- Email: [email protected]
Human T cell development: Lessons from patients lacking MHC class II expression
109
Abstracts
P592
THE TCR REPERTOIRE OF TUMOR-INFILTRATION LYMPHOCYTES FROM RENAL CELL CARCINOMA, A POSSIBLE ASSOCIATION BETWEEN THE RESTRICTED USAGE OF V BETA 4 GENE AND HLA-B51
Xue-Yi l.i", Yin Wang', Zhong-Chen Zhenlt and Kuang-Yen Chou' 'Shanghai Institute of Immunology, Shanghai Second Medical University; ItShanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai, China Tumor-infiltration lymphocytes (TlLs) were isolated from tissues of renal cell carcinoma (RCC) on which HLA class I antigens were assayed to be normally expressed. To examine the possible skewing of T cell receptor (TCRl repertoire, quantitative PCR was performed for cDNAof TILs from three RCC patients by using twenty-two 5' V beta primers and one isotope-labelled 3' C beta primer, And an invariant C alpha gene segment was co-amplified as an internal reference. The restricted usage of TCR V beta genes in TILs were quantitatively determined in comparison with the TCR repertoire in PBMCs of same patient. Expressed as percentage of cprn, the relative intensity of PCR amplification of V beta 4 gene in TlLs from patient 1 and 2 were found as 22and 12-fold higher than those in PBMCs, respectively. As to patient 3, the V beta 7 was expressed in the TILs as 14-fold higher than that in autologous PBMCs. Besides, some of other V beta genes were detected with higher percentages both in TILs and PBMCs, such as V beta 3, 2, 6 in patients 1,2,3, respectively. More interestingly, HLA-B61, an allele with low frequency of only 5% in the ethnic group the patients belong to, was serologically detected positive both for patients 1 and 2, who happend to share the expended TIL clone with TCR V beta 4 gene. In order to verify the connection between V beta 4 and B61 , an enlarged sample of RCC is under examination.
P594
H1IIUlN T CBLL DKVllLOPIlBIIT, CLASS II BXPRESSION.
LBSSONS FROM PATIBIlTS LACKING HIIC
Peter van den Elsen, Judy Henwood, Anette van Boxel-Dezaire, Ron Schipper. Martijn den Hoedt; , Ad Peijnenburg, Ozden Sanal, Fugen Ersoy, Ger Rijkers, Ben Zegers, Jaak Vossen, Maarten van 'rot and Gail Hawes. Department of Immunohematology and Blood Hospital Le Lden , Le i.den , The Netherlands.
Bank,
University
Patients with an MHC class II deficiency (bare lymphocyte syndrome) provide an unique opportunity to study development of the human T cell receptor repertoire. The lack of MHC class II antigen expression in these patients resulted in a strong reduction in the numbers of CD4+CDS- peripheral T cells. These peripheral CD4+CDS- T cells were not restricted in the employment of TCR V- and J -gene segments but the expressed TCRs manifested different CDR3 amino acid incorporation profiles resulting in altered charge and hydropathicity properties of the presumed antigen binding site (CDR3). Moreover, the lack of MHC class II antigen expression had altered the CD4+CDS'skewing patterns of TCR V-gene families which can be found in normals. In conclusion, the lack of MHC class II antigen expression had a profound impact on the CD4+CDS- TCR V-gene skewing patterns and on the amino acid profiles of the CDR3 in CD4+CDS- T cells isolated from MHC class II-deficient patients. The altered amino acid profi Les result in significant changes to the local environment of those CDR3s, which would have implications for antigen binding, and may reflect T cells that have escaped the normal selection processes in the thymus, where the affinity and avidity of the TCR interaction with MHC are thought to dictate those selection processes.
P596
HOST-SPECIFIC DONORHELPER T-CELLPRECURSORS AS PREDIcroRSOF ACUTE GYHD AND RELAPSE IN HLA-IDENTICALSIBLINGS BONE MARROW TRANSPLANTATION (BMT>_ ~ S. LeGouveHo, M. Kuentz, C. Cordonnter, F. Bemaudin, F. Beaujean, F.Roudot, J.P. Farcet, J.P. Vemant. Department' of Immunobiology andHematology, CHU Henri Mondor, Creell, France. It was recently suggested that hi.sh frequency of host Specific donor helper T-cell ~rsors (HLTp) could be predictive oT significant acute sr:a:ft versus host disease
(aGVHD). Wehaveretrospectively studiedthe frequency of HTLp by a simplified limiting dilution analysis to determine their predictive value in aGVHD and relapse. An increasing number of donor peripheral-blood mononuclear cells (PBMC) were cultured for 48 hours with a fix number of irradiated receiver PBMC. HfLE frequency was evaluated by the production of IL2 in culture supernatant, estimatea by fheproliferation of theCfLL2, lL2 dependant, coilline.Thirty-two patient' with hematological malignancies having received an HLA identical siblings BMf were tested for their pre-8MT host specificdonor HTLp frequencies. We correlated the HTtp frequency with the post graft occurence of aGVHD and relapse. HTLp f!equencies are significantly higher in patients who experienced aGVHD:2:grai::llI(n :::: 14) versus others (n = 18> (p '"' 0,026)and in those woo did not relapse In =19)versus those whodid (n =13)(p =O,(lOO4). The probability of relapse when the HTLJ' frequency was higher and lower than 1/300.000 was 16~ and 88lfQ respectively in the Kaplan Meier and was
~:i~~fin~Jo~~f~~~t~:Jf values,
predictive of aCVHD and relapse, should
~~~~~:md:~h=~;;i~:th~~i~~~r:~VHD~~~t~ia~,:~opmentof protocols
P593
TCRPV Allelic Frequencies in AfriClIDS and Caucasians:
Molecular Medicine Unit,Leeds University, Leeds, UK Q) Infectious DiseaseUnit,University College, London OJ Dept. ofMedicine, University of Zambia At least 12 of the 50 TCR ~V genesegments haveallelic variants. Differencesin allelic frequencies between ethnicgroup'havebeeureported for three of thesePV 6S7, PV20S1aod pV 3S1. Wehavestudied the useofPV 3S1aod PV2S1 allelesin a population of50 Zambiansand correlatedthe results with the percentageof ~V3 and PV2 positiveCD4and CD8 T Cells. Theresults showmarked differencesbetween the Zambian and British populations. ThepV3S1 • 1 allele frequency is 0.8 among Zambiansand 0.5 among the Britishindividuals studied. This is accompanied by the expectedlowfrequency of PV3"" CD4+ cellssincethisalleleis knownto be associated with lower~V3+CD4+ cellsnumbers The~V2S"2 allelefrequencies are shownin the tablebelow:TCRpV2SI' allele Frequency Zambian British 01 0.13 0.50 02 0.815 0.35 03 0.01 0.13 (I)
Linkage disequilibriumbetween PV2 and pV3 alleleswas not fbund in eitherpopulation. The pV3 alleles haveidentical coding sequences. The PV201 and 02 allelesdiffer by a singleaminoacidat position10 which is notin a putativeantigenbinding region. Thus differences in antigenic specificity are unlikely to account for the markeddifferences in alleles presentin thesetwopopulations.
P595 IMMUNE RESPONSES IN TAP DEFICIENT PATIENTS de la Salle Henri' Peyrat Marie A.,t Arnold Daniele.s Donato Lionel,' Rammensee Hans Georg,§ Cazenave Jean-Pierre' Hanau Daniel,' Bonneville
Marc.t Tongio Marie-Marthe" ETS Strasbourg, France"; INSERM U2i I , Nantes, Francer; Cancer Research Institute, Heidelberg, Germany§. Two siblings with a peptide transporter (TAP) deficiency were recently described. They were not unusually susceptible to viral infections and had low but substantial numbers of (X~ CD8+ T cells. Most of the cell surface expressed class I molecules are HLA-B products. Analysis of two peptides eluted from class I molecules expressedby TAP-deficient EBY B lymphoblastoid cell lines indicated thar both were derived from sequences of cytosolic proteins and presented by HLA-B molecules. Diversity of the TCR repertoire of fl~ CD8+ T cells was extensive using TCR V-region specific mAbs. Contrary to observations in class l-negative mice, most of flP CD8+ T cell clones studied were nonreactive against cells expressing normal levels of HLA alleles of Ihe TAPdeficient patients. However, one cytotoxic CD8+ T cell clone recognizing an EBY antigen presented by HLA-B molecules On TAP-deficient cells was found, thus providing evidence that fl~ CD8+ T cells can mediate immunity against viral infections in these patients. In contrast to healthy individuals, 50% of the up CDS+ T cells were CD1Ib+, a marker of memory T cells. During the two last
years, one patient originalIy healthy, started to suffer from pulmonary bacterial
infections. As the diseaseprogressed, expansion of yo and CD8+ fl~ T cells was observed. Thus, although only a limited number of up CD8+ T cells can
mature, these cells can be efficiently recruited in immune responses, and mediate HLA class l-resrricred cytotoxic defense against viral infections.
P597
IMGT, THE IMMUNOGENETICS DATABASE
Lefranc Marie-Paule" Bodmer Julia', MOiler Wemer', Giudicelli Yeronique " Busin Chantal" Marsh Steven', Chaume Denis' and Malik Ansae' 'UGM, Montpellier, France 'ICRF, London, UK 3 IFG, KOln, Germany 'CNUSC, Montpellier, France 'EMBL-EBI, Cambridge, UK The ImMunoGeneTics (IMGT) database is an integrated database which consists of the UGM database, containing Immunoglobulin (Ig) and T cell receptor (TcR) gene information, and the MHC/HLA database containing MHC encoded gene information. lMGT comprises expertly annotated sequence and alignment tables. IMGT main goals are to establish a common data access to all Immunogenetics data, including sequences, Oligonucleotide primers, gene map and other genetic data of Ig, TcR and MHC molecules, from all species, and to provide a graphical user friendly data access. IMGT will have important implications in medical research (repertoire in autoimmune diseases, AIDS, leukemias, lymphomas), therapeutical approaches (antibody engineering), genome diversity and genome evolution studies. IMGT is funded by the Groupement de Recherches et d'Etudes sur les G'momes, the Region Languedoc-RoussiIlon and the European Union BloMed 1 programme. Access; IMGTlUGM-DB contains 9.319 Ig and TcR sequences (3.000 fully annotated) from 61 different species and is freely available on the CNUSC ~ server hltp:l1Imgt.cnusc.fr:8104 (Mosaic or Netscape). Contact [email protected] and [email protected]. 'Annotated sequences are available on the EBI anonymous ftp server 'flp.ebi.ac.uk folder Ipub/databaseslimgt. Contact; [email protected]. 'HLA sequence alignments are available at URL hllp:llwww.icnetlaxp/tla/ index.htm!. Contact: [email protected]. IMGT coordinator; Marie-Paule Lefranc, [email protected].
Abstracts
P592
THE TCR REPERTOIRE OF TUMOR-INFILTRATION LYMPHOCYTES FROM RENAL CELL CARCINOMA, A POSSIBLE ASSOCIATION BETWEEN THE RESTRICTED USAGE OF V BETA 4 GENE AND HLA-B51
Xue-Yi l.i", Yin Wang', Zhong-Chen Zhenlt and Kuang-Yen Chou' 'Shanghai Institute of Immunology, Shanghai Second Medical University; ItShanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai, China Tumor-infiltration lymphocytes (TlLs) were isolated from tissues of renal cell carcinoma (RCC) on which HLA class I antigens were assayed to be normally expressed. To examine the possible skewing of T cell receptor (TCRl repertoire, quantitative PCR was performed for cDNAof TILs from three RCC patients by using twenty-two 5' V beta primers and one isotope-labelled 3' C beta primer, And an invariant C alpha gene segment was co-amplified as an internal reference. The restricted usage of TCR V beta genes in TILs were quantitatively determined in comparison with the TCR repertoire in PBMCs of same patient. Expressed as percentage of cprn, the relative intensity of PCR amplification of V beta 4 gene in TlLs from patient 1 and 2 were found as 22and 12-fold higher than those in PBMCs, respectively. As to patient 3, the V beta 7 was expressed in the TILs as 14-fold higher than that in autologous PBMCs. Besides, some of other V beta genes were detected with higher percentages both in TILs and PBMCs, such as V beta 3, 2, 6 in patients 1,2,3, respectively. More interestingly, HLA-B61, an allele with low frequency of only 5% in the ethnic group the patients belong to, was serologically detected positive both for patients 1 and 2, who happend to share the expended TIL clone with TCR V beta 4 gene. In order to verify the connection between V beta 4 and B61 , an enlarged sample of RCC is under examination.
P594
H1IIUlN T CBLL DKVllLOPIlBIIT, CLASS II BXPRESSION.
LBSSONS FROM PATIBIlTS LACKING HIIC
Peter van den Elsen, Judy Henwood, Anette van Boxel-Dezaire, Ron Schipper. Martijn den Hoedt; , Ad Peijnenburg, Ozden Sanal, Fugen Ersoy, Ger Rijkers, Ben Zegers, Jaak Vossen, Maarten van 'rot and Gail Hawes. Department of Immunohematology and Blood Hospital Le Lden , Le i.den , The Netherlands.
Bank,
University
Patients with an MHC class II deficiency (bare lymphocyte syndrome) provide an unique opportunity to study development of the human T cell receptor repertoire. The lack of MHC class II antigen expression in these patients resulted in a strong reduction in the numbers of CD4+CDS- peripheral T cells. These peripheral CD4+CDS- T cells were not restricted in the employment of TCR V- and J -gene segments but the expressed TCRs manifested different CDR3 amino acid incorporation profiles resulting in altered charge and hydropathicity properties of the presumed antigen binding site (CDR3). Moreover, the lack of MHC class II antigen expression had altered the CD4+CDS'skewing patterns of TCR V-gene families which can be found in normals. In conclusion, the lack of MHC class II antigen expression had a profound impact on the CD4+CDS- TCR V-gene skewing patterns and on the amino acid profiles of the CDR3 in CD4+CDS- T cells isolated from MHC class II-deficient patients. The altered amino acid profi Les result in significant changes to the local environment of those CDR3s, which would have implications for antigen binding, and may reflect T cells that have escaped the normal selection processes in the thymus, where the affinity and avidity of the TCR interaction with MHC are thought to dictate those selection processes.
P596
HOST-SPECIFIC DONORHELPER T-CELLPRECURSORS AS PREDIcroRSOF ACUTE GYHD AND RELAPSE IN HLA-IDENTICALSIBLINGS BONE MARROW TRANSPLANTATION (BMT>_ ~ S. LeGouveHo, M. Kuentz, C. Cordonnter, F. Bemaudin, F. Beaujean, F.Roudot, J.P. Farcet, J.P. Vemant. Department' of Immunobiology andHematology, CHU Henri Mondor, Creell, France. It was recently suggested that hi.sh frequency of host Specific donor helper T-cell ~rsors (HLTp) could be predictive oT significant acute sr:a:ft versus host disease
(aGVHD). Wehaveretrospectively studiedthe frequency of HTLp by a simplified limiting dilution analysis to determine their predictive value in aGVHD and relapse. An increasing number of donor peripheral-blood mononuclear cells (PBMC) were cultured for 48 hours with a fix number of irradiated receiver PBMC. HfLE frequency was evaluated by the production of IL2 in culture supernatant, estimatea by fheproliferation of theCfLL2, lL2 dependant, coilline.Thirty-two patient' with hematological malignancies having received an HLA identical siblings BMf were tested for their pre-8MT host specificdonor HTLp frequencies. We correlated the HTtp frequency with the post graft occurence of aGVHD and relapse. HTLp f!equencies are significantly higher in patients who experienced aGVHD:2:grai::llI(n :::: 14) versus others (n = 18> (p '"' 0,026)and in those woo did not relapse In =19)versus those whodid (n =13)(p =O,(lOO4). The probability of relapse when the HTLJ' frequency was higher and lower than 1/300.000 was 16~ and 88lfQ respectively in the Kaplan Meier and was
~:i~~fin~Jo~~f~~~t~:Jf values,
predictive of aCVHD and relapse, should
~~~~~:md:~h=~;;i~:th~~i~~~r:~VHD~~~t~ia~,:~opmentof protocols
P593
TCRPV Allelic Frequencies in AfriClIDS and Caucasians:
Molecular Medicine Unit,Leeds University, Leeds, UK Q) Infectious DiseaseUnit,University College, London OJ Dept. ofMedicine, University of Zambia At least 12 of the 50 TCR ~V genesegments haveallelic variants. Differencesin allelic frequencies between ethnicgroup'havebeeureported for three of thesePV 6S7, PV20S1aod pV 3S1. Wehavestudied the useofPV 3S1aod PV2S1 allelesin a population of50 Zambiansand correlatedthe results with the percentageof ~V3 and PV2 positiveCD4and CD8 T Cells. Theresults showmarked differencesbetween the Zambian and British populations. ThepV3S1 • 1 allele frequency is 0.8 among Zambiansand 0.5 among the Britishindividuals studied. This is accompanied by the expectedlowfrequency of PV3"" CD4+ cellssincethisalleleis knownto be associated with lower~V3+CD4+ cellsnumbers The~V2S"2 allelefrequencies are shownin the tablebelow:TCRpV2SI' allele Frequency Zambian British 01 0.13 0.50 02 0.815 0.35 03 0.01 0.13 (I)
Linkage disequilibriumbetween PV2 and pV3 alleleswas not fbund in eitherpopulation. The pV3 alleles haveidentical coding sequences. The PV201 and 02 allelesdiffer by a singleaminoacidat position10 which is notin a putativeantigenbinding region. Thus differences in antigenic specificity are unlikely to account for the markeddifferences in alleles presentin thesetwopopulations.
P595 IMMUNE RESPONSES IN TAP DEFICIENT PATIENTS de la Salle Henri' Peyrat Marie A.,t Arnold Daniele.s Donato Lionel,' Rammensee Hans Georg,§ Cazenave Jean-Pierre' Hanau Daniel,' Bonneville
Marc.t Tongio Marie-Marthe" ETS Strasbourg, France"; INSERM U2i I , Nantes, Francer; Cancer Research Institute, Heidelberg, Germany§. Two siblings with a peptide transporter (TAP) deficiency were recently described. They were not unusually susceptible to viral infections and had low but substantial numbers of (X~ CD8+ T cells. Most of the cell surface expressed class I molecules are HLA-B products. Analysis of two peptides eluted from class I molecules expressedby TAP-deficient EBY B lymphoblastoid cell lines indicated thar both were derived from sequences of cytosolic proteins and presented by HLA-B molecules. Diversity of the TCR repertoire of fl~ CD8+ T cells was extensive using TCR V-region specific mAbs. Contrary to observations in class l-negative mice, most of flP CD8+ T cell clones studied were nonreactive against cells expressing normal levels of HLA alleles of Ihe TAPdeficient patients. However, one cytotoxic CD8+ T cell clone recognizing an EBY antigen presented by HLA-B molecules On TAP-deficient cells was found, thus providing evidence that fl~ CD8+ T cells can mediate immunity against viral infections in these patients. In contrast to healthy individuals, 50% of the up CDS+ T cells were CD1Ib+, a marker of memory T cells. During the two last
years, one patient originalIy healthy, started to suffer from pulmonary bacterial
infections. As the diseaseprogressed, expansion of yo and CD8+ fl~ T cells was observed. Thus, although only a limited number of up CD8+ T cells can
mature, these cells can be efficiently recruited in immune responses, and mediate HLA class l-resrricred cytotoxic defense against viral infections.
P597
IMGT, THE IMMUNOGENETICS DATABASE
Lefranc Marie-Paule" Bodmer Julia', MOiler Wemer', Giudicelli Yeronique " Busin Chantal" Marsh Steven', Chaume Denis' and Malik Ansae' 'UGM, Montpellier, France 'ICRF, London, UK 3 IFG, KOln, Germany 'CNUSC, Montpellier, France 'EMBL-EBI, Cambridge, UK The ImMunoGeneTics (IMGT) database is an integrated database which consists of the UGM database, containing Immunoglobulin (Ig) and T cell receptor (TcR) gene information, and the MHC/HLA database containing MHC encoded gene information. lMGT comprises expertly annotated sequence and alignment tables. IMGT main goals are to establish a common data access to all Immunogenetics data, including sequences, Oligonucleotide primers, gene map and other genetic data of Ig, TcR and MHC molecules, from all species, and to provide a graphical user friendly data access. IMGT will have important implications in medical research (repertoire in autoimmune diseases, AIDS, leukemias, lymphomas), therapeutical approaches (antibody engineering), genome diversity and genome evolution studies. IMGT is funded by the Groupement de Recherches et d'Etudes sur les G'momes, the Region Languedoc-RoussiIlon and the European Union BloMed 1 programme. Access; IMGTlUGM-DB contains 9.319 Ig and TcR sequences (3.000 fully annotated) from 61 different species and is freely available on the CNUSC ~ server hltp:l1Imgt.cnusc.fr:8104 (Mosaic or Netscape). Contact [email protected] and [email protected]. 'Annotated sequences are available on the EBI anonymous ftp server 'flp.ebi.ac.uk folder Ipub/databaseslimgt. Contact; [email protected]. 'HLA sequence alignments are available at URL hllp:llwww.icnetlaxp/tla/ index.htm!. Contact: [email protected]. IMGT coordinator; Marie-Paule Lefranc, [email protected].