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119 Simplified human whole blood assay for measurement of dust mite specific gamma interferon production in vitro
118
119
IMMUNOGENIC EPITOPES OF THE p55 CHAIN OF THE INTERLEUKIN 2 (ILZ) RECEPTOR AND THEIR RELATIONSHIP TO IL2 BINDING. JT W&g, E Schott, A Aruffo, B Seed, EM Sabga, D Camerini, RB Colvin,Boston,MA IL2 provides a critical signal for activated T CL?ll.S. The high affinity IL2 receptor (IL2R) consists of a p55 chain and a p75 chain which individually comprise the low affinity and intermediate affinity receptors. Certain monoclonal antibodies (MAB) to the p55 chain have been able to inhibit graft rejection. We raised 8 MABs to this chain by immunizing a CB6Fl mouse with mouse L cells transfected with the human TAC cDNA, in which'the p55 chain is the only human antigen. Cytofluorometric staining and competitive inhibition assays with these MABs and an extensive workshop panel revealed 3 major immunogenic epitopes on the p55 ILLR chain with several subepitopes. Epitope A maps the same region as the TAC MABs. MAB binding to epitope C did not interMABs to both epitopes A fere with ILj2kinding. I-IL2 binding to the high affinity and B block IL2R in a predominately noncompetitive manner with those to epitope B also showing a competitive component. The likely explanation for the noncompetitive inhibition is that these MABs may prevent the formation of the high affinity receptor by the p55 and p75 chains. Under low affinity binding condition, MABs to both epitopes competitively inhibit the binding of IL2. these studies are consistent with the Together, hypothesis that the ILZR interchain joining region is close to the IL2 binding site. In view of the fact that no one has been able to directly crosslink the two chains, it remains plausible that IL2 may serve as the major bridge.
120
Simplified Human Whole Blood Assay for Measurement of Dust Mite Specific Gamma Interferon Production In Vitro. Maadhava Ellaurie, . ., Arye Rubinstein, M.D. and David L Rosenstrelch, M.D. Bronx, New York 10&l.
121
RECOtATION V.,
CIBP,
OF PLATCLCT E17CCTm
LA&AI&, In8tltut Pascour
~DNCTIONB
BY T CELLS.
L.~arcbh”dn.s
dc LILLE
(Trance),
The dcmonrtra~ion of effector functions 01 platelets in paratlrlc d3aerses rose th.? quc$llOr, 01 their posrlbla ruyulatlon by T c-ells. Normal human plstelccr trcaIed rlth cu)turr fuper”a~a”ta from st~mulatcd CD4+ T c~?lla drvcloppcd the c.sp.SCiCy 10 kill the lsrvsc of 5’. ma”sonf Jn the bhwncc of IgE antlhody. 1hC ncu~rallratio” by mo”oclonal anti-IFNV sntjbody of rhlr 1”ducl”g effect, the ~~r‘~so”c-c 01 IFN-f in the s”f~2rndtanL. and fjnslly ~hc dlrocL inducer effect of locomblnant IFN-1 clearly ldon~lllud thin lymphokinc as one 01 Lhc implicared fdaoz. The in vlvo lclavbnce of this O~IYCC wan ntvdlod in the I&L model i the passive ~ranalcr of normal rb~ plrrrlera rreated with Iat recombinant IFN-# to normal Byngrnsic rcciplcnrr on Lho day of chsllenqo lnfecrlon I& to L hiQh doqxco of prolectlo”. Morcovcr IFN-ice” also bet on the tqE-dependent. plsrelet dcl1vlLy by cnhrncjnq rho Iqf: recepror c~p~ussion on the plc,rclor mrmbr~nt. Srlmuldrod CDBt T cell rupcrnalan~s conlaincd (I factor able LO lrlhibit the Iq8-defw,du”l platelet cytotoxjcjty towdrd Lhe ysraaitja Jsrvac. The I~roducL~on UC oxyqmn m~caholitos by plstelats In rn lqz-onllIqE rosc!+ion WLI, likcwico, aLronqly jnhibitcd. This PlatOlOr AcLivfty Lymphokine (PASL) was idCntlfle0 a. a polypcpcid0 of 15-10 kD *‘Jth a pl of 4.6. ThO fn vfvo ralevsncc Of PASL could he rstbbllrhod by d complrtr rholjtlon of the protectjon normally conforr*a toward a chal1erq.s infection by LtiP
Sopprcsrivc
l”travenuu* normal
paujve ,LtLS,
afLcr
trbnsfor
01 ylaLc1ct.s
PAS-trcarmont.
from of
inmum
LO
trsosferrcd
platclCts.
We have developed a simplified microassay for measurement of dust mite specific i interferon production in vitro using diluted, unseparated humanTlood. 1 interferon in 72 hr. culture supernatants was measured using a solid phase radioimmunoassay (Centocor Labs). Maximum production in allergic patients occurred between 25 and 50 ug/ml of mite antigen while higher concentrations resulted in decreased levels. Maximum antigen stimulated levels ranged from 3130u/ml. Cells from a number of patients produced high levels of T interferon in the absence Both baseline and antigen of any stimulant. stimulated levels were highest in the group of mite allergic patients. Non-mite allergic individuals exhibited significantly lower mite specific responses, and the normal group exhibited the lowest mean response. Immunotherapy with mite extract resulted in a loss of mite specific 7 interferon production in approximately 60% of allergic patients. Treatment of patients with small doses of oral corticosteroids (5-15mg/day prednisone) obliterated both baseline and antigen stimulated 1 interThese results indicate that feron production. the whole blood assay coupled with the lymphokine radioimnunoassay is a convenient, rapid and sensitive method for measuring cell mediated and responses to immunoimmunity to allergens, therapy or drug treatment, that can be easily adapted to testlng large numbers of patients.
198
PREFERENTIAL DIFFERENTIATION OF DIFFERENT HEMATOPOIETIC CELLS FROM'HUMAN BONE MARROW CELLS BY RECOMBINANT HUMAN INTERLEUKINS. H. Saito, M.D., Baltimore, MD., K. M. Leiferman, M.D., Rochester, M.D., Boston, MA., N. Arai, MN., A. M. Dvorak, M.D., Palo Alto, CA., T. Ishizaka, M.D., Baltimore, MD. differentiation of hematopoietic --In vitro cells by recombinant human interleukins (rIL) was investigated. Mononuclear cells from umbilical cord blood (CB), or light density cells of bone marrow (BM) from both mastocytosis patients and normal individuals were cultured in the presence of rIL-5, rIL-4 or r IL-3. Recombinant IL-5 induced selective differentiation of eosinophils in both CB and BM cell cultures. After 3 weeks culture with IL-S, 80 to 90% of the nonadherent cells were eosinophils. Ultrastructural examination showed that the majority of the eosinophils were eosinophilic myelocytes. IL-4 induced selective differentiation and proliferation of OKT 3+ T cells. After 2 to 3 weeks culture of CB and BM cells with rIL-4, OKT 3+ T cells comprised 85 to 95% of the total nonadherent cells. Profiles of hematopoietic cells developed by IL3 suggested that this lymphokine was a multipotential growth factor. Basophils, eosinophils, neutrophils and macrophages of multiple differentiation stages developed by IL-3 in both BM and CB cell cultures within two weeks. After 4 weeks, however, eosinophils became the majority of nonadherent cells. No human mast cell developed by rIL-3 or by IL-3 plus IL-4, suggesting that differentiation of human mast cells in vitro may require some additional growth ?&tor from non-T cells.
119
IMMUNOGENIC EPITOPES OF THE p55 CHAIN OF THE INTERLEUKIN 2 (ILZ) RECEPTOR AND THEIR RELATIONSHIP TO IL2 BINDING. JT W&g, E Schott, A Aruffo, B Seed, EM Sabga, D Camerini, RB Colvin,Boston,MA IL2 provides a critical signal for activated T CL?ll.S. The high affinity IL2 receptor (IL2R) consists of a p55 chain and a p75 chain which individually comprise the low affinity and intermediate affinity receptors. Certain monoclonal antibodies (MAB) to the p55 chain have been able to inhibit graft rejection. We raised 8 MABs to this chain by immunizing a CB6Fl mouse with mouse L cells transfected with the human TAC cDNA, in which'the p55 chain is the only human antigen. Cytofluorometric staining and competitive inhibition assays with these MABs and an extensive workshop panel revealed 3 major immunogenic epitopes on the p55 ILLR chain with several subepitopes. Epitope A maps the same region as the TAC MABs. MAB binding to epitope C did not interMABs to both epitopes A fere with ILj2kinding. I-IL2 binding to the high affinity and B block IL2R in a predominately noncompetitive manner with those to epitope B also showing a competitive component. The likely explanation for the noncompetitive inhibition is that these MABs may prevent the formation of the high affinity receptor by the p55 and p75 chains. Under low affinity binding condition, MABs to both epitopes competitively inhibit the binding of IL2. these studies are consistent with the Together, hypothesis that the ILZR interchain joining region is close to the IL2 binding site. In view of the fact that no one has been able to directly crosslink the two chains, it remains plausible that IL2 may serve as the major bridge.
120
Simplified Human Whole Blood Assay for Measurement of Dust Mite Specific Gamma Interferon Production In Vitro. Maadhava Ellaurie, . ., Arye Rubinstein, M.D. and David L Rosenstrelch, M.D. Bronx, New York 10&l.
121
RECOtATION V.,
CIBP,
OF PLATCLCT E17CCTm
LA&AI&, In8tltut Pascour
~DNCTIONB
BY T CELLS.
L.~arcbh”dn.s
dc LILLE
(Trance),
The dcmonrtra~ion of effector functions 01 platelets in paratlrlc d3aerses rose th.? quc$llOr, 01 their posrlbla ruyulatlon by T c-ells. Normal human plstelccr trcaIed rlth cu)turr fuper”a~a”ta from st~mulatcd CD4+ T c~?lla drvcloppcd the c.sp.SCiCy 10 kill the lsrvsc of 5’. ma”sonf Jn the bhwncc of IgE antlhody. 1hC ncu~rallratio” by mo”oclonal anti-IFNV sntjbody of rhlr 1”ducl”g effect, the ~~r‘~so”c-c 01 IFN-f in the s”f~2rndtanL. and fjnslly ~hc dlrocL inducer effect of locomblnant IFN-1 clearly ldon~lllud thin lymphokinc as one 01 Lhc implicared fdaoz. The in vlvo lclavbnce of this O~IYCC wan ntvdlod in the I&L model i the passive ~ranalcr of normal rb~ plrrrlera rreated with Iat recombinant IFN-# to normal Byngrnsic rcciplcnrr on Lho day of chsllenqo lnfecrlon I& to L hiQh doqxco of prolectlo”. Morcovcr IFN-ice” also bet on the tqE-dependent. plsrelet dcl1vlLy by cnhrncjnq rho Iqf: recepror c~p~ussion on the plc,rclor mrmbr~nt. Srlmuldrod CDBt T cell rupcrnalan~s conlaincd (I factor able LO lrlhibit the Iq8-defw,du”l platelet cytotoxjcjty towdrd Lhe ysraaitja Jsrvac. The I~roducL~on UC oxyqmn m~caholitos by plstelats In rn lqz-onllIqE rosc!+ion WLI, likcwico, aLronqly jnhibitcd. This PlatOlOr AcLivfty Lymphokine (PASL) was idCntlfle0 a. a polypcpcid0 of 15-10 kD *‘Jth a pl of 4.6. ThO fn vfvo ralevsncc Of PASL could he rstbbllrhod by d complrtr rholjtlon of the protectjon normally conforr*a toward a chal1erq.s infection by LtiP
Sopprcsrivc
l”travenuu* normal
paujve ,LtLS,
afLcr
trbnsfor
01 ylaLc1ct.s
PAS-trcarmont.
from of
inmum
LO
trsosferrcd
platclCts.
We have developed a simplified microassay for measurement of dust mite specific i interferon production in vitro using diluted, unseparated humanTlood. 1 interferon in 72 hr. culture supernatants was measured using a solid phase radioimmunoassay (Centocor Labs). Maximum production in allergic patients occurred between 25 and 50 ug/ml of mite antigen while higher concentrations resulted in decreased levels. Maximum antigen stimulated levels ranged from 3130u/ml. Cells from a number of patients produced high levels of T interferon in the absence Both baseline and antigen of any stimulant. stimulated levels were highest in the group of mite allergic patients. Non-mite allergic individuals exhibited significantly lower mite specific responses, and the normal group exhibited the lowest mean response. Immunotherapy with mite extract resulted in a loss of mite specific 7 interferon production in approximately 60% of allergic patients. Treatment of patients with small doses of oral corticosteroids (5-15mg/day prednisone) obliterated both baseline and antigen stimulated 1 interThese results indicate that feron production. the whole blood assay coupled with the lymphokine radioimnunoassay is a convenient, rapid and sensitive method for measuring cell mediated and responses to immunoimmunity to allergens, therapy or drug treatment, that can be easily adapted to testlng large numbers of patients.
198
PREFERENTIAL DIFFERENTIATION OF DIFFERENT HEMATOPOIETIC CELLS FROM'HUMAN BONE MARROW CELLS BY RECOMBINANT HUMAN INTERLEUKINS. H. Saito, M.D., Baltimore, MD., K. M. Leiferman, M.D., Rochester, M.D., Boston, MA., N. Arai, MN., A. M. Dvorak, M.D., Palo Alto, CA., T. Ishizaka, M.D., Baltimore, MD. differentiation of hematopoietic --In vitro cells by recombinant human interleukins (rIL) was investigated. Mononuclear cells from umbilical cord blood (CB), or light density cells of bone marrow (BM) from both mastocytosis patients and normal individuals were cultured in the presence of rIL-5, rIL-4 or r IL-3. Recombinant IL-5 induced selective differentiation of eosinophils in both CB and BM cell cultures. After 3 weeks culture with IL-S, 80 to 90% of the nonadherent cells were eosinophils. Ultrastructural examination showed that the majority of the eosinophils were eosinophilic myelocytes. IL-4 induced selective differentiation and proliferation of OKT 3+ T cells. After 2 to 3 weeks culture of CB and BM cells with rIL-4, OKT 3+ T cells comprised 85 to 95% of the total nonadherent cells. Profiles of hematopoietic cells developed by IL3 suggested that this lymphokine was a multipotential growth factor. Basophils, eosinophils, neutrophils and macrophages of multiple differentiation stages developed by IL-3 in both BM and CB cell cultures within two weeks. After 4 weeks, however, eosinophils became the majority of nonadherent cells. No human mast cell developed by rIL-3 or by IL-3 plus IL-4, suggesting that differentiation of human mast cells in vitro may require some additional growth ?&tor from non-T cells.