ࡱ > > @ = , bjbj
.6 h h $ [ [ [ [ [ o o o o o L $ , [ , [ [ A [ [ N pi/ o | : : W 0 L [ 8 , , ! : Examining the percentage of Tregs and effector cells in the periphery and plasmacytoid DC in CX3CR1-/- and WT hearts:
We measured the frequency of Tregs (CD4+CD25+Foxp3+), CD4 effector cells (CD4+CD44highCD62low), and CD8 effector cells (CD8+CD44highCD62low) in recipient spleens and LNs (draining and non-draining) at days 5 and 14 following transplantation of CX3CR1-/- or WT hearts. Furthermore, we performed an MLR using splenocytes from the recipients of CX3CR1-/- or WT heart allografts as responders, stimulated with splenocytes from WT C57BL/6 mice. Given that the level of maturity of DC or that a specific DC subpopulation may have a role in the prolongation of heart allograft survival when CX3CR1-/- hearts are transplanted, extracted DC from CX3CR1-/- and WT hearts were examined for their level of expression of maturity markers, which showed no difference among the two groups ADDIN EN.CITE Thompson200290190190117Thompson, A. G.Thomas, R.Centre for Immunology and Cancer Research, Princess Alexandra Hospital, University of Queensland, Brisbane, Australia.Induction of immune tolerance by dendritic cells: implications for preventative and therapeutic immunotherapy of autoimmune diseaseImmunol Cell Biol509-19806AnimalsAntigen-Presenting Cells/immunologyAutoantigens/immunologyAutoimmune Diseases/*prevention & control/*therapyDendritic Cells/*immunologyHuman*Immune Tolerance*ImmunotherapyInflammation/immunology/prevention & controlMice2002Dec12406384http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=124063841. We also examined the percentage of plasmacytoid cells in CX3CR1-/- hearts, which were found to be tolerogenic ADDIN EN.CITE Ochando200621562156215617Ochando, J. C.Homma, C.Yang, Y.Hidalgo, A.Garin, A.Tacke, F.Angeli, V.Li, Y.Boros, P.Ding, Y.Jessberger, R.Trinchieri, G.Lira, S. A.Randolph, G. J.Bromberg, J. S.Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, New York 10029-6574, USA.Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized graftsNat Immunol652-62762006Jun16633346http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=166333462. There was no difference in the percentage of plasmacytoid (identified as CD11c+CD11b-Gr-1+B220+) or myeloid DC in the CX3CR1-/- and WT hearts by FACS analysis (data not shown). Due to extremely low numbers of heart DC found in the heart and the consequent difficulty in obtaining adequate cell numbers to use in an MLR assay, we extracted CD11c+ cells from the spleens of CX3CR1-/- and WT mice to stimulate allogeneic BALB/c cells in our MLR. Our data indicate that both types of DC were able to bring about equal proliferation of allogeneic T cells (data not shown). These data suggest that there is no intrinsic defect in CX3CR1-/- DC.
Histology of allografts: CX3CR1-/- and WT heart allografts were recovered at day 5 post-transplantation from BALB/c recipients for histological examination of acute rejection. Tissue sections of WT allografts demonstrated massive mononuclear cell infiltration and myocyte necrosis. In contrast, we observed less cellular infiltration and more viable myocytes in CX3CR1-/- grafts (Figure S1). The rejection score of CX3CR1-/- grafts was also significantly less than that of WT grafts (0.9 0.1 vs. 3.2 0.2, p<0.01). Examination of CX3CR1-/- and WT heart allografts at the time of rejection (day 18 and 10, respectively) showed a similar pattern of acute cellular rejection with no significant vascular lesions that are usually characteristic of humoral rejection.
The effect of pertussis toxin on heart tissue DC: Because CX3CR1 requires pertussis toxin-sensitive G protein-signaling to induce migration, pertussis toxin (ptx) should disrupt its function. We tested our hypothesis that the generation of heart tissue DC is dependent upon the function of CX3CR1 in its regulation of the migration of monocytes into heart tissue (followed by monocyte differentiation into heart DC); as such, treatment with ptx should have less of an effect in reducing the number of heart DC when CX3CR1-/- mice are treated than when WT mice are treated. We therefore determined the DC content of CX3CR1-/- and WT hearts extracted from mice injected with ptx. Leukocytes isolated by percoll density gradient centrifugation were gated on CD45, and CD11c+ expression was then assessed. Following treatment with ptx, the percentage of DC in the WT group decreased from 13% in nave untreated WT hearts (Figure S2b) to 4% in the treated group (Figure S2d), whereas in the CX3CR1-/- group, the DC percentage remained almost unchanged (Figures S2a and S2c, from 8% to 7%, n=3/group, p<0.03). Total numbers of heart CD11c+ cells before and after ptx treatment are shown in Figure S2e. These data suggest that migration of monocytes to the heart and their subsequent transformation to heart tissue DC (dictating heart DC content) are controlled by the CX3CR1-dependent pathway.
Measurement of serum alloantibodies: Serum was collected from recipients of CX3CR1-/- and WT allografts treated with or without Flt3L. Following a 1:16 dilution of serum incubated with nave C57BL/6 splenocytes, we stained with mouse anti-IgG1 and anti-IgG2a antibody. We found markedly different levels of alloantibody in CX3CR1-/- vs. WT allograft recipients (3.67% and 19.17% IgG1; 4.9% and 12.92% IgG2a, for CX3CR1-/- and WT, respectively), demonstrating that indirect allorecognition is impaired in the absence of donor CX3CR1. Furthermore, production of alloantibody by recipients of Flt3L-treated CX3CR1 and WT allografts increased as compared to untreated allografts in both groups (33.09% and 35.79% IgG1; 35.17% and 44.03% IgG2a for Flt3L-treated CX3CR1-/- and WT, respectively). These data are in accordance with the reports that dDC are capable of instigating both direct and indirect allorecognition ADDIN EN.CITE Womer200128102810281017Womer, K. L.Sayegh, M. H.Auchincloss, H., Jr.Laboratory of Immunogenetics and Transplantation, Renal Division, Brigham and Women'sHospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.Involvement of the direct and indirect pathways of allorecognition in tolerance inductionPhilos Trans R Soc Lond B Biol Sci639-473561409AnimalsHumansIsoantigens/*immunologyTransplantation Tolerance/*immunology2001May 2911375067http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1137506714.
References:
ADDIN EN.REFLIST 1. Thompson AG, Thomas R. Induction of immune tolerance by dendritic cells: implications for preventative and therapeutic immunotherapy of autoimmune disease. Immunol Cell Biol. 2002;80:509-519.
2. Ochando JC, Homma C, Yang Y, et al. Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts. Nat Immunol. 2006;7:652-662.
] ^ d g w z C D q t !
$
%
'
/
| } r s t u ȻȻȻȻì hD hZP h1=9 hZP H*j hZP U hZP H*hZP hk h hk H*h hk hwu h?5+ hZP 5H* h 5h?5+ hZP 5 hk 5 B w a" 0+ 1+ =+ , , , , $0d ^`0a$gdZP
$d a$gdZP
$d G$ a$gdu4 m$ $d a$gdu4 m$
U V e i % 1 a d s ! ! ! ! `! a! ! ! `" a" " " Ǻ h0/ h0/ 5h0/ hpr hu4 H*hZ hu4 H*hu4 hu4 B*H*ph hu4 B*ph hu4 hu4 5B*ph ho 5B*ph
hZP NH h?5+ hZP 5hTT hZP H*hTT hZP hZP H*hZP hZP B*ph 3" " " # # $ $ \% _% t% w% % % % % % % *+ ++ -+ .+ /+ 0+ 1+ =+ >+ P+ Q+ , , , ǿ hZP j hZP Uh1=9 hZP 5hu4 hr hwu H*j hr hwu Uhwu hr hwu hbA hwu 5h2 hwu H*h2 hwu , 1h/ =!"#$% b 6 6 6 6 6 6 6 6 6 v v v v v v v v v 6 6 6 6 6 6 > 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 h H 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 2 0 @ P ` p 2 ( 0 @ P ` p 0 @ P ` p 0 @ P ` p 0 @ P ` p 0 @ P ` p 0 @ P ` p 8 X V ~ PJ _HmH nH sH tH D ` D N o r m a l CJ _HaJ mH nHsH tH D A D D e f a u l t P a r a g r a p h F o n t R i R T a b l e N o r m a l 4
l 4 a ( k ( N o L i s t L B L ?5+ B o d y T e x t $d d d [$\$a$ 5 D D D B a l l o o n T e x t CJ OJ QJ aJ PK ! [Content_Types].xmlj0Eжr(Iw},-j4 wP-t#bΙ{UTU^hd}㨫)*1P' ^W0)T9<l#$yi};~@(Hu*Dנz/0ǰ$X3aZ,D0j~3߶b~i>3\`?/[G\!-Rk.sԻ..a濭? PK ! ֧ 6 _rels/.relsj0}Q%v/C/} (h"O
= C?hv=Ʌ%[xp{۵_Pѣ<1H0ORBdJE4b$q_6LR7`0̞O,En7Lib/Seе PK ! ky theme/theme/themeManager.xmlM
@}w7c(Ebˮ CAǠҟ7՛K
Y,
e.|,H,lxɴIsQ}#Ր ֵ+!,^$j=GW)E+&
8 PK ! P theme/theme/theme1.xmlYOo6w toc'vuر-MniP@I}úama[إ4:lЯGRX^6؊>$!)O^rC$y@/yH*)UDb`}"qۋJחX^)I`nEp)liV[]1M<OP6r=zgbIguSebORD۫qu gZo~ٺlAplxpT0+[}`jzA V2Fi@qv֬5\|ʜ̭NleXdsjcs7f
W+Ն7`gȘJj|h(KD-
dXiJ؇(x$(:;˹!I_TS1?E??ZBΪmU/?~xY'y5g&/ɋ>GMGeD3Vq%'#q$8K)fw9:ĵ
x}rxwr:\TZaG*y8IjbRc|XŻǿI
u3KGnD1NIBs
RuK>V.EL+M2#'fi~Vvl{u8zH
*:(W☕
~JTe\O*tHGHY }KNP*ݾ˦TѼ9/#A7qZ$*c?qUnwN%Oi4=3ڗP
1Pm\\9Mؓ2aD];Yt\[x]}Wr|]g-
eW
)6-rCSj
id DЇAΜIqbJ#x꺃6k#ASh&ʌt(Q%p%m&]caSl=X\P1Mh9MVdDAaVB[݈fJíP|8քAV^f
Hn-"d>znNJ ة>b&2vKyϼD:,AGm\nziÙ.uχYC6OMf3or$5NHT[XF64T,ќM0E)`#5XY`פ ;%1U٥m;R>QDDcpU'&LE/pm%]8firS4d7y\`JnίIR3U~7+#mqBiDi*L69mY&iHE=(K&N!V.KeLDĕ{D vEꦚdeNƟe(MN9ߜR6&3(a/DUz<{ˊYȳV)9Z[4^n5!J?Q3eBoCMm<.vpIYfZY_p[=al-Y}Nc͙ŋ4vfavl'SA8|*u{-ߟ0%M07%<ҍ PK !
ѐ ' theme/theme/_rels/themeManager.xml.relsM
0wooӺ&݈Э5
6?$Q
,.aic21h:qm@RN;d`o7gK(M&$R(.1r'JЊT8V"AȻHu}|$b{ P8g/]QAsم(#L[ PK- ! [Content_Types].xmlPK- ! ֧ 6 + _rels/.relsPK- ! ky theme/theme/themeManager.xmlPK- ! P theme/theme/theme1.xmlPK- !
ѐ ' theme/theme/_rels/themeManager.xml.relsPK ]