Demetris, AJ

Duquesnoy, R

Fung, J

Markus, B

Saidman, S

Starzl, T

Zeevi, A

English

Name not available

Role of HLA Class II-specific alloreactive T cells in biliary epithelium injury associated with liver transplant rejection

D-Scholarship@Pitt

----------------------- ----_._--------------,-Role of HLA Class II-Specific Alloreactive T Cells in Biliary Epithelium Injury Associated with Liver Transplant Rejection I ,/ S. Saidman. R Markus, AJ. Demetris. 1. Fung, A. Zeevi, T. Starzt, and R. Duquesnoy Ahstract: In this study, we report the establishment of lymphocyte cultures from liver allograft biopsies, and correlate their primed lymphocyte testing (PLT) speci-ficity towards HLA class I andlor class II donor antigens with clinicopathological findings. Early posttransplant hiopsies generally yielded -more clac;s I-specific cells than later biopsies. There appears to be no correlation of hepatocellular enzyme levels (SGOT or SGm with PLT specificity. However. the levels ofGGTP and AP. which arc biliary enzymes, were increased in patients whose biopsies yielded class II-specific rather than class 1-specific cells. There was also a trend towards more • Sp,inl""-v",I"l' New Yo"l 198M Immunnh"""l (t( HLA V,llutuc II: Immunt'l!cnctk .. and ei~ftMKK"tKDmpatahilily testmg (I'Ll, anO Dy cell-meotatea lymplluly:'l:' \'-I\,IL}. Often they demonstrate restricted specificity patterns against a limited number of donor HLA ant igens and this alloreactivity can be blocked with HLA-specific mono-clonal antibodies against appropriate determinants. The aim of this study was to evaluate the association of the HLA class I and class II allospecificity of lymphocytes grown from liver allograft biopsies with clinical, bio-chemical. and histopathologic findings. T lymphocytes were propagated from portions of per-cutaneous liver biopsies taken at times of liver allograft dysfunction. or from removed allografts. The tissue was divided into small segments and cultured in microculture wells in the presence of 200 III of recombinant IL2 as described previously (I). After approximately 2 weeks. sufficient cells were obtained for PLT assays against cryopreserved donor splenocytes. Class 1- andlor class 11-specific alloreactivity was determined by testing an infor-mat ive panel ofHLA-typed lymphocytes andlor blocking with anti-class I and class II monoclonal antibodies (3). Liver tissue from the same biopsy core or from the same region of the liver (in failed allografts) was submit-ted for histopathologic review with special emphasis on the presence of a mononuclear portal inflammatory infil-trate associated with ductual epithel ial and venular endo-thelial damage. findings considered to be important in hepatic rejection (4). Information about the clinical and biochemical data was obtained from the medical charts and included serum levels of total and direct bilirubin, glutamate oxaloacetic transaminase (SOan. glutamate pyruvate transaminase (SGPT), alkaline phosphatase (AP). and y-glutamyl transpeptidase (GGTP). All values were recorded from the day prior to obtaining the biopsy. Statist ical analysis of relevant data was performed by the nonparametric statistical tests of Mann-Whitney and Kruskal-Wallis using the SPSSPC software package (5). For 35 oul of 67 lymphocyte cultures established we could assess allospecificity towards HLA class I (n = 8). mixed class I and II (n = 15) or HLA class II antigens 534 damage to bile duct epithelium in ~iopsics Y·ielding class II-specific cells. These resull~ are probably related to the interaction of class lI-specific T cells with bile duct epithelium, which has been shown to express class II HLA antigens. Although liver transplantation has become an accepled treatment for a variety of end-stage liver diseases, the mechanisms ofliver transplant rejection are 5t ill obscure. A novel approach to learn more about the immunopath-ology of transplant rejection is the propagation and characterization of lymphocytes directly from the 533 o 'Cl ... I Cla .. lIn Cia .. n Class I VS. Class U p=O.017 Figure 1. Serum levels of the biliary enzymes alkaline phos-phatase (AP) and y-glutamyl transpeptidase (GGfP) from liver transplant recipients on the day prior to liver biopsy. mati~9i~ are grouped according to whether their biopsies grew.lympho-cytes reactive against only class I antigens, a combination of class I and II antigens. or only class II antigens. Bars indicate the median levels for each group. The p values are for the com-parison of median levels in the group yielding only class 1-reactive cells versus median levels in the group yielding only class II-reactive cells. using the Mann-Whitney rank sum test. (n = 12). As shown in Table I. class I-specific cells were generated from biopsies obtained earlier during the post-transplant time period than those yielding class II-specific cells. This is concordant with previous data from heart transplant biopsies showing that class 1-specific cells are more predominant in earlier biopsies whereas class II-specific cells are generally found in later biopsies (6). No statistically significant differences were found in the bilirubin, soar, and SGPT values between the groups yielding class 1- andlor class lI-specific cells, although a trend towards higher levels in the group of biopsies yielding class II-specific cells was noted. In contrast, significant differences were found for levels of AP and GGTP, both of which are biliary enzymes. Figure I shows that the median levels of serum AP were almost twice as high in the class II group as in the class 1 group (i2 versus 170 IU/ml, normal range < 100 IU/ml). Intermediate levels of AP were found in the group of biopsies yielding a mixture of class 1- and 11-specific cells. Similarly, the median GGTP levels were approximately 2.5 times higher in the class II group than in the class I group (365 versus 142 UI/ml. normal range males <44; females <32 IU/ml). €I Spri"l",-Ve,lq New \\o,k 1988 Immunobiology .... HLA Vulume II: Immunogenetics tlnd ei~toCDompalahilDty 3 Tahle I. HLA-Assnl'iatcd Alloreactivity of Livcr Biopsy-Grown Lymphoq"tc Cultures Spccifkity Class I Class I + II Class II Nn. of Cultures 8 15 12 Jst Posttransplant Day of Biopsy Median 8.5* 1TK~ 29.1J!1 Range 4-48 2-237 5-753 '"Class 1 versus class II differences for all biopsies (n = 20). p = 0.076: for biopsies with rejection (n = 16). P = 0.003. tThiny-five of 67 hiopsy-grown lymphocyte cultures showed class I andfor class II HLA-spccific reactivity. allograft. Previous studies have shown that activated T-cell cultures can be generated from liver allograft biopsies by incubation with recombinant IL2 (I ,2). Such cultured lymphocytes frequently exhibit alloreactivity towards donor cells as measured by primed lymphocyte testing (PLT) and by cell-mediated lympholysis (CML). Often they demonstrate restricted specificity patterns against a limited number of donor HLA antigens and this alloreactivity can be blocked with HLA-specific mono-clonal antibodies against appropriate determinants. The aim of this study was to evaluate the association of the HLA class I and class II allospecificity of lymphocytes grown from liver allograft biopsies with clinical, bio-chemical. and histopathologic findings. T lymphocytes were propagated from portions of per-cutaneous liver biopsies taken at times of liver allograft dysfunction. or from removed allografts. The tissue was divided into small segments and cultured in microculture wells in the presence of 200 III of recombinant IL2 as described previously (I). After approximately 2 weeks. sufficient cells were obtained for PLT assays against cryopreserved donorsplenocytes. Class 1- and/or class 11-specific a\1oreactivity was determined by testing an infor-mat ive panel ofHLA-typed lymphocytes and/or blocking with anti-class I and class II monoclonal antibodies (3). Liver tissue from the same biopsy core or from the same region of the liver (in failed allografts) was submit-ted for histopathologic review with special emphasis on the presence of a mononuclear portal inflammatory infil-trate associated with ductual epithelial and venular endo-thelial damage. findings considered to be important in hepatic rejection (4). Information about the clinical and biochemical data was obtained from the medical charts and included serum levels of total and direct bilirubin. glutamate oxaloacetic transaminase (SOOT). glutamate pyruvate transaminase (SOPT), alkaline phosphatase (AP). and y-glutamyl transpeptidase (OGrP). All values were recorded from the day prior to obtaining the biopsy. Stat ist ical analysis of relevant data was performed by the nonparametric statistical tests of Mann-Whitney and Kruskal-Wallis using the SPSSPC software package (5). For 35 out of 67 lymphocyte cultures established we could assess allospecificity towards HLA class I (n = 8). mixed class I and II (n = 15) or HLA class II antigens 534 ,/ AP l~fol IVIL 1\00 700 eoo 500 400 300 200 100 + • 1583 ::$:,1053 -I + o Cl: •• I Cla. I/D Clu. n GGTP IVIL 3000 1000 eoo aoo 700 aoo 500 .00 300 200 100 Class I vs. Class II p=o.oa o '-:Cl ..... =.I----,;Cl"-... =-"1 r.;U,.-----,;Cl"-.""'n::-n" Class I VB. Class II p=O.017 Figure I. Serum levels of the biliary enzymes alkaline phos-phatase (AP) and y·glutamyl transpeptidase (aarp) from liver transplant recipients on the day prior to liver biopsy. Patients are grouped according to whether their biopsies grew lympho-cytes reactive against only class 1 antigens. a combination of class I and II antigens. or only class II antigens. Bars indicate the median levels for each group. The p values are for the com-parison of median levels in the group yielding only class 1-reactive cells versus median levels in the group yielding only class II-reactive cells. using the Mann-Whitney rank sum test. (n = 12). As shown in Table I. class I-specific cells were generated from biopsies obtained earlier during the post-transplant time period than those yielding class 11-specific cells. This is concordant with previous data from heart transplant biopsies showing that class 1-specific cells are more predominant in earlier biopsies whereas class II-specific cells are generally found in later biopsies (6). No statistically significant differences were found in the bilirubin. SOOT, and SOPT values between the groups yielding class 1- and/or class II-specific cells. although a trend towards higher levels in the group of biopsies yielding class II-specific cells was noted. In contrast, significant differences were found for levels of AP and GarP, both of which are biliary enzymes. Figure 1 shows that the median levels of serum AP were almost twice as high in the class II group as in the class I group ~O versus 170 IU/ml, normal range < 100 lUlml). intermediate levels of AP were found in the group of biopsies yielding a mixture of class 1- and 11-specific cells. Similarly, the median OGrP levels were approximately 2.5 times higher in the class II group than in the class I group (365 versus 142 Ul/ml, normal range males <44; females <32 IU/ml). €l Springer·Verlag New V.>rk 1988 Immunobiol,,!,Y of HLA \bIumc II: Immunogenetics and Hi,to .... ompalabilily 3 /\ ./ Other studies have shown that increased GafP levels reflect injury to the biliary epithelium (7). Thus greater elevations of GafP (and probably also AP) levels in the class II group may suggest that a cellular infiltrate of class II-specific cells in the liver allograft is associ-ated with more injury to the biliary epithelium. Normal biliary epithelium expresses class [ HLA antigens, but no or very low levels of class II antigens (lW). In con-trast. increased expression of class II HLA antigens is frequently observed in liver allografts. especially during rejection (10). The expression of class II antigens on biliary epithelial cells can be induced by Iymphokines released by activated T cells. such as y interferon (II). During rejection. the biliary epithelium is often sur-rounded by infiltrating lymphocytes (12.13). Our studies have shown a marked trend towards increased bile duct damage in biopsies yielding class II-specific lymphocyte cultures as compared to biopsies growing class [-specific cells (3). These data suggest that a cellular infiltrate of class 11-specific alloreactive T cells may lead to injury of the bile duct epithelium. At the same time. an increased expres-sion of class II HLA antigens may render the biliary epithelium a preferred target in the cellular rejection of the liver allograft. Acknowledgment. This work was supported by NIH grants AI 23467 and HL 36416. B. Markus was a recipi-ent of a Fellowship Grant from the Deutsche Forschungs-gemeinschaft. References I. Fung JJ. Zeevi A. Starzl TE. Demetris AJ. Iwatsuki S. Duquesnoy RJ. Functional characterization of infiltrating T lymphocytes in human hepatic allografts. Hum Immunol 1986:16:182. 2. Markus BH. Fung JJ. Zeevi A. Starzl TE. Demelris N. Duquesnoy Rl. Anal}'5is of T lymphocytes infiltrating human hepatic allografts. Transplant Proc 1987; 19:2470. 3. Markus BH. Demetris Ai. ~m~K Saidman SL. Fung JJ. Zeevi A. et 81. A1loreactive T lymphocytes cul-tured from liver transplant biopsies: Associations of HLA specificitf~th clinicopathological findings. Clin Trans-plantKoyn1~ . 4. Demetris N. Jaffe R. Stam TE. Pathol Annu. in press. 5. Norusis MJ. SPSS/PC+ for the IBM PC/XT/AT. SPSS Inc .• Chicago. 1986. 6. Zeevi A. Fung J. Zerbe T. Kaufman C. Rabin B. Griffith B • et al. Allospecificity of activated T cells grown from endo-myocardial biopsies from heart transpla!lt patients. Trans-plantation 1986;41:620. 7. Leonard TB. Neptun DA. Popp JA. Serum gamma glutamyl transferase as a specific indicator of bile duct lesions in the rat liver. Am J Pathol 1984; 1/6:262. 8. Lautenschlager I. Hayry P. Expression of the major his-tocompatibility complex antigens on different liver cell-ular components in rat and man. Scand J Immunol 1981; 14:421. 9. Daar AS. Fuggle SV. Fabre JW. ling A. Morris PJ. The detailed distribution of MHC Class II antigens in normal human organs. Transplantation 1984;38:293. 10. Demetris N. Lasky S. van Thiel DH, Starzl TE. Whiteside T. Induction of DR/la antigens in human liver allografts. Transplantation 1985;5:504. I I. Demetris AJ. Markus B. Saidman S. Fung J. Nalesnik M. Makowka L. et al. Establishment of primary cultures of human biliary epithelium and induction of class II MHC antigens by interferon gamma. Transplant Proc. in press. 12. Porter KA. Pathology of the orthotopic homograft and het-erograft. In: Starzl TE (ed): Experience in Hepatic Trans-plantation. Philadelphia. WB Saunders. 1969, p 422. 13. Fennell RH. Ductular damage in liver transplant rejection: Its similarity to that of primary biliary cirrhosis and graft versus host disease. Palhol Annu 1981; 16(2):289. Author Affiliations S. Saidman. A.J. a~m~trisK A. urn. and R. Duqllt'Snoy. Department of Pathology. University of Pittsburgh. Pitts-burgh. PA. USA; B. Markus. J. Fung. and T. Starzl. Depart-ment of Surgery. University of Pittsburgh. miti~burghK PA. USA 

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Role of HLA Class II-specific alloreactive T cells in biliary epithelium injury associated with liver transplant rejection

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