Islet-infiltrating B-Cells in Nonobese Diabetic Mice Predominantly Target Nervous System Elements

Jorge Carrillo, Maria Carmen Puertas, Aurora Alba, Rosa Maria Ampudia, Xavier Pastor, Raquel Planas, Nadal Riutort, Nuria Alonso, Ricardo Pujol-

Borrell, Pere Santamaria, Marta Vives-Pi, and Joan Verdaguer

Presented by Lei Lin and Aron Airall

Structure of the Presentation

• Background

• Main Question

• Methods

• Figures and Tables

• Conclusion

Background• NOD mice develop diabetes similar to human type 1 diabetes

• 8.3-NOD mice = NOD mice whose B cells expressing a transgenic diabetogenic TCR (from pathogenic CD8 T cells that infiltrate islets in pre-diabetic NOD mice) specific for beta cell. So, these mice develop accelerated form of autoimmune diabetes

• Type 1 diabetes is characterized by selective destruction of pancreatic beta cells by the patient’s own immunity

• Islet-infiltrating T-cells are major effectors of beta-cell damage in type 1 diabetes

• B cells also play a critical role in initiation and progression of type 1 diabetes. They capture beta-cell autoAgs via cell surface Igs and by presenting these Ags to autoreactive T cells

• Other islet cells may also be targets (autonomous nervous system) of the autoimmune response

Main Question of the Paper

• Question: What is the antigenic repertoire of islet-infiltrating B-cells in non-obese diabetic mice (NOD mice)

• Answer: predominant B cell response against nervous system elements

Methods• diabetes-prone (NOD) and diabetes resistant (NOR) mice

• NOD RAG-2-/- develop no diabetes or insulitis.

• 2 models (NOD X NOR) F1 & (8.3-NOD X NOR) F1

• Pancreatic islets isolated cultured in media

• 12 hrs later, islet-infiltrating mononuclear cells migrating into the culture media fused with the NS-1 myeloma cell line

• Fused cell lines cultured in HAT media for 2 weeks

• Growing hybridomas screened for Ab production

• Immunofluorescence staining for Ab

• 33 Fusions, obtained 352 hybridomas, 74 were Ab-secreting

Table 1

Fig.1 A & Table 2

• a-j RAG-2 -/- mice

(no diabetes or insulitis)

• k-m Hep- 2 cell line n-o Crithidi luciliae

(Presence of mAbs to nuclear Ags and native DNA were confirmed by staining Hep-2 and Crithidia luciliae )

• NOD: 54 hybridomas, 12 of which Ab-secreting, 5 of which recognized pancreatic tissue components other than beta-cells, 2 of which (d,e,f, exocrine and i,extracellular connective tissue) produced mAbs reacting to exocrine tissue/ extracellular matrix. 3 of the 5 produce Abs specific for the same pancreatic tissue elements. These elements react with the anti-peripherin, anti-neurofilament 200, and/or anti-GFAP Abs. Peripherin, neurofilament 200, and GFAP are nervous system components=> specificity for nervous system.

• a) intraislet; b) neuroendocrine; c) mixed; d-f) exocrine; g & h) nervous system; i) extracellular connective tissue; j) tissue -ve control; k &n) nuclear anti-DNA; l) nuclear dots staining the nucleus; m & o) -ve control of Hep-2 and Crithidia luciliae.

• Secondary Abs = Goat anti-mouse IgM+G+A labeled with FITC

Fig. 1A & Table 2

• 8.3 NOD: 151 hybridomas, 18 Ab-secreting, 12 secreted Abs specific for pancreatic tissues. 4 of the 12 produced mAbs specific for an islet Ag that colocalize with peripherin, neurofilament 200, and/ or GFAP

=> suggesting reactivity against pancreatic nervous system.

• Remaining 7 of the 12 produced mAbs specific for exocrine tissue, cell nucleus, or extracellular connective tissue.

Ag specificity of B cells recruited to pancreatic tissue in non-diabetes-prone mice is also for nervous system

components of the pancreas

• In (NOD X NOR) F1 & (8.3-NOD X NOR) F1 mice, the largest proportion of Ab-secreting hybridomas produced mAbs specific for nervous system Ags.

• In (NOD X NOR) F1 mice, 11 of 17 Ab-secreting hybridomas specific for pancreatic tissues, 9 of the 11 produced Abs that colocalized with peripherin, neurofilament 200, and/or GFAP, only 1 recognized pancreatic islets.

• In & (8.3-NOD X NOR) F1 mice, 24 of 27 Ab-secreting hybridomas produced mABs specific for pancreatic tissues, 12 of the 24 recognized nervous system elements, only 3 recognized pancreatic islets.

• => indicated that predominant anti-nervous tissue element B cell response in NOD mice also occurs in mice in which islet beta cell destruction is limited.

• => predominant recruitment of nervous tissue specific B cells to pancreatic islets in diabetogenesis is dissociated from islet beta cell destruction

Fig. 1 B & C

Abs from the hybridomas are specific for insulin, somatostatin, and glucagon

A - intraislet

B - neuroendocrine

C - mixed (islet)

H173- colocalize with Abs specific for GFAP, neurofilament, and peripherin

2 nervous system staining patterns:

H184- colocalize with Abs specific for GFAP, neurofilament, and peripherin

H122- colocalize only partially with Abs specific for GFAP

Table 2

-52 Ab secreting hybridomas tested, 20 produced IgM mAb, 3 IgG1, 9 IgG2c, 19 IgG2b, and 1 IgA.

-IgM is the prevailing isotype for hybridomas restricted to exocrine, nuclear, and extracellular connective tissues => they originated from B cells producing polyreactive natural Abs.

-Most islet and nerve specific hybridomas secreted IgG2b, IgG2c, and IgG1 Abs => they derived from precursors that had undergone class switch recombination

=> Most islet-associated, Ab-secreting hybridomas specific for pancreatic islets and nervous system derive from B cells that have undergone Ig class switch recombination

Antigenic specificity of B cells penetrating the islet

is not restricted to the pancreas

Results

• Table 3 demonstrated that antigenic specificity of B cells penetrating the islet is not restricted to the pancreas.

• Antibody-secreting hybridomas negative for pancreas tissue was tested vs other NOD.RAG-2-/- mice organs.

• A total of 7/22 (31.8%) pancreas were negative to antibody secreting hybridomas directed against several other moue organs.

• In particular 3/7 (42.9%) reacted with brain and other tissues, whereas the 2/7 (28.6%) were exclusively nervous system elements (Carrillo et al 2004).

Results

Results• Figure 2 gives further evidence that antigenic specificity

of B cells penetrating the islet was not restricted to the pancreas.

• Cryosections of NOD.RAG-2-/- mice parotid, mixed salivary, and bronchial glands, thyroid, brain, stomach, and kidney were stained using a monoclonal antibody delegate for each sectioning pattern.

• Monoclonal antibodies with exocrine or reticular connective tissue staining properties reacted with all tested tissues.

• Similarly, monoclonal antibodies displayed nervous system staining properties to most tissues analyzed indicating that they were directed vs endogenous antigens of the nervous system.

• In particular, monoclonal antibodies with β cell specificity cross reacted with only a few stomach and kidney cells completely (Carrillo et al 2004).

Results

Results

• Figure 3 demonstrated that cross reactivity of monoclonal antibodies to similar antigens in rats and humans, indicating that the intrapancreatic B-cell response that takes place during initiation and/or progression of type 1 diabetes is directed against antigenic determinants conserved across species (Carrillo et al 2004).

Discussion• In mice, Th-1 humoral responses were dominated by

antibodies IgG3, IgG2a, and possibly IgG2c isotypes. • Alternatively Th2 responses were dominated by IgG1 and

IgE antibodies, whereas Th3 responses were dominated by IgG2b and IgA antibodies.

• There was also evidence of autoreactive B-cells, Th1 (IgG2c), Th2 (IgG1), and Th3 (IgG2b) complexes penetrating islet reticular connective tissue.

• These results were consistent with the complex cytokine profiles of islet-associated T-cells in 8.3-NOD, 8.3-NOR, and 8.3-F1 mice, indicating an active B-cell response vs pancreatic nervous system antigens at the earliest stages of diabetogenesis (i.e., in diabetes resistant mice).

• It is beyond the scope of this paper as to whether the intraislet immune response against pancreatic nervous tissue elements also occurs in human type 1 diabetic patients or whether this response is key to diabetogenesis (Carrillo et al 2004).

Discussion• It is speculative to think that their study was not a peculiarity of

NOD mice. Moreover, an early loss of islet sympathetic nerves during the course of diabetes has also been observed in BioBreeder diabetic rats (26).

• To discover whether islet sympathetic nerves are damaged during the autoimmune destruction of islet B-cells, sections of pancreas from BioBreeder (BB) diabetic rats were immunostained using antibodies against vesicular monoamine transporter 2 (VMAT2), a marker of sympathetic nerve terminals.

• They found a marked decrease in the VMAT2-positive fiber area in the islets of BB rats that had been diabetic for only 1-2 weeks compared with their nondiabetic controls.

• In contrast, there was no significant decrease in the VMAT2-positive fiber area in the exocrine pancreas in these early diabetic BB rats.

• Furthermore, streptozotocin-diabetic rats showed no decrease in VMAT2-positive fiber area in their islets compared with controls.

Discussion• The classical diabetic autonomic neuropathy (DAN) that

eventually occurs in the heart was not present in BB diabetic rats at this early stage as evidenced by normal cardiac VMAT2 immunostaining and normal cardiac norepinephrine content.

• Also, in contrast to DAN, this islet neuropathy did not worsen with duration of diabetes. These data provided evidence of unrecognized early sympathetic islet neuropathy.

• Because eSIN occurs selectively in the islet, rapid in onset, and associated with autoimmune but not chemically induced diabetes, it is distinct from DAN in location, time course, and mechanism.

• Nevertheless, cross reactivity of these murine antibodies for both rat and human pancreas tissue indicates the existence of conserved antigenic determinants across species.

• Presently, the molecular nature of these antigenic protein determinants is unknown (data not shown) (26).

Clinical Implications

• Sera of 94 type1 diabetic patients were tested for the presence of complement-fixing sympathetic ganglia (CF-SG) antibodies. In cross-sectional analysis (0-43 yr), 22% had detectable CF-SG antibodies. • Participants at high risk for T1D were also studied. Group 1 (4-64 yr) islet cell antibody-positive (ICA+) prediabetic, 10/19 (53%) were CF-SG+; group 2 (6-14 yr) ICA-prediabetic (first-degree relatives of T1D with either transient hyperglycemia, impaired OGT, and/or first-phase insulin release after I.V GT testing), 4/9 (44%) were CF-SG+ (2/4 ICA- CF-SG+ subjects have progressed to type 1 diabetes); group 3 (1.5-43 yr) ICA+ T1D (< or = to 1 yr duration) 6/10 (60%) were CF-SG+; and group 4 (8-59 yr) ICA- T1D (< or = to 1 yr duration), 2/11 (18%) were CF-SG+.

Clinical Implications• Postural blood pressure and simultaneous CF-SG antibody measurements were performed in 28 T1D subjects. • The drop in systolic blood pressure was greater in the CF-SG+ subjects (P less than .05), and the frequency of CF-SG was greater in the mean to -2SD group (P less than .03) when data were analyzed within mean +/- 2SD of the normal blood pressure response (45).

Clinical Implications

• There is also evidence that the immune system may play a role in the pathogenesis of autonomic neuropathy in T1D.

• The presence of autoantibodies to sympathetic and parasympathetic nervous structures and their correlation with other typical autoantibodies in well-characterised diabetic populations, with or without diabetic neuropathy, and normal subjects was investigated.

• Indirect immunofluorescent complement-fixation technique was used, with monkey adrenal gland, rabbit cervical ganglia and vagus nerve as substrates.

• Patients with symptomatic autonomic neuropathy 33% were positive for at least one autoantibody (20% anti-sympathetic ganglia, 10% anti-vagus nerve and 13% anti-adrenal medulla).

Clinical Implications• The frequency of having one or more antibodies to nervous

tissues and the prevalence of anti-cervical ganglia antibodies were significantly higher in the neuropathic patients than in the diabetic control subjects with disease of similar duration and in the normal subjects (p < 0.05).

• Patients without complications with diabetes of shorter duration 33% were positive for at least one autoantibody (13% anti-ganglia, 13% anti-vagus nerve and 13% anti-adrenal medulla). No correlation was found with other tissue autoantibodies, including islet cell antibodies.

• They concluded that nervous tissue autoantibodies are associated with symptomatic autonomic neuropathy. Anti-sympathetic ganglia and anti-vagus nerve antibodies seem to be more disease-specific.

• Therefore patients presenting with diabetes of shorter duration testing positive for these autoantibodies may represent pre-neuropathic patients (47).

Clinical Implications• Yet still another group evaluated the incidence of

autonomic nervous system autoantibodies (ANS) in nondiabetic family members of T1D diabetics.

• 24 families, including 45 nondiabetic parents and 53 nondiabetic siblings of a T1D proband were studied.

• 101 nondiabetic population control subjects were also studied.

• Stored sera from nondiabetic family members and control subjects were tested for complement-fixing (CF) adrenal medullary antibodies (CF-ADM), sympathetic ganglia antibodies (CF-SG), and vagus nerve antibodies (CF-V) by indirect immunofluorescence.

• HLA-DR3 and -DR4 typing was performed on 42 nondiabetic family members and 104 diabetic subjects.

Clinical Implications

• One or more CF-ANS were in 45 of 93 (40%) nondiabetic family members compared to 2/70 (2.8%) control subjects.

• CF-SG were in 28/92 (30%) family members compared to 0/101 control subjects (P = 0.0001). CF-V were in 25 of 95 (26%) family members compared to 0 of 76 control subjects (P = 0.0001).

• CF-ADM were in 10 of 83 (12%) family members compared to 2 of 70 (2.8%) control subjects (P = 0.056).

• There was no HLA-DR3 or HLA-DR4 association with ANS.

• Subclinical autonomic dysfunction was demonstrated in 3 of 4 family members with autoantibodies compared to 0 of 4 family members without autoantibodies (49).

Clinical Implications• To elucidate whether GAD-ab were associated with

diabetic autonomic neuropathy and/or complement fixing antibodies against sympathetic ganglia, adrenal medulla, and vagus nerve, another group examined 133 diabetic patients (95 with T1D).

• GAD-ab were determined by a radioligand binding assay whereas sympathetic ganglia antibodies, adrenal medulla antibodies, vagus nerve, and ICA were evaluated by indirect immunofluorescence assays.

• Autonomic nerve function was evaluated by objective tests (heart rate reactions to deep breathing and to tilt). Out of 133 patients, GAD-ab was detected in 36 patients, all of whom had type 1 diabetes. The frequency of GAD-ab was similar (38%) in T1D with and without signs of autonomic neuropathy (21/55 vs 15/40).

Clinical Implications

• Also, there were no significant associations between GAD-ab and autonomic nerve antibodies; GAD-ab were detected in 9/21 (43%) of patients with and in 27/112 (24%) of patients without sympathetic ganglia antibodies, in 5/15 (33%) of patients with and 31/118 (26%) without adrenal medulla antibodies, and in 5/15 (33%) with and 31/118 (26%) of patients without vagus nerve antibodies.

• The frequency of ICA, however, was significantly increased in patients with sympathetic ganglia antibodies compared with those without sympathetic ganglia antibodies (10/21 [48%] vs 21/112 [19%]; p < 0.01).

• In conclusion, GAD-ab were neither associated with disturbed autonomic nerve function nor with antibodies against autonomic nerve structures (48).

Conclusion

• Finally, in conclusion we describe a set of antibody-secreting hybridomas from islet-infiltrating B-cells of diabetes prone and resistant mice.

• Our presentation showed that most antibody body secreting, islet secreting B cells recognized antigens expressed by nervous cellular elements of the pancreas and provide existence of an active lymphocyte response against these cellular elements early in diabetogenesis.

• This work gives substantial evidence that nervous system elements of islets of Langerhans are important targets of the diabetogenic autoimmune response (Carrillo et al 2004).