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4 heterozygous IDDM patients
Abstracts
201 from the same patients and controls was digested with BamHI and analyzed by Southern blotting. Hybridization with a DQ/3 probe revealed the allelic 12 and 3.7 kb restriction fragments known to characterize DQw3 alleles. The RFLPs generated when compared to the protein patten obtained from the same cells showed that in each case the 12 kb fragment correlated with the DQw3 B2 specificity and the 3.7 kb fragment with DQw3 B1 specificity. These preliminary results suggest that it may be possible to identify specific RFLPs that correspond to allelic HLA-DQ gene products. The results also suggest that the high frequency DQw3 B2 (11/12) among IDDM patients and the occurence of particular haplotypes resulting from the association of DQw3 B2 genes with either DR4 B1 or B3 genes may be contributing factors for the development of IDDM.
DETECTION OF IN VIVO ALLOREACTIVITYAND ALLOIMMUNIZATION BY LIMITING DILUTION ANALYSIS. E.M. Mickelson, P. Polchi, M.A. Bean, C. Schommer, and J.A. Hansen; Puget Sound Blood Center," The Fred Hutchinson Cancer Research Center: The Virginia Mason Research Center, Seattle, WA
The cellular immune status of patients undergoing donor-specific transfusion (DST) prior to renal grafting and of patients undergoing allogeneic marrow transplantation from either an HLA identical sibling or a haploidentical donor was studied by limiting dilution analysis (LDA) and T-cell cloning. Using LDA, the alloreactive precursor frequencies of proliferative (ARPp) and cytolytic (ARPc) T cells were determined pre- and post-DST and during the early postmarrow transplant period (days 15-30 post-BMT). In patients receiving DST who developed a positive lymphocytotoxic cross-match (LCM) against the donor, a concurrent increase in ARPp frequency of up to five times pre-DST levels was detected by LDA. Patients who were not sensitized by DST (neg LCM) either demonstrated no significant increase in ARPp or a complex LDA curve indicating loss of first-order kinetics. The latter is consistent with the appearance of activated T suppressor cells. In patients receiving HLA-identical BMT, significant increases in ARPp frequency post-transplant vs. pretransplant are observed. The proliferative T cells were presumably directed against non-HLA determinants present in the chimeric patient but absent in the donor. In patients receiving HLAnonidentical BMT, complex changes in ARP frequency were observed posttransplant. In one patient mismatched with his haploidentical donor for HLA-A and B, two kinds of cytolytic T-cell clones (CTL) were isolated: one set of CTL specific for a public class I determinant present in the recipient; and one set specific for a polymorphic "autologous" determinant. These studies indicate that LDA and T-cell cloning can be used to monitor alloimmune reactivity in vivo after transfusion and transplantation and that these approaches should lead to a better understanding of the biological basis of tolerance and rejection. HYBRID DQ MOLECULES IN DR3/4 HETEROZYGOUS IDDM PATIENTS. Barbara S. Nepom, David Schwarz,Jerry Palmer, and Gerald T. Nepom; Genetic Systems Corporation, Seattle, WA Transcomplementation of c~ and/3 genes generates Ia molecules which consist of mixed dimers in the F1 offspring, potentially leading to a unique immune response distinct from either parent. This mechanism has been postulated to occur in some human diseases associated with a heterozygous HLA state, such as the well-known association of insulin-dependent diabetes mellitus (IDDM) with HLA-DR3/4. We report here evidence for the formation of hybrid DQ
202
Abstracts molecules by transcomplementation in IDDM patients. After immunoprecipiration by DQw3-specific MoAb 17.15, 2D-PAGE analysis of D Q antigens from DR3/4 heterozygous patients demonstrates that two types of D Q molecules occur: a D Q w 3 / 3 chain associating with a DQw3 a chain, and a D Q w 3 / 3 chain associating with a DQw2 c~ chain. The identity of ce and 13 chains comprising these hybrid molecules is confirmed by HPLC peptide map analysis. Several characteristic peptide peaks identify D Q ce chains and discriminate between DQw2 and DQw3. Both were found associated with DQw3/3. Furthermore, we show that this transcomplementation is not unique among IDDM patients but also occurs in their HLA identical nondiseased siblings.
CHARACTERIZATION OF AN UNUSUAL DR1 HAPLOTYPE FOUND IN RHEUMATOID ARTHRITIS. Parvin Merryman, Richard Hayes, John Schwenzer, Sicy Lee, Takami Matsuyama, Peter Gregersen, Antonio Nunez-Roldan, Jack Silver, and Robert Winchester; Hospital for Joint Diseases, New York, N Y In the course of examining the molecular basis of susceptibility to rheumatoid arthritis (RA), a distinctive subset of DR1 haplotypes was identified at higher frequency among individuals with RA than in ethnically matched controls. This D R 1 haplotype was distinguishable from the majority of DR1 haplotypes because it encodes a reactivity with the monoclonal antibody 109d6. Previous protein microsequencing analysis suggested that this determinant was encoded by the DR/31 gene. In order to determine the molecular basis for this unusual DR1 haplotype, we prepared a c D N A library from a lymphoblastoid cell line transformed from an RA patient who was H L A - A 3 0 , C w 4 , B 3 5 , D R 1 , D Q w l / A32,Cw4,B44,DR blank. Twenty DR/31 c D N A clones were isolated and characterized. Two different c D N A sequences were found. One is identical to the sequence of a DR/31 c D N A clone previously isolated from a DR3,6 cell line. The other is similar but not identical to the DR/31 sequence of a c D N A clone isolated from the DR1 homozygous cell line LG2; eight nucleotide differences resulting in three amino acid substitutions (51 Glu --+ Thr 85 val --+ ala and 86 Gly ---> val) were observed between these DR1/3 chains. These differences very likely are responsible for the preferential reactivity of some DR1/3 chains with the monoclonal antibody 109d6. Expression experiments in COS7 cells are presently in progress to confirm this hypothesis.
DNA RESTRICTION FRAGMENT PATTERNS DEFINING HLA HAPLOTYPES ASSOCIATED WITH INSULIN-DEPENDENT DIABETES. M. Segall, J. Barbosa, and F.H. Bach; Immunobiology Research Center, University of Minnesota, Minneapolis. MN Insulin-dependent diabetes (IDD) is positively associated with DR3 and DR4 and negatively associated with DR2. Positive associations with DR1 and DRw8 have also been reported. We have previously demonstrated that the D-region haplotype, D R 2 - M N 2 , which is significantly increased in DR2 + IDD patients as compared to DR2 + controls, has a D Q beta D N A restriction fragment (RF) pattern identical, except for one fragment, to that of D R 1 - D w l , and different from that of DR2-Dw2. These data suggest that the D R 1 - D w l D Q beta RF pattern, which we have called D Q w l . 1 to distinguish it from the patterns seen with other D Q w l + haplotypes, may be a marker for a susceptibility factor(s) for IDD. DRw6 and DRw8, two specificities which are in linkage disequilibrium with D Q w l , are also increased in frequency in the Minnesota I D D population.
201 from the same patients and controls was digested with BamHI and analyzed by Southern blotting. Hybridization with a DQ/3 probe revealed the allelic 12 and 3.7 kb restriction fragments known to characterize DQw3 alleles. The RFLPs generated when compared to the protein patten obtained from the same cells showed that in each case the 12 kb fragment correlated with the DQw3 B2 specificity and the 3.7 kb fragment with DQw3 B1 specificity. These preliminary results suggest that it may be possible to identify specific RFLPs that correspond to allelic HLA-DQ gene products. The results also suggest that the high frequency DQw3 B2 (11/12) among IDDM patients and the occurence of particular haplotypes resulting from the association of DQw3 B2 genes with either DR4 B1 or B3 genes may be contributing factors for the development of IDDM.
DETECTION OF IN VIVO ALLOREACTIVITYAND ALLOIMMUNIZATION BY LIMITING DILUTION ANALYSIS. E.M. Mickelson, P. Polchi, M.A. Bean, C. Schommer, and J.A. Hansen; Puget Sound Blood Center," The Fred Hutchinson Cancer Research Center: The Virginia Mason Research Center, Seattle, WA
The cellular immune status of patients undergoing donor-specific transfusion (DST) prior to renal grafting and of patients undergoing allogeneic marrow transplantation from either an HLA identical sibling or a haploidentical donor was studied by limiting dilution analysis (LDA) and T-cell cloning. Using LDA, the alloreactive precursor frequencies of proliferative (ARPp) and cytolytic (ARPc) T cells were determined pre- and post-DST and during the early postmarrow transplant period (days 15-30 post-BMT). In patients receiving DST who developed a positive lymphocytotoxic cross-match (LCM) against the donor, a concurrent increase in ARPp frequency of up to five times pre-DST levels was detected by LDA. Patients who were not sensitized by DST (neg LCM) either demonstrated no significant increase in ARPp or a complex LDA curve indicating loss of first-order kinetics. The latter is consistent with the appearance of activated T suppressor cells. In patients receiving HLA-identical BMT, significant increases in ARPp frequency post-transplant vs. pretransplant are observed. The proliferative T cells were presumably directed against non-HLA determinants present in the chimeric patient but absent in the donor. In patients receiving HLAnonidentical BMT, complex changes in ARP frequency were observed posttransplant. In one patient mismatched with his haploidentical donor for HLA-A and B, two kinds of cytolytic T-cell clones (CTL) were isolated: one set of CTL specific for a public class I determinant present in the recipient; and one set specific for a polymorphic "autologous" determinant. These studies indicate that LDA and T-cell cloning can be used to monitor alloimmune reactivity in vivo after transfusion and transplantation and that these approaches should lead to a better understanding of the biological basis of tolerance and rejection. HYBRID DQ MOLECULES IN DR3/4 HETEROZYGOUS IDDM PATIENTS. Barbara S. Nepom, David Schwarz,Jerry Palmer, and Gerald T. Nepom; Genetic Systems Corporation, Seattle, WA Transcomplementation of c~ and/3 genes generates Ia molecules which consist of mixed dimers in the F1 offspring, potentially leading to a unique immune response distinct from either parent. This mechanism has been postulated to occur in some human diseases associated with a heterozygous HLA state, such as the well-known association of insulin-dependent diabetes mellitus (IDDM) with HLA-DR3/4. We report here evidence for the formation of hybrid DQ
202
Abstracts molecules by transcomplementation in IDDM patients. After immunoprecipiration by DQw3-specific MoAb 17.15, 2D-PAGE analysis of D Q antigens from DR3/4 heterozygous patients demonstrates that two types of D Q molecules occur: a D Q w 3 / 3 chain associating with a DQw3 a chain, and a D Q w 3 / 3 chain associating with a DQw2 c~ chain. The identity of ce and 13 chains comprising these hybrid molecules is confirmed by HPLC peptide map analysis. Several characteristic peptide peaks identify D Q ce chains and discriminate between DQw2 and DQw3. Both were found associated with DQw3/3. Furthermore, we show that this transcomplementation is not unique among IDDM patients but also occurs in their HLA identical nondiseased siblings.
CHARACTERIZATION OF AN UNUSUAL DR1 HAPLOTYPE FOUND IN RHEUMATOID ARTHRITIS. Parvin Merryman, Richard Hayes, John Schwenzer, Sicy Lee, Takami Matsuyama, Peter Gregersen, Antonio Nunez-Roldan, Jack Silver, and Robert Winchester; Hospital for Joint Diseases, New York, N Y In the course of examining the molecular basis of susceptibility to rheumatoid arthritis (RA), a distinctive subset of DR1 haplotypes was identified at higher frequency among individuals with RA than in ethnically matched controls. This D R 1 haplotype was distinguishable from the majority of DR1 haplotypes because it encodes a reactivity with the monoclonal antibody 109d6. Previous protein microsequencing analysis suggested that this determinant was encoded by the DR/31 gene. In order to determine the molecular basis for this unusual DR1 haplotype, we prepared a c D N A library from a lymphoblastoid cell line transformed from an RA patient who was H L A - A 3 0 , C w 4 , B 3 5 , D R 1 , D Q w l / A32,Cw4,B44,DR blank. Twenty DR/31 c D N A clones were isolated and characterized. Two different c D N A sequences were found. One is identical to the sequence of a DR/31 c D N A clone previously isolated from a DR3,6 cell line. The other is similar but not identical to the DR/31 sequence of a c D N A clone isolated from the DR1 homozygous cell line LG2; eight nucleotide differences resulting in three amino acid substitutions (51 Glu --+ Thr 85 val --+ ala and 86 Gly ---> val) were observed between these DR1/3 chains. These differences very likely are responsible for the preferential reactivity of some DR1/3 chains with the monoclonal antibody 109d6. Expression experiments in COS7 cells are presently in progress to confirm this hypothesis.
DNA RESTRICTION FRAGMENT PATTERNS DEFINING HLA HAPLOTYPES ASSOCIATED WITH INSULIN-DEPENDENT DIABETES. M. Segall, J. Barbosa, and F.H. Bach; Immunobiology Research Center, University of Minnesota, Minneapolis. MN Insulin-dependent diabetes (IDD) is positively associated with DR3 and DR4 and negatively associated with DR2. Positive associations with DR1 and DRw8 have also been reported. We have previously demonstrated that the D-region haplotype, D R 2 - M N 2 , which is significantly increased in DR2 + IDD patients as compared to DR2 + controls, has a D Q beta D N A restriction fragment (RF) pattern identical, except for one fragment, to that of D R 1 - D w l , and different from that of DR2-Dw2. These data suggest that the D R 1 - D w l D Q beta RF pattern, which we have called D Q w l . 1 to distinguish it from the patterns seen with other D Q w l + haplotypes, may be a marker for a susceptibility factor(s) for IDD. DRw6 and DRw8, two specificities which are in linkage disequilibrium with D Q w l , are also increased in frequency in the Minnesota I D D population.