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Biochemical characterization of human 1-A subregion-like molecules
152
Annual AACHT Meeting, 1982 alone. The Leu-2 + precursors of this activated amplifier population could not be removed by treatment with a pool of cytotoxic monoclonai antibodies to HLADR antigen and thus appeared to become DR positive with activation. The activation of this suppressive subpopulation required Leu-3 + cells (suppressor/ inducer cells) in the primary cultured. Furthermore, i , the absence of fresh Leu2 + cells in the second culture, little or no suppression was observed. These results suggest that at least two distinct populations of Leu-2 + cells are required i0r maximal suppression of an immune response and that immunoregulatory circuits analogous to those described in the mouse exist in man.
LIMITING DILUTION ANALYSIS OF A N T I G E N REACTIVE CELLS SENSITIZED IN VITRO. Howard Gebel, John Scott, Curtis Parvin, and Glenn Rody; Washington
University, School of Medicine attd Barnes Hospital St. Louis Limiting dilution analysis was used to estimate the clone size of human peripheral blood T lymphocytes that proliferate following primary and secondary in vitro sensitization with keyhole limpet hemocyanin (KLH). T lymphocytes from 12 subjects were cultured with autologous adherent cells + KLH for 8 days (primary culture), pulsed with ~H-thymidine for 18 hr, and assayed on day 9. Several T cell concentrations (1 × 10s-2 x 10~') were cultured in replicates of 24, and the frequency of antigen reactive cells (ARC) was estimated by the Poisson statistic. The ARC of T cells from the 12 subjects ranged from 1:24,800 to 1:52,631. The degree of proliferation in standard cultures did not correlate with ARC frequency, i.e., a high (or low) proliferative response did not correlate with a high (or low) ARC frequency, lflymphocytes from the same subject were retested several times, the range ~f the standard proliferative response was broad (5,00023,000 cpm). In contrast, the ARC frequency was relatively consistent (1:37,000 to 1:41,000). T lymphocytes from 5 subjects were also primed for 12 days, then restimulated with fresh adherent cells + KLH for 4 days (secondary culture). The ARC frequency of primed cells increased to 1:1,123 to 1:7,247 indicating that T cell clones reactive to KLH had expanded during primary culture. Finally, we observed that the percentage of positively responding cultures was dramatically reduced when lymphocytes from 5/12 subjects were cultured at 2 x 10C'/ml compared to I x 10C'/ml (13% and 98%, respectively). However, removal o f T 8 + T cells before culture did not alter the inhibitory effect of high cell concentrations. ARC estimates in humans may be a preferred technique for assessing genetic low or high responders. BIOCHEMICAL CHARACTERIZATION OF H U M A N I-A SUBREGION-LIKE MOLECULES. Sanna M. Goyert and Jack Silver; Michigan State Unit'ersity. East Lansing.
Michigan Although two groups of la molecules, designated I-A and I-E, have been identified in mice, only one group, known as HLA-DR, has been found in man. Human DR molecules are homologous to the routine l-E molecules on the basis of amino acid sequence data. We have recently identified the human and marmoset equivalents to the murine I-A molecules (Nature 294:266, 1981) using a monoclonal antibody designated SG171. Additional studies have allowed us to draw the following conclusions. (1) SG171 is a polymorphic antibody, i.e., it binds I-A molecules found on DR7 cell lines but not on DR1, DR2, or DR3. This observation, combined with the observation that the I-A molecules of three different
Abstracts
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DR7 cell lines are identical suggest that, as in mice, the genees) fi~r th~~ b A molecule is tightly linked to the gene(s) or the DR {l-E-like~ moiecuk-~ ~2~ A rabbit antibody prepared by immunization with purified marmosc.t i-A n~g~k-cuk.~ reacts with I-A molecules from human cell lines other than DR7. ~3~ H u m ~ D R and I-A molecules, although quite distinct on the basis of am~m~ ~cM ~g-o quence and peptide map data, have very similar two-dimensiona*.| gel V,~tern~ The large subunits of I-A and D R molecules are virtually identical in size ~*sd charge; the small subunits, although distinguishable by charge, are ,~early ~e~,~:M in size. The similarity in size of the D R and I-A a- and/~subunits m ~ c s it like~ that others have isolated I-A molecules, but have mistakenly identified them ~g HLA-DR. S U B I D I V I S I O N OF B27 BY USE OF A L Y M P H O C Y T O T O X i C M O N O C L O N A L A N T I B O D Y , B27M2. F. Carl Grumet, Linda Fish, Brian M. Fendly, Steven Foung, and Edgar G. Engleman; Department of Pathology. Stanford University. Stanford. California A monoclonal antibody, anti-B27M2, which subdivides B27, is described, f)¢ ~ healthy B27 + individuals tested with this antibody by standard lymphocyto¢~xicity, 87% were B27M2 + and 13% were B27M2 -. Ethnic groups included C~ucasian, Black, Japanese, Chinese, and Mexican-American. B27 ÷ families, c~:h B27M2 + or B 2 7 M 2 - , showed expected segregation for each variant. U ~ k e anti-B27M 1, the first reported anti-B27 monoclonal antibody, anti-B27M2 ~k~:~ not cross-react with B7 but does recognize (3/3) B47 ÷ individuals. F~ab'~: ~t:rom alloantisera) and anti-beta-2-microglobulin blocking confirmed specificity: for the B27 antigen. By indirect radioimmunoassay with Epstein-Barr v i r u s - t r a n s f o ~ lymphoblastoid cell lines, B27 + B27M2 + (by cytotoxicity) cells bound an¢iB27M1, anti-B27M2, and anti-HLA (W6/32). In contrast B27 ÷ B2"~M2 ceUs bound equivalent amounts of anti-B27M 1 and W6/32 but not anti-B27M2. T h e ~ data suggest that the B27 antigen possesses at least two distinct epitopes: common B27M 1 epitope and a second epitope, B27M2, present on most ~at not all B27 molecules. A study of the relation between these B27 var/ants ~ B27-related spondyloarthropathies has been initiated and should prove of interest in further refining and dissecting the association between B27 and disea~- susceptibility. A N E W D R I ~ f P I N G M E T H O D U S I N G M O N O C L O N A L ANTI-T CELL A N ~ I B O D Y (TM 1) A N D U N F R A C T I O N A T E D PERIPHERAL BLOOD LYMPHOCYTES. F.C. Grumet, Sheryl Pask, Dolly B. Ness, Brian M. Fendly, and Edgar G. Englermm:
Department of Pathology, Stanford University. Stanford. California A simple new method for DR typing has been deveJoped in which no isoL~on or differential staining of B/T cells is required. The method, which is sim/l~r to routine ABC typing uses TM 1, an IgM monoclonal antibody cytotoxic to T but not B cells. Peripheral blood lymphocytes are isolated from as much as 3-d~yo old ACD whole blood !-,~, Ficoll-Hypaque, and then stored in liquid nitrogen. After thawing, cells are l,,iJeled with carboxy-fluorescein diacetate, relayered over Ficoll-Itypaque and then resuspended in medium at 5 x lff'/ml. Cell suspension (1 Mwell) is added to routine 60 or 72 well D R typing tr~ys preloaded with sera (1 Mwell). After ½ hr (37°C), IA of diluted T M | is to each well, and another ½ hr incubation is followed by addition of lO g/~eU of the usual complement. Two hours later (20°C) each well is read ~only k~ce
Annual AACHT Meeting, 1982 alone. The Leu-2 + precursors of this activated amplifier population could not be removed by treatment with a pool of cytotoxic monoclonai antibodies to HLADR antigen and thus appeared to become DR positive with activation. The activation of this suppressive subpopulation required Leu-3 + cells (suppressor/ inducer cells) in the primary cultured. Furthermore, i , the absence of fresh Leu2 + cells in the second culture, little or no suppression was observed. These results suggest that at least two distinct populations of Leu-2 + cells are required i0r maximal suppression of an immune response and that immunoregulatory circuits analogous to those described in the mouse exist in man.
LIMITING DILUTION ANALYSIS OF A N T I G E N REACTIVE CELLS SENSITIZED IN VITRO. Howard Gebel, John Scott, Curtis Parvin, and Glenn Rody; Washington
University, School of Medicine attd Barnes Hospital St. Louis Limiting dilution analysis was used to estimate the clone size of human peripheral blood T lymphocytes that proliferate following primary and secondary in vitro sensitization with keyhole limpet hemocyanin (KLH). T lymphocytes from 12 subjects were cultured with autologous adherent cells + KLH for 8 days (primary culture), pulsed with ~H-thymidine for 18 hr, and assayed on day 9. Several T cell concentrations (1 × 10s-2 x 10~') were cultured in replicates of 24, and the frequency of antigen reactive cells (ARC) was estimated by the Poisson statistic. The ARC of T cells from the 12 subjects ranged from 1:24,800 to 1:52,631. The degree of proliferation in standard cultures did not correlate with ARC frequency, i.e., a high (or low) proliferative response did not correlate with a high (or low) ARC frequency, lflymphocytes from the same subject were retested several times, the range ~f the standard proliferative response was broad (5,00023,000 cpm). In contrast, the ARC frequency was relatively consistent (1:37,000 to 1:41,000). T lymphocytes from 5 subjects were also primed for 12 days, then restimulated with fresh adherent cells + KLH for 4 days (secondary culture). The ARC frequency of primed cells increased to 1:1,123 to 1:7,247 indicating that T cell clones reactive to KLH had expanded during primary culture. Finally, we observed that the percentage of positively responding cultures was dramatically reduced when lymphocytes from 5/12 subjects were cultured at 2 x 10C'/ml compared to I x 10C'/ml (13% and 98%, respectively). However, removal o f T 8 + T cells before culture did not alter the inhibitory effect of high cell concentrations. ARC estimates in humans may be a preferred technique for assessing genetic low or high responders. BIOCHEMICAL CHARACTERIZATION OF H U M A N I-A SUBREGION-LIKE MOLECULES. Sanna M. Goyert and Jack Silver; Michigan State Unit'ersity. East Lansing.
Michigan Although two groups of la molecules, designated I-A and I-E, have been identified in mice, only one group, known as HLA-DR, has been found in man. Human DR molecules are homologous to the routine l-E molecules on the basis of amino acid sequence data. We have recently identified the human and marmoset equivalents to the murine I-A molecules (Nature 294:266, 1981) using a monoclonal antibody designated SG171. Additional studies have allowed us to draw the following conclusions. (1) SG171 is a polymorphic antibody, i.e., it binds I-A molecules found on DR7 cell lines but not on DR1, DR2, or DR3. This observation, combined with the observation that the I-A molecules of three different
Abstracts
|~
DR7 cell lines are identical suggest that, as in mice, the genees) fi~r th~~ b A molecule is tightly linked to the gene(s) or the DR {l-E-like~ moiecuk-~ ~2~ A rabbit antibody prepared by immunization with purified marmosc.t i-A n~g~k-cuk.~ reacts with I-A molecules from human cell lines other than DR7. ~3~ H u m ~ D R and I-A molecules, although quite distinct on the basis of am~m~ ~cM ~g-o quence and peptide map data, have very similar two-dimensiona*.| gel V,~tern~ The large subunits of I-A and D R molecules are virtually identical in size ~*sd charge; the small subunits, although distinguishable by charge, are ,~early ~e~,~:M in size. The similarity in size of the D R and I-A a- and/~subunits m ~ c s it like~ that others have isolated I-A molecules, but have mistakenly identified them ~g HLA-DR. S U B I D I V I S I O N OF B27 BY USE OF A L Y M P H O C Y T O T O X i C M O N O C L O N A L A N T I B O D Y , B27M2. F. Carl Grumet, Linda Fish, Brian M. Fendly, Steven Foung, and Edgar G. Engleman; Department of Pathology. Stanford University. Stanford. California A monoclonal antibody, anti-B27M2, which subdivides B27, is described, f)¢ ~ healthy B27 + individuals tested with this antibody by standard lymphocyto¢~xicity, 87% were B27M2 + and 13% were B27M2 -. Ethnic groups included C~ucasian, Black, Japanese, Chinese, and Mexican-American. B27 ÷ families, c~:h B27M2 + or B 2 7 M 2 - , showed expected segregation for each variant. U ~ k e anti-B27M 1, the first reported anti-B27 monoclonal antibody, anti-B27M2 ~k~:~ not cross-react with B7 but does recognize (3/3) B47 ÷ individuals. F~ab'~: ~t:rom alloantisera) and anti-beta-2-microglobulin blocking confirmed specificity: for the B27 antigen. By indirect radioimmunoassay with Epstein-Barr v i r u s - t r a n s f o ~ lymphoblastoid cell lines, B27 + B27M2 + (by cytotoxicity) cells bound an¢iB27M1, anti-B27M2, and anti-HLA (W6/32). In contrast B27 ÷ B2"~M2 ceUs bound equivalent amounts of anti-B27M 1 and W6/32 but not anti-B27M2. T h e ~ data suggest that the B27 antigen possesses at least two distinct epitopes: common B27M 1 epitope and a second epitope, B27M2, present on most ~at not all B27 molecules. A study of the relation between these B27 var/ants ~ B27-related spondyloarthropathies has been initiated and should prove of interest in further refining and dissecting the association between B27 and disea~- susceptibility. A N E W D R I ~ f P I N G M E T H O D U S I N G M O N O C L O N A L ANTI-T CELL A N ~ I B O D Y (TM 1) A N D U N F R A C T I O N A T E D PERIPHERAL BLOOD LYMPHOCYTES. F.C. Grumet, Sheryl Pask, Dolly B. Ness, Brian M. Fendly, and Edgar G. Englermm:
Department of Pathology, Stanford University. Stanford. California A simple new method for DR typing has been deveJoped in which no isoL~on or differential staining of B/T cells is required. The method, which is sim/l~r to routine ABC typing uses TM 1, an IgM monoclonal antibody cytotoxic to T but not B cells. Peripheral blood lymphocytes are isolated from as much as 3-d~yo old ACD whole blood !-,~, Ficoll-Hypaque, and then stored in liquid nitrogen. After thawing, cells are l,,iJeled with carboxy-fluorescein diacetate, relayered over Ficoll-Itypaque and then resuspended in medium at 5 x lff'/ml. Cell suspension (1 Mwell) is added to routine 60 or 72 well D R typing tr~ys preloaded with sera (1 Mwell). After ½ hr (37°C), IA of diluted T M | is to each well, and another ½ hr incubation is followed by addition of lO g/~eU of the usual complement. Two hours later (20°C) each well is read ~only k~ce