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AND CHARACTERIZATION OF PANCREATIC FROM THE NOD MOUSE.

T LYMPHOCYTES

Thomas W.H. Kay, Iain L. Campbell and Leonard C. Harrison, Walter & Elii Hall Institute of Medical Research, Royal Melbourne Hospital PO 3050, Victoria, AustraIia T lymphocytes mediating beta cell destruction in the NOD mouse are likely to be localized in the pancreas. We have purikd up to 2 x lo6 T lymphocytes per pancreas by collagena~e digestion and passage over nylon wed. Pancreatic T lymphocytes from NOD-WEHI mice, which have an incidence of spontaneous diites less than 5%, were compared before and after the intraperitoneal administration of300mg/kg

cyclophosphamide.ThistreatmentinducesTlyinphocytedependentbetace~destructionandh There wasa5-foldincreaseinpancreaticTlymphocytes 7-12days aftercyclophosphamide: initially there isa majorityofCD4lymphocytesbutbyday12CD8lymphocytespredominate.. Purity (W of T lymphocytes CD4zCD8 Dayfollowing recoveredtpancreas (x 10b5) viable cells) IdO cyclophosphamide 1.22 0 4.6 50.2 1 1.4 1.33 20.4 2.0 24.8 l-70 3 3.2 2-56 5 39.4 7 13.5 36.2 266 22.4 1.26 10 79.2 12 14.0 68.6 O-85 In contrast, the CD$CDS ratio of spleen lymphocytes remained greater than 1.7 throughout the period of study. Of cells bearing the Thy-l marker 5-30% were negative for both CD4 and CDS, the highest proportion being found at day 5. Other cells isolated were predominantly macrophages, together with some islet endocrine cells. Haemopoietic precursor cells which differentiated invitro into granulocytes and macrophages were also present 7 days after cyclophosphamide. We therefore demonstrate differential changes in CD4 and CD8 bearing T lymphocytes associated with beta cell desaucrion in cyclophosphamide injected NOD-WEHI mice. These changes do not occur in the spleen and would not be detectable in histological sections of the pancreas. The presence of gmnulocyte and macrophage

precursorsindicates thatthesecellsmayalsoheinvolvedin this modelofbetacelldestruction.

CULTURE-ISOLATED ISLET ALLOGRAE'TS ARE NONIMMUNOGENICAND NOT SUSCEPTIBLETO HOST AUTOIMMUNEDIABETES IN THFiBB/Wor RAT. Robert J. Ketchum, Orion D. Hegre, Albert Enriquez and Janet R. Serie. Dept. of Cell Bio. & Neuro.. 321 Church St SE, Universityof Minnesota,Minneapolis,MN 55455. Neonatal rat islets derived by culture-isolationwere allografted and found to be nonimmunogenic. Non-islet pancreatic componentstransplantedwith islets cause graft rejection. Immunostaininq of islets failed to demonstrate major histocompatibility complex (MHC) class II positivity, while staining of MIiCclass II on non-islettissue was clearly evident. Approximately200 culture-isolated islets, from Wistar/Furtb(W/F, Rtl") and/or Fischer-344 (F-344, Rtl'), were transplanted to the kidney capsule of 40 day old

74

Abstracts

prediabeticBB/Wor rats (Rtlu). Grafts remained in situ at least 25 days prior to sacrifice. In recipients not developinghyperglycemiaand in grafts examined immediatelyafter onset of hyperglycemia (+350 mg/dl), viable p cells were present in F-344 and W/F grafts with only minor lymphocyticaccumulation. In recipients with established hyperglycemia (14+ days) and ketosis, F-344 grafts containeddegranulatedp cells with no evidence of rejection; W/F grafts displayedextensivep cell damage and insulitis. Three hyperglycemicBB/Wor rats were given 1000 to 3000 F-344 islets in an attempt to reverse hyperglycemia. One animal showed ameliorationof hyperglycemia; two animals returnedto normoglycemia, and remained so for SO+ days post-transplantation.Removal of graft-bearing kidney resulted in a return to the diabetic state. Histological examination revealed end-stage islets in the pancreas, and intact alloislets with no evidence of immune cell infiltration at the graft site. These results demonstrate the non-immunogenicnature of culture-isolated islets, and support the hypothesisthat absence of MHC class II-positive antigen presenting cells results in reduced immunogenicityand enhanced islet allograft survival. These results also indicate that cultureisolatedislet allograftsare immunologicallysilent in the BB/Wor rat, are capable of reversingBB/Wor autoimmunediabetes, and are not a target for autoimmuneislet damage. (work supportedby NIH DK32237)

CIAMEKCNED IN THE NOD NICE - FAILURE TO SUPPHBSS INSULITIS E.F.Lam+er, P.Pozzilli,A.Signore,A.Williarns, K.Mansfield,E.A.M.Gs.le Dept. Diabetes& Iamnxqenetics, St.Bartholomew'sHospital,London ECl, UK, City Hospital,Leipzig,GDR,EndocrinolcgiaI, Universityof Rome, Italy The incidenceof diabetes in our colony of non obese diabetic (NOD)mice is bout 60% in females and 20% in males at the age of 30 weeks, but nearly ~11 animals develop insulitis. Imnotherapy has been used in NOD mice to suppress the pathologicalprocess and to prevent diabetes. In the present study Ciamexone (CMK)was given orally to NOD mice from the age of 7 weeks (group A : placebo; B : 0.3nq/daily;C : l.timg/daily).The animals were sacrificedat the age of 14 weeks before the onset of diabetes,pancreata revved and examined by light microscopy. Islets were counted and calculatedper area of tissue on each section , the number of small islets and the severityof insulitiswere determined. No significantdiffesences in then 1:of islet could be observefJbetween the groups (A: 62.0-10.6; p B B: 64.8-3.2 ; C: 40.9-14.0 islets/lOOrmn ). Similarly,the percentqge of small isietswas not affefted by the different treatments (A: 46.3-8.5%, B: 41.4-3.9 %, C: 35.9-10.7% ). Finally,CMK did +not influencz the severityof the insulitis: (periinsulitig: group A: 9+6-2.3%,B: 10*5-2.3%, C:6.5-2.5%; moderate insylitis:A: 11.773.8%B: 15.9-3+4%, C:11.9-3.9 8; severe insulitis:A: 22.1-8.1 %, B: 20.1-5.0 %, C: 42.9-10.7%N.S.). These figuresare similar to those found in the natural course of the disease in our colony. In conclusion:CMK had no effect on the extent of insulitisin NOD mice. However, as not all animals having insulitisdevelop diabetes, we still do not know whether CMX may influence the progression to hyperglycemia by reducingantibodyproduction. CMK is known to suppress humoral imnity and thereforewe are investigatingthis aspect and whether the onset of hyparglycaemiamay be delayed by the treatment.

GENETIC CONTROL OF y-INTERFERON-INDUCIBLE PROTEINS IN NOD ISLET CULTURES. Edward Leiter and Greg Christianson, The Jackson Laboratory, Bar Harbor, ME 04609,USA. Inappropriate expression of Class II MHC molecules on surfaces of B-cells has been proposed to be of pathogenic significance for the onset of diabetes. Induction of these antigens by y-interferon (IFN) on cultured