In this thesis I have focused on acute rejection as this process is easily monitored in our cardiac allografting model. Moreover, the problem of acute rejection has to be solved before chronic reaction can become an issue. ...

Zie: Summary

Raue, Hans-Peter,

Raue, Hans-Peter

University of Groningen Research Database

  
 University of Groningen
Simultaneous transplantation and intrathymic tolerance induction
Raue, Hans-Peter
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
1998
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Raue, H-P. (1998). Simultaneous transplantation and intrathymic tolerance induction s.n.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 11-02-2018
In chapter 1 a short overview of the history of transplantation tolerance is given
followed by a discussion of t l-re various mechanisms currently thought to rnediate
tolerance and the ways to induce transplantation tolerance. Section 1.1 ends rvith a
review of the literature on intrathymic tolerance induction. Section 1.2 introduces our
practical work. Current immunosuppressive protocols are very successful in
preventing or abrogating acute rejection. Hor.r'ever, these regimens have a number of
serious side effects (e.g. increased susceptibility to infection, increased risk of neoplastic
disease and renal and myocardiai toxicity). These problems could be prevented by the
induction of donor-specific tolerance which r,r.'ould eliminate the need for long-term
immunosuppression. One of the protocols with potential clinical application is
tolerance induction via intrathymic inoculaiion with donor-type antigens and short
term immunosuppression with antilymphocyte serum (ALS). However, the obiigatory
interval between tolerance indr.rction and the actual transplantation precludes clinical
use. We have tried to create an intrathymic tolerance induction protocol with clinical Lry
adding a short course of cyclosporin A (CsA) treatment to allow us to perform
tolerance induction and iransplantation simtrltaneously.
In section 2.L the results with our simultaneous transplantation and intrathymic
tolerance induction (STITTI) protocol are described. This protocol is highly efficient
(> 90"/,,) and can be used to induce tolerance for cardiac allografts in several different
fully MHC disparate ('high' responder) rat strains. As the STITTI protocol allows us to
perform tolerance induction simultaneously with the actual transplantation it may have
clinical potential.
Section 2.2 describes our skin transplant experiments. The unmodified STITTI protocol
does not induce tolerance to skin allografts in the PVG to AO rat strain combination.
However, substitution of keratinocytes for splenocytes in the intrathymic inoculum
does induce long-term acceptance of skin allografts in approximately 50% of the
recipients. The lower efficiency is probably due to the higher immunogenicity of skin as
cornpared to heart allografts. To investigate wether tolerance could 'spread' to new
antigens we transplanted donor-type skin grafts onto long-term cardiac allograft
acceptors. As approximately 50% of these allografts are accepted lorrg-term and thc
unmodified STITTI protocol does not induce tolerance to skin allografts we concluded
that spreading had indeed occurred.
As the cardiac aliograft in our rnodel is transplanted heterotopically the beating of the
graft does not indicate if it is fully Íunctional and could support life. To evaluate the
status of the cardiac allograft rve performed histological studies on grafts explanted at
different t imes after tolerance induction and transplantation. Results from this study
are reported in chapter 3. Early after transplantation and tolerance induction a
parenchymatous type infi ltrate is observed rvhich is indistinguishable from the
162
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infi ltrate seen in rejecting grafts. Later the infi l trate is more peripheral, blood vessel
associated, and there has been a shift from CD8* T cells and macrophages to CD4* T
cells and B cells. We aiso rroted a gradual deterioration of the heart t issue but are
unsure wether this is due to the inflammation or the blood vessel associated pathology
(chronic rejection) resuiting in an insufficient oxygen supply.
Tr.t'o of the most important questions remaining at this time were: (i) how is tolerance
induced, and (ii) wirat mechanism is responsible for the maintenance of tolerance. To
investigate the first of these two questions we evaluated the survival of allogerreic
splenocytes in the recipient's thymus with and without immunosuppression. Results
presented in chapter 4 indicate that allogeneic splenocytes injected into the thymus of a
recipient treated with our standard ALS + CsA regimen are present only in a restricted
area for a lirnited time (< 28 days). The allogeneic splenocytes are eliminated by a
reaction in which large numbers of recipient B cells and macrophages are observed.
However, no polymorphonr-rclear neutrophiis were seen. Recipient B cells and
macrophages coliocate rvith the allogenic cells. There is some morphological damage to
the thymus due to either the (very limited) graft versus host reaction or the
'inflammatory' response. In contrast, allogeneic cells are eiiminated much faster and
with much more damage to the thymus in the ALS treated and non immunosuppressed
animais while congenic cells survive at least 35 days with no sign of any reaction.
The reaction to the injection of allogenic cells may in turu induce a regulatory response
to prevent damage to the thymus. We tirink this regulatory response is peripheralized,
becomes thymus independent and subsequently is responsíble for the maintenance of
tolerance.
Chapter 5 deals with the investigation of the mechanism responsible for the
maintenance of tolerance. Results presented in section 5.L show that in animals treated
according to the STITTI protocol the proliferative response early (up to 90 days) after
tolerance induction is non-specificaily depressed but not absent after stimulation with
donor antigens. This indicates that the induced tolerance is not mediated solely by an
central (i.e. intrathymic) me.chanism.
Results from the experiments reported irr section 5.2 show that long-term acceptance of
cardiac allografts indr,rced with the STITTI protocol can be transferred to sublethally
irradiated naive syngeneic recipients using splenocytes, thoracic duct lymphocytes or
purif ied T cells. Therefore the mechanism mediating tolerar-rce is probably a mechanism
of active suppressior-r. Furthermore, the cell mediating'toierance' is most l ikely a
recirculating CD4 positive T cell.
Transferred celis comprise onlv a small percentage of the peripheral population in the
(secondary) recipients. As it is unlikely that donor derived celis are solely responsibie
for the tolerance seen in these animals we postulated that the recipient's peripheral
population has become functionally tolerant, thereby fulfilling the criteria for
1 6 3
' infectious tolerance'. We tested wether the peripheral population of the (secorrdarl.)
recipient had become tolerant by again attempting to transfer tolerance to new naive
(tertiary) recipient. This is indeed possible. We therefore postulate that functionai
tolerance inducec-l by the STITTI protocol is mediated by a rnechanism of active
suppression with the hallmarks of infectious tolerance'.
These results are discussed in the light of current literature in chapter 6.
Conclusions
. Simultaneous transplantation and intrathymic tolerance induction (STITTI) protocol
induces stable long-lasting tolerance.
. While the STITTI protocol compietely prevents or abrogates acute rejection there is
still chronic rejection which might be prevented by providing the 'right' antigens in the
thymus.
. Tolerance induction is possible in recipients predisposed to a cellular (Th1) response
but becomes difficult wherr recipients are predisposed to a humoral (Th2) response.
. Tolerance is not mediated by a central (i.e. intrathyrnic) mechanism but instead by an
mechanism of active suppression with 'infectious' properties.
. Tolerance is probably mediated by a CDS recirculating T cell.
Furthermore, the literature has provided evidence for the following:
. Toierance is probably mediaied by a CD45RC RT6"CD4-CD8- recirculating T cell.
. Intrathymic tolerance induction is not dependent upon either MHC class I or MHC
class II antigens.
. Intrathyn-ric tolerance induction can prevent and re'u,erse cellular autoimmunity and
may do the same for humoral autoimmunity.
. Intrathymic tolerance induction may also tolerize the humoral response and can
prevent hyperacute rejection.
Despite its current drawbacks (i.e. an extra operation to deliver the intr.rthymic
inoculum and chronic rejection of the graft) rr.'e feel that the simultaneous
transplantation and intrathymic tolerance induction protocol has clinical potential.
7.2 Sar
1 6 4


Simultaneous transplantation and intrathymic tolerance induction

University of Groningen Digital Archive

ARTS repository - University of Groningen

   University of GroningenSimultaneous transplantation and intrathymic tolerance inductionRaue, Hans-PeterIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1998Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Raue, H-P. (1998). Simultaneous transplantation and intrathymic tolerance induction. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 16-07-2023In chapter 1 a short overview of the history of transplantation tolerance is givenfollowed by a discussion of t l-re various mechanisms currently thought to rnediatetolerance and the ways to induce transplantation tolerance. Section 1.1 ends rvith areview of the literature on intrathymic tolerance induction. Section 1.2 introduces ourpractical work. Current immunosuppressive protocols are very successful inpreventing or abrogating acute rejection. Hor.r'ever, these regimens have a number ofserious side effects (e.g. increased susceptibility to infection, increased risk of neoplasticdisease and renal and myocardiai toxicity). These problems could be prevented by theinduction of donor-specific tolerance which r,r.'ould eliminate the need for long-termimmunosuppression. One of the protocols with potential clinical application istolerance induction via intrathymic inoculaiion with donor-type antigens and shortterm immunosuppression with antilymphocyte serum (ALS). However, the obiigatoryinterval between tolerance indr.rction and the actual transplantation precludes clinicaluse. We have tried to create an intrathymic tolerance induction protocol with clinical Lryadding a short course of cyclosporin A (CsA) treatment to allow us to performtolerance induction and iransplantation simtrltaneously.In section 2.L the results with our simultaneous transplantation and intrathymictolerance induction (STITTI) protocol are described. This protocol is highly efficient(> 90"/,,) and can be used to induce tolerance for cardiac allografts in several differentfully MHC disparate ('high' responder) rat strains. As the STITTI protocol allows us toperform tolerance induction simultaneously with the actual transplantation it may haveclinical potential.Section 2.2 describes our skin transplant experiments. The unmodified STITTI protocoldoes not induce tolerance to skin allografts in the PVG to AO rat strain combination.However, substitution of keratinocytes for splenocytes in the intrathymic inoculumdoes induce long-term acceptance of skin allografts in approximately 50% of therecipients. The lower efficiency is probably due to the higher immunogenicity of skin ascornpared to heart allografts. To investigate wether tolerance could 'spread' to newantigens we transplanted donor-type skin grafts onto long-term cardiac allograftacceptors. As approximately 50% of these allografts are accepted lorrg-term and thcunmodified STITTI protocol does not induce tolerance to skin allografts we concludedthat spreading had indeed occurred.As the cardiac aliograft in our rnodel is transplanted heterotopically the beating of thegraft does not indicate if it is fully Íunctional and could support life. To evaluate thestatus of the cardiac allograft rve performed histological studies on grafts explanted atdifferent t imes after tolerance induction and transplantation. Results from this studyare reported in chapter 3. Early after transplantation and tolerance induction aparenchymatous type infi ltrate is observed rvhich is indistinguishable from the162i rrfil trassoclcells aU N S U I(chrorTwo cinduc,investq n  I  e n .Preserr e n i n i rarea fcreacticHowe,macrolthe thy'inflanwith rranimalThe rerro PrevbecomrtoleranChaptemainteaccorditoierandono r ;centralResultscardiacirradiatpurif ie<of activrecirculTransfe(secondfor the ipopuialryi.5ltFP 4rF?q\r-:e ls grYent to mediatel ends with a2 introduces our;sfui inve a number ofI risk of neoplasticrrevented by thefnr  lnno-term' . - ' ' . - ^ ' ó  - . - - ' - -ication is;ens and shorter, ihe obligatory'recludes ciinical:oj lvith clinical byperformn t r e Í l r r r m i righly efficient:veral differentocol allorn's us to:ation it may havei STITTI protocolcornbination.ric inoculuml% of theenicity of skin asead' to newallograftrrm and thewe concludedh o : l i n , r  n f  + h orvaluate the; explanted atn this studytiorr am theinfi ltrate seen in rejecting grafts. Later the infi l trate is more peripheral, blood vesselassociated, and there has been a shift from CD8* T cells and macrophages to CD4* Tcells and B cells. We aiso rroted a gradual deterioration of the heart t issue but areunsure wether this is due to the inflammation or the blood vessel associated pathology(chronic rejection) resuiting in an insufficient oxygen supply.Tr.t'o of the most important questions remaining at this time were: (i) how is toleranceinduced, and (ii) wirat mechanism is responsible for the maintenance of tolerance. Toinvestigate the first of these two questions we evaluated the survival of allogerreicsplenocytes in the recipient's thymus with and without immunosuppression. Resultspresented in chapter 4 indicate that allogeneic splenocytes injected into the thymus of arecipient treated with our standard ALS + CsA regimen are present only in a restrictedarea for a lirnited time (< 28 days). The allogeneic splenocytes are eliminated by areaction in which large numbers of recipient B cells and macrophages are observed.However, no polymorphonr-rclear neutrophiis were seen. Recipient B cells andmacrophages coliocate rvith the allogenic cells. There is some morphological damage tothe thymus due to either the (very limited) graft versus host reaction or the'inflammatory' response. In contrast, allogeneic cells are eiiminated much faster andwith much more damage to the thymus in the ALS treated and non immunosuppressedanimais while congenic cells survive at least 35 days with no sign of any reaction.The reaction to the injection of allogenic cells may in turu induce a regulatory responseto prevent damage to the thymus. We tirink this regulatory response is peripheralized,becomes thymus independent and subsequently is responsíble for the maintenance oftolerance.Chapter 5 deals with the investigation of the mechanism responsible for themaintenance of tolerance. Results presented in section 5.L show that in animals treatedaccording to the STITTI protocol the proliferative response early (up to 90 days) aftertolerance induction is non-specificaily depressed but not absent after stimulation withdonor antigens. This indicates that the induced tolerance is not mediated solely by ancentral (i.e. intrathymic) me.chanism.Results from the experiments reported irr section 5.2 show that long-term acceptance ofcardiac allografts indr,rced with the STITTI protocol can be transferred to sublethallyirradiated naive syngeneic recipients using splenocytes, thoracic duct lymphocytes orpurif ied T cells. Therefore the mechanism mediating tolerar-rce is probably a mechanismof active suppressior-r. Furthermore, the cell mediating'toierance' is most l ikely arecirculating CD4 positive T cell.Transferred celis comprise onlv a small percentage of the peripheral population in the(secondary) recipients. As it is unlikely that donor derived celis are solely responsibiefor the tolerance seen in these animals we postulated that the recipient's peripheralpopulation has become functionally tolerant, thereby fulfilling the criteria for1 6 3' infectious tolerance'. We tested wether the peripheral population of the (secorrdarl.)recipient had become tolerant by again attempting to transfer tolerance to new naive(tertiary) recipient. This is indeed possible. We therefore postulate that functionaitolerance inducec-l by the STITTI protocol is mediated by a rnechanism of activesuppression with the hallmarks of infectious tolerance'.These results are discussed in the light of current literature in chapter 6.Conclusions. Simultaneous transplantation and intrathymic tolerance induction (STITTI) protocolinduces stable long-lasting tolerance.. While the STITTI protocol compietely prevents or abrogates acute rejection there isstill chronic rejection which might be prevented by providing the 'right' antigens in thethymus.. Tolerance induction is possible in recipients predisposed to a cellular (Th1) responsebut becomes difficult wherr recipients are predisposed to a humoral (Th2) response.. Tolerance is not mediated by a central (i.e. intrathyrnic) mechanism but instead by anmechanism of active suppression with 'infectious' properties.. Tolerance is probably mediated by a CDS recirculating T cell.Furthermore, the literature has provided evidence for the following:. Toierance is probably mediaied by a CD45RC RT6"CD4-CD8- recirculating T cell.. Intrathymic tolerance induction is not dependent upon either MHC class I or MHCclass II antigens.. Intrathyn-ric tolerance induction can prevent and re'u,erse cellular autoimmunity andmay do the same for humoral autoimmunity.. Intrathymic tolerance induction may also tolerize the humoral response and canprevent hyperacute rejection.Despite its current drawbacks (i.e. an extra operation to deliver the intr.rthymicinoculum and chronic rejection of the graft) rr.'e feel that the simultaneoustransplantation and intrathymic tolerance induction protocol has clinical potential.7.2 Sar1 6 4

English

   University of GroningenSimultaneous transplantation and intrathymic tolerance inductionRaue, Hans-PeterIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1998Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Raue, H-P. (1998). Simultaneous transplantation and intrathymic tolerance induction. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 30-10-2023In chapter 1 a short overview of the history of transplantation tolerance is givenfollowed by a discussion of t l-re various mechanisms currently thought to rnediatetolerance and the ways to induce transplantation tolerance. Section 1.1 ends rvith areview of the literature on intrathymic tolerance induction. Section 1.2 introduces ourpractical work. Current immunosuppressive protocols are very successful inpreventing or abrogating acute rejection. Hor.r'ever, these regimens have a number ofserious side effects (e.g. increased susceptibility to infection, increased risk of neoplasticdisease and renal and myocardiai toxicity). These problems could be prevented by theinduction of donor-specific tolerance which r,r.'ould eliminate the need for long-termimmunosuppression. One of the protocols with potential clinical application istolerance induction via intrathymic inoculaiion with donor-type antigens and shortterm immunosuppression with antilymphocyte serum (ALS). However, the obiigatoryinterval between tolerance indr.rction and the actual transplantation precludes clinicaluse. We have tried to create an intrathymic tolerance induction protocol with clinical Lryadding a short course of cyclosporin A (CsA) treatment to allow us to performtolerance induction and iransplantation simtrltaneously.In section 2.L the results with our simultaneous transplantation and intrathymictolerance induction (STITTI) protocol are described. This protocol is highly efficient(> 90"/,,) and can be used to induce tolerance for cardiac allografts in several differentfully MHC disparate ('high' responder) rat strains. As the STITTI protocol allows us toperform tolerance induction simultaneously with the actual transplantation it may haveclinical potential.Section 2.2 describes our skin transplant experiments. The unmodified STITTI protocoldoes not induce tolerance to skin allografts in the PVG to AO rat strain combination.However, substitution of keratinocytes for splenocytes in the intrathymic inoculumdoes induce long-term acceptance of skin allografts in approximately 50% of therecipients. The lower efficiency is probably due to the higher immunogenicity of skin ascornpared to heart allografts. To investigate wether tolerance could 'spread' to newantigens we transplanted donor-type skin grafts onto long-term cardiac allograftacceptors. As approximately 50% of these allografts are accepted lorrg-term and thcunmodified STITTI protocol does not induce tolerance to skin allografts we concludedthat spreading had indeed occurred.As the cardiac aliograft in our rnodel is transplanted heterotopically the beating of thegraft does not indicate if it is fully Íunctional and could support life. To evaluate thestatus of the cardiac allograft rve performed histological studies on grafts explanted atdifferent t imes after tolerance induction and transplantation. Results from this studyare reported in chapter 3. Early after transplantation and tolerance induction aparenchymatous type infi ltrate is observed rvhich is indistinguishable from the162i rrfil trassoclcells aU N S U I(chrorTwo cinduc,investq n  I  e n .Preserr e n i n i rarea fcreacticHowe,macrolthe thy'inflanwith rranimalThe rerro PrevbecomrtoleranChaptemainteaccorditoierandono r ;centralResultscardiacirradiatpurif ie<of activrecirculTransfe(secondfor the ipopuialryi.5ltFP 4rF?q\r-:e ls grYent to mediatel ends with a2 introduces our;sfui inve a number ofI risk of neoplasticrrevented by thefnr  lnno-term' . - ' ' . - ^ ' ó  - . - - ' - -ication is;ens and shorter, ihe obligatory'recludes ciinical:oj lvith clinical byperformn t r e Í l r r r m i righly efficient:veral differentocol allorn's us to:ation it may havei STITTI protocolcornbination.ric inoculuml% of theenicity of skin asead' to newallograftrrm and thewe concludedh o : l i n , r  n f  + h orvaluate the; explanted atn this studytiorr am theinfi ltrate seen in rejecting grafts. Later the infi l trate is more peripheral, blood vesselassociated, and there has been a shift from CD8* T cells and macrophages to CD4* Tcells and B cells. We aiso rroted a gradual deterioration of the heart t issue but areunsure wether this is due to the inflammation or the blood vessel associated pathology(chronic rejection) resuiting in an insufficient oxygen supply.Tr.t'o of the most important questions remaining at this time were: (i) how is toleranceinduced, and (ii) wirat mechanism is responsible for the maintenance of tolerance. Toinvestigate the first of these two questions we evaluated the survival of allogerreicsplenocytes in the recipient's thymus with and without immunosuppression. Resultspresented in chapter 4 indicate that allogeneic splenocytes injected into the thymus of arecipient treated with our standard ALS + CsA regimen are present only in a restrictedarea for a lirnited time (< 28 days). The allogeneic splenocytes are eliminated by areaction in which large numbers of recipient B cells and macrophages are observed.However, no polymorphonr-rclear neutrophiis were seen. Recipient B cells andmacrophages coliocate rvith the allogenic cells. There is some morphological damage tothe thymus due to either the (very limited) graft versus host reaction or the'inflammatory' response. In contrast, allogeneic cells are eiiminated much faster andwith much more damage to the thymus in the ALS treated and non immunosuppressedanimais while congenic cells survive at least 35 days with no sign of any reaction.The reaction to the injection of allogenic cells may in turu induce a regulatory responseto prevent damage to the thymus. We tirink this regulatory response is peripheralized,becomes thymus independent and subsequently is responsíble for the maintenance oftolerance.Chapter 5 deals with the investigation of the mechanism responsible for themaintenance of tolerance. Results presented in section 5.L show that in animals treatedaccording to the STITTI protocol the proliferative response early (up to 90 days) aftertolerance induction is non-specificaily depressed but not absent after stimulation withdonor antigens. This indicates that the induced tolerance is not mediated solely by ancentral (i.e. intrathymic) me.chanism.Results from the experiments reported irr section 5.2 show that long-term acceptance ofcardiac allografts indr,rced with the STITTI protocol can be transferred to sublethallyirradiated naive syngeneic recipients using splenocytes, thoracic duct lymphocytes orpurif ied T cells. Therefore the mechanism mediating tolerar-rce is probably a mechanismof active suppressior-r. Furthermore, the cell mediating'toierance' is most l ikely arecirculating CD4 positive T cell.Transferred celis comprise onlv a small percentage of the peripheral population in the(secondary) recipients. As it is unlikely that donor derived celis are solely responsibiefor the tolerance seen in these animals we postulated that the recipient's peripheralpopulation has become functionally tolerant, thereby fulfilling the criteria for1 6 3' infectious tolerance'. We tested wether the peripheral population of the (secorrdarl.)recipient had become tolerant by again attempting to transfer tolerance to new naive(tertiary) recipient. This is indeed possible. We therefore postulate that functionaitolerance inducec-l by the STITTI protocol is mediated by a rnechanism of activesuppression with the hallmarks of infectious tolerance'.These results are discussed in the light of current literature in chapter 6.Conclusions. Simultaneous transplantation and intrathymic tolerance induction (STITTI) protocolinduces stable long-lasting tolerance.. While the STITTI protocol compietely prevents or abrogates acute rejection there isstill chronic rejection which might be prevented by providing the 'right' antigens in thethymus.. Tolerance induction is possible in recipients predisposed to a cellular (Th1) responsebut becomes difficult wherr recipients are predisposed to a humoral (Th2) response.. Tolerance is not mediated by a central (i.e. intrathyrnic) mechanism but instead by anmechanism of active suppression with 'infectious' properties.. Tolerance is probably mediated by a CDS recirculating T cell.Furthermore, the literature has provided evidence for the following:. Toierance is probably mediaied by a CD45RC RT6"CD4-CD8- recirculating T cell.. Intrathymic tolerance induction is not dependent upon either MHC class I or MHCclass II antigens.. Intrathyn-ric tolerance induction can prevent and re'u,erse cellular autoimmunity andmay do the same for humoral autoimmunity.. Intrathymic tolerance induction may also tolerize the humoral response and canprevent hyperacute rejection.Despite its current drawbacks (i.e. an extra operation to deliver the intr.rthymicinoculum and chronic rejection of the graft) rr.'e feel that the simultaneoustransplantation and intrathymic tolerance induction protocol has clinical potential.7.2 Sar1 6 4

University of Groningen

   University of GroningenSimultaneous transplantation and intrathymic tolerance inductionRaue, Hans-PeterIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1998Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Raue, H-P. (1998). Simultaneous transplantation and intrathymic tolerance induction. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019In chapter 1 a short overview of the history of transplantation tolerance is givenfollowed by a discussion of t l-re various mechanisms currently thought to rnediatetolerance and the ways to induce transplantation tolerance. Section 1.1 ends rvith areview of the literature on intrathymic tolerance induction. Section 1.2 introduces ourpractical work. Current immunosuppressive protocols are very successful inpreventing or abrogating acute rejection. Hor.r'ever, these regimens have a number ofserious side effects (e.g. increased susceptibility to infection, increased risk of neoplasticdisease and renal and myocardiai toxicity). These problems could be prevented by theinduction of donor-specific tolerance which r,r.'ould eliminate the need for long-termimmunosuppression. One of the protocols with potential clinical application istolerance induction via intrathymic inoculaiion with donor-type antigens and shortterm immunosuppression with antilymphocyte serum (ALS). However, the obiigatoryinterval between tolerance indr.rction and the actual transplantation precludes clinicaluse. We have tried to create an intrathymic tolerance induction protocol with clinical Lryadding a short course of cyclosporin A (CsA) treatment to allow us to performtolerance induction and iransplantation simtrltaneously.In section 2.L the results with our simultaneous transplantation and intrathymictolerance induction (STITTI) protocol are described. This protocol is highly efficient(> 90"/,,) and can be used to induce tolerance for cardiac allografts in several differentfully MHC disparate ('high' responder) rat strains. As the STITTI protocol allows us toperform tolerance induction simultaneously with the actual transplantation it may haveclinical potential.Section 2.2 describes our skin transplant experiments. The unmodified STITTI protocoldoes not induce tolerance to skin allografts in the PVG to AO rat strain combination.However, substitution of keratinocytes for splenocytes in the intrathymic inoculumdoes induce long-term acceptance of skin allografts in approximately 50% of therecipients. The lower efficiency is probably due to the higher immunogenicity of skin ascornpared to heart allografts. To investigate wether tolerance could 'spread' to newantigens we transplanted donor-type skin grafts onto long-term cardiac allograftacceptors. As approximately 50% of these allografts are accepted lorrg-term and thcunmodified STITTI protocol does not induce tolerance to skin allografts we concludedthat spreading had indeed occurred.As the cardiac aliograft in our rnodel is transplanted heterotopically the beating of thegraft does not indicate if it is fully Íunctional and could support life. To evaluate thestatus of the cardiac allograft rve performed histological studies on grafts explanted atdifferent t imes after tolerance induction and transplantation. Results from this studyare reported in chapter 3. Early after transplantation and tolerance induction aparenchymatous type infi ltrate is observed rvhich is indistinguishable from the162i rrfil trassoclcells aU N S U I(chrorTwo cinduc,investq n  I  e n .Preserr e n i n i rarea fcreacticHowe,macrolthe thy'inflanwith rranimalThe rerro PrevbecomrtoleranChaptemainteaccorditoierandono r ;centralResultscardiacirradiatpurif ie<of activrecirculTransfe(secondfor the ipopuialryi.5ltFP 4rF?q\r-:e ls grYent to mediatel ends with a2 introduces our;sfui inve a number ofI risk of neoplasticrrevented by thefnr  lnno-term' . - ' ' . - ^ ' ó  - . - - ' - -ication is;ens and shorter, ihe obligatory'recludes ciinical:oj lvith clinical byperformn t r e Í l r r r m i righly efficient:veral differentocol allorn's us to:ation it may havei STITTI protocolcornbination.ric inoculuml% of theenicity of skin asead' to newallograftrrm and thewe concludedh o : l i n , r  n f  + h orvaluate the; explanted atn this studytiorr am theinfi ltrate seen in rejecting grafts. Later the infi l trate is more peripheral, blood vesselassociated, and there has been a shift from CD8* T cells and macrophages to CD4* Tcells and B cells. We aiso rroted a gradual deterioration of the heart t issue but areunsure wether this is due to the inflammation or the blood vessel associated pathology(chronic rejection) resuiting in an insufficient oxygen supply.Tr.t'o of the most important questions remaining at this time were: (i) how is toleranceinduced, and (ii) wirat mechanism is responsible for the maintenance of tolerance. Toinvestigate the first of these two questions we evaluated the survival of allogerreicsplenocytes in the recipient's thymus with and without immunosuppression. Resultspresented in chapter 4 indicate that allogeneic splenocytes injected into the thymus of arecipient treated with our standard ALS + CsA regimen are present only in a restrictedarea for a lirnited time (< 28 days). The allogeneic splenocytes are eliminated by areaction in which large numbers of recipient B cells and macrophages are observed.However, no polymorphonr-rclear neutrophiis were seen. Recipient B cells andmacrophages coliocate rvith the allogenic cells. There is some morphological damage tothe thymus due to either the (very limited) graft versus host reaction or the'inflammatory' response. In contrast, allogeneic cells are eiiminated much faster andwith much more damage to the thymus in the ALS treated and non immunosuppressedanimais while congenic cells survive at least 35 days with no sign of any reaction.The reaction to the injection of allogenic cells may in turu induce a regulatory responseto prevent damage to the thymus. We tirink this regulatory response is peripheralized,becomes thymus independent and subsequently is responsíble for the maintenance oftolerance.Chapter 5 deals with the investigation of the mechanism responsible for themaintenance of tolerance. Results presented in section 5.L show that in animals treatedaccording to the STITTI protocol the proliferative response early (up to 90 days) aftertolerance induction is non-specificaily depressed but not absent after stimulation withdonor antigens. This indicates that the induced tolerance is not mediated solely by ancentral (i.e. intrathymic) me.chanism.Results from the experiments reported irr section 5.2 show that long-term acceptance ofcardiac allografts indr,rced with the STITTI protocol can be transferred to sublethallyirradiated naive syngeneic recipients using splenocytes, thoracic duct lymphocytes orpurif ied T cells. Therefore the mechanism mediating tolerar-rce is probably a mechanismof active suppressior-r. Furthermore, the cell mediating'toierance' is most l ikely arecirculating CD4 positive T cell.Transferred celis comprise onlv a small percentage of the peripheral population in the(secondary) recipients. As it is unlikely that donor derived celis are solely responsibiefor the tolerance seen in these animals we postulated that the recipient's peripheralpopulation has become functionally tolerant, thereby fulfilling the criteria for1 6 3' infectious tolerance'. We tested wether the peripheral population of the (secorrdarl.)recipient had become tolerant by again attempting to transfer tolerance to new naive(tertiary) recipient. This is indeed possible. We therefore postulate that functionaitolerance inducec-l by the STITTI protocol is mediated by a rnechanism of activesuppression with the hallmarks of infectious tolerance'.These results are discussed in the light of current literature in chapter 6.Conclusions. Simultaneous transplantation and intrathymic tolerance induction (STITTI) protocolinduces stable long-lasting tolerance.. While the STITTI protocol compietely prevents or abrogates acute rejection there isstill chronic rejection which might be prevented by providing the 'right' antigens in thethymus.. Tolerance induction is possible in recipients predisposed to a cellular (Th1) responsebut becomes difficult wherr recipients are predisposed to a humoral (Th2) response.. Tolerance is not mediated by a central (i.e. intrathyrnic) mechanism but instead by anmechanism of active suppression with 'infectious' properties.. Tolerance is probably mediated by a CDS recirculating T cell.Furthermore, the literature has provided evidence for the following:. Toierance is probably mediaied by a CD45RC RT6"CD4-CD8- recirculating T cell.. Intrathymic tolerance induction is not dependent upon either MHC class I or MHCclass II antigens.. Intrathyn-ric tolerance induction can prevent and re'u,erse cellular autoimmunity andmay do the same for humoral autoimmunity.. Intrathymic tolerance induction may also tolerize the humoral response and canprevent hyperacute rejection.Despite its current drawbacks (i.e. an extra operation to deliver the intr.rthymicinoculum and chronic rejection of the graft) rr.'e feel that the simultaneoustransplantation and intrathymic tolerance induction protocol has clinical potential.7.2 Sar1 6 4

Simultaneous transplantation and intrathymic tolerance induction

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