Hematopoietic Regeneration Is Suppressed by the GvHD Condition in a Haplo-Identical Bone Marrow Transplant Model.

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Abstract 1343Poster Board I-365Acute graft-versus-host disease (aGVHD) remains a major obstacle for successful allogeneic bone marrow transplantation (BMT). Pancytopenia is frequently observed in the patients with aGVHD and contributes to poor prognosis. However, the mechanisms for pancytopenia are not well understood. To study how hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) are affected by aGVHD, we have examined the kinetics and functional alterations of HSC and HPC in a haplo-identical BMT model, in which parent lymphocytes were transferred into F1 offerspings (P1→F1) for induction of aGVHD. 6×107 splenocytes together with 5×106 bone marrow nucleated cells (BMNCs) of B6.SJL (B6, CD45.1+) were transplanted into lethally irradiated B6 x Balb/c F1 mice (CB6F1, CD45.1/.2). Same numbers of BMNCs without splenocytes were transplanted into the irradiated recipients as controls. In this aGVHD model, the average pathological score for aGVHD was 3.2±0.3 at day 14 after transplantation. The donor-derived hematopoietic cells (CD45.1+) were isolated for phenotypic analyses and functional assays at different time points. As expected, reduced peripheral blood cell counts and bone marrow (BM) cellularity were observed in the aGVHD hosts. aGVHD hosts showed more severe deficiencies [(0.95±0.2) colony forming unit (CFU) /105 BMNCs and 1 cobblestone area forming cell (CAFC) /106 BMNCs] than the controls without aGVHD [(1.75±0.3) CFU /105 BMNCs and 1 CAFC/3×105 BMNCs]. The frequency of HSC-enriched population (CD150+CD48−Lin−) in the aGVHD hosts [(1.05±0.38)x10−5] was significantly lower than that in the controls [(1.97±0.21)x10−5] at day 14 (P=0.03). When normalized to the bone marrow cellularity, the absolute yield of this population was decreased 2.89-fold in the aGVHD hosts than that in the control group. To measure the long-term engraftment of HSCs isolated from aGVHD hosts, we performed the secondary BMT, in which 2 × 106 of donor-derived BMNCs (CD45.1+) isolated from the aGVHD hosts or control animals at day 14 after the induction of aGVHD were transplanted into lethally irradiated CB6F1 recipients. Unexpectedly, the multilineage engraftment of the hematopoietic cells from the aGVHD hosts was 2 times more than that of the control group [(21.24±4.21)% vs (9.14±2.54)%,P<0.01]. Due to the decreased frequency of HSC in aGVHD hosts before sBMT, the repopulation potential per HSC was thus not compromised. To explore the mechanisms of such suppression on hematopoiesis in the aGVHD model, we quantified the T cell subset that has been thought to be the major cellular cause for aGVHD. Compared with control, the CD3+CD8+ subset was significantly expanded in the BM of aGVHD host from the 5th day after aGVHD induction [(59±8)% vs (5±1.2)%]. At day 14, the percent of CD3+CD8+ population increased to 67% and stabilized around 62% within 3 months. Moreover, the level of cytokine IFN-γ dramatically increased in the aGVHD hosts [(163±12.86) ng/mL vs (98±7.64) ng/mL]. The same trend was observed with TNF-α. These data suggested that the alloreactive T cells may entail a severe aGVHD reaction and be responsible for the defective regeneration of hematopoiesis. Finally, we utilized BrdU incorporation to track the proliferation of hematopoietic primitive cells (Lin−Sca-1+) in vivo. In aGVHD hosts, about (34.7±4.2)% Lin−Sca-1+ cells were BrdU+ at 16h, while ∼20% in control remained BrdU+. Donor-derived Lin−Sca-1+ cells did not show an increased rate of apoptosis in aGVHD hosts. Since Lin−Sca-1+ population is capable of proliferation to generate the progenies to reconstitute hematopoiesis, the data shows that under the aGVHD condition, the defective donor hematopoiesis was not associated with increased apoptosis and instead, the progenitor cells are more active in cell cycle, ultimately resulting in hematopoietic exhaustion. In summary, our current work provides definitive evidence for the inhibition of normal hematopoietic regeneration by aGVHD and its plausible underlying mechanisms, thereby having important implications for clinical management of aGVHD. DisclosuresNo relevant conflicts of interest to declare.