DCCT) studies suggest that patients with such a profile benefit from a reduced progression to complications [42].

We observed an interesting effect of the antibody combination therapy on invasion of the inflammatory infiltrate into the substance of an islet. While both treated and control groups of NOD mice showed a large proportion of the islets affected by insulitis, there was an important qualitative difference in the integrity of the islets between the treated and the control groups. Unlike those in the untreated NOD mice, including the normoglycaemic 16-week-old controls, the islets of the treated group appeared to be protected from intra-islet infiltration. This has been observed in other studies and has been referred to as ‘respectful insulitis’[43]. The islets are surrounded by a peri-islet basal membrane thought to play a role in protecting islets from invasion by inflammatory cells [44]. It is likely that only primed, effector T cells acquire the phenotype that enables them to cross this peri-islet barrier. Thus T cell priming, probably occurring in the local lymph nodes, could the turning-point in diabetes pathology, resulting in a change from non-invasive peri-insultis to invasive, destructive insulitis. In a transplant model we have shown previously that this antibody combination therapy, which targets activated, primed T cells, can achieve the removal of the primed T cells and reversal of the immune system of the transplanted recipient to a naive, non-sensitized state [6]. It is feasible that similar effects occur in the diabetic NOD mice in which the putative removal of primed T cells results in reversal to a prediabetic naive state.

Although diabetic mice were assigned randomly to control or treated groups and experimental groups were performed in parallel, our control untreated groups showed a higher starting glucose compared to treated groups (Figs 1a,b and 3a,b). This could have had an influence on the outcomes, thus emphasizing the effects of the therapy. However, analysis of the extensive published data strongly supports our findings that diabetic NOD mice with blood glucose above 13·9 mmol/l (250 mg/dl) almost never revert spontaneously to a normoglycaemic state [25,45,46]. This was evident in a study where none of the 150 untreated diabetic mice with recent-onset diabetes became normoglycaemic despite the supporting use of insulin [45]. Similarly, Fiorina et al. reported that none of the newly hyperglycaemic NOD mice in their control untreated group (n = 10) reverted to normoglycaemia [25]. Notably, the levels of blood glucose in NOD mice at the time of hyperglycaemia/diabetes diagnosis observed in this study was highly variable, and similar to our data [25]. Furthermore, the initial level of blood glucose did not correlate with the ability of subsequent therapy to restore normoglycaemia [25].

Histological analyses (islet infiltration scores) were performed using the normoglycaemic controls at younger age, when islet pathology is much less pronounced than in the older, hyperglycaemic treatment groups. Thus, the controls are set in such a way that therapy needs to achieve a striking improvement in the histology of islets in order to allow for the detection of therapeutic effects. By taking both sets of experiments into account, we provide strong evidence for the therapeutic potential of this antibody combination in both early and advanced stages of the diabetes in NOD mice.

Regulatory T cells (Tregs) [forkhead box protein 3 (FoxP3+) cells] are believed to play a protective role in autoimmune diabetes [47,48], thus we have stained pancreatic sections and examined their numbers in islets (approximately 10 islets per mouse were chosen at random for histological analysis, four to seven mice per group). Tregs in the islets of normoglycaemic NOD mice represented a small proportion of the total CD3+ T cell infiltrate (typically 1–3 FoxP3+ cells per islet, approximately 4% of the total CD3+ cells). Following the onset of diabetes, Treg numbers in islets showed a trend for decrease (typically <1 FoxP3+ cell per islet). This trend was reversed with therapy, which appears to restore Treg numbers close to prediabetic levels (1–3 FoxP3+ cell per islet). However, the absolute numbers of FoxP3+ cells in the islets of NOD mice were too low for conclusive interpretation of the data.

We have attempted to examine the numbers of effector T cells in the islets; however, due to interference by the antibody therapy, we were unable to determine the proportion of CD25+FoxP3 T cells present. The expression of the CD69 activation marker is transient, and thus not suitable for the detection of effector T cells in the islets (data not shown). It is further possible that the key functional change is in the ability of the effector T cells to be regulated [49], as has also been suggested in man [50].

In conclusion, we have shown that an antibody combination therapy consisting of CD8-, CD25- and CD70-specific antibodies can reverse extant autoimmune diabetes in NOD mice. This study provides an insight into the potential of using synergistic antibody combinations to treat autoimmune diabetes. The availability of clinically approved antibodies, such as the anti-CD25 currently used in transplantation, could greatly expedite the translation of our findings into the clinic.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

S.J. is the recipient of a JDRF grant 5-2007-330. The authors acknowledge the support of the MRC Centre for Transplantation. T.A. is the recipient of a Croatian Science Foundation PhD studentship stipend. T.A. acknowledges the support of the Alma Matris Alumni Croaticae UK. The authors are grateful to Angela Giorgini for her kind help in designing experiments. The authors would also like to thank Drs Ksenija Marjanovic and Marko Ivanovic (Department of Pathology, Clinical Hospital Centre, Osijek, Croatia), who kindly provided equipment and offered their expertise in histological analysis and islet scoring.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References