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Graft-versus-host disease (GvHD) is a major complication afterallogeneic bone-marrow transplantation (BMT). In this processdonor T cells recognize major histocompatibility complex(MHC) and their associated peptides on host-derived antigen-presenting cells (APCs), which results in tissue damage1. Thehistopathology of acute GvHD is selective epithelial damage inthe target organs: skin, gut and liver2,3. Host APC activation ofdonor T cells leads to their proliferation and differentiation intoeffector T cells. Thus, it seems that T-cell receptors and MHC in-teractions are also required in the effector phase of GvHD, be-cause alloantigen specific cytotoxic T lymphocytes (CTLs)contribute to experimental GvHD (refs. 4,5).

The relationships between T-cell subsets (CD4+ or CD8+) andMHC alloantigenic targets are well described6. When donors andrecipients differ only at MHC class II loci, CD4+ cells alone causelethal GvHD, whereas CD8+ cells are responsible for GvHD afterBMT across only MHC class I differences. Although MHC class Iis present on virtually all nucleated cells, MHC class II expres-sion is primarily limited to APCs. During GvHD, MHC class IImolecules aberrantly express on epithelial and endothelial targetcells7–9, and this aberrant MHC expression is generally assumedto be essential for target cell damage in GvHD. Here we testedwhether MHC class II or class I alloantigen expression on targetcells is required for organ damage of GvHD and subsequent mor-tality. Using bone-marrow (BM) chimeras expressing MHC classII or class I alloantigens only on BM-derived APCs but not onnonhematopoietic target cells such as epithelium, endotheliumand parenchyma, we found that acute GvHD did not require al-loantigen expression on host target cells.

MHC II–/– mice are resistant to CD4-mediated acute GvHDWe first tested the requirement for the expression of MHC classII antigens to stimulate allogeneic CD4+ T cells in vivo. Carboxy-fluorescein diacetate succinimidyl ester (CFSE)-labeled T cells

from B6.C-H2bm12 (bm12) donors were transferred to B6 micethat had received 13 Gy total body irradiation (TBI) and that dif-fer from donor bm12 mice at a single MHC class II allele6. Flow-cytometric analysis of spleens 3 days after transfer (Fig. 1a)showed that 42% of CFSE-labeled T cells from bm12 donor micehad progressed through at least 1 cell division in allogeneic wild-type (WT) B6 recipients (bm12 → B6), whereas only 1% did so inallogeneic MHC II–/– B6 recipients (bm12 → MHC II–/–). 16% ofdonor T cells underwent ‘homeostatic’ divisions after syngeneictransfer (B6 → B6). This lymphopenia-driven homeostatic prolif-eration of CD4+ T cells requires T-cell receptor and MHC peptideinteractions10 and was not seen in MHC II–/– mice, which resultedin lower CD4+ cell numbers in MHC II–/– recipients than in syn-geneic controls (Fig. 1b). bm12 donor T-cell expansion was con-firmed in B6.Ly-5a recipients (CD45.1+) where more than 95% ofsplenic CD4+ T cells were of donor (CD45.2+) origin (data notshown). Expansion of bm12 CD4+ T cells in WT B6 recipientswas associated with an increased expression of CD25 and CD49(Fig. 1c) and an increased serum level of interferon-γ (IFN-γ) (Fig.1d), findings that were absent in MHC II–/– recipients. WhenMHC II–/– or B6 mice were given 13 Gy TBI and then transplantedwith BM and T cells from bm12 donor mice, WT B6 recipientsexperienced 100% mortality from GvHD by day 7, whereas 0%mortality was observed in MHC II–/– B6 recipients (Fig. 1e).Clinical GvHD scores in MHC II–/– recipients were also equivalentto syngeneic controls (data not shown).

APC MHC class II expression stimulates CD4+ T cellsThe above experiments showed that MHC II–/– mice were resis-tant to CD4-dependent GvHD. To test the requirement for theexpression of MHC class II on host APCs in this disease process,we generated BM chimeras expressing MHC class II on BM-de-rived APCs but lacking MHC class II on target cells. We did thisby reconstituting lethally irradiated MHC II–/– mice with B6 BM

Acute graft-versus-host disease does not require alloantigenexpression on host epithelium

TAKANORI TESHIMA1, RAINER ORDEMANN1, PAVAN REDDY1, SVETLANA GAGIN1, CHEN LIU2,KENNETH R. COOKE1 & JAMES L.M. FERRARA1

1Departments of Internal Medicine and Pediatrics, University of Michigan Cancer Center,Ann Arbor, Michigan, USA

2Department of Pathology, University of Florida College of Medicine, Gainesville, Florida, USACorrespondence should be addressed to J.L.M.F.; email: ferrara@umich.edu

Alloantigen expression on host antigen-presenting cells (APCs) is essential to initiate graft-ver-sus-host disease (GvHD); therefore, alloantigen expression on host target epithelium is alsothought to be essential for tissue damage. We tested this hypothesis in mouse models of GvHDusing bone-marrow chimeras in which either major histocompatibility complex class I or class IIalloantigen was expressed only on APCs. We found that acute GvHD does not require alloanti-gen expression on host target epithelium and that neutralization of tumor necrosis factor-α andinterleukin-1 prevents acute GvHD. These results pertain particularly to CD4-mediated GvHDbut also apply, at least in part, to CD8-mediated GvHD. These results challenge current para-digms about the antigen specificity of GvHD effector mechanisms and confirm the central rolesof both host APCs and inflammatory cytokines in acute GvHD.

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cells (B6 → MHC II–/– chimeras). Four months later, flow-cyto-metric analysis of splenic dendritic cells isolated from thesemice showed complete replacement of splenic dendritic cells bydonor BM cells with MHC class II expression on more than 95%of CD11c+ dendritic cells as described11 (data not shown).Identically treated B6 → B6 chimeras were created as controls.Chimeras were re-irradiated with 13 Gy TBI and injected with 4 × 106 CFSE-labeled T cells from bm12 donors. Analysis of thespleen three days later showed equally robust cell division andsignificant proliferation of CD4+ bm12 donor T cells in both B6→ MHC II–/– and B6 → B6 chimeric recipients compared withsyngeneic B6 → [B6 → B6] controls (Fig. 2a and b). No CD8+

T-cell expansion was observed, confirming the stimulation ofonly CD4+ donor T cells in this system (data not shown). Serumlevels of IFN-γ at day 5 were markedly elevated in both B6 →MHC II–/– and B6 → B6 chimera recipients compared with syn-geneic controls (Fig. 2c). Alloantigens on host APCs alone aretherefore sufficient to induce CD4+ T-cell expansion and cy-tokine secretion.

GvHD does not require epithelial MHC class II alloantigenWe next tested whether donor CD4+ T-cell activation could in-duce GvHD in the absence of MHC class II on nonhematopoi-etic target cells. Similar to the experiments described above, we

created B6 → MHC II–/– and B6 → B6 chimeras and four monthslater we transplanted them with BM and T cells from bm12donors after 13 Gy TBI. GvHD was highly severe in B6 → B6 re-cipients of bm12 T donors; all recipients died at day 5 of BMT incontrast to 90% survival of recipients of syngeneic B6 donors(Fig. 3a). GvHD was equally severe in B6 → MHC II–/– recipientsof bm12 T cells as in B6 → WT recipients and all died by day 7,consistent with the equivalent donor CD4+ T-cell activation andproliferation observed in these chimeras. Clinical GvHD scoresthat were assessed on day 5 in B6 → MHC II–/– recipients ofbm12 donor cells (6.4 ± 0.5) were equivalent to those of B6 →WT recipients of bm12 T cells (6.4 ± 0.1), and they were signifi-cantly greater than in both sets of two syngeneic controls (3.4 ±0.2; P < 0.05). GvHD pathology scores of the liver and small in-testine in allogeneic B6 → WT and B6 → MHC II–/– recipients onday 5 after BMT were significantly greater than those in corre-sponding syngeneic recipients (Fig. 3b). Liver histology showedstandard histological features of acute GvHD, including mildmononuclear cell infiltrates in bile ducts and portal triads, aci-dophilic bodies and endothelialitis3 (Fig. 3c and d). These

a b c

d e

Fig. 1 MHC II–/– mice are resistant to CD4-mediated GvHD. a, CFSE-la-beled splenic T cells from either bm12 or B6 mice were transferred intolethally irradiated mice. Splenocytes collected 3 d after transfer were com-bined and analyzed (n = 3 per group). Cell division determined by FITC-CFSE is shown for CD4+ cells. b–d, 6 d after transfer of donor T cells,splenocytes were analyzed by flow cytometry for cell expansion and serumwas collected (n = 3 per group). Numbers of splenic CD4+ T cells (b), per-centage of CD25+ or CD49+ cells in CD4+ T cells (c), and serum levels of IFN-γ (d). �, B6 → B6; �, bm12 → B6; �, bm12 → MHC II–/–. Data repre-sent mean ± s.d. *, P < 0.05 (� versus �); **, P < 0.05 (� versus �). e, Lethally irradiated MHC II–/– mice were injected with BM and T cells frombm12 donors and survival was monitored. �, B6 → B6 (n = 11); �, bm12→ B6 (n = 7); �, bm12 → MHC II–/– (n = 10). ***, P < 0.001 (� versus �).

a

b c

Fig. 2 Host APCs alone are sufficient to stimulate allogeneic CD4+ T cells.a, Chimeras were created by irradiating either WT or MHC II–/– B6 mice andthen injecting with WT B6 BM cells. 4 mo later, chimeras were re-irradiatedand injected with CFSE-labeled splenic T cells from either bm12 or B6donor mice. Splenocytes collected 3 d after transfer were combined (n = 3per group) and analyzed. Cell division determined by FITC-CFSE is shownfor CD4+ cells. b and c, 5 d after transfer of donor T cells, splenocytes wereanalyzed by flow cytometry for CD4 and serum was collected (3 mice pergroup). Total CD4+ T cells per spleen (b) and serum levels of IFN-γ (c). �, B6→ [B6 → B6]; �, bm12 → [B6 → B6]; �, bm12 → [B6 → MHC II–/–]. Datarepresent mean ± s.d. UD; undetectable. *, P < 0.05 (� versus �); **, P < 0.01 (� versus �).

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changes were, if anything, more prominent in B6 → MHC II–/–

recipients that lacked MHC class II expression on target cells(Fig. 3b). Histopathology of small intestine also showed signifi-cant changes of GvHD in B6 → MHC II–/– recipients of bm12 T cells, including villous atrophy with epithelial apoptosis (Fig.3e and f). Mice receiving 13 Gy TBI and no BM survived morethan 10 days (data not shown), ruling out graft failure as a causeof this early mortality12.

To confirm the absence of a requirement for alloantigen onGvHD target cells, we created a second set of chimeras B6 →bm12 in a similar fashion. In this model, host target cells haveMHC class II syngeneic to the bm12 donors but host APCs haveMHC class II allogeneic to the donors. GvHD was equally severe

in B6 → B6 and B6 → bm12 recipients of bm12 T cells and bothgroups died of GvHD by day 10 after BMT (Fig. 3g). These resultsconfirm the sufficiency of alloantigens on host APC alone to in-duce GvHD mortality.

We next examined the survival of host APCs after BMT. Sixdays after allogeneic BMT of bm12 donors (CD45.2+) into con-geneic B6.Ly-5a (CD45.1+) recipients, flow-cytometric analysis ofsplenocytes demonstrated that 1.1 ± 0.1 % of host dendritic cells(defined as CD45.1+CD11c+CD3–B220–NK1.1–Gr-1–) survived atthis time. These results demonstrate that a small number of hostdendritic cells still survived 6 days after allogeneic BMT. EffectorT cells require continued stimulation with antigens to survive13.We therefore investigated whether adoptively transferred, acti-

vated CD4+ T cells stimulated by alloantigens couldcause GvHD in the absence of alloantigen. MHCII–/– mice were conditioned with 13 Gy TBI and weretransplanted with bm12 BM cells and 5 × 106 naiveor in vivo primed CD4+ bm12 T cells. GvHD was notobserved in either group by all parameters tested,including mortality, clinical score, weight changeand pathology (Table 1). This result suggested thatalloantigen expression is needed on the small num-ber of host APCs that survive irradiation and thatstill reside in target organs or secondary lymphoid

Table 1 In vivo primed CD4+ T cells fail to induce GvHD in MHC II–/– mice

bm12 CD4+ T cells Mortality Clinical Weight Pathology scoresscores change

Liver Intestinenaive 1/5 3.6 ± 0.1 27 ± 1 % 1.0 ± 0.6 6.5 ± 0.3in vivo primed 0/5 3.1 ± 0.4 25 ± 4 % 1.4 ± 0.4 5.2 ± 0.5

B6 mice were lethally irradiated and injected with 2 × 106 T cells from bm12 mice. 6 d later, 5 × 106 CD4+

T cells negatively isolated from spleen were transferred to lethally irradiated MHC II–/– mice. Control MHCII–/– recipients were injected with 5 × 106 CD4+ T cells isolated from naive bm12 mice. 7 d after transfer,GvHD was assessed.

a b c

d e f

gFig. 3 CD4-mediated GvHD does not require MHC class II alloantigen expression on host epithelium.BM chimeras were re-irradiated and were injected with BM and splenic T cells from either bm12 or B6donors. a, Survival after BMT. �, B6 → [B6 → B6] (n = 10); �, bm12 → [B6 → B6] (n = 6); �, B6 → [B6→ MHC II–/–] (n = 4); �, bm12 → [B6 → MHC II–/–] (n = 7). b, Liver and small intestine samples were ob-tained on day 5 post-BMT for pathological analysis. Coded slides (n = 4 per group) were scored semi-quantitatively (see Methods). �; B6 → [B6 → B6]; �, bm12 → [B6 → B6]; �, B6 → [B6 → MHC II–/–]; �,bm12 → [B6 → MHC II–/–]. c–f, Histology of the liver (c and d) and small intestine (e and f) from syn-geneic (c and e) and allogeneic (d and f) B6 → MHC II–/– recipients. Endothelialitis of a hepatic vein (ar-rowheads) and hepatocytes undergoing necrosis (arrows) in (d). Apoptotic crypt cells (arrows) in f. g, Chimeras were created by irradiating B6 and bm12 mice and injecting with T cell–depleted BM cellsfrom B6 donor mice. Four months later, GvHD was induced by injecting bm12 T cells. Survival afterBMT. �, B6 → [B6 → B6] (n = 6); �, bm12 → [B6 → B6] (n = 6); �, bm12 → [B6 → bm12] (n = 6). *, P < 0.05; **, P < 0.001 compared with corresponding syngeneic control for a, b and g.

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tissue, which are critical to the continued activation of donorCD4+ T cells and the induction of acute GvHD.

Neutralization of inflammatory cytokines prevents GvHDThe results above demonstrated that cellular cytotoxicity medi-ated by CTLs were not required for GvHD target damage, and wehypothesized that cytotoxicity mediated by inflammatory cy-tokines would have a major role in this GvHD model as in theother models14,15. Inflammatory cytokines are produced duringGvHD when mononuclear cells are primed by donor T-cell IFN-γand stimulated by lipopolysaccharide (LPS)14. Serum levels ofLPS, which correlate well with the degree of small bowel injury16,were equally high in B6 → MHC II–/– recipients and B6 → B6 re-cipients of bm12 cells 5 days after BMT (Fig. 4a). Serum levels oftumor necrosis factor (TNF)-α and interleukin-1β (IL-1β) werealso equivalently elevated (Fig. 4b and c), demonstrating thathost APCs can efficiently activate inflammatory effectors ofGvHD in the absence of alloantigens on target cells.

To determine whether the production of inflammatory cy-tokines was causally related to GvHD mortality in this model, weneutralized TNF-α and IL-1 with a combination of dimerichuman soluble TNF-α receptor p80–IgG1 Fc fusion protein(TNFR–Fc) and hamster anti-mouse type 1 IL-1 receptor (αIL-1R-M147)16. 0% of B6 → MHC II–/– recipients treated with diluentsurvived, whereas 80% of mice treated with the cytokine in-hibitors survived at day 40 (P < 0.05) (Fig. 4d). Clinical GvHDscores of the surviving recipients were also significantly reducedin the group receiving the blockade of TNF-α and IL-1 (P < 0.05)

(Fig. 4e). Evaluation of donor T cells in spleens five days afterGvHD induction in B6 → MHC II–/– recipients showed that donorCD4+ T-cell expansion was not suppressed by the cytokine block-ade (Fig. 4f), confirming the effects of the blockade on the effec-tor phase rather than activation phase of GvHD. The samecombination of TNFR–Fc and αIL-1R improved survival and clinical GvHD scores in the second model (B6 → bm12) (Fig. 4g and h). Clinical scores and weight loss of B6 → bm12 re-cipients of bm12 T cells were not statistically different from syn-geneic controls, confirming that GvHD mortality is primarilymediated by inflammatory effectors without direct interactionsof effectors and target cells or the expression of alloantigen onhost target cells.

CD8-mediated GvHD does not require epithelial MHC class IWe next studied whether MHC class I alloantigen expression onhost target epithelium was similarly unnecessary for acute GvHDin a well-characterized mouse model of GvHD directed to a sin-gle MHC class I alloantigen (bm1 → B6). bm1 mice differ fromB6 mice only by a mutation in the Kb class I antigen6. In thisdonor/recipient strain combination, GvHD is mediated by CD8+

T cells and mortality is slower than in the bm12 → B6 straincombination1,17. We created B6 → bm1 and B6 → B6 chimeras byreconstituting lethally irradiated bm1 and B6 mice, respectively,with 5 × 106 T cell–depleted BM cells from B6 donors. Fourmonths later, these chimeras were re-irradiated with 13 Gy TBIand transplanted with 5 × 106 T cell–depleted BM cells and 2 × 106 CD8+ T cells from bm1 donors. bm1 CD8+ T cells pro-

b c d

e f g h

Fig. 4 Neutralization of inflammatory cytokines prevents CD4+ Tcell–mediated GvHD. Mice were transplanted as in Fig. 3. a–c, Mice werekilled on day 5 (n = 3 per group) and serum levels of LPS (a), TNF-α (b) andIL-1β (c) were measured. �, B6 → [B6 → B6]; �, bm12 → [B6 → B6]; �,B6 → [B6 → MHC II–/–]; �, bm12 → [B6 → MHC II–/–]. Data represent mean± s.d. UD, undetectable, *, P < 0.05 (� versus �); **, P < 0.01 (� versus�). d–f, BM chimeras were re-irradiated and injected with BM and T cellsfrom either bm12 or B6 mice. B6 → MHC II–/– chimeras were injected in-traperitoneally with either a combination of TNFR–Fc and αIL-1R or diluentas in Methods. Survival (d) and clinical GvHD scores (e) after BMT weremonitored. �, B6 → [B6 → B6] (n = 4); �, bm12 → [B6 → MHC II–/–]

treated with diluent (n = 4); �, bm12 → [B6 → MHC II–/–] treated withTNFR–Fc and αIL-1R (n = 5). f, Spleens were collected 5 d after BMT anddonor CD4+ T-cell expansion was determined by a flow-cytometric analysis(n = 3 per group). �, B6 → [B6 → B6]; �, bm12 → [B6 → MHC II–/–]treated with diluent; �, bm12 → [B6 → MHC II–/–] treated with TNFR–Fcand αIL-1R. g and h, Additional chimeras were re-irradiated, transplantedand injected with either TNFR–Fc and αIL-1R or diluent as above. Survival(g) and clinical GvHD scores (h) after BMT were monitored. �, B6 → [B6→ B6] (n = 6); �, bm12 → [B6 → bm12] treated with diluent (n = 6); �,bm12 → [B6 → bm12] treated with TNFR–Fc and αIL-1R (n = 6). *, P < 0.05; **, P < 0.01 (� versus �).

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duced 82% mortality by day 50 in B6 → B6 chimeras expressingallogeneic MHC class I on both APCs and target cells, whereas allB6 → B6 chimeric recipients of syngeneic B6 CD8+ T cells sur-vived (Fig. 5a). bm1 CD8+ T cells caused lethal GvHD in 42% ofB6 → bm1 chimeric recipients that did not express MHC class Ialloantigens on target epithelium, a significant decrease com-pared with 82% mortality in B6 → B6 recipients (Fig. 5a and b).Although mortality was delayed in B6 → bm1 chimeric recipi-ents of bm1 CD8+ T cells, histological analyses of the liver, intes-tine and skin showed standard pathological features of acuteGvHD (Fig. 5c). Analysis of the thymus demonstrated equally re-duced numbers of double-positive thymocytes in both B6 → B6and B6 → bm1 recipients of bm1 CD8+ T cells (Fig. 5d). Thus theabsence of MHC class I alloantigens on target epithelial cells sig-nificantly reduced the onset of acute GvHD mortality, but in-duced equally severe target organ damage in surviving mice. Wenext determined whether inflammatory cytokines were also ef-fector molecules of acute GvHD in this CD8-dependent GvHDmodel. Neutralization of TNF-α and IL-1 with a combination ofTNFR–Fc and αIL-1R from day –2 to day 35 again completely pre-vented acute GvHD by all parameters tested (mortality, clinicalscores and pathology) (Fig. 5).

DiscussionAlloantigen expression on host APCs is essential1 for the activa-tion phase of acute GvHD. The effector phase of GvHD is also be-lieved to be antigen-specific and to require alloantigenexpression on target cells. Our results challenge long-held as-sumptions about the antigen specificity of GvHD effector mech-anisms; we demonstrate that the damage to GvHD target organsdoes not require alloantigen expression on epithelial and non-hematopoietically derived target cells. The lack of requirementfor alloantigen expression pertains to both CD4-dependent andCD8-dependent GvHD, but is particularly true of CD4-depen-dent GvHD damage that was mediated primarily by cytokines.This antigen-independent effector mechanism may help to ex-

plain how CD4+ T cells damage host target epithelium that nor-mally do not express MHC class II. Current paradigms postulatethat MHC class II aberrantly expressed in target organs duringGvHD renders them vulnerable to attack by CD4+ effectorcells7–9. Our results suggest that aberrant MHC class II expressionis the result of tissue inflammation, probably from systemic IFN-γ production. Our results agree with a study of experimentalautoimmune diabetes demonstrating that CD4+ CTL-mediatedkilling of islet cells is associated with cytokine-induced upregula-tion of Fas on islet cells independent of their MHC class II ex-pression18.

Effector mechanisms of acute GvHD are thought to includeboth CTL-mediated and cytokine-mediated cytotoxicity. A sec-ond cytolytic pathway during GvHD is an inflammatory cascadegenerated when mononuclear cells are stimulated to secreteTNF-α and other cytokines by LPS, which enters into systemiccirculation from damaged gut mucosa19. Thus, host APCs aloneduring GvHD can activate the inflammatory cytokine cascade,and mortality and morbidity of GvHD was suppressed whenboth TNF-α and IL-1 were neutralized. This reduction of GvHDwas due to the suppression of the effector phase rather than theafferent phase of GvHD because cytokine blockade did not sup-press donor T-cell expansion early after BMT as described16.

Initial studies in experimental GvHD models suggested thatcytotoxicity mediated by CTLs have a central role of GvHD mor-tality and target organ injury4,5. Studies of CD4-dependentGvHD induced by a single MHC class II disparity betweendonors and recipients have shown that inhibition of eitherFas/FasL pathways20 or perforin pathways21 resulted in reducedmortality. In contrast, we show that cytokine-mediated cytotox-icity has a central role in CD4-dependent GvHD. This differencemay be due to the difference in the endpoint of study. In previ-ous studies, mortality of the recipients was caused by marrowaplasia in a ‘graft-versus-hematopoiesis’ reaction, which is pri-marily mediated by FasL-dependent mechanisms22. However,this graft-versus-hematopoiesis reaction does not reflect clinical

a b

c d

Fig. 5 CD8-mediated GvHD does not require MHCclass I alloantigen expression on host epithelium.Chimeras were created by irradiating either B6 or bm1mice and then injecting with B6 T cell–depleted BMcells. 4 mo later, chimeras were re-irradiated and in-jected with T cell–depleted BM and CD8+ T cells fromeither bm1 or B6 donors. a and b, Survival (a) andclinical GvHD scores (b) after BMT are shown. �, B6→ [B6 → B6] treated with control (n = 9); �, bm1 →[B6 → B6] treated with control (n = 11); �, bm1 Tcell–depleted BM → [B6 → bm1] treated with control(n = 6); �, bm1 → [B6 → bm1] treated with control (n= 12); �, bm1 → [B6 → bm1] treated with TNFR–Fcand αIL-1R (n = 12). *, P < 0.05 (� versus �, � and �).c and d, 45 d after BMT, liver, intestine, and skin sam-ples (c) were collected for pathological analysis, andthymus (d) was collected and DP thymocytes wereenumerated. Coded slides (n = 4–6 per group) werescored semi-quantitatively as in Methods. Scores of in-testine are the sum of those of small intestine andlarge intestine. �, B6 → [B6 → B6]; �, bm1 → [B6 →B6]; �, bm1 T cell–depleted BM → [B6 → bm1]treated with control; , bm1 → [B6 → bm1] treatedwith control; �, bm1 → [B6 → bm1] treated withTNFR–Fc and αIL-1R. *, P < 0.05 (� versus �; versus

); **, P < 0.05 (� versus ).

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GvHD after myeloablative conditioning in which hosthematopoiesis is eradicated and donor-derived hematopoieticstem cells reconstitute recipient hematopoiesis. In this commonclinical scenario, GvHD mortality is mediated by damage to vis-ceral target organs such as the liver and gastrointestinal tract. Itis now clear that cytotoxic double-deficient CD4+ T cells (lackingboth FasL and perforin) can mediate GvHD after myeloablativeconditioning23,24. Our studies extend those observations andidentify that inflammatory cytokines have a central role in CD4-dependent GvHD.

In the effector phase, a cognate interaction between the anti-gen-specific T-cell receptors on CTLs and peptide antigen pre-sented on the MHC molecule of target cells is required forperforin/granzyme-mediated cytotoxicity, but is less significantfor Fas-mediated cytotoxicity25. CD8+ CTLs kill their targetsusing perforin/granzyme, Fas/FasL and inflammatory cytokinepathways. Acute GvHD mediated by CD8+ T cells did not requirealloantigen expression on target epithelium, although acuteGvHD mortality was more rapid when epithelial targets ex-pressed the alloantigen. Neutralization of both TNF-α and IL-1completely prevented GvHD target organ injury in chimeric re-cipients lacking alloantigens on target cells. These resultsdemonstrate that CD8-dependent GvHD is mediated by bothantigen-non-specific cytokines and antigen-specific cellular ef-fectors, and they confirm the delayed mortality observed inCD8-dependent GvHD models using perforin- or granzyme-defi-cient donors4,5,20.

Accumulating evidence in experimental GvHD suggests thatFas/FasL-mediated cytotoxicity is an important effector pathwayin hepatic GvHD (refs. 4,15,26), and our results contrast withprevious studies in which administration of anti-TNF-α mono-clonal antibodies failed to reduce hepatic GvHD (refs. 15,27).Differences in dosing and schedule of cytokine neutralizationmay account for these discrepancies; only four doses of mono-clonal antibodies were given in one study15 and therapy wasstarted on day 8 in another study27. This is in contrast to thelong-term administration (day –2 to day 35) in our study, whichalso neutralized IL-1. Notably, the reduction of hepatic GvHDobserved in our study was associated with markedly decreasedlymphocyte infiltrates in the liver. Perhaps blockade of inflam-matory cytokines also prevents chemokine-mediated effector-cell recruitment to target organs (for example, prevention ofTNF-α induction of macrophage inflammatory protein-1α thatrecruits CCR5+ CD8+ T cells into the liver during GvHD)28–30.

In summary, our results demonstrate the following for acuteGvHD: host APCs are sufficient in both activation and effectorphase; alloantigen expression on host target cells is not alwaysrequired, and therefore the effector phase is not antigen specific;and inflammatory cytokines mediate mortality and target de-struction. These conclusions are particularly true for CD4-medi-ated acute GvHD but also apply, at least in part, forCD8-mediated disease. Our results have important clinical im-plications as donor CTLs are important effectors of beneficialgraft-versus-leukemia (GVL) effects31 in patients receiving BMTfor hematological malignancies. Elective blockade of inflamma-tory cytokines may thus be a strategy to preserve GVL while re-ducing toxicity of GvHD.

MethodsMice. Female C57BL/6 (B6, KbDbI-AbI-Eb, CD45.2+), B6.Ly-5a (CD45.1+),B6.C-H2bm12 (bm12, KbDbI-Abm12I-Eb, CD45.2+) and B6.C-H2bm1 (bm1,Kbm1DbI-AbI-Eb, CD45.2+) mice were purchased from the Jackson

Laboratories (Bar Harbor, Maine). bm12 and bm1 mice possess mutantMHC class II and class I alleles, respectively, that differ from B6 mice. B6-background MHC II–/– mice32 (B6.129-Abbtm1N5: CD45.2+) were purchasedfrom Taconic (Germantown, New York). The age range of mice used asBMT donor and recipients was 9–16 wk. All animal protocols were ap-proved by the University Committee on Use and Care of Animals at theUniversity of Michigan.

Generation of BM chimera. To create BM chimeras, mice received 13 GyTBI (137Cs source) split into 2 doses separated by 3 h to minimize gastroin-testinal toxicity, and then injected intravenously with 5 × 106 T cell–de-pleted BM cells from donor mice on day 0. T-cell depletion was performedby incubating BM cells with Thy-1.2 MicroBeads and the autoMACS(Miltenyi Biotec, Bergisch Gladbach, Germany) separation. Mice werehoused in sterilized microisolator cages and received autoclaved hyperchlo-rinated drinking water for the first 3 wk after BMT, and filtered water there-after.

GvHD induction. 4 mo after the generation of BM chimeras, GvHD was in-duced according to a standard protocol as described33. For GvHD inductionto MHC class II alloantigen, mice were re-irradiated with 13 Gy TBI, splitinto 2 doses, and injected with 4 × 106 nylon wool-purified splenic T cellsand 5 × 106 BM cells on day 0. For GvHD induction to MHC class I alloanti-gen, mice received 2 × 106 CD8+ splenic T cells with 5 × 106 CD4-depletedand natural killer T cell–depleted BM cells from bm1 donors after 13 Gy TBI.CD8+ T cells were negatively isolated by using CD4, DX5, MHC class II, andCD11b MicroBeads and the autoMACS following nylon wool purification ofT cells from splenocytes. For analysis of cell division, splenic T cells were la-beled with CFSE (Molecular Probes, Eugene, Oregon) as described34 andthen transferred into irradiated recipients.

Systemic and histopathological analysis of GvHD. Survival after BMT wasmonitored daily and the degree of clinical GvHD was assessed weekly by ascoring system which sums changes in 5 clinical parameters: weight loss,posture, activity, fur texture and skin integrity (maximum index = 10) as de-scribed35. This score is a more sensitive index of GvHD severity than weightloss alone in multiple murine models35. Acute GvHD was also assessed bydetailed histopathological analysis of liver and intestine, two primary GvHDtarget organs as described36,37. Slides were coded without reference tomouse type or prior treatment status and examined systematically by a sin-gle pathologist (C.L.) using a semiquantitative scoring system36,37.

Blockade of inflammatory cytokine. TNFR–Fc and αIL-1R were suppliedand used as recommended by Immunex (Seattle, Washington)16. Micewere injected intraperitoneally with TNFR–Fc (100 µg/day) and αIL-1R (200µg/day) on days –2, –1 and 0 and then on alternate days up to day 20 inCD4-dependent GvHD models and up to day 35 in CD8-dependent GvHDmodel as described16. Mice in control groups received injections of controlhuman or hamster immunoglobulin.

Flow-cytometric analysis. A flow-cytometric analysis was performed usingFITC-, PE- or allophycocyanin-conjugated monoclonal antibodies to mouseCD45.1, CD3ε, CD4, CD8α, CD11c (HL3), CD25, CD49 and I-Ab (BDPharmingen, San Diego, California). Cells were preincubated with 2.4G2monoclonal antibodies to block FcγR, and were then incubated with the rel-evant monoclonal antibodies for 30 min at 4 °C. Finally, cells were washedtwice with 0.2% BSA in PBS, fixed with 1% paraformaldehyde in PBS andanalyzed by EPICS Elite ESP cell sorter (Beckman-Coulter, Miami, Florida).Irrelevant IgG2a/b monoclonal antibodies were used as a negative control. 1 × 104 live events were acquired for analysis.

Enzyme-linked immunosorbent assay (ELISA). ELISA for IFN-γ, IL-1β, IL-2,IL-4 and TNF-α (BD Pharmingen) were performed as described38. For deter-mination of LPS concentration in serum, the Limulus amebocyte lysate (LAL)assay (BioWhittaker, Walkersville, Maryland) was performed as described14.Samples and standards were run in duplicate. Unit is relative to the UnitedStates reference standard EC-6.

Statistical analysis. Survival curves were plotted using Kaplan–Meier esti-mates. The Mann–Whitney U-test was used for the statistical analysis of in

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NATURE MEDICINE • VOLUME 8 • NUMBER 6 • JUNE 2002 581

ARTICLES

vitro data and clinical scores while the Mantel–Cox log-rank test was usedto analyze survival data. P < 0.05 was considered statistically significant.

AcknowledgmentsThis work was supported by NIH grants CA39542 (to J.L.M.F.) and HL03565-05 (to K.C.).

Competing interests statementThe authors declare that they have no competing financial interests.

RECEIVED 13 MARCH; ACCEPTED 17 APRIL 2002

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