Lack of Host Bone Marrow-Derived Interleukin-12 Increased the Incidence of Allograft Rejection in Allogeneic Bone Marrow Transplantation

Abstract Abstract 2960 Background: Donor cell engraftment following allogeneic bone marrow transplantation (BMT) is affected by several factors, including immunological major histocompatibility complex (MHC) barriers, the intensity of the conditioning regimen, and the content of T-cells in the graft. The current model for engraftment in allogeneic BMT is that host dendritic cells (DCs) activate donor T-cells which promote engraftment by eliminating radio-resistant cytotoxic host immune cells, especially natural killer (NK) cells and T-cells. To explore the interaction between donor T-cell and host antigen-presenting cells (APC) in engraftment in allogeneic BMT, we focused on the role of interleukin-12 (IL-12), a key cytokine produced mainly by DCs that drives the development of donor type 1 helper T cells (Th1) and type 1 cytotoxic T lymphocytes (Tc1). Methods: Radiation chimeras with >95% donor chimerism were created by transplanting 5 × 106 bone marrow (BM) cells from IL-12 knock out (IL-12 KO) or wild type (WT) B6 (H-2Kb, CD45.2) donors into congenic BL6 Pepboy (B6.SJL-PtprcaPep3b/BoyJ, H-2Kb, CD45.1) mice following lethal 11 Gy irradiation. A second allogeneic BMT was conducted 2 months later using MHC mismatched FVB (H-2q, CD45.1), BA.B10 (H-2Kk, CD45.2, CD90.1) or B10.BR (H-2Kk, CD45.2, CD90.2) donor cells. In vivo bioluminescent imaging (BLI) was performed to analyze the number and in vivo distribution of luciferase+ donor T-cells. The whole-body bioluminescent signal was used as a marker of the donor T cell expansion. Engraftment of donor myeloid cells was determined by flow cytometry using mAbs for specific leukocyte markers expressed on donors and recipients (CD45.1, CD45.2, H-2Kb). Intracellular cytokine expression (IL-4, IL-10, IFN-g) by donor CD4+ and CD8+ T cells was analyzed by flow cytometry. Results: WT BL6→BL6 radiation chimeras recipients showed greater expansion of luciferase+ donor T-cells compared with IL-12 KO BL6→BL6 radiation chimeras recipients and FVB→FVB syngeneic recipients at early time point (2 wks) following 9 Gy re-irradiation and transplantation of 3 × 105 luciferase+ FVB-L2G85 T-cells in combination with 5 × 106 T cell depleted (TCD) BM cells from FVB mice following (Fig 1). At 4 weeks post transplant, more WT BL6→BL6 radiation chimeras achieved myeloid engraftment than IL-12 KO BL6→BL6 radiation chimera recipients(75.0% versus 33.3% respectively, p = 0.086), and the former group had better erythroid engraftment than the latter group (RBC 8.65 ± 1.88 × 1012/L versus 5.67 ± 2.22 × 1012/L respectively, p = 0.011). However, when FVB, WT BL6→BL6 or IL-12 KO BL6→BL6 radiation chimeras recipients were conditioned with a larger dose of irradiation prior to the second transplantation (10 Gy) and received a larger dose of donor T-cells (5 × 105), both the WT BL6→BL6 and IL-12 KO BL6→BL6 radiation chimeras recipients achieved full donor engraftments (85.7% versus 87.5% respectively, p = NS). Donor T cells in allogeneic BMT recipients were Th1/Tc1 polarized, there were no differences in frequencies and total numbers of Th1/Tc1 donor CD4+ and CD8+ T cells comparing recipients of WT BL6→BL6 and IL-12 KO BL6→BL6 radiation chimeras. In spite of an increased irradiation dose and larger number of donor T-cells in the second transplant regimen, no increase in graft versus host disease (GVHD) clinical scores and GVHD-mortality were observed in the recipients of WT BL6→BL6 radiation chimeras compared with recipients of IL-12 KO BL6→BL6 radiation chimeras. Conclusion: These data support a role for host BM-derived IL-12 in facilitating engraftment in allogeneic BMT following a reduced dose (9 Gy) radiation. The lack of host BM-derived IL-12 expression led to allograft rejection. Rejection could be overcome by increasing the dose of pre-transplant irradiation and the content of donor T-cells without causing lethal GVHD. As the main source of host BM-derived IL-12, recipient APC thus play an important role in donor T-cell activation. As has been previously demonstrated in a murine BMT model, the addition of IL-12 in the peri-transplant period helped to separate graft versus leukemia effects from the GVHD-promoting activity of donor T-cells (Yang, 1997). Patients predicted to be high risk of graft failure may benefit from treatment strategies that contribute to production of IL-12 during the early phases of hematopoietic engraftment. Disclosures: No relevant conflicts of interest to declare.

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Donor T-cell alloreactivity against host thymic epithelium limits T-cell development after bone marrow transplantation

Blood ◽

10.1182/blood-2006-07-034157 ◽

2007 ◽

Vol 109 (9) ◽

pp. 4080-4088 ◽

Cited By ~ 58

Author(s):

Mathias M. Hauri-Hohl ◽

Marcel P. Keller ◽

Jason Gill ◽

Katrin Hafen ◽

Esther Pachlatko ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Bone Marrow Transplantation ◽

T Cell ◽

Marrow Transplantation ◽

Allogeneic Bone Marrow Transplantation ◽

Allogeneic Bone Marrow ◽

Thymic Epithelial Cells ◽

Allogeneic Bone ◽

Donor T Cells

Abstract Acute graft-versus-host disease (aGVHD) impairs thymus-dependent T-cell regeneration in recipients of allogeneic bone marrow transplants through yet to be defined mechanisms. Here, we demonstrate in mice that MHC-mismatched donor T cells home into the thymus of unconditioned recipients. There, activated donor T cells secrete IFN-γ, which in turn stimulates the programmed cell death of thymic epithelial cells (TECs). Because TECs themselves are competent and sufficient to prime naive allospecific T cells and to elicit their effector function, the elimination of host-type professional antigen-presenting cells (APCs) does not prevent donor T-cell activation and TEC apoptosis, thus precluding normal thymopoiesis in transplant recipients. Hence, strategies that protect TECs may be necessary to improve immune reconstitution following allogeneic bone marrow transplantation.

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Host Reactive Donor T Cells Are Associated With Lung Injury After Experimental Allogeneic Bone Marrow Transplantation

Blood ◽

10.1182/blood.v92.7.2571.2571_2571_2580 ◽

1998 ◽

Vol 92 (7) ◽

pp. 2571-2580 ◽

Cited By ~ 7

Author(s):

Kenneth R. Cooke ◽

Werner Krenger ◽

Geoff Hill ◽

Thomas R. Martin ◽

Lester Kobzik ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Bone Marrow Transplantation ◽

Lung Injury ◽

Allogeneic Bone Marrow Transplantation ◽

Allogeneic Bone ◽

Allogeneic Bmt ◽

Donor T Cells ◽

Ifn Γ

Noninfectious lung injury is common after allogeneic bone marrow transplantation (BMT), but its association with acute graft-versus-host disease (GVHD) is unclear. Using a murine BMT system where donor and host differ by multiple minor histocompatibility (H) antigens, we investigated the nature of lung injury and its relationship both to systemic GVHD and host-reactive donor T cells. Lethally irradiated CBA hosts received syngeneic BMT or allogeneic (B10.BR) T-cell–depleted (TCD) bone marrow (BM) with and without the addition of T cells. Six weeks after BMT, significant pulmonary histopathology was observed in animals receiving allogeneic BMT compared with syngeneic controls. Lung damage was greater in mice that received allogeneic T cells and developed GVHD, but it was also detectable after TCD BMT when signs of clinical and histologic acute GVHD were absent. In each setting, lung injury was associated with significant alterations in pulmonary function. Mature, donor (Vβ6+and Vβ3+) T cells were significantly increased in the broncho-alveolar lavage (BAL) fluid of all allogeneic BMT recipients compared with syngeneic controls, and these cells proliferated and produced interferon-γ (IFN-γ) to host antigens in vitro. These in vitro responses correlated with increased IFN-γ and tumor necrosis factor-α (TNF-α) in the BAL fluid. We conclude that alloreactive donor lymphocytes are associated with lung injury in this allogeneic BMT model. The expansion of these cells in the BAL fluid and their ability to respond to host antigens even when systemic tolerance has been established (ie, the absence of clinical GVHD) suggest that the lung may serve as a sanctuary site for these host reactive donor T cells. These findings may have important implications with regard to the evaluation and treatment of pulmonary dysfunction after allogeneic BMT even when clinical GVHD is absent.

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Analysis of T-cell repopulation after allogeneic bone marrow transplantation: significant differences between recipients of T-cell depleted and unmanipulated grafts

Blood ◽

10.1182/blood.v87.9.3984.bloodjournal8793984 ◽

1996 ◽

Vol 87 (9) ◽

pp. 3984-3992 ◽

Cited By ~ 173

Author(s):

E Roux ◽

C Helg ◽

F Dumont-Girard ◽

B Chapuis ◽

M Jeannet ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Allogeneic Bone Marrow Transplantation ◽

Cell Receptor ◽

Allogeneic Bone ◽

Receptor Repertoire ◽

Donor T Cells ◽

Circulating T Cells ◽

Cell Repopulation

We have studied the repopulation of the T-cell compartment in 27 patients transplanted with bone marrow from an (HLA)-identical sibling. Significant differences were found between recipients of unmanipulated and T-cell depleted grafts. Analysis of the T cells by a method based on amplification of minisatellite DNA regions showed that without depletion > 99.9% of the clones responding to a mitogenic stimulus after transplantation were of donor origin. In contrast, when the graft had been depleted with Campath-1M plus complement, a significant part of the T cells cloned during the first weeks after transplantation comprised of recipient T cells that had survived the preconditioning. This mixed population of low numbers donor and recipient T cells (19 +/- 31/mm3 at day 14) expanded rapidly (predominantly CD8+ T cells) during the first 2 months, without a significant change of the ratio of recipient/donor T cells. In 11 of 17 evaluable patients a late wave ( > 9 months) of donor T cells occurred. As a consequence, T-cell chimerism changed in favor of donor T cells and the CD4/CD8 ratio that had been reversed ( < 0.5) after the first expansion, normalized (1.5 +/- 0.51). Analysis of the T-cell receptor repertoire showed that in recipients of a T-cell depleted graft, the recipient as well as the donor T cells that repopulated the peripheral T-cell pool during the first month, were the progeny of a limited number of precursors. Because without depletion, when larger numbers of donor T cells had been cotransfused with the marrow, the repertoire was much more diverse, these data show that immediately after transplantation, the peripheral pool is repopulated primarily through expansion of circulating T cells.

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Critical and Opposing Effects of T Cell-Derived TNFα and Interferon-γ on IL-17 Induction Assessed in Vitro and in Vivo Following Allogeneic Bone Marrow Transplantation

Blood ◽

10.1182/blood.v112.11.3482.3482 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3482-3482

Author(s):

Minghui Li ◽

Kai Sun ◽

Mark Hubbard ◽

Doug Redelman ◽

Angela Panoskaltsis-Mortari ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Th17 Cells ◽

Allogeneic Bone Marrow Transplantation ◽

Allogeneic Bone ◽

Allogeneic Bmt

Abstract IL-17-producing CD4 T cells (Th17) are a recently identified T helper subset that plays a role in mediating host defense to extracellular bacteria infections and is involved in the pathogenesis of many autoimmune diseases. In vitro induction of IL-17 in murine CD4+ T cells has been shown to be dependent on the presence of the proinflammatory cytokines TGF-β and IL-6 whereas IFNγ can suppress the development of Th17 cells. In the current study, we examined the roles of TNFα and IFNγ on IL-17 production by purified T cells in vitro and in vivo after allogeneic bone marrow transplantation (BMT). We present findings that expression of TNFα by the T cell itself is necessary for optimal development of Th17 under in vitro polarizing conditions. A novel role for T cell-derived TNFα in Th17 induction was observed when in vitro polarization of Tnf−/−CD4+ T cells resulted in marked reductions in IL-17+CD4+ T cells compared to Tnf+/+CD4+ T cells. In marked contrast, T cell-derived IFNγ markedly inhibited Th17 development as more IL-17+CD4+ T cells were found in Ifnγ−/−CD4+ T cells than in Ifnγ+/+CD4+ T cells, and of particular interest was the dramatic increase in IL-17+CD8+ cells from Ifnγ−/− mice. To determine if T cell-derived TNFα or IFNγ can regulate Th17 development in vivo we examined the differentiation of alloreactive donor T cells following allogeneic BMT. We have found that donor-derived Th17 cells can be found in lymphoid tissues and GVHD-affected organs after allogeneic BMT. However, transfer of Tnf−/− CD4+ T cells after allogeneic BMT resulted in marked reductions in Th17 cells in the spleen (18×103 vs 7×103, P<0.05). In agreement with the in vitro data and in contrast to what was observed with transfer of Tnf−/− CD4+ T cells, transfer of donor Ifnγ−/− T cells resulted in marked increases in not only IL-17+CD4+ but also IL-17+CD8+ T cells infiltrating the liver (7×103 vs 14×103, P<0.05; 4×104 vs 12.5×104, P<0.05). These results suggest that the donor T cell-derived TNFα and IFNγ opposingly regulate IL-17 induction of both CD4+ and CD8+ T cells in vitro and after allogeneic BMT which correlates with GVHD pathology.

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Visualizing Allogeneic Bone Marrow (BM) Graft Rejection.

Blood ◽

10.1182/blood.v104.11.43.43 ◽

2004 ◽

Vol 104 (11) ◽

pp. 43-43

Author(s):

Patricia Taylor ◽

Angela Panoskaltsis-Mortari ◽

Randolph Noelle ◽

Alexander Rudensky ◽

Jonathan Serody ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

The Body ◽

Allogeneic Bone Marrow ◽

Therapeutic Interventions ◽

Organ Distribution ◽

Allogeneic Bone ◽

Allogeneic Bmt

Abstract The process by which bone marrow is rejected by host T cells has not been able to be directly visualized to date. To study the process of allogeneic bone marrow rejection and the effects of therapeutic interventions, we created 2 models that allow us to 1) quantify the specific expansion of host-type alloreactive T cells early post-transplant in response to allogeneic BM infusion and to 2) image host T cells in vivo during BM rejection. For the first model, 2C and TEa lymph node (LN) cells were adoptively transferred into syngeneic C57BL/6 (B6) Rag deficient mice on d-2. 2C CD8+ and TEa CD4+ T cell receptor transgenic T cells are reactive against BALB/c alloantigen. Mice were irradiated with 200 cGy on d-1 and BALB/c BM was infused on d0. Controls included mice that received 2C/TEa LN cells but no BM. Ten days later, spleen analysis revealed that 2C CD8+ and TEa CD4+ T cells had expanded 322-fold and 33-fold (ave of 6 exp.), respectively, in mice receiving BALB/c BM compared to controls that did not receive BM. Expanded T cells were activated as determined by flow cytometric parameters and cell surface antigens. Data indicate that host alloreactive T cell expansion was inhibited by >95% by combined, but not single, costimulatory pathway blockade. Studies are in progress to analyze in vitro host anti-donor responses of adoptively transferred T cells. To visualize the response of host T cells to donor BM in vivo, we developed a rejection model for imaging involving the adoptive transfer of green fluorescent protein (GFP) T cells (obtained from GFP transgenic mice) into syngeneic non-GFP B6 recipients immediately following sublethal irradiation (500 cGy). Allogeneic BALB/c or syngeneic B6 BM was infused the following day. The syngeneic BMT controls allowed for the distinction of homeostatic vs alloreactive expansion of GFP+ T cells. Transplanted mice not receiving GFP T cells served as negative controls to verify lack of autofluorescence. Cohorts were imaged d4 to d18 post BMT. By d4, low numbers of GFP+ cells were evident in femoral BM cavity, peripheral and mesenteric LNs, spleen, Peyer’s patches (PP) and to a lesser extent, lung. By d7, massive expansion of GFP+ cells could be visualized throughout the body of recipients of allogeneic BM. LNs, spleen, PP (peri-follicular area) and BM cavity increased dramatically in GFP intensity from d4 to d7. On d7 to d14, there were large foci of GFP+ cells in the lung, liver, skin, gingiva, kidney, uterus, and colon in allogeneic BMT recipients. Compared with allogeneic BMT recipients, syngeneic BMT recipients had greatly reduced numbers of GFP+ T cells in lymphoid organs and only rare cells were noted in liver, kidney, skin, BM and gingiva. In both allogeneic and syngeneic BMT recipients, lengths of ileum were diffusely infiltrated while other sections contained discrete foci of GFP+ cells. These imaging data provide a vivid illustration of the massive expansion and multi-organ distribution of host anti-donor T cells in vivo. Recent generation of GFP+ 2C and GFP+ TEa mice will permit the imaging of alloantigen-specific T cells during a rejection response. Additional imaging experiments are planned to study the fate of GFP+ BM transferred to allogeneic recipients under conditions of engraftment vs rejection. These models provide a unique platform for the testing of therapeutic interventions.

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Factors governing the activation of adoptively transferred donor T cells infused after allogeneic bone marrow transplantation in the mouse

Blood ◽

10.1182/blood-2006-09-048124 ◽

2007 ◽

Vol 109 (10) ◽

pp. 4564-4574 ◽

Cited By ~ 17

Author(s):

Nadira Durakovic ◽

Vedran Radojcic ◽

Mario Skarica ◽

Karl B. Bezak ◽

Jonathan D. Powell ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Bone Marrow Transplantation ◽

Marrow Transplantation ◽

Allogeneic Bone Marrow Transplantation ◽

Mixed Chimerism ◽

Allogeneic Bone ◽

Donor T Cells ◽

Antigen Presenting

Abstract Murine models of bone marrow transplantation were used to study the mechanisms governing the activation of donor lymphocyte infusions (DLIs) manifesting as lymphohematopoietic graft-versus-host (LH-GVH) and graft-versus-leukemia (GVL) reactivities. We demonstrate here that established mixed chimerism influences the potency of DLI-mediated alloreactivity only in the MHC-mismatched but not MHC-matched setting. In the MHC-matched setting, high levels (≥ 40%) of residual host chimerism correlated negatively with DLI-mediated alloreactivity irrespective of the timing of their administration, the donor's previous sensitization to host antigens, or the level of residual host APCs. In vivo administration of Toll-like receptor (TLR) ligands was required to maximize DLI-mediated LH-GVH and GVL reactivities in chimeras with low levels (≤ 15%) of residual host chimerism. In contrast, coadministration of DLI with antigen-presenting cell (APC) activators was insufficient to augment their LH-GVH response in the presence of high levels of host chimerism unless the host's T cells were transiently depleted. Together, these results show the cardinal influence of donor-host incompatibility on DLI-mediated GVH responses and suggest that in MHC-matched chimeras, the induction of optimal alloreactivity requires not only donor T cells and host APCs but also TLR ligands and in the presence of high levels of host chimerism depletion of host T cells.

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HDAC11 Inhibits T-Cell Response to Alloantigens and Reduces Gvhd In Mice.

Blood ◽

10.1182/blood.v116.21.1466.1466 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1466-1466

Author(s):

Dapeng Wang ◽

Fengdong Cheng ◽

Yu Yu ◽

Kenrick Semple ◽

Lirong Peng ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Allogeneic Bone Marrow Transplantation ◽

T Cell Response ◽

Allogeneic Bone ◽

Allogeneic Bmt ◽

Significant Difference

Abstract Abstract 1466 Background: Histone acetyltransferases and histone deacetylases (HDAC) regulate gene expression through acetylation-deacetylation of histones. HDACs are the target of a family of compounds known as HDAC inhibitors, which have been shown to suppress pro-inflammatory cytokines and reduce acute graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) effect after allogeneic bone marrow transplantation (BMT) in mice. However, the role of individual HDAC members in the development of GVHD is not clear. Recently, HDAC11, the newest member of the HDAC family has emerged as an important transcriptional regulator of inflammatory responses in antigen-presenting cells (APCs)1. Here, we evaluated the role of HDAC11 on APCs and T cells in the allogeneic BMT setting in mice with genetic disruption of HDAC11. Method: Proliferation of wild-type (WT) and HDAC11 knock-out (KO) T cells in response to allogeneic antigens was compared by [H3] thymidine incorporation assay. Using the same method, we also tested the antigen presentation ability of WT and HDAC11 KO APCs. For in vivo studies, we used a clinical relevant mouse model of BMT: C57BL/6 (B6) ® BALB/c. To evaluate the role of HDAC11 in the function of T cells and APCs, WT and KO mice on B6 background were used as donors and recipients, respectively. Recipient survival was monitored daily and GVHD symptom was evaluated at least twice a week. HDAC11 KO mice were supplied by Merck and Co., Inc. Results: In vitro, HDAC11 KO T cells proliferated stronger than WT T cells under the stimulation of allogeneic APCs. Recipients of HDAC11 KO T cells lost significantly more body weight (p < 0.05), and died significantly sooner than those of WT T cells (p < 0.01). The pathologic score of KO recipients was higher than that of WT recipients in each of GVHD target organs including lung, liver, small intestine and colon. Mechanistically, we found that there were significantly more total and IFNγ-producing donor T cells in the recipients of KO cells than those of WT cells (p < 0.05). Collectively, HDAC11 KO T cells have higher activity in response to alloantigens in vitro and induced more severe GVHD in vivo compared to WT T cells. In contrast, KO and WT APCs had a similar ability to stimulate allogeneic T cells in vitro, and no significant difference in GVHD development was observed in WT or KO recipients after allogeneic BMT. Conclusion: HDAC11 negatively regulates T-cell function, but has no significant effect on APC function. This finding provides a rationale to promote T-cell immunity or tolerance by inhibiting or enhancing HDAC11, respectively. 1 Villagra et al. Nature Immunology, 10:92-100, 2009. Disclosures: No relevant conflicts of interest to declare.

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Host Reactive Donor T Cells Are Associated With Lung Injury After Experimental Allogeneic Bone Marrow Transplantation

Blood ◽

10.1182/blood.v92.7.2571 ◽

1998 ◽

Vol 92 (7) ◽

pp. 2571-2580 ◽

Cited By ~ 79

Author(s):

Kenneth R. Cooke ◽

Werner Krenger ◽

Geoff Hill ◽

Thomas R. Martin ◽

Lester Kobzik ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Bone Marrow Transplantation ◽

Lung Injury ◽

Allogeneic Bone Marrow Transplantation ◽

Allogeneic Bone ◽

Allogeneic Bmt ◽

Donor T Cells ◽

Ifn Γ

Abstract Noninfectious lung injury is common after allogeneic bone marrow transplantation (BMT), but its association with acute graft-versus-host disease (GVHD) is unclear. Using a murine BMT system where donor and host differ by multiple minor histocompatibility (H) antigens, we investigated the nature of lung injury and its relationship both to systemic GVHD and host-reactive donor T cells. Lethally irradiated CBA hosts received syngeneic BMT or allogeneic (B10.BR) T-cell–depleted (TCD) bone marrow (BM) with and without the addition of T cells. Six weeks after BMT, significant pulmonary histopathology was observed in animals receiving allogeneic BMT compared with syngeneic controls. Lung damage was greater in mice that received allogeneic T cells and developed GVHD, but it was also detectable after TCD BMT when signs of clinical and histologic acute GVHD were absent. In each setting, lung injury was associated with significant alterations in pulmonary function. Mature, donor (Vβ6+and Vβ3+) T cells were significantly increased in the broncho-alveolar lavage (BAL) fluid of all allogeneic BMT recipients compared with syngeneic controls, and these cells proliferated and produced interferon-γ (IFN-γ) to host antigens in vitro. These in vitro responses correlated with increased IFN-γ and tumor necrosis factor-α (TNF-α) in the BAL fluid. We conclude that alloreactive donor lymphocytes are associated with lung injury in this allogeneic BMT model. The expansion of these cells in the BAL fluid and their ability to respond to host antigens even when systemic tolerance has been established (ie, the absence of clinical GVHD) suggest that the lung may serve as a sanctuary site for these host reactive donor T cells. These findings may have important implications with regard to the evaluation and treatment of pulmonary dysfunction after allogeneic BMT even when clinical GVHD is absent.

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Donor T Cells Lacking IFN-Gamma Receptor Expression Enhance Donor Myeloid Engraftment and Decrease Early Acute Graft-Versus-Host Disease Mortality Following Allogeneic Bone Marrow Transplantation

Blood ◽

10.1182/blood.v112.11.3514.3514 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3514-3514

Author(s):

Kai Sun ◽

Minghui Li ◽

Mark A. Hubbard ◽

Lisbeth A. Welniak ◽

William J. Murphy

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Allogeneic Bone Marrow Transplantation ◽

Allogeneic Bone Marrow ◽

Allogeneic Bone ◽

Ifn Gamma ◽

Graft Versus Host ◽

Gamma Receptor ◽

Donor T Cells

Abstract Lack of engraftment, graft-versus-host disease (GVHD) and tumor relapse are major issues that affect clinical outcome after allogeneic bone marrow transplantation (allo-BMT). Donor T cells in the graft present a dilemma in allo-BMT due to both the beneficial and deleterious clinical effects they can exert. Therefore, modulation of donor T cell function may represent a potential therapeutic approach in allo-BMT. In murine allo-BMT models, It has been demonstrated that donor T cell-derived IFN-gamma is required for optimal graft-versus-tumor (GVT) responses but it also plays complex roles by exhibiting both protective and pathogenic effects in GVHD development. We therefore investigated the role of IFN-gamma responsiveness by the donor T cells in allo-BMT through the use of IFN-gamma receptor deficient (IfnR−/−; RKO) mice. In these experiments, recipient BALB/c (H2d) mice received lethal total body irradiation. Irradiation was followed by the infusion of graded doses of T cell-depleted (TCD) allogeneic bone marrow cells (5 or 15 × 106) intravenously from major histocompatibility complex (MHC)-disparate wild type (WT) C57BL/6 (H2b, IfnR+/+) mice, with or without 0.5 × 106 T cells (or 3 × 106 splenocytes as a source of allogeneic T cells) from either WT (H2b, IfnR+/+) or RKO mice. Compared with transplantation of WT TCD-BMCs with T cells from a WT donor, we found that transplantation of WT TCD-BMCs with RKO T cells resulted in marked and significant increases in myeloid engraftment as determined by CFU-GM, peripheral neutrophil and platelet counts during the early phase of allo-BMT. Consistently, a significant increase serum G-CSF was also found on day 7 after allo-BMT with this group. This enhanced myeloid engraftment by RKO T cells occurred only in murine allo-BMT but not syngeneic BMT models, indicating it is a property of alloreactivity. Interestingly, no significant differences in donor T cell engraftment, nor in the percentage of Treg, Th17, CD4+ or CD8+ T cell subsets and IFN-gamma+ T cells, were observed in spleen. However, on day 7 after allo-BMT, significantly fewer donor T cells were observed in the gut in the recipient of RKO T cells compared to recipients of WT T cells. This reduction of donor T cells in gut was associated with a significant decrease in early acute GVHD lethality. This novel finding suggests transplantation of T cells lacking IFN-gamma receptors resulted in less donor T cell homing to the gut during the early phase of allo-BMT. We next addressed the ability of donor RKO T cells to provide GVT responses. A20 tumor-bearing BALB/c (H2d) mice were transplanted with WT C57BL/6 TCD-BMC. Compared with transplantation of 1 × 106 WT T cells with WT TCD-BMC, co-transplantation of 1 × 106 RKO T cells with WT TCD-BMC resulted in less GVHD-related mortality with greater anti-tumor effects. Taken together, these observations suggest that targeting IFN-gamma receptor signaling on donor T cells may help to improve the efficacy of allo-BMT by promoting donor myeloid engraftment and decreasing early acute GVHD lethality with greater GVT potential due to altered lymphocyte homing.

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Epigenetic Control of GvHD and Gvl Using the Hypomethylating Agent Azacitidine.

Blood ◽

10.1182/blood.v114.22.2447.2447 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2447-2447

Author(s):

Jaebok Choi ◽

Julie Ritchey ◽

John F. DiPersio

Keyword(s):

Clinical Trials ◽

T Cells ◽

T Cell ◽

Ex Vivo ◽

Foxp3 Expression ◽

Allogeneic Bone ◽

Allogeneic Bmt ◽

Hypomethylating Agent ◽

Donor T Cells

Abstract Abstract 2447 Poster Board II-424 Allogeneic bone marrow transplantation (BMT) represents the most effective treatment for patients with high risk and relapsed hematologic malignancies. One of the major complications of allogeneic BMT is graft-versus-host disease (GvHD), which is caused by donor T cells reacting against host antigens. These same alloreactive donor T cells can provide a beneficial ¡°graft-versus-leukemia¡± (GvL) effect as well resulting in reduction in leukemia relapse. Because regulatory T cells (Tregs) have been shown to suppress GvHD while preserving GvL, their use in the allogeneic transplant setting provides a promising strategy to treat GvHD. However, three major obstacles prevent their routine use in human clinical trials: 1) the low circulating numbers of Tregs in peripheral blood, 2) loss of suppressor activity following ex vivo expansion and 3) the lack of Treg-specific markers to purify ex vivo expanded Tregs. Foxp3 is a forkhead transcription factor which is both exclusively expressed in Tregs and, when overexpressed in conventional effector T cells (Teff), can convert these Teff into functionally suppressive Treg-like T cells. The Foxp3 locus is unmethylated in Tregs while highly methylated and silenced in all other T cells. Several groups have shown that the hypomethylating agent azacitidine (AzaC) induces FOXP3 expression in non-Tregs. Furthermore, we have shown that treatment of anti-CD3/CD28 antibody-coated bead-activated CD4+CD25- T cells with AzaC results in robust and prolonged (>7 days) expression of FOXP3. AzaC-induced FOXP3 expression is also associated with a potent Treg-like suppressive phenotype in vitro. Thus, we hypothesize that AzaC treatment of mice after allogeneic BMT will dramatically mitigate GvHD while preserving GvL via transcriptional regulation of Foxp3 in alloreactive Tconv. In murine T-cell depleted BMT model (B6¡æBalb/c; 900 cGy TBI) with delayed infusion of conventional T cells (Tconv) (2 ×106) at day 11 post BMT, followed by subcutaneous treatment of AzaC (2 mg/kg at days 15, 17, 19, and 21 post BMT), we found that AzaC dramatically suppressed GvHD caused by allogeneic donor T cells while maintaining donor (H2Kb) engraftment of all lineages. We found that the AzaC group had significantly higher FOXP3+ Tregs than in PBS control group and that these Tregs were derived from donor T cells, suggesting that the suppression of GvHD was mediated by AzaC-induced Tregs. We further tested whether AzaC treatment of mice transplanted with allogeneic T cells preserve GvL while mitigating GvHD. Using the same murine allogeneic BMT model, Click Beetle Red luciferase-expressing A20 leukemia cells (BALB/C derived) were injected with T-cell depleted BM and 10 × 106 Tconv and in vivo bioluminescence imaging was performed to assess tumor burden in vivo post transplant. We found that AzaC treatment mitigated GvHD without abrogating GvL (Fig. 1) or donor engraftment. Thus, the adminstration of hypomethlating agents like AzaC in vivo after allogeneic stem cell transplant dramatically reduces GvHD while maintaining both donor engraftment and a potent GvL effect providing the foundations for future clinical trials. Disclosures: DiPersio: Genzyme Corporation: Honoraria.

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