Suppression of insulitis and diabetes in B cell-deficient mice treated with streptozocin: B cells are essential for the TCR clonotype spreading of islet-infiltrating T cells

Shiori Kondo1, Isao Iwata1, Keizo Anzai4, Tomoyuki Akashi1, Shigeharu Wakana5, Kumiko Ohkubo4, Hitoshi Katsuta3, Junko Ono4, Takeshi Watanabe3, Yoshiyuki Niho1 and Seiho Nagafuchi1,2

1 Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences,
2 Department of Medical Technology, School of Health Sciences, and
3 Department of Molecular Immunology, Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
4 Department of Clinical Medicine, School of Medicine, Fukuoka University, Fukuoka 814-0133, Japan
5 Molecular Analysis Unit, Central Institute for Experimental Animals, Kawasaki 216-0001, Japan

Correspondence to: S. Nagafuchi or S. Kondo, Department of Medical Technology, School of Health Sciences, and the Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In order to clarify the role of B cells in the development of insulitis and diabetes, B cell-deficient (B) mice treated with streptozocin (STZ) were studied. The extent of insulitis and the cumulative incidence of diabetes were significantly suppressed in B mice (P < 0.0001), indicating that B cells are crucial for the progression of insulitis and diabetes. Accumulation of both CD4+ T cells and B cells was observed in islets of B+ mice, while CD4+ T cells but not B cells were found in B mice. A few CD8+ T cells and macrophages were detectable in both types of mice. The immunohistochemical study did not reveal any change in the subpopulations of infiltrating lymphocytes except for the absence of B cells in the B mice. TCR Vß gene repertoire usage of islet-infiltrating T cells was restricted to some extent in the B+ or B mice, but there was no significant difference between the B+ and B mice, suggesting that the initial islet-reactive T cell response can occur in the absence of B cells. In contrast, TCR clonotype spreading of islet-infiltrating T cells was significantly suppressed in B mice compared with B+ mice (P < 0.0001). These data suggest that initial priming of T cells is not impaired and TCR Vß repertoire usage is not limited by the lack of B cells, while B cells are important essentially for the spreading of islet-infiltrating clonal T cells in autoimmune diabetic mice induced with STZ.

Keywords: B Lymphocytes, diabetes, insulitis, single-stranded conformation polymorphism, TCR clonotype


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The loss of insulin-producing pancreatic ß cells due to autoimmunity leads to insulin-dependent diabetes mellitus (IDDM) (1). Several autoimmune animal models of IDDM, such as non-obese diabetic (NOD) mice (2), BioBreeding (BB) rats (3) and multiple low doses of streptozocin (STZ)-induced diabetes (4), have demonstrated pathologies analogous to human diseases, including islet-associated mononuclear cell infiltration (insulitis) and autoantibodies reactive to a number of ß cell components in the case of NOD mice (5).

STZ, originally isolated as an antibiotic from Streptomyces achromogenes, possesses not only anti-tumor, but also oncogenic and diabetogenic properties (6). A single high dose (e.g. 100mg/kg body wt) of STZ caused ß cell necrosis and diabetes within 24 h. On the other hand, Like and Rossini reported that multiple low-dose injections of STZ in susceptible mice induced pancreatic insulitis and delayed the onset of hyperglycemia (4). Insulitis and diabetes induced with multiple low doses of STZ is one of the excellent experimental animal models of autoimmune diabetes (6,7), and the AKR/J mouse is one of inbred mouse strains which can be induced for insulitis and diabetes (8).

Several lines of evidence have been suggested that T cells mediate the process of autoimmune insulitis: (i) many antibodies against the surface molecules of lymphocytes have been found to inhibit STZ-induced insulitis and diabetes, such as antibodies to CD3 (9,10), CD4, CD8 (11), Thy-1 (12, 13), TCR Vß8 (14), IL-2 receptor (15), MHC class II H-2A and H-2E (1618) and LFA-1 (19); (ii) cytokine gene expression was detectable in the islets from mice treated with STZ (20,21) and mAb against IFN-{gamma} prevented the disease (21); (iii) a variety of immunosuppressive agents have been found to inhibit the disease (22,23); (iv) some co-stimulatory molecules such as B7-1, CTLA-4, but not B7-2, increased susceptibility to the disease (24); and (v) athymic (nude) mice have proved to be relatively resistant to disease induction (25,26).

There are now numerous data accumulated in both normal and pathologic immune responses which suggest preferential, if not exclusive, activation of T cells expressing particular TCR Vß gene segments (2729). Recognition of MHC-bound peptides by T cells is conferred primarily via the complementarity-determining region-3 encoded by TCR V(D)J junctional sequences (27,30). If the first detectable islet infiltrates harbor T cells with specificity for relatively few antigens, this might be reflected by restricted TCR Vß gene repertoire usage, which makes contact with the peptide. TCR Vß gene repertoire usage of islet-infiltrating T cells was restricted in the case of young NOD mice (31,32), while diverse TCR Vß elements were expressed in islet infiltrates, as insulitis progressed (3335). These data suggest that early islet-infiltrating T cells might recognize a single or a few autoantigens and have a restricted (oligoclonal) TCR repertoire, and that TCR Vß gene usage was successfully established along with the progression of insulitis. However, it remains uncertain whether the spreading of TCR Vß usage is derived from diversification of the restricted antigen-specific T cell clone(s) or from the accumulation of T cells reactive to various antigens.

Although T cells have been shown to play a major role in the pathogenesis of autoimmune diabetes, recent studies have shown that B cells may also play an essential role in the development of autoimmune insulitis and diabetes (36-39). We have already reported the significant accumulation of B cells in the region of insulitis in AKR/J mice treated with STZ (8) and also in NOD mice (36). Surprisingly, development of diabetes has been shown to be completely prevented in B cell-deficient NOD mice, indicating that B cells also play a major role in the pathogenesis of IDDM, at least in NOD mice (3739). We have previously shown that B cells contribute to both progression of insulitis and development of diabetes in NOD mice (37), but initiation of autoimmunity mediated by priming of autoreactive T cells with autoantigens occurs even in the absence of B cells. Consistently, Epstein et al. presented successful T cell priming in B cell-deficient mice against various antigens. They suggested that priming of T cells can be mediated by specialized cells such as macrophages and/or dendritic cells, while antigen presentation by B cells might serve distinct immunological functions (40). In contrast, Serreze et al. reported that insulitis was scarcely detected in B cell-deficient NOD mice (38) and suggested that B cells are essentially important for the initiation of insulitis as well as the development of diabetes in NOD mice. In addition, recent studies have shown that B cells are not necessary for the development of experimental allergic encephalitis (41,42). Therefore, the role of B cells in the initiation of autoimmunity and/or the expansion of autoimmune responses seems to be controversial and remains to be elucidated.

In the present study, we studied the development of insulitis and diabetes in low-dose STZ-treated B cell-deficient (B) AKR/J mice, and examined TCR Vß gene usage and T cell clonotypes in infiltrates in islets. We found that initial T cell response in islets occurred in the absence of B cells, whereas TCR clonotype spreading of islet-infiltrating T cells was significantly impaired in B AKR/J mice, indicating that B cells play a crucial role in the spreading of autoreactive clonal T cells, and in the progression of insulitis and diabetes induced with STZ.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Male heterozygous mutant C57BL/6 mice (µMT) with a targeted disruption of the membrane exon of the Ig µ chain gene (kindly provided by Dr Daisuke Kitamura, Science University of Tokyo) (43) were backcrossed to female AKR/J mice (Seiwa Experimental Animal, Oita, Japan) for 10 generations. Then the mice were intercrossed to generate homozygous B cell-deficient AKR/J mice. All mice were kept under specific pathogen-free conditions in the Kyushu University Animal Center, and had free access to sterilized water and pelleted food throughout the experimental period except 4 h before the i.p. glucose tolerance test (IPGTT). Male mice at 10–12 weeks of age were used at the start of experiments.

Confirmation of MHC haplotype and B cell deficiency
MHC class II was typed by PCR using the tail DNA. All mice were routinely checked for lack of B cells in peripheral blood by flow cytometric analysis after staining of peripheral blood mononuclear cells with phycoerythrin-labeled anti-B220 (Dainippon, Osaka, Japan) and FITC-labeled anti-CD3{varepsilon} (PharMingen, San Diego, CA) mAb. The µMT allele was confirmed by PCR (37).

Induction of insulitis and diabetes with STZ
STZ (Sigma, St. Louis, MO) was stored at –20°C. Solutions were freshly made daily using cold 0.01 M citrate buffer, pH 4.5, and were used after passing through a 0.45 µm Millipore filter within 5 min of dissolution. STZ was injected i.p. with doses of 40 mg/kg of body wt, in a volume of 0.3 ml, daily for 5 days (day 1–5).

Blood glucose determinations
To determine the development of diabetes, non-fasting blood glucose levels were measured weekly (on days 8, 15, 22 and 29) and the glucose tolerance test (i.p. injection of 0.2 ml of 20% glucose/mouse or 1.33 g glucose/kg body wt) was also performed weekly (on days 9, 16, 23 and 30). Blood samples were collected from tail vessels and assayed for glucose concentration using the Glutest sensor (Sanwa-Kagaku, Nagoya, Japan) on a Glutest E.

Histologic examination
Pancreata were collected on days 14, 21, 28 and 35, and embedded in Tissue Tek (Miles, Elkhart, IN), snap frozen in liquid nitrogen and stored at -80°C. For light microscopy, 5 µm cryosections at 50 µm intervals were stained with hematoxylin & eosin. All islets were evaluated and the insulitis scores were determined as follows: 0, no intraislet mononuclear cell infiltration; 1, mild peri-insuler mononuclear cell infiltration (granulation of <30%); 2, intraislet moderate mononuclear cell infiltration (granulation of <50%); 3, severe to massive cell infiltration (granulation of >50%). Adjacent sections were used for immunohistochemical studies as described below.

Immunohistochemical study
For the immunohistochemical study, the following antibodies were used: rat anti-mouse CD4 (L3T4) (clone RM4-5) mAb which reacts with helper/inducer T lymphocytes, and rat anti-mouse CD8a (Lyt-2) (clone 53-6.7) mAb which reacts with suppressor/cytotoxic T lymphocytes, both purchased from PharMingen (San Diego, CA); rat anti-mouse F4/80 mAb (clone A3-1) which mainly reacts with mouse monocytes/macrophages, purchased from BMA (Augst, Switzerland); and affinity-purified rabbit anti-mouse IgM (µ chain specific) antibody, purchased from Zymed (San Francisco, CA). The 5 µm cryosections of pancreata were dried in room air for 30 min, acetone fixed for 30 min and again air dried for 30 min. After washing in PBS (pH 7.4), the appropriately diluted antibody for the specific antigen was overlaid for 60 min, then the preparations were washed 3 times in PBS and incubated with biotinylated goat anti-rat or anti-rabbit IgG (Dako Japan, Kyoto) for 50 min, washed 3 times in PBS, and finally incubated with avidin and biotinylated horseradish complex for 60 min. After another wash in PBS, they were exposed to diaminobenzidine and H2O2.

Isolation of islets of Langerhans
The technique used was adapted from the protocol previously reported (44). Briefly, 5 ml collagenase solution (Wako, Osaka, Japan; 1 mg/ml in Hanks' solution) was injected into the common bile duct of a mouse anesthetized with diethyl ether, with the aid of a 30 gauge needle, and then the fully expanded pancreas with collagenase solution was carefully dissected free of adjacent pancreatic lymph nodes and other tissues such as fat. The pancreas was incubated in a dish for 20 min at 37°C, then pipetted and washed in Hanks' solution 2 times. Exocrine tissues and debris were removed from islets by use of the Ficoll gradient, and islets were hand-picked with the aid of an inverted microscope and a 2 µl micropipetter. In general, between 40 and 80 islets were harvested from each pancreas.

RT-PCR
Total RNA was extracted from islets, lymphocytes in pancreatic lymph nodes (LN) and spleen from each mouse using ISOGEN (Nippon Gene, Tokyo, Japan), according to the manufacturer's instructions. Mice were sacrificed at day 7 (2 days after the fifth injection of STZ). Total RNA (1 µg) was converted to cDNA with reverse transcriptase (SuperScript; Gibco/BRL, Gaithersburg, MD) and oligo(dT) primer at 42°C for 50 min in 20 µl. cDNA from each mouse was amplified by PCR with a common Cß primer, one of the Vß subfamily- specific primers, dNTP and Taq DNA polymerase (rTaq; Takara, Otsu, Japan) for 40 cycles (94°C for 1 min, 56°C for 1 min and 72°C for 1 min) in a Thermal Cycler 480 (Perkin-Elmer Cetus, Norwalk, CT). The sequences of the Vß and Cß primers were as follows: Vß1, 5'-TTCGAAATGAGACGGTGCCC-3'; Vß2, 5'-AGAGGTCAAATCTCTTCCCG-3'; Vß3, 5'-CTTCAGCAAATAGACATGAC-3'; Vß4, 5'-TGGACAATCAGACTGCCTCA-3'; Vß5.1, 5'-GAGATAAAGGAAACCTGCCC-3'; Vß5.2, 5'-GAGACAAAGGATTCCTACCC-3'; Vß6, 5'-CGACAGGATTCAGGGAAAGG-3'; Vß7, 5'-ATACAGGGTCTCACGGAAGA-3'; Vß8.1, 5'-AGACCAAGCCAAGAGAATTCCCTC-3'; Vß8.2, 5'-CATAT- GGTGCTGGCAGCACT-3'; Vß8.3, 5'-CATATGGTGC TGGCA- ACCTT-3'; Vß9, 5'- ACAGGGAAGCTGACACTTTT-3'; Vß10, 5'-AATCAAGTCTGTAGAGCCGG-3'; Vß11, 5'-GGAGTCCCTGA- CTTACTTTC-3'; Vß12, 5'-AAGATGGTGGGGCTTTCAAG-3'; Vß13, 5'-TCTATAACAGTTGCCCTCGG-3'; Vß14, 5'-CCTCC- AGCAACTCTTCTACT-3'; Vß15, 5'-CGCCTCAAA AGGCATT- TGAA-3'; Vß16, 5'-CAGCAGATGGAGTTTCTGGT-3'; Vß17, 5'-ACAGACTTGGTCAAGAAGAG-3'; Vß18, 5'-GTTCCAGGAACAGAGCTTGA-3'; Vß19, 5'-GAAACCGGGAGAAGAACTCA-3' Cß, 5'-TTGATGGCTCAAACAAGGAGACC-3'.

PCR products were electrophoresed through a 2% agarose gel and stained with ethidium bromide. The size of each product was ~200 bp.

SSCP analysis
PCR products were once concentrated by ethanol precipitation (5:1), then diluted (1:5) with loading dye, heat denatured and electrophoresed in non-denaturing 5% polyacrylamide gel containing 10% glycerol. The Vß-amplified fragments were run for 90 min at 35 W constant power for detection of a single or smear band. The DNA was transferred to nylon membrane (Hybond-N), hybridized to a {gamma}-32P-labeled internal common Cß oligonucleotide probe (5'-AGGATCTGAGAA- ATGTGACT-3') at 53°C for 2 h using QuikHyb (Stratagene, La Jolla, CA). Autoradiograms were prepared after washing the Southern blots as follows; once in 2xSSC and 0.1% SDS at room temperature for 15 min, then once in 0.1% SSC and 0.1% SDS at room temperature for 20 min.

Statistical analysis
Statistical significance was determined with a two-way repeated-measures ANOVA in the analysis of cumulative incidence of diabetes. To compare the insulitis score between B+ mice and B mice, a Mann–Whitney U-test was used. Statistical significance in the analysis of T cell clonotypes was determined with a two-way factorial ANOVA.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation and phenotyping of B AKR/J mice
To obtain progeny homozygous for the µMT mutation on the background of AKR/J, µMT/+ B6 mice were backcrossed to + /+ AKR/J mice 10 times to obtain µMT /+ mice with the AKR/J background. µMT/+ AKR/J males and females were intercrossed, and the littermates were assessed for their genetic background and also for the absence of mature B cells. The mice were genotyped for µMT and wild-type alleles by the PCR reaction (Fig. 1AGo). No B220+ mature B cells were detected in the peripheral blood of µMT/µMT (B) AKR/J (Fig. 1BGo). The MHC of these mice was confirmed to be of the H-2k haplotype (data not shown). Throughout this study, µMT /+ (B+) AKR/J males were used as control littermates carrying mature B cells.




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Fig. 1. Generation of B cell-deficient AKR/J mice. µMT/+ B6 mice were backcrossed to +/+ AKR/J mice 10 times. µMT/+ (B+) AKR/J mice were intercrossed and µMT/µMT (B) AKR/J mice were obtained. (A) Genotyping for µMT and wild-type alleles by PCR. Homozygocity of the µMT allele was determined in µMT/µMT (B) AKR/J mice. (B) Flow cytometric analysis revealed the lack of B cells in peripheral blood from B mice.

 
Development of insulitis and diabetes in B+ and B- AKR/J mice
The development of diabetes was followed for 6 weeks. Diabetes was diagnosed when blood glucose levels exceeded 250 mg/dl by IPGTT. As shown in Fig. 2Go, at day 36 (5 weeks), the incidence of diabetes in B+ mice was 22 of 27 (81.5%). On the other hand, it was 19 of 45 (42.2%) in B mice (significance of the difference was defined by P < 0.0001 in a two-way repeated-measures ANOVA). The extent of insulitis was histologically scored. The insulitis was detected in B mice. Percentage of islets scored as grade 3 was 21% (21 of 100) in B+ mice, although it was only 7% (seven of 100) in B mice (significance of the difference was defined by P < 0.001 in a Mann–Whitney U-test) (Table 1Go). Histologically, B mice did also develop insulitis, although the extent of insulitis was less severe than it was in B+ mice (Fig. 3Go). Thus, diabetes (hyperglycemia) as well as insulitis developed in both B and B+ mice, but both the extent of insulitis and cumulative incidence of diabetes were reduced in B mice compared with the control littermates with B cells (Table 1Go and Fig. 2Go).



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Fig. 2. Cumulative incidence of diabetes in the control B+ and B mice. Every week IPGTT was performed for 6 weeks. Mice with blood glucose readings >250 mg/dl were diagnosed diabetic. Cumulative incidence of diabetes dissociated in B+ ({circ}) and B ({square}) mice. At day 36, the incidence of diabetes was 22 of 27 (81.5%) and 19 of 45 (42.2%) in B+ and B mice respectively. Cumulative incidence of diabetes was reduced significantly in B mice (P < 0.0001).

 

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Table 1. Incidence and grading of insulitis in B- mice and in B+ control mice
 


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Fig. 3. Representative histopathology of insulitis in B and B+ mice. Islet-infiltrating cells were not found in these two islets from B mice (A). Severe insulitis in B+ mice was presented (B). Although mild to severe insulitis was also detected in other sections in B mice, the extent of insulitis in B mice was lower than that in B+ mice.

 
Immunohistochemical study
Moderate insulitis lesions of the same extent derived from B+ and B mice were selected and immunnohistochemically stained to examine the subpopulations of islet-infiltrating lymphocytes. Predominant accumulation of CD4+ T cells (Fig. 4CGo) and B cells (Fig. 4GGo) with a small admixture of CD8+ T cells (Fig. 4EGo) was observed, and F4/80+ macrophages (Fig. 4IGo) were also present around the inflammatory islets in B+ mice. In B mice, the inflammatory lesion was composed of similar subpopulations of infiltrating lymphocytes but without B cells (Fig. 4D, F, H and JGo). The immunohistochemical study did not reveal any change in the subpopulations of infiltrating lymphocytes except for the absence of B cells in B mice.



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Fig. 4. Immunohistochemical studies of insulitis from B+ and B mice. Serial sections of islets with the same extent of insulitis from B+ (A, C, E, G and I) and B (B, D, F, H and J) mice were immunohistochemically stained (x200). In control islets from B+ mice, predominant accumulation of CD4+ cells (C) and surface IgM+ B cells (G) was observed, while only a few CD8+ T cells (E) were admixed. A few F4/80+ macrophages were also detected around the islet (I). In islets of B mice, a predominant accumulation of CD4+ T cells (D) was observed, but not that of B cells (H), while only a few CD8+ T cells (F) and F4/80+ macrophages were infiltrated around the islet (J).

 
TCR Vß gene repertoire usage in islet
TCR Vß gene repertoire usage in islet infiltrates of STZ-treated B+ and B mice was determined by RT-PCR. In the analyses using several individuals, TCR Vß gene repertoire usage was restricted to some extent in B+ or B mice, but there was no significant difference between the B+ and B mice, and no specific TCR Vß gene usage was observed in both animals (Fig. 5A and BGo). It is known that, in AKR mice, the specific TCR Vß repertoire such as Vß12 is deleted by superantigen. FACS analyses of splenocytes revealed that the forbidden subpopulation of T cells with Vß12 was indeed under- represented, but present, in the B AKR mouse (0.34%) compared to B C57BL/6 mouse (4.13%). Moreover, sequencing of amplified fragments of TCR Vß12 derived from the inflammatory islets in the B AKR mice revealed that these fragments indeed consisted of Vß 12 sequences (data not shown). Thus, the small but significant number of so called `forbidden' T cell clones was present in the inflammatory islets in the B as well as B+ AKR mice.




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Fig. 5. TCR Vß gene usage in islet infiltrates from B+ and B mice by RT-PCR. Mice were sacrificed at day 7 (2 days after the fifth injection of STZ). (A) Representative patterns of TCR Vß gene usage in total islets from single B+ or B mice are shown. (B) Percentages (number of mice expressing the TCR Vß/total number of mice studiedx100) of TCR Vß usage in the pancreatic infiltrates in B+ (n = 8) and B (n = 7) mice are presented. B+ and B mice showed similar patterns of TCR Vß gene usage.

 
Spreading of TCR clonotype in islets
By the RT-PCR analysis employed in previous studies and the present study, however, it was difficult to distinguish each clonal T cell population. Therefore, in order to define the role of epitope-reactive specific clonal T cells, RT-PCR followed by SSCP analysis was used in this study. By this method, amplified CDR3 DNA originating from one T cell clone can be visualized as a single band in SSCP gel, while amplified DNA from a diverse T cell population exhibits a smear pattern composed of innumerable faint bands (50). T cells from islets of each mouse were analyzed by RT-PCR/SSCP by using PCR primers covering all Vß families. SSCP analysis revealed that oligoclonal T cells were detected as distinct bands in islet infiltrates both in B+ and B mice (Fig. 6AGo). No clonal TCR Vß expression in lymphocytes in pancreatic LN (Fig. 6AGo) or spleens (data not shown) taken from both B+ and B mice was detectable. Taken together, it was suggested that the specifically primed and oligoclonal T cells accumulated exclusively in the islets. Although distinct bands were detected in B mice, as well as in B+ mice (Fig. 6AGo), the number of the distinct bands in the PCR products determined by SSCP analysis significantly decreased in B mice compared to B+ mice (significance of the difference was defined by P < 0.0001 in a two-way factorial ANOVA) (Fig. 6BGo). These data indicate that TCR Vß gene repertoire usage was not limited even in the absence of B cells; however, TCR clonotype spreading of islet-infiltrating T cells in B mice was significantly suppressed compared to that in B+ mice.




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Fig. 6. Clonotype analysis of TCR Vß repertoire diversity of islet infiltrates from B+ and B mice by SSCP analysis. Mice were sacrificed at day 7. (A) Representative results of SSCP analysis for islets of B+ (n = 3) and B mice (n = 3). Each band corresponds to the single TCR clonotype. No band was detected and the smearing pattern was seen in control pancreatic LN from B+ and B mice. (B) A comparison between B+ and B mice in diversity of T cell clonotypes in islets by SSCP analysis. `Number of distinct bands' is the summation of visible bands in the SSCP analysis. Black bars represent the mean numbers of bands of B+ control mice (n = 7), while white bars represent those of B mice (n = 5). Data are means ± SE. Significantly fewer bands (T cell clones) were detected in islets of B mice than in those of B+ mice (P < 0.0001).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The general consensus that autoimmune diabetes is T cell-mediated autoimmunity has been well documented (1,13,14,2527); however, recent studies have also shown that B cells play an important role in the pathogenesis of autoimmune diabetes (3739,46). In the present study, AKR/J (H-2k) mice treated with multiple low doses of STZ, another excellent animal model of autoimmune diabetes (8), were examined. It was found that the BAKR/J mice treated with multiple low doses of STZ did develop insulitis, although the extent of insulitis was significantly suppressed compared with that in the B+ mice, along with the suppression of diabetes development. These observations are consistent with the previous notion that BNOD mice developed a suppressed level of insulitis associated with the prevention of diabetes (37). Immunohistochemical study revealed that CD4+ cells and a few CD8+ T cells infiltrated into islets in B mice. Taken together, it was confirmed that B cells are not critical for the initiation of insulitis, but are rather, required for the further progression of insulitis. The role of B cells in the priming or proliferation of T cells in B cell-deficient mice has been controversial. Mice rendered B cell deficient by treatment with rabbit anti-mouse IgM (anti-µ) antibodies from birth fail to respond when primed with soluble protein antigens in complete Freund's adjuvant, as measured by T cell proliferation when challenged with antigen in vitro, suggesting that B cells appeared to be crucial for T cell priming in vivo (47). Consistently, Falcone et al. found that splenocytes from B cell-deficient mice have no spontaneous responses to 65 kDa glutamate decarboxylase in vitro (48). On the contrary, it was reported that B cells are not required for T cell stimulation (49). In addition, Epstein et al. demonstrated that B cells are not critical for either CD4 or CD8 T cell priming in any of the systems including assays of T cell proliferation and cytokine production in responses to protein antigens, T cell killing to minor and major histocompatibility antigens, skin rejection, and the in vitro and in vivo responses to shistosoma eggs (40). As shown by the present study, autoreactive T cell priming, which would lead to the initiation of insulitis, appeared to be successfully achieved and the TCR Vß gene repertoire usage was well obtained even in the absence of B cells, indicating that B cells are not required for the initiation of autoimmune diabetes, at least in STZ-treated AKR mice.

The immunologic mechanisms by which the progression of insulitis is mediated by B cells remain to be clarified. One possibility is that the expansion of autoreactive T cells induced by proliferative cytokines as IL-2 and/or cytokine balance, i.e. the Th1/Th2 ratio of reactive T cells, may be crucial for the progression of insulitis. Autoimmune-mediated insulitis was believed to be a `Th1 dominant disease' supported by the evidence that mAb against IFN-{gamma} prevents the disease (21). Therefore, B cells may possibly play a role in the enhanced Th1 dominant disease in this case. Another possibility is the repertoire spreading of antigen-reactive T cell populations mediated by B cells, since autoimmune diabetes associated with insulitis has been associated with polyclonal T cell response (33) and B cells are known to possess the capability of antigen presentation to stimulate T cells. We therefore examined the possible mechanisms involved in this animal model.

In the histopathologic study of insulitis, accumulation of both CD4+ T cells and B cells was observed in islets of B+ mice, while CD4+ T cells but not B cells infiltrated in B mice. A few CD8+ T cells and macrophages were detectable in both mice. The immunohistochemical study did not reveal any change in the subpopulations of infiltrating lymphocytes except for the absence of B cells in the B mice. In addition, we further studied cytokine gene expression of islet infiltrates to examine whether skewing of the immune response was operative in the insulitis lesion in B mice. Cytokine profiles in the insulitis lesion indicated that both IFN-{gamma} and IL-4 were produced in B mice as well as in B+ mice 2 days after the fifth STZ injection (data not shown), suggesting that the Th1/Th2 cytokine balance was not altered in the B mice. Taken together, it can be suggested that B cells played the role in the augmentation of anti-islet autoimmunity; however, the cytokine balance shift, i.e. the change of the Th1/Th2 ratio of reactive T cells, was not likely to play a major role in the suppression of autoimmune diabetes in B mice treated with STZ.

There are many previous studies in which the TCR repertoire was analyzed in STZ-induced autoimmune diabetes. In C57BL/KsJ mice treated with multiple low doses of STZ, which also causes IDDM, TCR Vß8.2+ T cells played a very important role at an early stage of insulitis and anti-Vß8.2 antibody inhibited the progression of STZ-induced insulitis (14). However, as shown by this study, no specific TCR Vß gene usage was observed in insulitis in B+ or B AKR mice. Therefore, it is not likely that T cells bearing a specific TCR Vß population is essential for the progression of insulitis and development of diabetes in this autoimmune diabetes model. In addition, since B mice could successfully use a wide range of TCR Vß gene repertoire as could B+ mice, it was suggested that T cell priming could occur in the absence of B cells. This initial T cell recognition of specific antigens may be mediated by professional antigen-presenting cells such as dendritic cells and/or macrophages, since these populations are active in B mice (40). We further analyzed the infiltration of each clonal T cells bearing specific TCR Vß by using SSCP analysis. As shown in the present study, although TCR Vß usage was well obtained in B mice, the number of TCR clonotype of islet-infiltrating T cells as assessed by PCR/SSCP was significantly reduced along with the suppression of the development of diabetes in B mice. Taken together, it was suggested that not only professional antigen-presenting cells such as dendritic cells and macrophages but also B cells might be essentially important for antigen presentation in the antigen-specific recognition of T cells, and the B cell-dependent efficient antigen recognition would result in the spreading of autoreactive clonal T cells, leading to the progression of insulitis and the development of diabetes in AKR/J mice treated with STZ.

Alternatively, antibody-mediated and/or antibody-dependent cell-mediated cytotoxicity responses mediated by B cells might be important for the accelerated damage of islet ß cells. However, no autoreactive antibody, including islet cell antibody or islet cell surface antibody, has been detected in STZ-induced insulitis and diabetes mice (6), and therefore this mechanism is probably not involved in the pathogenesis of this autoimmune animal model.

In conclusion, we have shown that B cells play an important role in the progression of insulitis and diabetes in mice treated with multiple low doses of STZ. B cells are essentially important for the clonotype spreading of islet-infiltrating T cells. Although B cell-mediated antigen presentation and/or co-stimulatory signals to T cells may be involved in the pathogenesis of STZ-induced autoimmune insulitis and diabetes, further studies are required to elucidate the mechanisms of B cell-dependent autoimmune diabetes.


    Acknowledgments
 
This work was supported in part by a grant from Kyushu University Interdisciplinary Programs in Education and Projects in Research Development.


    Abbreviations
 
IDDM insulin-dependent diabetes mellitus
IPGTT i.p. glucose tolerance test
LN lymph node
NOD non-obese diabetic
SSCP single-stranded conformation polymorphism
STZ streptozocin

    Notes
 
Transmitting editor: D. Kitamura

Received 2 November 1999, accepted 31 March 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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