- Email: [email protected]
Formation and kinetics of MHC class I-ovalbumin peptide complexes on murine dendritic cells following an ovalbumin peptide-pulse
ESDR I JSID I SID Abstracts
S16
0091
0094
OFFUNCTIONAL IL18 BYDIFFERENT SUBTYPES OF MuRlNEDENDRlTlC CELLS- DC-DERIVED IL-18ENHANCES IlrlZ-DEPENDENT THl-DEVEL0PMENT.s.
FASiFAS
PRODUCTION
H. Jonukif, E. Scbmm.’ G. Mliller. H. Yamauelu.’ M. Ku”moto,* J Know and A H E* Clinical Research U”it, Dept. of Der”latol * and +I”st. of Im”l”nol, “Ill”. of Ma&, Mamz, FRG, and *Fuji& Inst., Hayashdxra Sxwbem. Lab, Inc., Okaya”va, Japan ILL* is a recently described cytokine that shares hologic activities v&b n-12 in dnnng the development of Tbt-fyp T-cells As dendrltic cells are very potent inducera of T-cell probferaao” and differe”t~atio” w aondered whether they utdize IL-,8 as a faxor driving Thl-development We demonstrateby Northern blot and RT-PCR that various subtypesof murine DC coma” a-1 8 mRNA When supematants of various DC-subtypes were uutyzed for prodwtm” of IL-t8 protein. IL-18 production was detected in an IL-1 I-specific ELISA and functionally assessadin IL-18 bioasaays In a tramganc rrnwe systan, v&w about 70% of naive peripheral T-calls contain a” Ovrdbummspectic T-cell receptor (OVA-TCltm) we further demonstrate that DC-derived IL-18 patennates IL 12-dependent Thl-develcqme”t. Naive T cells and b”x-DC r\ere prepared &,m the OVA-TCRm. !,C wre pulsed wit,! OVA and wed for repetitive rwnds ofT cell-stimulation I” the ab+e”ce or presence ofanti-ll-t8, ar,t,-Lt2 or both Whereas no IL-4 wk( detected 10 tbe ~upema‘mts thrc@ou, the expximent, strong IFN-y-prcdwtion was observedafter the third restimulabon. This release of IPN? was completely abrogated by addition of anti-IL-t2, whereas additro” of rmtl-IL-18 had no efIst in the third restmwlation Hewer, in the fourth restimulation IL-18 release by DC bad a much more pronwneed e&t
on the induction of lFN_y-release than E-12, aa addition of anti-IL-18 wa8 able to
LIGAND-MEDIATED
LANGERHANS e hm’i
Sh’
Yamanashi
APOPTOSIS
CELLS IN DRAINING
OF
ANTIGEN-BEARING
LYMPH NODES. Uxuyoshl
Kawamura’~‘.
1: Lkparbnent of !alnat010gy. ad ’ Y * K Medical University, Yananashi, Japan. 2: Deparment of Immunology,
Tokyo, Japan. Although functional roles of Langerhans cells (LC) as APC in the draning lymph “ode have been well mvesttgated, little is know” about the fate of LC after the antigenJuntendo Umversity,
presentation to T ccUs. We investigated whether epidermal LC in vim
or the
Ag-bearing
LC in viva underwent apoptosis in a FasiFasL-dependent manner. We demonstrated that murine epidermal LC constitutively expressed Fas antigen, and their expression was upregulated by the addition of IFN-y in cul~res. Epidermal LC underwent apoptosis by ligation of the Fas antigen using recombinant soluble Fas I&and (FasL) in the presence of IFNy, but not by FasL alone. Antigen @g-bearing LC that migrated info the draining
about 20% of
lymph nodes and were idenufied as fluorescent cells after skin painting wtb FITC also
IFN-y productirm. Addition of both antisera in combmatio” abrogated IFNrpm&tio” mmpletely To qport tbess fi”d”ags, DC deriwd from tL-12 &co Mimala we ured for stimulano” Although thee a”“nah prcduced normal levels of U-18, no IFi.+prcdwtion was detectable a&x the fourth ressmulatirm. In raazate the data demonstrate that subtvws of DC are able to release ftmcticmal IL18 that is able to induce IFNy prcdwtm” and augments Tht-differentiation m primed T cells Honever, IL- 18 by itself is unable to imtiate Thl+di@erentation in “atw T-cells.
expressed the Fas antigen. Interestingly, clearanceof the Ag-bearing LC was significantly delayed ad the ratio of apoptotic cells was reduced in Fas-deficient Ipr and FasL-deficient
0092
0095
DIFFERENTIAL EFFECTS OF CD101 AND CD80/CD86 IN T CELL ACTIVATION THROUGH SKJN DENDRITIC CELLS. Anne and. INSERM U448 and Deparbnent of Dermatology, Paris XII University. CrCteil, France.
INDUCTION OF MATURE DENDRITIC CELLS FROM A MURINE EPIDERh4AL CELL LINE (XS52) USING M’IERLEUKIN-4. E‘Tohuo Yun&. Dermatology Branch, National Cancer Institute, Bethesda, MD, USA Langerhans cells (LC) have been shown to play a critical role in the generation of immune responses to haptens and proteins, and a” important role in DNA-bawd inixnxtawous vaccination. Our goal is u) aansfect dendritic cells (DC) ia vim and use them for in viva vaccination. However, only limited numbers of LC can be isolated from skin. We therefore determined whether, in tie presence of various cytokintx such as GM-CSF. TNF-a or IL-4, a dendrhic cell line, XS52, isolated from fetal cpidernus of BALB/c mice by X” et al (J Immunol 154:2697,1995) could undergo maturation comparable to culered LC. Phenotypic analysis revealed that XS52 cells are Class II low, CDS+, CDIIc-, CD13-, CD14+, CD71+, and F4/80+. n-2, IL-7, M-CSF, and Stem Cell Factor had no stgnificant effect on the expression of XS52 surface molecules. However, when lOng/ml of IL4 was added to XS52 growth media for 9 days (IL4XS). the cells became Class II high, CD5 low, CDllc+, CD13+, CD14., CD71 low, and F4/80 low. Expression of costinmIatory molecules including CD40, CD80, and CDS6 was also markedly enhanced In addition, IL4XS induced markedly enhanced antigen-speciiic T cell proliferation after incubation with protein antigens compared to XS52. TNF-a and ILlp did not induce phenotypic changes in XS52, but when added to L-4, significantly enhanced expression of many DC-bke maturation markers and thou abdity to aawate naive allogeneic T cells (greater than with IL4 alone). ‘I%“&we have succeeded in generating mature dendritic cells from XS52, a celI line thought to represent progenxors of dendritic cells We anticipate that the use of cytokine-treated XS52 cells wdl facilitate the ex w&in viva use of dendriric cells for immunizauon.
block up to 75% of the ,FN-y-release I” contrast anti-ILlZmAb
alone only lccked
-_
Activahon of T lymphocytes by antigen-presenting cells requires co-stimulatory signals in addition to the mimar~ simal provided bv the mm~ement of the T cell receptor. CD101 molecule is a 146 kDa Gpe 7 tra&embr& proteinco&aining seven immunoglobuline type Ig-V domains in the extracellular portion. Using different monoclonal antibodies (mAb) 8827, BA27. and w-1, we have previously shown that CD101 is expressed on a major subpopulation of HLA-DR t, CDla +, CDlc + skin dendritic cells (DC), and on activated T cells. We further studied the functional role of CD101 and its relation to costimulatory molecules. Anti-CD101 mAbs BB27 and “7.1 inhibited the activation of T cells by skin DC, both in allogeneic and soluble antigen specific reactions. The prolikratio” of T lymphocytes was reduced by 55 to 75 % in the presence of anti-CD101mAb, by 40% in the presence of anti-CDRO/CDB6mAb, and by W-99% in the presence af both mAbs, indicating a synergistic effect. CD101 higgering on ski” CC induced signal transduction as cross-linking of CD101 with anti-CD101 mAb resulted in a modulation of surface molecules on ski” DC: upregulation of HLA-DR and downregulation of CD& I” order to study the role of CD101 ligand on T cell activation, CHO or 3T3 cells were hansfected with CD101 cDNA and assessed for their shmtiting capacity T cells proliferated in response of soluble anti-CD3 mAb in presence of CD86 trmsfected cells
which induced a 3-4 fold increase in ‘H-TdR incorporation by T cells compared induced by the same “umber of mock transfected c&s. Ln contrast CDllJl
to that transfected cells did
not induce a significant increase m 3H-TdR incorporation by T cells compared to that induced by the same “umber of mock hansfected cells. Furthermore there was no proliferation of T cells in response of anti-CD3 mAb and the a~sociaho” of the CD101 and CD86 hansfectd cells, indicating that CD101 cells gave a” inhibitory signal. These rew1t.s indicate that CD101 molecule mediates a negative signal whereas CD80/CD86 molecules provide via CD28 a positive signal on Tell activation.
gld mice at 2 days after skin painting. ‘Lowefmdmgs suggest that a substantial population of LC after A&presentation may undergo apoptosis by the Fas/FasL system through “pregulation of tbe Fas expression by IFNy derived from activated T cells.
DENDRITTC
S&&a&&
0093
0096
DENDRITIC CELL APOFTOSIS DURING ANTIGEN-SPECIFIC INTFJUCTION WITI3 lxM+ T CELL% Hirow?dyBer Deparmmt ofDe”m‘ology,
FORMATlON AND KtNETlCS OF MHC CLASS COVALBUMIN PEPTIDE COMPLEXES ON MURINE DENDRITIC CELLS FOLLOWING AN OVALBUMIN PEPTIDE-PULSE I$& RO&,er’. Jon&h*. G era Id S c h uw. Manfred Lutz ‘, ‘NuRfeM Depariment of Surgery, Uniwmity of Oxford, Oxford, U.K.; “Deparbnent of Dermatology, Unive&y of Erlangen-Nuremberg,Erla”gan, Germany Recent studies of human de”dri& cells (DC) generated in vitro have unravelled a stdtdngly shoti half-life ,Tl/2 <{Oh) of MHC cla8s I moleades eve” on mature DC. Thii implies ineffective antigen presentation to CDS+ T cells following loading and reinjection of DC with MHC class r&f&ad pepifdas e.g. for therapeutical induction of immunity to tumcis or iti&“. Asthe generation of human DC follows variaMe and in part suboptimal prolocols we sought to adressthk crudal issue usi”g well defined CC populations and novel technology. To tiis end we generated DC fro”, m”d”e bone-marrow wfth GM-CSF, and pulsed them wim the OVA pep&de SIINFEKL. which binds exclus&ly to C MHC class I molecules. for various psrtcds of time and st diirent cowentrations. Thereafter we monitored the s-c MHCpept!de complex (compared ti tots1 surface MHC c&s I molecules) by the antibody 25. Df 18 that speoiffcaliy d&&s Kb SIINFEKL complexes. and determined its k$“etics on mature and immature dendridc cells + TNF alpha during the p@de pulse. Pulsing wlvl fpM peptide WBS sficient for mahlre DC, whereas speci@z MHC I-peptie complexes could be detected III imm8tllre DC only at 10pM. At 1 WpM no full ~‘dturation vd6 reached. yet. Optimal peptide loading was reached after 6 to 8 hours Of ps@e pulse. and was comparable in e.?rum-free versus serumcontaining medium. Pulse-chase experimwds revealed a two times longer half-life of MHC cl1 I pepSde complexes on mature compared to fmmature DC. If mature DC were exposed to TNF alpha awing the pulse Re half-life was further douMed whereas immature DC ehwiad M) such effect Our experiments were aimed at the optimization of pephds load&&, of MHC c&a I molecules on DC, and unraveled that (truly and fully) mature DC notably afta TNF alpha exposure express spechlc MHC-cfass for polonged perk&s of time. These findings may have important impliitkms for the us8 of pepfide-loaded DC in tumor immunotherapy.
UnhwsitvofTexasSonthweSmn Medical
Schml. Dallas, USA, and Deparmmt of ~logy,*
I
Icom~lems
S16
0091
0094
OFFUNCTIONAL IL18 BYDIFFERENT SUBTYPES OF MuRlNEDENDRlTlC CELLS- DC-DERIVED IL-18ENHANCES IlrlZ-DEPENDENT THl-DEVEL0PMENT.s.
FASiFAS
PRODUCTION
H. Jonukif, E. Scbmm.’ G. Mliller. H. Yamauelu.’ M. Ku”moto,* J Know and A H E* Clinical Research U”it, Dept. of Der”latol * and +I”st. of Im”l”nol, “Ill”. of Ma&, Mamz, FRG, and *Fuji& Inst., Hayashdxra Sxwbem. Lab, Inc., Okaya”va, Japan ILL* is a recently described cytokine that shares hologic activities v&b n-12 in dnnng the development of Tbt-fyp T-cells As dendrltic cells are very potent inducera of T-cell probferaao” and differe”t~atio” w aondered whether they utdize IL-,8 as a faxor driving Thl-development We demonstrateby Northern blot and RT-PCR that various subtypesof murine DC coma” a-1 8 mRNA When supematants of various DC-subtypes were uutyzed for prodwtm” of IL-t8 protein. IL-18 production was detected in an IL-1 I-specific ELISA and functionally assessadin IL-18 bioasaays In a tramganc rrnwe systan, v&w about 70% of naive peripheral T-calls contain a” Ovrdbummspectic T-cell receptor (OVA-TCltm) we further demonstrate that DC-derived IL-18 patennates IL 12-dependent Thl-develcqme”t. Naive T cells and b”x-DC r\ere prepared &,m the OVA-TCRm. !,C wre pulsed wit,! OVA and wed for repetitive rwnds ofT cell-stimulation I” the ab+e”ce or presence ofanti-ll-t8, ar,t,-Lt2 or both Whereas no IL-4 wk( detected 10 tbe ~upema‘mts thrc@ou, the expximent, strong IFN-y-prcdwtion was observedafter the third restimulabon. This release of IPN? was completely abrogated by addition of anti-IL-t2, whereas additro” of rmtl-IL-18 had no efIst in the third restmwlation Hewer, in the fourth restimulation IL-18 release by DC bad a much more pronwneed e&t
on the induction of lFN_y-release than E-12, aa addition of anti-IL-18 wa8 able to
LIGAND-MEDIATED
LANGERHANS e hm’i
Sh’
Yamanashi
APOPTOSIS
CELLS IN DRAINING
OF
ANTIGEN-BEARING
LYMPH NODES. Uxuyoshl
Kawamura’~‘.
1: Lkparbnent of !alnat010gy. ad ’ Y * K Medical University, Yananashi, Japan. 2: Deparment of Immunology,
Tokyo, Japan. Although functional roles of Langerhans cells (LC) as APC in the draning lymph “ode have been well mvesttgated, little is know” about the fate of LC after the antigenJuntendo Umversity,
presentation to T ccUs. We investigated whether epidermal LC in vim
or the
Ag-bearing
LC in viva underwent apoptosis in a FasiFasL-dependent manner. We demonstrated that murine epidermal LC constitutively expressed Fas antigen, and their expression was upregulated by the addition of IFN-y in cul~res. Epidermal LC underwent apoptosis by ligation of the Fas antigen using recombinant soluble Fas I&and (FasL) in the presence of IFNy, but not by FasL alone. Antigen @g-bearing LC that migrated info the draining
about 20% of
lymph nodes and were idenufied as fluorescent cells after skin painting wtb FITC also
IFN-y productirm. Addition of both antisera in combmatio” abrogated IFNrpm&tio” mmpletely To qport tbess fi”d”ags, DC deriwd from tL-12 &co Mimala we ured for stimulano” Although thee a”“nah prcduced normal levels of U-18, no IFi.+prcdwtion was detectable a&x the fourth ressmulatirm. In raazate the data demonstrate that subtvws of DC are able to release ftmcticmal IL18 that is able to induce IFNy prcdwtm” and augments Tht-differentiation m primed T cells Honever, IL- 18 by itself is unable to imtiate Thl+di@erentation in “atw T-cells.
expressed the Fas antigen. Interestingly, clearanceof the Ag-bearing LC was significantly delayed ad the ratio of apoptotic cells was reduced in Fas-deficient Ipr and FasL-deficient
0092
0095
DIFFERENTIAL EFFECTS OF CD101 AND CD80/CD86 IN T CELL ACTIVATION THROUGH SKJN DENDRITIC CELLS. Anne and. INSERM U448 and Deparbnent of Dermatology, Paris XII University. CrCteil, France.
INDUCTION OF MATURE DENDRITIC CELLS FROM A MURINE EPIDERh4AL CELL LINE (XS52) USING M’IERLEUKIN-4. E‘Tohuo Yun&. Dermatology Branch, National Cancer Institute, Bethesda, MD, USA Langerhans cells (LC) have been shown to play a critical role in the generation of immune responses to haptens and proteins, and a” important role in DNA-bawd inixnxtawous vaccination. Our goal is u) aansfect dendritic cells (DC) ia vim and use them for in viva vaccination. However, only limited numbers of LC can be isolated from skin. We therefore determined whether, in tie presence of various cytokintx such as GM-CSF. TNF-a or IL-4, a dendrhic cell line, XS52, isolated from fetal cpidernus of BALB/c mice by X” et al (J Immunol 154:2697,1995) could undergo maturation comparable to culered LC. Phenotypic analysis revealed that XS52 cells are Class II low, CDS+, CDIIc-, CD13-, CD14+, CD71+, and F4/80+. n-2, IL-7, M-CSF, and Stem Cell Factor had no stgnificant effect on the expression of XS52 surface molecules. However, when lOng/ml of IL4 was added to XS52 growth media for 9 days (IL4XS). the cells became Class II high, CD5 low, CDllc+, CD13+, CD14., CD71 low, and F4/80 low. Expression of costinmIatory molecules including CD40, CD80, and CDS6 was also markedly enhanced In addition, IL4XS induced markedly enhanced antigen-speciiic T cell proliferation after incubation with protein antigens compared to XS52. TNF-a and ILlp did not induce phenotypic changes in XS52, but when added to L-4, significantly enhanced expression of many DC-bke maturation markers and thou abdity to aawate naive allogeneic T cells (greater than with IL4 alone). ‘I%“&we have succeeded in generating mature dendritic cells from XS52, a celI line thought to represent progenxors of dendritic cells We anticipate that the use of cytokine-treated XS52 cells wdl facilitate the ex w&in viva use of dendriric cells for immunizauon.
block up to 75% of the ,FN-y-release I” contrast anti-ILlZmAb
alone only lccked
-_
Activahon of T lymphocytes by antigen-presenting cells requires co-stimulatory signals in addition to the mimar~ simal provided bv the mm~ement of the T cell receptor. CD101 molecule is a 146 kDa Gpe 7 tra&embr& proteinco&aining seven immunoglobuline type Ig-V domains in the extracellular portion. Using different monoclonal antibodies (mAb) 8827, BA27. and w-1, we have previously shown that CD101 is expressed on a major subpopulation of HLA-DR t, CDla +, CDlc + skin dendritic cells (DC), and on activated T cells. We further studied the functional role of CD101 and its relation to costimulatory molecules. Anti-CD101 mAbs BB27 and “7.1 inhibited the activation of T cells by skin DC, both in allogeneic and soluble antigen specific reactions. The prolikratio” of T lymphocytes was reduced by 55 to 75 % in the presence of anti-CD101mAb, by 40% in the presence of anti-CDRO/CDB6mAb, and by W-99% in the presence af both mAbs, indicating a synergistic effect. CD101 higgering on ski” CC induced signal transduction as cross-linking of CD101 with anti-CD101 mAb resulted in a modulation of surface molecules on ski” DC: upregulation of HLA-DR and downregulation of CD& I” order to study the role of CD101 ligand on T cell activation, CHO or 3T3 cells were hansfected with CD101 cDNA and assessed for their shmtiting capacity T cells proliferated in response of soluble anti-CD3 mAb in presence of CD86 trmsfected cells
which induced a 3-4 fold increase in ‘H-TdR incorporation by T cells compared induced by the same “umber of mock transfected c&s. Ln contrast CDllJl
to that transfected cells did
not induce a significant increase m 3H-TdR incorporation by T cells compared to that induced by the same “umber of mock hansfected cells. Furthermore there was no proliferation of T cells in response of anti-CD3 mAb and the a~sociaho” of the CD101 and CD86 hansfectd cells, indicating that CD101 cells gave a” inhibitory signal. These rew1t.s indicate that CD101 molecule mediates a negative signal whereas CD80/CD86 molecules provide via CD28 a positive signal on Tell activation.
gld mice at 2 days after skin painting. ‘Lowefmdmgs suggest that a substantial population of LC after A&presentation may undergo apoptosis by the Fas/FasL system through “pregulation of tbe Fas expression by IFNy derived from activated T cells.
DENDRITTC
S&&a&&
0093
0096
DENDRITIC CELL APOFTOSIS DURING ANTIGEN-SPECIFIC INTFJUCTION WITI3 lxM+ T CELL% Hirow?dyBer Deparmmt ofDe”m‘ology,
FORMATlON AND KtNETlCS OF MHC CLASS COVALBUMIN PEPTIDE COMPLEXES ON MURINE DENDRITIC CELLS FOLLOWING AN OVALBUMIN PEPTIDE-PULSE I$& RO&,er’. Jon&h*. G era Id S c h uw. Manfred Lutz ‘, ‘NuRfeM Depariment of Surgery, Uniwmity of Oxford, Oxford, U.K.; “Deparbnent of Dermatology, Unive&y of Erlangen-Nuremberg,Erla”gan, Germany Recent studies of human de”dri& cells (DC) generated in vitro have unravelled a stdtdngly shoti half-life ,Tl/2 <{Oh) of MHC cla8s I moleades eve” on mature DC. Thii implies ineffective antigen presentation to CDS+ T cells following loading and reinjection of DC with MHC class r&f&ad pepifdas e.g. for therapeutical induction of immunity to tumcis or iti&“. Asthe generation of human DC follows variaMe and in part suboptimal prolocols we sought to adressthk crudal issue usi”g well defined CC populations and novel technology. To tiis end we generated DC fro”, m”d”e bone-marrow wfth GM-CSF, and pulsed them wim the OVA pep&de SIINFEKL. which binds exclus&ly to C MHC class I molecules. for various psrtcds of time and st diirent cowentrations. Thereafter we monitored the s-c MHCpept!de complex (compared ti tots1 surface MHC c&s I molecules) by the antibody 25. Df 18 that speoiffcaliy d&&s Kb SIINFEKL complexes. and determined its k$“etics on mature and immature dendridc cells + TNF alpha during the p@de pulse. Pulsing wlvl fpM peptide WBS sficient for mahlre DC, whereas speci@z MHC I-peptie complexes could be detected III imm8tllre DC only at 10pM. At 1 WpM no full ~‘dturation vd6 reached. yet. Optimal peptide loading was reached after 6 to 8 hours Of ps@e pulse. and was comparable in e.?rum-free versus serumcontaining medium. Pulse-chase experimwds revealed a two times longer half-life of MHC cl1 I pepSde complexes on mature compared to fmmature DC. If mature DC were exposed to TNF alpha awing the pulse Re half-life was further douMed whereas immature DC ehwiad M) such effect Our experiments were aimed at the optimization of pephds load&&, of MHC c&a I molecules on DC, and unraveled that (truly and fully) mature DC notably afta TNF alpha exposure express spechlc MHC-cfass for polonged perk&s of time. These findings may have important impliitkms for the us8 of pepfide-loaded DC in tumor immunotherapy.
UnhwsitvofTexasSonthweSmn Medical
Schml. Dallas, USA, and Deparmmt of ~logy,*
I
Icom~lems