Demetris, AJ

Fu, F

Fung, JJ

Gao, L

Li, Y

Lu, L

Noyola, H

Qian, S

Rao, AS

Starzl, TE

Thomson, AW

English

PubMed

Name not available

The use of MHC class I or class II "knock out" mice to investigate the role of these antigens in allosensitization

D-Scholarship@Pitt

IIZUKA ET Al 'CR analysis. ere so-called :ather, thl:\c he technIcal experinL' n I alated U'"1g Ihis methlld not so high ility of CD4 f the causal . phenome_ lsplantalion .<57. 199~ :ev 101:1,)'1 SasaZUKI :ss. Il)l)~. i' ):163.19l)3 ,plantatHlll I "rLThe Use of MHC Class I or Class II "Knock Out" Mice to Investigate -;;: the Role of These Antigens in Allosensitization ,jf;'" ~ .";:",S. Qian. F. Fu, Y. Li. L. Gao. L. Lu. H. Noyola. A.S. Rao, A.W. Thomson. A.J. Demetris. T'l\S~rzl, -,t', and J.J. Fung \1 '\ , '''",T. ;' ACCELERATED and hyperacute rejections in presen-. . sitized transplant patients constitute major causes of -:: early allograft loss. Analysis of the distinct role of various :~, transplantation antigens in the process of sensitization is therefore important in further understanding and subse-.... ~-quently preventing the occurrence of such events. The :.~. recent availability of Hknock out"' mice. which do not ~. express major histocompatibility complex (MHC) class I or ':<cIass II molecules. 1.1 has provided a tool to analyze the role :;" Of these antigens in the initiation of immune responses to ';<- allosensitization. In this study. we have presensitized mice .).. " with skin grafts from either class I or class II MHC-deficient donors, and then examined the sensitization status of the .,. recipients by subsequent heart or liver grafting. The cellular ii and humoral immune responses of the sensitized recipients '~.r , :-~::::d~ETHODS .,. Animals and Surgical Procedures > Male 10- to 12-week-old inbred C57BUI0 (BIO. H-:!'). C57BU6 ;., (86, H-:!'), BIO.BR (H-2"). C3H (H-2"l. b2m (H-:!'. class I <)I.. deficient). and BALB/c (H-?) mice were purchased from the Jackson Laboratory (Bar Harbor. Me). C2D (H-t'. class II defi-cient) mice were purchased from GenPharm International (Moun-lain View. Calif). Onhotopic liver transplantation was performed . as described previously.J Abdominal heart transplantation was ~;' carried out as described elsewhere.' Total cessation of palpable contraction of the heart graft was defined as rejection. Allosensi-tization by skin grafting was done 14 to 21 days prior to heart or liver transplantation. by implanting a full-thickness tail skin graft on the dorsal side of the reciplcnt trunk. Mixed Lymphocyte Reaction (MLR) Equal numbers (2 x lOS) of responder and y-irradiated stimulator apleeo cells were cultured at 37°C in 5% CO~ in air and pulsed with 1 /loCi of [JH)TdR for the last 18 hours of a 4-day culture period. The cells were harvested onto glass fiber filters and [3H)TdR Uptake was measured by liquid scintillation counting. Results were Clpressed as mean counts per minute (cpm) :: 1 SD. Lymphocyte-Mediated Cytotoxicity Assay Freshly isolated spleen cells were uscd as effectors. The EL4 (H·t') lymphoma cell line (ATCC. Rockville. Md) or Con A-activated 810 splc~n cells were used as targets. The PSIS (H-?') tumor cell line or Con A-activated BALB/c~ spleen cells were used as third-party target controls. The target cells were labeled with Na~ siCrO. (NEN. Boston. Mass). washed. and plated at 4 x \OJ cells per 200 lAUwell at different Err ratios. Killing bv CYtotoxic T celis was quantitated by determining the percent~sp~cifi'c 51Cr release over a 4-hour period. Antibody-Dependent Cellular CytotOXIcity (ADCC) Antibody titers were measured by modification of method de· scribed previouslv.' Briefly. seriallv diluted and decomplemented sera were Incubated with target cells (5 x l(5) in microtller plates for I hour. The cells were then washed and incubated for 30 minutes with 100 p.L of baby rabbit complement. At the end of this incubation the cells were washed and incubated for an additional :1 hours in HBSS supplemented with 20 p.L of MIT (Sigma Chemical Co. St Louis. Mo). Dimethylsulfoxide (150 p.L) was then added to each well. and the optical density was measured using a kinetiC microplate reader (Molecular Dcviccs. Menlo Park. Calif). RESULTS Skin graft-induced sensitization was tested in a fully allo-geneic BIO -+ C3H liver transplantation model. As previ-ously reported. B 10 liver allografts were spontaneously accepted and survived> 100 days in C3H recipients without any immunosuppressive therapy." However. after skin pre-sensitization. the liver grafts were uniformly rejected in 4 to 5 days. Kinetic studies demonstrated that donor skin grafts took at least 2 weeks to fully sensitize the recipients. and sensitization status was stable for up to 3 months. As shown in Table I. B10 heart or liver grafts. when transplanted into C3H recipients that had received a skin graft from B10. B6. or C2D mice 14 to 21 days earlier. were rejected in an accelerated fashion: however. this phenom-enon was not observed in recipients that had been presen-sitized with either b2m. BlO.BR. or BALB/c skin grafts. These results suggest that a skin graft expressing donor-specific MHC class II antigens alone and/or donor specific non-MHC-histocompatibility antigens (non-MHC-HA) is incapable of sensitizing the recipient sufficiently to induce accelerated rejection of a subsequently transplanted whole organ. On the other hand. skin grafts expressing only donor MHC class I antigens were able to induce significant sensitization in vivo. as evidenced by the accelerated rejec-tion of the subsequently transplanted heart or liver al-lografts. The immune status of the skin-sensitized C3H mice was From the Pittsburgh Transplantation Institute. University of Pittsburgh Medical Center. Pittsburgh. Pennsylvania. Supported in part by NIH Grant OK 29961. Address reprint requests to Shiguang Oian. MD. Pittsburgh Transplantation Institute. University of Pittsburgh Medical Cen-ter. E1540 Biomedical Science Tower. 200 Lothrop Street. Pittsburgh. PA 15213. © 1995 by Appleton & Lange 0041-1345/95/$3.001 + 0 475 Ttansplantation Proceedings, Vol 27. No 1 (February), 1995: pp 475-476 476 OIAN. FU. LI ET AL Table 1. Survival of 810 Heart or Uver Allografts In Skln-Preaensitized C3H Recipients SUl'VIVal (MSn (days) Skin donor Sensitization loci Heart l.iVef B. 8. 8. 8. 9 (8) 35. >100, >100, >100, >100 (>100) 810 MHC. non-MHC-HA 4. 4, 5. 5. 5 (5) 4, 4, 4, 4, 5 (4) 86 MHC 3. 3, 4. 4, 5 (4) 4. 5. 5. 5. 8 (5) b2m " 7, 7, 7, 8. B. 9 (7. 5) 20.35, >100. >100, >100, >100 (>1001 C2D 3. 3, 4. 4, 4 (4) 3. 4, 4. 4. 4 (4) 810.BR Non-MHC-HA 6, 7,7,7. 7.8 (7) 21.28, >100. >100, >100, >100 (>100) 8ALB/c Third-party B, 5, 9. 9. 9, 9 (9) 15.51. >100, >100. >100, >100 (>100) tested in vitro MLR. CI'L. and ADCC assavs, An elevated level of H-i'-specific cytotoxicity was detected in fresh splenocytes isolated from C3H recipients that had received skin grafts from either B1O. B6. or C2D mice. but not from b2m, BlO.BR. or BALB/c donors. Furthennore. this ele-vated donor-specific cytotoxicity correlated well with the in vivo observations in which accelerated rejection of a subse-quently transplanted BlO heart or liver allograft was ob-served. MLR and ADCC assays did not correlate with these in vivo findings, although b2m skin grafts induced a similar level of donor-specific ADCC in response to skin allografts from C2D mice (Table 1). DISCUSSION It has been demonstrated that presensitization with fully disparate donor skin grafts can lead to accelerated rejection of a subsequently transplanted heart or liver allograft. The sensitizing antigens appear to be restricted to MHC, since BlO.BR skin (which shares the same non-MHC-HA with B1O) does not lead to accelerated rejection of either B10 heart or liver grafts. Within the MHC loci, mismatched class I antigens appear to be crucial in sensitizing the recipients leading to accelerated rejection of heart and liver grafts. whereas class II antigens alone are inefficient in generating sufficient sensitization to cause subsequent ac-celerated rejection. It has long been debated whether MHC class I and class II antigens play different roles in the immune response to allografts.7 In the present study, presensitization by C2D skin grafts (MHC class II deficient) led to generation of CI'L. while exposure to b2m skin (class I deficient) did not. This correlated with the pattern of rejection seen in a subsequently transplanted vascularized whole organ. These results confirm previous findings that the accelerated ("sec-ond set") rejection is caused primarily by presensitized cytotoxic T cellss in contrast to the primary ("first set") rejection which is initiated mainly by the donor MHC class II antigens which are known to stimulate helper T cells. 'J Furthermore, pre sensitization with b2m and C2D skin grafts induced a similar level of donor-specific cytotoxic antibody response: however. sensitization with class I-deli-cient skin grafts failed to induce accelerated rejection of the subsequently transplanted whole organ. This suggests that the presence of donor-specific cytotoxic antibodies does not correlate with accelerated rejection. In fact. the presence 01 circulating donor-specific MHC class II antibodies hav\: been shown to be unlikely mediators of accelerated or hyperacute rejection and. in some circumstances. they have been documented to prolong allograft survival. IU It is interesting to note that, in clinical histocompatibility test-ing, the presence of donor-specific class II antibody is not considered a contraindication for transplantation. REFERENCES 1. Zijilstra M. Bix M. Smister NE. et al: Nature (Lond) 344:742. 1990 2. Grusby MJ. Johnson RS. Papaioannon YE. et al: Science (Washington. DC) 253:1417. 1991 3. Qian S. Fung JJ. Demetris AJ. et al: Transplantation 52:562. 1991 4. Ono K. Lindsey ES: J Thorac Cardiovasc Surg 57:225. 19t1'l 5. Terasaki PI. Bernoco D. Park MS. et al: Am J Clin Pathol 69: 103. 1978 6. Qian S. Demetris AJ. Murase N. et al: Hepatology 19:916. 1994 7. Fishbein JM. Rosengard BR. Gianello P. et al: Transplanta-tion 57:1303, 1994 8. Haisch CEo Deepe RM. Gordon DA. et al: Transplant Proc 15:1148, 1983 9. Campos L Naji A. Dei! BC. et al: American Society of Transplant Surgeons (Chicago. 1lI). 1994. P 191 10. Harrv p. von Willebrand E. Parthenas E: Immunol Rev 77:85, 1984' ............ -----------------1 

The use of MHC class I or class II 'knock out' mice to investigate the role of these antigens in allosensitization

http://d-scholarship.pitt.edu/5127/1/31735062125491.pdf

The use of MHC class I or class II 'knock out' mice to investigate the role of these antigens in allosensitization

Abstract

Similar works

Full text

Available Versions

Name not available

Name not available

D-Scholarship@Pitt