Donor CD4+ T-cell production of tumor necrosis factor alpha significantly contributes to the early proinflammatory events of graft-versus-host disease

Experimental Hematology 35 (2007) 155–163

Donor CD4þ T-cell production of tumor necrosis factor alpha significantly contributes to the early proinflammatory events of graft-versus-host disease Patricia Ewinga, Sandra Miklosa, Krystyna M. Olkiewiczb, Gunnar Mu¨llera, Reinhard Andreesena, Ernst Hollera, Kenneth R. Cookeb, and Gerhard C. Hildebrandta a

Department of Hematology and Oncology, University of Regensburg Medical School, Regensburg, Germany; bThe Department of Pediatrics, University of Michigan, Ann Arbor, Mich., USA (Received 31 May 2006; revised 14 September 2006; accepted 21 September 2006)

Objective. Tumor necrosis factor alpha (TNFa) is an old foe in allogeneic bone marrow transplantation (allo-BMT) promoting acute graft-versus-host disease (aGVHD). We investigated to what extent donor T cells contribute to TNFa production. Methods. Lethally irradiated B6D2F1 mice were transplanted with bone marrow (BM) and T cells from syngeneic B6D2F1 or allogeneic B6 donors and assessed for cytokine production, aGVHD, and survival. Results. Analysis of serum TNFa kinetics in recipients of allogeneic B6 wild-type BM and wild-type T cells revealed that TNFa levels peaked around day 7 after allo-BMT, whereas TNFa was undetectable in syngeneic controls. TNFa was produced by both host and donor cells. Further exploration showed that specifically donor CD4+ but not CD8+ T cells were the primary donor cell source of TNFa at this early time point; numbers of TNFa expressing splenic CD4+ T cells were higher than CD8+ T cells 7 days after allo-BMT, and maximal serum TNFa levels were detected following allo-BMT with only CD4+ T cells compared to levels found in allogeneic recipients of only wild-type CD8+ or to only CD4+ TNFaL/L T cells. Concurrent with increased TNFa levels, early clinical aGVHD and mortality were more severe following allo-BMT with either wild-type CD4+ and CD8+ or CD4+ T cells only. Conclusion. Our data demonstrate that in addition to residual host cells donor CD4+ T cells significantly contribute to the proinflammatory cytokine milieu engendered early after alloBMT through the production of TNFa. These findings support strategies focusing on TNFa neutralization as primary treatment for aGVHD. Ó 2007 International Society for Experimental Hematology. Published by Elsevier Inc.

Allogeneic bone marrow transplantation (allo-BMT) is the only curative treatment option for a variety of malignant and nonmalignant diseases [1]. However, its efficacy is counterbalanced by the risk of acute graft-versus-host disease (aGVHD), which represents the most frequent and potentially lethal complication of allo-BMT. The incidence of aGVHD varies from 20 to 70% depending on conditioning regimen intensity, the stage of primary disease, the age of the transplant recipient, and histocompatibility differences between the donor and the recipient [2–7]. Damage to Offprint requests to: Gerhard C. Hildebrandt, M.D., Department of Hematology and Oncology, University of Regensburg Medical School, Franz-Josef-Strauß Allee 11, D-93053 Regensburg, Germany; E-mail: [email protected]

host tissues during aGVHD is caused by inflammatory cytokines and cellular cytotoxicity involving TRAIL, Fas/ FasL, and perforin/granzyme [8–13]. Previous studies have demonstrated an important role for tumor necrosis factor (TNFa) in the pathogenesis of aGVHD in both the experimental [9,13–20] and clinical settings [21–23]. Increased serum TNFa levels precede major complications following clinical allo-BMT [21–23], whereas early TNFa release has been shown to be causally related to both early gastrointestinal toxicity and aGVHD mortality in experimental allo-BMT models [16,19,20]. The actions of TNFa are mediated by two receptors: a 55- to 60-kDa type I receptor (TNFRI; p55/60; CD120a) and a 75to 80-kDa type II receptor (TNFRII; p75/80; CD120b) [24–28]. These two receptors are coexpressed in almost

0301-472X/06 $–see front matter. Copyright Ó 2007 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 10.1016/j.exphem.2006.09.012

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every cell although in different levels. In human studies, increased levels of sTNFRI correlate with aGVHD and other acute transplant-related complications [29], and TNFRI expression on host target tissues has been shown to be critical for early aGVHD development and mortality after alloBMT, but not for aGVHD target organ pathology in animal models [18]. Recently, Schmaltz et al. demonstrated a role for donor T cell–derived TNFa in aGVHD pathophysiology. Recipients of allogeneic TNFa/ T cells showed improved survival, most evident during the first 10 days after transplant, and a reduction in aGVHD injury of the small and large bowel, but not of liver and skin [9]. However, the authors were unable to detect systemic TNFa levels in recipients of allogeneic TNFaþ/þ T cells, so the precise contribution of donor CD4þ and/or CD8þ T cells in the proinflammatory milieu engendered early after BM transplantation remains unclear. In this study, we focused on the contribution of either donor T-cell subset in comparison to host cells to TNFa production after allo-BMT. By using TNFa/ mice as allogeneic donors in various mixing experiments and transplanting enriched T-cell subsets only, we were able to show that in addition to residual host cells, donor CD4þ T cells are an important source of TNFa in aGVHD development and thereby significantly influence early mortality after allo-BMT.

Materials and methods Mice and bone marrow transplantation Female C57BL/6 (H-2b), B6Ly5.2 (H-2b), LP/J (H-2b), B6D2F1 (H-2bxd), B6.129-Tnfrsf1atm1Mak/J (H-2b; TNFRI/, p55 deficient) [30], B6129SF2/J (H-2b; TNFaþ/þ), and B6;129S6Tnf tm1Gkl (H-2b; TNFa/) [31] were purchased from the Jackson Laboratories (Jax; Bar Harbor, ME, USA), from the Frederick Cancer Research and Development Center (National Cancer Institute; Frederick, MD, USA), or from Charles River Laboratories (Sulzbach, Germany). Animals used were between 10 and 20 weeks old. All experiments performed in the United States were approved by the University of Michigan Committee on the Use and Care of Animals and those conducted in Germany were according to German animal protection laws. Mice were transplanted according to a standard protocol as described previously [32]. When using the major histocompatability complex (MHC)-matched, minor histocompatibility complex–different LP/J / B6 system, B6 wild-type and B6 TNFRI/ recipients received 13 Gy of total body irradiation (TBI; 137Cs source) prior to transplant followed by the infusion of 5  106 bone marrow (BM) cells and 2  106 splenic T cells from either syngeneic (B6) or allogeneic (LP/J) donors. T-cell purification was performed by magnetic-bead-separation using CD4 and CD8 MicroBeads and the autoMACS system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer’s protocol with O85% of cells obtained being positive for CD4 or CD8 surface antigens (data not shown). Percentages of purified CD4þ and CD8þ T cells did not significantly differ between do-

nors. Transplanted mice were cared for as previously described [33]. In experiments using the B6 / B6D2F1 transplantation system, host mice received routinely 11 or in some experiments 14-Gy TBI (USA: 137Cs source; Germany: accelerator, 150 cGy/ min) followed by the infusion of 5  106 BM and 2  106 T cells (CD4þ only, CD8þ only, or CD4þ CD8þ). T cells in these experiments were either purified by magnetic bead separation using CD4 and CD8 MicroBeads and the autoMACS system or by using miniMACS columns (Miltenyi Biotec GmbH). Cell surface phenotype and intracellular cytokine analysis To analyze cell surface phenotype, cells were stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (MoAbs) to CD4, CD8; with phycoerythrin (PE)-conjugated MoAbs to CD4, CD8, TNFa; or with allophycocyanin (APC)-conjugated MoAbs to CD4, CD8 for flow cytometric analysis. Intracellular cytokine staining was performed using a commercially available kit (BD Pharmingen, San Diego, CA, USA; no. 559311) according to the manufacturer’s protocol. All MoAbs were purchased from BD Pharmingen. Three-color flow cytometric analysis of 1  104 cells was performed using a FACSCalibur (BD Biosciences, San Jose, CA, USA). The FACScan was calibrated using FITC-, PE-, and APC-conjugated nonspecific immunoglobulin G antibodies. Clinical aGVHD and survival Survival was monitored daily until day þ35 after transplantation, and aGVHD clinical scores were assessed weekly by a scoring system incorporating five clinical parameters: weight loss, posture (hunching), mobility, fur texture, and skin integrity, as described elsewhere [34]. Serum cytokine analysis and splenic T-cell expansion after BM transplantation Animals were exsanguinated 7 days after transplantation, and blood samples were collected in 1.5 mL Eppendorf tubes and centrifuged at 5000 rpm for 5 minutes. Serum was harvested for subsequent analysis for TNFa and interferon gamma (IFN-g) by enzyme-linked immunosorbent assay (ELISA). Then, spleens were harvested at the same time point, and single-cell suspensions were generated from individual animals. Splenocytes were subsequently counted and stained for CD4, CD8, and TNFa. Measurement of cytokine levels by ELISA Serum concentrations of specific cytokines were measured in serum using ELISA kits for IFN-g (OptEIA; BD Pharmingen) and TNFa (Quantikine M; R&D Systems, Minneapolis, MN, USA). Assays were performed according to the manufacturer’s protocol. Assay sensitivity was less than 7.5 pg/mL for IFN-g and less than 3.0 pg/mL for TNFa. Statistical considerations All values are expressed as the means 6 SEM. Statistical comparisons between groups were completed using the parametric independent sample t-test with five or more animals per group and using the Mann-Whitney test if n ! 5 animals per group. The Wilcoxon rank-test was used for analyzing survival data.

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Results Donor T cells and residual host cells significantly contribute to systemic TNFa levels early after allo-BMT Early after allo-BMT, TNFa can be produced by host cells, by donor cells, or by both. To further determine the contribution of donor cell subsets as well as of host cells to systemic TNFa levels, we chose to use a haploidentical murine BM transplantation system (parent into F1). In the first set of experiments, we determined the time point of peak TNFa serum levels after allo-BMT. Lethally irradiated B6D2F1 mice were transplanted with BM and splenic T cells from either syngeneic B6D2F1 or allogeneic B6 animals. TNFa serum levels were measured on days 0, 3, 7, 14, and 35. As expected, significant amounts of TNFa were not detectable in syngeneic recipients throughout the entire time period. In contrast, TNFa levels in allogeneic recipients abruptly increased beginning day 3 and peaked on day 7 before rapidly decreasing until day 14 and then slowly until day 35 (Fig. 1A). In light of these results, day 7 after transplantation was chosen as time point of analysis for all further experiments. We next separated donor versus host cell production of TNFa by using genetically modified donor mice, which lack the capability to produce TNFa [31]. In this set of experiments, B6D2F1 mice received syngeneic BM transplantation or allo-BMT from either TNFaþ/þ or TNFa/ donors. A third experimental group received allogeneic TNFaþ/þ BM cells mixed with TNFa/ T cells and a fourth experimental group was transplanted with allogeneic TNFa/ BM cells mixed with TNFaþ/þ T cells. Despite the inability of donor cells to produce TNFa, day þ7 serum TNFa levels remained significantly higher in recipients of allo TNFa/ BM and TNFa/ T cells compared to syngeneic controls, suggesting that TNFa was also produced by host cells (Fig. 1B). In addition, a significant role for donor cells in the production of TNFa was suggested as serum levels in these animals were still significantly lower than in animals receiving allogeneic wild-type BM and wild-type T cells (Fig. 1B). Furthermore, allo-BMT with wild-type BM and TNFa/ T cells resulted in a comparable reduction in serum TNFa, whereas the administration of TNFa/ BM and wild-type T cells did not decrease TNFa levels (Fig. 1B). In contrast to serum TNFa levels, serum levels of IFN-g as a marker cytokine produced by activated T cells after allo-BMT did not differ between allogeneic groups (data not shown). Collectively, these results demonstrate that both residual host cells and donor T cells contribute to peak TNFa levels after allo-BMT. TNFa from donor CD4þ but not CD8þ T cells is associated with early mortality after allo-BMT As both CD4þ and CD8þ T cells can express TNFa, we next determined whether CD4þ or CD8þ T cells are the major donor source of TNFa in this BM transplantation

Figure 1. Donor T cells significantly contribute to systemic TNFa levels early after allo-BMT. (A): Lethally irradiated B6D2F1 mice were transplanted as described in Materials and methods (syngeneic, -; allogeneic wild-type, C), and serum TNFa levels were determined on days 0, þ3, þ7, þ14, and þ35 after BM transplantation. Data are presented as mean 6 SEM and combined from two experiments; n 5 6 to 10 per group. (B): Lethally irradiated B6D2F1 mice received BM transplantation from syngeneic (white), allogeneic TNFa/ (light gray), or allogeneic TNFaþ/þ (black) donors. Additional allogeneic groups received either TNFaþ/þ BM mixed with TNFa/ T cells (dark gray) or TNFa/ BM mixed with TNFaþ/þ T cells (hatched). Serum TNFa levels were assessed on day 7. Data are presented as mean 6 SEM; n 5 3 to 5 per group; *p ! 0.05, black bar and hatched bars versus light gray and dark gray bars.

system. First, using congenic B6 donors, which express the CD45.1 allele for the polymorphic region of the common leukocyte antigen (CD45), and B6D2F1 recipients expressing the CD45.2 allele, we demonstrated that T cells expanding in the spleen 7 days after lethal irradiation and allo-BMT were nearly all of donor origin (CD4: 88.4 6 1.7; CD8: 99.6 6 0.1; Fig. 2A). We then transplanted lethally irradiated B6D2F1 mice with syngeneic, allogeneic wild-type or allogeneic TNFa/ BM and T cells and examined T-cell expansion in the spleen on day 7. Both TNFaþ/þ and TNFa/ donor CD4þ and CD8þ T cells expanded to a comparable degree after allo-BMT with the percentage of CD8þ T cells being slightly higher than CD4þ T cells (Fig. 2B). In contrast, flow cytometric analysis for

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Figure 2. Donor CD4þ and not CD8þ T cells are the main producers of donor-derived TNFa early after allo-BMT. (A): Donor (CD45.1)–host (CD45.2) chimerism of spenic T cells 7 days after allo-BMT was determined by using congenic B6 donor mice expressing the CD45.1 allele of the common leukocyte antigen (CD45). Data presented are from one experiment; n 5 4. (B–D): Lethally irradiated B6D2F1 mice received BM transplantation as described in Materials and methods and splenic T-cell expansion was determined on day 7. Although donor CD8þ T cells expand slightly more than CD4þ T cells (B), relative (C) and absolute (D) numbers of TNFa-expressing cells were higher for CD4þ than for CD8þ T cells. Data are presented as mean 6 SEM and are from one of three comparable experiments; n 5 3 to 5 per group. (E): Lethally irradiated B6D2F1 received syngeneic BM transplantation (white) or BM from allogeneic TNFaþ/þ donors supplemented with allogeneic TNFaþ/þ CD4þ (black), TNFaþ/þ CD8þ (light gray), or TNFa/ CD4þ (dark gray). Data are presented as mean 6 SEM; n 5 3 to 5 per group. (F): Lethally irradiated B6D2F1 mice received allo-BMT with CD4þ and CD8þ T cells (black solid line), CD4þ T cells only (gray line), or CD8þ T cells only (black dotted line). Syngeneic controls were also included (100% survival by day 35; data not shown). Survival was monitored for 35 days after BM transplantation. Data are presented as mean 6 SEM or % survival and are from one of three comparable experiments; n 5 3 to 5 per group.

intracellular TNFa revealed that both relative percentages and absolute numbers of TNFaþ cells as well as the cellular mean intensity as a parameter for tumor necrosis factor production per cell (CD4: 117.4 6 3.4 vs CD8: 70.7 6 5.0; mean intensity !30 5 tumor necrosis factor-negative) were higher for the CD4þ T-cell fraction (Fig. 2C and D). Moreover, significant numbers of residual host TNFa producing T cells were not detectable following allo-BMT using TNFa/ donor cells (Fig. 2C). Collectively, these data suggest that CD4þ T cells rather than CD8þ T cells are the predominant source of donor T cell–derived TNFa early after allo-BMT. To specifically evaluate this possibility, B6D2F1 mice received allogeneic BM and either wildtype or TNFa/ purified CD4þ or CD8þ T cells only. Higher serum TNFa levels were detected on day 7 following BM transplantation with only purified allogeneic

wild-type CD4þ T cells compared to those measured in mice receiving only allogeneic wild-type CD8þ T cells and to recipients of only allogeneic CD4þ TNFa/ T cells (Fig. 2E). Similar findings were also noted on day 5 after allo-BMT when mice were conditioned with 14-Gy TBI (data not shown). In addition, TNFa serum levels in allogeneic recipients of CD8þ TNFaþ/þ T cells were still higher than in recipients of TNFa/ CD4þ and CD8þ T cells or of TNFa/ CD4þ T cells alone. Finally, we determined whether increases in TNFa levels early after BM transplantation correlated with early mortality. B6D2F1 mice were treated with 14-Gy TBI and then received syngeneic BM transplantation or allogeneic BM supplemented with either splenic CD4þ and CD8þ T cells, CD4þ T cells alone, or CD8þ T cells alone. In the setting of increased TNFa levels, allogeneic recipients of

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wild-type CD4þ and CD8þ as well as recipients of allogeneic CD4þ T cells demonstrated significant early mortality, whereas animals receiving allogeneic CD8þ T cells did not (Fig. 2F). TNFRI/ recipients show an early reduction of clinical aGVHD and an improvement of survival after allo-BMT In a final set of experiments, we set out to determine whether TNFa:TNFR interactions contribute to the development of aGVHD in an MHC-matched model (that more closely reflects the clinical scenario following an allo-BMT using a ‘‘matched’’ unrelated donor). As TNFRI is the high affinity receptor for soluble TNFa (sTNFa) [35] mediating most of the proinflammatory effects of this cytokine [30,36–40], we now specifically evaluated the role of TNFRI in this context. Lethally irradiated B6 wild-type mice or B6 mice deficient in TNFRI were transplanted with BM and splenic T cells from either syngeneic B6 or allogeneic LP/J donor mice as described in Materials and methods, and the development of clinical aGVHD and survival were closely monitored. As shown in Figure 3, severe clinical aGVHD developed in B6 wild-type recipients in comparison to syngeneic controls resulting in significant mortality beginning 1 week after transplant (Fig. 3A–C). In contrast, overall survival was improved in TNFRI/ recipients (Fig. 3A), and the improved outcomes were specifically related to a reduction in early mortality (between 5 and 10 days) after allo-BMT and not to a reduction in that mortality seen during the cellular effector phase of aGVHD later after allo-BMT (Fig. 3B). Therefore, TNFa:TNFRI interactions seem critical to early mortality after allo-BMT.

Discussion aGVHD is the most important complication after alloBMT. The pathophysiology of aGVHD is complex and in-

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volves both donor T-cell and monocyte/macrophage effectors and the production of inflammatory cytokines [41]. In particular, previous studies have demonstrated a role for TNFa in the pathogenesis of aGVHD both in experimental and clinical settings [9,13–23]. TNFa not only leads to direct tissue damage through apoptosis and necrosis but also is responsible for the initiation of several inflammatory events by enhancing alloantigen presentation, alloantigenspecific T-cell responses, and effector cell recruitment to aGVHD target organs [17,32,42]. TNFa is produced by both host and donor cells [9,12,16]. We now show that in addition to residual host cells, donor CD4þ but not CD8þ T cells are a critical source of TNFa in early aGVHD development, associated with early mortality after allo-BMT. The demonstration, that increased serum TNFa levels are associated with the development of clinical aGVHD, was reported for the first time by Holler et al. [22,43]. The time course analysis of serum TNFa in patients receiving allo-BMT revealed similar kinetics to those seen in our murine model with peak levels of TNFa noted at day þ8 and day þ7, respectively (Fig. 1A [16]). Because at this time tissue macrophages and related cells are not yet eliminated by pretransplant conditioning and can persist for prolonged intervals after BM transplantation [44–47], serum TNFa levels measured in allogeneic recipients of both TNFa/ BM and TNFa/ T cells most likely reflect the production from residual host cells (Fig. 1B). The early role of host cell–derived TNFa at this time is not clearly defined. Although previous results from our group showed that increases in TNFa during pretransplant conditioning, especially TBI, were predictive for the induction and severity of early aGVHD [16,22,43], a direct causative relationship between host TNFa and aGVHD has to be questioned. Using lethally irradiated TNFa/ recipients in a fully MHC-mismatched murine BM transplantation model, Welniak et al. did not see any differences in survival or aGVHD of the small and large intestine or the liver compared to

Figure 3. TNFRI-deficient recipients show an early reduction of systemic aGVHD and improved survival after MHC-matched, minor histocompatibility complex–mismatched allo-stem cell transplantation. BM and T cells from MHC-matched, minor histocompatibility complex–different LP/j donor mice were transplanted into allogeneic B6 wild-type (–, black) or B6 TNFRI/ recipients (—, gray) as described in Materials and methods. Syngeneic B6 wild-type controls (----, white) received BM and T cells from B6 wild-type donors. (A): Overall survival was significantly improved in allogeneic TNFRI/ recipients (—, gray) when compared to allogeneic wild-type controls (–, black). (B): Five-day interval analysis of mortality shows that aGVHD-related deaths in TNFRI/ recipients (gray) were especially reduced between 5 and 10 days after allo-BMT (vs allogeneic wild-type controls, black). (C) Clinical aGVHD severity was assessed weekly by changes in weight, fur texture, skin integrity, posture, and mobility as described in Materials and methods. Data presented are combined from four comparable experiments; n 5 21 to 38 per group; *p ! 0.05.

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TNFaþ/þ recipients after allo-BMT, suggesting that host TNFa is not required for the development of lethal aGVHD in mice that undergo myeloablative conditioning and alloBMT [48]. In addition, in our study, no significant amounts of systemic TNFa were detectable in syngeneic controls at any time point after BM transplantation (Fig. 2A), suggesting that conditioning toxicity alone is not sufficient to induce TNFa production in host cells. Rather, other costimuli like the large amounts of IFN-g, which are produced early during aGVHD and which can prime macrophages to secrete TNFa [49], and/or the translocation of microbial products like lipopolysaccharide (LPS) across a damaged gastrointestinal tract are also required. Furthermore, in the light of reduced intensity conditioning regimens the question must be raised whether decreased early conditioning toxicity to host tissues results in an even more prominent shift from host to donor TNFa contribution in aGVHD pathophysiology. It is generally thought that TNFRI is the dominant receptor in TNFa biology by being the high-affinity receptor for sTNFa [35] and mediating most of the proinflammatory effects of this cytokine [30,36-40]. TNFRI deficiency or TNFRI blockade through p55-specific monoclonal antibodies has been shown to be associated with resistance to endotoxic shock but enhanced susceptibility to infection with certain bacteria such as Listeria monocytogenes [30,50–52]. A role for TNFRI has further been described in eliciting a fulminant alloreactive T-cell response [17,53] and in the development of aGVHD early after allo-stem cell transplantation [18]. Our results using TNFR-deficient mice as BM transplant recipients are consistent with those reported by Speiser and colleagues. This group demonstrated a reduction in early mortality after allo-stem cell transplantation in TNFRI/-deficient recipients, although no differences in target organ injury of liver and gut were seen [18]. The beneficial effect on survival was most evident in lethally irradiated animals and thereby suggests that TNFa:TNFRI signaling is particularly important during the early phase of aGVHD, which involves both conditioning related toxicity, a deleterious cascade of inflammatory cytokine release, and activation of host antigen-presenting cells [54–56]. The mortality rate of allogeneic TNFRI/ recipients in our study was also reduced during this early phase, whereas during the cellular effector phase later on, when TNFa serum levels have subsided, both clinical aGVHD scores and mortality did not significantly differ from controls (Fig. 3C). Next to cell contact–dependent cytotoxic pathways such as Fas/FasL, perforin/granzyme, and TRAIL [8,11,57], donor T cells can mediate aGVHD target organ injury through the secretion of inflammatory cytokines [9]. Previously, allo-BMT with donor T cells lacking the ability to produce TNFa resulted in a significant survival benefit early after transplantation while preserving the graft versus leukemia effect [9]. As TNFa/ T cells do not differ from wild-

type T cells with respect to alloantigen-specific proliferation, IFN-g production or cytotoxic T-cell function [9,32], their inability to produce TNFa can be seen as the likely cause for improved survival despite the fact that the authors were unable to measure serum TNFa and directly confirm this association [9]. Our data support and extend these findings, as early TNFa levels in recipients of allogeneic TNFaþ/þ T cells were significantly increased in comparison to animals receiving TNFa/ T cells irrespective of the donor BM type (Fig. 1B). In addition, CD4þ rather than CD8þ cells were the predominant donor T-cell source of systemic TNFa levels and had a direct impact on mortality during the first days after allo-BMT (Fig. 2). These findings are supported by various studies: Teshima et al. also demonstrated that depletion of CD4þ T cells from the donor cell inoculum effectively eliminated early aGVHD mortality, whereas depletion of CD8þ T cells did not [58]. In addition, the same group described serum TNFa levels between day 5 and day 7 after BM transplantation and survival kinetics using murine BM transplantation systems across isolated MHC class I (bm1 / B6) or isolated MHC class II (bm12 / B6) mismatches [13,59], in which aGVHD is dependent on an alloreactive CD8þ or CD4þ T-cell response, respectively. TNFa levels were higher and aGVHD-related mortality occurred more rapidly in the presence of an isolated MHC class II mismatch involving a CD4þ T-cell response compared to an isolated MHC class I mismatch with a predominantly CD8þ T-cell response [13,59]. However, the important effects of CD4þ T cell–derived sTNFa on mortality may be most prominent during early aGVHD development, as at later time points, during the cellular effector phase of aGVHD, alloantigen-specific CD8þ cytotoxic T cells more and more infiltrate aGVHD target organs and initiate injury directly in a cell contact– dependent manner using various cytotoxic pathways [8,11,57,60,61]. This is supported by unpublished findings from our group, that using the same murine allo-BMT model, the transplantation of TNFa/ donor cells did not significantly affect target organ aGVHD of either small and large bowel or liver at later time points. In contrast, aGVHD target organ injury to the lung was significantly reduced in recipients of allogeneic TNFa/ CD8þ and TNFa/ CD4þ T cells or of allogeneic TNFaþ/þ CD4þ T cells but not in animals receiving allo-BMT with TNFaþ/þ CD8þ T cells. As CD8þ T cells predominantly express membrane-bound TNFa [60,61] and as alveolar epithelial cells are uniquely sensitive to membrane-bound TNFa-mediated injury [62], donor CD8þ TNFa therefore may be specifically critical to lung injury after allo-BMT. In addition to donor T cells, donor BM-derived accessory cells can contribute to aGVHD severity and survival [20,32]. Allo-BMT with LPS-resistant BM cells resulted in increased survival and less severe systemic aGVHD compared to recipients of BM transplantation with LPSsensitive cells irrespective of the LPS sensitivity of donor

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T cells [20]. This model, wherein more robust aGVHD (100% mortality by day 7) and significantly higher TNFa levels (ng/mL vs. pg/mL) were induced by fourfold fewer T cells (0.5  106 vs 2  106), focused solely on the ability of donor cells to produce TNFa in response to LPS. It is likely that these BM transplantation parameters and the accompanying engraftment kinetics of donor cells revealed the contribution of donor accessory cells in this system. This hypothesis is supported by subsequent findings from our group showing that allogeneic recipients of BM cells from either TNFa-deficient or LPS-resistant donors also developed less severe lung injury 6 weeks after BM transplantation when complete turnover of alveolar macrophage to donor cells was apparent both functionally and phenotypically [32,46,63]. In conclusion, this study confirms the important role of TNFa in the systemic inflammation observed early following allo-BMT. Specifically our data demonstrate that alloreactive donor lymphocytes and host macrophages each contribute to elevated TNFa levels early after allo-BMT and that TNFa production by donor CD4þ but not CD8þ cells is critical to the development of early aGVHD and subsequent early mortality seen in allo-BMT recipients. These results, in conjunction with recent clinical data showing that etanercept, a fusion protein capable of neutralizing TNFa, appears to be an effective option for the initial treatment of aGVHD [64], suggest that strategies targeting the effects of TNFa early after BM transplantation may be promising in the future.

6.

7.

8. 9.

10.

11.

12.

13.

14.

15.

Acknowledgments Dr. Cooke is an Amy Strelzer-Manasevit Scholar of the National Marrow Program, a Fellow of the Robert Wood Johnson, Harold Amos Medical Faculty Development Program, a Leukemia and Lymphoma Society Scholar in Clinical Research, and the recipient of a Clinical Scientist Award in Translational Research from the Burroughs Wellcome Fund. Dr. Hildebrandt is a Max Eder Scholar of the Deutsche Krebshilfe e.V.

16.

References

19.

1. Ferrara JL, Deeg HJ. Graft-versus-host disease. N Engl J Med. 1991; 324:667–674. 2. Storb R, Deeg HJ, Whitehead J, et al. Methotrexate and cyclosporine compared with cyclosporine alone for prophylaxis of acute graft versus host disease after marrow transplantation for leukemia. N Engl J Med. 1986;314:729–735. 3. Storb R, Deeg HJ, Fisher L, et al. Cyclosporine v methotrexate for graft-v-host disease prevention in patients given marrow grafts for leukemia: long-term follow-up of three controlled trials. Blood. 1988;71: 293–298. 4. Storb R, Deeg HJ, Pepe M, et al. Methotrexate and cyclosporine versus cyclosporine alone for prophylaxis of graft-versus-host disease in patients given HLA-identical marrow grafts for leukemia: long-term follow-up of a controlled trial. Blood. 1989;73:1729–1734. 5. Storb R, Deeg HJ, Pepe M, et al. Graft-versus-host disease prevention by methotrexate combined with cyclosporin compared to methotrexate

17.

18.

20.

21.

22.

23.

161

alone in patients given marrow grafts for severe aplastic anaemia: long-term follow-up of a controlled trial. Br J Haematol. 1989;72: 567–572. Ratanatharathorn V, Nash RA, Przepiorka D, et al. Phase III study comparing methotrexate and tacrolimus (prograf, FK506) with methotrexate and cyclosporine for graft-versus-host disease prophylaxis after HLA-identical sibling bone marrow transplantation. Blood. 1998; 92:2303–2314. Nash RA, Antin JH, Karanes C, et al. Phase 3 study comparing methotrexate and tacrolimus with methotrexate and cyclosporine for prophylaxis of acute graft-versus-host disease after marrow transplantation from unrelated donors. Blood. 2000;96:2062–2068. Schmaltz C, Alpdogan O, Kappel BJ, et al. T cells require TRAIL for optimal graft-versus-tumor activity. Nat Med. 2002;8:1433–1437. Schmaltz C, Alpdogan O, Muriglan SJ, et al. Donor T cell-derived TNF is required for graft-versus-host disease and graft-versus-tumor activity after bone marrow transplantation. Blood. 2003;101:2440– 2445. Via C, Nguyen P, Shustov A, Drappa J, Elkon K. A major role for the Fas pathway in acute graft-versus-host disease. J Immunol. 1996;157: 5387–5393. Hattori K, Hirano T, Miyajima H, et al. Differential effects of anti-Fas ligand and anti-tumor necrosis factor-a antibodies on acute graft-versus-host disease pathologies. Blood. 1998;91:4051–4055. Nestel FP, Price KS, Seemayer TA, Lapp WS. Macrophage priming and lipopolysaccharide-triggered release of tumor necrosis factor alpha during graft-versus-host disease. J Exp Med. 1992;175:405– 413. Teshima T, Ordemann R, Reddy P, et al. Acute graft-versus-host disease does not require alloantigen expression on host epithelium. Nat Med. 2002;8:575–581. Brown GR, Lindberg G, Meddings J, Silva M, Beutler B, Thiele D. Tumor necrosis factor inhibitor ameliorates murine intestinal graftversus-host disease. Gastroenterology. 1999;116:593–601. Hill GR, Ferrara JL. The primacy of the gastrointestinal tract as a target organ of acute graft-versus-host disease: rationale for the use of cytokine shields in allogeneic bone marrow transplantation. Blood. 2000; 95:2754–2759. Hill GR, Crawford JM, Cooke KR, Brinson YS, Pan L, Ferrara JL. Total body irradiation and acute graft-versus-host disease: the role of gastrointestinal damage and inflammatory cytokines. Blood. 1997; 90:3204–3213. Hill GR, Teshima T, Rebel VI, et al. The p55 TNF-alpha receptor plays a critical role in T cell alloreactivity. J Immunol. 2000;164: 656–663. Speiser DE, Bachmann MF, Frick TW, et al. TNF receptor p55 controls early acute graft-versus-host disease. J Immunol. 1997;158: 5185–5190. Piguet PF, Grau GE, Allet B, Vassalli PJ. Tumor necrosis factor/cachectin is an effector of skin and gut lesions of the acute phase of graft-versus-host disease. J Exp Med. 1987;166:1280–1289. Cooke KR, Hill GR, Crawford JM, et al. Tumor necrosis factor- alpha production to lipopolysaccharide stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease. J Clin Invest. 1998;102:1882–1891. Remberger M, Ringden O, Markling L. TNF alpha levels are increased during bone marrow transplantation conditioning in patients who develop acute GVHD. Bone Marrow Transplant. 1995;15:99–104. Holler E, Kolb HJ, Moller A, et al. Increased serum levels of tumor necrosis factor alpha precede major complications of bone marrow transplantation. Blood. 1990;75:1011–1016. Holler E, Kolb HJ, Hintermeier-Knabe R, et al. The role of tumor necrosis factor alpha in acute graft-versus-host disease and complications following allogeneic bone marrow transplantation. Transplant Proc. 1993;25:1234–1236.

162

P. Ewing et al./ Experimental Hematology 35 (2007) 155–163

24. Brockhaus M, Schoenfeld HJ, Schlaeger EJ, Hunziker W, Lesslauer W, Loetscher H. Identification of two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies. Proc Natl Acad Sci U S A. 1990;87:3127–3131. 25. Loetscher H, Pan YC, Lahm HW, et al. Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor. Cell. 1990;61: 351–359. 26. Dembic Z, Loetscher H, Gubler U, et al. Two human TNF receptors have similar extracellular, but distinct intracellular, domain sequences. Cytokine. 1990;2:231–237. 27. Lewis M, Tartaglia LA, Lee A, et al. Cloning and expression of cDNAs for two distinct tumor necrosis factor receptors demonstrate on receptor is species-specific. Proc Natl Acad Sci U S A. 1991;88: 2830–2834. 28. Schall TJ, Lewis M, Koller KJ, et al. Molecular cloning and expression of a receptor for human tumor necrosis factor. Cell. 1990;61: 361–370. 29. Antin JH, Weisdorf D, Neuberg D, et al. Interleukin-1 blockade does not prevent acute graft-versus-host disease: results of a randomized, double-blind, placebo-controlled trial of interleukin-1 receptor antagonist in allogeneic bone marrow transplantation. Blood. 2002;100: 3479–3482. 30. Pfeffer K, Matsuyama T, Kundig TM, et al. Mice deficient for the 55kD tumor necrosis factor receptor are resistant to endotoxin shock, yet succumb to L. monocytogenes infection. Cell. 1993;73:457–467. 31. Pasparakis M, Alexopoulou L, Episkopou V, Kollias G. Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J Exp Med. 1996;184: 1397–1411. 32. Hildebrandt GC, Olkiewicz KM, Corrion LA, et al. Donor-derived TNF-alpha regulates pulmonary chemokine expression and the development of idiopathic pneumonia syndrome after allogeneic bone marrow transplantation. Blood. 2004;104:586–593. 33. Cooke K, Gerbitz A, Hill G, et al. LPS antagonism reduces graft-versus-host disease and preserves graft-versus-leukemia activity after experimental bone marrow transplantation. J Clin Invest. 2001;107: 1581–1589. 34. Cooke KR, Kobzik L, Martin TR, et al. An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation. I. The roles of minor H antigens and endotoxin. Blood. 1996;88:3230– 3239. 35. Grell M, Wajant H, Zimmermann G, Scheurich P. The type 1 receptor (CD120a) is the high-affinity receptor for soluble tumor necrosis factor. Proc Natl Acad Sci U S A. 1998;95:570–575. 36. Tartaglia LA, Ayres TM, Wong GH, Goeddel DV. A novel domain within the 55 kd TNF receptor signals cell death. Cell. 1993;74: 845–853. 37. Tartaglia LA, Rothe M, Hu YF, Goeddel DV. Tumor necrosis factor’s cytotoxic activity is signaled by the p55 TNF receptor. Cell. 1993;73: 213–216. 38. Neumann B, Machleidt T, Lifka A, et al. Crucial role of 55 kilodalton TNF receptor in TNFa induced adhesion molecule expression and leukocyte organ infiltration. J Immunol. 1996;156:1587–1593. 39. Peschon JJ, Torrance DS, Stocking KL, et al. TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation. J Immunol. 1998;160:943–952. 40. Tartaglia LA, Goeddel DV. Two TNF receptors. Immunol Today. 1992;13:151–153. 41. Reddy P. Pathophysiology of acute graft-versus-host disease. Hematol Oncol. 2003;21:149–161. 42. Laster SM, Wood JG, Gooding LR. Tumor necrosis factor can induce both apoptic and necrotic forms of cell lysis. J Immunol. 1988;141: 2629–2634.

43. Holler E, Kolb HJ, Mittermueller J, et al. Modulation of acute graftversus-host disease after allogeneic bone marrow transplantation by tumor necrosis factor a (TNFa) release in the course of pretransplant conditioning: role of conditioning regimens and prophylactic application of a monoclonal antibody neutralizing human TNFa (MAK 195F). Blood. 1995;86:890–899. 44. Perreault C, Pelletier M, Belanger R, et al. Persistence of host Langerhans cells following allogeneic bond marrow transplantation: Possible relationship with acute graft-versus-host disease. Br J Haematol. 1985;60:253–260. 45. Nakata K, Gotoh H, Watanabe J, et al. Augmented proliferation of human alveolar macrophages after allogeneic bone marrow transplantation. Blood. 1999;93:667–673. 46. Hildebrandt GC, Duffner UA, Olkiewicz KM, et al. A critical role for CCR2/MCP-1 interactions in the development of idiopathic pneumonia syndrome after allogeneic bone marrow transplantation. Blood. 2004;103:2417–2426. 47. Matute-Bello G, Lee JS, Frevert CW, et al. Optimal timing to repopulation of resident alveolar macrophages with donor cells following total body irradiation and bone marrow transplantation in mice. J Immunol Methods. 2004;292:25–34. 48. Welniak LA, Kuprash DV, Tumanov AV, et al. Peyer patches are not required for acute graft-versus-host disease after myeloablative conditioning and murine allogeneic bone marrow transplantation. Blood. 2006;107:410–412. 49. Gifford GE, Lohmann-Matthes M-L. Gamma interferon priming of mouse and human macrophages for induction of tumor necrosis factor production by bacterial lipopolysaccharide. J Natl Cancer Inst. 1987; 78:121–124. 50. Rothe J, Lesslauer W, Lotscher H, et al. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature. 1993;364:798–802. 51. Rothe J, Mackay F, Bluethmann H, Zinkernagel R, Lesslauer W. Phenotypic analysis of TNFR1-deficient mice and characterization of TNFR1-deficient fibroblasts in vitro. Circ Shock. 1994;44: 51–56. 52. Sheehan KC, Pinckard JK, Arthur CD, Dehner LP, Goeddel DV, Schreiber RD. Monoclonal antibodies specific for murine p55 and p75 tumor necrosis factor receptors: identification of a novel in vivo role for p75. J Exp Med. 1995;181:607–617. 53. Brown GR, Thiele DL. Enhancement of MHC class I-stimulated alloresponses by TNF/TNF receptor (TNFR)1 interactions and of MHC class II-stimulated alloresponses by TNF/TNFR2 interactions. Eur J Immunol. 2000;30:2900–2907. 54. Ferrara JL. Cytokine dysregulation as a mechanism of graft-versushost disease. Curr Opin Immunol. 1993;5:794–799. 55. Antin JH, Ferrara JLM. Cytokine dysregulation and acute graft-versushost disease. Blood. 1992;80:2964–2968. 56. Ferrara JLM, Abhyankar S, Gilliland DG. Cytokine storm of graftversus-host disease: a critical effector role for interleukin-1. Transplant Proc. 1993;25:1216–1217. 57. Via CS, Nguyen P, Shustov A, Drappa J, Elkon KB. A major role for the Fas pathway in acute graft-versus-host disease. J Immunol. 1996; 157:5387–5393. 58. Teshima T, Hill GR, Pan L, et al. IL-11 separates graft-versusleukemia effects from graft-versus-host disease after bone marrow transplantation. J Clin Invest. 1999;104:317–325. 59. Min CK, Maeda Y, Lowler K, et al. Paradoxical effects of interleukin18 on the severity of acute graft-versus-host disease mediated by CD4þ and CD8þ T-cell subsets after experimental allogeneic bone marrow transplantation. Blood. 2004;104:3393–3399. 60. Monastra G, Cabrelle A, Zambon A, et al. Membrane form of TNF alpha induces both cell lysis and apoptosis in susceptible target cells. Cell Immunol. 1996;171:102–110.

P. Ewing et al./ Experimental Hematology 35 (2007) 155–163 61. Kinkhabwala M, Sehajpal P, Skolnik E, et al. A novel addition to the T cell repertory. Cell surface expression of tumor necrosis factor/ cachectin by activated normal human T cells. J Exp Med. 1990;171: 941–946. 62. Zhao MQ, Stoler MH, Liu AN, et al. Alveolar epithelial cell chemokine expression triggered by antigen-specific cytolytic CD8(þ) T cell recognition. J Clin Invest. 2000;106:R49–R58.

163

63. Cooke KR, Hill GR, Gerbitz A, et al. Hyporesponsiveness of donor cells to lipopolysaccharide stimulation reduces the severity of experimental idiopathic pneumonia syndrome: potential role for a gut-lung axis of inflammation. J Immunol. 2000;165:6612–6619. 64. Uberti JP, Ayash L, Ratanatharathorn V, et al. Pilot trial on the use of etanercept and methylprednisolone as primary treatment for acute graftversus-host disease. Biol Blood Marrow Transplant. 2005;11:680–687.