2880 Diabetes Volume 64, August 2015

Anne Jörns,1,2 Ümüs Gül Ertekin,1 Tanja Arndt,1 Taivankhuu Terbish,1 Dirk Wedekind,3 and Sigurd Lenzen1

TNF-a Therapy in Combination With the T-Cell–Specific Antibody Anti-TCR Reverses the Diabetic Metabolic State in the LEW.1AR1-iddm Rat

Diabetes 2015;64:2880–2891 | DOI: 10.2337/db14-1866

Anti–tumor necrosis factor-a (TNF-a) therapy (5 mg/kg b-cell death (1–3). The infiltrate in the pancreatic islets is body weight), alone or combined with the T-cell–specific composed of different immune cell types, especially macro- antibody anti–T-cell receptor (TCR) (0.5 mg/kg body phages and T cells (2–4). Upon activation, the immune cells weight), was performed over 5 days immediately after release proinflammatory cytokines and other cytotoxic disease manifestation to reverse the diabetic metabolic mediators, causing b-cell apoptosis (3,5). state in the LEW.1AR1-iddm rat, an animal model of hu- Effective prevention strategies require a combination man . Only combination therapy starting therapy (6–8) to target the proinflammatory cytokines pro- at blood glucose concentrations below 15 mmol/L re- duced in the different immune cell types. Of crucial impor- stored normoglycemia and normalized C-peptide. In- tance is in this context the proinflammatory cytokine tumor b creased -cell proliferation and reduced apoptosis led necrosis factor-a (TNF-a). It is expressed in all immune cell to a restoration of b-cell mass along with an immune types infiltrating the pancreatic islets in patients with T1D ISLET STUDIES cell infiltration–free pancreas 60 days after the end as well as in the spontaneous mouse (NOD mouse) and rat of therapy. This combination of two , anti- (BB, Komeda, and LEW.1AR1-iddm rats) models (3,9). TCR/CD3, as a cornerstone compound in anti–T-cell Cumulative evidence from studies in a variety of other therapy, and anti–TNF-a, as the most prominent and a autoimmune diseases supports the contention that the effective therapeutic antibody in suppressing TNF- fl action in many autoimmune diseases, was able to re- in ammatory process in the affected organs always goes – – verse the diabetic metabolic state. With increasing along with a high TNF-a expression (9 13). Anti TNF-a blood glucose concentrations during the disease pro- therapy is therefore an established therapeutic principle – fl gression, however, the proapoptotic pressure on the (14 16) in rheumatoid arthritis, in ammatory bowel dis- residual b-cell mass increased, ultimately reaching eases, lupus erythematosus, Sjögren syndrome, psoriasis, a point where the reservoir of the surviving b-cells was and Hashimoto thyroiditis (10,15,17). insufficient to allow a restoration of normal b-cell mass Anti–TNF-a monotherapy in T1D is ineffective (18–21) through regeneration. The present results may open and may even aggravate the disease (22,23). It is probably a therapeutic window for reversal of diabetic hyper- not surprising in an autoimmune disease such as T1D, in glycemia in patients, worthwhile of being tested in which the pancreatic b-cells are so extremely vulnerable clinical trials. (24), that these cells not only become dysfunctional but are also quickly destroyed. As has been documented in diabetic LEW.1AR1-iddm Type 1 diabetes (T1D) is an immune cell–mediated disease (IDDM) rats, as well as in pancreases from patients with that causes insulin dependence due to selective pancreatic T1D (3), the islet-infiltrating immune cells produce and

1Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany This article contains Supplementary Data online at http://diabetes 2Centre for Anatomy, Hannover Medical School, Hannover, Germany .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-1866/-/DC1. 3 Institute for Laboratory Animal Science, Hannover Medical School, Hannover, A.J. and Ü.G.E. contributed equally to this work. Germany © 2015 by the American Diabetes Association. Readers may use this article as Corresponding author: Sigurd Lenzen, [email protected]. long as the work is properly cited, the use is educational and not for profit, and Received 9 December 2014 and accepted 11 March 2015. the work is not altered. diabetes.diabetesjournals.org Jörns and Associates 2881 release, along with (IL)-1b, TNF-a as the dom- previously described (36). To identify changes in the inating proinflammatory cytokine (3,5,25). pancreatic islets, one biopsy was collected on the day Combination therapies, at variance from monothera- of T1D manifestation, immediately before the start of pies, have shown promising results in T1D (25–29) therapy, and another at the end of therapy; the remaining and other autoimmune diseases (11,14,15). We there- pancreas, comprising head and tail, pancreas-draining fore investigated in the IDDM rat, an animal model of lymph nodes, and serum, were collected 60 days after humanT1D,whichmirrorsthe human T1D situation the end of therapy (25). Tissue specimens were fixed for most closely (3,5,30,31), whether a combination therapy microscopic analyses (25,36). Blood glucose concentrations of anti–TNF-a, which suppresses TNF-a release from all were determined daily (Glucometer Elite; Bayer, Leverkusen, immune cells, with anti–T-cell receptor (TCR), which Germany). Serum C-peptide was analyzed with a rat-specific suppresses proinflammatory cytokine release from ac- ELISA (Mercodia, Uppsala, Sweden) and serum cytokine pro- tivated T cells, can be successful. Anti-TCR is an anti- tein concentrations with a multiplex immunoassay kit (Bio- body against T cells that binds to an epitope of the Rad, Munich, Germany) (25,36). a/b-chains of the TCR (32). It is comparable in action Morphological Analyses to anti-CD3, which binds to the ´-chain of the CD3 Serial sections were stained with the avidin-biotin com- molecule (33). Both antibodies bind noncovalently to plex method or the double-immunofluorescence tech- the TCR/CD3 complex and modulate T cells by affect- nique with primary antibodies for b-cells and immune ing the trans-signaling pathway (32,33). Anti-TCR is cells (3,36). The antibodies against TNF-a and T cells used in rats because anti-CD3 is available for mice recognized epitopes other than those targeted by the (33) and humans (33,34) but not for rats (25,35). We antibodies used for therapy. b-Cell apoptosis was quan- show in this study that this combination therapy tified by TUNEL (Roche, Mannheim, Germany), as well scheme is capable of successfully reversing the diabetic as proliferation by a double staining with Ki67 antibody metabolic state. (COOH-terminal; Acris, Herford, Germany) and insulin – RESEARCH DESIGN AND METHODS (D3E7; AbD Serotec). At each time point, 25 40 pan- creatic islets were studied (25,36). A minimum of 1,000 Animals b-cells were counted in the proliferation and apoptosis Congenic rats (for details see http://www.mh-hannover analyses (25,36). Islet infiltration was scored as de- .de/3642.html) were bred and maintained as described scribed (25). The b-cell mass, identified by insulin and (25,30). Experimental procedures were approved by the GLUT2 staining on sequential sections, was calculated District Government of Hannover (LAVES, nos. 33-42502- by the ratio of the b-cell area (mm2)tothewholepan- 05/958 and 509.6-42502-03/684). creatic area, determined four times in distances of 100 Experimental Groups mm each and multiplied by the pancreas weight. Anal- Four experimental groups were studied. Group 1 (n =5) yses were performed using an Olympus BX61 micro- comprised healthy, normoglycemic IDDM rats without scope (25,36). therapy; group 2 (n = 12) comprised acutely diabetic In Situ RT-PCR IDDM rats treated for 5 consecutive days with rat- Pancreatic sections from all experimental groups were specificanti–TNF-a (5 mg/kg body weight [bw] i.v.; pro- placed on three-chamber slides. The in situ RT-PCR vided by Janssen Research & Development, Spring analysis was performed on a special thermal cycler (MJ House,PA).Group3(n = 12) comprised diabetic rats Research, Waltham, MA) (25,36). Primer sequences are treated for 5 consecutive days with anti–TNF-a and in provided in Supplementary Table 2. addition with anti-TCR (0.5 mg/kg bw i.v.) (R73; AbD Serotec, Munich, Germany). Group 4 (n =5)comprised Real-time RT-PCR untreated diabetic IDDM rats. All therapies were started The quality of the mRNA isolated from pancreas- within 1 day after T1D onset at blood glucose concen- draining lymph nodes was controlled with the Experion trations .10mmol/L.Animalsingroups1and4re- electrophoresis system (Bio-Rad). mRNA was quanti- ceived 0.9% NaCl solution. Anti-TCR causes slight fied in real-time RT-PCR reactions performed with the lymphocyte reduction in the periphery (,20%), which DNA Engine Opticon fluorescence detection system disappears within 1 week after cessation of therapy (MJ Research) (36). Primer sequences are provided in (25). Anti–TNF-a was without effect and also did not Supplementary Table 3. further reduce the lymphocyte count in combination Statistics therapy with anti-TCR (data not shown). The CD4-to- Results are presented as mean values 6 SEM. Compari- CD8 T-cell ratios were not affected by any of the treat- sons among the different therapy groups and the normo- ments (Supplementary Table 1). glycemic or diabetic control rats were analyzed with the Pancreatic Biopsies and Tissue Processing Mann-Whitney U test and ANOVA, and correlation coef- Biopsies of tissue (30 mg) were obtained from the pan- ficients were calculated according to Pearson with the creas tail of each animal under isoflurane anesthesia as Prism 5 program (GraphPad Inc., San Diego, CA). 2882 TNF-a/Anti-TCR Therapy Reverses Diabetic State Diabetes Volume 64, August 2015

RESULTS both parameters in the diabetic rats. At the day of T1D Metabolic Effects of Therapy With Anti–TNF-a Alone or manifestation, the rats that were responsive to the com- Combined With Anti-TCR bination therapy (8 of 12) showed significantly more than Anti–TNF-a (5 mg/kg bw, for 5 days) was administered to a 4-fold increase of the proliferation rate (Fig. 3A), and IDDM rats within 1 day after T1D manifestation (blood the apoptosis rate analyzed by TUNEL increased more glucose .10 mmol/L), alone or combined with anti-TCR than 15-fold (Fig. 3B) compared with normoglycemic con- (0.5 mg/kg bw, for 5 days). Combination therapy resulted trol rats. In the nonresponding rats (4 of 12), the pro- in an instantaneous and sustained return to normoglycemia liferation rate also increased 4-fold (Fig. 3A), but the within 1 day after the start of therapy in 8 of 12 rats (Fig. apoptosis rate increased even more than 20-fold (Fig. 3B). 1A), defined as blood glucose ,10 mmol/L, compared with The 12 rats on anti–TNF-a monotherapy, all nonres- the nonresponding rats. Rats (n = 12) with anti–TNF-a ponding irrespective of the blood glucose concentration at therapy alone after T1D manifestation remained perma- start of therapy (Fig. 2B), showed a 5-fold increase of the nently hyperglycemic (.15.0 mmol/L) (Fig. 1A), and se- proliferation rate (Fig. 3A) at the day of T1D manifesta- rum C-peptide levels were not preserved (Fig. 1C). In the tion and a 17-fold increase of the apoptosis rate (Fig. 3B). animals responding to combination therapy, normoglyce- Immediately after the end of combination therapy, the mia achieved at the end of the 5-day therapy period was proliferation rate was still as high as before the start of further maintained for another 60 days in the absence of therapy in the responding rats (Fig. 3A). The apoptosis therapy (Fig. 1A). Thereby, combination therapy achieved rate was strongly reduced, being close to that of healthy glucose levels identical to those of healthy, normoglyce- control animals (Fig. 3B). mic control rats (5.4 6 0.2 vs. 5.1 6 0.1 mmol/L). Rats In the nonresponding rats, proliferation and apoptosis (n = 4) not responding to combination therapy and di- rates were very low after anti–TNF-a therapy and after abetic rats without therapy showed no return to normo- combination therapy due to the nearly complete b-cell glycemia and needed insulin supplementation soon after loss in the islets (Fig. 3A and B). the start of therapy (Fig. 1A and B). Healthy control rats At 60 days after the end of therapy, rats successfully stayed normoglycemic during the entire observation pe- treated with the combination therapy showed virtually riod (Fig. 1B). normal proliferation and apoptosis rates, as in healthy In the successfully treated animals with the combina- controls (Fig. 3A and B). All b-cells were lost after anti– tion therapy, serum C-peptide concentrations increased TNF-a monotherapy (Fig. 3A and B) and after combina- (Fig. 1C). At the end of therapy and more so 60 days later, tion therapy in the nonresponding rats (Fig. 3A and B). C-peptide was significantly (P , 0.05) higher with com- Changes in Proliferation-to-Apoptosis Ratios in b-Cells bination therapy than with anti–TNF-a monotherapy. Calculation of the proliferation-to-apoptosis ratios This continuous increase reached about two-thirds of con- revealed an increase to values in the range of the healthy trol values (726 6 79 vs. 1023 6 41 pmol/L) at the end of control rats only in rats responding to therapy, but the the observation period (Fig. 1C). The untreated diabetic increase was persistent only after combination therapy rats had C-peptide concentrations ,20% at the time of but not anti-TCR monotherapy. Anti–TNF-a monother- T1D manifestation (Fig. 1C). C-peptide remained low af- apy did not even cause a transient ratio increase (Supple- ter anti–TNF-a monotherapy and in animals nonrespon- mentary Table 4). sive to combination therapy (Fig. 1C). A detailed analysis revealed interesting results. Six Infiltration Score rats in the monotherapy group had blood glucose values On the day of T1D manifestation, before the start of at the start of therapy between 10.0 and 15.0 mmol/L therapy, the infiltration score of the islets was .3 (Fig. (12.5 6 0.5 mmol/L), and the other six values were 3C). The score was reduced to values of ;2 at the end of above 15.0 mmol/L (16.7 6 0.4 mmol/L) (Fig. 2A). But combination therapy for the responding and nonrespond- in none of the 12 rats did monotherapy restore b-cell ing animals (Fig. 3C). At 60 days after the end of combi- mass. In the two subgroups with combination therapy, nation therapy, the infiltration score in the pancreases thebloodglucoseconcentrationswerealsosignificantly with the regenerated b-cells was reduced to ,0.1, with (P , 0.05) different. In all rats that responded to com- only single nonactivated macrophages being left in the bination therapy, blood glucose values were below responding animals (Fig. 3C). In the nonresponding 15 mmol/L (11.4 6 0.3 mmol/L; n =8)butwereabove rats, islet infiltration was absent after anti–TNF-a as 15 mmol/L in all nonresponding rats (16.4 6 0.4 mmol/L; well as after combination therapy due to the complete n =4)(Fig.2B). loss of b-cells (Fig. 3C).

Morphometric Quantification of Therapy Effects on b-Cell Mass b fi -Cells and Pancreatic Islet In ltration On the day of T1D manifestation, before the start of Changes of Proliferation and Apoptosis Rates in b-Cells therapy, the b-cell mass of the pancreases in all rats was The analyses of the percentage changes, as presented in reduced to approximately one-third of the value in the Fig. 3A and B, revealed drastic increases of the rates for control rats (Fig. 3D). Immediately after the end of diabetes.diabetesjournals.org Jörns and Associates 2883

Figure 1—Effects of combination therapy with anti–TNF-a (5 mg/kg bw) for 5 days and anti-TCR (0.5 mg/kg bw) for 5 days and effects of monotherapy with anti–TNF-a (5 mg/kg bw) for 5 days on the metabolic profile of IDDM rats after T1D manifestation. A: Blood glucose concentration (mmol/L) changes are shown for rats responding to the combination therapy (n = 8) and for rats nonresponding to the monotherapy (n = 12). B: Blood glucose concentration (mmol/L) changes are shown for healthy (n = 5) rats and untreated diabetic (n =5) rats. The first dashed line at day 0 indicates the start of therapy, and the second dashed line at day 5 indicates the end of therapy. C: Serum C-peptide concentration changes (pmol/L) are shown for rats responding (n = 8) and not responding (n = 4) to the combination of anti–TNF-a and anti-TCR and for nonresponding rats with anti–TNF-a monotherapy (n = 12) at different times during the observation period. Data are mean values 6 SEM. *P < 0.05 for combination therapy compared with monotherapy. 2884 TNF-a/Anti-TCR Therapy Reverses Diabetic State Diabetes Volume 64, August 2015

glucose value ,15 mmol/L or .15 mmol/L at the start of therapy. However, the percentage of insulin-positive b-cells that were positive in addition for caspase 3 and perforin 1 was approximately three times higher when the blood glucose concentration at the start of therapy was .15 mmol/L (Table 1), indicating an ac- celerated b-cell death rate of the residual b-cells in the islets of the pancreases of diabetic rats with blood glucose .15 mmol/L. The correlation coefficients between an increase of blood glucose and an increase of caspase 3 and perforin 1 were r = 0.947 and r = 0.953, respectively (both P , 0.001), for the values (n = 24) in Table 1. Changes of the Immune Cell Infiltration Pattern in Pancreatic Islets in Response to Therapy On the day of T1D manifestation, a strong immune cell infiltration composed of 45% CD8 T cells, 5% CD4 T cells, 40% CD68 macrophages, and 10% of other immune cell types was observed in the islets in responding and nonresponding rats, with a concomitant loss of b-cells (Fig. 4A–D and Supplementary Fig. 1A and B, respectively) (3). Immediately after the end of combination therapy with anti–TNF-a and anti-TCR, immune cell infiltration was still substantially reduced by .50% in responding animals compared with the situation before the start of therapy (Fig. 4A–D). Immune cell infiltration was also reduced in the nonresponding rats; however, this went along with alossofb-cells (Supplementary Fig. 1A and B). At 60 days Figure 2—Relation between blood glucose concentration (mmol/L) after the end of therapy, islet immune cell infiltration was at the start of therapy and b-cell mass (mg) 60 days after end of – A entirely absent after combination therapy in the responding therapy in IDDM rats after therapy with anti TNF-a ( ) and combi- A–D nation therapy with anti–TNF-a and anti-TCR (B). b-Cell mass was rats (Fig. 4 ) in pancreases with a normal islet b-cell mass significantly (P < 0.05) higher in animals with starting blood glucose not different from that in healthy control animals. In pan- concentrations <15 mmol/L compared with those >15 mmol/L. In creases of nonresponding rats with islets lacking b-cells, only the two subgroups with combination therapy, the blood glucose occasional single nonactivated immune cells were observed concentrations were also significantly different. (Supplementary Fig. 1A and B). Gene Expression Changes in Activated Immune Cells in Pancreatic Islets During Therapy anti–TNF-a monotherapy, the pancreatic b-cell mass was Immune cells were activated in diabetic rats. This was reduced even more, to ,10%. But immediately after the documented by high gene expression levels of the end of successful combination therapy, the pancreatic proinflammatory cytokines, Tnf and Il1b (Fig. 5A and B), b-cell mass had more than doubled from the value before as well as of the CD8 marker, Gzmb, in the infiltrating therapy, reaching in the responding animals .80% of immune cells (Fig. 5C), and of the proapoptotic enzyme, the value in the healthy control rats (Fig. 3D). At 60 Casp3, in the b-cells (Fig. 5D) before start of therapy. At days after the end of combination therapy, the pancre- 60 days after the end of combination therapy, an expres- atic b-cell mass had attained normal values in these rats sion of these genes was virtually completely lost in the (Fig. 3D). In the nonresponding animals, b-cell mass noninfiltrated islets of the responding rats (Fig. 5A-D). In was further reduced strongly already during therapy, both groups nonresponding to therapy, a few activated and b-cells were completely lost 60 days thereafter immune cells were observed, but only in the perivascular (Fig. 3D). space, which was not surprising in view of the complete b C D Increased Rate of b-Cell Demise at High Blood -cell loss (Supplementary Fig. 1 and ). Glucose Concentrations Changes of Pro- and Anti-Inflammatory Cytokines and A deeper analysis of the situation immediately before the T-Cell Markers in the Circulation and in Pancreas- start of therapy revealed that the percentage of insulin- Draining Lymph Nodes During Therapy positive b-cells in the pancreases was comparably high In the animals responding to combination therapy, the in all therapy groups (Fig. 3D), irrespective of a blood serum protein concentrations of the proinflammatory diabetes.diabetesjournals.org Jörns and Associates 2885

Figure 3—Morphometric analyses of b-cells and immune cells in IDDM rats after successful and nonsuccessful combination therapy with anti–TNF-a and anti-TCR or after nonsuccessful therapy with anti–TNF-a alone after T1D manifestation. Changes in the rate of proliferation (A), apoptosis (B), islet infiltration scores (C), and pancreas b-cell mass (D). Rats were treated with anti–TNF-a alone or combined with anti- TCR, leading to a group of animals responding with a reversal of T1D or to a group of nonresponding animals with a sustained diabetic state compared with healthy control rats (n = 4) and untreated rats with T1D for 1 day (n = 4). Measurements were performed before therapy, at the end of therapy, and at 60 days after the end of therapy. Data are mean values 6 SEM. *P < 0.05 vs. healthy control rats, which showed no signs of immune cell infiltration. Numbers of pancreases analyzed as given in Fig. 1. Islet infiltration was graded according to the following score: stage 0, no immune cell infiltration (control); stage 1, immune cell infiltration restricted to the periphery; stage 2, weak invasive immune cell infiltration; stage 3, strong invasive immune cell infiltration; stage 4, complete b-cell loss without infiltration.

cytokines TNF-a and IL-1b were as low as in healthy combined with anti-TCR, the concentrations of the control animals (Fig. 6A and B). Both in animals non- anti-inflammatory cytokine IL-10 were significantly responding to monotherapy and combination therapy, (P , 0.01) increased, irrespective of therapy success TNF-a and IL-1b serum protein concentrations remained (Fig. 6F). significantly (P , 0.01) increased (Fig. 6A and B). The The same changes of the pro- and anti-inflammatory proinflammatory cytokine -g (IFN-g) was un- cytokines as well as of the CD8 T-cell markers granzyme B changed in all groups compared with the control groups and perforin 1 on the level of gene (Supplementary Table (Fig. 6C). The increased levels of the immune cell–activating 5) and protein (Supplementary Table 6) expression were cytokine IL-2 in diabetic rats decreased only in animals observed in the immune cells in the pancreas-draining responding to combination therapy with a significant lymph nodes 60 days after the end of therapy in the (P , 0.05) reduction compared with the animals responding and the nonresponding rats. nonresponsive to the therapies (Fig. 6D). Only after successful combination therapy was the protein con- DISCUSSION centration of the anti-inflammatory cytokine IL-4 in- This therapy combines two antibodies, anti-TCR directed creased, reaching levels close to those of the healthy against the TCR/CD3 complex, as a cornerstone com- rats (Fig. 6E). After anti–TNF-a therapy, alone and pound in anti–T-cell therapy, and anti–TNF-a, the most 2886 TNF-a/Anti-TCR Therapy Reverses Diabetic State Diabetes Volume 64, August 2015

Table 1—Number of double-positive b-cells expressing either insulin plus caspase 3 or insulin plus perforin 1 in pancreatic islets of IDDM rats immediately before start of therapy Nonresponding group Nonresponding group Responding group after Nonresponding group after therapy with after therapy therapy with anti–TNF-a after therapy with anti–TNF-a anti–TNF-a with anti–TNF-a and anti-TCR and anti-TCR (glucose ,15 mmol/L) (glucose .15 mmol/L) (glucose ,15 mmol/L) (glucose .15 mmol/L) Double-positive b-cells n =6 n =6 n =8 n =4 Insulin plus caspase 3 3.4 6 0.1 10.6 6 0.3* 3.1 6 0.1 10.1 6 0.1* Insulin plus perforin 1 3.7 6 0.2 11.4 6 0.3* 3.3 6 0.2 10.8 6 0.3* The percentages (%) of double-positive b-cells (for insulin plus caspase 3 and for insulin plus perforin 1, respectively) are depicted in relation to the total number of insulin-positive b-cells at the start of therapy. The quantitative analyses were performed in the pancreatic biopsy specimens (immediately after T1D manifestation and before the start of therapy) of all groups in the infiltrated islets. In the monotherapy group, 1,028 b-cells were counted in the nonresponding group (glucose ,15 mmol/L) and 1,021 were counted in the nonresponding group (glucose .15 mmol/L). In the combination therapy group, 1,173 b-cells were counted in the responding group (glucose ,15 mmol/L) and 1,007 were counted in the nonresponding group (glucose .15 mmol/L). Data are mean values 6 SEM. *P , 0.01 vs. the group with glucose ,15 mmol/L in the monotherapy and combination therapies, respectively. effective therapeutic antibody in many autoimmune dis- strategy in reversing diabetic hyperglycemia. This eases, based on considerations of the pathophysiological was documented by normalization of glycemia, serum understanding of the disease process of T1D in the endo- C-peptide, and b-cell mass. At the same time, immune crine pancreas (3). This combination provided therapy cell infiltration in the islets was completely abolished. success without inclusion of other compounds such as Monotherapy with anti–TNF-a was not successful immunomodulatory agents (i.e., anti–IL-1b, proinsulin, in the diabetic animals, confirming earlier studies IL-10, fingolimod) (25,28,37). (18,46). Anti-TCR monotherapy did also not yield The idea behind this therapy was that a combination of a sustained success under the same therapy regimen, antibodies is required to reverse autoimmunity in the as we reported in detail in a previous study in this rat T1D state, because none of the antibodies is able to model (25). Even the transient reduction of hypergly- target all proinflammatory cytokines in the different cemia in 3 of 12 animals was not sustained (25), a phe- immune cell types of the infiltrate. Therefore, mono- nomenon also observed in human studies with anti-CD3 therapies have proven not to be particularly successful (33,34) and confirmed in the current study by the com- (23,38–40) even with the most promising antibodies, parative calculations of the apoptosis-to-proliferation anti-CD3 in humans and mice and in analogy anti-TCR ratios under the different treatment regimens (Supple- in rats (25,27,28,41–44). We report here a successful mentary Table 4). combination therapy for T1D in the IDDM rat, an animal Interestingly, reversal of the T1D state was achieved model of human T1D (25), using two antibodies with only in those animals with a blood glucose concentration different target profiles. ,15 mmol/L, whereas the one-third of the T1D animals The proinflammatory cytokine TNF-a, expressed in the with a blood glucose concentration .15 mmol/L at the infiltrating immune cells, is known to be a key player in start of combination therapy remained diabetic (Fig. 2). the pathology of many autoimmune diseases, including This observation (Fig. 2) documents strikingly how impor- T1D (9,12,13). However, although a monotherapy with tant an early start of combination therapy is, as soon as anti–TNF-a is an established successful therapy in a num- possible after diagnosis of the disease, as also concluded ber of autoimmune diseases (15–17), anti–TNF-a is not in recent reviews (8,23). The explanation for this obser- effective in T1D treatment (18–23). Anti–TNF-a neutralizes vation is provided by the experiments reported in Table 1, TNF-a, produced by the immune cells infiltrating the target showing that the percentage of insulin-positive b-cells, organ during the inflammatory process (11,14,15,45), which were in addition also positive for caspase 3 and and the combination of anti–TNF-a with anti-TCR also for perforin 1, was approximately three times higher at prevents the release of IL-1b, the other critical proin- the start of therapy, when blood glucose concentrations flammatory cytokine for the disease process (3), from were .15 mmol/L, than at blood glucose concentrations the activated T cells (25). In addition, we found that suc- below this threshold (Table 1). This demonstrates that cessful combination therapy also suppressed other inflam- with increasing blood glucose concentrations, the percent- matory mediators, such as perforin 1 and granzyme B, in age of dying insulin-positive b-cells increases (47). This Tcells. correlation is highly significant. It also explains that at a starting blood glucose concentration .15 mmol/L, the Beneficial Effects of Combination Therapy number of the remaining healthy insulin-positive b-cells The results of the current study show that anti–TNF-a is insufficient to secure a regenerative capacity, allowing therapy combined with anti-TCR, started immediately af- the generation of a sufficient number of new b-cells to ter T1D manifestation in the rats (30), was an effective regain normoglycemia through replication (48,49). This diabetes.diabetesjournals.org Jörns and Associates 2887

Figure 4—Immune cell infiltration in pancreatic islets of IDDM rats after successful combination therapy with anti–TNF-a and anti-TCR. A–D: b-Cells (green) and immune cells (red) were examined in islets from animals successfully treated with the combination of anti–TNF-a and anti-TCR after T1D manifestation. The amount of b-cells and immune cells in the first biopsy specimen (collected before start of therapy) was representative for spontaneous disease development without therapy. The second biopsy was collected at the end of therapy. The results obtained in the first and second biopsy samples were compared with the immune cell–free islets in the same pancreases obtained after 60 days without therapy. Islets were immunostained for insulin (Ins) (green) and CD8 T cells (red) (A and C) or CD68 macrophages (red) and counterstained with DAPI (blue) (B and D). Erythrocytes were identified by yellow color through autofluorescence in the red and green channel. A, B at low and C, D at high magnification; numbers of pancreases analyzed as given in Fig. 1. 2888 TNF-a/Anti-TCR Therapy Reverses Diabetic State Diabetes Volume 64, August 2015

Cytokine Profile Changes as Biomarkers for Success of Combination Therapy The changes in different cytokines and chemokines in the circulation, as observed under therapy in the current study, mirrored the corresponding changes in the infiltrated islets and thus represent reliable biomarkers to monitor disease progression and therapeutic success in this T1D rat model. This could not be documented so farinpatientswithT1D(50).Inanexperimentalap- proach, such as the one used in the IDDM rat in the present and in previous studies for mono- and combina- tion therapies (25,36), pancreatic tissue could be directly accessed for b-cell analyses by biopsies. Restitution of normoglycemia and serum C-peptide induced by b-cell mass restoration in the pancreas correlated with a normal- ization of the cytokine and chemokine protein expression profile in serum and pancreas-draining lymph nodes. This was documented by a reduction of the increased levels of the proinflammatory cytokines TNF-a and IL-1b and the chemokine (C-C motif) ligand-2 (MCP-1) as well as the T-cell markers perforin 1 and granzyme B to the normal range typical for the healthy state along with an increase of the anti-inflammatory cytokine IL-4. This profile normalization in the circulation not only accompanied the loss of pancreatic islet infiltration by activated immune cells but also went along with a nor- malization of the cytokine and chemokine protein ex- pression profile in the pancreas-draining lymph nodes. Thus, changes in the protein expression profile in serum are reliable biomarkers for documentation of therapy success achieved in the endocrine pancreas and in the neighboring lymph nodes.

Conclusions The results can be summarized as follows:

1. A sufficient number of intact b-cells is required to allow a reversal of the diabetic state through proliferation of existing b-cells. 2. Therefore, an immediate start of therapy after T1D Figure 5—Immune cell activation detected by gene expression in diagnosis is necessary to secure therapy success. representative pancreatic islets of IDDM rats after successful com- fl bination therapy with anti–TNF-a and anti-TCR. Gene expression 3. The proin ammatory cytokines TNF-a and IL-1b, both levels (in situ RT-PCR) were determined for proinflammatory cyto- highly expressed in the activated immune cells of the kines Tnf (A), Il1b (B), the CD8 marker Gzmb (C), and the apoptotic islet infiltrate in the T1D pancreas, are targeted, marker Casp3 (D). Islets from rats that had returned to normogly- thereby securing therapy success of this combination. cemia after combination therapy with anti–TNF-a and anti-TCR were compared with islets from untreated acutely diabetic animals. 4. The results show convincingly that antibodies, which The dashed lines highlight the borders of the islets. Photomicro- are not effective alone, can reverse T1D in combination graphs are representative for 80 islets analyzed in each group. therapy successfully. Numbers of pancreases analyzed as given in Fig. 1. Combination therapy might thus be an attractive option inthefuturebecausemonotherapypreventionstudies have been ineffective not only in the IDDM rat model capacity of the b-cells to regenerate and normalize b-cell but also in patients with T1D (30,38–40), as advocated mass in rats with blood glucose ,15 mmol/L could be by several scientists recently (6–8). Because the potential recovered under combination therapy but not under to optimize therapy success through dose increases of monotherapy, convincingly emphasizing the crucial im- anti-CD3 and anti-TCR is limited due to T-cell reduction portance of the antibody combination for therapy success. in the circulation, which develops under this therapy, diabetes.diabetesjournals.org Jörns and Associates 2889

Figure 6—Cytokine pattern in serum of IDDM rats after successful combination therapy with anti–TNF-a and anti-TCR or after non- successful therapy with anti–TNF-a alone after T1D manifestation. Changes in protein concentrations of cytokines measured by multiplex analysis were examined: TNF-a (A), IL-1b (B), IFN-g (C), IL-2 (D), IL-4 (E), and IL-10 (F). Results after therapy with anti–TNF-a alone or combined with anti-TCR, subdivided by responding and nonresponding groups, were compared with those from healthy control rats and untreated acutely diabetic rats. Cytokine protein concentrations (pg/mL) are expressed as mean values 6 SEM. A–F: The dotted lines show changes in the proinflammatory and anti-inflammatory cytokines compared with the healthy control. *P < 0.01 vs. the untreated diabetic controls; #P and **P < 0.05 vs. untreated diabetic control rats. Number of animals as given in Fig. 1.

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