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Identification of the human minor histocompatibility antigen HA-1
464
Neudmmunology
25 June 1997 - Poster presentations
Materlalo and hbthodr: Prompted by their ability to inhibit T- as well as B-cell functions both MNAs have been evaluated for their effects on the in vivo lymphoproliferation that occurs after challenge with allogeneic cells in a local GvH reaction in Lewis x Brown Norway (LBN) FI hybrid rats by measuring the enlargement of the draining popliteal lymph node (PLN) and they have been investigated as to their disease modifying activity on an acute lethaf GvHD in B6C3Fl mice and on a chronic autoimmune GvHD in B6D2Fl hybrid mice. Result% In the PLN assay an oral administration of various concentrations (7.5 to 50 rn@kg) of both MNAs on day 0, dose-dependently inhibited the localized GvH reaction and suppressed the lymph node hyperplasia. Both MNAs acted also therapeutically in this model when they were given dunng an ongoing alloreactivity as late as on day 4 after challenge. The response to the allogeneic cells and the PLN swelling was always inhibited, independent to the time when the nodes were harvested. In the model of an acute lethal GvHD the treatment of the GvH-B6C3Fi mice with vadous concentrations (2.5 to 20 mg/kg/day) of the MNAs shortly after induction of the disease on days 3 to 12 resulted in a dose-dependently improved survival rate. With 20 mg/kdday mortality of this life-threatening GvHD was completely prevented. Also other parameters like splenomegaly, erythrocyte counts and hematocrtte values were strongly suppressed. Treatment of sensitized GvH-BDFl hybrtd mice in the chronic autoimmune-like model with 30 mg&g’day of the MNAs, given on days 6 to 36 after GvH induction resulted in an improved survfval rate, lowered the percentage of animals with lymphadenopathy and splenomegaly, reduced the levels of autoantibodies and the hiah serum titres of IaE. IaGI. and laG2a. and inhibited the development of glom~mlonephntis. Concluelon: These results presented here clearly indicate that both MNA 279 and MNA 715 can inhibit ongoing aberrant immune responses in animals suffering from GvHD and it should be justified to apply these drugs for bone marrow transplantation and the fight against GvHD.
P 5 19 16 jTTJ
Identification of the human minor hlstocompatlbility antigen HA-1
J.M.M. den Haan ‘, W. Wang*, L.R. Meadows3, E. Blokland ‘, of C. Reinhardus ‘, V.H. Engelhard *, D.F. Hunt3, E. Goulmy ‘. ’DePr. lmmunohaematology and Bloodbank, University Hcspid Leiden; RC Leiden, The Netherlands, *Dept. of MicmWobgy and Beime Carter Center for Immunology Research, University of Virginia, VA, USA, 3Dept. of Chemistw University of Virginia, VA, USA Introduction: Disparity for the minor His&compatibility antigen (mHag) HA-1 between HLA-matched bone marrow donor and recipient is corretated with an increased risk for Graft-versus-Host-Disease. This mHag is recognized by HlAA2.1 restricted HA-I specific cytotoxic T cell clones (CTL). but the peptide sequence of HA-1 and the protein encoding this peptide is unknown. Using the HA-1 specific T cell clones we set out to identify the HA-I peptide. Method: HLAA2.1 molecules were purified from HA-1 expressing EVBBLCL by affinity chromatography. The HlAA2.1 bound peptides were eluted and separated from the MHC molecules by acid treatment and a 10 kD filtration step. The peptides were fractionated by reverse phase HPLC and the fractions were tested for HA-1 activity in a cytotoxicity assay with the HA-l specific CTL. Two fractions with HA-1 activity were purified after 2 rounds of HPLC fractionation. These fractions were analysed with microcapillary HPLC/electrospray ionisation tandem mass spectrometry and candidates were selected for sequencing. Resu,ults:Candidate peptide with m/z 513 was sequenced and the synthetic peptide sensitized target cells for lysis by the HA-1 specific CTL. A data base search revealed a sequence with one amino acid difference. Polyrnerase chain reaction (PCR) primers were developed based on this protein in the databank. PCR were performed on HA-1 positive and negative EBV-BLCL and the PCR product was sequenced. Results indicated that the found protein encoded the mHag HA-l. Conclusion:The mHag HA-1 has been sequened and the protein encoding this mHag has been identified. These results might enable the development of diagnostic tools for prevention of Graft-versus-Host Disease and the treatment of leukemia relapses after bone marrow transplantation.
children. These data led us to evaluate changes in serum cytokines and soluble receptors in children with and without acute GVHD. Yatsrlalsand Methods:Sixteen children received allogeneic BMT from HLA identical donors. Profytaxis for GVHD was performed with cyclosportn A, methotrexate and immunoglobulins. Serum IFN gamma, TNF alfa, lL-6, slL-2R, sTNF I, sTNF II concentrations were determined by ELISA Nedgenid in period D-7 to D+64. Serum concentrations of cytokines and soluble receptors in children with GVHD /n = 7/were compared with levels in children without/n = 91. Reeults: Mean serum IL-2R concentrations increased significantly in children with acute GVHD /D + 14 to D + 42, GVHD manifestation D + 16.7 engraftment D + 12.4/hut did not change significantly in children without GVHD /engraftment D + 16.1/. Similarly serum sTNF I and sTNF II increased significantiy only in children with GVHD/D + 7 to D + 70, resp. D + 16 to D + 70/ Significant correlation between sTNF I and sTNF II was found, but not between sTNF and IL-2R. Serum IL-6 concentration increased significantly in both groups of patients immediately after BMT /D + 2/, serum TNF alfa and IFN gamma concentrations did not change significantly in patfents with and without GVHD. Concluston: Peak serum concentrations of slL-2R, sTNF I, sTNF II were found a few days /weeks/ after detection of engraftment /by molecular biology methods/. These receptors may be used for monitoring acute GVHD in children after allogeneic BMT.
m -
MLC reactivltv can be further differentiated bv cytokine measurements
Willemijn M. Wissink, Arnold van der Meer, Jos Ruiter, Wil Ailebes en Ina Joosten. TIsnsplant Serology Laboratory, Blood TransfusionService, University Hospital Nijmegen, The Netherlands Introduction: The value of the mixed lymphocyte culture (MLC) for donor-recipient matching is much debated. Consequently, additional cellular tests (HTLp and CTLp assays) are now assessed on their predictive value for GVHD or allograft rejection, but these am laborious. Already cytokines (as a measure for ThliThP responses) have been associated with transplant failurelsuccess. It is now apparent that simply measudng proliferation after MLC does not provide sufficient information on the nature of the alloreactivity. By extending the standard MLC with addltionel cytoklne measurements we may gain insight in the potential direction of the expected alloresponw and whether or not this reactivity is acceptable by our standards. Material and Methods: We have anafysed effector-stlmulator combinations exhibiting different degrees of HLA matching. Standard one-way MLC, as well as HTLp and CTLp frequency analysts was performed. IFNy, IL-4 and IL-10 were measured after one-way MLC using a sandwich ELISA technique. Irradiated pool cells and irradiated control cells were taken as positve and negative control respectively. Results and Concluelon: The results so far revwl that the HLA class I and II MM combinations, chosen to validate the system, show distinct cytoktne production patterns while all showing high SI values in MLC. Class II MM appear the main stimulus for cytokine production. In combinations with isolated class I MM the levels are very low. In meet MM combinations the Thl cytokine pattern (high ratio IFNy: 114(ILlO)) is predominant. The degree of mismatch appears not predictive of the nature of the alloresponw, we SW both Thl-like and ThP-like responses (as measured by IFNy I IL4 ratio on day 5 of culturing). In the combinations with a shift from a Thl to a more Th2 like response higher levels of IL-10 are found. The HTLp and CTLp frequencies not always correlate with the cytokine profiles. The cytokine measurements can differentiate between MLC combinations where HiA matching, MLC proliferation and the HTLp/CTLp assay don’t. This will be further investigated. We aim to do so by retrospective analysis of BMT donor -recipient combinations.
09:00-18:30/12:00-14:00
j-Y-Iq P 5 19 17
Serum concentrations of the soluble 11-2, TNF I and TNF II receptors for monltorlng acute graft-versus-host dlsease In children
Forum lounges
P.5.20
Neuroimmunology
P.5.20.01
Autoentibodles to brain tlssue in rat sers
,
J. Vaviinec’, D. PospfSilova*, J. Stal)i' V. Vavra’, L. Honzatkova3, 2. Sieglova 3. ’2nd Pediatric Department, Prague, Czech RepuWic,*Institute of Immunobg~ Prague, Czech Republic, 32nd medical Faculty; Institute of Hematology and Blood Transfusion, Prague, Czech Republic Introduction: Acute GVHD is perceived as donor T-cell mediated reaction and inflammatory cytokines play a important rote in activating of these cells. Elevated serum soluble IL-2 receptor concentration have been reported in adults with GVHD after allogeneic BMT, but limited number of information is available in
I. Staiikova, L. Prokesova, S. Trojan. First Faculty of Medicine, Charles lJniversi& Prague, Czech Republic Introduotlon: The increased levels of autoantibodies against neural tissue can serve as a marker of anoxic damage of the brain. Antibodies to neural tissue were tested in normal and anoxic rats.
Neudmmunology
25 June 1997 - Poster presentations
Materlalo and hbthodr: Prompted by their ability to inhibit T- as well as B-cell functions both MNAs have been evaluated for their effects on the in vivo lymphoproliferation that occurs after challenge with allogeneic cells in a local GvH reaction in Lewis x Brown Norway (LBN) FI hybrid rats by measuring the enlargement of the draining popliteal lymph node (PLN) and they have been investigated as to their disease modifying activity on an acute lethaf GvHD in B6C3Fl mice and on a chronic autoimmune GvHD in B6D2Fl hybrid mice. Result% In the PLN assay an oral administration of various concentrations (7.5 to 50 rn@kg) of both MNAs on day 0, dose-dependently inhibited the localized GvH reaction and suppressed the lymph node hyperplasia. Both MNAs acted also therapeutically in this model when they were given dunng an ongoing alloreactivity as late as on day 4 after challenge. The response to the allogeneic cells and the PLN swelling was always inhibited, independent to the time when the nodes were harvested. In the model of an acute lethal GvHD the treatment of the GvH-B6C3Fi mice with vadous concentrations (2.5 to 20 mg/kg/day) of the MNAs shortly after induction of the disease on days 3 to 12 resulted in a dose-dependently improved survival rate. With 20 mg/kdday mortality of this life-threatening GvHD was completely prevented. Also other parameters like splenomegaly, erythrocyte counts and hematocrtte values were strongly suppressed. Treatment of sensitized GvH-BDFl hybrtd mice in the chronic autoimmune-like model with 30 mg&g’day of the MNAs, given on days 6 to 36 after GvH induction resulted in an improved survfval rate, lowered the percentage of animals with lymphadenopathy and splenomegaly, reduced the levels of autoantibodies and the hiah serum titres of IaE. IaGI. and laG2a. and inhibited the development of glom~mlonephntis. Concluelon: These results presented here clearly indicate that both MNA 279 and MNA 715 can inhibit ongoing aberrant immune responses in animals suffering from GvHD and it should be justified to apply these drugs for bone marrow transplantation and the fight against GvHD.
P 5 19 16 jTTJ
Identification of the human minor hlstocompatlbility antigen HA-1
J.M.M. den Haan ‘, W. Wang*, L.R. Meadows3, E. Blokland ‘, of C. Reinhardus ‘, V.H. Engelhard *, D.F. Hunt3, E. Goulmy ‘. ’DePr. lmmunohaematology and Bloodbank, University Hcspid Leiden; RC Leiden, The Netherlands, *Dept. of MicmWobgy and Beime Carter Center for Immunology Research, University of Virginia, VA, USA, 3Dept. of Chemistw University of Virginia, VA, USA Introduction: Disparity for the minor His&compatibility antigen (mHag) HA-1 between HLA-matched bone marrow donor and recipient is corretated with an increased risk for Graft-versus-Host-Disease. This mHag is recognized by HlAA2.1 restricted HA-I specific cytotoxic T cell clones (CTL). but the peptide sequence of HA-1 and the protein encoding this peptide is unknown. Using the HA-1 specific T cell clones we set out to identify the HA-I peptide. Method: HLAA2.1 molecules were purified from HA-1 expressing EVBBLCL by affinity chromatography. The HlAA2.1 bound peptides were eluted and separated from the MHC molecules by acid treatment and a 10 kD filtration step. The peptides were fractionated by reverse phase HPLC and the fractions were tested for HA-1 activity in a cytotoxicity assay with the HA-l specific CTL. Two fractions with HA-1 activity were purified after 2 rounds of HPLC fractionation. These fractions were analysed with microcapillary HPLC/electrospray ionisation tandem mass spectrometry and candidates were selected for sequencing. Resu,ults:Candidate peptide with m/z 513 was sequenced and the synthetic peptide sensitized target cells for lysis by the HA-1 specific CTL. A data base search revealed a sequence with one amino acid difference. Polyrnerase chain reaction (PCR) primers were developed based on this protein in the databank. PCR were performed on HA-1 positive and negative EBV-BLCL and the PCR product was sequenced. Results indicated that the found protein encoded the mHag HA-l. Conclusion:The mHag HA-1 has been sequened and the protein encoding this mHag has been identified. These results might enable the development of diagnostic tools for prevention of Graft-versus-Host Disease and the treatment of leukemia relapses after bone marrow transplantation.
children. These data led us to evaluate changes in serum cytokines and soluble receptors in children with and without acute GVHD. Yatsrlalsand Methods:Sixteen children received allogeneic BMT from HLA identical donors. Profytaxis for GVHD was performed with cyclosportn A, methotrexate and immunoglobulins. Serum IFN gamma, TNF alfa, lL-6, slL-2R, sTNF I, sTNF II concentrations were determined by ELISA Nedgenid in period D-7 to D+64. Serum concentrations of cytokines and soluble receptors in children with GVHD /n = 7/were compared with levels in children without/n = 91. Reeults: Mean serum IL-2R concentrations increased significantly in children with acute GVHD /D + 14 to D + 42, GVHD manifestation D + 16.7 engraftment D + 12.4/hut did not change significantly in children without GVHD /engraftment D + 16.1/. Similarly serum sTNF I and sTNF II increased significantiy only in children with GVHD/D + 7 to D + 70, resp. D + 16 to D + 70/ Significant correlation between sTNF I and sTNF II was found, but not between sTNF and IL-2R. Serum IL-6 concentration increased significantly in both groups of patients immediately after BMT /D + 2/, serum TNF alfa and IFN gamma concentrations did not change significantly in patfents with and without GVHD. Concluston: Peak serum concentrations of slL-2R, sTNF I, sTNF II were found a few days /weeks/ after detection of engraftment /by molecular biology methods/. These receptors may be used for monitoring acute GVHD in children after allogeneic BMT.
m -
MLC reactivltv can be further differentiated bv cytokine measurements
Willemijn M. Wissink, Arnold van der Meer, Jos Ruiter, Wil Ailebes en Ina Joosten. TIsnsplant Serology Laboratory, Blood TransfusionService, University Hospital Nijmegen, The Netherlands Introduction: The value of the mixed lymphocyte culture (MLC) for donor-recipient matching is much debated. Consequently, additional cellular tests (HTLp and CTLp assays) are now assessed on their predictive value for GVHD or allograft rejection, but these am laborious. Already cytokines (as a measure for ThliThP responses) have been associated with transplant failurelsuccess. It is now apparent that simply measudng proliferation after MLC does not provide sufficient information on the nature of the alloreactivity. By extending the standard MLC with addltionel cytoklne measurements we may gain insight in the potential direction of the expected alloresponw and whether or not this reactivity is acceptable by our standards. Material and Methods: We have anafysed effector-stlmulator combinations exhibiting different degrees of HLA matching. Standard one-way MLC, as well as HTLp and CTLp frequency analysts was performed. IFNy, IL-4 and IL-10 were measured after one-way MLC using a sandwich ELISA technique. Irradiated pool cells and irradiated control cells were taken as positve and negative control respectively. Results and Concluelon: The results so far revwl that the HLA class I and II MM combinations, chosen to validate the system, show distinct cytoktne production patterns while all showing high SI values in MLC. Class II MM appear the main stimulus for cytokine production. In combinations with isolated class I MM the levels are very low. In meet MM combinations the Thl cytokine pattern (high ratio IFNy: 114(ILlO)) is predominant. The degree of mismatch appears not predictive of the nature of the alloresponw, we SW both Thl-like and ThP-like responses (as measured by IFNy I IL4 ratio on day 5 of culturing). In the combinations with a shift from a Thl to a more Th2 like response higher levels of IL-10 are found. The HTLp and CTLp frequencies not always correlate with the cytokine profiles. The cytokine measurements can differentiate between MLC combinations where HiA matching, MLC proliferation and the HTLp/CTLp assay don’t. This will be further investigated. We aim to do so by retrospective analysis of BMT donor -recipient combinations.
09:00-18:30/12:00-14:00
j-Y-Iq P 5 19 17
Serum concentrations of the soluble 11-2, TNF I and TNF II receptors for monltorlng acute graft-versus-host dlsease In children
Forum lounges
P.5.20
Neuroimmunology
P.5.20.01
Autoentibodles to brain tlssue in rat sers
,
J. Vaviinec’, D. PospfSilova*, J. Stal)i' V. Vavra’, L. Honzatkova3, 2. Sieglova 3. ’2nd Pediatric Department, Prague, Czech RepuWic,*Institute of Immunobg~ Prague, Czech Republic, 32nd medical Faculty; Institute of Hematology and Blood Transfusion, Prague, Czech Republic Introduction: Acute GVHD is perceived as donor T-cell mediated reaction and inflammatory cytokines play a important rote in activating of these cells. Elevated serum soluble IL-2 receptor concentration have been reported in adults with GVHD after allogeneic BMT, but limited number of information is available in
I. Staiikova, L. Prokesova, S. Trojan. First Faculty of Medicine, Charles lJniversi& Prague, Czech Republic Introduotlon: The increased levels of autoantibodies against neural tissue can serve as a marker of anoxic damage of the brain. Antibodies to neural tissue were tested in normal and anoxic rats.