B lymphocytes not required for progression from insulitis to diabetes in non-obese diabetic mice

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1 Immunology and Cell Biology (2001) 79, Research Article B lymphocytes not required for progression from insulitis to diabetes in non-obese diabetic mice BRETT CHARLTON, MING DA ZHANG and ROBYN M SLATTERY John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory, Australia Summary Previous studies have implicated B lymphocytes in the pathogenesis of diabetes in the non-obese diabetic (NOD) mouse. While it is clear that B lymphocytes are necessary, it has not been clear at which stage of disease they play a role; early, late or both. To clarify when B lymphocytes are needed, T lymphocytes were transferred from 5-week-old NOD female mice to age-matched NOD/severe combined immunodeficiency (SCID) recipient mice. NOD/SCID mice, which lack functionally mature T and B lymphocytes, do not normally develop insulitis or insulin-dependent diabetes melitus (IDDM). The NOD/SCID mice that received purified T lymphocytes from 5-week-old NOD mice subsequently developed insulitis and diabetes even though they did not have detectable B lymphocytes. This suggests that while B lymphocytes may be essential for an initial priming event they are not requisite for disease progression in the NOD mouse. Key words: major histocompatibility complex, non-obese diabetic mice, severe combined immunodeficiency, T lymphocytes. Introduction The non-obese diabetic (NOD) mouse is a murine model of human insulin-dependent diabetes melitus (IDDM) characterized by the spontaneous development of diabetes as a result of lymphocytic islet infiltration and subsequent β cell destruction. The MHC class II regions of the NOD mouse encode a non-functional Eα gene and a unique Aβ gene that results in an unusual MHC class II phenotype. 1 Major histocompatibility complex class II genes are expressed on a restricted population of cells; principally these are APC, such as activated macrophages and dendritic cell types. Major histocompatibility complex class II is also expressed on B lymphocytes, which probably function as APC as well as immunoglobulin-producing cells. This APC function of B lymphocytes may be partly responsible for the T-cell reactivity of NOD mice to self β cell antigens, such as GAD65. 2,3 It has also been shown that NOD mice deficient in B lymphocytes, as a result of backcrossing the Igµ null/ null phenotype onto the NOD background, rarely develop diabetes 4 6. While the majority of these mice do not develop hyperglycemia, many have insulitis, and while only a small proportion spontaneously develop hyperglycemia, cyclophosphamide (CY) administration induces hyperglycemia in a significant proportion. 7 Correspondence: Dr RM Slattery, John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra, ACT 2601, Australia. robyn.slattery@anu.edu.au Received 21 May 2001; accepted 28 June Non-obese diabetic mice treated with antibody to IgM from early after birth also have a depleted B lymphocyte compartment and do not develop diabetes. 8 Other studies have shown that purified T cells from diabetic NOD mice can transfer diabetes even when recipients have been depleted of B lymphocytes by antibody treatment. 9 Other studies hinted that B lymphocytes may not be necessary for disease progression in NOD/severe combined immunodeficiency (SCID) recipients. 10 Based on these findings it was originally suggested that B lymphocytes may not be necessary in the disease process after their involvement in the initiation of the autoimmune response. It has been suggested that the role of B lymphocytes in NOD mice may be in the initiation of T-cell responses to β cell-specific antigens (e.g. GAD65). 2,3 Contrary to these findings it has recently been suggested that B lymphocyte/cd4 + T-cell interactions are necessary for the progression of established insulitis to β cell destruction 7 and that impaired CD4 + -activation-induced cell death results from this. 11 In order to separate the role of B lymphocytes in the initial stages of disease and in the progression from insulitis to β cell destruction we used an adoptive transfer model in NOD/SCID recipients. T cells were purified from 35-day-old NOD mice and were transferred to NOD/SCID recipients. NOD/SCID mice do not have mature T or B cells 12 and do not develop insulitis or diabetes. 13 We found that insulitis and diabetes did develop in these NOD/SCID recipients despite the apparent absence of B lymphocytes. This suggests that there is not a requirement for B lymphocytes after 35 days, during which time the disease progresses from insulitis to diabetes.

2 598 B Charlton et al. Materials and Methods Mice Non-obese diabetic/scid, NOD/Denv and (AKR NOD)F1 mice were obtained from the Animal Breeding Establishment, Australian National University. Mice were contained in microisolation cages and maintained on a standard diet and autoclaved water. All animal experimentation protocols were approved by the Australian National University Animal Experimentation Ethics Committee and were carried out according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. T-cell purification and transfer T cells were enriched from the pooled lymph nodes of 35-day-old female NOD mice by nylon wool separation. The enriched T cells were then incubated with antibodies against HSA (M1/69) and B220 (RA3-6B2) and antibody-coated cells were removed using magnetic beads (Advanced Magnetics, Cambridge, MA, USA). The remaining purified T cells were typically >97% CD4 + and CD8 +, and <0.5% mouse Ig + or B Purified T cells ( ; 2 3 donors) were then injected i.v. into 35-day-old female NOD/SCID recipients. In a few mice the recipients were reconstituted with T cells from (AKR NOD)F1 donors or splenocytes from the same F1 donors. Flow cytometry Blood, lymph node or spleen cells were stained with antibodies to CD4, CD8, mouse Ig (Caltag, San Francisco, CA, USA), B220 and H2Kk (Pharmingen, San Diego, CA, USA). Stained populations were analysed on a FACScan (Becton-Dickinson, San Jose, CA, USA). Assessment of diabetes Urine glucose was assessed bi-weekly using Tes-Tape (Lilly, Indianapolis, IN, USA). Blood glucose levels of glycosuric animals were then tested using a One Touch II glucometer (Lifescan, Milpitas, CA, USA). Consecutive readings of >16 mmol/l were considered confirmation of diabetes. Cyclophosphamide administration Cyclophosphamide (Farmitalia Carla Erba, Hawthorn, Vic., Australia) was dissolved in saline and injected i.p. at a dose of 250 mg/kg. Blood glucose was tested on day 17 after CY administration. Histology Pancreatic tissue was sectioned on at least three levels and stained with haematoxylin and eosin. The insulitis in each islet was then assigned a score for comparison of severity among groups. 14 Immunohistochemistry was performed on frozen sections using biotin-conjugated or unconjugated primary antibodies and a second step of streptavidin-conjugate or species-specific conjugated antibodies. Results Purified T lymphocytes from 5-week-old NOD mice induce diabetes in NOD/SCID recipients Eleven female NOD/SCID mice were followed to 220 days of age after receiving NOD T cells at 5 weeks of Figure 1 Representative histological section of pancreas from a non-obese diabetic/severe combined immunodeficiency (NOD/ SCID) NOD T-cell recipient mouse at age (a) 220 days and (b) an age-matched female NOD. age. Eight of eleven (73%) recipients developed diabetes by 220 days of age, which is consistent with the cumulative incidence of diabetes in the background NOD colony (Fig. 1). Islets in the diabetic NOD/SCID T-cell recipient mice appeared similar to those observed in recently diabetic NOD mice (i.e. mainly end-stage or massively infiltrated islets; Fig. 2). Those mice that did not become diabetic had widespread insulitis.

3 Diabetes develops without B lymphocytes 599 Figure 2 Diabetes incidence in female non-obese diabetic/ severe combined immunodeficiency (NOD/SCID) recipients of purified female NOD T lymphocytes (n = 11). No nonreconstituted NOD/SCID mice develop diabetes. NOD/SCID recipients of purified T lymphocytes are deficient in B lymphocytes Fluorescence-activated cell sorter analysis of lymphocytes from the NOD T-cell recipient NOD/SCID mice at 220 days showed less than 0.5% B220 bright or mig + cells in the spleen, blood or lymph nodes (Fig. 3). Further, staining of frozen sections from these mice showed no more mig or B220 positive cells than control NOD/SCID mice that had not been reconstututed with T lymphocytes (Fig. 4). To further assess for B-lymphocyte contamination in this transfer model, F1 T cells from H2Kk + donors were also purified and transferred to three NOD/SCID mice to allow the detection of markers not present in the NOD/SCID background. In these NOD/SCID recipients all CD3 +, CD4 + and CD8 + cells coexpressed H2Kk, demonstrating that T cells in the recipients were donor derived (data not shown). Notably, B lymphocytes (<0.5%) could not be detected, based on the absence of both H2Kk-expressing non T cells and H2Kk-expressing B220 + cells. Cyclophosphamide accelerates diabetes in NOD/SCID recipients of purified T lymphocytes A group of five NOD/SCID mice given purified NOD T lymphocytes were treated with CY (300 mg/kg) 6 weeks after cell transfer (at 77 days of age). All five mice became hyperglycemic by day 17 after CY treatment. No B lymphocytes could be detected by FACS or immunohistochemistry in any of these NOD/SCID recipients. Discussion It has been reported that the absence of B lymphocytes protects NOD mice from developing diabetes. The protection afforded by a B lymphocyte deficiency is not absolute and Figure 3 Representative FACS profiles of lymph node cells stained for CD4 and B220 from a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) recipient of purified NOD T lymphocytes (top) and a normal NOD donor (bottom). some mice do develop insulitis and diabetes, either spontaneously or when precipitated by CY administration. 7 The mechanism of protection afforded by the absence of B lymphocytes in NOD mice is not known. Two suggested explanations have been proposed: (i) that B lymphocytes may be necessary to initiate the response of autoreactive T cells to the key autoantigen GAD65, 2,3 which then triggers a cascading immune response to other β cell antigens; 15 and (ii) that NOD B lymphocytes are unable to induce activation-induced cell death (AICD) in autoreactive CD4 + T cells, unlike B lymphocytes from non-autoimmune strains. 11 To test whether B lymphocytes are necesary for disease progression in NOD mice, NOD T cells were purified from 35-day-old NOD mice and transferred into NOD/SCID recipient mice. Both insulitis and diabetes occurred in NOD/SCID recipients of purified NOD T cells. In addition, less than 0.5% B cells were detectable in T-cell recipient NOD/SCID mice, suggesting that B cells are not required for disease progression. A lack of requirement for B lymphocytes in NOD mice has previously been reported only for disease transfer using diabetic donor T cells. 9 While the existence of a small percentage of B lymphocytes in the NOD/SCID recipients cannot be ruled out, this marked deficiency would be expected to have an impact on disease progression if B lymphocytes were requisite. Despite this, the diabetes incidence was no different from that of the B-lymphocyte competent parent colony in either timing or

4 600 B Charlton et al. Figure 4 Frozen sections of spleens stained for B220 positive cells. Spleen from (a) normal non-obese diabetic (NOD) mouse (b) normal non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse and (c) NOD/SCID recipient of purified NOD T lymphocytes. overall incidence. Furthermore, 6 weeks after T-cell transfer a group of recipients were given CY to induce disease progression 14 and all of the CY-treated T-lymphocyte recipient NOD/SCID mice became hyperglycemic within 17 days, and again B lymphocytes were not detectable by FACS. In a recent study using chimeric NOD mice in which the B lymphocytes had an absence of IAg7 on their surface, it was reported that the progression of insulitis to diabetes was halted. 7 The authors suggested that B lymphocytes were essential for disease progression through the event termed checkpoint 2; 16 a checkpoint that allows the insulitis to become destructive. 7 The data presented here suggest that this is not the case. It is not apparent what important differences may exist in the T cell to NOD/SCID transfer model compared with the IAg7 null B lymphocyte chimeric model used by Noorchashm et al. 7 One significant difference may be that NOD/SCID mice have a higher number of monocytes than NOD mice. Furthermore, the class II negative B-lymphocyte chimeric NOD mice may carry potentially protective MHC class I genes and surrounding genes. These genes have previously been shown to be associated with protection from IDDM. 17 It was also recently reported that B lymphocytes are needed for sialitis and thyroiditis to develop in congenic NOD mice. 18 However, our studies suggest that B lymphocytes are not necesary for the progression of sialitis in NOD mice because the NOD/SCID recipients developed sialitis to a similar extent as that seen in normal NOD mice. Thus, despite the absence of B lymphocytes from the age of 5 weeks, autoreactive T cells alone are capable of causing disease progression to hyperglycemia. This suggests that islet-reactive T cells do not require B lymphocytes after an initial priming event, presumably dependent on B cells, has occurred. A similar observation was published at the time of manuscript preparation. 19 Taken together the data suggest that the development of diabetes in the NOD mouse is dependent on B lymphocytes in the first 5 weeks of life, but not after that time. The nature of the B-lymphocyte role is still unknown, although initiation of a β-cell specific T-cell response appears likely. 2,3 However, it will be of immense value to determine exactly what role B lymphocytes play in the early stages of disease. Acknowledgements This work was supported by grants from the Juvenile Diabetes Foundation International (JDFI). B Charlton was the recipient of a Diabetes Australia Fellowship. RM Slattery was the recipient of a JDFI postdoctoral fellowship. References 1 Acha-Orbea H, McDevitt HO. The first external domain of the nonobese diabetic mouse class II, I-A beta chain is unique. Proc. Natl Acad. Sci. USA 1987; 84: Serreze DV, Fleming SA, Chapman HD, Richard SD, Leiter EH, Tisch RM. B lymphocytes are critical antigen-presenting cells for the initiation of T cell-mediated autoimmune diabetes in nonobese diabetic mice. J. Immunol. 1998; 161: Falcone M, Lee J, Patstone G, Yeung B, Sarvetnick N. B lymphocytes are crucial antigen-presenting cells in the pathogenic autoimmune response to GAD65 antigen in nonobese diabetic mice. J. Immunol. 1998; 161: Serreze DV, Chapman HD, Varnum DS et al. B lymphocytes are essential for the initiation of T cell-mediated autoimmune diabetes: analysis of a new speed congenic stock of NOD.Ig mu null mice. J. Exp. Med. 1996; 184: Akashi T, Nagafuchi S, Anzai K et al. Direct evidence for the contribution of B cells to the progression of insulitis and the development of diabetes in non-obese diabetic mice. Int. Immunol. 1997; 9: Yang M, Charlton B, Gautam AM. Development of insulitis and diabetes in B cell-deficient NOD mice. J. Autoimmun. 1997; 10: Noorchashm H, Lieu YK, Noorchashm N et al. I-Ag7-mediated antigen presentation by B lymphocytes is critical in overcoming a checkpoint in T cell tolerance to islet beta cells of nonobese diabetic mice. J. Immunol. 1999; 163: Noorchashm H, Noorchashm N, Kern J, Rostami SY, Barker CF, Naji A. B-cells are required for the initiation of insulitis and sialitis in nonobese diabetic mice. Diabetes 1997; 46: Bendelac A, Boitard C, Bedossa P, Bazin H, Bach JF, Carnaud C. Adoptive T cell transfer of autoimmune nonobese diabetic mouse diabetes does not require recruitment of host B lymphocytes. J. Immunol. 1988; 141: Slattery RM, Heath WR, Miller JFAP, Charlton B. Failure of a protective MHC molecule to delete autoreactive T cells in

5 Diabetes develops without B lymphocytes 601 autoimmune diabetes. Proc. Natl Acad. Sci. USA 1993; 90: Noorchashm H, Moore DJ, Noto LE et al. Impaired CD4 T cell activation due to reliance upon B cell-mediated costimulation in nonobese diabetic (NOD) mice. J. Immunol. 2000; 165: Prochazka M, Gaskins HR, Shultz LD, Leiter EH. The nonobese diabetic scid mouse: model for spontaneous thymomagenesis associated with immunodeficiency. Proc. Natl Acad. Sci. USA 1992; 89: Christianson SW, Shultz LD, Leiter EH. Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice. Relative contributions of CD4+ and CD8+ T-cells from diabetic versus prediabetic NOD.NON-Thy-1a donors. Diabetes 1993; 42: Charlton B, Bacelj A, Slattery RM, Mandel TE. Cyclophosphamide-induced diabetes in NOD/WEHI mice. Evidence for suppression in spontaneous autoimmune diabetes mellitus. Diabetes 1989; 38: Tian J, Olcott AP, Hanssen LR, Zekzer D, Middleton B, Kaufman DL. Infectious Th1 and Th2 autoimmunity in diabetesprone mice. Immunol. Rev. 1998; 164: Katz JD, Wang B, Haskins K, Benoist C, Mathis D. Following a diabetogenic T cell from genesis through pathogenesis. Cell 1993; 74: Hattori M, Yamato E, Itoh N et al. Cutting edge: homologous recombination of the MHC class I K region defines new MHClinked diabetogenic susceptibility gene(s) in nonobese diabetic mice. J. Immunol. 1999; 163: Braley-Mullen H, Yu S. Early requirement for B cells for development of spontaneous autoimmune thyroiditis in NOD. H-2h4 mice. J. Immunol. 2000; 165: Chiu PP, Serreze DV, Danska JS. Development and function of diabetogenic T-cells in B-cell-deficient nonobese diabetic mice. Diabetes 2001; 50: