Induction Of Acute Graft-versus-host Disease Research Articles

CTL-Promoting Effects of IL-21 Counteract Murine Lupus in the Parent→F1 Graft-versus-Host Disease Model.

IL-21 promotes B cell and CTL responses in vivo, conferring IL-21 with a role in both humoral and cellular responses. Because CTL can target and eliminate autoreactive B cells, we investigated whether IL-21R signaling in CD8 T cells would alter the expansion of autoreactive B cells in an autoimmune setting. We addressed this question using the parent→F1 murine model of acute and chronic (lupus-like) graft-versus-host disease (GVHD) as models of a CTL-mediated or T-dependent B cell-mediated response, respectively. Induction of acute GVHD using IL-21R-deficient donor T cells resulted in decreased peak donor CD8 T cell numbers and decreased CTL effector function due to impaired granzyme B/perforin and Fas/Fas ligand pathways and a phenotype of low-intensity chronic GVHD with persistent host B cells, autoantibody production, and mild lupus-like renal disease. CTL effector maturation was critically dependent on IL-21R signaling in Ag-specific donor CD8, but not CD4, T cells. Conversely, treatment of DBA/2J→F1 chronic GVHD mice with IL-21 strongly promoted donor CD8 T cell expansion and rescued defective donor anti-host CTLs, resulting in host B cell elimination, decreased autoantibody levels, and attenuated renal disease, despite evidence of concurrently enhanced CD4 help for B cells and heightened B cell activation. These results demonstrate that, in the setting of lupus-like CD4 T cell-driven B cell hyperactivity, IL-21 signaling on Ag-specific donor CD8 T cells is critical for CTL effector maturation, whereas a lack of IL-21R downregulates CTL responses that would otherwise limit B cell hyperactivity and autoantibody production.

C‐Rel is an essential transcription factor for the development of acute graft‐versus‐host disease in mice

Transcription factors of the Rel/NF-κB family are known to play different roles in immunity and inflammation, although the putative role of c-Rel in transplant tolerance and graft-versus-host disease (GVHD) remains elusive. We report here that T cells deficient for c-Rel have a dramatically reduced ability to cause acute GVHD after allogeneic bone marrow transplantation using major and minor histocompatibility mismatched murine models. In the study to understand the underlying mechanisms, we found that c-Rel(-/-) T cells had a reduced ability to expand in lymphoid organs and to infiltrate in GVHD target organs in allogeneic recipients. c-Rel(-/-) T cells were defective in the differentiation into Th1 cells after encountering alloantigens, but were enhanced in the differentiation toward Foxp3(+) regulatory T (Treg) cells. Furthermore, c-Rel(-/-) T cells had largely preserved activity to mediate graft-versus-leukemia response. Taken together, our findings indicate that c-Rel plays an essential role in T cells in the induction of acute GVHD, and suggest that c-Rel can be a potential target for therapeutic intervention in allogeneic hematopoietic cell transplantation in the clinic.

T-Bet Is Critical for the Development of Acute Graft-Versus-Host Disease Through Controlling T Cell Differentiation and Function

Abstract 452 Background:Allogeneic hematopoietic cell transplantation (HCT) offers great promise for the treatment of hematologic malignancies. However, HCT benefits are frequently offset by graft-versus-host disease (GVHD). Donor T cells can differentiate into Th1 or Th17 subset that contribute to GVHD. The T-box transcription factor T-bet is important for promoting the differentiation of naïve CD4+T cells into Th1 phenotype, while simultaneously inhibiting Th2 and Th17 lineage commitment. Published data indicate that donor T cells deficient for IFN-γ induce exacerbated GVHD. In contrast, our recent study showed that T cells deficient for T-bet were impaired in the induction of GVHD. Given T-bet is a master regulator for the differentiation into Th1 cells that produce IFN-γ, the underlining mechanisms accounted for the distinct outcomes caused by T-bet- versus IFN-γ-deficient donor T cells are not clear. Method:We evaluated the roles of T-bet and IFN-γ in acute GVHD induced by naïve CD4+ T cells or polarized Th17 cells using murine allogeneic bone marrow transplantation (allo-BMT) model. WT, T-bet knockout (KO) and IFN-γ KO mice on C57BL/6 (B6) background were used as donors, and lethally irradiated BALB/c mice were used as recipients. Pathologic analysis and serum cytokine detection were done 14 days after adoptive transfer of WT, T-bet–/–, and IFN-γ–/– CD4+ T cells. Using microarray technology, gene expression profile on donor T cells was analyzed 7 days after adoptive transfer by sorting donor-derived CD4+ T cells from the recipients of WT, T-bet–/– or IFN-γ–/– CD4+ T cells. Results:We compared the ability of WT, T-bet–/–, and IFN-γ–/– CD4 T cells in the induction of acute GVHD. In the comparison with WT cells, IFN-γ–/– CD4 T cells caused similar or even more severe GVHD as expected. In sharp contrast, T-bet–/– CD4 T cells induced much ameliorated GVHD, as significantly higher survival and less body weight loss were observed in the recipients of T-bet–/–T cells. Pathology study on GVHD target organs showed that recipients of T-bet–/– donor T cells had markedly reduced T cell infiltration and tissue damage in liver, gut, and skin, when compared with those of WT or IFN-γ–/– T cells. Reduced GVHD in the recipients of T-bet–/– T cells was consistent with significantly lower levels of pathogenic cytokines IFN-γ, TNF-α, and IL-2 but higher IL-10 (anti-inflammatory), IL-6 (Th17 related) and IL-4 (Th2 related) in serum as compared with those in the recipients of WT T cells. Mechanistic studies in vitro revealed that T-bet–/– CD4 T cells expressed significantly lower levels of IFN-γ, CXCR3 (Th1 specific chemokine receptor) and CD122 (T cell activation marker), but higher levels of IL-17 (Th17 cytokine) and CCR6 (Th17 specific chemokine receptor) compared with WT CD4 T cells, indicating that T-bet–/– T cells impaired in differentiating into Th1 cells and instead into Th17 cells. Given Th17 subset only is capable of causing GVHD and T-bet–/– T cells are prone to Th17-differentiation, we assessed the role of T-bet or IFN-γ in the development of GVHD by comparing the pathogenicity of in vitro polarized WT, T-bet–/– and IFN-γ–/– Th17 cells. While IFN-γ–/– Th17 cells had a comparable ability to cause GVHD compared with WT Th17 cells, T-bet–/– Th17 cells had reduced pathogenicity, and caused ameliorated GVHD. Furthermore, microarray analysis identified genes that are regulated by T-bet but independent of IFN-γ, including Cxcr3, Ccr5, Ccl3, Ccl4, Klrc1, Klrd1, Nkg7 and Pdcd1, which may explain the compromised ability of T-bet−/− not IFN-γ–/–T cells in the induction of GVHD. Conclusions:We conclude that T-bet is required for Th1 differentiation and optimal function of Th17 cells, and it can also control T cell infiltration into GVHD target organs by regulating chemokines and their receptors. Thus, failure in Th1 generation, migration and reduced activity of polarized Th17 cells are likely accounted for impaired ability of T-bet−/− CD4 T cells in the induction of acute GVHD. The current study suggests that targeting T-bet or regulating its downstream effectors independent of IFN-γ may be a promising strategy to control GVHD after allogeneic HCT in clinic. Disclosures:No relevant conflicts of interest to declare.

PI3K-Independent Signaling Pathways Contribute to ICOS-Mediated T-Cell Costimulation in Acute Graft-Versus-Host Disease in Mice.

AbstractAbstract 3003ICOS provides an important costimulation to promote T-cell activation and function. Using a knock-in mouse strain, termed ICOS-YF, in which the cytoplasmic tail of ICOS cannot activate phosphoinositide 3-kinase (PI3K), we have shown that ICOS-PI3K signaling axis is critical for the generation of follicular helper T cells. We also observed that, in both CD4+ and CD8+ T cells, ICOS could potentiate TCR-mediated calcium flux in a PI3K-independent manner in vitro. Although ICOS can potentiate TCR-mediated calcium flux independent of PI3K, its biological significance is unclear. To address this question, we studied the function of ICOS-YF T cells in comparison with ICOS wild-type (WT) and knock-out (KO) T cells in MHC-mismatched bone marrow transplantation (BMT) models. Severity of acute graft-versus-host disease (GVHD) was evaluated based on recipient survival, body weight change, and pathologic scores. Consistent with the data previously published by us and others, ICOS KO T cells had significantly reduced ability to cause acute GVHD as compared to WT T cells. We further observed that YF T cells were significantly more capable in causing GVHD than KO T cells, but less capable than WT T cells. Mechanically, the levels of serum TNFa and IFNg were similar in the recipients of YF or KO T cells, but significantly lower than those of WT T cells. However, on the per-cell basis, YF CD8+ T cells expressed similar levels of intracellular IFNg as WT T cells, but significantly higher than KO T cells. We further compared the ability of CD4+ or CD8+ T cells alone in the induction of acute GVHD, and found that CD4+ T cells from YF and KO mice were similarly impaired in their capacity to induce acute GVHD. In contrast, the pathogenic capacity of CD8+ T cells from YF mice was comparable to that of WT cells, whereas KO CD8+ T cells were significantly less pathogenic. These results suggest that although both CD4+ and CD8+ T cells depend on ICOS costimulation, the downstream signaling pathways they utilize are distinct: CD4+ T cells depend on ICOS-PI3K signaling whereas CD8+ T cells are more dependent on PI3K-independent pathways, probably calcium signaling. Taken together, our study reveals a complexity in ICOS signaling mechanisms in T cell activation and GVHD induction. Disclosures:No relevant conflicts of interest to declare.

Pretransplant CSF-1 therapy expands recipient macrophages and ameliorates GVHD after allogeneic hematopoietic cell transplantation

Acute graft-versus-host disease (GVHD) results from the attack of host tissues by donor allogeneic T cells and is the most serious limitation of allogeneic hematopoietic cell transplantation (allo-HCT). Host antigen-presenting cells are thought to control the priming of alloreactive T cells and the induction of acute GVHD after allo-HCT. However, whereas the role of host DC in GVHD has been established, the contribution of host macrophages to GVHD has not been clearly addressed. We show that, in contrast to DC, reducing of the host macrophage pool in recipient mice increased donor T cell expansion and aggravated GVHD mortality after allo-HCT. We also show that host macrophages that persist after allo-HCT engulf donor allogeneic T cells and inhibit their proliferation. Conversely, administration of the cytokine CSF-1 before transplant expanded the host macrophage pool, reduced donor T cell expansion, and improved GVHD morbidity and mortality after allo-HCT. This study establishes the unexpected key role of host macrophages in inhibiting GVHD and identifies CSF-1 as a potential prophylactic therapy to limit acute GVHD after allo-HCT in the clinic.

The role of antigen-presenting cells in triggering graft-versus-host disease and graft-versus-leukemia

After allogeneic blood or bone marrow transplantation, donor T cells interact with a distorted antigen-presenting cell (APC) environment in which some, but not all, host APCs are replaced by APCs from the donor. Significantly, host APCs are required for the priming of acute graft-versus-host disease (GVHD). Donor APCs play a lesser role in the induction of acute GVHD despite their predicted capacity to cross-present host antigens. In contrast, donor APCs may play a role in perpetuating the tissue injury observed in chronic GVHD. Host APCs are also required for maximal graft-versus-leukemia responses. Recent studies have suggested potential strategies by which the continued presence of host APCs can be exploited to prime strong donor immunity to tumors without the induction of GVHD.

Evidence of Graft-versus-Tumor Effect in Refractory Metastatic Neuroblastoma

There are several reports describing graft-versus-tumor (GVT) effects in patients with solid tumors (1). Recently, GVT effects against advanced pancreatic cancer were added in this journal (2). To our knowledge, GVT effects in patients with high-risk neuroblastoma have never been reported and it has been believed that the effects are questionable in neuroblastoma (3). Here, we report eradication of tumor cells following the induction of acute graft-versus-host disease (GVHD) in a child with refractory metastatic neuroblastoma. The clearance and early detection of minimal residual disease (MRD) were confirmed by flow cytometric analysis of CD9+CD56+CD45− bone marrow cells, a population we previously showed that is consistent with neuroblastoma cells (4). A 7-year-old girl had a history of disseminated stage IV neuroblastoma with N-myc amplification. She had a recurrence of the tumor with the elevation of urinary vanillylmandelic acid two years after tandem highdose chemotherapy with autologous peripheral blood stem cell rescue. Magnetic resonance imaging showed bilateral multiple massive paraaortic metastases. I-123-meta-iodobenzylguanidine (MIBG) scan revealed to metastases of generalized lymph nodes and bone marrow, and abdominal mass. Bone marrow aspiration revealed presence of CD9+CD56+CD45− neuroblastoma cells (Fig. 1, B–i). Clustering of neuroblastoma cells in bone marrow was confirmed by immunohistochemistry using anti-tyrosine hydroxylase monoclonal antibody (Fig. 1, C–i) (5). Three courses of conventional chemotherapy and topotecan-based chemotherapy were relatively effective, reducing the tumor mass size by 30 percent (Fig. 1A). She received allogeneic peripheral blood stem cell transplant from an HLA-compatible sister after total surgical resection of the mass and intraoperative 20 Gy irradiation of tumor bed to achieve complete remission (Fig. 1, B-ii and C-ii). On day +40 the minimal residual tumor in bone marrow reappeared (Fig. 1, B-iii and C-iii), suggesting a recurrence of neuroblastoma, although urinary vanillylmandelic acid, magnetic resonance imaging, and MIBG scan were not positive. At least two slides were examined. It was reported that the sensitivity of morphological and histologic analysis is approximately 1 per 103–4 cells (6). Flow cytometric analysis could detect occult neuroblastoma cells at a level of 1 per 104–5 cells (4).FIGURE 1.: Induction of a graft-versus-neuroblastoma effect. (A) A patient with recurrent refractory neuroblastoma received allogeneic peripheral blood stem cell transplantation. A graft-vs.-neuroblastoma effect was induced by discontinuing cyclosporine A. (B) The expression of CD9 antigen on gated CD56+/CD45− is shown in histograms. The percentage of CD9+CD56+CD45− cells detected in bone marrow pooled samples at the recurrence (i), before transplantation (ii), day 40 after transplantation (iii) and two weeks after withdrawing cyclosporine A (iv). Total number of cells analyzed was 4x104 in (i) and 2×106 in (ii)-(iv). (C) Immunohistochemistry of bone marrow with anti-tyrosine hydroxylase (TH) monoclonal antibody provided the detection of neuroblastoma cells. Typical neuroblastoma cell clustering is shown in (i) and a TH-positive cell with a round nucleus is shown in (iii; arrow). Negative staining is confirmed in (ii) and (iv) taken from at least two sites of bone marrow. CsA, cyclosporine A; VMA, vanillylmandelic acid; HVA, homovanillic acid.Resolution of bone marrow metastasis was observed in association with the development of grade III acute GVHD two weeks after withdrawing cyclosporine A (Fig. 1, B-iv and C-iv). Acute GVHD was successfully treated and she remained free of MRD for 30 months after transplantation, when she relapsed with left tibia bone involvement. Bone marrow involvement was not observed. At this time, she was placed on tacrolimus and prednisolone for extensive type of chronic GVHD. Our observation here is a first reported definite evidence of graft-versus-neuroblastoma effect, which was confirmed by disappearance of tumor cells after the induction of acute GVHD. Target structure in GVT effects is unknown. However, minor histocompatibility antigen may be a possible target. Clinical experience on a large scale would be required to determine the clinical efficacy of GVT effects in patients with neuroblastoma. ACKNOWLEDGMENTS This work was supported by a Research Grant on Human Genome, Tissue Engineering (H17-014) from the Japanese Ministry of Health, Labor & Welfare. Masahiro Hirayama Eiichi Azuma Mariko Araki Yoshihiro Komada Department of Pediatrics and Cell Transplantation Mie University School of Medicine Mie, Japan Atsuko Nakagawa Division of Pathology Department of Clinical Laboratory Medicine National Center for Child Health and Development Tokyo, Japan

Recent Advances in the Treatment of Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) is a lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT) where immunocompetent donor T cells attack the genetically disparate host cells. The predominant symptoms of acute GVHD occur in the skin, liver, and intestine. Induction of acute GVHD can be divided into three phases: recipient conditioning, donor T cell activation, and effector cell-mediated GVHD. Chronic GVHD usually appears up to 100 days after HSCT and is characterized by symptoms similar to those observed for autoimmune disease. It is possible that chronic GVHD is the result of autoreactive T cells that escaped negative selection due to damage to the thymus from conditioning regimens, acute GVHD, and/or age related atrophy. Recent advances in the understanding of the basic mechanisms involved in GVHD pathophysiology have led to new strategies designed to block GVHD. This review focuses on recent developments in the treatment of GVHD, including insights gained from our own experimental studies.

Role of perforin in controlling B-cell hyperactivity and humoral autoimmunity.

To determine the role of perforin-mediated cytotoxic T lymphocyte (CTL) effector function in immune regulation, we studied a well-characterized mouse model of graft-versus-host disease (GVHD). Induction of acute GVHD using perforin-deficient donor T cells (pfp-->F1) initially resulted in features of acute GVHD, e.g., engraftment of both donor CD4(+) and CD8(+) T cells, upregulation of Fas and FasL, production of antihost CTL, and secretion of both Th1 and Th2 cytokines. Despite fully functional FasL activity, pfp donor cells failed to totally eliminate host B cells, and, by 4 weeks of disease, cytokine production in pfp-->F1 mice had polarized to a Th2 response. Pfp-->F1 mice eventually developed features of chronic GVHD, such as increased numbers of B cells, persistence of donor CD4 T cells, autoantibody production, and lupuslike renal disease. We conclude that in the setting of B- and T-cell activation, perforin plays an important immunoregulatory role in the prevention of humoral autoimmunity through the elimination of both autoreactive B cells and ag-specific T cells. Moreover, an ineffective initial CTL response can evolve into a persistent antibody-mediated response and, with it, the potential for sustained humoral autoimmunity.

Involvement of both donor cytotoxic T lymphocytes and host NK1.1+ T cells in the thymic atrophy of mice suffering from acute graft-versus-host disease.

It has been reported that a dramatic decrease in the number of thymocytes (thymic atrophy) in mice suffering from acute graft-versus-host disease (GVHD) is ascribed to glucocorticoids. In this study, we examined the possibility that cellular immune responses may thus be involved in thymic atrophy. In contrast to chronic GVHD mice, acute GVHD (C57BL/6 X DBA/2) F1 (BDF1) hybrid mice, which were injected intravenously with both spleen and lymph node cells from C57BL/6 mice, showed a dramatic decrease in the number of CD4 CD8 double-positive thymocytes 2 or 3 weeks after the induction of GVHD. A flow cytometric analysis revealed the donor-derived T cells with either CD4 or CD8 molecules to infiltrate the thymus of the mice undergoing acute GVHD for 10 days. In a cytolytic assay, such thymus-containing cells exhibited a cytolytic activity specific to the host cells. In addition, anti-H-2d cytolytic T cells showed a high level of cytolytic activity against BDF1 (H-2bXd) thymocytes, whereas they also showed a low level of cytolytic activity against C57BL/6 (H-2b) thymocytes, thus suggesting that the thymus-infiltrating donor-derived T cells killed the host thymocytes through both anti-H-2d-specific and non-specific mechanisms. Interestingly, a flow cytometric analysis revealed both the percentage and the absolute cell number of host-derived NK1.1+ CD3+ cells to increase in the thymus of mice suffering from acute GVHD for 10 days. In addition, they also showed the cytolytic activity against YAC-1 cells and the mRNA expression of interleukin-12 (IL-12) in the thymus to be also significantly augmented on day 7 after the induction of acute GVHD. Collectively, our results indicate that the cellular immune responses such as donor cytotoxic T lymphocytes and host NK1.1+ T cells are therefore involved in the thymic atrophy of mice suffering from acute GVHD.

Involvement of Fas-Mediated Apoptosis in the Hematopoietic Progenitor Cells of Graft-Versus-Host Reaction-Associated Myelosuppression

The influence of graft-versus-host (GVH) reaction on the host hematopoietic cells clinically manifests itself both as adverse reactions in transfusion-associated GVH disease (GVHD) and as a therapeutic graft-versus-leukemia (GVL) effect in either donor lymphocytes transfusion (DLT) or allogeneic bone marrow (BM) transplantation. We examined the effect of GVH reaction on the host hematopoiesis in the murine parent-into-F1 (P1 --> F1) model of GVHD. The systemic transfer of 5 x 10(7) of C57BL/6 (B6) splenocytes into (B6xDBA/2)F1 mice (BDF1), which results in acute GVHD, reduced the peripheral blood cell counts, the number of BM cells, and colony-forming unit-granulocyte macrophage (CFU-GM), whereas the injection of 10(8) of DBA/2 cells into BDF1, which results in chronic GVHD, did not affect hematopoiesis 2 weeks after the transfer. To clarify the mechanism of such myelosuppression, we examined the Fas expression in both hematopoietic progenitor cells as well as whole BM cells. The Fas expressions in each fraction significantly increased in BDF1 mice 2 weeks after the induction of acute GVHD, whereas no such effects were observed in the BDF1 mice with chronic GVHD. Furthermore, when such BM cells were incubated with anti-Fas antibody (Jo2), which induces apoptosis through Fas, the fraction of apoptotic cells increased and the number of CFU-GM decreased significantly. The in vivo administration of neutralizing anti-FasL antibody into BDF1 mice receiving with B6 spleen cells thus protected the host mice from BM failure. These results indicate that the functional expression of Fas on hematopoietic cells plays an essential role in the myelosuppressive effect of GVHD.

Host intestinal intraepithelial γδ T lymphocytes present during acute graft‐versus‐host disease in mice may contribute to the development of enteropathy

We reported that T cell receptor (TcR) gamma delta intestinal intraepithelial lymphocytes (i-IEL) of host origin increased transiently, then decreased drastically at the early stage of non-irradiated acute graft-versus-host disease (GVHD) in mice. We investigated the role of the TcR gamma delta i-IEL of host origin in the pathogenesis of the intestinal lesions that occur during acute GVHD. The acute GVHD was induced in mice which had been depleted of TcR gamma delta by in vivo administration of hamster monoclonal antibody (mAb) against TcR gamma delta. Although the degree of splenomegaly after the induction of acute GVHD in mice treated with anti-TcR gamma delta mAb was similar to that in control mice treated with hamster anti-2,4,6-trinitrophenyl mAb, infiltration of donor-derived T cells into the epithelium, and mitosis and apoptosis of crypt cells in the intestinal mucosa were dramatically suppressed in these mice. This suggest that host TcR gamma delta T cells in i-IEL contribute to the development of enteropathy in acute GVHD in mice.

Bone Marrow Graft Engineering by Counterflow Centrifugal Elutriation: Results of a Phase I-II Clinical Trial

In an attempt to reduce the incidence and severity of acute graft-versus-host disease (GVHD), we have decreased the number of bone marrow (BM) lymphocytes in the donor marrow graft before bone marrow transplantation (BMT) using counterflow centrifugal elutriation (CCE). In a phase I-II clinical trial, 23 patients received lymphocyte-depleted BM allografts from their HLA-identical, mixed lymphocyte culture (MLC)-nonreactive sibling donors. The patients entered in the study were deemed to be at high risk for treatment failure on the basis of age (greater than 30 years; median, 39 years) and the result of our skin explant assay predictive of acute GVHD. Patients predicted not to develop acute GVHD by this assay were excluded from this study. All patients received a standard lymphocyte dose of 0.5 x 10(6) morphologic lymphocytes per kilogram ideal body weight (IBW) in the marrow graft and were maintained on cyclosporine A (CsA) immunosuppression for 170 days after BMT. Prompt hematopoietic recovery occurred in 22 of 23 patients with a median time to an absolute neutrophil count (ANC) greater than or equal to 500/microL of 21 days. Donor cell engraftment was subsequently verified by cytogenetic and/or DNA analysis in all of 21 evaluable patients. No patient developed systemic acute GVHD. Only five (22%) developed cutaneous GVHD (clinical stage 1) that required steroid treatment, including one patient who failed to engraft. The median follow-up of the patients enrolled in this study is 14 months (range, 5 to 20 months). Actuarial survival 1 year after BMT is 83%. Thus, in two consecutive clinical trials using CCE to deplete donor BM of alloreactive lymphocytes (1.0 x 10(6) versus 0.5 x 10(6) lymphocytes/kg), we have demonstrated that the procedure does not interfere with BM engraftment and is effective in decreasing the incidence and severity of acute GVHD. Furthermore, comparison of these studies has revealed a differential dose response relationship between the number of graft lymphocytes, protection of engraftment, and induction of acute GVHD. Although there appears to be a modest relationship between lymphocyte dose and time to hematopoietic recovery, the 50% reduction in lymphocyte dose from that used in our previous trial resulted in a marked decrease in acute GVHD without compromising engraftment.

Bone marrow graft engineering by counterflow centrifugal elutriation: results of a phase I-II clinical trial

In an attempt to reduce the incidence and severity of acute graft- versus-host disease (GVHD), we have decreased the number of bone marrow (BM) lymphocytes in the donor marrow graft before bone marrow transplantation (BMT) using counterflow centrifugal elutriation (CCE). In a phase I-II clinical trial, 23 patients received lymphocyte- depleted BM allografts from their HLA-identical, mixed lymphocyte culture (MLC)-nonreactive sibling donors. The patients entered in the study were deemed to be at high risk for treatment failure on the basis of age (greater than 30 years; median, 39 years) and the result of our skin explant assay predictive of acute GVHD. Patients predicted not to develop acute GVHD by this assay were excluded from this study. All patients received a standard lymphocyte dose of 0.5 x 10(6) morphologic lymphocytes per kilogram ideal body weight (IBW) in the marrow graft and were maintained on cyclosporine A (CsA) immunosuppression for 170 days after BMT. Prompt hematopoietic recovery occurred in 22 of 23 patients with a median time to an absolute neutrophil count (ANC) greater than or equal to 500/microL of 21 days. Donor cell engraftment was subsequently verified by cytogenetic and/or DNA analysis in all of 21 evaluable patients. No patient developed systemic acute GVHD. Only five (22%) developed cutaneous GVHD (clinical stage 1) that required steroid treatment, including one patient who failed to engraft. The median follow-up of the patients enrolled in this study is 14 months (range, 5 to 20 months). Actuarial survival 1 year after BMT is 83%. Thus, in two consecutive clinical trials using CCE to deplete donor BM of alloreactive lymphocytes (1.0 x 10(6) versus 0.5 x 10(6) lymphocytes/kg), we have demonstrated that the procedure does not interfere with BM engraftment and is effective in decreasing the incidence and severity of acute GVHD. Furthermore, comparison of these studies has revealed a differential dose response relationship between the number of graft lymphocytes, protection of engraftment, and induction of acute GVHD. Although there appears to be a modest relationship between lymphocyte dose and time to hematopoietic recovery, the 50% reduction in lymphocyte dose from that used in our previous trial resulted in a marked decrease in acute GVHD without compromising engraftment.