Total donor chimerism with bone marrow GVHD after multivisceral transplantation
Authors: Vincent Alcazer, Assia Eljaafari, Laure Lebras, Valérie Dubois, Adriana Plesa, Mohamad Sobh, Arnaud Hot, Jérôme Dumortier, Olivier Boillot, Cécile Chambrier, Mauricette Michallet
Journal: American Journal of Hematology (2018)

A 62-year-old woman was admitted to our institution for a small intestine volvulus with intestinal necrosis. After 6 months of parenteral nutrition, she developed a severe intestinal failure-associated liver disease for which a multivisceral transplantation including liver, stomach, pancreas, spleen, small intestine and colon was performed. The donor was ABO compatible with an incompatibility in Rhesus E (E+ donor vs E- recipient). Human leukocyte antigen (HLA) typing by PCR-SSP revealed a full mismatch (1A, 2B, 2DR, and 2DQ). A Cross match assay was positive and pre-formed antibodies directed against donor's HLA antigens B55 and DR4 were identified. Two months after transplantation, a pancytopenia was observed with a marked neutropenia (<0.5 G/L). A former bone marrow (BM) aspiration was performed, showing that the 3 cell-lineages were well represented and regenerative without any hemophagocytosis. Pancytopenia was considered multifactorial, mainly related to malnutrition, antivirals, and immunosuppressives therapies. While discontinuation of valganciclovir and cotrimoxazole associated with Granulocyte Colony-stimulating factor (G-CSF) injections allowed white and red blood count improvement, a severe thrombocytopenia (<50 G/L) persisted. Methylprednisolone was started at 1 mg/kg per day without any improvement leading to the introduction of intravenous immunoglobulin at 1 g/kg for 2 days, which allowed reaching a sustained platelet count of >50 G/L. Cytopenias progressively recovered, allowing progressive tapering of corticosteroids. Two years after transplantation, the patient developed an isolated, acute, marked normocytic, and regenerative anemia resistant to transfusions. An active hemolysis was detected with undetectable haptoglobin and a positive direct antiglobulin test, strongly positive for anti C3d and weakly positive for anti IgG. Indirect antiglobulin test showed high levels of autoantibodies directed against erythrocytes (anti A, D, C, E, and e). Further analysis revealed that the patient blood group had changed from A Rh D+ C+ E− c+ e+ K− to A Rh D+ C+ E+ c+ e+ K−, without any recent transfusion during this period. The complete erythrocyte phenotype confirmed the presence of a double population since 2 distinct antigens Jka (JK1) and s (MNS4), were detected, corresponding to the donor and recipient blood group, respectively. Short tandem repeat analysis of chimerism by PCR-SSP revealed a total donor chimerism on both blood and BM, with 0% recipient cells on total blood, 0.2% on CD3+ peripheral cells and 0.85% on CD34+ BM cells. In this context of macrochimerism, a BM graft-vs-host disease (GVHD) was suggested to account for the anemia and previous cytopenias. Supplementary chimerism analyses confirmed the recipient origin of BM–mesenchymal stem cells (MSCs) and skin fibroblasts used as control and the donor origin of CD3+ BM-T cells. To assess the cytotoxicity of donor T cells against recipient BM-MSC, a cytotoxic assay was performed. Effectors T cells were harvested from BM and cultured with 10UI/ml rIL-2 (Thermo Fischer Scientific, Waltham, USA) for 8 days, and labeled for CD8 (FITC, Beckman Coulter, Brea, USA) and HLA-DR (PE-A, Beckman Coulter) and analyzed by flow cytometry before use. Host MSC were harvested from BM aspiration, expanded in Alpha Minimum Essential Medium (MEM) (Stemcell technologies, Grenoble, France) with 10% fetal bovine serum (FBS) (ThermoFIscher Scientific). As controls, recipient or allogeneic (full HLA-mismatch) skin fibroblasts were used as targets. Host fibroblasts were harvested from a skin biopsy and expanded in Dulbecco's Modified Eagle Medium (DMEM) (ThermoFIscher Scientific) with 10% FBS. Targets were labeled with BADTA reagent (PerkinElmer, Villepinte, France), washed, and incubated with effectors. The released ligand was revealed with Europium fluorescent chelator DELFIA (PerkinElmer). Cytotoxic assays showed a strong cytolysis of BM–MSC by BM-T cells (Figure 1A). As control, recipient but not allogeneic skin fibroblasts were lysed by BM-T cells, demonstrating the specificity of BM-T cells towards recipient HLA antigens. Corticosteroids (1 mg/kg daily) and intravenous immunoglobulins (1 g/kg for 2 days) allowed an increase in the red blood count without long-lasting effect. Weekly Rituximab perfusions were eventually prescribed and efficiently stabilized anemia. The decrease of both BM-MSC cytolysis and HLA-DR expression on BM-CD8 T cells argued for a Rituximab-mediated therapeutic effect on BM-GVHD (Figure 1B,C). Sustained total donor chimerism allowed progressive corticosteroid tapering with tacrolimus maintenance for BM-GVHD. The patient unfortunately developed successive infectious episodes including disseminated candidiasis and septic shock. The patient was admitted in August 2010 in intensive care for an encephalopathy with a cerebral thrombophlebitis and died in September 2010 from cerebral hemorrhage. At our knowledge, this is the third case of spontaneous macrochimerism after solid organ transplantation reported in literature. Collins et al. reported in 1993, the case of donor-derived hematopoiesis in a liver-transplant recipient.1 Alexander et al.2 reported a complete hematopoietic chimerism in a 9-year-old girl after a liver transplantation. In both cases, the transplantation included the liver, an organ well-known to contain hematopoietic stem cells (HSCs), as a result of foetal liver hematopoiesis. In our case, the spleen was also transplanted. The presence of HSC in spleen has been first shown in cadaveric donors. Although containing few HSC in the steady state, it has been shown that macrophages can retain them in the spleen in an inflammatory context. This mechanism may be a part of the so-called “extramedullary hematopoiesis”, which contributes to leukocyte oversupply at sites of inflammation and has been described in other diseases such as atherosclerosis or myocardial infarction. These 3 cases underline the possibility of a total donor chimerism in the absence of myelosuppressive or intensive lymphosuppressive treatments nor co-infusion of donor HSC. The mixed red cell population, in contrast with a predominant donor chimerism within white cells, is reminiscent of previous observations in patients benefiting from a combined kidney and BM transplantation.3 These patients exhibited a mixed donor/host blood type despite full donor chimerism in peripheral blood. The low-intensity conditioning used may have allowed retention of hematopoietic progenitor and stem cells, similarly to our case, which can be targeted by donor's T cells. With their HSC nursing function and their immunomodulatory effect, MSC are being extensively studied particularly in the context of BM-GVHD.4 Importantly, as opposed with BM-HSC, MSC are known to remain of host origin irrespective of the conditioning regimen and graft source, and are thus a putative target source for GVHD. Whereas as yet in human, only a loss of BM-osteoblasts (resulting in decreased numbers of CD19+ but increased CD3+ cells5) has been shown to be associated with certain forms of chronic GVHD, herein we have observed that host BM-MSC can be directly targeted by BM T cells of donor origin. Interestingly, we have observed a Rituximab-mediated inhibition of BM GVHD. Whereas anti-CD20 therapy has shown its efficacy in patients with chronic refractory GVHD where uncontrolled B-cell activation is a main feature, B cell activation was not likely to be present in our clinical case, since BM-aplasia concerned both B and T cells. Thus, in addition to Christopeit et al.6 previous report, our study suggests a beneficial effect of Rituximab in this situation. The authors would like to thank all their respective department who participated in the patient's care. The authors declare that they have no conflicts of interest with the contents of this article.