Engraftment Failure After Non-Myeloablative Hematopoietic Stem Cell Transplantation: Donor CD4 Cells Interfere with Residual Host Cells and Can Trigger Rejection of the Graft.

Abstract 3528

Poster Board III-465

In the last decade the field of allogeneic hematopoietic cell transplantation has made significant strides in the reduction of morbidity and mortality using non-myeloablative conditioning. However, with reduced conditioning intensity the risk of allograft rejection has increased due to the persistence of host cells that mediate host-vs-graft alloreactivity or that occupy niches in the host hematopoietic microenvironment. Here we studied hematopoietic reconstitution and chimerism after non-myeloablative total body irradiation (TBI: 4 Gy [sublethal]; 7 Gy [lethal]) or total lymphoid irradiation (TLI: 17×240 cGy) in a MHC-matched mouse model with known high resistance to engraftment. BALB.K (H-2K) hosts received FACS-purified hematopoietic stem cells (HSC: cKIT+Thy1.1loSca1+Lin-) alone or supplemented with T cells (TC) from AKR/J (H-2K) donors. Peripheral blood (PB), bone marrow (BM), and spleen (SP) were analyzed 2 and 4 weeks (w) post transplant (pTX), and PB chimerism was followed beyond day (d) 100. Mice conditioned with lethal TBI survived, when grafts contained HSC only, and became mixed chimeric for TC, but converted to primarily donor type for B cells and the myeloid lineage. When grafts were supplemented with TC, chimerism converted to full donor type, but hosts developed fulminant acute GVHD. In contrast, sublethal TBI resulted in only mild symptoms of GVHD and mice recovered rapidly. Recipients of HSC alone or HSC+CD8 TC became mixed chimeras in all lineages, even long-term (>100d). However, mice given grafts of either HSC with total TC, or HSC+CD4 TC remained host type for T and B cells, and achieved only low levels of donor engraftment in the myeloid lineage. These findings suggested that the addition of peripheral donor CD4 TC resulted in powerful lymphoid suppression as well as retardation of myeloid engraftment. Indeed at 2w pTX recipients of HSC+CD4 TC displayed severe lymphopenia (<5% of live cells [vs >40% in recipients of HSC alone]) and B cell reconstitution was the most severely affected (6% vs 75% of lymphocytes). Graft suppression was also evidenced by absolute cytopenia as mice given HSC+CD4 TC vs HSC alone, had reduced cellularity in BM (median 3.8×10^∧6 [n=7] vs 13.3 ×10^∧6 cells [n=4]; p=0.0003) and spleen (median 4.4×10^∧6 vs 14×10^∧6 cells; p=0.03). Suppression of hematopoiesis by CD4 TC grafts was accompanied by an expansion of donor and host CD4 TC, each comprising ∼6% of BM cells as compared with <1% in groups that received pure HSC or were only irradiated sublethally. PMA-stimulated donor, but not host CD4 TC secreted high levels of IFNγ (30-50%) in the BM. This IFNγ expression far exceeded the levels of IFNγ measured in CD4 TC of the spleen from treated (HSC+CD4) mice as well as in CD4 TC of BM or spleen from transplanted, irradiated or unmanipulated control mice (3-15%). The proportion of NK cells in the BM was also substantially increased in recipients of HSC+CD4 TC vs recipients of HSC only or sublethal TBI controls (median 14% vs <1%), as were the absolute NK cell numbers (6.3×10^∧5 vs <0.4 ×10^∧5 cells). NK cells were derived from both donor and host in recipients of HSC, but solely of host type in recipients of HSC+CD4 TC. Findings similar to those observed in the 4 Gy TBI-treated mice given donor CD4 TC with their graft were noted when mice underwent non-myeloablative TLI for conditioning: while recipients of HSC alone were mixed chimeras in their lymphoid and myeloid lineages [n=4], mice that received HSC supplemented with splenocytes or total TC failed to engraft with T and B cells, had marginal myeloid engraftment [n=12], but had increased proportions of host NK cells in the PB. In conclusion: Our data show in the setting of non-myeloablative conditioning transplantation of purified HSC alone or with CD8 TC results in superior immune reconstitution and donor chimerism as compared to grafts of HSC+CD4 or HSC+total TC. These results are of broad significance as it is generally believed that TC augment donor HSC engraftment. The way in which CD4 TC retard engraftment appears to be by activation of inflammation within the BM and expansion of host lymphoid populations that may mediate resistance. IFNγ, which is a known regulator of innate and acquired immune responses, may be central in activating host CD4 TC and enhancing NK cell mediated rejection of the graft and/or suppression of donor hematopoiesis.