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229 Simplified method of HLA-Dr typing using the PCR
196 Abstracts
229
SlMPLIFIED METHOD OF HLA-DR . TYPING USING . THE PCR. N Shinomiva. MD. M Shmgg, va, MD. L R
231
Freidhoff. M.S. and DG Marsh. Ph.& Ba&more, MD. It has been reported that certain IUA-D-encoded specificities are associatedwith specific immune responsesto particular allergens. The amplification of specific DNA sequences by the polymerase chain reaction (PCR), with dot-blotting using sequence-specificoligonucleotides (SSOs), has provided a new approach for typing HLA-D alleles. We have developed a simplified method which facilitates this procedure, which will be usedfor first HLA and Allerev studv to be included in the 1991Histocompatibility W&kshoi. The HLA-D data will be correlated with specific IgE and I& Abs to >lO highly purified allergens. Atdpic pati&ts’ W&s were separatedand lysed by boiling. Second-exon HLA-DRB segments of the releasedDNA were amplified by PCR with primers DRBS’-I (ACCGGATCG’MCITGTCCCCICAG14CA1s) and DRB-3’-1 (283CTCGCCICIGCACIGTIAAGC263) designed in our laboratory. Inosine (I) was included at positions known to exhibit allelic polymorphism in expressed DRB eenes. The Denultimate 3’-residue of DRB3!-I coincides Gith a polykorphic residue sharedby all known expressed DRB genes, but not by pseudogenes. Specific alleles in a PCR-amplified sample were determined by dot-blot hybridization with 70 32P-labeledSSOsfrom the Histocompatibility Workshop and our lab. A conserved “framework” SSO was used to control for the Ievel of amplification, and a seriesof dilutions of a mixture of several patients’ amplified DRB secondexons was used to standardize the procedure. Quantitation of the procedure was achieved using an automated scanner(Betagen), from which the data was analyzed by computer. These newly designed primers allowed amplification of the all known expressed alleles of DRBI. B3. B4 and B5 loci. but no useudopenes. This rauid. and reprodbcible assay (and related &says hr DQA, DQB’, DPA and DPB) will be valuable in HLA-D typing 15OO+ patients for the 1991 HLA and Allergy study.
230
CROSSREACTIVITY OF T CELL EPITOPES IN Amb a V AND Amb t V. &JJhu Ph.D.. I Qg#&&j&
ALLERGIC HUMAN T CELL REACTIVITY TO MILTIPlf FORMS OF Amb a I, A MAJOR ALLERGEN GF SHORT ._ RAGWEED. A.D.. C.M. m ti S., S.. J.P. Noraenstern.PhJl,_&.-L, Greenstein., Cambridge, MA. The major allergen of short ragweed i&wr~.ia artemiisifolia), Amb a I, formerly called Antfqen E, has recently been shown by clor,iny and sequencing studies to be composed of a faml.ly of four related proteins, Amb a 1.1, i.?, 1.3 and 1.4. The identity in primary amino acid sequence among the four family members ranges from T3% to 87%. Recombinant Amb a .I proteins were produced in bacteria and were shown to react with TqE from ragweed-allergic patients on Western blots, consistent with the notion that a;: four forms of Amb a I are allergenic in humans. Sir,ce T ceil help is required for IgE production, :t is also of interest to examine the reactivity of 7' cells from ragweed-allergic patients to the fcur fzrms of Amb a I. Using ragweed pollen extractspecific T cell lines, a study of 1:he reac?;ivi:y of the T cells to the four forms 02 .Q&J a i is underway. Preliminary results show !-hat al1 four forms are capable of stiaulatln~ r_he proliferation of pollen-reactive T cells, 2vm5d I proteins appear to be the major 'i cell-reactive proteins present in crude pollen extract, based on the magnitude of the proliferative responses to crude extract compared to recovhinant ~mb a I. A study of the relative potency of the four forms of Amb a I in the activation of hurar. 'I' cells is underway. In further experiments, tke r CR!: cross-reactivity among the four forms nf recombinant Amb a I will be determined. Preliminary results show a high level of .zrossamong the four forms of &ti a 1. The reactivity areas of the AI& a T molecules confer-i-?ng 1 ceil cress-reactivity will be examined.
232
LYMPHOCYTE PROLIFERATIVE RESPONSES TO PURIFIED ANTIGENS OF ASPERGILLUS. P.S.Hutcheaon,
Ph D.. J,L.
Cambridge, MA, Montreal, Canada. Amb a V (Ra5S) and Amb 1 V (Ra5G) are two small allergens purified from short and giant ragweed pollen, respectively. These two proteins share 50% amino acid homology with eight cysteines located in the identical positions. However, human and animal antibodies generated to Amb a Vor Amb f V have been reported to have little or no crossreactivity. We have mapped the T cell epitopes of Amb a V and Amb t V by the stimulation of antigen specific T cell hybridomas with overlapping peptide sets. The epitopes mapped using T cell hybtidomas were confirmed by murine T cell proliferation studies. Two overlapping peptides from each allergen stimulated T cell responses. There are eight amino acids shared at the overlapping region of the two peptides. Five of these eight residues near the amino terminus are identical. Crossreactivity studies have shown that peptides containing T cell epitopes from Amb a Vstimulated T cells obtained from animals primed with Amb t V. These results indicated the crossreactivities of these two allergens at the T cell level. We are currently investigating the effects of MHC haplotype on the T cell epitopes of Amb a Vand Amb t V.
(AF) are recognized by lymphocytes of allergic bronchopuhnonary aspergikks (ABPA) patients, lymphocyte transformations were performed using purified antigens Asp tl and mitogillin. Asp fl is an 18 kD antigen immunoprecipitated from cultures of AF. Asp fI showed 95% homology to mitogillin, a cytotoxin produced by Asoerpilfus restrictus. Both Asp fl and mitogiIlin caused >90% inhibition of protein &thesis in a rabbiireticdocyte lysate assay. When prolifckions were performed, none of the ABPA patients or controls tested had a positive stimulation index (>2) with mitogiltin stimulation. In fact, mitogillin had an inhibitory or toxic effect on cellular proliferation. Conversely, 3/4 ABPA patients demonstrated a strong stimulation index to lOfig/ml Asp fl [x SI=6.8) and S@ml Asp fl (x X=7.8). 2/4 controls showed a posiiive SI, although the response was not as great (x Sl=2.5 and x SI=1.7 at 10 and S&/ml, respectively). We tesled the ability of these two antigens to inhibit ongoing lymphocyte rescxmses to Candida and tetanus toxoid. Mitogillin (lO&mi) strohgly inhibited ongoing responses in 4 &PA patients (x 85.4% inhibition for Candida and.x 69.6% inhibition ior tetank), and in 4 controls (x 87.6% inhibition for Candida and x 87.5% inh*&ition for tetanus). In comparison, Asp il (lO~g/ml) inhibited to a much lesser extent ongoing Candida responses in i/3 AEtPA patients (14.2% inhibition), ongoing tetanus responses in 2/3 AEPA patients (4X6%, 10.4% inhibition), ongoing Candida responses in 2/3 controls (X5.3%, 72.7% inhibition) and ongoing tetanus responses in 313controls (x 52.6% inhibition). Although mitogillin and Asp fi show homology and are both inhibitors of protein synthesis, they act quite differently in their ability to stimulate lymphocytes and inhibit ongoing proliferative responses in vitro.
229
SlMPLIFIED METHOD OF HLA-DR . TYPING USING . THE PCR. N Shinomiva. MD. M Shmgg, va, MD. L R
231
Freidhoff. M.S. and DG Marsh. Ph.& Ba&more, MD. It has been reported that certain IUA-D-encoded specificities are associatedwith specific immune responsesto particular allergens. The amplification of specific DNA sequences by the polymerase chain reaction (PCR), with dot-blotting using sequence-specificoligonucleotides (SSOs), has provided a new approach for typing HLA-D alleles. We have developed a simplified method which facilitates this procedure, which will be usedfor first HLA and Allerev studv to be included in the 1991Histocompatibility W&kshoi. The HLA-D data will be correlated with specific IgE and I& Abs to >lO highly purified allergens. Atdpic pati&ts’ W&s were separatedand lysed by boiling. Second-exon HLA-DRB segments of the releasedDNA were amplified by PCR with primers DRBS’-I (ACCGGATCG’MCITGTCCCCICAG14CA1s) and DRB-3’-1 (283CTCGCCICIGCACIGTIAAGC263) designed in our laboratory. Inosine (I) was included at positions known to exhibit allelic polymorphism in expressed DRB eenes. The Denultimate 3’-residue of DRB3!-I coincides Gith a polykorphic residue sharedby all known expressed DRB genes, but not by pseudogenes. Specific alleles in a PCR-amplified sample were determined by dot-blot hybridization with 70 32P-labeledSSOsfrom the Histocompatibility Workshop and our lab. A conserved “framework” SSO was used to control for the Ievel of amplification, and a seriesof dilutions of a mixture of several patients’ amplified DRB secondexons was used to standardize the procedure. Quantitation of the procedure was achieved using an automated scanner(Betagen), from which the data was analyzed by computer. These newly designed primers allowed amplification of the all known expressed alleles of DRBI. B3. B4 and B5 loci. but no useudopenes. This rauid. and reprodbcible assay (and related &says hr DQA, DQB’, DPA and DPB) will be valuable in HLA-D typing 15OO+ patients for the 1991 HLA and Allergy study.
230
CROSSREACTIVITY OF T CELL EPITOPES IN Amb a V AND Amb t V. &JJhu Ph.D.. I Qg#&&j&
ALLERGIC HUMAN T CELL REACTIVITY TO MILTIPlf FORMS OF Amb a I, A MAJOR ALLERGEN GF SHORT ._ RAGWEED. A.D.. C.M. m ti S., S.. J.P. Noraenstern.PhJl,_&.-L, Greenstein., Cambridge, MA. The major allergen of short ragweed i&wr~.ia artemiisifolia), Amb a I, formerly called Antfqen E, has recently been shown by clor,iny and sequencing studies to be composed of a faml.ly of four related proteins, Amb a 1.1, i.?, 1.3 and 1.4. The identity in primary amino acid sequence among the four family members ranges from T3% to 87%. Recombinant Amb a .I proteins were produced in bacteria and were shown to react with TqE from ragweed-allergic patients on Western blots, consistent with the notion that a;: four forms of Amb a I are allergenic in humans. Sir,ce T ceil help is required for IgE production, :t is also of interest to examine the reactivity of 7' cells from ragweed-allergic patients to the fcur fzrms of Amb a I. Using ragweed pollen extractspecific T cell lines, a study of 1:he reac?;ivi:y of the T cells to the four forms 02 .Q&J a i is underway. Preliminary results show !-hat al1 four forms are capable of stiaulatln~ r_he proliferation of pollen-reactive T cells, 2vm5d I proteins appear to be the major 'i cell-reactive proteins present in crude pollen extract, based on the magnitude of the proliferative responses to crude extract compared to recovhinant ~mb a I. A study of the relative potency of the four forms of Amb a I in the activation of hurar. 'I' cells is underway. In further experiments, tke r CR!: cross-reactivity among the four forms nf recombinant Amb a I will be determined. Preliminary results show a high level of .zrossamong the four forms of &ti a 1. The reactivity areas of the AI& a T molecules confer-i-?ng 1 ceil cress-reactivity will be examined.
232
LYMPHOCYTE PROLIFERATIVE RESPONSES TO PURIFIED ANTIGENS OF ASPERGILLUS. P.S.Hutcheaon,
Ph D.. J,L.
Cambridge, MA, Montreal, Canada. Amb a V (Ra5S) and Amb 1 V (Ra5G) are two small allergens purified from short and giant ragweed pollen, respectively. These two proteins share 50% amino acid homology with eight cysteines located in the identical positions. However, human and animal antibodies generated to Amb a Vor Amb f V have been reported to have little or no crossreactivity. We have mapped the T cell epitopes of Amb a V and Amb t V by the stimulation of antigen specific T cell hybridomas with overlapping peptide sets. The epitopes mapped using T cell hybtidomas were confirmed by murine T cell proliferation studies. Two overlapping peptides from each allergen stimulated T cell responses. There are eight amino acids shared at the overlapping region of the two peptides. Five of these eight residues near the amino terminus are identical. Crossreactivity studies have shown that peptides containing T cell epitopes from Amb a Vstimulated T cells obtained from animals primed with Amb t V. These results indicated the crossreactivities of these two allergens at the T cell level. We are currently investigating the effects of MHC haplotype on the T cell epitopes of Amb a Vand Amb t V.
(AF) are recognized by lymphocytes of allergic bronchopuhnonary aspergikks (ABPA) patients, lymphocyte transformations were performed using purified antigens Asp tl and mitogillin. Asp fl is an 18 kD antigen immunoprecipitated from cultures of AF. Asp fI showed 95% homology to mitogillin, a cytotoxin produced by Asoerpilfus restrictus. Both Asp fl and mitogiIlin caused >90% inhibition of protein &thesis in a rabbiireticdocyte lysate assay. When prolifckions were performed, none of the ABPA patients or controls tested had a positive stimulation index (>2) with mitogiltin stimulation. In fact, mitogillin had an inhibitory or toxic effect on cellular proliferation. Conversely, 3/4 ABPA patients demonstrated a strong stimulation index to lOfig/ml Asp fl [x SI=6.8) and S@ml Asp fl (x X=7.8). 2/4 controls showed a posiiive SI, although the response was not as great (x Sl=2.5 and x SI=1.7 at 10 and S&/ml, respectively). We tesled the ability of these two antigens to inhibit ongoing lymphocyte rescxmses to Candida and tetanus toxoid. Mitogillin (lO&mi) strohgly inhibited ongoing responses in 4 &PA patients (x 85.4% inhibition for Candida and.x 69.6% inhibition ior tetank), and in 4 controls (x 87.6% inhibition for Candida and x 87.5% inh*&ition for tetanus). In comparison, Asp il (lO~g/ml) inhibited to a much lesser extent ongoing Candida responses in i/3 AEtPA patients (14.2% inhibition), ongoing tetanus responses in 2/3 AEPA patients (4X6%, 10.4% inhibition), ongoing Candida responses in 2/3 controls (X5.3%, 72.7% inhibition) and ongoing tetanus responses in 313controls (x 52.6% inhibition). Although mitogillin and Asp fi show homology and are both inhibitors of protein synthesis, they act quite differently in their ability to stimulate lymphocytes and inhibit ongoing proliferative responses in vitro.