A.W. Thomson

D.H. Lynch

F.G. Chambers

Jenkinson

L. Lu

Matzinger

McCarthy

P. Hershberger

Rastellini

S. Qian

Starzl

Steinman

Suss

T.E. Starzl

Thomas

Thomson

W.A. Rudert

Y. Li

Zheng

PubMed

Crossref

Blocking of the B7-CD28 pathway increases apoptosis induced in activated T cells by in vitro-generated CD95L (FasL) positive dendritic cells

Lu, L

Qian, S

Hershberger, P

Rudert, WA

Li, Y

Chambers, FG

Starzl, TE

Lynch, DH

Thomson, AW

D-Scholarship@Pitt

Blocking of the B7 -CD28 Pathway Increases Apoptosis Induced in Activated T Cells by In Vitro-Generated CD95L (FasL) Positive Dendritic Cells L. Lu, S. Qian, P. Hershberger, W.A. Rudert, Y. U, F.G. Chambers, T.E. Starzl, D.H. Lynch, and A.W. Thomson D ENDRITIC cells (DC) that express high levels of MHC class II and costimu!atory molecules are the most potent antigen-presenting cells and the only popula-tion that can activate naive T cells. 1 Evidence has also accumulated, however, that DC can playa role in tolerance induction. Thus, thymic DC induce central tolerance to MHC antigens.: The ubiquitous presence of donor DC in long-surviving human and animal organ allograft recipients suggests that DC may hold the key to the induction of transplantation tolerance.3.4 We have reported that co-stimulatory molecule-deficient DC induce alloantigen-spe-cific unresponsiveness in vitro,S and prolong allograft sur-vival.°.7 The mechanisms by which tolerance can be induced by DC, however, remain unclear. A recent report indicates that CD8+ Fas-L + (CD95L +) mouse lymphoid tissue DC can induce apoptosis in activated T cells in vitro.s The present study was undertaken to evaluate the capacity of in vitro propagated DC to induce allogeneic T-cell apoptosis and to determine the possible regulatory roles of CD95 (Fas/Apo-I) ligand and B7 molecules expressed on DC. MATERIALS AND METHODS Mouse hone marrow (BM) cells from BIO.BR. B6. and B6-gld mice were cultured with GM-CSF+ IL-4 for 5 days as dcscrihed.' then further pUrified either on metrizamide columns or hy two-color sorting with MAhs directed against DEC 205 and B7-2 (C086)." The phenotype of these cultured DC was confirmed hy How cytometry and morphology. Their potent allostimulatory activity was confirmed hy MLR. The apoptotic activity of naive and activated BIO.BR and BlO T cells was analyzed by DNA fragmen-tation'" and JAM assay" respectively. and by in situ nick-end labeling (TUNEL). Fas L mRNA exprcssion by DC was deter-mined hy RT-PCR. Fas L protein on DC was detected hy flow cytometry and immunocytochemical staining USing mouse Fas-Fe fusion protein (Immunex. Seattle. Wash). or polvclonal rabhit anti-Fas L antihody (N-:!O; Santa Cruz Biotechnolo~yK Santa Cruz. Calif). Ihat recognizes the ke~ l<:rmlnus of Fas L. In addition. Fas L was detected hy Western hlotting using anti-Fas L ..\b. The fusion protell1 IlllTL,\4-11! (from Dr Peter S. Linsley. Bristol t-.1Yers SL1uihh. Scattle. Wash) \\;IS used in blockin!,! experIments to de!ernlll1e the mle ot 137 molecules in the rq.:ulation "I DC· induccd T cell apopt"'''. 0041-1315/97/$17.00 PII S0041-1345(96)00424-1 1094 RESULTS AND DISCUSSION As described previously,9 the purity of GM-CSF+ IL-4 stimulated DC defined by morphology and cell surface immunophenotype was 90% to 95%. These cells were highly efficient inducers of primary allogeneic T cell re-sponses in MLR. Their allostimulatory activity, however, was inhibited in a dose-dependent manner by the addition of mCTLA4-Ig at the start of cultures. The expression of message for Fas L by cultured DC was confirmed by RT-PCR analysis. Levels of Fas L mRNA were similar to those expressed by Con A-activated T cells. Flow cytometric analysis using Fas-Fc showed that the DC were positive for Fas L. Further immunocytochemical analysis of permeabil-ized cells using anti-Fas LAb supported the RT-PCR data and gave reactivity: normal spleen cells < Con A blasts = cultured DC. The addition of a competitive peptide, which corresponded to amino acids 2-19 at the NH2-terminus of Fas L and cross-reacted with mouse Fas L, totally inhibited staining of DC with anti-Fas L antibody, verifying the specificity of the antibody reaction with the cells. 12 To test the apoptosis-inducing function of the cultured Fas L + DC, Fast- Jurkat T cells were used as targets in a 18 hour JAM assay. DC propagated from wild-type (B6) mice induced 43.8 ::t 1.4% DNA fragmentation at 5: 1 ratio (E:T). This was reduced to 27.5 :': 2.7% by adding Fas-Fc fusion protein (10 JLg/mL) at the start of the culture. The DC grown from FasL-deflcient 86-gld mice induced only 1.5 ::t 0.5% DNA fragmentation at the same E:T ratio. These results indicated the functional expres-sion of Fas L on DC. 810.BR DC induced comparable low levels of apoptosis hoth in syngeneic or allogeneic From the Thomas E. Starzl Transplantation Institute and the Departments of Surgery. PediatriCS. and Molecular Genetics and Biochemistry. UniverSity of Pittsburgh. Pennsylvania and Immu-nex Corporation. Seattle. Washington (D.H.L.) .. This study was supported by National Institutes of Health grants DK 49745-01A1 and DK 29961-14. Address reprint requests to Dr A.W. Thomson. W 1544 Bio-medical Science Tower. UniverSity of Pittsburgh Medical Center. Pittsburgh. PA 15213. @ 1997 by Elsevier Science Inc. 655 Avenue of the Americas. New York. NY 10010 Transolantatton Proceedings. 29. 1094-1095 (1997) , I I I l I \ t t . \ ----------------------- --------------------APOTOSIS INDUCED IN ACTIVATED T CELLS Con A-activated normal T cells (14.4 :!: 0.8% DNA fragmentation in 48-hour ConA-BI0 T cells and 12.2 :!: 1.7% in ConA-BlO.BR T cells. respectively). Naive T cells were resistant to apoptosis induced by DC. Inter-estingly, CTLA4-lg (200 ng/mL), which totally inhibited T cell proliferative responses in MLR, enhanced DNA fragmentation of Con A activated target cells by 2- to 4-fold. These data indicated that CD28 costimulation could protect activated T cells from apoptosis and that the apoptotic effect of DC was not MHC restricted. Compared with DC from wild-type (B6) mice, DC from B6-g1d mice induced higher allostimulatory responses in MLR, but a lower incidence of apoptosis in activated T cells by TUNEL assay. However, similarly increased levels of apoptosis were induced by B6-DC and gld-DC effectors when the CD28/B7 pathway was blocked by CTLA4-lg. It therefore appears that Fas/Fas L is not the only molecular pathway via which DC can induce apoptosis, (TNF-medi-ated apoptosis is one potential mechanisml3) and that CD28 costimulation may play an important counter-regu-lating role in T cell survival. 1095 REFERENCES 1. Steinman RM: Annu Rev Immunol 9:271, 1991 2. Jenkinson EJ. Jhitray p, Kingston R. et al: Transplantation 39:331. 1982 3. Starzl TE, Demetris Al, Murase N, et al: Lancet 339:1579, 1992 4. Thomson A W, Lu 1.., Murase N, et al: Stem Cells 13:622, 1995 5. Lu 1.., McCaslin D. Starzl TE, et al: Transplantation 60:1539. 1995 6. Fu F. Li Y. Qian S, et al: Transplantation 1996, 62:659. 1996 7. RasteUini C. Lu 1.., Ricordi C. et al: Transplantation 60:1366. 1995 8. Suss G, Shortman K: 1 Exp Med 183:1789. 1996 9. Lu 1.., Rudert WA, Qian S, et al: 1 Exp Med 182:379. 1995 10. McCarthy SA, Cacchione RN, Mainwaring MS. et al: Trans-plantation 54:543, 1992 11. Matzinger P: 1 Immunol Methods 145:185, 1991 12. Thomas SG, Thomas B, Sharon MF, et al: Science 270:1189, 1995 13. Zheng 1.., Fisher G, Miller RE, et al: Nature 377:348, 1995 

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Blocking of the B7-CD28 pathway increases apoptosis induced in activated T cells by in vitro generated CD95L (Fas L) positive dendritic cells

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Blocking of the B7-CD28 pathway increases apoptosis induced in activated T cells by in vitro-generated CD95L (FasL) positive dendritic cells

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