Pathogenic mechanisms of Acute Graft versus Host Disease

Patogenia da Doença do Enxerto Contra o Hospedeiro Aguda

Artigo Especial / Special Article

Pathogenic mechanisms of Acute Graft versus Host Disease

James L.M. Ferrara

Graft-versus-host-disease (GVHD) is the major complication of allogeneic Bone Marrow Transplant (BMT). Older BMT recipients are a greater risk for acute GVHD after allogeneic BMT, but the causes of this association are poorly understood. Using well-characterized murine BMT models we have explored the mechanisms of increased GVHD in older mice. GVHD mortality and morbidity, and pathologic and biochemical indices were all worse in old recipients. Donor T cell responses were significantly increased in old recipients both in vivo and in vitro when stimulated by antigen-presenting cells (APCs) from old mice. In a haploidential GVHD model, CD4⁺ donor T cells mediated more severe GVHD in old mice. We confirmed the role of aged APCs in GVHD using bone marrow chimera recipient created with either old or young bone marrow. APCs from these mice also stimulated greater responses from allogeneic cells in vitro. In a separate set of experiments we evaluated whether alloantigen expression on host target epithelium is essential for tissue damage induced by GVHD. Using bone marrow chimeras recipients in which either MHC II or MHC I alloantigen was expressed only on APCs, we found that acute GVHD does not require alloantigen expression on host target epithelium and that neutralization of tumor necrosis factor-alpha and interleukin-1 prevents acute GVHD. These results pertain to CD4-mediated GVHD and to a lesser extent in CD8-mediated GVHD, and confirm the central role of most APCs as well as inflammatory cytokines.

Keywords: Bone marrow transplant, cytokine, T lymphocyte, gastrointestinal tract, antigen presenting cells

Introduction

Graft Versus Host Disease (GVHD) is a life threatening complication of allogeneic bone marrow transplantation (BMT), an important therapy for a number of hematologic diseases. In its acute phase, GVHD involves activation of proinflammatory cytokine cascades and immune effector cells that result in target tissue damage (1, 2). Multiple cellular populations and cytokines interact in a complex process that ultimately results in apoptotic injury in target organs (skin, gut, liver) and systemic disease. Shlomchik and colleagues have shown that host-derived antigen presenting cells (APCs) play a key role in the initiation of acute GVHD (3). Advanced age of the BMT recipient is an important determinant of GVHD severity (4), and the increased risk of GVHD is one factor that often excludes older patients from consideration for allogeneic BMT.

We evaluated the effect of recipient age in well-established murine models of GVHD elicited by both major and minor histocompatibility antigen differences between donors and recipients. Young (2 months), old (14 - 16 months) and very old (22 months) recipient mice were irradiated with 11 Gy TBI and transplanted with bone marrow (BM) and T cells from semiallogeneic recipients as published (5). We found that recipient age was a continuous variable with respect to GVHD severity. Severe GVHD developed in very old recipients with 100% mortality by day 11. In old mice mortality was slightly but significantly delayed, and it was further delayed in young recipients of allogeneic BMT, 50% of which were alive on day 50. Similar results were observed in two other donor/recipient strain combinations.

Increased GVHD was also observed in old compared to young recipients after a variety of conditioning regimens, including cyclophosphamide alone. Recipient mice were treated with 100 mg/kg Cytoxan at day ¾2 and ¾1 and transplanted with 5 x 10⁶ BM cells and 20 x 10⁶ semiallogeneic spleen cells. Day 40 survival in 16 month old BMT recipients was significantly decreased compared to young mice (0% vs. 83%, P = 0.01). Thus, increased GVHD mortality and morbidity in aged recipients was neither strain nor conditioning dependent.

Accelerated GVHD in old mice was confirmed by histopathologic analysis of the small and large intestine in the haploidential BMT model. Acute GVHD histologic features (diffuse apoptosis, lymphocytic infiltrate, brush border loss, mucosal sloughing into the lumen, epithelial degeneration and crypt regeneration) were summed to produce a semiquantitative pathology index as previously described (6). Damage to the gastrointestinal (GI) tract was twice as severe in older recipients. Serum levels of LPS and TNFa were also significantly increased in older recipients, consistent with the pathologic data (5). Thus by all clinical, pathologic and biochemical indices, acute GVHD was worse in old recipients.

We next analyzed donor T cell expansion in the spleens of BMT recipients four days after transplant. Using donors cells that could be identified by FACs, we observed significantly increased number of donor T cells (CD4⁺, CD8⁺, CD45.1⁺) that correlated with advancing recipient age, mainly due to the increase of donor CD4⁺ T cells. Flow cytometric analysis of intracellular IFN-g, an important cytokine mediator of acute GVHD, showed a twofold increase in the number of IFN-g expressing CD4⁺ donor T cells in the spleens of old recipients (5).

To evaluate the role of donor CD4⁺ T cells with respect to the severity of GVHD in old recipients, we depleted either CD4⁺ or CD8⁺ T cells from donor semiallogeneic splenic T cells with mAb coupled to magnetic beads. Depletion of CD4⁺ T cells from the donor cell inoculum effectively eliminated GVHD mortality and both young and old recipients of CD8⁺ cells and they survived the entire observation period (5). By contrast, old recipients of CD4⁺ T cells experienced significantly greater mortality than young recipients. CD4⁺ donor T cells are therefore the principal effectors in acute GVHD severity in this haploidential mouse model and are responsible for the increased GVHD in old recipients. We also compared the ability of APCs from freshly isolated recipient splenocytes to stimulate naïve semiallogeneic donor responders in vitro. Donor T cells proliferated more rapidly and produced greater amounts of IFN-g and IL-2 to stimulator cells from old animals in two responder/stimulator combinations. We also compared the allostimulatory capacity of BM-derived DCs from young versus old mice and found that DCs from old mice induced greater proliferation of allogeneic T cells compared to DCs from young mice. TNF-a and IL-12, known to play important roles in augmenting alloreacitve T cell responses, were also produced in significantly greater amounts by old DCs after LPS stimulation.

To investigate whether aged APCs were sufficient to induce more severe GVHD, we generated bone marrow chimeras in which cells of hematopoietic lineages were derived from old mice and non-hematopoietic cells (including all epithelial GVHD targets) were from young mice (5). We hypothesized that if increased age of APCs were the primary cause of increased GVHD severity, then [old ® young] chimeras with old APCs should develop more intense disease than [young ® young] chimeras despite the identical age of the GVHD target tissues. Our results confirmed this hypothesis, and GVHD was significantly more severe after allogeneic BMT in [old ® young] chimeras than in [young ® young] chimeras. Analysis of CFSE labeled donor T cells in vivo at day 3 post BMT confirmed greater CD4⁺ donor T cell expansion in response to old APCs in vivo. These data indicate that old APCs are sufficient to induce an increased response from allogeneic donor T cells and cause more severe GVHD irrespective of the age of the recipient target organs.

These studies demonstrate the central importance of APCs in the pathogenesis of acute GVHD. Previously, the primary hypotheses regarding increased GVHD in elderly recipients involved the altered function of the immune system of old recipients, increased load of bacterial and viral antigens with age, and reduced repair capacity of aged tissues. The process of thymic involution (decreased thymic size and function) that occurs with increasing age has long been thought to be related to increased GVHD in the elderly (11). Decreased thymic function in aged recipients might amplify acute GVHD by reducing host resistance to donor T cell expansion. As a result of thymic involution, the export of naïve T cells from the thymus decreases and consequently shifts the ratio of naïve/memory T cells, which is associated with a decline in proliferation and IL-2 production after mitogen stimulation (12-14). This decline in T cell function with age could be considered a type of acquired immunodeficiency that makes older patients more susceptible to a GVH reaction. GVHD itself also damages the thymus, resulting in a loss of negative selection and emergence of host reactive clones (15-17).

A second possibility for more severe GVHD in aged recipients is the increase in viral or bacterial antigenic exposure with age. Clinical acute GVHD is associated with viral infections, particularly CMV, and viral antigens are well known targets for T cell responses (18-21). This phenomenon is an unlikely explanation for our observations because all recipients have been housed in specific pathogen free conditions their entire lives and all animals tested were negative for the entire panel of infectious pathogens.

A second issue related to exposure of infectious agents relates to bacterial gut flora. Bacterial lipopolysaccharide (LPS) crosses a damaged gut mucosa amplifying systemic GVHD in animal models, and blockade of LPS or selective bowel decontamination can reduce clinical GVHD and improve survival (22-25). Some clinical studies suggest that bacterial overgrowth of the small bowel increases with age although these studies are small and may suffer from selection bias (26, 27). Even if such bacterial loading holds possible clinical relevance, the BM chimeras experiments rule out this mechanism as an explanation for more severe GVHD in this experimental model.

An issue related to the titer of viral and/or bacterial antigens is the decreased ability of aged tissue to repair. Several studies show increased cardiotoxicity, neurotoxicity and mucositis after chemotherapy in the elderly (28). The molecular mechanisms for such decreased repair may involve telomere shortening which correlates with the remaining life span of somatic cells (29). The reduced ability of aged GI mucosa to repair itself may have a similar effect as bacterial overgrowth in the GI tract ¾ more LPS and inflammatory mediators can translocate to the systemic circulation in aged mice because repair is slower, thus amplifying cytokine dysregulation and the severity of GVHD. Again, the differences observed between young BM chimeras and old BM chimeras effectively rule out this explanation. All epithelial target tissues remain of the same age in the chimera recipients, whereas only the hematopoietic system, including APCs, differ in age.

In our next series of experiments, we evaluated the requirement for MHC alloantigen to be present on host target tissues during GVHD. We first tested the requirement for the expression of MHC class I antigens to stimulate allogeneic CD4⁺ T cells in vivo. Donor mice received 13 Gy total body irradiation (TBI) and were transplanted with allogeneic BM and T cells from donor bm12 mice that differ at a single MHC class II allele (7). When II ^-/- or normal recipient mice were given 13 Gy TBI and then transplanted with BM and T cells from bm12 donor mice, normal recipients experienced 100% mortality from GVHD by day 7, whereas 0% mortality was observed in II^-/- recipients (8). To test the requirement for the expression of MHC class II on host APCs in GVHD we generated BM chimeras expressing MHC class II on BM-derived APCs but lacking MHC class II on target cells by reconstituting lethally irradiated II^-/- mice with B6 BM cells ( [B6®II^-/-] chimeras). Four months later, flow cytometric analysis of splenic DCs isolated from these animals showed complete replacement of splenic DCs by donor BM cells with MHC class II expression on >95% of CD11c⁺ DCs. Identically treated [B6 ® B6] chimeras were created as controls. [B6 ® II^-/-] and [B6 ® B6] chimeras were transplanted with BM and T cells from bm 12 donors after 13 Gy TBI. As expected, GVHD was very severe in [B6 ® B6] recipients of bm12 T donors: all recipients died at day 5 after BMT in contrast to 90% survival of recipients of syngeneic B6 donors. GVHD was equally severe in [B6 ® II^-/-] recipients of bm12 T cells as in [B6 ® wt] recipients and all died by day 7. GVHD pathology scores of the liver and small intestine in allogeneic [B6 ® wt] and [B6 ® II^-/-] recipients on day 5 post-BMT were significantly greater than those in corresponding syngeneic recipients. Liver histology showed standard histological features of acute GVHD included mild mononuclear cells infiltrates in bile ducts and portal triads, acidophilic bodies, and endothelialitis, which is characteristic of severe GVHD (9). These changes were, if anything, more prominent in [B6 ® II^-/-] recipients that lacked MHC class II expression on target cells. Small intestine histopathology also showed significant changes of GVHD in [B6 ® II^-/-] recipients of bm12 T cells, including villous atrophy with epithelial apoptosis. Mice receiving 13 Gy TBI and no BM survived more than 10 days (data not shown), ruling out graft failure as a cause of this early mortality as described (10).

Alloantigen expression on host APCs is essential (3) for the activation phase of acute GVHD. The effector phase of GVHD is also believed to be antigen specific and to require alloantigen expression on target cells. The results of our second set of experiments challenge long held assumptions about the antigen specificity of GVHD effector mechanisms: we demonstrate for the first time that the damage to GVHD target organs does not require alloantigen expression on epithelial and nonhematopoietically derived target cells. The lack of requirement for alloantigen expression pertains to both CD4-dependent and CD8-dependent GVHD, but is particularly true of CD4-dependent GVHD damage that was mediated primarily by cytokines. This antigen-independent effector mechanism may help to explain how CD4⁺ T cells damage host target epithelium that normally do not express MHC class II molecules. Current paradigms postulate that MHC class II aberrantly expressed in target organs during GVHD renders them vulnerable to attack by CD4⁺ effector cells (30-32). Our results suggest that aberrant MHC class II expression is the result of tissue inflammation, probably from systemic IFN-g production.

Effector mechanisms of acute GVHD are thought to include both CTL-mediated and cytokine-mediated cytotoxicity. A second cytolytic pathway during GVHD is an inflammatory cascade generated when mononuclear cells are stimulated to secrete TNF-a and other cytokines by LPS which enters into systemic circulation from damaged gut mucosa (22). Thus host APCs alone during GVHD can activate the inflammatory cytokine cascade, and mortality and morbidity of GVHD was dramatically suppressed when both TNF-a and IL-1 were neutralized. This reduction of GVHD was due to the suppression of the effector phase rather than the afferent phase of GVHD because cytokine blockade did not suppress donor T cell expansion early after BMT as previously described (33).

In the effector phase of GVHD, a cognate interaction between the antigen-specific T cell receptor (TCR) on CTLs and peptide antigen presented on the MHC molecule of target cells is required for perforin/granzyme-mediated cytotoxicity, but is less significant for Fas-mediated cytotoxicity (34). CD8⁺ CTLs kill their targets using perforin/granzyme, Fas/FasL, and inflammatory cytokine pathways. Surprisingly, acute GVHD mediated by CD8⁺ T cells did not require alloantigen expression on target epithelium, although acute GVHD mortality was more rapid when epithelial targets expressed the alloantigen. Neutralization of both TNF-a and IL-1 completely prevented GVHD target organ injury in chimeric recipients lacking alloantigens on target cells. These results demonstrate that CD8-dependent GVHD is mediated by both antigen non-specific cytokines and antigen specific cellular effectors and they confirm the delayed mortality observed in CD8-dependent GVHD models using perforin or granzyme deficient donors (35-37).

In summary, our results demonstrate that for acute GVHD: [1] host APCs are sufficient in both activation and effector phases; [2] alloantigen expression on host target cells is not required, and therefore the effector phase is not antigen specific; [3] inflammatory cytokines mediate mortality and target destruction. These conclusions are true particularly for CD4-mediated acute GVHD but also apply, at least in part, for CD8-mediated disease. Our results have important clinical implications because donor CTLs are important effectors of beneficial graft-versus-leukemia effects (38) in patients receiving BMT for hematologic malignancies, and elective blockade of inflammatory cytokines may thus be a strategy to preserve graft-versus-leukemia while reducing toxicity of GVHD.

Patogenia da Doença do Enxerto Contra o Hospedeiro Aguda

Resumo

A Doença do Enxerto Contra o Hospedeiro (DECH) é considerada a complicação mais importante no transplante de medula óssea alogênico. Pacientes idosos apresentam maior risco para DECH apesar de se desconhecer as causas desta ocorrência. Utilizando modelos murinos de TMO, investigamos os prováveis mecanismos envolvidos na DECH em camundongos com idade mais avançada. As taxas de mortalidade e morbidade na DECH e os índices patológicos e bioquímicos foram todos piores nos camundongos receptores mais idosos. As respostas das células T dos doadores foram significativamente elevadas nos receptores idosos tanto in vitro com in vivo quando estimuladas por células apresentadoras de antígenos (APCs) derivadas de camundongos idosos Em um modelo de DECH haploidêntico, as células T CD4+ do doador mediaram a DECH mais grave em camundongos idosos. Nós confirmamos o papel das APCs envelhecidas na DECH utilizando receptores com quimeras de medulares originárias de medula derivadas de camundongos jovens e idosos. As APCs destes camundongos também estimularam maiores respostas de células T alogênicas in vitro. Em diferentes experimentos, avaliamos se a expressão dos aloantígenos do epitélio do hospedeiro é importante para o dano tecidual induzido pela DECH. Usando receptores de quimeras de medula óssea nos quais os alonatígenos MHC I ou MHC II foram expressos somente nas APCs, nós verificamos que a DECH aguda não requer expressão de aloantígenos no epitélio alvo do hospedeiro e que neutralização do fator de necrose tumoral alfa e da interleucina-1 previne a DECH aguda. Estes resultados são pertinentes com a DECH aguda mediada pelas células T CD4+ e em menor grau com a DECH mediada pelas células T CD8+ e confirma o papel central das APCs bem como das citocinas inflamatórias na DECH.

Palavras-chave: Transplante de medula óssea, citocinas, linfócito T, trato gastrointestinal, células apresentadoras de antígenos

5. Ordemann R. Enhanced Allostimulatory Actvity of Host Antigen-Presenting Cells in Old Mice Intesnifies Acute Graft-versus-Host Disease. JCI (2002).

Recebido: 10/06/2002

Aceito: 15/06/2002

Blood and Marrow Transplant Program, Division of Hematology/Oncology, Departments of Internal Medicine and Pediatrics, University of Michigan Cancer Center

Correspondence to: James L.M. Ferrara

University of Michigan Cancer Center

1500 E. Medical Center Drive. Ann Arbor, MI 48109-0942

Phone: [phone]. E-mail: [e-mail]

• 1. Antin JH, Ferrara JLM. Cytokine dysregulation and acute graft-versus-host disease Blood 1992: 80; 2964-2968.
• 2. Ferrara JL. Pathogenesis of acute graft-versus-host disease: cytokines and cellular effectors J Hematother Stem Cell Res 2000: 9; 299-306.
• 3. Shlomchik WD et al. Prevention of graft versus host disease by inactivation of host antigen-presenting cells Science 1999: 285; 412-5.
• 4. Sullivan K, Storb R, Buckner D, Fefer A. Graft-versus-Host Disease as Adoptive Immunotherapy in Patients with Advanced Hematologic Neoplasms N. Engl. J. Med. 1989: 320; 828-834.
• 6. 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; 2754-9.
• 7. Korngold R, Sprent J. T cell subsets in graft-vs.-host disease. in Graft-vs.-Host Disease: Immunology, Pathophysiology, and Treatment (eds. Burakoff SJ, Deeg HJ, Ferrara J, Atkinson K) 31-50 (Marcel Dekker, New York, 1990).
• 8. Teshima T. Acute Graft-versus-Host Disease does not Require Alloantigen Expression on Host Epithelium. Nature Medine (2002).
• 9. Crawford JM. Graft-versus-host disease of the liver. in Graft-versus Host Disease (eds. Ferrara JLM, Deeg HJ, Burakoff SJ) 315-336 (Marcel Dekker, New York, 1997).
• 10. Paris F. et al. Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice Science 2001: 293; 293-7.
• 11. Storb R, Thomas ED. Graft-versus-host disease in dog and man: the Seattle experience Immunol Rev 1985: 88; 215-38.
• 12. Roberts-Thomson IC, Whittingham S, Youngchaiyud U, Mackay IR. Ageing, immune response, and mortality. Lancet 1974: 2; 368-70.
• 13. Lerner A, Yamada T, Miller RA. Pgp-1hi T lymphocytes accumulate with age in mice and respond poorly to concanavalin A Eur J Immunol 1989: 19; 977-82.
• 14. Utsuyama M et al. Differential age-change in the numbers of CD4+CD45RA+ and CD4+CD29+ T cell subsets in human peripheral blood. Mech Ageing Dev 1992: 63; 57-68.
• 15. Van den Brink MR, Moore E, Ferrara JL, Burakoff SJ. Graft-versus-host-disease-associated thymic damage results in the appearance of T cell clones with anti-host reactivity Transplantation 2000: 69, 446-9.
• 16. Holländer GA, Widmer B, Burakoff SJ. Loss of normal thymic repertoire selection and persistence of autoreactive T cells in graft vs. host disease J. Immunol. 1994: 152; 1609-1617.
• 17. Fukushi N et al. Thymus: a direct target tissue in graft-versus-host reaction after allogeneic bone marrow tranpslantation that results in abrogation of induction of self-tolerance Proc. Natl. Acad. Sci. USA 1990: 87; 6301-6305.
• 18. Grundy JE, Shanley JD, Shearer GM. Augmentation of graft-versus-host reaction by cytomegalovirus infection resulting in interstitial pneumonitis Transplantation 1985: 39; 548-53.
• 19. Ringden O. Viral infections and graft-vs.-host disease in Graft-vs.-Host Disease (eds. Burakoff SJ, Deeg HJ, Ferrara J, Atkinson K) 467 (Marcel Dekker, New York, 1990).
• 20. Hagglund H et al. Risk factors for acute graft-versus-host disease in 291 consecutive HLA-identical bone marrow transplant recipients Bone Marrow Transplantation 1995: 16; 747-753.
• 21. Riddell SR, Greenberg PD. Principles for adoptive T cell therapy of human viral diseases Annu Rev Immunol 1995: 13; 545-86.
• 22. 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.
• 23. Hill GR et al. Total body irradiation and acute graft versus host disease. The role of gastrointestinal damage and inflammatory cytokines Blood 1997: 90; 3204-3213.
• 24. Cooke KR 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; 1581-9.
• 25. Beelen DW et al. Evidence that sustained growth suppression of intestinal anaerobic bacteria reduces the risk of acute graft-versus-host disease after sibling marrow transplantation Blood 1992: 80; 2668-2676.
• 26. Lewis SJ, Potts LF, Malhotra R, Mountford R. Small bowel bacterial overgrowth in subjects living in residential care homes. Age Ageing 1999: 28; 181-5.
• 27. Lipski PS, Kelly PJ, James OF. Bacterial contamination of the small bowel in elderly people: is it necessarily pathological? Age Ageing 1992: 21; 5-12.
• 28. Balducci L, Extermann M. Cancer chemotherapy in the older patient: what the medical oncologist needs to know Cancer 1997: 80; 1317-22.
• 29. Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990: 345; 458-60.
• 30. Lampert IA, Suitters AJ, Chisholm PM. Expression of Ia antigen on epidermal keratinocytes in graft-versus-host disease Nature 1981: 293; 149-50.
• 31. Mason DW, Dallman M, Barclay AN. Graft-versus-host disease induces expression of Ia antigen in rat epidermal cells and gut epithelium Nature 1981: 293; 150-151.
• 32. Barclay AN, Mason DW. Induction of Ia antigen in rat epidermal cells and gut epithelium by immunological stimuli J Exp Med 1982: 156; 1665-76.
• 33. Hill GR et al. Differential roles of IL-1 and TNF-alpha on graft-versus-host disease and graft versus leukemia. J Clin Invest 1999: 104; 459-67.
• 34. Santamaria P. Effector lymphocytes in autoimmunity. Curr Opin Immunol 2001: 13; 663-9.
• 35. Braun YM, Lowin B, French L, Acha-Orbea H, Tschopp J. Cytotoxic T cells deficient in both functional Fas ligand and perforin show residual cytolytic activity yet lose their capacity to induce lethal acute graft-versus-host disease J Exp Med 1996: 183; 657-661.
• 36. Baker MB, Altman NH, Podack ER, Levy RB. The role of cell-mediated cytotoxicity in acute GVHD after MHC-matched allogeneic bone marrow transplantation in mice J Exp Med 1996: 183; 2645-2656.
• 37. Graubert TA, DiPersio JF, Russell JH, Ley TJ. Perforin/granzyme-dependent and independent mechanisms are both important for the development of graft-versus-host disease after murine bone marrow transplantation. J. Clin. Invest. 1997: 100; 904-911.
• 38. Truitt RL, Johnson BD, McCabe C, Weiler MB. Graft versus leukemia. in Graft-vs-Host Disease (eds. Ferrara JLM, Deeg HJ, Burakoff SJ) 385 (Marcel Dekker, Inc., New York, 1997).

Publication Dates

• Publication in this collection
  14 Jan 2003
• Date of issue
  2002