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Bone marrow transplantation with CD34 selected cells : The role of CD34 different epitopes expression
Abstracts
P483 EXAMINING HEMATOLOGICAL MARROW TRANSPLANTATION
89
PATIENTS PRIOR TO BONE
B.Beli6;D.Pejin~ N.Stefanovi6~ A.Beli6~
R.JOVanovi6 1 1 Institute of Blood Transfusion,Novi Sad 2 Faoulty of Medioine. Centre for Transplantation Clinio for Hematology,Novi Sad, Yugoslavia At the Institute of Blood Transfusion in Novi Sad 86 patients with various hematologioal diseases {aplastio anemia,aoute and ohronio leukemia, malignant lymphoma)were examined during the period January 1986 to August 1995.248 family members (fathers, mothers, sisters and brothers)were examined.The following prooedures were used: determination of blood groups ABO,Rh,MNS,Kell.Duffy, Kidd and Lutheran.determination of HLA antigen for A, Band C locus. in identical donors cross match was done for D and DR antigen locus. the HLA system as well as MLC.Out of 86 patients. 28 patients had identical bone marrow donors and 10 patients underwent bone marrow transplantation. 8 patients had positive answers and remissions, while chronio comlications suoh as: chronic GVHD. relapses and late rejection significantly influenoed the final outcome of the transplantation.
P485
RECOVERY OF IMMUNE CELLS AFTER ALLOGENEIC BONE MARROW TRANSPLANTATION USING DIFFERENT CONDmONING REGIMENS
P484
THE IMPORTANCE OF NON.HLA rnX·8) AU.oANTIGENS IN RELATED BMT ~, A.PalliiD)'i, E.Gy6di, K...Rajczy, R.Dme:s. -T.MlIlszi, ~.Krivlin. Gy.Periayi
Nad. bl.of HaemaIoJogy. Blood Transfusioo aDd 1nummo1usr. Butbpcst, ·Szt.Usz16 Hotp. BJdape!ll. Huoaary
Non-HLA rminor ft ) histocompatibility antigens (mH) might have an additional role in the induction of OVH reactions. especially in genotypically identical sibling pairs. One representative of the n.oo-HLA antigens may be the TLX-B, whicb appears to be associated with the CD46 glikoprotein and functionally associaled with the FeRIi. Using 10 alloimmune sera collected from donors af"" planned immunization, 5 dilf""",' TLXB "clusters" were established 00 Ihe pane] of tOO unrelated individuals. Family studies revealed that TLX-B alleles inberited independently fl'Olll HLA alleles. 5ince anti-TLX-8 antibodies with FcRJI blocking feature incb:ed by transfUsion bas been shown 10 have a beneficial effect 011 kidney graft survival and OIl the wteome of pregnancy, it seemed reasonably to perform a study on TLX-B malChing in lite 8MT program focusing on lite OYH am! OVL complicatioos. Thirty HLA identical sib pairs were typed for TLXB antigens. Haematological diseases were as follows: CML (0=12). AML (0=12). ALL (0=2). SAA (0=4). Cooditiooing proUlCOls were Cyclopbospbamid. ARA-{: am! DBM or TRI and eyelopltosplwDid. MTX and Cyclosporin-A were used for OVH preventioo. Only 9 fl'Olll 30 HLA identical sib pain were TLX-B identical as well. Tv.u patients (22%) of thmx are iII complete retnission without any sign for GVHD. 5 patients had acute OYHD. 2 died shonly after I1llIlSpiantatiOO. TLX-B antig_ were dilf"""'t in 21 sibpain. 17 of the patienLs are alive. 4 patients died. Seven patients developed clinically significant .... te OVHD. 2 of them died. OVHD was no< - . c d in 14 cases (66%), 3 patients relapsed (2 of them died) and 11 patients (52%) are in complete remissioo. CNchrsiM: aJthwgh the number of cases is very limited. it seems that disparity in TLX-B alleles in HLA identical sib pairs has a favourite effect for avoiding the GVHD a550ciated 'Wi.tb the indllclioo of cenain suppressive mecbaoism similar to the "beeefieial transfusion effect" in kidney transplantation. This wort was supporteclin part by Hungarian Miniill}' of WcD&rc (ETI'~T227/1993), by Ihc Enropean FOUDdatioo for Sctcnces, Arta aud Culture, Paris-Amstcrdam.
P486
IHE DIVERSITY OF HUIWI IlEKAIOPOIEIIC SIEH/PROGElHIOR CELLS'! I.l1O--COLOR FLOW CYIOMEIRIC ANALYSIS OF THE DlFFERENl FUNCTIONAL SUBPOPULAIIONS OF C03(+ HEIlAIOPOIEIIC SIEMlPROCENlIOR CELLS RICKED FROM IIUIIAN BONE !WlIlOW
Katahn Piil6czi, Janos Milosevits, Peter IDes, Robin Ezsi, Robert Denes, Anna Pores, Eva Torbagyi, Endre Kelemen, Gy6z6 Petranyi
XI Yoq:-Zhf .. Zhang Shuanl-XI,. 10ftl Fen-Hua, Wei Wen, and Tanl Pei-Hsien Department of IumunolOlY' Arfitated 307 Hospital, Beljinl' China,. 100039
National Institute of Haematology and Immunology, Budapest, Hungary
In hematopoiesi •• hUll8ft eM4 protein is a strict deveJopmental stage-specifio antigen that marks bematopoietic stelllprogenitor cells, suuesttina tha.t it plays essential role in hematopoiests. More recent Iy, t t has been demonstrated that hematopoietic cells expressing CD34 antigen constitute var ious heterogeneous eeJ I populaHons in which each C03(+ subset was associated "i tb commitment to a particular Ilneaae. and differed !rOIl other's in reconstituting hematopoiesis. In this report. we have assessed the different funotional subpopulation of CDU+ hematopoietio stemiprOienitor enriohed 'rOlD hu_n bone II&rrow uslftK Isolex™ 50 system according to the strategy on immunolPll&lletio separat ion of positive setect Ion, C004+ oell population of high pur UyO 80~O was analyzed by Deans of double staining prooedure of fluorescein conjugated monoolonal antibodies on the two-color FACS440.Eigbt cell subsets or parlfied C034+ hematopoietlo stem/progenitor cells have been defined by undertaking a comparative coexpeess ing of CDl1.CD45, COOS and HLA-DR antigens on C034+ hematopoietic stemiprOieni tor cells. The frequences of var lous subsets in three
Conditioning regimens for bone marrow transplantation (BMT) are important in determining transplant outcome. A radiation-free protocol containing Mitobromtol (DBM), Cytarabine (Are-C) and Cyclophosphamide (Cy) was used for conditioning of patients with chronic myeloid leukemia (CML). To determine the immunological effect of the DBM/Ara-C/Cy conditioning, the recovery of peripheral blood lymphocytes was examined after allogeneic BMT for patients with CML in comparison with TBIICy conditioning. The lymphocyte subsets of eleven DBM-patienlll were followed and analyzed periodically (30-90day., 4-12 month. and >13 months) using ten monoclonal antibodies and flow cytometry. Decreased percentage of Iota! T cells as well as CD4+ and CD8+ subpcpulations, significantly decreased T cell activation and increased proportion of TCRyo+ cells were found 10 be characteristic in the early posursnsplant period in the DBM group. Early recovery and consistently higher percentage of B cells were observed for the whole follow-up period of patients receiving DBM conditioning. A high proportion of NK cells was observed in all transplant recipients. Those finding. suggest that the characteristic pattern of recovering lymphocytes is associated with the lack of severe transplant related clinical complications following DBM/Ara-C/Cy conditioning.
P487 -BONE MARROW TRANSPLANTATION WITH CD34 SELECTED CELLS: THE ROLE OF CD34 DIFFERENT EPITOPES EXPRESSION. Letellier C. (I), Fizet D. (1), Bouzgarrou R. (1), Reiffers J. (2). Vezon G. (I). (I) Laboratoire de Culture Cellulaire - C.R.T.S. - Bordeaux (2) Service des Maladies du Sang - Hopital Haut Leveque - Pessac CD34+ cells were selected from patients's CSS willl various systems : magnetic beads. antibodies affinity, ... It is kwoned that results depend of numerous factors :: number of WBC, contamination wiII1 RBC, number of CD34 cells in platelets concentrates. We obtain a mean yield between 50 - 60 % pure CD34+ suspension. However for some patients this yield was very low <: 20 %. Aside the differents reasons described above, we were interestedin fluorescence intensityby flow cytometry which is correlated with antigen expresssion : bad results are always obtained when fluorescence located in low chanels near the negative cootroL CD34 molecule is composed of three differents epitopes (Class 1, 2. 3) which expression outside the cell membran will be modified by paIhological conditions or treatmeat (like IFN). We have to discover their functions and their relations with HLA-DR, DP Antigens which act in cellular activities.
j Ilustratlve are as rollowlogs, 1l.CD3H/CD1I- and C03H/CD1l+ (23.(3Z-58.80'7. versus 33.(0'7.-88.80'7.1, 2). CD3H/CD45- and CD3H/Cl)(5+ (80.80Z-U.50 versus 8.IOZ-ll.20'7.l; 3). C03H/CD33- and CD3H/CD33+ (20.40'7.-80.80'7. versus 1(.80'7. -8(.80), (I. CD3H/DR- and CD3H/DR+ ( 8.30'7.-11.00'7. venus 82.80'7.-85.50'7.).
ImmunolOlical double color staining of lCiSS-APAAP was developed and a lsc used to further analyse 1he CD34+ eel l subsets as above-mentiorted,1he r85QI18 were very stili lllr to those obtained by FACS440. Our data indicate that CD34+ hematopoietic oell fraotion. is tar from heinl a uniforll oell population, and lIllIly works reaardlng biological properties and .rowth regulation of CDS,H hematopoietic cell subsets are betng carried out.
P483 EXAMINING HEMATOLOGICAL MARROW TRANSPLANTATION
89
PATIENTS PRIOR TO BONE
B.Beli6;D.Pejin~ N.Stefanovi6~ A.Beli6~
R.JOVanovi6 1 1 Institute of Blood Transfusion,Novi Sad 2 Faoulty of Medioine. Centre for Transplantation Clinio for Hematology,Novi Sad, Yugoslavia At the Institute of Blood Transfusion in Novi Sad 86 patients with various hematologioal diseases {aplastio anemia,aoute and ohronio leukemia, malignant lymphoma)were examined during the period January 1986 to August 1995.248 family members (fathers, mothers, sisters and brothers)were examined.The following prooedures were used: determination of blood groups ABO,Rh,MNS,Kell.Duffy, Kidd and Lutheran.determination of HLA antigen for A, Band C locus. in identical donors cross match was done for D and DR antigen locus. the HLA system as well as MLC.Out of 86 patients. 28 patients had identical bone marrow donors and 10 patients underwent bone marrow transplantation. 8 patients had positive answers and remissions, while chronio comlications suoh as: chronic GVHD. relapses and late rejection significantly influenoed the final outcome of the transplantation.
P485
RECOVERY OF IMMUNE CELLS AFTER ALLOGENEIC BONE MARROW TRANSPLANTATION USING DIFFERENT CONDmONING REGIMENS
P484
THE IMPORTANCE OF NON.HLA rnX·8) AU.oANTIGENS IN RELATED BMT ~, A.PalliiD)'i, E.Gy6di, K...Rajczy, R.Dme:s. -T.MlIlszi, ~.Krivlin. Gy.Periayi
Nad. bl.of HaemaIoJogy. Blood Transfusioo aDd 1nummo1usr. Butbpcst, ·Szt.Usz16 Hotp. BJdape!ll. Huoaary
Non-HLA rminor ft ) histocompatibility antigens (mH) might have an additional role in the induction of OVH reactions. especially in genotypically identical sibling pairs. One representative of the n.oo-HLA antigens may be the TLX-B, whicb appears to be associated with the CD46 glikoprotein and functionally associaled with the FeRIi. Using 10 alloimmune sera collected from donors af"" planned immunization, 5 dilf""",' TLXB "clusters" were established 00 Ihe pane] of tOO unrelated individuals. Family studies revealed that TLX-B alleles inberited independently fl'Olll HLA alleles. 5ince anti-TLX-8 antibodies with FcRJI blocking feature incb:ed by transfUsion bas been shown 10 have a beneficial effect 011 kidney graft survival and OIl the wteome of pregnancy, it seemed reasonably to perform a study on TLX-B malChing in lite 8MT program focusing on lite OYH am! OVL complicatioos. Thirty HLA identical sib pairs were typed for TLXB antigens. Haematological diseases were as follows: CML (0=12). AML (0=12). ALL (0=2). SAA (0=4). Cooditiooing proUlCOls were Cyclopbospbamid. ARA-{: am! DBM or TRI and eyelopltosplwDid. MTX and Cyclosporin-A were used for OVH preventioo. Only 9 fl'Olll 30 HLA identical sib pain were TLX-B identical as well. Tv.u patients (22%) of thmx are iII complete retnission without any sign for GVHD. 5 patients had acute OYHD. 2 died shonly after I1llIlSpiantatiOO. TLX-B antig_ were dilf"""'t in 21 sibpain. 17 of the patienLs are alive. 4 patients died. Seven patients developed clinically significant .... te OVHD. 2 of them died. OVHD was no< - . c d in 14 cases (66%), 3 patients relapsed (2 of them died) and 11 patients (52%) are in complete remissioo. CNchrsiM: aJthwgh the number of cases is very limited. it seems that disparity in TLX-B alleles in HLA identical sib pairs has a favourite effect for avoiding the GVHD a550ciated 'Wi.tb the indllclioo of cenain suppressive mecbaoism similar to the "beeefieial transfusion effect" in kidney transplantation. This wort was supporteclin part by Hungarian Miniill}' of WcD&rc (ETI'~T227/1993), by Ihc Enropean FOUDdatioo for Sctcnces, Arta aud Culture, Paris-Amstcrdam.
P486
IHE DIVERSITY OF HUIWI IlEKAIOPOIEIIC SIEH/PROGElHIOR CELLS'! I.l1O--COLOR FLOW CYIOMEIRIC ANALYSIS OF THE DlFFERENl FUNCTIONAL SUBPOPULAIIONS OF C03(+ HEIlAIOPOIEIIC SIEMlPROCENlIOR CELLS RICKED FROM IIUIIAN BONE !WlIlOW
Katahn Piil6czi, Janos Milosevits, Peter IDes, Robin Ezsi, Robert Denes, Anna Pores, Eva Torbagyi, Endre Kelemen, Gy6z6 Petranyi
XI Yoq:-Zhf .. Zhang Shuanl-XI,. 10ftl Fen-Hua, Wei Wen, and Tanl Pei-Hsien Department of IumunolOlY' Arfitated 307 Hospital, Beljinl' China,. 100039
National Institute of Haematology and Immunology, Budapest, Hungary
In hematopoiesi •• hUll8ft eM4 protein is a strict deveJopmental stage-specifio antigen that marks bematopoietic stelllprogenitor cells, suuesttina tha.t it plays essential role in hematopoiests. More recent Iy, t t has been demonstrated that hematopoietic cells expressing CD34 antigen constitute var ious heterogeneous eeJ I populaHons in which each C03(+ subset was associated "i tb commitment to a particular Ilneaae. and differed !rOIl other's in reconstituting hematopoiesis. In this report. we have assessed the different funotional subpopulation of CDU+ hematopoietio stemiprOienitor enriohed 'rOlD hu_n bone II&rrow uslftK Isolex™ 50 system according to the strategy on immunolPll&lletio separat ion of positive setect Ion, C004+ oell population of high pur UyO 80~O was analyzed by Deans of double staining prooedure of fluorescein conjugated monoolonal antibodies on the two-color FACS440.Eigbt cell subsets or parlfied C034+ hematopoietlo stem/progenitor cells have been defined by undertaking a comparative coexpeess ing of CDl1.CD45, COOS and HLA-DR antigens on C034+ hematopoietic stemiprOieni tor cells. The frequences of var lous subsets in three
Conditioning regimens for bone marrow transplantation (BMT) are important in determining transplant outcome. A radiation-free protocol containing Mitobromtol (DBM), Cytarabine (Are-C) and Cyclophosphamide (Cy) was used for conditioning of patients with chronic myeloid leukemia (CML). To determine the immunological effect of the DBM/Ara-C/Cy conditioning, the recovery of peripheral blood lymphocytes was examined after allogeneic BMT for patients with CML in comparison with TBIICy conditioning. The lymphocyte subsets of eleven DBM-patienlll were followed and analyzed periodically (30-90day., 4-12 month. and >13 months) using ten monoclonal antibodies and flow cytometry. Decreased percentage of Iota! T cells as well as CD4+ and CD8+ subpcpulations, significantly decreased T cell activation and increased proportion of TCRyo+ cells were found 10 be characteristic in the early posursnsplant period in the DBM group. Early recovery and consistently higher percentage of B cells were observed for the whole follow-up period of patients receiving DBM conditioning. A high proportion of NK cells was observed in all transplant recipients. Those finding. suggest that the characteristic pattern of recovering lymphocytes is associated with the lack of severe transplant related clinical complications following DBM/Ara-C/Cy conditioning.
P487 -BONE MARROW TRANSPLANTATION WITH CD34 SELECTED CELLS: THE ROLE OF CD34 DIFFERENT EPITOPES EXPRESSION. Letellier C. (I), Fizet D. (1), Bouzgarrou R. (1), Reiffers J. (2). Vezon G. (I). (I) Laboratoire de Culture Cellulaire - C.R.T.S. - Bordeaux (2) Service des Maladies du Sang - Hopital Haut Leveque - Pessac CD34+ cells were selected from patients's CSS willl various systems : magnetic beads. antibodies affinity, ... It is kwoned that results depend of numerous factors :: number of WBC, contamination wiII1 RBC, number of CD34 cells in platelets concentrates. We obtain a mean yield between 50 - 60 % pure CD34+ suspension. However for some patients this yield was very low <: 20 %. Aside the differents reasons described above, we were interestedin fluorescence intensityby flow cytometry which is correlated with antigen expresssion : bad results are always obtained when fluorescence located in low chanels near the negative cootroL CD34 molecule is composed of three differents epitopes (Class 1, 2. 3) which expression outside the cell membran will be modified by paIhological conditions or treatmeat (like IFN). We have to discover their functions and their relations with HLA-DR, DP Antigens which act in cellular activities.
j Ilustratlve are as rollowlogs, 1l.CD3H/CD1I- and C03H/CD1l+ (23.(3Z-58.80'7. versus 33.(0'7.-88.80'7.1, 2). CD3H/CD45- and CD3H/Cl)(5+ (80.80Z-U.50 versus 8.IOZ-ll.20'7.l; 3). C03H/CD33- and CD3H/CD33+ (20.40'7.-80.80'7. versus 1(.80'7. -8(.80), (I. CD3H/DR- and CD3H/DR+ ( 8.30'7.-11.00'7. venus 82.80'7.-85.50'7.).
ImmunolOlical double color staining of lCiSS-APAAP was developed and a lsc used to further analyse 1he CD34+ eel l subsets as above-mentiorted,1he r85QI18 were very stili lllr to those obtained by FACS440. Our data indicate that CD34+ hematopoietic oell fraotion. is tar from heinl a uniforll oell population, and lIllIly works reaardlng biological properties and .rowth regulation of CDS,H hematopoietic cell subsets are betng carried out.