Perforin/granzyme-dependent and independent mechanisms are both important for the development of graft-versus-host disease after murine bone marrow transplantation. T A Graubert, … , J H Russell, T J Ley J Clin Invest. 1997;100(4):904-911. https://doi.org/10.1172/JCI119606. Research Article Graft-versus-host disease (GvHD) is the major limiting toxicity of allogeneic bone marrow transplantation. T cells are important mediators of GvHD, but the molecular mechanisms that they use to induce GvHD are controversial. Three effector pathways have been described for cytotoxic T lymphocytes: one requires perforin and granzymes, the second Fas (APO-1; CD95) and its ligand. Thirdly, secreted molecules (e.g., TNF-alpha, gamma-IFN) can also mediate cytotoxicity. Together, these mechanisms appear to account for virtually all cytotoxicity induced by activated CTL in standard in vitro lytic assays. Using transplants across histocompatibility barriers, we were able to analyze the contributions of these effector molecules to cell-mediated cytotoxicity in vivo in a GvHD model. We found that Fas ligand is an important independent mediator of class II-restricted acute murine GvHD, while perforin/granzyme-dependent mechanisms have only a minor role in that compartment. In contrast, perforin/ granzyme-dependent mechanisms are required for class I-restricted acute murine GvHD, while Fas ligand is not. The perforin/granzyme pathway may therefore represent a novel target for anti-GvHD drug design. In support of this approach, we provide additional data suggesting that specific perforin/granzyme inhibitors should not adversely affect hematopoietic recovery after transplantation. Find the latest version: https://jci.me/119606/pdf Perforin/granzyme-dependent and Independent Mechanisms are Both Important for the Development of Graft-versus-host Disease After Murine Bone Marrow Transplantation Timothy A. Graubert,* John F. DiPersio,* John H. Russell,‡ and Timothy J. Ley* *Division of Bone Marrow Transplantation and Stem Cell Biology, Department of Internal Medicine and Department of Genetics; and ‡Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, Missouri 63110-1093 Abstract dence that T cells mediate GvHD (1). T cell depletion, how- ever, is associated with an increased risk of graft failure (2) and Graft-versus-host disease (GvHD) is the major limiting tox- relapse of malignancy after transplantation (3). Efforts to ex- icity of allogeneic bone marrow transplantation. T cells are plain the molecular basis of T cell cytotoxicity are motivated important mediators of GvHD, but the molecular mecha- by a desire to develop more precise strategies for manipulating nisms that they use to induce GvHD are controversial. Three donor T cells, while preserving a favorable balance between effector pathways have been described for cytotoxic T lym- their beneficial and harmful effects in the host. phocytes: one requires perforin and granzymes, the second Murine bone marrow transplant models developed in the Fas (APO-1; CD95) and its ligand. Thirdly, secreted mole- past decade have demonstrated that CD4ϩ and CD8ϩ cyto- cules (e.g., TNF-␣, ␥-IFN) can also mediate cytotoxicity. To- toxic T lymphocytes (CTL) can both mediate lethal GvHD (4, gether, these mechanisms appear to account for virtually all 5). These effector populations induce GvHD syndromes with cytotoxicity induced by activated CTL in standard in vitro similar natural histories and dose-response relationships. In lytic assays. Using transplants across histocompatibility bar- vitro experiments, however, suggest that two distinct cytotoxic riers, we were able to analyze the contributions of these ef- mechanisms exist and may be used differently in CD4ϩ and fector molecules to cell-mediated cytotoxicity in vivo in a CD8ϩ CTL. Both pathways culminate in the apoptotic death GvHD model. We found that Fas ligand is an important in- of target cells. dependent mediator of class II–restricted acute murine The first apoptotic mechanism is the granule exocytosis GvHD, while perforin/granzyme-dependent mechanisms have pathway. In this model, the specific recognition and binding of a only a minor role in that compartment. In contrast, perforin/ CTL to a susceptible target cell leads to discharge of the lympho- granzyme-dependent mechanisms are required for class cyte’s secretory granules into the extracellular space. One of I–restricted acute murine GvHD, while Fas ligand is not. the granule components is perforin, which undergoes Caϩϩ- The perforin/granzyme pathway may therefore represent a dependent polymerization on the target cell membrane, creat- novel target for anti-GvHD drug design. In support of this ing a complement-like lesion (6, 7). This membrane damage approach, we provide additional data suggesting that spe- is not sufficient to cause apoptotic death of the cell (8), but cific perforin/granzyme inhibitors should not adversely af- may allow other granule constituents (including the granzymes) fect hematopoietic recovery after transplantation. (J. Clin. to gain access to the target cell cytoplasm. There, the gran- Invest. 1997. 100:904–911.) Key words: graft-versus-host zymes trigger a series of biochemical events that result in apop- disease • hematopoiesis • perforin • granzyme B • Fas tosis (9–13). CTL from mice deficient in perforin have a se- vere defect in their ability to induce apoptosis (14–17). Introduction Granzyme B is required for the early phase of perforin-depen- dent cytotoxicity (18). Bone marrow transplantation across major or minor histocom- The second pathway involves the Fas/Fas ligand interac- patibility barriers carries a significant risk of death from graft- tion. Activated CTL upregulate expression of Fas ligand versus-host disease (GvHD).1 The elimination of GvHD by (FasL), a membrane-bound protein with homology to TNF depleting bone marrow grafts of T cells provided strong evi- (19). Binding of FasL to its receptor (APO-1; CD95), present on the surface of target cells, triggers apoptosis through a Address correspondence to Timothy J. Ley, M.D., Washington Uni- death domain on the cytoplasmic tail of the Fas receptor via a versity Medical School, Division of Bone Marrow Transplantation series of docking proteins. These proteins transmit the death and Stem Cell Biology, Campus Box 8007, 660 South Euclid Avenue, signal through intermediates that include cysteine proteinases St. Louis, MO 63110-1093. Phone: 314-362-8831; FAX: 314-362-9333; of the caspase family (20–24). Mice with a naturally occurring E-mail: [email protected] loss of function mutation of FasL (gld/gld) accumulate double Received for publication 24 February 1997 and accepted in revised negative (CD4ϪCD8Ϫ) T cells in the periphery (25) due to a form 21 May 1997. failure of activation-induced cell death of mature T cells (26, 27). Reduced cytotoxicity of gld/gld effectors has demon- 1. Abbreviations used in this paper: BFU-E, burst-forming unit eryth- strated the importance of a Fas/FasL-dependent mechanism of roid; CFU-GM, colony-forming unit granulocyte macrophage; CTL, cytotoxicity in vitro (28), particularly in CD4ϩ CTL (28–30). cytotoxic T lymphocytes; CFU-S8, day 8 spleen colony-forming unit; FasL, Fas ligand; GvHD, graft-versus-host disease. Allogeneic transplants from mice doubly deficient in per- forin and FasL (31), but not FasL alone, have been shown to J. Clin. Invest. cause less mortality from GvHD. Similarly, the onset of lethal © The American Society for Clinical Investigation, Inc. GvHD is delayed using perforin-deficient effectors, while he- 0021-9738/97/08/0904/08 $2.00 patic and cutaneous pathology is absent after transfer of FasL- Volume 100, Number 4, August 1997, 904–911 deficient effectors in transplants across an MHC-matched allo- http://www.jci.org geneic barrier (32). These studies, however, did not define the 904 Graubert et al. T cell compartments affected by the mutations. We previously (Sigma Chemical Co., St. Louis, MO) and 50 U/ml rhIL-2 for 48 h) reported that granzyme B is an important mediator of cytotox- were used as a source of positive control RNA; T cells acquire a CTL icity in vivo mediated by CD8ϩ, but not CD4ϩ CTL (33). This phenotype under these culture conditions, as characterized by signifi- study was conducted to define further the role of perforin/ cant upregulation of granzymes A and B (40) and perforin (41) ex- granzyme-dependent and independent mechanisms in the de- pression. Negative control samples (no RNA) were processed in par- allel. Specific end-labeled DNA probes (100,000 total cpm) were used velopment of GvHD across class I and class II barriers. We to detect correctly processed human ␤-actin and granzyme B mRNAs found that a perforin/granzyme-independent mechanism (i.e., as previously described (39). The perforin probe was generated by the Fas system) is an important mediator of GvHD across a PCR from genomic K562 (human erythroleukemia cell) DNA. The class II barrier in this model. The perforin/granzyme pathway fragment was end-labeled at a BamH1 site; correctly processed per- does not contribute significantly to class II-restricted GvHD, forin mRNA protects a 413-nucleotide exon 2 probe fragment from but it is essential for class I-restricted GvHD. This observation S1 nuclease digestion. suggests to us that inhibition of the perforin/granzyme path- Acute GvHD model. Lymphocytes were isolated from the me- way may represent a novel target for GvHD therapy. senteric lymph nodes of sex-matched littermate donors after passage Until recently, the expression of perforin and granzyme B through a stainless steel mesh. T cell subsets were then prepared as had not been detected outside the lymphoid compartment (34, previously described (33). In brief, antibody-mediated complement lysis was performed by adsorbing antibodies to B cells (MoAb J11d) 35). Berthou, et al. (36), however, found evidence of perforin and CD4ϩ cells (MoAb RL.172) or CD8ϩ cells (MoAb 3.155) at 4ЊC and granzyme B expression in KG1a (human acute myeloge- for 30 min at a concentration of 107 lymphocytes/ml in RPMI 1640 ϩ nous leukemia) cells and in CD34 peripheral blood cells from (Sigma Chemical Co., St.