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HLA Class II expression and cellular immunity
Abstracts
134
P733 ASSOCIATIONBETWEENLONG TERMKIDNEY GRAFTSURVIVAL AND '!HE PRESENCE
OF PRE 'IRANSPLANT CYTOTOXIC AHTI-II\..l AND/OR NON-IlIlC "Fc-GAllllA-1lII
BLOCKING"(AJm.TlX) ALLOAN11BODY
Agnes Padinyi, Eva GyM, KalaIin Rajczy, Ferenc Perner,Gl'6Z6G. Petrartli NationalII'6li1!AeofHaematology,Blood TransfIJsionand Immunology,Budapest,HungaJy
SevenII explanationsareknown to unde!sfaIld the "beneficialtransfusioo effect" 00 kidney lJa/l survival, mostof them based 00 the presence of vaJious alloanlibodieswith 'bIccking"~ 00 certain in Yilro immune function.Todarifytherole of antibodieswith erythroeyte-an1ibodyIt8lIt&intibilion lEAl) funclioo lD4l00gtsrm(nine yeaI$) SlJI'Iivif1lkidney graft recipienlslIlllI8 analysedretrospectivelywith regaJlltothepresenceofEAI (Fc-gamma-RII bIockif1l) a cyIoloxicHLA antibody induced by Jl'e transpIan1 transfusion. Previous studies rEiYealed thatlransfusioo iOOuoed EAt antibody may have Sjlllcifi<;il¥ 10 TLXlCD46IMCP aIIoantigsns. A st.pBIia graft SlIml (65%19 yeaI$) was fOl.lld in the presence of EAI aIloanIibodycomparedtograft SUlvival in theabsaIlce oflhis antibody (40%19 years). Further analysis showed theflJIlowingsurvival IlIlesin reIaIion to thecombined appeal1Ince of HLA cytotoxicand EAI b1cckingantibody:EAt pes. HLA neg. 67%19 yeaI$; EAI pos., HLA pes. 50'019 years; EAI neg., HLA pes. 0%19 years; EAI neg. HLA neg. 40%19 years. There was S1rikif1l1ow lJa/l faillnin thefirst 3-6monthsinpatienlswith EAI antibody.Taldf1l intoconsiderationthat theHLA BlOR mismatching gradein all various groups was thesame and no considerable differencewas found inassociationto lJa/l SUlvival, thepresence or absenceof EAt blocking (alpha-TlX) antibody solely seems to have superior (;j( additional effect on graft SllNival as comparedtoHLA matd1ng.The immunogeneticpre!lIQUisiteconcerningKLA and TLX a1lotypes matCtingbetween transfusiondonaand recipientto indu;:e the"SlJIlIlI"l'SSiveimmLmregUlalion" is_ted.
P735 PANEL REACTIVE ANTIBODIES POST-KIDNEY TRANSPLANTATION
P734
Usefulness of post-transplantmonitoring by Flow crossmatch in the prevention of graft loss in a LAD renal recipient. Gopal Krishnan, Nassar Youssef, Darri.eI Cook and John Capalli. Our Lady of Lourdes Madical Canter, Camdan, NJ, USA and The Cleveland Clinic Foundation, Cleveland, OK,USA_ We present herewith the evidence for a successful transplantationemploying post-transplantcross-
match technique. Mr"XII
,
an Afro-American male received a living
related kidney from his sister who had only IB, IDA mismatched antigens. Although the transplantwas technically successful,
signs of acute accelerated rejection were noticed on the I
third day. FK-506 raplaced CyA immunosuppressant.OKT3 therapy was initiated and continued for 14 days. Although creatinine level dropped to 2.5 mg%, the patient had to be admitted again within 3 days. Inspi.t.e of treatments with ATG, steroid pulsa, prostaglandinEdrip and cytogam/ganciclovir for CMV, the rejection could not be reversed. Post-transplantcrossmatch against donor T and B lymphocytes by microlymphocytotoxicity test did not reveal any positive reaction. However flow cytometry crossmatch showed a strong binding of the serum to donor B lymphocytes. The antibody was of 19G type. Plasmapherasis( 5X) reduced the binding of the serum to B lymphocytes severalfold. At this time, the patiant's creatinine stabilized at 3.0 mg%. The patient has been clinically doing well 12 months post-transplant.Since the Flow PRA indicated a low percentage(4%) with no specificity against a panel of T cells, the antibody that was responsibla for the initial rejections '\-ias proDobly of class II antibody of 19G type.
P736
IMMUNOLOGIC MONITORING IN PATIENTS WITH AUTOLOGOUS BONE MARROW TRANSPLANTATION
MongkolsukTasanee 1, SumethkulVasant 2,SujirachatoKanchana 1, ehlewsilpPimol1. 1Histocompatibility & ImmunogeneticsLaboratory,SirikitMedical Centerand 2Departmentof Medicine,Facultyof Medicine, RamathibodiHospital,Bangkok,Thailand.
Bubnova LUdmila, Rozanova Olga, Glazanova Tatiana, Pavlova Irina, Moiseev Sergey,AbdulkadyrovKudrat Institute ofHematology& Transfusiology,St.Petersburg, Russia.
This study was aimed to evaluate the panel reactive antibodies (PRA) post kidney transplantation(KT). A total number of 90 KT recipientsconsistedof 71 male and 19 female patients. Thirty-two haploidenticaland 3 HLA-identicalpairs for livingrelated KT and 55 cadaveric KT with 3-6 mismatched antigenswere included in this study. The an\llysis revealed that there were 2 out of 69 (2.89%) patientswith no episodeof rejectionhad PRA-T and or PRA-B > 80% Pre-KT, while they were 5.79% and 23.19%for Post-KT. No patientin 21 cases with KT rejection had Pre-KT-PRA-T and -B > 80%. There were significantincreaseof antibodiesPost-KT rejection,whichwere 28.57% for Post-KT -PRA-T and -B. None of 3 cases with g raft failure (GF) from chronicrejectionhad Pre-KT-PRA-T and -B >20% and only one of them had Post-KT-PRA-T " 80%. Nodonor apeclfic HLA antibodywas found among this groupof patients.Althoughantibody to donor HLA antigens was not observed in these patients, the increase of PRA-T and -B Post-KT mayindicate the immunological reactionresultingin GF.
Peripheral blood lymphoid subsets have been determined in 16 patients with acute leukemia during autologous bone marrow transplantation (ABMT). 9 pts had event free survival (1st group), 7 pts had early relapse (2nd group). Lymphoid subsets' levels differed insignificantly in both groups taken before conditioning, performed on standard TACC or TAACC protocols. But 30 days after ABMT CD3+ and CD4+ decreased 1,2 times, and CD8+ -1,6 times in 2nd group compared to those in 1st group. Later, up to 60-90th day, CD3+ and CD4+ levels became similiar in pts of both groups, and they nearly returned to initial (pre-transplant) level, while CD8+ remained significantlylower in 2nd group. CD22+ levels were similiar in both groups studied. Thus, more marked deficiencyof T-cells have been found in pts with early relapse after ABMT comparing to event free survival. SerumTNFCt level have been determined in 11 pts : 9 - 1st group described above and 2 - with ABMT performed in early relapse. Though basicTNFCt level in 1st group varied widely (0,0381,8 ng/ml),it increased significantly after conditioning in all cases.
P737
P738 AUTOLOGOUS BMf MONITORING BY PCR AND
HLA CLASS II EXPRESSIONAND CELLULARIMMUNITY Fizet D. (I), Letellier C.(I), Perel Y. (2), Reiffers J. (3), Vezon G. (I). (I) Laboratoire de Culture Cellulaire - C.R.T.S.Bordeaux (2) Hopital Pediatrique- CHU Bordeaux (3) Service des Maladies du Sang - Hopital Haut Leveque - Pessac HLA-DR - DP mismatchare known to induceproliferativeresponseon MLR I, but several autors havedemonstratedDP differencedoes not influence GVH. Some MLR arenegative with I or 2HLA-DPmismatch orpositive with 0 HLA-DP mismatch. We have noticed that for the same patient, MLR Ibecame more positive (RR = 40 to 89).To understandif the expressionrate of the MHC Class II Ag have an effect on MLRresponse, we have aoalyse these Ag by flow cytomerry. Theexpressionof HLA-DP aod HLA-DR was stable in donors(DR; 23,8 ± 8,7 % ;DP : 18,6 ± 9,8 %)and not altered bycryopreservation.However. imponantfluctuations of rateexpressionof the MHC ClassII Ag were noticed in patients (DR: 24,3 ± 19,8 %); DP; 23,3 ± 19,6 %) studied at various stages ot their diseases: duringInterferontreatmentfor some CML, fullremissionafter induction therapy or intensive combinationtherapies for ALL and AML. The rate 0 the expressionof HLA-DRaod HLA-DPseems to have aninfluence on RR value more cases to The MHC ClassII Ag are stimulatorsAg during MLR I but may car be mainly consideredas activation Ag andconsequentlycan be determinant for the proliferative response.
ELECTROPHORESIS
CAPILLARY
ComeauFrancois,Merel Patrick, Dupin Brigitte, Francois-XavierMahon, Reiffers Josy, Vezon Gerard, Etablissementde TransfusionSanguine d' Aquitaine, Bordeaux et unite de greffe de moelle, HopitalHaut-Leveque,Pessac, France. Amplification of VNTR and STR are now widely used forcomparison of recipient and donor genetic material in Bone Marrow transplantation monitoring. Moreover documentation of STR Loci on the X chromosome allows now to design molecular tests for Autologous BMf monitoring. Resolution of STR alleles is however labor intensive and difficult to appreciate quantitatively. For this reason we have developed a clonality assessment analysis by capillaryelectrophoresis(CE). DNAs from female patients are amplified by PCR at the HumARA Locus for heterozygoty statement. Heterozygotes female patients DNAs atdiagnostic and after graft are then digested with methylation a sensitive restriction enzyme, amplified at HumARA and analyzed with a PACEILIF 5510 capillary electrophoresis system from BeckmanInstruments. Clonality of 51 women has been analyzed. A control study from 36 hematologically healthy women indicates a rate of 13,8% skewed methylation. IS patients at diagnostic and at several times after autologous bone marrowtransplantationwere analyzed for clonality. This study shows a predominant clonal status at diagnostic, and a polyclonality after hematopoietic reconstitution.in favor of a complete remission. It thus appears that clonality assessment can be a potential diagnostic autologous for BMT monitoring and that CE with its simplicity of use, speed and high level of sensitivity will bring the quantitative aspect to this new level of information.
134
P733 ASSOCIATIONBETWEENLONG TERMKIDNEY GRAFTSURVIVAL AND '!HE PRESENCE
OF PRE 'IRANSPLANT CYTOTOXIC AHTI-II\..l AND/OR NON-IlIlC "Fc-GAllllA-1lII
BLOCKING"(AJm.TlX) ALLOAN11BODY
Agnes Padinyi, Eva GyM, KalaIin Rajczy, Ferenc Perner,Gl'6Z6G. Petrartli NationalII'6li1!AeofHaematology,Blood TransfIJsionand Immunology,Budapest,HungaJy
SevenII explanationsareknown to unde!sfaIld the "beneficialtransfusioo effect" 00 kidney lJa/l survival, mostof them based 00 the presence of vaJious alloanlibodieswith 'bIccking"~ 00 certain in Yilro immune function.Todarifytherole of antibodieswith erythroeyte-an1ibodyIt8lIt&intibilion lEAl) funclioo lD4l00gtsrm(nine yeaI$) SlJI'Iivif1lkidney graft recipienlslIlllI8 analysedretrospectivelywith regaJlltothepresenceofEAI (Fc-gamma-RII bIockif1l) a cyIoloxicHLA antibody induced by Jl'e transpIan1 transfusion. Previous studies rEiYealed thatlransfusioo iOOuoed EAt antibody may have Sjlllcifi<;il¥ 10 TLXlCD46IMCP aIIoantigsns. A st.pBIia graft SlIml (65%19 yeaI$) was fOl.lld in the presence of EAI aIloanIibodycomparedtograft SUlvival in theabsaIlce oflhis antibody (40%19 years). Further analysis showed theflJIlowingsurvival IlIlesin reIaIion to thecombined appeal1Ince of HLA cytotoxicand EAI b1cckingantibody:EAt pes. HLA neg. 67%19 yeaI$; EAI pos., HLA pes. 50'019 years; EAI neg., HLA pes. 0%19 years; EAI neg. HLA neg. 40%19 years. There was S1rikif1l1ow lJa/l faillnin thefirst 3-6monthsinpatienlswith EAI antibody.Taldf1l intoconsiderationthat theHLA BlOR mismatching gradein all various groups was thesame and no considerable differencewas found inassociationto lJa/l SUlvival, thepresence or absenceof EAt blocking (alpha-TlX) antibody solely seems to have superior (;j( additional effect on graft SllNival as comparedtoHLA matd1ng.The immunogeneticpre!lIQUisiteconcerningKLA and TLX a1lotypes matCtingbetween transfusiondonaand recipientto indu;:e the"SlJIlIlI"l'SSiveimmLmregUlalion" is_ted.
P735 PANEL REACTIVE ANTIBODIES POST-KIDNEY TRANSPLANTATION
P734
Usefulness of post-transplantmonitoring by Flow crossmatch in the prevention of graft loss in a LAD renal recipient. Gopal Krishnan, Nassar Youssef, Darri.eI Cook and John Capalli. Our Lady of Lourdes Madical Canter, Camdan, NJ, USA and The Cleveland Clinic Foundation, Cleveland, OK,USA_ We present herewith the evidence for a successful transplantationemploying post-transplantcross-
match technique. Mr"XII
,
an Afro-American male received a living
related kidney from his sister who had only IB, IDA mismatched antigens. Although the transplantwas technically successful,
signs of acute accelerated rejection were noticed on the I
third day. FK-506 raplaced CyA immunosuppressant.OKT3 therapy was initiated and continued for 14 days. Although creatinine level dropped to 2.5 mg%, the patient had to be admitted again within 3 days. Inspi.t.e of treatments with ATG, steroid pulsa, prostaglandinEdrip and cytogam/ganciclovir for CMV, the rejection could not be reversed. Post-transplantcrossmatch against donor T and B lymphocytes by microlymphocytotoxicity test did not reveal any positive reaction. However flow cytometry crossmatch showed a strong binding of the serum to donor B lymphocytes. The antibody was of 19G type. Plasmapherasis( 5X) reduced the binding of the serum to B lymphocytes severalfold. At this time, the patiant's creatinine stabilized at 3.0 mg%. The patient has been clinically doing well 12 months post-transplant.Since the Flow PRA indicated a low percentage(4%) with no specificity against a panel of T cells, the antibody that was responsibla for the initial rejections '\-ias proDobly of class II antibody of 19G type.
P736
IMMUNOLOGIC MONITORING IN PATIENTS WITH AUTOLOGOUS BONE MARROW TRANSPLANTATION
MongkolsukTasanee 1, SumethkulVasant 2,SujirachatoKanchana 1, ehlewsilpPimol1. 1Histocompatibility & ImmunogeneticsLaboratory,SirikitMedical Centerand 2Departmentof Medicine,Facultyof Medicine, RamathibodiHospital,Bangkok,Thailand.
Bubnova LUdmila, Rozanova Olga, Glazanova Tatiana, Pavlova Irina, Moiseev Sergey,AbdulkadyrovKudrat Institute ofHematology& Transfusiology,St.Petersburg, Russia.
This study was aimed to evaluate the panel reactive antibodies (PRA) post kidney transplantation(KT). A total number of 90 KT recipientsconsistedof 71 male and 19 female patients. Thirty-two haploidenticaland 3 HLA-identicalpairs for livingrelated KT and 55 cadaveric KT with 3-6 mismatched antigenswere included in this study. The an\llysis revealed that there were 2 out of 69 (2.89%) patientswith no episodeof rejectionhad PRA-T and or PRA-B > 80% Pre-KT, while they were 5.79% and 23.19%for Post-KT. No patientin 21 cases with KT rejection had Pre-KT-PRA-T and -B > 80%. There were significantincreaseof antibodiesPost-KT rejection,whichwere 28.57% for Post-KT -PRA-T and -B. None of 3 cases with g raft failure (GF) from chronicrejectionhad Pre-KT-PRA-T and -B >20% and only one of them had Post-KT-PRA-T " 80%. Nodonor apeclfic HLA antibodywas found among this groupof patients.Althoughantibody to donor HLA antigens was not observed in these patients, the increase of PRA-T and -B Post-KT mayindicate the immunological reactionresultingin GF.
Peripheral blood lymphoid subsets have been determined in 16 patients with acute leukemia during autologous bone marrow transplantation (ABMT). 9 pts had event free survival (1st group), 7 pts had early relapse (2nd group). Lymphoid subsets' levels differed insignificantly in both groups taken before conditioning, performed on standard TACC or TAACC protocols. But 30 days after ABMT CD3+ and CD4+ decreased 1,2 times, and CD8+ -1,6 times in 2nd group compared to those in 1st group. Later, up to 60-90th day, CD3+ and CD4+ levels became similiar in pts of both groups, and they nearly returned to initial (pre-transplant) level, while CD8+ remained significantlylower in 2nd group. CD22+ levels were similiar in both groups studied. Thus, more marked deficiencyof T-cells have been found in pts with early relapse after ABMT comparing to event free survival. SerumTNFCt level have been determined in 11 pts : 9 - 1st group described above and 2 - with ABMT performed in early relapse. Though basicTNFCt level in 1st group varied widely (0,0381,8 ng/ml),it increased significantly after conditioning in all cases.
P737
P738 AUTOLOGOUS BMf MONITORING BY PCR AND
HLA CLASS II EXPRESSIONAND CELLULARIMMUNITY Fizet D. (I), Letellier C.(I), Perel Y. (2), Reiffers J. (3), Vezon G. (I). (I) Laboratoire de Culture Cellulaire - C.R.T.S.Bordeaux (2) Hopital Pediatrique- CHU Bordeaux (3) Service des Maladies du Sang - Hopital Haut Leveque - Pessac HLA-DR - DP mismatchare known to induceproliferativeresponseon MLR I, but several autors havedemonstratedDP differencedoes not influence GVH. Some MLR arenegative with I or 2HLA-DPmismatch orpositive with 0 HLA-DP mismatch. We have noticed that for the same patient, MLR Ibecame more positive (RR = 40 to 89).To understandif the expressionrate of the MHC Class II Ag have an effect on MLRresponse, we have aoalyse these Ag by flow cytomerry. Theexpressionof HLA-DP aod HLA-DR was stable in donors(DR; 23,8 ± 8,7 % ;DP : 18,6 ± 9,8 %)and not altered bycryopreservation.However. imponantfluctuations of rateexpressionof the MHC ClassII Ag were noticed in patients (DR: 24,3 ± 19,8 %); DP; 23,3 ± 19,6 %) studied at various stages ot their diseases: duringInterferontreatmentfor some CML, fullremissionafter induction therapy or intensive combinationtherapies for ALL and AML. The rate 0 the expressionof HLA-DRaod HLA-DPseems to have aninfluence on RR value more cases to The MHC ClassII Ag are stimulatorsAg during MLR I but may car be mainly consideredas activation Ag andconsequentlycan be determinant for the proliferative response.
ELECTROPHORESIS
CAPILLARY
ComeauFrancois,Merel Patrick, Dupin Brigitte, Francois-XavierMahon, Reiffers Josy, Vezon Gerard, Etablissementde TransfusionSanguine d' Aquitaine, Bordeaux et unite de greffe de moelle, HopitalHaut-Leveque,Pessac, France. Amplification of VNTR and STR are now widely used forcomparison of recipient and donor genetic material in Bone Marrow transplantation monitoring. Moreover documentation of STR Loci on the X chromosome allows now to design molecular tests for Autologous BMf monitoring. Resolution of STR alleles is however labor intensive and difficult to appreciate quantitatively. For this reason we have developed a clonality assessment analysis by capillaryelectrophoresis(CE). DNAs from female patients are amplified by PCR at the HumARA Locus for heterozygoty statement. Heterozygotes female patients DNAs atdiagnostic and after graft are then digested with methylation a sensitive restriction enzyme, amplified at HumARA and analyzed with a PACEILIF 5510 capillary electrophoresis system from BeckmanInstruments. Clonality of 51 women has been analyzed. A control study from 36 hematologically healthy women indicates a rate of 13,8% skewed methylation. IS patients at diagnostic and at several times after autologous bone marrowtransplantationwere analyzed for clonality. This study shows a predominant clonal status at diagnostic, and a polyclonality after hematopoietic reconstitution.in favor of a complete remission. It thus appears that clonality assessment can be a potential diagnostic autologous for BMT monitoring and that CE with its simplicity of use, speed and high level of sensitivity will bring the quantitative aspect to this new level of information.