NK Cells Efficiently Prevent Engraftment of Donor Stem Cells after Tolerigenic Bone Marrow Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3025-3025 ◽  
Author(s):  
Leslie Kean ◽  
Kelly Hamby ◽  
Thomas Pearson ◽  
Christian Larsen
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Nk Cells ◽  
Mixed Chimerism ◽  
Low Doses ◽  
Donor Bone Marrow ◽  
Donor Specific Tolerance ◽  
Specific Tolerance

Abstract Introduction: Immunologic tolerance remains an elusive goal of transplantation. In mice, mixed-chimerism and donor-specific tolerance can be induced by blocking the CD28/CD40L T-cell costimulatory pathways after bone marrow transplant (BMT). However, large doses of marrow (~1x109 cells/kg) are required, and these regimens have not yet been successfully translated to clinical practice. There is a growing body of evidence that NK cells may play a central role in the failure of low doses of donor bone marrow to engraft, but the mechanisms underlying NK alloreactivity remain to be determined. Methods: (1) BMT in the presence of CD28/CD40L T cell costimulation blockade was performed using C57BL/6 (B6) recipients and Balb/C donor bone marrow. The role of host-anti-donor NK alloreactivity in preventing engraftment was determined by specifically depleting B6 NK cells. The contribution of the NK cell-surface receptor, LFA1 to NK alloreactivity was determined with the anti-LFA1 blocking antibody M17/5.2. (2) An in vivo NK alloreactivity assay was developed that should allow the investigation of the mechanism of NK alloreactivity and the molecular mediators of this process. In this assay, CFSE-labeled B6 splenocytes were adoptively transferred into B6xBalbC F1 progeny. As such, alloreactivity was specifically mediated by NK cells. NK alloreactivity was measured flow-cytometrically by the disappearance of the CFSE-labeled B6 population. Results: Transient depletion of recipient NK cells resulted in increased donor stem cell survival and the induction of stable mixed-chimerism and tolerance despite BMT with low doses (≤2x106 cells) of donor bone marrow. This effect was specific to allogeneic donor cells: depletion of NK cells did not increase engraftment of syngeneic bone marrow. Blocking the adhesion molecule, LFA-1 recapitulated the effects of whole-scale NK depletion. Newly emergent NK cells exhibited significantly lower expression of the donor-specific activating receptor, Ly49D, and these NK cells did not exhibit in vivo alloreactivity. These results suggest that the NK repertoire in the mixed-chimeric setting exhibited donor-specific tolerance. Using the in vivo hybrid resistance NK alloreactivity assay, we measured 80% NK-specific target killing 8 days after adoptive transfer. Significantly less killing occurred at 2, 4, and 6 days. Pre-sensitizing the recipient for 4 days increased the efficiency of killing—from 50% to 80%, suggesting a potent activation phenomenon required for efficient NK allorecognition and/or cytotoxicity. Implications: These results reveal the importance of NK alloreactivity in the acquisition of mixed-chimerism after BMT at limiting stem cell doses, and suggest that clinical approaches to tolerance-induction transplantation may require mechanisms to control NK alloreactivity.

Long-term mixed chimerism after immunologic conditioning and MHC-mismatched stem-cell transplantation is dependent on NK-cell tolerance

Blood ◽  
2005 ◽  
Vol 106 (6) ◽  
pp. 2215-2220 ◽  
Author(s):  
Geert Westerhuis ◽  
Wendy G. E. Maas ◽  
Roel Willemze ◽  
René E. M. Toes ◽  
Willem E. Fibbe
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Nk Cells ◽  
Bone Marrow Cells ◽  
Nk Cell ◽  
Mixed Chimerism ◽  
Cell Tolerance ◽  
Donor Cells ◽  
Marrow Cells

Abstract T-cell tolerance is mandatory for major histocompatibility complex (MHC)-mismatched stem-cell transplantation without cytoreduction. Here, we used a cytotoxicity assay based on the infusion of differentially carboxyfluorescein succinimidyl ester (CFSE)-labeled syngeneic and donor splenocytes to determine the survival of donor cells in vivo. In vivo cytotoxicity data showed that treatment with anti-CD40 ligand monoclonal antibody in combination with a low dose of MHC-mismatched bone marrow cells was sufficient to induce T-cell tolerance. However, CFSE-labeled donor cells were still eliminated. A similar elimination pattern was observed in T-cell and natural killer T-cell (NKT-cell)-deficient mice, suggesting the involvement of natural killer (NK) cells. Indeed, in vivo NK-cell depletion resulted in a prolonged survival of CFSE-labeled donor cells, confirming the role of NK cells in this process. Transplantation of a megadose of MHC-mismatched bone marrow cells was required for a complete survival of CFSE-labeled donor cells. This NK-cell tolerance was donor specific and was associated with mixed chimerism. Additional NK-cell depletion significantly enhanced engraftment and allowed long-term chimerism after transplantation of a relatively low dose of donor bone marrow cells. These data demonstrate the importance of NK cells in the rejection of MHC-mismatched hematopoietic cells and may explain the high numbers of bone marrow cells required for transplantation over MHC barriers. (Blood. 2005;106:2215-2220)

scholarly journals Effect of recombinant human macrophage colony-stimulating factor in irradiated murine recipients of T-cell-depleted allogeneic or non- depleted syngeneic bone marrow transplants

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1636-1642 ◽  
Author(s):  
BR Blazar ◽  
SL Aukerman ◽  
DA Vallera
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Nk Cells ◽  
Graft Rejection ◽  
Tnf Alpha ◽  
Colony Stimulating Factor ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Recombinant macrophage colony-stimulating factor (rM-CSF), which reacts exclusively with cells of monocyte lineage, was evaluated in the murine bone marrow (BM) transplant setting for in vivo effects on recipient survival, hematologic recovery, and engraftment. Two types of fully allogeneic donors were selected based on the expression (BALB/c), or lack of expression (DBA/1), of hybrid hematopoietic histocompatibility (Hh1) antigens. These antigens are established targets for monocyte and/or natural killer (NK) cell-mediated graft rejection. Irradiated C57BL/6 mice were used as recipients for all experiments. Recipients of T-cell-depleted (TCD) BALB/c BM and a 14-day continuous subcutaneous infusion of 16.8 micrograms/d rM-CSF (n = 30) showed a significant decrease in donor cell engraftment as compared with recipients of donor BM administered pumps delivering saline. These mice administered rM-CSF also displayed significantly reduced levels of circulating leukocytes (predominantly lymphocytes) on day 14 posttransplant (compared with saline controls). Neither engraftment effects nor leukocyte effects were observed when C57BL/6 recipients were administered Hh1 nonexpressing TCD DBA/1 BM cells (n = 30), suggesting that the monocyte/macrophage population is important in long-term alloengraftment in certain donor-recipient strain combinations in which donor Hh1 antigens can serve as target antigens for host effector cells, but are not important in strain combinations in which they are not recognized. Circulating tumor necrosis factor alpha (TNF alpha) levels measured at two time periods during rM-CSF infusion were not elevated. Thus, the reduction in alloengraftment is not likely to be directly related to TNF alpha. However, in vivo elimination of NK cells in the BALB/c into C57BL/6 model prevented the impairment of engraftment mediated by rM-CSF. Thus, rM-CSF-mediated inhibition of alloengraftment is contingent on the presence of host NK cells with antidonor reactivity. Survival was unaffected when rM-CSF was administered in either allogeneic BM transplant model, but was significantly reduced when rM-CSF was administered to C57BL/6 recipients of syngeneic BM transplants. These data are the first analyzing the effects of rM-CSF in murine allogeneic BM transplantation and extend our previous studies using the BALB/c into C57BL/6 model in which in vivo infusions of recombinant granulocyte-macrophage CSF, but not recombinant granulocyte-CSF, lead to decreases in alloengraftment. These data show that rM-CSF-induced stimulation of monocytes may increase BM graft rejection in instances in which NK cells are involved in the rejection process. These data may have future clinical implications for the use of rM-CSF in allogeneic BM transplantation.

Platelet Engraftment (PE) in AML Patients Receiving HLA Matched Sibling Donor Allogeneic Bone Marrow Transplant (ABMT) Correlates with Inhibitory HLA Ligand Groups for Killer Immunoglobulin-Like Receptors (KIRs).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 836-836
Author(s):  
Ronald Sobecks ◽  
Edward J. Ball ◽  
Lisa Rybicki ◽  
Stacey Brown ◽  
Jaroslaw Maciejewski ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Nk Cells ◽  
Median Time ◽  
Nk Cell ◽  
Multivariable Analysis ◽  
Sibling Donor ◽  
Cell Dose ◽  
Hla Ligands

Abstract The interaction of KIRs with target cell HLA class I molecules regulates the activity of NK cells and some T cell populations. KIR interactions are reported to influence allogeneic hematopoietic stem cell transplant outcomes, particularly for AML. We have previously reported that AML pts homozygous for C1 or C2 have superior survival and lower relapse rates than do pts heterozygous for C1/C2. However, the influence of KIR matching on PE has not been well described. We therefore analyzed the KIR ligand phenotypes of 60 AML pts who received HLA identical sibling donor myeloablative ABMT from 4/9/97 – 11/5/03. The median age was 45 years (range, 8–62 yrs). At transplant a minority (40%) were in CR. All pts received a busulfan/cyclophosphamide based preparative regimen and T-cell replete bone marrow as their stem cell source. Patient HLA KIR ligands were categorized as: 1) HLA-Cw group C1- or C2 - homozygous vs C1/C2 heterozygous; and 2) HLA-Bw4 (positive or negative) (reviewed in Farag et al Blood100:1035, 2002). Kaplan Meier estimates of median time to PE >20 K/μL and >50 K/μL were 23 and 30 days, respectively. PE was next assessed in relation to the inhibitory HLA KIR ligand group expressed. PE >20 K/μL was superior for those C1 or C2 homozygous (n=26) compared to C1/C2 heterozygotes (n=34) (median 21 vs 26 days, p=0.049) and 31 Bw4 negative pts had superior PE compared to 29 Bw4+ pts (median 21 vs 30 days, p=0.012); these findings remained significant in multivariable analysis. A similar analysis performed for PE >50 K/μL found that Bw4 negative pts had superior PE compared to Bw4 + pts (median 26 vs 38 days, p=0.015); this remained significant in multivariable analysis. 57 cases had KIR genotyping performed for those KIRs with established HLA ligands and there were no cases in which the donor did not have at least one inhibitory KIR gene specific for expressed HLA ligands. Age at transplant, number of prior chemotherapy regimens, disease status at transplant and CMV status were not predictive of PE. Since both C1/C2 heterozygosity and Bw4+ status correlated with reduced early PE, the possible interaction of these two variables was next investigated. The analysis for PE >20 K/μL and >50 K/μL suggested an additive effect: pts lacking expression of both these variables had the most rapid PE, while those who expressed both variables had the slowest PE. Those who expressed only 1 of the variables had an intermediate time to PE as shown below: HLA KIR Ligand Status N Median CD34+ cell dose (x10^6/kg) Median Total Nucleated Cell Dose (x10^8/kg) Median time to PE>20K/μL Median time to PE >50K/μL C1/C2 and Bw4 negative 14 2.02 2.64 19 days 25 days C1/C2 positive and Bw4 negative OR C1/C2 negative and Bw4 positive 29 1.92 2.62 23 days 29 days C1/C2 and Bw4 positive 17 1.87 2.60 31 days 41 days p= 0.54 p=0.82 p=0.003 p=0.003 These results may suggest that donor NK cells control host effector cells that delay PE. When minimal opportunity for inhibitory KIR engagement exists (C1/C2 negative, Bw4 negative) maximal NK cell control is expected and rapid PE ensues. When maximal opportunity for inhibitory KIR engagement exists (C1/C2 positive, Bw4 positive) donor NK cell controlling activity would be reduced, leading to delayed PE.

Graft-Versus-Host Tolerance in Dogs Following T-Cell Depleted Bone Marrow Transplantation with Absorbed ATG or CD6-Antibody.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2338-2338
Author(s):  
Julia Zorn ◽  
Hans Jochem Kolb
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
T Cell ◽  
Nk Cells ◽  
Marrow Transplantation ◽  
Graft Versus Host ◽  
Host Tolerance ◽  
Rabbit Complement

Abstract Graft-versus-host disease (GvHD) is the major obstacle of allogeneic stem cell transplantation. Depletion of T-cells from the graft reduces the risk of GvHD, but results in a higher risk of leukemia relapse. Adoptive immunotherapy with donor lymphocyte transfusion (DLT) has been shown to control leukemia in patients after T-cell depleted allogeneic stem cell transplantation. However, GvHD may occur, if DLT is given too early after transplantation. In canine models of DLA-identical and DLA-haploidentical bone marrow transplantation, we compared different methods of T-cell depletion (TCD) and investigated the potential of DLT at different times after transplantation to induce GvHD. T-cell depletion was performed either with absorbed anti-thymocyte globuline (aATG) or with a combination of CD6-antibody and baby rabbit complement. ATG was absorbed with erythrocytes, liver, kidney and spleen for eliminating antibodies against stem cells. CD6-antibody (M-T606) and rabbit complement depleted T-cells effectively without affecting hematopoietic progenitor cells. Unlike aATG, monoclonal CD6-antibody spares natural killer (NK) cells and some CD8-positive cells. Treatment of bone marrow with aATG prevented GvHD in 9 dogs following DLA-identical transplantation. DLT on days 1 and 2 or 21 and 22 induced fatal GvHD in two dogs each. However, it did not induce GvHD when given on days 61 and 62 and later. In DLA-haploidentical bone marrow recipients, non-manipulated marrow produced fatal GvHD in all dogs (n=7), whereas marrow treated with aATG (vol:vol 1:100 and 1:200) produced fatal GvHD in 5 out of 16 dogs only. CD6-depletion prevented GvHD in 3 of 3 DLA-haploidentically transplanted dogs. DLT produced fatal GvHD in one dog each, when given on day 3, 7 or 14 after CD6-depleted haploidentical bone marrow transplantation. However, it produced fatal GvHD in only 2 of 4 dogs transfused on day 20 post grafting. Thus, DLT could be given earlier in DLA-haploidentical animals transplanted with CD6-depleted marrow than in DLA-identical animals transplanted with aATG treated marrow without producing GvHD. These findings support the hypothesis that graft-versus-host tolerance can be induced earlier with grafts not depleted of NK cells. NK cells in the graft may inactivate host dendritic cells necessary for the induction of GvHD. In grafts depleted with aATG, NK cells are depleted as well, because aATG still retains broad specificity despite extensive absorptions. This leaves host DCs unaffected. Transfused donor T-cells encountering this environment will thus be activated which results in severe GvHD. In contrast, monoclonal CD6-antibody spares NK cells, so that donor lymphocytes cannot be activated by host DCs at the time of DLT and thus won’t trigger GvHD. CD6-depletion is the preferred method if adoptive immunotherapy with DLT is planned.

Pentostatin Facilitates Fully MHC-Disparate Murine Mini-Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3539-3539
Author(s):  
Jacopo Mariotti ◽  
Kaitlyn Ryan ◽  
Paul Massey ◽  
Nicole Buxhoeveden ◽  
Jason Foley ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Suppression ◽  
Graft Rejection ◽  
Mixed Chimerism ◽  
Post Transplant

Abstract Abstract 3539 Poster Board III-476 Pentostatin has been utilized clinically in combination with irradiation for host conditioning prior to reduced-intensity allogeneic hematopoietic stem cell transplantation (allo-HSCT); however, murine models utilizing pentostatin to facilitate engraftment across fully MHC-disparate barriers have not been developed. To address this deficit in murine modeling, we first compared the immunosuppressive and immunodepleting effects of pentostatin (P) plus cyclophosphamide (C) to a regimen of fludarabine (F) plus (C) that we previously described. Cohorts of mice (n=5-10) received a three-day regimen consisting of P alone (1 mg/kg/d), F alone (100 mg/kg/d), C alone (50 mg/kg/d), or combination PC or FC. Combination PC or FC were each more effective at depleting and suppressing splenic T cells than either agent alone (depletion was quantified by flow cytometry; suppression was quantified by cytokine secretion after co-stimulation). The PC and FC regimens were similar in terms of yielding only modest myeloid suppression. However, the PC regimen was more potent in terms of depleting host CD4+ T cells (p<0.01) and CD8+ T cells (p<0.01), and suppressing their function (cytokine values are pg/ml/0.5×106 cells/ml; all comparisons p<0.05) with respect to capacity to secrete IFN-g (13±5 vs. 48±12), IL-2 (59±44 vs. 258±32), IL-4 (34±10 vs. 104±12), and IL-10 (15±3 vs. 34±5). Next, we evaluated whether T cells harvested from PC-treated and FC-treated hosts were also differentially immune suppressed in terms of capacity to mediate an alloreactive host-versus-graft rejection response (HVGR) in vivo when transferred to a secondary host. BALB/c hosts were lethally irradiated (1050 cGy; day -2), reconstituted with host-type T cells from PC- or FC-treated recipients (day -1; 0.1 × 106 T cells transferred), and challenged with fully allogeneic transplant (B6 donor bone marrow, 10 × 106 cells; day 0). In vivo HVGR was quantified on day 7 post-BMT by cytokine capture flow cytometry: absolute number of host CD4+ T cells secreting IFN-g in an allospecific manner was ([x 106/spleen]) 0.02 ± 0.008 in recipients of PC-treated T cells and 1.55 ± 0.39 in recipients of FC-treated cells (p<0.001). Similar results were obtained for allospecific host CD8+ T cells (p<0.001). Our second objective was to characterize the host immune barrier for engraftment after PC treatment. BALB/c mice were treated for 3 days with PC and transplanted with TCD B6 bone marrow. Surprisingly, such PC-treated recipients developed alloreactive T cells in vivo and ultimately rejected the graft. Because the PC-treated hosts were heavily immune depleted at the time of transplantation, we reasoned that failure to engraft might be due to host immune T cell reconstitution after PC therapy. In an experiment performed to characterize the duration of PC-induced immune depletion and suppression, we found that although immune depletion was prolonged, immune suppression was relatively transient. To develop a more immune suppressive regimen, we extended the C therapy to 14 days (50 mg/Kg) and provided a longer interval of pentostatin therapy (administered on days 1, 4, 8, and 12). This 14-day PC regimen yielded CD4+ and CD8+ T cell depletion similar to recipients of a lethal dose of TBI, more durable immune depletion, but again failed to achieve durable immune suppression, therefore resulting in HVGR and ultimate graft rejection. Finally, through intensification of C therapy (to 100 mg/Kg for 14 days), we were identified a PC regimen that was both highly immune depleting and achieved prolonged immune suppression, as defined by host inability to recover T cell IFN-g secretion for a full 14-day period after completion of PC therapy. Finally, our third objective was to determine with this optimized PC regimen might permit the engraftment of MHC disparate, TCD murine allografts. Indeed, using a BALB/c-into-B6 model, we found that mixed chimerism was achieved by day 30 and remained relatively stable through day 90 post-transplant (percent donor chimerism at days 30, 60, and 90 post-transplant were 28 ± 8, 23 ± 9, and 21 ± 7 percent, respectively). At day 90, mixed chimerism in myeloid, T, and B cell subsets was observed in the blood, spleen, and bone marrow compartments. Pentostatin therefore synergizes with cyclophosphamide to deplete, suppress, and limit immune reconstitution of host T cells, thereby allowing engraftment of T cell-depleted allografts across MHC barriers. Disclosures: No relevant conflicts of interest to declare.

Effect of a Novel Nucleoside Analogue, Triciribine Phosphate (TCN-P) on Murine Acute Graft-Vrs-Host Disease (aGvHD)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2977-2977
Author(s):  
Edward Dela Ziga ◽  
Jaebok Choi ◽  
Mark Needles ◽  
Julie Ritchey ◽  
John F. DiPersio
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nucleoside Analogue ◽  
Foxp3 Expression ◽  
Negative Control ◽  
Jurkat Cells ◽  
Acute Graft

Abstract Abstract 2977 BACKGROUND: The successful establishment of donor registries and development of improved conditioning regimens among others, has led to the increased use of hematopoietic stem cell transplant (HSCT) as a key component in the treatment of some malignant and benign hematopoietic/lymphoid disorders as well as some metabolic disorders. Although a potential curative therapy for many hematologic diseases, allogeneic stem cell transplantation is associated with considerable morbidity and mortality primarily from acute graft-versus-host disease (aGvHD). Furthermore, graft-versus-leukemia (GVL) mediated by donor T cells can be abrogated with T cell depletion or suppression in vivo resulting in disease relapse with treatment of aGvHD. Moreso, modern therapies for aGvHD are limited and often toxic, thus there is a need for novel treatments and approaches that control aGvHD without compromising GVL. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor has been shown to decrease the severity of aGvHD (Reddy et al, PNAS 2004) through its effect on pro-inflammatory cytokines while maintaining GVL in a murine GvHD model. Also, previous work from our lab demonstrated that treatment of mice with the hypomethylating agent azacitidine (AzaC) after allogeneic HSCT mitigates aGvHD while preserving GVL by inducing FOXP3 expression in activated non-T regulatory cells (Choi et al, Blood 2010). However, the myelosuppression mediated by AzaC is a potential limitation that results in delayed donor engraftment. This led us to explore alternate options for single or combination drug therapy in the treatment of aGvHD. We screened a library of 2000 chemical agents obtained from the National Institutes of Health. Screening resulted in a single hit identified as Triciribine phosphate (TCN-P), an Akt inhibitor with structural similarity to the nucleoside analogue AzaC. In this experiment, a Foxp3 promoter-luciferase construct was designed and transfected into Jurkat cells. Cells were incubated for 2 days and then treated with three concentrations (0.1uM, 1umM and 10uM) of each chemical agent in the library. Bioluminescence imaging (BLI) was done on day 4 with AzaC as positive control (Choi et al, Blood 2010) and PBS as negative control. Only wells treated with TCN-P 10uM showed a signal, suggesting luciferase activity secondary to the Foxp3-promoter activation. We therefore hypothesized that TCN-P as a single agent or in combination with SAHA and or AzaC would mitigate GvHD by inducing FOXP3 without interfering with engraftment or immune reconstitution. METHODS: Using a C57BL/6(H2b) into Balb/c (H2d) murine MHC mismatch bone marrow transplant (BMT) model, we transplanted 5 × 106 T cell-depleted (TCD) bone marrow cells obtained from C57BL/6 (H2b, CD45.1+) mice into Balb/c (H2d, CD45.2+) mice after 900cGy of TBI. Delayed donor infusions of 2 × 106 pan-T cells/mouse obtained from FOXP3/GFP KI: B6 CD45.2+ H2b mice were infused on Day +11 in order to induce GvHD. Azacitidine 2mg/kg, SAHA 35mg/kg and TCN-P 10mg/kg were injected intraperitoneally every other day from Day +15 to Day +21(total of 4 doses). Acute GvHD was assessed by a standardized scoring developed by Cooke and Ferrara. (Blood, 1996) RESULTS : 1. Using our Foxp3-reporter system, both AzaC and TCN-P induced significant luciferase expression in Jurkat cells. SAHA had no effect. 2. Only AzaC but neither SAHA nor TCN-P induced significant Foxp3 expression in WT bead activated T cells. 3. In vivo, both AzaC 2mg/kg and TCN-P 10mg/kg but not SAHA 35mg/kg significantly improved survival of mice with less weight loss and clinical signs of aGvHD in a MHC mismatched aGvHD model. CONCLUSION: A novel nucleoside analogue TCN-P that was previously FDA approved for treatment of multiple myeloma and structurally related to AzaC, induces Foxp3 using a luciferase reporter construct in Jurkat cells and improves survival in mice after MHC mismatched allogeneic transplant. Though the 100 day survival between TCN-P and PBS (as negative control) in our murine aGvHD model was not quite statistically significant, the findings suggest a therapeutic potential for TCN-P and possibly other Akt inhibitors in the mitigation of aGvHD. Disclosures: No relevant conflicts of interest to declare.

MyD88-Dependent Expansion of An Immature GR-1+CD11b+ Population From Donor Bone Marrow Reduces Early Mortality After Allogeneic Hematopoietic Stem Cell Transplantation,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4011-4011
Author(s):  
Ji-Young Lim ◽  
Gyeongsin Park ◽  
Hyewon Youn ◽  
Eun-Young Choi ◽  
Dae-Chul Jeong ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Early Mortality ◽  
Hematopoietic Stem ◽  
Target Organs ◽  
Donor Bone Marrow ◽  
Donor T Cells

Abstract Abstract 4011 Graft-versus-host disease (GVHD) is a common complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with anti-inflammatory activity. MyD88 is a cytoplasmic adaptor molecule essential for integrating and transducing the signals generated by the toll-like receptor (TLR) family. Activation of inflammatory signaling through MyD88, presumably through ligation of multiple TLRs, plays a key role in the expansion of MDSCs. We therefore investigated how the MyD88-dependent expansion of MDSCs from donor bone marrow (BM) contributes to protection of acute GVHD. To test this, we employed an intestinal GVHD murine model, C57BL/6 (H-2b) → B6D2F1 (H-2b/d), which differs at major and minor histocompatibility loci. Lethally irradiated recipient mice were transplanted with wild-type (WT) or MyD88 knock out (KO) mice T cell-depleted (TCD)-BM together with WT spleen T cells. Morbidity and mortality of GVHD was significantly worse in recipients of MyD88 KO TCD-BM with higher intestinal pathologic grading. Animals that underwent syngeneic HSCT did not show early mortality regardless of presence of MyD88 in BM, which ruled out myelosuppression-associated toxicity. The expression of Gr-1+CD11b+ in blood, mesenteric lymph nodes and liver on day 13 was significantly reduced in the recipients of MyD88 KO TCD-BM compared with those of WT TCD-BM while the percentage of donor T cells infiltrating colon and liver was significantly higher. In parallel, the percentages of donor T cells to undergo apoptosis in response to alloantigens in vivo were significantly decreased in recipients of MyD88 KO TCD-BM. Injection of MDSCs from BM of non-tumor bearing donor markedly inhibited GVHD lethality in recipients of MyD88 KO TCD-BM. Moreover, in vivo administration of lipopolysaccharide (LPS), a TLR ligand, to donor mice expanded GR-1+CD11b+ in BM with enhanced expression of MyD88 mRNA. Recipients of TCD-BM from WT mice injected LPS showed attenuated GVHD severity as measured by weight loss and survival compared to those of TCD-BM from WT mice injected diluent. In summary, MyD88-dependent expansion of GR-1+CD11b+ population from donor TCD-BM appears to be critical for survival after allo-HSCT. Incomplete expansion of GR-1+CD11b+ population in target organs correlates with decreased apoptosis and increased infiltration of donor T cells into the target organs. Disclosures: No relevant conflicts of interest to declare.

Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1999-1999
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Benedetta Nicolini ◽  
Nadim Mahmud ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Committed T lymphocyte stem cells of rats. Characterization by surface W3/13 antigen and radiosensitivity.

1981 ◽  
Vol 154 (4) ◽  
pp. 1164-1177 ◽  
Author(s):  
M J Dyer ◽  
S V Hunt
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Gamma Irradiation ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Cell Activity ◽  
Rat Bone

The existence of stem cells committed to the T lymphoid lineage was deduced from studying how rat T and B stem cells differ in their expression of membrane W3/13 antigen and in their susceptibility in vivo to gamma irradiation. Stem cell activity of rat bone marrow and fetal liver was measured in long-term radiation chimeras using B and T cell alloantigenic surface markers to identify the progeny of donor cells. Monoclonal mouse anti-rat thymocyte antibody W3/13 labeled approximately 40% of fetal liver cells and 60-70% of young rat bone marrow cells (40% brightly, 25% dimly). Bright, dim, and negative cells were separated on a fluorescence-activated cell sorter. All B and T lymphoid stem cells in fetal liver were W3/13 bright, as were B lymphoid stem cells in bone marrow. W3/13 dim bone marrow had over half the T cell repopulating activity of unseparated marrow but gave virtually no B cell repopulation. In further experiments, the radiosensitivity of endogenous B and T lymphoid stem cells was determined by exposing host rats to between 4.5 and 10 Gy of gamma irradiation before repopulation with genetically marked marrow. The results depended on whether chimerism was assayed before day 50 or after day 100. At early times, a radioresistant T stem cell was indicated, whose activity waned later. Thus committed T stem cells of rats carry moderate amounts of W3/13 antigen and are more radioresistant but less permanently chimeragenic than the stem cells that regenerate B lymphocytes.

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