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Specific inhibition of graft rejection by soluble MHC superdimers
149
Abstracts
0809 SPECIFIC INHIBITIO"l Of' GRAFf REJECTION BY SOLUBLE MHC SUPERDIMERS. Jonathan P. Schneck Joan Sechler Sean M. O'Herrin Joan G. Bieler, Nicholas Barnes and Amy Rosenberg. Food and Drug Administration, Bethesda, MD, 20892 and Johns Hopkins School of Medicine, Baltimore, MD. 21205. We have previously described a genetically engineered soluble divalent analog of an MHC molecule (soluble superdimer). This protein, H-2KblIgG, was generated as a fusion protein between the extracellular domains of a murine class I polypeptide, II-2K b, and an immunoglohulin heavy chain polypeptide. Soluble M HC superdimers inhibit lysis of target cells by alloreactive cytotoxic T cells at nanomolar concentrations. A direct binding assay showed high affinity hinding, ICsoof 1.2 oM, between the H-2KblIgG molecule and an II-2Khspecific allorcactive T cell clone. To analyze the influence of H-2Kh/lgG on ill vivo responses, we have studied the effect of H-2K blIgG on skin graft rejection. Anti-H-2Kb specific responses were studied by cngrafting skin from C57B1/6 mice (H-2K b) onto H_2K bm l recipients. Median survival umes increased by 7-10 days when animals were injected with H-2K"/lgG, 000 ug/dose, every other day for a total of only six doses; several animals demonstrated long term graft survival, up to 60 days. There was no influence of H-2K b/lgG on graft rejection mediated by either a different class I MHC molecule, H-2D', or by a full MHC mismatch. We are currently studying the mechanism of prolongation of graft survival using in vitro MLR derived tron: treated mice. Tbese experiments demonstrate selective in vivo immunosuppression of grafts rejection using soluble MHC superdimers, Thus soluble MHC superdimers effectively regulate in vivo ,...llospecific immune responses.
0810 "MATURE" NEURONS BLOCK
IFtI-GAMMA IImUCTIOU OF liLA CLASS II
011 ASTROCYTES
J. Boucraut,n. S'te i nachne l de r , P. Del nae , H.Gola, II. ueker-Je , D. Bernard. Lab. Immunology, Fac. Timone, Lab. of Ireur-ob i c l ogy , CURS UPR A9024-Harsei 1] e. France. MPI J Marti ner i ed, Nun i ch , All emagne , Human astrocytes in vi tro consti tuvely expr-esaeo lILA cJ
0811
P812
ALLELIC VARIANTS OF MHC CLASS II MOLECULES CAN ACT AS PARTIAL AGONISTS OF ANTIGEN-SPECIFIC T CELL RESPONSES
HLA-DR ON JURK'AT AND U937 CELL LINES
Derek G, Doherty", David M. Koellet, William W. Kwok*, Susan Masewicz*, Mary Ellen Domeier*, and Gerald T. Nepom*t
*Virginia Mason Research Center and the tUniversity of Washington School of Medicine, Seattle, WA 98101, USA TCR engagement of a peptide-MHC complex can trigger activation of various effector functions including T cell proliferation, cytolysis and different patterns of cytokine release. Analogs of antigenic peptides with amino acid substitutions at TCR contact residues can act as partial TCR agonists, eliciting some but not all effector functions. In contrast to these previously described "altered peptide ligands", we describe partial agonist effects of allelic variants of HLA-DR ("altered self ligands") in association with a specific peptide response by a human T cell clone. T cell clone ELS4.34 responds to the HSV VPI6 peptide 393-405 presented by several different DR alleles in proliferation assays, in close correlation with the relative binding avidity of the peptide for DR. In contrast, T cell cytotoxicity is limited to a small number of restricting alleles which all share a common amino acid sequence, ILEDE, at DR~ positions 67-71. Using a panel of APes expressing DR molecules that were in vitromutagenized, we demonstrate that residues lle67, Asp70 and Glu7l are required for a cytolytic response of ELS4.34 to this peptide. These results demonstrate that allelic variants of HLA-DR molecules can act as partial agonists of T cell responses to a particular peptide and that specific residues on the e-helical region of DR~ modulate T cell recognition that results in proliferative or cytotoxic responses. The findings may have important implications for the roles of MHC molecules in positive and negative thymocyte selection and in the generation of T cell responses that mediate autoimmune disease.
Zahra Amilcghofran
Shuaz University of Medical Sciences, Shiraz, Iran. CD53 is a pan-leucoc.yte antigen
which spans the lJldSrIla
membrane four times and is a member of transmenbrano I..
superfamiJy(TM4SF). The precise function of TM4SF is not known but data suggest a role in the regulation of cell development. proliferation and activatton. Modulation of HLA-DR and IL-2R on Jut-kat and U937 cell lines were analyzed after addition of a specific monoclonal anttbody (m Ab ) raised against CD53 molecule Into the culture. Binding of this mAb (designated as SU14 ) to Jurkat cells results in a significant increase in the expression of HLA-DR and IL-2R. These data indicate that SU14 mAb binding lnducesexpress!onofactivation markers on the surface of Jurkat cells. Addition of SU14 mAb into the culture of Jurkat cells was also followed by an increase in the expression of CD44 molecule. In a similar experiment on U937 cell line an increase in the expression of HLA-DR and CRI molecules was observed.
P814
P813
INl£TICJ
Chatterjee Moitreyee* Agral'a I Suraksha
lIgarv.a1 ShYMl S.
Department of r-.'ecllcal Genetics, Sanja~ Ganctli Post E~~~~e_ ~n2s6~N~.t1N8tA~ical Sc Iences , ebareli Road, Co'ltlinatorial
interaction of
transcription factors
is
evol vlng as one of the rm.i6r mechenism by Vlhlch a IIml ted
ntnber of
CD53 MONOCLONAL ANTIBODY INDUCES EXPRESSION OF
transcription' factors rewlate expression of
~~~~~e d~~~9nt 5~r~I~1~~'i"ci.:dn~er:l.fa lexf~Jc"er~y~[g~
particular gene and rmy also effect differential regullItion of di Herent genes by two very similar carpounds e.g., Interferon alpha and ganmo. We have observed that only IFN gamna 15 able to effect an increase In transcrifltion of the beta globin gene In K562 cells
~~~et~~~cr~t1~~1na~~I~~~n~:r~~~~~tC~~s~ class I 9!lne transcription "RP9"rs to be directly related to Interferon mediated nuclear translocat Ion of p65; ldenti fled to be II menner of the rei farnll y of protooncogenes in this study. IFN gM1T'8, leads to enhllnced expression of a RXR beta like protein In K562 cells. We propose that, IFN gMlT'ft mediated beta globin gene transcription may be a resuI t of synergistic Inter acUon of p65 and RXR beta transcription factors.
Cytolytic Lymphocytes Inactivate RNA Viruses in Infected Target cells in the Absence of Perforin. Grace Hommel-Berrey, Zacharie Brahmi, Department of Medicine, Indiana University Medical Center, Indianapolis, IN 46202. We have investigated the ability of human LAX cells to control the production of an RNA virus, vesicular stomatitis virus (VSV). in infected target cells using the plaque formation assay as an index of infectivity. LAX cells significantly inhibited VSV production as compared to
~~~~~~;~·U9;~ e:;:~~~:ew~:~~~~e~i~~~hRf:_::;d~:r:~~'3
to 6 h post infection and chased with HEM supplemented with cold uridine and cytidine. VSV-infected U937 were incuba.ted with
~i~:~~~vi~~ ~~Bt~e~e~~~:d~heG~::~;~i~~a~:;:·~~c~:;e~H_
labelled RNA in VSV-infected cells WilS degraded in 3 h. Northern analysis of RNA extracted from infected cell. with a VSV N eDNA probe confirmed that viral transcripts were degraded. In addition, LAX cells with the serine protease inhibitor 3. 4-dichloroisocoumarin (DCI). blocked RNA degradation in both un infected and VSV-infected U937' whereas 4-(2-aminoethyl) benzenesulfonylfluoride (ABBSI') had no effect. The addition of IPN-y, TNF-a, anti-Fas mAB, granzyme A, and highly purified human perforin to normal and infected U937 did not induce RNA degradation. In contrast, isolated granzyme B eelectively triggered RNA degradation in VSVinfected cells in a dose- (ED~o 250 ng/ml) and time-dependent :manner. Proteolytic inaetivat1.on of granzyme B blocked this activity. Furthermore, granzyme B was able to suppress PPU .produced by infected U9J7 cells by 60'. Our da.ta suqqeet. that. granzyme B is both necessary and sufficient to reduoe vsv produotion by decrea8inq t.he level of viral tran8- cripts within infected U937 cells. Preliminary results using HIV infected targets will also be discussed.
Abstracts
0809 SPECIFIC INHIBITIO"l Of' GRAFf REJECTION BY SOLUBLE MHC SUPERDIMERS. Jonathan P. Schneck Joan Sechler Sean M. O'Herrin Joan G. Bieler, Nicholas Barnes and Amy Rosenberg. Food and Drug Administration, Bethesda, MD, 20892 and Johns Hopkins School of Medicine, Baltimore, MD. 21205. We have previously described a genetically engineered soluble divalent analog of an MHC molecule (soluble superdimer). This protein, H-2KblIgG, was generated as a fusion protein between the extracellular domains of a murine class I polypeptide, II-2K b, and an immunoglohulin heavy chain polypeptide. Soluble M HC superdimers inhibit lysis of target cells by alloreactive cytotoxic T cells at nanomolar concentrations. A direct binding assay showed high affinity hinding, ICsoof 1.2 oM, between the H-2KblIgG molecule and an II-2Khspecific allorcactive T cell clone. To analyze the influence of H-2Kh/lgG on ill vivo responses, we have studied the effect of H-2K blIgG on skin graft rejection. Anti-H-2Kb specific responses were studied by cngrafting skin from C57B1/6 mice (H-2K b) onto H_2K bm l recipients. Median survival umes increased by 7-10 days when animals were injected with H-2K"/lgG, 000 ug/dose, every other day for a total of only six doses; several animals demonstrated long term graft survival, up to 60 days. There was no influence of H-2K b/lgG on graft rejection mediated by either a different class I MHC molecule, H-2D', or by a full MHC mismatch. We are currently studying the mechanism of prolongation of graft survival using in vitro MLR derived tron: treated mice. Tbese experiments demonstrate selective in vivo immunosuppression of grafts rejection using soluble MHC superdimers, Thus soluble MHC superdimers effectively regulate in vivo ,...llospecific immune responses.
0810 "MATURE" NEURONS BLOCK
IFtI-GAMMA IImUCTIOU OF liLA CLASS II
011 ASTROCYTES
J. Boucraut,n. S'te i nachne l de r , P. Del nae , H.Gola, II. ueker-Je , D. Bernard. Lab. Immunology, Fac. Timone, Lab. of Ireur-ob i c l ogy , CURS UPR A9024-Harsei 1] e. France. MPI J Marti ner i ed, Nun i ch , All emagne , Human astrocytes in vi tro consti tuvely expr-esaeo lILA cJ
0811
P812
ALLELIC VARIANTS OF MHC CLASS II MOLECULES CAN ACT AS PARTIAL AGONISTS OF ANTIGEN-SPECIFIC T CELL RESPONSES
HLA-DR ON JURK'AT AND U937 CELL LINES
Derek G, Doherty", David M. Koellet, William W. Kwok*, Susan Masewicz*, Mary Ellen Domeier*, and Gerald T. Nepom*t
*Virginia Mason Research Center and the tUniversity of Washington School of Medicine, Seattle, WA 98101, USA TCR engagement of a peptide-MHC complex can trigger activation of various effector functions including T cell proliferation, cytolysis and different patterns of cytokine release. Analogs of antigenic peptides with amino acid substitutions at TCR contact residues can act as partial TCR agonists, eliciting some but not all effector functions. In contrast to these previously described "altered peptide ligands", we describe partial agonist effects of allelic variants of HLA-DR ("altered self ligands") in association with a specific peptide response by a human T cell clone. T cell clone ELS4.34 responds to the HSV VPI6 peptide 393-405 presented by several different DR alleles in proliferation assays, in close correlation with the relative binding avidity of the peptide for DR. In contrast, T cell cytotoxicity is limited to a small number of restricting alleles which all share a common amino acid sequence, ILEDE, at DR~ positions 67-71. Using a panel of APes expressing DR molecules that were in vitromutagenized, we demonstrate that residues lle67, Asp70 and Glu7l are required for a cytolytic response of ELS4.34 to this peptide. These results demonstrate that allelic variants of HLA-DR molecules can act as partial agonists of T cell responses to a particular peptide and that specific residues on the e-helical region of DR~ modulate T cell recognition that results in proliferative or cytotoxic responses. The findings may have important implications for the roles of MHC molecules in positive and negative thymocyte selection and in the generation of T cell responses that mediate autoimmune disease.
Zahra Amilcghofran
Shuaz University of Medical Sciences, Shiraz, Iran. CD53 is a pan-leucoc.yte antigen
which spans the lJldSrIla
membrane four times and is a member of transmenbrano I..
superfamiJy(TM4SF). The precise function of TM4SF is not known but data suggest a role in the regulation of cell development. proliferation and activatton. Modulation of HLA-DR and IL-2R on Jut-kat and U937 cell lines were analyzed after addition of a specific monoclonal anttbody (m Ab ) raised against CD53 molecule Into the culture. Binding of this mAb (designated as SU14 ) to Jurkat cells results in a significant increase in the expression of HLA-DR and IL-2R. These data indicate that SU14 mAb binding lnducesexpress!onofactivation markers on the surface of Jurkat cells. Addition of SU14 mAb into the culture of Jurkat cells was also followed by an increase in the expression of CD44 molecule. In a similar experiment on U937 cell line an increase in the expression of HLA-DR and CRI molecules was observed.
P814
P813
INl£TICJ
Chatterjee Moitreyee* Agral'a I Suraksha
lIgarv.a1 ShYMl S.
Department of r-.'ecllcal Genetics, Sanja~ Ganctli Post E~~~~e_ ~n2s6~N~.t1N8tA~ical Sc Iences , ebareli Road, Co'ltlinatorial
interaction of
transcription factors
is
evol vlng as one of the rm.i6r mechenism by Vlhlch a IIml ted
ntnber of
CD53 MONOCLONAL ANTIBODY INDUCES EXPRESSION OF
transcription' factors rewlate expression of
~~~~~e d~~~9nt 5~r~I~1~~'i"ci.:dn~er:l.fa lexf~Jc"er~y~[g~
particular gene and rmy also effect differential regullItion of di Herent genes by two very similar carpounds e.g., Interferon alpha and ganmo. We have observed that only IFN gamna 15 able to effect an increase In transcrifltion of the beta globin gene In K562 cells
~~~et~~~cr~t1~~1na~~I~~~n~:r~~~~~tC~~s~ class I 9!lne transcription "RP9"rs to be directly related to Interferon mediated nuclear translocat Ion of p65; ldenti fled to be II menner of the rei farnll y of protooncogenes in this study. IFN gM1T'8, leads to enhllnced expression of a RXR beta like protein In K562 cells. We propose that, IFN gMlT'ft mediated beta globin gene transcription may be a resuI t of synergistic Inter acUon of p65 and RXR beta transcription factors.
Cytolytic Lymphocytes Inactivate RNA Viruses in Infected Target cells in the Absence of Perforin. Grace Hommel-Berrey, Zacharie Brahmi, Department of Medicine, Indiana University Medical Center, Indianapolis, IN 46202. We have investigated the ability of human LAX cells to control the production of an RNA virus, vesicular stomatitis virus (VSV). in infected target cells using the plaque formation assay as an index of infectivity. LAX cells significantly inhibited VSV production as compared to
~~~~~~;~·U9;~ e:;:~~~:ew~:~~~~e~i~~~hRf:_::;d~:r:~~'3
to 6 h post infection and chased with HEM supplemented with cold uridine and cytidine. VSV-infected U937 were incuba.ted with
~i~:~~~vi~~ ~~Bt~e~e~~~:d~heG~::~;~i~~a~:;:·~~c~:;e~H_
labelled RNA in VSV-infected cells WilS degraded in 3 h. Northern analysis of RNA extracted from infected cell. with a VSV N eDNA probe confirmed that viral transcripts were degraded. In addition, LAX cells with the serine protease inhibitor 3. 4-dichloroisocoumarin (DCI). blocked RNA degradation in both un infected and VSV-infected U937' whereas 4-(2-aminoethyl) benzenesulfonylfluoride (ABBSI') had no effect. The addition of IPN-y, TNF-a, anti-Fas mAB, granzyme A, and highly purified human perforin to normal and infected U937 did not induce RNA degradation. In contrast, isolated granzyme B eelectively triggered RNA degradation in VSVinfected cells in a dose- (ED~o 250 ng/ml) and time-dependent :manner. Proteolytic inaetivat1.on of granzyme B blocked this activity. Furthermore, granzyme B was able to suppress PPU .produced by infected U9J7 cells by 60'. Our da.ta suqqeet. that. granzyme B is both necessary and sufficient to reduoe vsv produotion by decrea8inq t.he level of viral tran8- cripts within infected U937 cells. Preliminary results using HIV infected targets will also be discussed.