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140 Human IgE binding factor produced by T cell hybridomas
SPONTANEOUS IgE FORMATIONIN VITRO BY ISOLATED B CELLS FROMPATIENTS WITH ATOPIC DERMATITIS. Donald McNeil, M.D., Linda F. Thompson, Ph.D., Michael H. Mellon, M.D., Robert S. Zeiger, M.D., Ph.D. and Hans L. Spiegelberg, M.D., La Jolla,
139
California Spontaneous IgE formation was stud--in vitro ied in B cell preparations from 6 patients with atopic dermatitis having serum IgE levels of 1800 to 31,000 IU/ml. Monocytes were depleted with a magnet after colloidal iron treatment and T cells were depleted by rosetting with neuraminidase-treated SRBC. To control for IgE preformed in vivo, cells were cultured with 10 pg cycloheximidelml and/or frozen and thawed on day 0 and cultured for 10 days. Supernatants from
control cultures contained 0.2-3 ng IgE/ml/106 cells while those from experimental cultures contained 1.1-40 ng IgE/ml after 10 days. The ratio of the concentration of IgE in experimental vs. control supernatants was 8.424.3 (mean+SD).
Supernatants
from cultured
depletion
T cells
of
but
B cells not
enriched
monocytes
by
contained
similar concentrations of IgE/ml (3-43 rig/ml), but control supernatants from such cultures contained
2-12
ng IgE/ml.
Thus,
a large
fraction
of
IgE preformed -___ in viva
appears to be associated with phagocytic cells in these cultures. The data indicate that lymphocyte preparations enriched for B and null cells from atopic dermatitis patients spontaneously synthesize significant amounts of IgE --in vitro and contain only small amounts of IgE preformed in vivo. Such -I_ isolated B cell preparations may be better suited for studying the regulation of IgE synthesis by
1DENTlFICATION OF THE MEMBRANE MOLECULES FROM RPM1 8866 CELLS REACTING WlTH A MONOCLONAL T. Nakajima, ANTIBODY (Mab) SPECIFIC TO FCER. Ph.D., Ph.D., A.H. Sehon, M.Sc., E. Rector, D.Sc. and G. Delespesse, M.D., Ph . b. , Wi nnipcg. Manitoba, Canada. The purpose of this srudy is to compare the membrane molecules from FCER bearing RPM1 8866 cells binding to IgE to those binding to d previously characterized mouse Mab specific to w~i-f surf‘ice FccR (Mab KE ). RPM1 8866 cells solubIliz.ed with NP40 and subseradiolabelled, adsorbed on Pansorbin and human IgGquently coupled to Sepharose-4B (lgG-Seph). The fraction was then reacted with IgE-Seph, Mab RE-Seph. ,ind or in the absence control Seph, in the presence of soluble IgE, Mab RE. and unrelated Mab. After incubation at 4'C, gels were washed, counted in SDS-PAGE for a gamma counter and processed Results conditions. analysis under reducing indicated that (1) two major bands with approximate MW of 45 kd and 80 kd were identified in the SDS-PAGE autoradiograms from IgE-Seph, Mab and (2) the binding and control-Seph, RE-Seph, of these 45 kd and 80 kd materials to IgE-Scph and to Mab RE-Seph was strongly reduced by both soluble IgE and Mab Rt but not by unrelated Mab as shown both by the SUS-PAGE autoradiograms and by measuring the radioactivity of the corrcsIt is concluded thnt immunoadsorbents. pending (i) Mab RE and IgE bind to the same two membrnnc molecules with HW of 45 and 80 kd ab previously (Ii) the same molrculcs reported by others, display some nonspecific affinity for Scplb~~rosc4B.
isotype-specific regulatory cells and cell-free factors than mononuclear cell preparations.
138
INCREASEDNUMBERSOF CIRCULATING LEU 2+ LELI llf
140
LYMPHOCYTES IN HYPER IGE STATES. D.Y.M. Leung, B.R. Smith. V. Monafo. R.S. Ceha, Boston, MA. Peripheral blood lymphocytes (PBL) bearing a high density of T8 or Leu 2a surface antigens represent suppressorlcytotoxic T lymphocytes. Recently, PBL bearing low density Leu 2 or T8 surface have been described. These dimly fluorescent Leu 2+ cells stain with t?ce NK marker, Leu 11, but not with the T cell markers, Leu 1 or Leu 4. Leu 2+ Leu 11+, but not Leu 2+ Leu 4+ cells manifest IiK activity against K562 cells. In this report PBL from patients with the hyper IgE syndrome (HIE;n=3), severe atopic dermatitis (AD;n=6) and normal donors (NL;n=8) were stained with fluorescein conjugated (FITC)Leu 2a. Fluorescent cells were enumerated on a B-D FACS IV cell sorter. Both patient populations had an abnormally low % PBL of brightly fluorescent LeuZ+ cells (HIE mean=7%; (~(0.01); AD mean=ll% (P(O.01) vs NL mean =19%). In contrast, patients with HIE and AD had a significantly higher % of dimly fluorescent Leu 2+cells than normal (HIE mean=7.1%, ~(0.01); AD mean= 9.0%; p< 0.01 vs NL mean=3.6%). Double immunofluorescence studies using phycoerythrin and
FITC antibody
conjugates
revealed
that
HUMAH IGE BINDING FACTOR PRODUCED BY T CELL HYBRIDOMAS. B.M. Stadler, J. Knutti
Miiller
and A.L.
de Week, Bern,
Switzerland Ha,nnan T cell hybridomas were produced by fusion of a mutant azaguanine-resist-
ant human T cell line (JURKAT) with human peripheral blood lymphocytes stimulated with PHA and expanded in interleukin 2. In the supernatant of such hybridoma cell cultures, a 60 KD glycoprotein could be isolated by affinity chromatography on Con A-Sepharose and further
purified
by HPLC.
This
protein
has the property of strongly binding IgE in a isotype-specific manner as revealed by a highly sensitive binding test on nitrocellulose membranes. The biological properties of this T cell factor are assessed by its effect on IgE synthesis in vitro from atopic PBL and from the U 266 cell line. The purified IgE binding factor was also evaluated in the in vivo IgE response of SJL and Balb/c mice, which had been shown earlier to be profoundly depressed by cruder supernatants. This hybridoma appears to be a suitable source for isolation of messenger RNA and production of the IgE binding factor by genetic engineering.
the in-
creased number of Leu 2+ cells in patients were Leu ll+ Leu I- Leu 4-. These results suggest that patients with HIE and severe AD have increased numbers of circulating Leu 2f which are non-T cells with NK phenoThe presence of circulating Leu 2 or T8+ type. NK cells in these patients may account for conflicting results obtained in the past by various investigators who have attempted to enumerate % T8+ or Leu 2-t T cells in HIE and AD.
139
139
California Spontaneous IgE formation was stud--in vitro ied in B cell preparations from 6 patients with atopic dermatitis having serum IgE levels of 1800 to 31,000 IU/ml. Monocytes were depleted with a magnet after colloidal iron treatment and T cells were depleted by rosetting with neuraminidase-treated SRBC. To control for IgE preformed in vivo, cells were cultured with 10 pg cycloheximidelml and/or frozen and thawed on day 0 and cultured for 10 days. Supernatants from
control cultures contained 0.2-3 ng IgE/ml/106 cells while those from experimental cultures contained 1.1-40 ng IgE/ml after 10 days. The ratio of the concentration of IgE in experimental vs. control supernatants was 8.424.3 (mean+SD).
Supernatants
from cultured
depletion
T cells
of
but
B cells not
enriched
monocytes
by
contained
similar concentrations of IgE/ml (3-43 rig/ml), but control supernatants from such cultures contained
2-12
ng IgE/ml.
Thus,
a large
fraction
of
IgE preformed -___ in viva
appears to be associated with phagocytic cells in these cultures. The data indicate that lymphocyte preparations enriched for B and null cells from atopic dermatitis patients spontaneously synthesize significant amounts of IgE --in vitro and contain only small amounts of IgE preformed in vivo. Such -I_ isolated B cell preparations may be better suited for studying the regulation of IgE synthesis by
1DENTlFICATION OF THE MEMBRANE MOLECULES FROM RPM1 8866 CELLS REACTING WlTH A MONOCLONAL T. Nakajima, ANTIBODY (Mab) SPECIFIC TO FCER. Ph.D., Ph.D., A.H. Sehon, M.Sc., E. Rector, D.Sc. and G. Delespesse, M.D., Ph . b. , Wi nnipcg. Manitoba, Canada. The purpose of this srudy is to compare the membrane molecules from FCER bearing RPM1 8866 cells binding to IgE to those binding to d previously characterized mouse Mab specific to w~i-f surf‘ice FccR (Mab KE ). RPM1 8866 cells solubIliz.ed with NP40 and subseradiolabelled, adsorbed on Pansorbin and human IgGquently coupled to Sepharose-4B (lgG-Seph). The fraction was then reacted with IgE-Seph, Mab RE-Seph. ,ind or in the absence control Seph, in the presence of soluble IgE, Mab RE. and unrelated Mab. After incubation at 4'C, gels were washed, counted in SDS-PAGE for a gamma counter and processed Results conditions. analysis under reducing indicated that (1) two major bands with approximate MW of 45 kd and 80 kd were identified in the SDS-PAGE autoradiograms from IgE-Seph, Mab and (2) the binding and control-Seph, RE-Seph, of these 45 kd and 80 kd materials to IgE-Scph and to Mab RE-Seph was strongly reduced by both soluble IgE and Mab Rt but not by unrelated Mab as shown both by the SUS-PAGE autoradiograms and by measuring the radioactivity of the corrcsIt is concluded thnt immunoadsorbents. pending (i) Mab RE and IgE bind to the same two membrnnc molecules with HW of 45 and 80 kd ab previously (Ii) the same molrculcs reported by others, display some nonspecific affinity for Scplb~~rosc4B.
isotype-specific regulatory cells and cell-free factors than mononuclear cell preparations.
138
INCREASEDNUMBERSOF CIRCULATING LEU 2+ LELI llf
140
LYMPHOCYTES IN HYPER IGE STATES. D.Y.M. Leung, B.R. Smith. V. Monafo. R.S. Ceha, Boston, MA. Peripheral blood lymphocytes (PBL) bearing a high density of T8 or Leu 2a surface antigens represent suppressorlcytotoxic T lymphocytes. Recently, PBL bearing low density Leu 2 or T8 surface have been described. These dimly fluorescent Leu 2+ cells stain with t?ce NK marker, Leu 11, but not with the T cell markers, Leu 1 or Leu 4. Leu 2+ Leu 11+, but not Leu 2+ Leu 4+ cells manifest IiK activity against K562 cells. In this report PBL from patients with the hyper IgE syndrome (HIE;n=3), severe atopic dermatitis (AD;n=6) and normal donors (NL;n=8) were stained with fluorescein conjugated (FITC)Leu 2a. Fluorescent cells were enumerated on a B-D FACS IV cell sorter. Both patient populations had an abnormally low % PBL of brightly fluorescent LeuZ+ cells (HIE mean=7%; (~(0.01); AD mean=ll% (P(O.01) vs NL mean =19%). In contrast, patients with HIE and AD had a significantly higher % of dimly fluorescent Leu 2+cells than normal (HIE mean=7.1%, ~(0.01); AD mean= 9.0%; p< 0.01 vs NL mean=3.6%). Double immunofluorescence studies using phycoerythrin and
FITC antibody
conjugates
revealed
that
HUMAH IGE BINDING FACTOR PRODUCED BY T CELL HYBRIDOMAS. B.M. Stadler, J. Knutti
Miiller
and A.L.
de Week, Bern,
Switzerland Ha,nnan T cell hybridomas were produced by fusion of a mutant azaguanine-resist-
ant human T cell line (JURKAT) with human peripheral blood lymphocytes stimulated with PHA and expanded in interleukin 2. In the supernatant of such hybridoma cell cultures, a 60 KD glycoprotein could be isolated by affinity chromatography on Con A-Sepharose and further
purified
by HPLC.
This
protein
has the property of strongly binding IgE in a isotype-specific manner as revealed by a highly sensitive binding test on nitrocellulose membranes. The biological properties of this T cell factor are assessed by its effect on IgE synthesis in vitro from atopic PBL and from the U 266 cell line. The purified IgE binding factor was also evaluated in the in vivo IgE response of SJL and Balb/c mice, which had been shown earlier to be profoundly depressed by cruder supernatants. This hybridoma appears to be a suitable source for isolation of messenger RNA and production of the IgE binding factor by genetic engineering.
the in-
creased number of Leu 2+ cells in patients were Leu ll+ Leu I- Leu 4-. These results suggest that patients with HIE and severe AD have increased numbers of circulating Leu 2f which are non-T cells with NK phenoThe presence of circulating Leu 2 or T8+ type. NK cells in these patients may account for conflicting results obtained in the past by various investigators who have attempted to enumerate % T8+ or Leu 2-t T cells in HIE and AD.
139