Inhibitory effect of a CD4-CDR3 peptide analog on graft-versus-host disease across a major histocompatibility complex-haploidentical barrier

A structure-based designed peptide has been engineered to exhibit the same molecular surface as a portion of the CDR3-like region in domain 1 of the murine CD4 molecule. Earlier in vitro experiments indicated that this analog, known as rD-mPGPtide, inhibited T-cell proliferation in mixed lymphocyte reactions and blocked activation of both normal CD4+ T cells and T-cell lines after T-cell receptor triggering. In addition, rD-mPGPtide proved to be a potent inhibitor in vivo of CD4+ T-cell- mediated experimental allergic encephalomyelitis disease in the SJL mouse model. In this current report, we have evaluated the potential of rD-mPGPtide for suppressing the development of graft-versus-host disease (GVHD) in an irradiated major histocompatibility complex (MHC)- haploidentical murine bone marrow transplantation (BMT) model [(B6 x DBA/2)F1-->(B6 x CBA)F1 (950 cGy)]. Our results indicated that early administration of rD-mPGPtide was effective in the inhibition of alloreactive responses of the donor T cells against the host and thus delayed or prevented the onset of GVHD. The median survival time of animals treated with rD-mPGPtide was enhanced as much as four-fold with as little as a single dose of peptide at the time of transplant. Decreased alloreactivity was indicated by phenotypic and functional analysis of positively selected thoracic duct lymphocytes 4 days after transplant and by histopathological examination of skin and gastrointestinal tissue samples 4 weeks later. Therefore, the administration of a CD4-CDR3 peptide is an efficacious approach against the development of GVHD during allogeneic BMT.     

Therapy for ongoing graft-versus-host disease induced across the major or minor histocompatibility barrier in mice with anti-CD3F(ab')2-ricin toxin A chain immunotoxin

A new pharmacologic agent, anti-CD3F(ab')2-ricin toxin A chain (RTA), was synthesized for the purpose of targeting T cells and as a means of treating established graft-versus-host disease (GVHD). The Fc region of anti-CD3 monoclonal antibody (MoAb) was removed to prevent its ability to activate T cells. The resulting F(ab')2 fragments were conjugated to deglycosylated RTA (dgRTA), a catalytic and potent phytotoxin. The resulting immunotoxin (IT) was potent (greater than 95% inhibition) and selective in inhibiting T-cell mitogenesis in vitro. In vivo, the IT depleted 80% of T cells in mice receiving bone marrow (BM) transplants. Transplantation in an aggressive acute GVHD model using C57BL/6 donor cells and H-2 disparate B10.BR recipients resulted in an infiltration of CD3-expressing cells and a median survival time (MST) of 20 to 30 days. A 5-day course of anti-CD3F(ab')2-RTA (30 micrograms/d intraperitoneally) beginning 7 days after GVHD induction was beneficial in treating established GVHD in these mice, as evidenced by significantly prolonged survival (MST, greater than 80 days), superior mean weight values, and improved clinical appearance. Neither intact anti-CD3, unconjugated anti-CD3 F(ab')2 fragments, nor a mixture of anti-CD4 and anti-CD8 MoAbs (which are highly effective in prophylactic models) were as effective. F(ab')2 fragments made from anti-Lyt-1 (the murine homologue of human anti-CD5) linked to RTA were also not effective, despite the fact that both anti-CD3F(ab')2-RTA and anti-Lyt- 1F(ab')2-RTA had similar half-lives of about 9 hours. The IT also increased MST in two aggressive models of GVHD across non-H-2 minor histocompatibility barriers, indicating that the usefulness of anti- CD3F(ab')2-dgRTA is not limited to a single-strain combination. This agent should be further investigated as an alternative to current strategies for treating steroid refractory GVHD.     

Prevention of murine graft-versus-host disease and bone marrow alloengraftment across the major histocompatibility barrier after donor graft preincubation with anti-LFA1 immunotoxin

We have investigated the effects of the in vitro depletion of LFA1 positive cytolytic T lymphocytes, natural killer (NK) cells, and monocytes on the afferent phase of graft-versus-host disease (GVHD). Lethal GVHD was induced across the murine major histocompatibility complex by injecting C57BL/6 (H-2b) bone marrow (BM) cells (a source of stem cells) and splenocytes (S) (a source of T cells) into lethally irradiated B10.BR (H-2k) recipients. Because anti-LFA1 does not bind complement (C') effectively, we conjugated anti-LFA1 alpha chain monoclonal antibody (MoAb) to ricin toxin A chain (RTA) as a means of facilitating target cell elimination. A 2-hour preincubation of C57BL/6 bone marrow/spleen (BMS) with anti-LFA1-RTA in the presence of ammonium chloride (a potentiator of immunotoxin toxicity), but not a control immunotoxin (IT), reduced CTL activity by greater than 2 logs, significantly reduced NK cell activity, and prevented B10.BR mice from developing GVHD. Depletion of target cells by toxin-labeled-MoAb and not the blockade of the LFA1 molecule by the anti-LFA1 MoAb accounted for our results, because incubating cells with IT in the absence of a potentiator had no effect on GVHD prevention. In contrast, C57BL/6 recipients of C3H BMS grafts only partially benefited from anti-LFA1-RTA preincubation, demonstrating that in this system, different cells not expressing LFA1 were involved in GVHD generation. The same findings observed with anti-LFA1-RTA preincubation were observed with preincubation with L-leucyl-L-leucine methyl ester, a chemical compound eliminating cytolytic cells, providing further support that GVHD induction in the C3H/HeJ into C57BL/6 system is not entirely mediated by classical cytolytic T cells. We next tested anti-LFA1-RTA in a model devised to measure its effect on alloengraftment (B10.BR recipients given lower doses of irradiation). Anti-LFA1-RTA BM preincubation selectively reduced alloengraftment in the model. This observation, combined with experiments showing that LFA1-RTA preincubation, but not anti-Thy 1.2 + C' or control IT preincubation, reduced colony-forming unit-spleen formation, indicates that anti-LFA1 alpha chain IT may remove accessory cells or stem cells critical to engraftment. Still, anti-LFA1-RTA may be useful for clinical GVHD prevention when combined with positive selection techniques designed to enrich for stem cells.     

Toxicity and efficacy of anti-T-cell ricin toxin A chain immunotoxins in a murine model of established graft-versus-host disease induced across the major histocompatibility barrier

Graft-versus-host disease (GVHD) was induced across the murine major histocompatibility complex by injecting C57BL/6 (H-2b) bone marrow and splenocytes into lethally irradiated B10.BR (H-2k) murine recipients. An immunotoxin (IT) composed of a pan T-cell monoclonal antibody called anti-Ly1 (the murine homologue to human anti-CD5) was conjugated to ricin toxin A chain (anti-Ly1-RTA) and used to treat recipient mice. In vitro, IT was as active as free RTA, bound selectively, and inhibited T-cell proliferation even in the absence of potentiators. Mice administered anti-Ly1-RTA in vivo during ongoing GVHD, at a dose of 10 micrograms/d for 5 days, showed lower numbers of splenic Thy1.2+ T cells and significantly improved survival as compared with mice given phosphate-buffered saline (PBS) or irrelevant control RTA IT. Protection was transient because GVHD and weight loss occurred when injections ceased. Survival could not be enhanced by crosslinking RTA30, a low oligosaccharide-containing fraction of purified RTA. Treatment with anti-Ly1-RTA caused a significant elevation in neutrophils, and higher doses were associated with mild hepatotoxicity. In contrast, infusion of identical doses and schedules of another pan T-cell immunotoxin, anti-Thy1.2-RTA, caused a significant decrease in lymphocytes, but not neutrophils; a precipitous increase in weight; a decrease in total plasma protein (TPP); and an increase in pleural and peritoneal effusions reminiscent of vascular leak syndrome (VLS). Although the toxic effects of anti-Thy1.2-RTA were too severe to show a survival advantage in a GVHD model, histopathologic studies showed a definite anti-GVHD effect. The most significant decline in GVHD as compared with the PBS-treated controls was observed in skin, and to a lesser extent, in liver and lung. To investigate the cause of IT toxicity, anti-Thy1.2-RTA was administered intraperitoneally to lethally irradiated B10.BR (H-2k) recipients of syngeneic bone marrow. These recipients showed the same weight gain, hypoproteinuria, and VLS observed in the GVHD model. Death occurred at higher anti-Thy1.2-RTA doses (30 or 50 micrograms/daily injections administered days 8 through 12 posttransplant). Anti-Thy1.2-RTA had a negligible effect on renal function, but histologic studies showed patchy dropout of the renal tubules. Treatment resulted in pulmonary vascular congestion, but there was no pathologic evidence of liver, brain, or colon toxicity. Weight gain was enhanced by irradiation because nonirradiated normal mice did not undergo such a precipitous weight increase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Targeting haematopoietic-specific minor histocompatibility antigens to distinguish graft-versus-tumour effects from graft-versus-host disease

Allogeneic stem cell transplantation (allo-SCT) and donor lymphocyte infusions can induce durable remission in patients with haematological malignancies through a graft-versus-tumour (GvT) effect. In human leukocyte antigen (HLA)-matched settings, this powerful immunotherapeutic effect is predominantly mediated by donor T cells directed at the recipient's minor histocompatibility antigens (mHags) presented on malignant cells. The mHags are short peptides excised from polymorphic regions of intracellular proteins, and are presented by HLA molecules to donor T cells. Several ubiquitously expressed mHags are involved not only in GvT but also in graft-versus-host disease (GvHD). However, a specific set of mHags is expressed exclusively by haematopoietic cells and their malignant counterparts. Targeting these haematopoietic mHags is an attractive strategy to induce specific GvT effects without increasing the risk of GvHD. This chapter will summarize the current efforts to identify therapeutically relevant haematopoietic mHags, and outline the strategies to apply mHag-based cellular immunotherapy to treat recurrent malignancies after allo-SCT.     

Genetics of graft-versus-host disease: The major histocompatibility complex

Graft-versus-host disease (GVHD) is a potentially life-threatening complication of allogeneic hematopoietic cell transplantation. Many genes are presumed to be involved in GVHD, but the best characterized genetic system is that of the human major histocompatibility complex (MHC) located on chromosome 6. Among the hundreds of genes located within the MHC region, the best known and characterized are the classical HLA genes, HLA-A, C, B, DRB1, DQB1, and DPB1. They play a fundamental role in T cell immune responses, and HLA-A, C, and B also function as ligands for the natural killer cell immunoglobulin-like receptors involved in innate immunity. This review highlights the state-of-the art in the field of histocompatibility and immunogenetics of the MHC with respect to genetic risk factors for GVHD.     

In vivo blockade of CD28/CTLA4: B7/BB1 interaction with CTLA4-Ig reduces lethal murine graft-versus-host disease across the major histocompatibility complex barrier in mice

We tested whether the in vivo infusion of recombinant, soluble CTLA4 fused with Ig heavy chains, as a surrogate ligand used to block CD28/CTLA4 T-cell costimulation, could prevent efficient T-cell activation and thereby reduce graft-versus-host disease (GVHD). Lethally irradiated B10.BR recipients of major histocompatibility complex disparate C57BL/6 donor grafts received intraperitoneal injections of human CTLA4-Ig (hCTLA4-Ig) or murine CTLA4-Ig (mCTLA4-Ig) in various doses and schedules beginning on day -1 or day 0 of bone marrow transplantation (BMT). In all five experiments, recipients of CTLA4-Ig had a significantly higher actuarial survival rate compared to mice injected with an irrelevant antibody control (L6) or saline alone. Survival rates in recipients of hL6 or PBS were 0% at 29 to 45 days post-BMT. In recipients of CTLA4-Ig, survival rates were as high as 63% mice surviving 3 months post-BMT. However, protection was somewhat variable and recipients of CTLA4-Ig were not GVHD-free by body weight, clinical appearance, and histopathologic examination. There were no significant differences in the survival rates in comparing injection dose, injection duration, or species of CTLA4-Ig (hCTLA4-Ig v mCTLA4- Ig). Splenic and peripheral blood flow cytometry studies of long-term hCTLA4-Ig-injected survivors showed a significant peripheral B-cell and CD4+ T-cell lymphopenia, consistent with GVHD. A kinetic study of splenic reconstitution was performed in mice that received hCTLA4-Ig and showed that mature splenic localized CD8+ T-cell repopulation was not significantly different in recipients of hCTLA4-Ig compared with hL6, despite the significant increase in actuarial survival rate in that experiment. These data suggest that the beneficial effect of hCTLA4-Ig on survival is not mediated by interfering with mature donor- derived T-cell repopulation post-BMT. Neither hCTLA4-Ig nor mCTLA4-Ig interfered with hematopoietic recovery post-BMT. We conclude that CTLA4- Ig (most likely in combination with other agents) may represent an important new modality for GVHD prevention.     

Evidence for two distinct mechanisms in acute fatal graft-versus-host reaction across minor histocompatibility barriers

The roles of Lyt-1+ and Lyt-2+ T cells in the mechanisms of minor histocompatibility graft-versus-host reaction (MiHL-GvHR), as well as the influence of the source tissues from which those T cells were drawn, have been examined. Using SJL/J recipients H-2 matched to B10.S donors, the responses obtained transplanting donor spleen cells alone, spleen cells mixed with marrow, or lymph nodes mixed with marrow, and treated with anti-Thy-1, anti-Lyt-1, and/or anti-Lyt-2 monoclonal antibodies (MABs) were compared. The results indicated that both Lyt-1+ and Lyt-2+ cells may contribute to MiHL-GvHR and that, at least in part, they may play separate roles. It was also found that when the T cells were derived from the spleen, as opposed to the lymph nodes, there were substantial differences between the observed GvHR survival patterns and in the relative influences of Lyt-1+ versus Lyt-2+ cells on the resultant survival. With the spleen transplant, the Lyt-1+ cells exerted a dominant influence, but with the lymph node transplant, the influence of Lyt-2+ cells was dominant. There was also evidence to suggest the possibility of a Lyt-1 helper-cell contribution to the MiHL-GvHR exhibited by this transplant combination. Finally, it was found that the relative influences of Lyt-1+ and Lyt-2+ cells on MiHL-GvHR were expressed at two distinctly places in the survival curves, the former being seen in the early phase of acute GvHR and the latter at a later phase of the acute response.     

Selective depletion of major and minor histocompatibility antigen reactive T cells: towards prevention of acute graft-versus-host disease

Development of acute graft-versus-host disease (aGVHD) following HLA-identical sibling bone marrow transplantation (BMT) remains a serious complication. A selective depletion of T cells has proved to be effective in preventing aGVHD but is associated with relapse and increased incidence of infection. As aGVHD is directed mainly against epithelial tissues we examined whether it would be feasible to selectively deplete T cells reactive with epithelial cells whilst preserving other specificities. Donor T cells which express HLA-DR, CD25, CD69 and CD71 activation markers after cocultivation with patient keratinocytes were depleted using magnetic cell separation techniques. Depletion of major as well as minor histocompatibility antigen activated T cells revealed a significant (P = 0.004 and P = 0.031, respectively) 10-fold decrease in the frequency of donor T lymphocyte precursors reactive with patient keratinocytes. The frequency reactive with third-party and patient peripheral blood mononuclear cells, including leukaemia cells, remained unchanged, supporting the notion that aGVHD and graft-versus-leukaemia (GVL) may be separable. This alloantigen-specific depletion may be used in matched unrelated as well as HLA-identical sibling BMT for reducing aGVHD whilst conserving GVL.     

IL-10-transduced bone marrow mesenchymal stem cells can attenuate the severity of acute graft-versus-host disease after experimental allogeneic stem cell transplantation

Recent data suggest that adult mesenchymal stem cells (MSCs) might enhance allogeneic hematopoietic engraftment and prevent graft-versus-host disease (GVHD) owing to their immunosuppressive nature. Using a murine model of acute GVHD, this study examined whether or not the immunosuppressive properties of MSCs could reduce the severity of experimental GVHD. The early injection of MSCs after transplant did not attenuate the severity of acute GVHD. Therefore, this study investigated whether or not the use of IL-10-transduced MSCs (IL-10 MSCs) could reduce the severity of acute GVHD. Lethally irradiated recipients were transplanted and injected with IL-10 MSCs, the MSC-expressing vector alone (vector MSCs), or the diluent (controls), respectively, on day +1. Compared with the vector MSCs or controls, there was a significantly lower mortality in the recipients of the IL-10 MSCs at day 50 after the transplant (percent survival, 0 or 10 vs 70%, P=0.0004 or 0.0064, respectively). The decrease in mortality was confirmed by the semi-quantitative GVHD score (P<0.05), and was associated with decreased serum levels of the pro-inflammatory cytokines, IFN-gamma, on day +7 (P=0.015). Therefore, beneficial effects on GVHD were observed when MSCs were engineered to express the anti-inflammatory cytokine, IL-10.     

Xenogeneic immunosuppression of human umbilical cord mesenchymal stem cells in a major histocompatibility complex-mismatched allogeneic acute graft-versus-host disease murine model

Mesenchymal stem cells (MSCs) hold great promise for treating immune disorders owing to their immunoregulatory capacity, but the mechanism remains controversial. As we show here, the mechanism of human umbilical cord mesenchymal stem cell (HUCMSC)‐mediated immunosuppression involves TGF‐β and indoleamine 2,3‐dioxygenase (IDO). In this study, we investigated the influence of xenogeneic HUCMSCs on acute graft‐versus‐host disease (aGVHD) in murine allogeneic bone marrow transplantation (BMT). In the HUCMSC‐treated group, lethally irradiated DBA/2(H‐2Kd) mice were adoptively transferred with expanded HUCMSCs, bone marrow (BM), and splenocytes (SCs) from C57BL/6 (H‐2Kb) mice. Recipients in the control group were transferred only BM and SCs. The two groups were compared in survival, weight, histopathologic specimens, and aGVHD scoring. In the HUCMSC‐treated group, 60% of the mice survived past day 30 after BMT, but in the control group, all mice died within 18 d. The mice treated with HUCMSCs exhibited light symptoms of aGVHD after day 30. The results suggest that xenogeneic HUCMSCs could alleviate aGVHD symptoms and prolong survival after allogeneic BMT. Our study suggests that in vitro expanded HUCMSCs might be used to inhibit severe aGVHD effectively in allogeneic hematopoietic cell transplantation clinically.     

Reduction of lethal graft-versus-host disease: transplantation of cultured murine bone marrow across minor histocompatibility differences

The murine bone marrow culture technique was used to prepare donor marrow for bone marrow transplantation across minor histocompatibility complex differences. Previous studies have shown that theta-positive cells are rapidly lost from such cultures and that transplantation of cultured marrow across major histocompatibility complex differences results in a delay in the development of lethal graft-v-host disease (GVHD). In this study, a total of 1 to 2 X 10(7) nonadherent cells (740 to 1560 CFUs [colony-forming units]) from three-day-old cultures were used as a source of donor marrow. Three strain combinations were evaluated; LP/J into C57BL/6; BIO.BR into CBA/J; and C57BL/6 into LP/J. Donor mice were immunized with recipient spleen cells prior to culture in order to increase the graft-v-host response. For LP/J marrow into C57BL/6 mice, 5 X 10(7) donor spleen cells transplanted along with the marrow were needed to induce lethal GVHD. However, lethal GVHD was seen without the addition of spleen cells for BIO.BR into CBA/J and C57BL/6 into LP/J strain combinations. Most animals receiving fresh marrow were dead of GVHD five weeks after transplantation. With the use of cultured marrow the three-month survival was 80%, 51%, and 93%, respectively, for LP/J into C57BL/6, BIO.BR into CBA/J, and C57BL/6 into LP/J strain combinations. Long-term donor engraftment in all recipient animals receiving cultured marrow was confirmed by analyzing hemoglobin polymorphisms between the strain combinations. These results demonstrate that in contrast to transplantation across major histocompatibility complex differences, the use of cultured cells for bone marrow transplantation across minor histocompatibility complex differences allows for engraftment while reducing the risk of lethal GVHD.     

Real-time T-cell profiling identifies H60 as a major minor histocompatibility antigen in murine graft-versus-host disease

Although CD8 T cells are thought to be a principal effector population of graft-versus-host disease (GVHD), their dynamics and specificity remain a mystery. Using a mouse model in which donor and recipient were incompatible at many minor histocompatibility antigens (minor H Ags), the CD8 T-cell response was tracked temporally and spatially through the course of GVHD. Donor CD8 T cells in the circulation, spleen, lung, and liver demonstrated virtually identical kinetics: rapid expansion and then decline prior to morbidity. Remarkably, up to one fourth of the CD8 T cells were directed against a single minor antigen, H60. Extreme H60 immunodominance occurred regardless of sampling time, site, and genetic background. This study is the first to analyze the T cells participating in GVHD in "real-time," demonstrates the exceptional degree to which immunodominance of H60 can occur, and suggests that such superdominant minor H Ags could be risk factors for GVHD.     

Crypt loss is a marker of clinical severity of acute gastrointestinal graft-versus-host disease

BACKGROUND: Crypt loss is a histological finding in acute gastrointestinal Graft-Versus-Host Disease (GI-GvHD) of undefined clinical significance. METHODS: Colonic crypt loss was graded in twenty-three patients treated for GI-GvHD following stem cell transplantation and then correlated with clinical parameters of disease severity and mortality. RESULTS: Crypt loss was present in 17/23 cases, and in 11/23 cases crypt loss was deemed severe by the presence of contiguous areas of crypt loss. Nine of 11 patients with severe crypt loss had daily stool volumes in excess of 1000 ml/day, while only 3/12 of those with minimal or no crypt loss had this level of severe diarrhea. All 11 patients with severe crypt loss had a pathologic appearance at endoscopy and 10/11 had steroid refractory disease. Diarrhea resolved in only 3/9 patients with severe crypt loss. Five out of 10 patients (50%) with severe crypt loss expired within 15 months of diagnosis. All five deaths were attributable to the progression of GvHD itself or infection in the presence of continued GI-GvHD. Conversely, only 1 of 12 patients (8%) with mild or no crypt loss had a death attributable to GvHD or infection. CONCLUSIONS: This study shows that severe colonic crypt loss predicts severe clinical GI-GvHD that is more likely to be refractory to steroid treatment. In addition, crypt loss severity appears associated with higher mortality related to GvHD. Crypt loss can serve as a tool to predict clinically severe GI-GvHD.     

DNA synthesis in human blood mononuclear cells correlates with severity of acute graft-versus-host disease

'Spontaneous' blood mononuclear cell DNA synthesis was studied in 83 bone marrow transplantation (BMT) recipients and 58 controls. Prior to BMT, patients with chronic myeloid leukemia had increased DNA synthesis, which decreased dramatically after conditioning and transplantation. After engraftment, patients with syngeneic marrow or allogeneic marrow without graft-versus-host disease (GVHD) had increased DNA synthesis compared to healthy controls. However, patients with acute GVHD (AGVHD) had a significantly increased DNA synthesis compared to patients without GVHD (p less than 0.001). DNA synthesis increased with increasing grade of AGVHD. Among patients with severe AGVHD, recipients of HLA-mismatched marrow had higher lymphocyte DNA synthesis at diagnosis of GVHD and maximum values compared to HLA-matched siblings (p less than 0.05). At diagnosis of GVHD, patients who developed grades II-IV GVHD with progressive disease had higher DNA synthesis, 23.9 +/- 4.0 x 10(3) c.p.m. (mean +/- SE) compared to 11.1 +/- 2.7 x 10(3) c.p.m. in patients in whom GVHD resolved (p less than 0.02). DNA synthesis during GVHD was lower in sheep erythrocyte rosette-forming cells (E-RFC) compared to enriched non-E-RFC. Herpes simplex virus, cytomegalovirus, bacterial septicemia and chronic GVHD had no major effect on lymphocyte DNA synthesis in these patients.     

Human marrow T cell dose correlates with severity of subsequent acute graft-versus-host disease

Sixteen patients with haematological malignancy received high-dose chemotherapy or chemotherapy and total body irradiation followed by an HLA-identical sibling marrow transplant from which the T lymphocytes had been depleted prior to infusion by incubation with an anti-CD2 anti-T cell antibody with (seven patients) or without (nine patients) an anti-CD8 anti-T cell antibody together with rabbit complement. Additionally, all patients received cyclosporin. The number of T cells present in the donor marrow was determined by limiting dilution analysis, and was found to correlate with the subsequent incidence and severity of acute graft-versus-host disease (GVHD). The number of T cells infused into patients with no acute GVHD or with minimal acute GVHD of the skin (skin rash present for 14 days or less) was 1.3 +/- 1.0 x 10(5)/kg, while the number infused into those with moderate acute GVHD or with skin acute GVHD present for 15 days or more was 12.3 +/- 11.5 x 10(5)/kg (p less than 0.001). Thus a dose of 10(5) (or less) T cells/kg was associated with minimal or no acute GVHD, while 10(6) T cells/kg (or more) caused significant disease.     

Histological changes of bile duct in experimental graft-versus-host disease across minor histocompatibility barriers II. Electron microscopic observations

ABSTRACT— Electron microscopic features of intrahepatic bile ducts of experimental mouse graft-versus-host disease (GVHD) across minor histocompatibility barriers were studied for 14 months after transplantation. In GVHD mice, the bile duct epithelial layer was consistently infiltrated by lymphoid cells and often accompanied by polymorphonuclear leukocytes, monocytes and rarely by plasma cells. The epithelial cells in close contact with and in the vicinity of these infiltrated cells showed a variety of degenerative changes, including darkness of the cytoplasm and the nucleus with shrunken, irregular contours, increase in the amount of endoplasmic reticulum and number of mitochondria, and formation of intracytoplasmic vesicles and diverticula, cytoplasmic blebs, and apoptopic bodies. Lymphocytes were in close contact with epithelial cells through a number of point-contacts and located in the lateral intercellular spaces and/or between the basement membranes and the epithelial cells. The localization of infiltrating lymphocytes beneath the epithelial cells with conspicuous detachment from the basement membranes strongly suggested a link with subsequent epithelial cell injury and death. The lymphoid cells had irregular cytoplasmic projections which occasionally extended into spaces created by retractions of the epithelial cell membranes, reflecting an activation of the lymphocytes. These findings support the notion that the bile duct lesions in GVHD across minor histocompatibility barriers are mediated by specifically sensitized lymphocytes against epithelial cell membrane antigens. From our previous finding that a large majority of the infiltrating lymphocytes had a phenotype of helper/inducer T cells, a putative role of these lymphocytes in the induction of the bile duct lesions is discussed.     

Histological changes of the liver in experimental graft-versus-host disease across minor histocompatibility barriers. VIII. Role of eosinophil infiltration

Abstract: Although eosinophil infiltrate has been recognized in hepatic graft-versus-host disease, its significance in relation to hepatic graft-versus-host disease lesions is unknown. In the present study, we analyzed hepatic eosinophil infiltration in relation to bile duct damage in experimental mouse graft-versus-host disease across minor histocompatibility barriers up to 14 months after transplantation. Portal eosinophil infiltration was found from 1 week after transplantation throughout the entire 14-month observation period. It was most striking during the early chronic stage of hepatic graft-versus-host disease between 2 to 7 months, with a peak at 5 months after transplantation. Microscopic and electron microscopic study revealed eosinophils infiltrated around the bile duct as well as in the bile duct epithelial layer. They were commonly found together with lymphocytes but were also occasionally found singly around the bile duct and in the bile duct epithelial layer. Bile duct epithelial cells in contact with and in the vicinity of eosinophils showed a variety of degenerative changes, occasionally associated with the presence of extracellular eosinophil granules. Bile duct epithelial cells with eosinophil infiltration just beneath the basement membrane frequently showed further characteristic severe degenerative changes with shedding or dropping-off into the lumen, which features were quite similar to those seen in the bronchial epithelium in asthma patients. These results indicate that not only lymphocytes but also eosinophils may be involved in the production of the bile duct injury in hepatic graft-versus-host disease, especially in its early chronic stage.     

Histological changes of the liver in experimental graft-versus-host disease across minor histocompatibility barriers. IV A study of lymphocyte-endothelial interaction

ABSTRACT— It has been reported in human hepatic graft-versus-host disease (GVHD) that an attachment of lymphocytes to vascular wall, the feature called “endothelialitis”, is the most important predictive histologic sign of GVHD. However, its precise nature and significance in GVHD are still unknown. We developed experimental mouse GVHD across minor histocompatibility barriers and examined the lesion during a 14-month period after transplantation. The lesion was transiently found, appearing first at 4 days after transplantation, reaching a maximal level at 2 weeks and disappearing 5 weeks after transplantation. Electron microscopically, an intimate interaction between lymphocyte and endothelial cell was demonstrated. Lymphocytes showed irregular cytoplasmic processes and pseudopods and were in close contact with endothelial cells. Lymphocytes frequently penetrated in between and under the endothehal cells, and migrated into the perivascular spaces. Immunohistochemical analysis revealed that the vast majority of lymphocytes attached to the endothelial cells are helper/inducer T cells, indicating the cardinal role of helper/inducer T cell in lymphocyte-endothelial cell interactions. These results, together with previous evidence of the presence of Ia antigens and an antigen-presenting ability of vascular endothelial cells, suggest that the attachment of lymphocytes to the vascular endothelial cells in the early course of GVHD may represent an in situ morphologic representation of antigen presentation by endothelial cells to helper T cells.