Syngeneic Graft-versus-host Disease Research Articles

Anti-IL-23p19 therapy inhibits the adoptive transfer of syngeneic graft-versus-host disease

Syngeneic graft-versus-host disease (SGVHD), a chronic inflammatory disease, develops following irradiation, syngeneic bone marrow transplantation (BMT) and treatment with the immunosuppressive agent cyclosporine A (CsA). We have shown that TH1 and TH17 cytokine responses are increased during the development of SGVHD. The current study was designed to further investigate the involvement of TH17 immunity in SGVHD-associated colitis. IL-23 is a TH17 cytokine responsible for maintaining the effector functions of TH17 cells. The administration of anti-mouse IL-23p19 was shown to significantly reduce the clinical symptoms of primary and secondary SGVHD-associated colitis resulting in a significant reduction in both TH1 and TH17 associated cytokine expression. These results demonstrate that the TH17-associated cytokine, IL-23, may prove to be a beneficial therapeutic target in the treatment of chronic colon inflammation.

Role of Oxidative Stress in the Colonic Complications of Murine Syngeneic Graft-versus-host Disease

Syngeneic graft-versus-host disease (SGVHD) develops in mice following lethal irradiation, reconstitution with syngeneic bone marrow (BM) and treatment with a short course of the immunosuppressive agent cyclosporine A (CsA). The development of SGVHD is a complex process resulting from the cooperative interaction of multiple effector cell populations and inflammatory mediators contributing to the pathogenesis of this inducible disease. Using gene ex- pression analysis, flow cytometric analysis and immunohistochemistry, time course studies revealed increased reactive oxygen and nitrogen species in the tissues of CsA-treated animals compared to bone marrow transplantation (BMT) con- trols during the induction of SGVHD (d0-21 post-BMT). Studies were undertaken to determine the effect of CsA-induced oxidative stress on the induction of SGVHD. In vivo treatment with the superoxide dismutase mimetic, manganese (III) meso-tetrakis (4-benzoic acid) porphyrin (MnTBAP), during (d0-21 post BMT), or after CsA therapy (>d21 post-BMT), caused a reduction in the development of clinical symptoms of SGVHD (weight loss, diarrhea). Interestingly, treatment with MnTBAP resulted in a significant reduction in the deposition of peroxynitrite in the colons of CsA-MnTBAP-treated versus control CsA-treated SGVHD animals. These studies suggest a role for oxidative stress in the development of mur- ine SGVHD.

A gut feeling about murine syngeneic GVHD

Murine syngeneic graft-versus-host disease (SGVHD) results in chronic colon and liver inflammation following syngeneic bone marrow transplantation (BMT) and treatment with the calcineurin inhibitor, cyclosporine A (CsA). SGVHD was initially thought to arise as a result of an autoreactive immune response, but more recently it has been shown that enhanced antimicrobial responses develop in SGVHD mice. Consequently, we performed studies to analyze the role of the microbiota in the development of murine SGVHD. Treatment with broad-spectrum antibiotics eliminated disease-associated inflammatory immune responses and pathology, linking the role of the microbiota and microbial-specific immunity to the development of murine SGVHD. In a broader context, these results bring into question the role that anti-microbial immune responses play in post-transplant immune pathologies that develop following allogeneic stem cell transplantation and use of calcineurin inhibitors.

Murine Syngeneic Graft-Versus-Host Disease Is Responsive to Broad-Spectrum Antibiotic Therapy

Murine syngeneic graft-versus-host disease (SGVHD) initiates colon and liver inflammation following lethal irradiation, reconstitution with syngeneic bone marrow transplantation, and therapy with the immunosuppressive agent cyclosporine A. Previous studies have demonstrated that the inducible disease is mediated by CD4(+) T cells with increased reactivity of peripheral and liver-associated lymphocytes against intestinal microbial Ags. In the current report, studies were performed to analyze the specificity of the CD4(+) T cell response of T cells isolated from diseased animals and to determine the in vivo role of the microbiota to the development of SGVHD. Increased major histocompatibility Ag (MHC) class II-restricted responsiveness of SGVHD CD4(+) T cells against microbial Ags isolated from the ceca of normal animals was observed. The enhanced proliferative response was observed in the CD62L(-) memory population of CD4(+) T cells. To determine the role of the bacterial microbiota in the development of murine SGVHD, control and CsA-treated bone marrow transplantation animals were treated with broad-spectrum antibiotics (metronidazole, ciprofloxacin) after transplantation. Cyclosporine A-treated animals that were given antibiotic therapy failed to develop clinical symptoms and pathological lesions in the target tissues characteristic of SGVHD. Furthermore, the reduction in intestinal bacteria resulted in the elimination of the enhanced antimicrobial CD4(+) T cell response and significantly reduced levels of the inflammatory cytokines, IFN-γ, IL-17, and TNF-α. The elimination of the disease-associated inflammatory immune responses and pathology by treatment with broad-spectrum antibiotics definitively links the role of the microbiota and microbial-specific immunity to the development of murine SGVHD.

Development of a T H17 immune response during the induction of murine syngeneic graft-versus-host disease

Syngeneic graft-versus-host disease (SGVHD) develops following lethal irradiation, reconstitution with syngeneic bone marrow (BM) and treatment with a 21 day course of the immunosuppressive agent cyclosporine A (CsA). Clinical symptoms of SGVHD appear 2–3 weeks post-CsA with inflammation occurring in the colon and liver. Previously we have demonstrated that CD4 + T cells and a T helper cell type 1 cytokine response (T H1) are involved in the development of SGVHD associated intestinal inflammation. Studies have recently discovered an additional T cell lineage that produces IL-17 and is termed T H17. It has been suggested that inflammatory bowel disease is a result of a T H17 response rather than a T H1 response. This study was designed to investigate T H17 involvement in SGVHD-associated colitis. Following induction of SGVHD, the levels of T H17 and T H1 cytokine mRNA and protein were measured in control and SGVHD animals. In vivo cytokine neutralization was performed to determine the role of the prototypic T H17 cytokine, IL-17, in the disease process. We found that during CsA-induced murine SGVHD there was an increase in both T H17 and T H1 mRNA and cytokines within the colons of diseased mice. The administration of an anti-mouse IL-17A mAb did not alter the course of disease. However, neutralization of IL-17A resulted in an increased production of IL-17F, a related family member, with an overlapping range of effector activities. These results demonstrate that in the pathophysiology of SGVHD, there is a redundancy in the T H17 effector molecules that mediate the development of SGVHD.

Association between chronic liver and colon inflammation during the development of murine syngeneic graft-versus-host disease.

The murine model of cyclosporine A (CsA)-induced syngeneic graft-versus-host disease (SGVHD) is a bone marrow (BM) transplantation model that develops chronic colon inflammation identical to other murine models of CD4(+) T cell-mediated colitis. Interestingly, SGVHD animals develop chronic liver lesions that are similar to the early peribiliary inflammatory stages of clinical chronic liver disease, which is frequently associated with inflammatory bowel disease (IBD). Therefore, studies were initiated to investigate the chronic liver inflammation that develops in the SGVHD model. To induce SGVHD, mice were lethally irradiated, reconstituted with syngeneic BM, and treated with CsA. All of the SGVHD animals that developed colitis also develop chronic liver inflammation. Liver samples from control and SGVHD animals were monitored for tissue pathology, RNA for inflammatory mediators, and phenotypic analysis and in vitro reactivity of the inflammatory infiltrate. Diseased animals developed lesions of intrahepatic and extrahepatic bile ducts. Elevated levels of mRNA for molecules associated with chronic liver inflammation, including mucosal cellular adhesion molecule -1, the chemokines CCL25, CCL28, CCR9, and T(H)1- and T(H)17-associated cytokines were observed in livers of SGVHD mice. CD4(+) T cells were localized to the peribiliary region of the livers of diseased animals, and an enhanced proliferative response of liver-associated mononuclear cells against colonic bacterial antigens was observed. The murine model of SGVHD colitis may be a valuable tool to study the entero-hepatic linkage between chronic colon inflammation and inflammatory liver disease.

Reduction of murine Syngeneic graft‐versus‐host disease (SGVHD) following treatment with the superoxide dismutase mimetic MnTBAP

Syngeneic graft‐vs‐host disease (SGVHD) is induced by reconstituting lethally irradiated mice with syngeneic bone marrow followed by a 21 day treatment with the immunosuppressive agent cyclosporine A (CsA). Symptoms of the disease appear 2–3 weeks following cessation of CsA therapy and disease‐associated inflammation occurs primarily in the colon and liver. Irradiation and CsA are both required for the development of SGVHD and both contribute to the production of oxidative stress. Time course studies revealed increased reactive oxygen and nitrogen species in CsA treated animals compared to BMT controls. Studies also demonstrated increased migration of CD4+T cells into the colon during CsA therapy. In addition, increased colon mRNA levels of proinflammatory cytokines (TNF‐α, IL‐12) and cell adhesion molecules (CAMs) were observed as early as day 14‐post BMT in colon of CsA treated animals. Since reactive oxygen species (ROS) are known to mediate CsA toxicity, studies were undertaken to determine the effect of CsA‐induced oxidative stress on the induction of SGVHD. Treatment with the antioxidant MnTBAP, during CsA therapy, caused a reduction in SGVHD development as well as decreased colon mRNA levels for iNOS, CAMs and proinflammatory cytokines. These studies suggest a role for oxidative stress in the development of intestinal pathology associated with murine SGVHD. Research support: NIH Grant P01‐CA092372.

Induction of Murine Syngeneic Graft-Versus-Host Disease by Cells of Recipient Origin

Syngeneic graft-versus-host disease (SGVHD) develops after lethal irradiation, reconstitution with syngeneic bone marrow (BM), and treatment with a 21-day course of the immunosuppressant cyclosporine A (CsA). Clinical symptoms of SGVHD appear 2-3 weeks after CsA treatment, with inflammation in the colon and liver. It has been demonstrated that CD4+ T cells and a T helper cell type 1 cytokine response (Th1) are involved in the development of SGVHD associated intestinal inflammation. The immune response associated with SGVHD is thought to be the result of the reconstitution of the recipient immune system with the syngeneic donor BM. However, definitive studies have not addressed this issue experimentally. To determine the origin of the effector cells that participate in SGVHD, C3H/HeN recipient mice were lethally irradiated and transplanted with BM from normal immunocompetent mice or from immunodeficient, severe combined immune deficient, or Rag-2-/- animals. CsA-treated animals, but not control animals, developed inflammation characteristic of SGVHD in the colon and liver regardless of the source of the donor marrow. Furthermore, immunologically, all CsA treated animals responded similarly with increased production of inflammatory cytokines and an increase in activated CD4+ T cells in the periphery and colon relative to controls. These results demonstrate that after lethal irradiation and in the absence of donor T cells, T cells of recipient origin can expand and mediate the induction of CsA-induced SGVHD.

Role of STAT1 Expression during Graft-Versus-Host Disease (GVHD) in the Gastrointestinal Tract: Association with Lamina Propria Cell Infiltration and Tissue Cytokine/Chemokine Expression.

The role of the IFN-g in the development of GVHD remains enigmatic. Whereas it has been shown that GVHD can occur in the absence of IFN-g, there are partially contradicting results how IFN-g can modulate GVHD. Thus, it has been suggested that blocking IFN-g might ameliorate gut GVHD in rodent BMT models and its direct cytopathic effects in human ex vivo skin explant models. However, there is also data supporting the notion that IFN-g is important for limiting GVHD by induction of activation induced cell death (AICD) in donor T cells. Furthermore, it has been demonstrated in preclinical BMT models that IFN-g may accelerate or mitigate GVHD depending on the conditioning intensity. We have recently demonstrated that mitigation of GVHD by treatment with the Histone deacetylase Inhibitor (HDACi) suberonylanilide hydroxamic acid (SAHA) is associated with early downregulation of STAT1 phosphorylation in the spleen and host epithelial GVHD target organs. To further understand the role of STAT1 in the development of GVHD we studied STAT1 and p-STAT1 (Tyr701) expression by immunohistochemistry in the GVHD target organs liver, small bowel and colon following induction of GVHD and correlated these findings with the presence of lamina propria (LP) lymphocytes, typical features of GVHD-induced tissue damage (crypt cell apoptosis, crypt regeneration) and expression of tissue cytokines/chemokines. GVHD was induced in the fully MHC mismatched BALB/c to B6 strain combination following lethal irradiation with 975 rad and animals were sacrificed on days +1, +3 and +6. As detected by western blots p-STAT1 expression became detectable on day +1 in the spleen and on days +3 in the liver, small bowel and colon. Compared to untreated controls immunohistochemical p-STAT1 staining became apparent on day +3 post-BMT in the small bowel and colon of syngeneic controls and GVHD animals. In syngeneic controls p-STAT1 expression decreased again on day +6. In contrast, a further significant increase in p-STAT1 staining was observed in animals with GVHD in the colon and small bowel on day +6. In the colon this significant increase in crypt cell p-STAT1 staining was associated with the presence of LP infiltrating lymphocytes and coincided with the maximal features of tissue damage (luminal sloughing, crypt destruction and crypt apoptosis). In line with these results IFN-g protein expression became detectable in colon tissue lysates on day +6 supporting the role of IFN-g producing infiltrating donor T cells in causing STAT1 activation and tissue damage. We conclude that in comparison to untreated controls STAT1 activation can be observed in the colon and small bowel starting on day +3 in animals with GVHD and syngeneic controls. Whereas pSTAT1 staining peaks at day +3 in syngeneic controls and declines thereafter, maximal STAT1 activation occurs in GVHD animals on day +6, coincides with detectable IFN-g expression and is accompanied by LP infiltration and features of severe GVHD-related tissue damage. To fully understand the role of IFN-g in the development of gut GVHD further studies are warranted to delineate the role of STAT1 dependent and independent signaling pathways in the development of GVHD.

T cells of recipient origin mediate the development of murine syngeneic graft-versus-host disease

Syngeneic graft-versus-host disease (SGVHD), characterized by a TH1 cytokine response, and intestinal inflammation develops following lethal irradiation, reconstitution with syngeneic bone marrow, and treatment for 21 days with cyclosporine A (CsA). It has generally been assumed that the T cell responses associated with SGVHD resulted from the reconstitution of irradiated recipients with donor bone marrow. However, to determine the origin of the effector cells that mediate SGVHD, syngeneic bone marrow from normal immunocompetent mice or immunodeficient mice were transferred into lethally irradiated recipients.

Antigen-specific T-lymphocyte responses in acute and chronic syngeneic graft-versus-host disease

Administration of cyclosporine (CyA) following autologous bone marrow transplantation elicits a T-lymphocyte autoaggression syndrome with remarkable similarity to acute and chronic graft-versus-host disease (GVHD). This syndrome termed syngeneic (S) GVHD is mediated by a restricted repertoire of autoreactive T cells that promiscuously recognize major histocompatibility complex (MHC) class II determinants in association with a peptide from the invariant chain (CLIP). The present studies evaluated and compared antigen-specific autoreactive T cells during acute and chronic SGVHD isolated ex vivo with a soluble MHC class II-immunoglobulin (sMHC class II-Ig) molecular construct. Two major subsets were detected that had overlapping specificity recognizing the MHC class II binding domain of CLIP but were differentially dependent on the N- and C-terminal flanking domains of this peptide. Both subsets were detected in acute and chronic SGVHD. Interestingly, the cytokine profiles of the CLIP-reactive T cells closely correlated with the onset and progression of disease. Levels of type 1 cytokines, particularly IFN-γ mRNA production assessed by quantitative polymerase chain reaction (PCR), were dominant during acute SGVHD, whereas chronic SGVHD was associated with type 2 cytokine mRNA production. Although there was a dramatic polarization with respect to cytokine production, only subtle changes in antigen specificity were detected. Of additional interest, autoreactive T cells producing IL-10 mRNA were detected in both acute and chronic SGVHD, suggesting that this cytokine may play an important but perhaps paradoxical role in both the onset and progression of this experimental autoaggression syndrome.

Functional divergence of antigen-specific T-lymphocyte responses in syngeneic graft-versus-host disease.

Administration of cyclosporine after autologous bone marrow transplantation elicits a T-lymphocyte autoaggression syndrome with remarkable similarity to graft-versus-host disease (GVHD). This syndrome, termed syngeneic GVHD (SGVHD), with both acute and chronic phases, is mediated by a restricted repertoire of autoreactive T cells that promiscuously recognize major histocompatibility complex (MHC) class II determinants in association with a peptide from the invariant chain (MHC class II-invariant chain peptide; CLIP). This study evaluated and compared antigen-specific autoreactive T cells during acute and chronic SGVHD ex vivo isolated with a soluble MHC class II immunoglobulin molecular construct. This approach allows for the direct assessment of the functional behavior of the effector T cells without potential modification by in vitro culture. Two major subsets were detected that had overlapping specificity recognizing the MHC class II-binding domain of CLIP but that were differentially dependent on the N- and C-terminal flanking domains of this peptide. The N- and C-terminal subsets were primarily associated with acute and chronic SGVHD, respectively. The cytokine profiles of the CLIP-reactive T cells, however, were most informative and closely correlated with the onset and progression of disease. Levels of type 1 cytokine, particularly interferon-gamma, messenger RNA (mRNA) production, assessed by quantitative polymerase chain reaction, were dominant during acute SGVHD, whereas chronic SGVHD was associated with type 2 cytokine mRNA production. Although there was a dramatic polarization with respect to cytokine production, only subtle changes in antigen specificity could be detected. Unexpectedly, the functional behavior within the antigen-specific effector cell populations is not fixed and seems to change as the disease progresses to the chronic phase. Concordant with the evolution of the effector T-cell response is a differential loss in B7.1 mRNA expression in the N-terminal CLIP-reactive T-cell subset that may reflect the regulation of this autoimmune response. Of additional interest, autoreactive T cells producing interleukin-10 mRNA were detected in both acute and chronic SGVHD, suggesting that this cytokine may play an important but perhaps paradoxical role in both the onset and progression of this experimental autoaggression syndrome.

Update on graft versus host disease.

Update on graft versus host disease.

Unexpected T-cell diversity in syngeneic graft-versus-host disease revealed by interaction with peptide-loaded soluble MHC class II molecules.

Administration of cyclosporine after syngeneic bone marrow transplantation elicits a systemic autoaggression syndrome termed syngeneic graft-versus-host disease (SGVHD). The effector T cells recognize a peptide from the invariant chain termed CLIP (MHC class II invariant chain peptide) presented on MHC class II molecules. Moreover, the N-terminal flanking region of CLIP interacts with the T-cell receptor (TcR) beta chain. The current study uses a novel approach to isolate and examine the responding T cells ex vivo. A soluble MHC class II molecule using immunoglobulin (Ig)G (MHC class II-Ig) as a scaffold was constructed. The MHC class II-Ig molecule loaded with different peptide variants of CLIP (lacking the N-terminal or C-terminal flanking regions of CLIP) was used to isolate antigen-specific T cells from animals with SGVHD by panning or by flow cytometric sorting. Two subsets of antigen specific T cells restricted by the N- and C-terminal flanking regions of CLIP can be isolated during acute SGVHD that express the Vbeta8.5 TcR determinant but secrete different cytokines (interferon [IFN]-gamma, interleukin [IL]-10) as detected by real-time quantitative polymerase chain reaction (PCR). Spectratyping of the complementarity-determining region 3 regions reveals that the N-terminal CLIP reactive population has greater diversity in both the CDR3 and J regions than the C-terminal CLIP reactive subset. Interestingly, the N-terminal CLIP reactive cells can be preferentially detected in the target tissues during acute SGVHD. However, only IFN-gamma messenger (m)RNA was detected in the tissues. The ability to isolate and examine antigen specific T cells ex vivo has revealed unexpected differences in TcR diversity and cytokine production in SGVHD.

Discoid Lupus Erythematosus in a Patient Receiving Cyclosporine for Liver Transplantation

Sir, total clearance of skin lesions could be obtained with a 5 months course of chloroquine 250mg daily. Primary biliary cirrhosis is associated with many immunological abnormalities and is therefore widely held to be an autoimmune disorder (1). Among these are chronic thyreoidDISCUSSION itis, rheumatoid arthritis, Sjogren’s syndrome, scleroderma, The coexistence of primary biliary cirrhosis and other autothe CREST syndrome and systemic lupus erythematosus immune disorders is well documented. However, to the best (2, 3). Here, we report a patient who developed discoid lupus of our knowledge this is the Ž rst report of DLE following erythematosus (DLE) during treatment with cyclosporine primary biliary cirrhosis. It is also of considerable interest that 6 years after liver transplantation for primary biliary cirrhosis. these lesions occurred despite immunosuppressive treatment. There are several reports in the literature showing cyclosporine to be poorly eVective in in uencing DLE at a dosage CASE REPORT up to 5.3mg/kg/day (4, 5). Similar to our observation, Di In November 1999, a 50-year-old woman presented with a Lernia & Bisighini described the onset of DLE in a patient 1-month history of sharply marginated bright-red discoid receiving this drug for recalcitrant psoriasis (6). plaques on the nose, right cheek and scalp, causing a 2-cm The fungal metabolite cyclosporine is a potent immunodiameter area of scarring alopecia. No other skin Ž ndings suppressive agent with a relatively selective action on T were present. Apart from photosensitivity, she did not suVer lymphocytes. However, paradoxical eVects on the immune from any other symptoms. A skin biopsy revealed hyperparaksystem by breaking self-tolerance may occur. Administration eratosis and plugging of hair follicles, epidermal atrophy and of this agent can elicit autoimmune diseases in certain animal inŽ ltrating lymphocytes causing an interface dermatitis. models by inhibiting clonal deletion in the thymus with a Immuno uorescence studies showed granular deposits of IgG consequent release of autoreactive T cells (7, 8). at the basement membrane zone (similar to a lupus band). In summary, it is intriguing to speculate that the predisposiThe patient had a 15-year history of primary biliary cirrhosis tion to an autoimmune disease with primary biliary cirrhosis, leading to liver failure and subsequent liver transplantation in on the one hand, and treatment with cyclosporine, on the 1993. Immunosuppressive therapy consisted of anti-thymocyte other, may have contributed to the development of DLE in globulins immediately after transplantation, a short course of this patient. Our observation also demonstrates that treatment glucocorticosteroids and maintenance therapy with cyclosporwith cyclosporine is poorly eVective in DLE. ine, which was given at a dosage of 175mg daily (3mg/kg of body weight) at the time of admission. Minor rises of liver REFERENCES enzymes in a cholestatic pattern suggestive of a recurrence of 1. Kaplan MM. Primary biliary cirrhosis. N Engl J Med 1996; primary biliary cirrhosis in the allograft settled spontaneously 335: 1570–1580. under this therapeutic regimen. Drug-induced hypertension 2. Hall S, Axelsen PH, Larson DE, Bunch TW. Systemic lupus was treated with a combination of lisinopril 20mg and hydroerythematosus developing in patients with primary biliary cirrhosis. chlorothiazide 25mg daily. The only other medications were Ann Intern Med 1984; 100: 388–389. ursodeoxycholic acid 300mg and calcitriol 0.5mg. 3. Nachbar F, Korting HC, HoVman RM, Kollmann M, Meurer M. At the time of skin biopsy, liver function tests showed a Unusual coexistence of systemic lupus erythematosus and primary cholestatic pattern with a three-fold rise of c-glutamyl transbiliary cirrhosis. Dermatology 1994; 188: 313–317. ferase (62 U/l; normal 6–19) and an elevated alkaline phos4. Heule F, van Joost T, Beukers R. Cyclosporine in the treatment of lupus erythematosus. Arch Dermatol 1986; 122: 973–974. phatase (180 U/l; normal 50–155); the anti-mitochondrial 5. Yell JA, Burge SM. Cyclosporin and discoid lupus erythematosus. antibody titre was 1:160. All other routine laboratory investiBr J Dermatol 1994; 131: 132–133. gations were within the normal range; anti-nuclear anti6. Di Lernia V, Bisighini G. Discoid lupus erythematosus during bodies, anti-neutrophil cytoplasmatic and anti-cardiolipin treatment with cyclosporine. Acta Derm Venereol 1996; 76: 87–88. antibodies could not be detected. 7. Sakaguchi N, Sakaguchi S. Causes and mechanism of autoimmune Treatment of DLE consisted of cryotherapy and a steroid disease: cyclosporin A as a probe for the investigation. J Invest cream (with little success) and subsequently hydroxychloroDermatol 1992; 98: 70S–76S. quine 200mg b.i.d. (6mg/ kg of body weight per day) in 8. Chen W, Thoburn C, Hess AD. Characterization of the pathogenic addition to a potent sunscreen. At a follow-up visit after 6 autoreactive T cells in cyclosporine-induced syngeneic graft-versushost disease. J Immunol 1998; 161: 7040–7046. months, the lesions were only in partial remission. However,

Complexity of effector mechanisms in cyclosporine-induced syngeneic graft-versus-host disease

Administration of the immunosuppressive drug cyclosporine after syngeneic or autologous bone marrow transplantation elicits a T-lymphocyte-dependent autoimmune syndrome similar to graft-versus-host disease (GVHD). The onset of this autoaggression syndrome, termed syngeneic GVHD, is associated with the development of a highly restricted repertoire of CD8+ autoreactive T cells that recognize a peptide from the invariant chain, termed CLIP, presented by major histocompatibility complex (MHC) class II molecules. Clonal analysis reveals 2 distinct subsets of autoreactive T cells defined by their activation requirement for either the N-terminal or the C-terminal flanking regions of CLIP and by their cytokine profile. The studies here reveal that the autoreactive T-cell clones requiring the N-terminal flanking region of CLIP produce type 1 cytokines (interferon [IFN]-gamma, interleukin [IL]-2, and tumor necrosis factor-alpha). In contrast, the autoreactive T-cell clones that require the C-terminal flanking region of CLIP produce type 2 cytokines (IL-4, IL-10, transforming growth factor-beta). As assessed in a local graft-versus-host reaction assay, the N-terminal flanking-restricted clones mediate changes consistent with acute GVHD, whereas the clones responsive to the C-terminal flanking region do not. Moreover, the autoreactive T-cell clones restricted by the C-terminal flanking region of CLIP ameliorate the pathogenic potential of the cells responsive to the N-terminal flanking region of CLIP. The mechanism accounting for this regulatory affect appears to be the downregulation of mRNA message for type 1 cytokines (IFN-gamma and IL-2). The C-terminal-restricted autoreactive T-cell clones, however, could manifest disease with dermal changes similar to those seen in chronic syngeneic GVHD, provided that IFN-gamma was present. Consistent with these observations was the demonstration that type 1 cytokines are preferentially detected during the acute phase of syngeneic GVHD, whereas type 2 cytokines dominate during the chronic phase. The results suggest that acute and chronic syngeneic GVHD is mediated by distinct autoreactive T cells, which are separated by their fine specificity for the CLIP-MHC class II complex and by their cytokine profiles. Biol Blood Marrow Transplant 2000;6(2):13-24.

Rejection of an MHC class II negative tumor following induction of murine syngeneic graft-versus-host disease.

Cyclosporin A (CsA) has been used clinically to induce graft-versus-host disease following autologous bone marrow transplantation in an attempt to destroy residual leukemia cells and reduce relapse. To analyze the antitumor potential of murine syngeneic graft-versus-host disease (SGVHD), C3H/HeN mice were lethally irradiated, reconstituted with T cell-depleted syngeneic bone marrow (ATBM) and treated with CsA for 21 days. Graft-versus-leukemia activity was assessed by challenging groups of olive oil-treated control ATBM (OO-ATBM) and CsA-treated (CsA-ATBM) mice 1 week after CsA therapy with graded doses of the syngeneic 38C13 B cell lymphoma. Following CsA treatment, up to 70% of CsA-ATBM developed SGVHD and more than 70% of the animals injected with 500 38C13 cells exhibited long-term survival (MST >80 days). In contrast, none of the OO-ATBM control mice developed SGVHD, and more than 75% of these mice died following injection of 500 38C13 tumor cells (MST = 34 days). Long-term survivors were not resistant to tumor challenge suggesting that tumor-specific immunity did not develop. Finally, class II negative 38C13 cells cultured in IL-4 or IL-10 were not inducible for MHC class II molecules, demonstrating that class II-independent antitumor mechanisms exist in SGVHD mice.

Autoimmune diseases and autoimmunity post-bone marrow transplantation.

BMT can both transmit and eliminate autoimmune diseases, and hence it has been suggested as an optional treatment for severe autoimmune conditions. In this communication we deal with the question of whether chronic GVHD is an autoimmune disease in itself, review the literature reports of autoimmune diseases following BMT in humans, and describe the autoimmune nature of the post-BMT state. Chronic GVHD, which is a frequent complication post-BMT, has clinical and pathogenic characteristics similar to autoimmune diseases, such as scleroderma and Sjogren's syndrome. Although the pathogenesis of chronic GVHD is not yet clear, thymic damage induced by acute GVHD may contribute to both the immunodeficiency and autoimmunity characterising chronic GVHD. A similar phenomenon is syngeneic GVHD, which results from an imbalance between autoreactive and autoregulatory lymphocytes. Additionally, other autoimmune diseases have been reported in post-BMT patients, and among these the most common are hypothyroidism, hyperthyroidism, myasthenia gravis and immune cytopenias. Although these diseases also occur also outside the post-BMT setting, they are unique with respect to pathogenesis (no association between myasthenia gravis and thymic pathology), diagnosis (symptoms of hyperthyroidism may be inadvertently related to other conditions), and prognosis (post-BMT autoimmune cytopenias may be fatal and treatment non-responsive). Nevertheless, many other autoimmune diseases have been reported after BMT, and these are mainly presented as case reports. Regarding the mechanism of post-BMT autoimmunity, the minority of cases stem from donor-related transfer of pathogenic lymphocytes or their progenitors, while most of the cases (either chronic GVHD or specific diseases) can be attributed to the immunologic imbalances characterising the post-BMT setting. The factors that may expose an individual to autoimmunity development post-BMT include genetic predisposition, an environmental factor such as CMV, and the nature of the donor who may aid in creating microchimerism and subsequently chronic GVHD and its related autoimmune manifestations.

Promiscuous recognition of major histocompatibility complex class II determinants in cyclosporine-induced syngeneic graft-versus-host disease: specificity of cytolytic effector T cells.

Administration of the immunosuppressive drug cyclosporine after syngeneic/autologous bone marrow transplantation paradoxically elicits a systemic autoimmune syndrome resembling graft-versus-host disease (GVHD). This syndrome, termed autologous or syngeneic GVHD, is associated with the development of a highly restricted repertoire of cytolytic T lymphocytes that promiscuously recognizes major histocompatibility complex class II determinants, including self. Vbeta8.5+CD8+ effector lymphocytes and T-cell clones were isolated from Lewis rats with cylosporine-induced syngeneic GVHD. The specificity of the effector T cells and T-cell clones was examined in vitro. The pathogenicity of the T-cell clones was confirmed in vivo using a local graft-versus-host reaction assay. Clonal analysis reveals that the pathogenic effector T cells recognize a peptide from the invariant chain termed CLIP in association with major histocompatibility complex class II determinants. Moreover, there appears to be an additional interaction between the N-terminal flanking region of CLIP and the Vbeta segment of the T cell receptor. The results suggest that recognition of this highly conserved peptide along with the additional interaction between the flanking region and the T cell receptor may account for the promiscuous activity of the autologous/syngeneic GVHD autoreactive T cells.

Prevention of graft-versus-host disease and the induction of transplant tolerance by low-dose UV-B irradiation of BM cells combined with cyclosporine immunosuppression.

GVHD is prevented and stable chimerism is induced in the rat BMT model by 700 J/m2 but not 100-500 J/m2 UV-B irradiation of allogeneic BM cells. Paradoxically, CsA which prevents GVHD in clinical BMT causes an aggressive autoimmune disease termed syngeneic GVHD in irradiated syngeneic BMT recipients after its withdrawal. Recently, we have shown that while 500-700 J/m2 UV-B irradiation of syngeneic BM cells combined with a 30-day course of CsA recipient immunosuppression impairs hemopoiesis due to lack of hemopoietic factors, a low dose of 100-300 J/m2 UV-B is effective in preventing CsA-induced autoimmune disease without endangering BM engraftment. This study extends these findings to the P-to-F1 hybrid and fully allogeneic rat BMT models and examines the effectiveness of low-dose UV-B irradiation of BM cells combined with a short course of CsA treatment in the prevention of GVHD and induction of transplant tolerance. Lethally gamma-irradiated (10.5 Gy) LBNF1 recipients of naive or UV-B irradiated (100-700 J/m2) BMT were treated with CsA (12.5 mg/kg/day) for 30 consecutive days after BMT. All lethally irradiated LBNF1 that did not receive BMT died in < 16 days, while animals transplanted with UV-B (700 J/m2) BMT survived > 1 year without GVHD. In contrast, all recipients of naive BMT died of lethal GVHD in < 50 days. Similarly, all recipients of naive BMT that received a 30-day course of CsA therapy developed severe GVHD with 60% mortality after cessation of CsA therapy. CsA-treated recipients of BMT irradiated with 700 J/m2 died between 12 and 25 days from failure of hemopoiesis. In contrast, CsA-treated recipients of 100-200 J/m2 UV-B irradiated BMT showed full BM engraftment without GVHD after cessation of CsA and survived > 1 year. These results were reproducible in the fully allogeneic UV-B BMT model. To test for donor-specific tolerance, the animals challenged 100 days after BMT with cardiac allografts accepted permanently (> 100 days) Lewis but not BN (non-BMT parental donor) cardiac allografts. Our results confirm that 700 J/m2 UV-B irradiation of BM cells combined with CsA recipient immunosuppression impairs the recovery capacity of stem cells while the use of lower UV-B (100-200 J/m2) is effective in preventing CsA-induced autoimmune disease without endangering BM engraftment and leads to induction of transplant tolerance.