Subclinical Graft-versus-host Disease Research Articles

Case Report: Liver as a Source of Hematopoietic Stem Cells After Liver Transplantation Following Hematopoietic Stem Cell Transplantation

We report a child with Fanconi anemia who, after hematopoietic stem cell transplantation (HSCT) complicated by acute graft-versus-host disease (GVHD), underwent orthotopic liver transplantation (OLT). Approximately 1 month after OLT, the presence of third-party genetic material from the liver donor was noted and in the next few weeks, the chimerism assessment revealed 100% liver donor leukocytes in the peripheral blood. The rapidly progressing GVHD with gut involvement resulted in patient’s death 6 months after OLT. The liver can act as a clinically significant source of hematopoietic stem cells, and the liver donor’s young age must be emphasized as potentially predisposing to this phenomenon. Transfer of OLT hematopoietic stem cells may not have clinical significance unless the patient is not immunocompetent or develops liver-transplantation associated GVHD, that can result in lymphocyte mediated elimination of original hematopoiesis. Patients with preexisting immunity disorder (such as primary or secondary immunodeficiency) might require intensified immunosuppressive therapy in peritransplant period as a prevention of liver-transplantation associated GVHD. Close monitoring of hematopoietic chimerism after OLT is warranted in patients at risk, because cytopenia or OLT hematopoiesis can reflect subclinical GVHD and further studies are necessary to elucidate this phenomenon.

Histopathological Grading of Oral Mucosal Chronic Graft-versus-Host Disease: Large Cohort Analysis.

Graft-versus-host disease (GVHD) can manifest as acute or chronic complications in patients after hematopoietic cell transplantation (HCT). Oral chronic GVHD (cGVHD) occurs in approximately 70% of HCT recipients and includes lichenoid-like mucosal reactions, restricted mouth opening, and salivary gland dysfunction. However, the underlying histopathological presentation remains to be validated in large cohorts. We characterized the histopathological features of oral mucosal cGVHD and devised a scoring model in a large patient cohort (n=112). Oral mucosal biopsy sections (n=303) with and without oral cGVHD were identified from archived and current HCT recipients with additional healthy controls. Histological screening was performed on hematoxylin and eosin-stained and periodic acid-Schiff-stained sections. A points-based grading tool (0 to 19, grade 0 to IV) was established based on intraepithelial lymphocytes and band-like inflammatory infiltrate, atrophic epithelium with basal cell liquefaction degeneration, including apoptosis, as well as separation of epithelium and pseudo-rete ridges. Validation involved 62 biopsy specimens, including post-HCT (n=47) and healthy (n=15) specimens. Remaining biopsy specimens (n=199) were blindly graded by 3 observers. Histological severity was correlated with clinical diagnostic and distinctive features, demonstrating a spectrum of individual patient severity, including frequent signs of subclinical GVHD in healthy mucosa. However, oral cGVHD presented with significantly higher (P < .001) scores compared with HCT controls, with moderate to high positive likelihood ratios for inflammatory infiltrate, exocytosis, and basal membrane alterations. The grade II-IV biopsy specimens demonstrated a histopathological diagnosis of active mucosal lichenoid-like cGVHD, highlighting the importance of correlating clinical presentation with the dynamic histopathological processes for improved patient stratification. In addition, this tool could be used for assessing treatments, pathological processes, and immune cellular content to provide further insight into this debilitating disease.

Increased splenic human CD4+:CD8+ T cell ratios, serum human interferon-γ and intestinal human interleukin-17 are associated with clinical graft-versus-host disease in humanized mice

Graft-versus-host disease (GVHD) is a frequent complication following allogeneic hematopoietic stem cell transplantation (HSCT) with current therapies limited to general immunosuppression. Humanized mouse models of GVHD are emerging as valuable intermediaries to allow translation of findings from allogeneic mouse models to humans to prevent and treat this disease, but such models require further characterization. In this study, humanized mice were generated by injecting immunodeficient non-obese diabetic severe combined immunodeficiency interleukin (IL)-2 receptor γ common chain null (NSG) mice with human peripheral blood mononuclear cells (hPBMCs). Clinical GVHD development was assessed using established scoring criteria (weight loss, posture, activity, fur texture and skin integrity). Differences between humanized NSG mice that developed clinical or subclinical GVHD were then compared. Both groups of mice demonstrated similar frequencies of human leukocyte engraftment. In contrast, mice that developed clinical GVHD demonstrated increased histological damage compared to mice with subclinical GVHD. Furthermore, mice with clinical GVHD exhibited increases in the splenic human CD4+:CD8+ T cell ratio, serum human interferon (IFN)-γ and intestinal human IL-17 expression compared to mice with subclinical GVHD. These cellular and molecular changes could be used as potential markers of disease progression in this preclinical model. This study also provides further insights into GVHD development which may be relevant to human HSCT recipients.

Thiol/Redox Metabolomic Profiling Implicates GSH Dysregulation in Early Experimental Graft versus Host Disease (GVHD)

Graft-versus-host disease (GVHD) is a common complication of allogeneic bone marrow transplantation (BMT). Upregulation of inflammatory cytokines precedes the clinical presentation of GVHD and predicts its severity. In this report, thiol/redox metabolomics was used to identify metabolic perturbations associated with early preclinical (Day+4) and clinical (Day+10) stages of GVHD by comparing effects in Syngeneic (Syn; major histocompatibility complex- identical) and allogeneic transplant recipients (Allo BMT) in experimental models. While most metabolic changes were similar in both groups, plasma glutathione (GSH) was significantly decreased, and GSH disulfide (GSSG) was increased after allogeneic compared to syngeneic recipient and non-transplant controls. The early oxidation of the plasma GSH/GSSG redox couple was also observed irrespective of radiation conditioning treatment and was accompanied by significant rise in hepatic protein oxidative damage and ROS generation. Despite a significant rise in oxidative stress, compensatory increase in hepatic GSH synthesis was absent following Allo BMT. Early shifts in hepatic oxidative stress and plasma GSH loss preceded a statistically significant rise in TNF-α. To identify metabolomic biomarkers of hepatic GVHD injury, plasma metabolite concentrations analyzed at Day+10 were correlated with hepatic organ injury. GSSG (oxidized GSH) and β-alanine, were positively correlated, and plasma GSH cysteinylglycine, and branched chain amino acids were inversely correlated with hepatic injury. Although changes in plasma concentrations of cysteine, cystathionine (GSH precursors) and cysteinylglycine (a GSH catabolite) were not significant by univariate analysis, principal component analysis (PCA) indicated that accumulation of these metabolites after Allo BMT contributed significantly to early GVHD in contrast to Syn BMT. In conclusion, thiol/redox metabolomic profiling implicates that early dysregulation of host hepatic GSH metabolism and oxidative stress in sub-clinical GVHD before elevated TNF-α levels is associated with GVHD pathogenesis. Future studies will probe the mechanisms for these changes and examine the potential of antioxidant intervention strategies to modulate GVHD.

The Impact of Early Viral Infections and Graft‐Versus‐Host Disease on Immune Reconstitution Following Paediatric Stem Cell Transplantation

Viral infections and graft-versus-host disease (GVHD) render an impact on both the clinical and immunological recovery following allogeneic hematopoietic stem cell transplantation (HSCT). We studied the recuperation of the immune defence after transplant in the paediatric setting and assessed the impact of early (<100 days post-HSCT) viral [cytomegalovirus (CMV), Ebstein-Barr virus (EBV) and adenovirus] reactivations/infections and GVHD. Fifty-one paediatric recipients of HSCT were enrolled. T cell recovery was evaluated on lymphocyte subpopulations using flow cytometry and functionally by measuring T cell excision circles (TRECs) and through the analysis of T lymphocyte responses to mitogens. B cell recovery was studied by flow cytometry and functionally by ELISPOT. Acute and mild chronic GVHD allowed for a brisk recovery of both cellular and humoral immunity while moderate to severe chronic graft-versus-host disease (cGVHD) associated with a significant, tampering effect on the immunological recovery after transplant. In the former group, the early viral reactivations/infections seemingly linked with a delayed recovery of T lymphocytes and low TRECs values. Moderate to severe cGVHD appears to associate with an impaired immunological recovery after HSCT. Early viral infections linked with prolonged T cell immunodeficiency and thymic dysfunction may be indicative of the presence of subclinical GVHD.

ED‐1 is a marker for activated macrophages and subclinical graft versus host disease in vascularized stem cell transplants

Acute and chronic graft‐versus‐host disease (GVHD) is a major complication of bone marrow transplantation (BMT). Overt histopathological findings of GVHD are well documented. However, histopathology of subclinical GVHD is uncertain. We are studying a new type of BMT that can facilitate tolerance induction without GVHD. We hypothesized that subclinical GVHD may be induced. The model is known as a vascularized bone marrow transplant (VBMT).Lewis‐Brown‐Norway (LBN) rats radiation. Rats then received either a cellular (cBMT, n=5) or VBMT (n=4). After 110 days, splenic histopathology was assessed by H&amp;E staining and digital image analysis.Then splenic specimens were studied using Immunohistochemical staining for ED‐1 macrophage cell surface antigen.Digital image analysis revealed very little hemosiderin present in the control spleens (mean ISI 0.86) and a large amount of hemosiderin in the cBMT spleens (mean ISI 11.75). The VBMT had an intermediate level of hemosiderin (mean ISI 8.27). All groups were significantly different (p&lt;0.05). Qualitatively, it was noted that increased amounts of ED‐1 positive macrophages were present in cBMT spleens and VBMT spleens undergoing subclinical GVHD, compared to normal spleens.Hemosiderin laden and activated macrophages may represent markers for sub‐clinical GVHD due to red blood cell destruction through opsonization associated with graft versus host disease.

Purified hematopoietic stem cell allografts reconstitute immunity superior to bone marrow

Antigen-specific immune responses are impaired after allogeneic hematopoietic cell transplantation (HCT). The events contributing to this impairment include host hematolymphoid ablation and donor cell regeneration, which is altered by pharmacologic immune suppression to prevent graft-versus-host disease (GVHD). A generally accepted concept is that graft T cell depletion performed to avoid GVHD yields poorer immune recovery because mature donor T cells are thought to be the major mediators of protective immunity early post-HCT. Our findings contradict the idea that removal of mature donor cells worsens immune recovery post-HCT. By transplantation of purified hematopoietic stem cells (HSC) compared with bone marrow (BM) across donor and recipient pairs of increasing genetic disparity, we show that grafts composed of the purified progenitor population give uniformly superior lymphoid reconstitution, both qualitatively and quantitatively. Subclinical GVHD by T cells in donor BM likely caused this lympho-depleting GVHD. We further determined in the major histocompatibility complex (MHC)-mismatched pairs, that T cell restricted proliferative responses were dictated by donor rather than host elements. We interpret these latter findings to show the importance of peripheral antigen presentation in the selection and maintenance of the T cell repertoire.

363: Early Detection of Severe Acute Graft-Versus-Host Disease using Plasma Markers of T-cell Activation

Severe acute graft-versus-host disease (GVHD) is a significant cause of transplant related mortality in patients that receive allogeneic hematopoietic stem cell transplantation (HSCT). Treatment of grade III and IV GVHD is frequently ineffective. Preemptive treatment based on the early detection of cytokines associated with T-cell activation and subclinical GVHD might be a more effective strategy, similar to the monitoring of CMV infection after HSCT.

Pure Hematopoietic Stem Cells Reconstitute Immunity in Allogeneic Hosts Superior to Bone Marrow: Immunosuppression by Subclinical Graft-Versus-Host Disease.

Antigen specific immune responses are known to be impaired following allogeneic hematopoietic cell transplantation (HCT). Some clinical studies suggest that graft T cell depletion for the prevention of graft-versus-host disease (GVHD) leads to poorer immune recovery, while other correlate GVHD with delayed immune reconstitution. Our studies sought to examine the degree to which the co-transplantation of GVHD-inducing mature cells mediated protective immunity post-HCT. We compared the transplant of FACS purified hematopoietic stem cells (HSC: cKit+Thy1.1loSca+Lin-) with bone marrow (BM) between congenic (BA to B6Ly5.2), minor-antigen mismatched (BA to BALB.B), haploidentical (BAxSWR F1 to BALB/cxSWR F1) and MHC-mismatched (BA to BALB/c) donor and host pairs. We show that grafts composed solely of purified HSC give uniformly superior lymphoid reconstitution across all mismatched pairs, both qualitatively and quantitatively. Although absolute blood lymphocytes counts were increased in recipients of BM compared to HSC, lymphoid reconstitution as measured by lymph node size, counts and architecture was significantly improved in the HSC groups regardless of minor or major mismatches between donor and host. Proliferative responses to the allele specific peptides of the antigen hen egg lysozyme were also significantly increased in the HSC as compared BM recipients (p=0.028), with fully MHC mismatched BM recipient cells showing almost no proliferative response. The use of MHC allele specific antigens also revealed that T cell responses post-HCT are dominated by donor-restricted elements. These data suggest that subclinical GVHD mediated by mature cells in the donor BM result in impaired immune reconstitution. While there may be an increase in absolute lymphocyte numbers, this increase does not correlate with an increase in immune cell function. These findings provide important insight into the benefits of purified HSC for preventing post-HCT infectious complication in addition to its known GVHD prophylaxis benefits. Our data highlights the benefit of transplanting a pure HSC population, as the relatively small number of T cells that are found in mouse bone marrow or that remain in the graft after T cell depletion in humans can still result in immune impairment due to subclinical GVHD.

Hemosiderin‐laden macrophages: a marker for sub‐clinical graft‐versus‐host‐disease (GVHD) after bone marrow transplantation

Introduction: Acute and chronic graft-versus-host disease (GVHD) is a major complication of bone marrow transplantation. Overt histopathological findings in GVHD are well documented. However, histopathology of subclinical GVHD is uncertain. We have been studying a new type of bone marrow transplant in rats that can facilitate tolerance induction without overt GVHD. It was hypothesized that subclinical GVHD may be induced. The model, known as a vascularized bone marrow transplant (iVBMT), involves the microsurgical transplantation of a bone marrow composite graft. Methods: Lewis-Brown-Norway (LBN) rats received 900 rads radiation. Rats then received either a conventional cellular (cBMT, n=5) or iVBMT (n=4). After 110 days, organ histopathology was studied by digital image analysis. Results: There was a significant increased concentration of hemosiderin found within the splenic of cBMT rats compared to iVBMT rats (mean ISI 11.75 vs. 8.27; p=0.009). Conclusion: The increased concentration of hemosiderin-laden macrophages in spleens of cBMT rats compared to iVBMT rats may represent sub-clinical GVHD in which red blood cells are being destroyed due to the disease process. No evidence of sub-clinical GVHD was present in the iVBMT group. This supports previous findings suggesting that an iVBMT modulates GVHD and induces tolerance through an undefined mechanism. Supported by American Heart Association Grant #0555795T. Dr Hewitt is recipient of the Ellen Marmer research fellowship from the AHA for 2006.

Influence of the additional injection of host-type bone marrow on the immune tolerance of minor antigen-mismatched chimeras: possible involvement of double-negative (natural killer) T cells.

It has previously been demonstrated that adding T cell-depleted (TCD) host bone marrow (BM) to an MHC-mismatched BM inoculum allows for induction of long-term stable chimeras without graft-versus-host disease (GVHD) even when non-TCD allogeneic BM was used. The present study was undertaken to investigate immune tolerance mechanisms in minor antigen-mismatched allogeneic BM chimeras when host-type BM was added to the BM inoculum. C3H (H2k, Thy 1.2, Mls 2a) recipients were conditioned with 9.5 gray (Gy) of total body irradiation. To exclude any interference with possible subclinical GVHD, 5x10(6) TCD AKR (H2k, Thy 1.1, Mls 1a) BM cells were injected with (syn + allo) or without (allo) 5x 10(6) TCD C3H BM cells. Chimerism, clonal deletion, and T lymphocyte subsets were scored using FACS and anti-mouse Thy, Vbeta6, Vbeta3, CD3, CD4, or CD8 monoclonal antibodies. The stability of tolerance was studied by investigating mixed lymphocyte reaction and cytotoxic T cell induction in chimeras after immunization with host, donor, or third-party (BALB/c) splenocytes. Breaking of chimerism was attempted by injecting nontolerant 40x10(6) host-type splenocytes 2 months after BM transplantation. Cytokines and Valpha14 mRNA were assayed using real time quantitative reverse transcriptase-polymerase chain reaction at 4 and 48 hr, respectively, after injection of nontolerant host-type splenocytes. Both groups of mice became long-term stable mixed chimeras without any clinical sign of GVHD. Neither group was able to produce antihost nor antidonor cytotoxic T cells, even after immunization. The addition of syngeneic BM to the allogeneic inoculum reduced the overall level of allogeneic chimerism (from approximately 70% or approximately 85% in peripheral blood lymphocytes and spleen, respectively, in allo chimeras versus approximately 35% and approximately 60% in syn + allo chimeras). Moreover, it resulted in complete clonal deletion of both host-reactive (Vbeta3) and donor-reactive (Vbeta6) lymphocytes in syn + allo chimeras in contrast to in allo chimeras, in which only donor-reactive lymphocytes were completely deleted. After nontolerant C3H splenocyte injection, high levels of interleukin 2 mRNA were produced and chimerism decreased in syn + allo chimeras. In contrast, in allo chimeras, this maneuver was followed by the production of higher levels of interleukin 4 and interferon-gamma, and of Valpha14 mRNA, as well as by the proliferation of CD3+CD4-CD8- (double-negative) T cells and by an increase of donor chimerism. The addition of host-type BM to the allogeneic inoculum has an influence on the level of chimerism, the extent of clonal deletion, and the reaction of chimeras after the injection of nontolerant host-type splenocytes. In the latter phenomenon, cytokine production and proliferation of Valpha14+ CD3+CD4-CD8- (double-negative, natural killer T) lymphocytes may be involved.