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Family segregation analysis of allospecific T cell clones
Abstracts
161 volunteers; studies of the B cell response showed that: (1) part of the response was directed against assembled topographic sites; (2) 8 0 % of the response was directed against determinants present on fragment A; (3) few determinants present on the C N B r peptides of B cross-react with determinant present on A. Together, these studies, which investigate the anti-DT human immune repertoire, indicate that large cross-reactives against D T determinants exist for both B and T cell responses.
THE DR/31 CHAIN BUT NOT THE DR/32 CHAIN PRESENTS ANTIGEN TO A DRw13 RESTRICTED INFLUENZA SPECIFIC T CELL CLONE. A. Haziot, J. Michon, C. Freidel, L. Degos, J.P. Levy, and D.J. Charron; Laboratoire d'Immunogdndtique Structurale 91. Boulevard de l'H6pital 75013, Paris, France Assignment to a discrete Ia molecule of the restriction element of a T cell clone is arduous in humans because of the strong linkage disequilibrium between the genes coding for the H L A - D products and the presence of epitopes shared by distinct molecules. We developed a proliferative (and cytotoxic) influenza specific T cell clone (COT C2) restricted to several DRw13 haplotypes. The molecular diversity of the DRw13 haplotypes is generated by the complex association of 3 /31 and 2 ]32 distincts D R / 3 chains. There is no D R / 3 chain common to all DRw13 haplotypes. T o characterize the D R molecule bearing the restriction element of C O T C2, biochemical and functional studies were undertaken on the same panel of DRw13 cells. Localization to the D R molecule of the restriction element was suggested by blocking experiments with anti-DR monomorphic MoAbs, while no anti-DQ MoAbs could inhibit the proliferative response. Furthermore, not only D R w 1 3 - D Q w l , but also D R w l 3 - D Q w 3 and D R w 1 3 D Q w blank cells, could function as APC. Only cells expressing the D R 131 chain called 6 B5 could present antigen to C O T C2 whatever the D R / 3 2 chain (6B3 and 6B4) associated with it on the DRw13 haplotype. Moreover, the cell could not function as APC when the 32 chain was expressed either alone or in association with another/31 chain. Evidence is thus provided for restriction of a T cell clone by only one of the two D R / 3 chains expressed in DRw13 haplotypes.
FAMILY SEGREGATION ANALYSIS OF ALLOSPECIFIC T CELL CLONES, Sandra RosenBronson, Armead H. Johnson, Robert J. Hartzman, and David D. Eckels; Division of Immunologic Oncology, Georgetown University, Washington D.C. We have studied the complexity and fine specificity of the H L A - D region using a panel of T cell clones (TLCs) generated against alloantigens associated with H L A - D R 1 - D R 8 . H L A - D R homozygous PBLs were stimulated with D R heterozygous PBLs in primary MLCs for 4 days. Primed cells were cloned by limiting dilution at 0.3 cells/well in the presence of 20% T C G F and irradiated stimulator cells. After preliminary analyses of expanded clones on stimulator panels, 89 representative clones were selected from preliminary studies for extensive testing against a panel of 200 unrelated stimulator cells. In addition to population studies, each clone was tested in proliferation assays with 14 families. Segregation patterns were analyzed to confirm or disprove haplotype associations indicated by population studies. Sequential lod scores were calculated to ascertain the likelihood that genes encoding epitopes detected by TLCs were linked to HLA genes. Based on the segregation patterns observed, four basic categories were identified: (1) clonal responses which segregated with the same H L A - D region haplotype in all informative pedigrees; (2) clonal responses which segregated with HLA in all pedigrees but not always with the same or related haplotypes; (3) clonal responses
162
Annual AACHT Meeting, 1985 which segregated with HLA in some families but which failed to segregate in others or produced equivocal results; and (4) clonal responses which did not segregate with HLA haplotypes. Thirty-one clones were shown to detect epitopes encoded by genes linked t o H L A ; some of these clones detected epitopes which segregated exclusively with one HLA haplotype in all families tested (see table;,. The lod score results o f 41 clones were inconclusive. Five clones had negative lod scores suggesting these clones detected epitopes encoded by genes not linked to HLA genes. In several cases family analysis disproved haplotype correlations indicated by population studies and thus were important in the complete c h a r acterization of such allospecific clones since without the family analysis, haplotyp~' associations would have been m a d e incorrectly for several clones. The data ob~ tained from family studies done in conjunction with population studies provided useful information in the characterization of HLA-D region epitopes. The number of clones detecting epitopes encoded by genes which segregated exclusively with one HLA haplotype in all families tested: DR1 6
DR2 9
DR3 4
DR4 0
DR5 0
DR6 0
DR7 3
DR8 2
HLA-D REGION SPEC1FIC T CELL CLONES DETECT THE COEXISTENCE OF HETEROGENEITY ALONG WITH INTERHAPLOTYPE HOMOGENEITY. Sandra Rosen-Bronson, Armead H. Johnson, Robert J. Hartzman, and David D, Eckels; Division of Immuno/ogic Oncolo~>
Georgetown University, Washington D,C. Evidence from serologic and cellular analysis as well as molecular genetic techniques and protein chemistry support the concept o f extreme complexity within the H L A - D region of the human major histocompatibility complex. In order to study the complexity and fine specificity of this region at the level of T cell recognition, a panel of T cell clones (TLCs) was generated against alloantigens associated with HLA-DR1 through DR8. H L A - D R homozygous PBLs were stimulated with D R heterozygous PBLs in primary MLCs for 4 days. Primed cells were cloned by limiting dilution at 0.3 cells/well in the presence of 20c~ T C G F and irradiated stimulator cells. Viable clones were tested in proliferation assays for allospecificity. Such primings produced approximately 800 clones: some r e o ognized H L A - D region associated specificities whereas others recognized only their original stimulator cell. Preliminary analysis of expanded clones on snmuIator panels showed alloantigenic complexity within groups of TLCs recognizing similar sets of D-region associated epitopes suggesting subtypes or "splits" ol D R alleles. Subsequently, 89 representative clones were selected from preliminary studies for extensive tesnng against 200 unrelated stimulator cells. Clone by-clone correlation analyses were done to ascertain whether any clones recograzed similar or identical epitopes. In addition, clonal reactivity patterns were correlated with known HLA specificities, Twelve groups of clones were identified with similar reactivity patterns using clone-by-clone correlation analysis. Some groups also shared a significant correlation with one D-region haplotype, others had no significant correlation with any defined D-region specificity. Groups of clones where identified that correlated with HLA-DR1,2,3,7, and 8 haplotypes. However, groups o f clones were not found with significant correlation to DR4.5, or 6 haplotypes. Although the reactivity of some clones correlated with these haplotypes m population studies, in family segregation analyses, the epitopes detected were encoded by genes that segregated with HLA but not consistentl~ with the expected DR4,5, or 6 haplotype. Such findings suggest DR4,5,6 haplotypes may be somehow different from the other haplotypes with respect to the epitopes recognized by T ceils. Further study of this question is needed to confirm these findings and to ascertain the nature o f the differences. The large panel o f allospeofic T cell clones used in these studies allowed a "macro" view of the.
161 volunteers; studies of the B cell response showed that: (1) part of the response was directed against assembled topographic sites; (2) 8 0 % of the response was directed against determinants present on fragment A; (3) few determinants present on the C N B r peptides of B cross-react with determinant present on A. Together, these studies, which investigate the anti-DT human immune repertoire, indicate that large cross-reactives against D T determinants exist for both B and T cell responses.
THE DR/31 CHAIN BUT NOT THE DR/32 CHAIN PRESENTS ANTIGEN TO A DRw13 RESTRICTED INFLUENZA SPECIFIC T CELL CLONE. A. Haziot, J. Michon, C. Freidel, L. Degos, J.P. Levy, and D.J. Charron; Laboratoire d'Immunogdndtique Structurale 91. Boulevard de l'H6pital 75013, Paris, France Assignment to a discrete Ia molecule of the restriction element of a T cell clone is arduous in humans because of the strong linkage disequilibrium between the genes coding for the H L A - D products and the presence of epitopes shared by distinct molecules. We developed a proliferative (and cytotoxic) influenza specific T cell clone (COT C2) restricted to several DRw13 haplotypes. The molecular diversity of the DRw13 haplotypes is generated by the complex association of 3 /31 and 2 ]32 distincts D R / 3 chains. There is no D R / 3 chain common to all DRw13 haplotypes. T o characterize the D R molecule bearing the restriction element of C O T C2, biochemical and functional studies were undertaken on the same panel of DRw13 cells. Localization to the D R molecule of the restriction element was suggested by blocking experiments with anti-DR monomorphic MoAbs, while no anti-DQ MoAbs could inhibit the proliferative response. Furthermore, not only D R w 1 3 - D Q w l , but also D R w l 3 - D Q w 3 and D R w 1 3 D Q w blank cells, could function as APC. Only cells expressing the D R 131 chain called 6 B5 could present antigen to C O T C2 whatever the D R / 3 2 chain (6B3 and 6B4) associated with it on the DRw13 haplotype. Moreover, the cell could not function as APC when the 32 chain was expressed either alone or in association with another/31 chain. Evidence is thus provided for restriction of a T cell clone by only one of the two D R / 3 chains expressed in DRw13 haplotypes.
FAMILY SEGREGATION ANALYSIS OF ALLOSPECIFIC T CELL CLONES, Sandra RosenBronson, Armead H. Johnson, Robert J. Hartzman, and David D. Eckels; Division of Immunologic Oncology, Georgetown University, Washington D.C. We have studied the complexity and fine specificity of the H L A - D region using a panel of T cell clones (TLCs) generated against alloantigens associated with H L A - D R 1 - D R 8 . H L A - D R homozygous PBLs were stimulated with D R heterozygous PBLs in primary MLCs for 4 days. Primed cells were cloned by limiting dilution at 0.3 cells/well in the presence of 20% T C G F and irradiated stimulator cells. After preliminary analyses of expanded clones on stimulator panels, 89 representative clones were selected from preliminary studies for extensive testing against a panel of 200 unrelated stimulator cells. In addition to population studies, each clone was tested in proliferation assays with 14 families. Segregation patterns were analyzed to confirm or disprove haplotype associations indicated by population studies. Sequential lod scores were calculated to ascertain the likelihood that genes encoding epitopes detected by TLCs were linked to HLA genes. Based on the segregation patterns observed, four basic categories were identified: (1) clonal responses which segregated with the same H L A - D region haplotype in all informative pedigrees; (2) clonal responses which segregated with HLA in all pedigrees but not always with the same or related haplotypes; (3) clonal responses
162
Annual AACHT Meeting, 1985 which segregated with HLA in some families but which failed to segregate in others or produced equivocal results; and (4) clonal responses which did not segregate with HLA haplotypes. Thirty-one clones were shown to detect epitopes encoded by genes linked t o H L A ; some of these clones detected epitopes which segregated exclusively with one HLA haplotype in all families tested (see table;,. The lod score results o f 41 clones were inconclusive. Five clones had negative lod scores suggesting these clones detected epitopes encoded by genes not linked to HLA genes. In several cases family analysis disproved haplotype correlations indicated by population studies and thus were important in the complete c h a r acterization of such allospecific clones since without the family analysis, haplotyp~' associations would have been m a d e incorrectly for several clones. The data ob~ tained from family studies done in conjunction with population studies provided useful information in the characterization of HLA-D region epitopes. The number of clones detecting epitopes encoded by genes which segregated exclusively with one HLA haplotype in all families tested: DR1 6
DR2 9
DR3 4
DR4 0
DR5 0
DR6 0
DR7 3
DR8 2
HLA-D REGION SPEC1FIC T CELL CLONES DETECT THE COEXISTENCE OF HETEROGENEITY ALONG WITH INTERHAPLOTYPE HOMOGENEITY. Sandra Rosen-Bronson, Armead H. Johnson, Robert J. Hartzman, and David D, Eckels; Division of Immuno/ogic Oncolo~>
Georgetown University, Washington D,C. Evidence from serologic and cellular analysis as well as molecular genetic techniques and protein chemistry support the concept o f extreme complexity within the H L A - D region of the human major histocompatibility complex. In order to study the complexity and fine specificity of this region at the level of T cell recognition, a panel of T cell clones (TLCs) was generated against alloantigens associated with HLA-DR1 through DR8. H L A - D R homozygous PBLs were stimulated with D R heterozygous PBLs in primary MLCs for 4 days. Primed cells were cloned by limiting dilution at 0.3 cells/well in the presence of 20c~ T C G F and irradiated stimulator cells. Viable clones were tested in proliferation assays for allospecificity. Such primings produced approximately 800 clones: some r e o ognized H L A - D region associated specificities whereas others recognized only their original stimulator cell. Preliminary analysis of expanded clones on snmuIator panels showed alloantigenic complexity within groups of TLCs recognizing similar sets of D-region associated epitopes suggesting subtypes or "splits" ol D R alleles. Subsequently, 89 representative clones were selected from preliminary studies for extensive tesnng against 200 unrelated stimulator cells. Clone by-clone correlation analyses were done to ascertain whether any clones recograzed similar or identical epitopes. In addition, clonal reactivity patterns were correlated with known HLA specificities, Twelve groups of clones were identified with similar reactivity patterns using clone-by-clone correlation analysis. Some groups also shared a significant correlation with one D-region haplotype, others had no significant correlation with any defined D-region specificity. Groups of clones where identified that correlated with HLA-DR1,2,3,7, and 8 haplotypes. However, groups o f clones were not found with significant correlation to DR4.5, or 6 haplotypes. Although the reactivity of some clones correlated with these haplotypes m population studies, in family segregation analyses, the epitopes detected were encoded by genes that segregated with HLA but not consistentl~ with the expected DR4,5, or 6 haplotype. Such findings suggest DR4,5,6 haplotypes may be somehow different from the other haplotypes with respect to the epitopes recognized by T ceils. Further study of this question is needed to confirm these findings and to ascertain the nature o f the differences. The large panel o f allospeofic T cell clones used in these studies allowed a "macro" view of the.