Donor BM Research Articles

Poor Performance Status, Chemorefractory Disease At Transplantation, and Umbilical Cord Blood As Donor Source Were Adverse Prognostic Factors for Overall Survival After Allogeneic Stem Cell Transplantation for Follicular Lymphoma: Retrospective Study of the Japan Society of Hematopoietic Stem Cell Transplantation (JSHCT) Lymphoma Working Group

Abstract 3093Allogeneic hematopoietic stem cell transplantation (allo SCT) is potentially curative treatment for relapsed follicular lymphoma (FL) that remains non-curative disease even with modern immunochemotherapy. Number of potential candidate of allo SCT for FL has been increasing with the development of reduced intensity conditioning (RIC) regimens and increased donor availability due to use of hematopoietic stem cell from unrelated donor including cord blood (CB). However, short-term and long-term transplantation-related complications are still major obstacles in applying this treatment. Recently, EBMT and CIBMTR reported a prognostic score of allo SCT for FL (Ann Oncol 2011;22 :Suppl 4, iv94). However, this score awaits validation. To elucidate prognostic factors for OS after allo SCT for FL, we conducted a retrospective study using the national registry data of the Japan Society of Hematopoietic Cell Transplantation (JSHCT). In total, 472 cases of allo SCT for FL performed from 2000 to 2008 were identified. 220 (46.6%) were male and the median age at allo SCT was 50 yo (range: 27–75). Forty six (9.7%) patients were 60 yo or older. Eighty two (17.3%) patients had previous history of autologous transplantation (ASCT). Stem cell source was BM or PB from related donor in 215 (45.5%), unrelated donor BM in 180 (38.1%), and unrelated donor umbilical cord blood (CB) in 77 (16.3%). Conditioning regimen was myeloablative (MAC) in 20.7% and reduced intensity (RIC) in 79.3%, respectively. Patients undergoing MAC were younger than those with RIC (45 vs 50 yo, P<0.01). T-cell depletion with ATG or ALG was performed in 7.5%. ECOG performance status (PS) at allo SCT was ≥ 2 in 10.7%. 71.5% had chemosensitive disease at allo SCT and disease status was CR in 38.9%. With a median follow up of 47.3 mo (range: 0.9–122) among survivors, overall survival rate at 4years after allo SCT (4y OS) was 52.6%. Patients who had CB transplantation had worse OS than those transplanted from related donor or unrelated BM (30.0%, 58.5%, 54.6%, P =1.35e-6, logrank). Additionally, in univariate analysis, PS ≥2, chemoresistant disease at allo SCT, older age, male sex were adverse prognostic factors for OS, while previous history of ASCT, time from diagnosis to allo SCT (<3y vs ≥3y), disease status at allo SCT (CR vs non CR), conditioning regimen (MAC vs RIC), and year of allo SCT (2000–2 vs 2003–5 vs 2006–8) were not. 4y OS of patients undergoing MAC and RIC were 46.2% and 54.0% (P =0.28), respectively. In multivariate analysis with proportional hazard modeling, PS ≥2 (HR 3.2, P =0.00015), chemorefractory disease (HR 2.7, P =0.039), and use of CB (HR 3.8, P =0.039) were independent adverse predictors of OS. Although risk factors that are incorporated in the proposed EBMT/CIBMTR risk score other than PS were not predictive of OS in the present analysis, we applied this score to the population of the present study. In the entire population, 4y OS were 61.0% and 38.5% (P =9.5e-08) in patients with the score <3 (n=294) and ≥3 (n=77), respectively. This score was predictive of OS in patients who underwent transplantation from related donor (65.4% vs 36.7%, P =3.77e-08) and from unrelated BM (59.0% vs 41.7%, P =0.0217). In conclusion, this retrospective study of JSHCT registry data showed that allo SCT for FL is a reasonable option for patients with relapsed FL when suitable donor is available. Poor PS, chemorefractory disease, and CB as donor source were adverse prognostic factors for OS. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Delayed Processing of Bone Marrow Samples Reveals a Prognostic Pattern of NME mRNA Expression in Cytogenetically Normal AML

Abstract 4904 Background:Prognostic molecular markers are expected to be of increasing value in stratifying treatment of patients with AML lacking cytogenetic abnormalities. While specific DNA-level mutations such as those in the FLT-3 or NPM1 genes clearly identify informative genotypes, phenotypic characterization of gene expression should be a more direct indicator of cell function. However, of the prognostic mRNA levels identified to date, such as BAALC, ERG or NME1/2, none appear to be superior to DNA mutation as indicators of prognosis. Because of the lability of mRNA expression, it is common practice to quantify levels only in freshly processed or direct-lysed samples in order to provide a snapshot of gene expression as close as possible to that of fresh tissue. However, characteristics of leukemic cells relevant to prognosis might become more apparent under challenging conditions, such as the stress environment developing in a bone marrow sample during transport or storage. The NME1 and -2 mRNAs have been reported to be prognostic indicators in AML and are multifunctional proteins coordinating metabolism, signalling and gene expression. To see how storage related stress affects the prognostic power of NME mRNA, we have assessed their prognostic relevance in CN-AML bone marrow samples which had originally been processed either directly, or following ≥ 2 days of transport from collaborating centers. Methods:A total of 78 archived CN-AML BMMNC samples were available for this analysis. All samples were taken at presentation from patients under 60ys treated within the AML 96 (OSHO #033) or AML 2002 (OSHO #061) protocols of the East German Study Group (OSHO). Of the 78 samples, 25 (32%) originated locally and had been freshly processed to cryopreserved MNC, while the remaining 53 (68%) had been submitted as bone marrow from other centers with an associated delay of 2–3 days. NME1 and NME2 mRNA levels were determined by triplicate qRT-PCR determinations normalized to RPLP0. The mean NME1 and NME2 expression values from each patient were expressed relative to the corresponding mean NME expression of 17 healthy donor BM samples (control group).To investigate directly the effects of delayed processing on NME mRNA expression, aliquots of 11 fresh CN-AML bone marrow samples were removed for analysis either immediately or following storage at room temperature for ≥ 2 days. Results:Both NME1 and -2 mRNA were increased relative to controls in 84% (21/25) of fresh samples with no prognostic relevance of expression above or below the median. Comparable results were obtained from a cohort of 59 freshly processed CN-AML samples from a separate study. In contrast, the transported samples yielded a heterogeneous pattern of NME mRNA expression above and below control levels, with a significant correlation between NME2 mRNA and event-free survival (p=0.009) independently of FLT-3ITD status. Direct analysis of aliquoted AML bone marrow samples confirmed that a delay in processing of ≥ 2 days resulted in a universal decrease in NME1 mRNA with variable changes in NME2 mRNA. Conclusion:NME1/2 mRNA levels are not indicative of prognosis in freshly processed bone marrow from CN-AML patients <60ys. However, delayed processing is associated with the development of an NME2 expression pattern with high prognostic relevance, maintenance of high NME2 in samples with reduced NME1 being a strong indicator of increased event-free survival. These results suggest that subjecting leukemic cells to defined challenges ex-vivo may reveal phenotypic properties of high relevance to treatment optimization. Disclosures:No relevant conflicts of interest to declare.

GM-CSF Signaling Abnormalities in Chronic Myelomonocytic Leukemia

Abstract 1713 Background:Chronic Myelomonocytic Leukemia (CMML) and Juvenile Myelomonocytic leukemia (JMML) are classified as MDS/MPN in the WHO classification system. Despite sharing clinical and histological features, CMML is characterized by a heterogeneous collection of molecular lesions while JMML is defined by well-established molecular aberrations clustered along the RAS pathway leading directly to GM-CSF hypersensitivity; a pathognomonic characteristic of JMML. Here we test whether a molecular signature for GM-CSF hypersensitivity in JMML, determined by the pSTAT5 activation assay, is also present in CMML and whether this signature clusters within a specific CMML subgroup. Methods:Cryopreserved bone marrow aspirates from 24 patients with newly diagnosed or relapsed CMML were obtained from the Moffitt Cancer Center Tissue Repository. Cells were thawed and rested in Stem Span H3000 with 10% FBS for 2 hours and then either starved for one hour in serum-free media, serum free group (n=12), or rested in Stem Span for an additional hour, serum group (n=12), prior to stimulation with G-CSF, IL-3, or GM-CSF for 15 minutes and then fixed and permeabilized with formaldehyde and methanol, as previously described. Samples were stained with an anti-pSTAT5(Y-694) antibody and analyzed by flow cytometry (Kotecha, Cancer Cell. 2009). Cells stained with isotype-control antibody were used to establish the threshold for basal STAT5 phosphorylation. Because STAT5 was constitutively phosphorylated in serum, and to a lesser extent in serum-free conditions, inducible cytokine activation was defined as the percentage of pSTAT5 positive cells above untreated samples in both CMML and healthy controls. A retrospective chart review was performed to obtain clinical variables including age, sex, WHO classification, Dusseldorf scoring system, MD Anderson scoring system, WBC, peripheral monocyte count, blast percentage, anemia, platelet count, splenomegaly, and metaphase cytogenetics. Results:The percentage of pSTAT5 responsive cells after G-CSF stimulation with doses up to 10 ng/ml was similar in cases and normal BM controls (p=0.14), whereas, a statistically significant increase in the percentage of inducible pSTAT5 positive cells was observed with GM-CSF 0.1 ng/ml (p=0.04), GM-CSF 1 ng/ml (p=0.02), and GM-CSF 10 ng/ml (p=0.01) in CMML BM cells compared to healthy donor BM cells, as shown in Figure 1. Using one standard deviation below the mean as a cut point, only 5 patients failed to show GM-CSF hypersensitivity in the serum (n=3) and serum-free groups (n=2), respectively. IL-3 and GM-CSF play similar roles in hematopoietic growth through the activation of JAK2/STAT5 and share a common beta-chain required for signaling. Signaling mediated by GM-CSF and IL3 converge to activate RAS and other downstream intermediates that regulate DNA synthesis, cell-cycle progression and suppression of apoptosis. The concentration of IL3 required to induce STAT5 phosphorylation was 10-fold greater than GM-CSF in CMML cells, but the percentage of cells responsive to IL3 was greater in CMML cases compared to controls at 10 ng/ml (p=0.02). Analysis of the percentage of GM-CSF hypersensitive cells and clinical parameters revealed no associations with age at onset, WHO classification, Dusseldorf scoring system, MD Anderson scoring system, blast percentage, anemia, platelet count, splenomegaly, or karyotype. The percentage of pSTAT5 positive cells with GM-CSF 0.1 ng/ml positively correlated with the total leukocyte (p=0.03) and total monocyte (p=0.02) count indicating that the JAK2/STAT5 signaling response is indicative of disease burden. [Display omitted] Conclusions:Based on the threshold for cytokine stimulation and percentage of cells that display pSTAT5 induction, CMML appears to preferentially utilize GM-CSF for survival and/or expansion. Although RAS mutations were not assessed, CMML cells were preferentially sensitive to GM-CSF in newly diagnosed cases independent of cytogenetic abnormalities suggesting that JMML and CMML share biological features of GM-CSF hypersensitivity. Disclosures:Padron:KaloBios Pharmaceuticals, Inc.: Research Funding. Bebbington:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership. Baer:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership.

Solution-processed small-molecule solar cells with 6.7% efficiency

Organic photovoltaic devices that can be fabricated by simple processing techniques are under intense investigation in academic and industrial laboratories because of their potential to enable mass production of flexible and cost-effective devices. Most of the attention has been focused on solution-processed polymer bulk-heterojunction (BHJ) solar cells. A combination of polymer design, morphology control, structural insight and device engineering has led to power conversion efficiencies (PCEs) reaching the 6-8% range for conjugated polymer/fullerene blends. Solution-processed small-molecule BHJ (SM BHJ) solar cells have received less attention, and their efficiencies have remained below those of their polymeric counterparts. Here, we report efficient solution-processed SM BHJ solar cells based on a new molecular donor, DTS(PTTh(2))(2). A record PCE of 6.7% under AM 1.5 G irradiation (100 mW cm(-2)) is achieved for small-molecule BHJ devices from DTS(PTTh(2))(2):PC(70)BM (donor to acceptor ratio of 7:3). This high efficiency was obtained by using remarkably small percentages of solvent additive (0.25% v/v of 1,8-diiodooctane, DIO) during the film-forming process, which leads to reduced domain sizes in the BHJ layer. These results provide important progress for solution-processed organic photovoltaics and demonstrate that solar cells fabricated from small donor molecules can compete with their polymeric counterparts.

High-Resolution Imaging and Antitumor Effects of GFP + Bone Marrow-Derived Cells Homing to Syngeneic Mouse Colon Tumors

Bone marrow-derived cells (BMDCs) participate in the growth and spread of tumors of the breast, brain, lung, and stomach. To date, there are limited reports of bone marrow involvement in colon cancer pathogenesis, but such findings would have the potential to generate novel treatments for colon cancer patients. We have established a mouse model for imaging BMDCs from whole tumor to single-cell resolution, whereby the bone marrow of lethally irradiated host animals is reconstituted with EGFP-expressing bone marrow cells from matched TgActb(EGFP) donors. The BM transplants yield mice with fluorescently labeled bone marrow, and so BMDCs can subsequently be monitored within a tumor through optical imaging. Successful BM reconstitution was confirmed at 8 weeks after transplantation, when surviving BALB/c mice were injected with CT26 mouse colon cancer cells. We find that up to 45% of cells dissociated from the tumors are GFP(+) and approximately 50% of Lin(+), CD11b(+), and CD3(+) cells express high levels of GFP. Notably, tumor growth is reduced in BM transplanted animals, compared with untransplanted host mice or EGFP-expressing BM donor mice. A needed next step is to separate the molecular and cellular (eg, T cells, NK cells, macrophages) bases of the antitumor effect of the BMDCs from any protumorigenic effect that could be subverted for therapeutic gain.

P02-108 - Bone marrow, depression” - transmission of psychiatric diseases through hematologic stem cell transplantation ? - Aetilogic considerations and a clinical case report

Allogeneous hematogenic stem cell transplantation (HSCT) is a curative therapeutic strategy for numerous benign and malignant hematologic diseases. The transplantation procedure is characterized by significant physical, social and psychological stressors, especially for the graft recipient. This can lead to depression and anxiety. Every HSCT represents a potential risk for transmission of known and unknown diseases. Hematologically transmissible diseases of the donor therefore are considered a contraindication. Recent animal experiments have demonstrated that psychiatric diseases can transmitted via HSCT from donor to recipient. So far there are no such observations in human patients.Here we report the case of a 33 year old male patient who, because of a rare chronic hypoplastic congenital aenemia, underwent HSCT. The BM donor was the brother of the patient and suffered from recurrent depressive disorder. Because of the short stature resulting from his underlying hematologic disease the patient had been in psychotherapy form several years. Depressive episodes had not been observed during this time. 10 weeks post HSCT the patient experienced a depressed mood and recurring anxiety attacs. The initiation of antidpressant medication was considered but not started due to the mild symptomatology.While depressive symptoms after HSCT are not infrequent because of the multiple stressors outlined above, we argue that in the light of the recent animals studies on the transmission of psychiatric disease via hematopoeitic stem cells in the present constellation a hematologic transmission of the mood disorder can also be condsidered.Acknowledment: To the colleagues of the Department of Pediatrics.

Drug rash with eosinophilia and systemic symptoms (DRESS) after an unrelated donor BM transplant

Drug rash with eosinophilia and systemic symptoms (DRESS) after an unrelated donor BM transplant

Bone Marrow Myeloid-Derived Suppressor Cells (MDSC) Inhibit Graft Versus Host Disease (GVHD) Via An Arginase-1 Dependant Mechanism That Is Upregulated by IL-13

AbstractAbstract 241 Background:Myeloid-derived suppressor cells (MDSC) are a well-defined population of cells that accumulate in the tissue of tumor-bearing animals and are known to inhibit immune responses. Here, we have developed a novel method by which we can generate MDSC from the BM of wild type mice. Our goals were to determine the effectiveness of these cells in inhibiting an allogeneic T-cell reaction, the mechanism by which this occurs, and the impact on the graft-versus-tumor (GVT) activity of donor T-cells. Results:Our results show that the incubation of WT BM with G-CSF and GM-CSF for a period of 4 days results in a population of cells predominately co-expressing CD11b and Gr1 (50%). Most of these cells have a monocytic phenotype of Ly6GloLy6C+ (73%) and also express other markers associated with MDSCs such as IL4Ra (64%), F4/80 (63%) and CD115 (43%). Such MDSC potently inhibited in vitro allogeneic T-cell responses and the addition of IL-13 to the MDSC culture enhanced their suppressive capacity (60% suppression by MDSC at the peak of the response, day 5 vs. 75% suppression by MDSC-IL13, P<0.001). Suppression was dependent on L-arginine depletion because only the addition excess L-arginine, but not tryptophan, resulted in a significant reduction in suppression (60% reduction at day 4, P=0.001). Adding IL-13 to the MDSC cultures resulted in arginase-1 upregulation (20 fold increase in expression over control BM for MDSC vs. 350 fold increase in MDSC-IL13, P<0.001). The arginase inhibitor, nor-NOHA, significantly reduced MDSC suppression in vitro. GNC2 kinase knockout T cells were resistant to suppression, consistent with their inability to sense L-arginine depletion. Although iNOS was upregulated 5-fold in MDSCs vs BM, IL13 addition did not further upregulate iNOS. Suppression was mostly contact-independent. In vivo, MDSC-IL13 derived from GFP transgenic BM migrated to sites of allopriming. MDSC-IL13 from luciferase transgenic donors increased over a 3 week period of time as assessed by bioluminescent imaging. Although both MDSC and MDSC-IL13 inhibited GVHD lethality, MDSC-IL13 were more effective at enhancing survival (MDSC vs. MDSC-IL13, P=0.001). When compared with untreated mice, GVHD inhibition in MDSC-IL13 treated mice was associated with: 1) Limited donor T-cell proliferation (CD4+CFSE−, 40% vs. 17%, P=0.01; CD8+CFSE−, 51% vs. 16%, P=0.01); 2) Decreased donor T-cell activation (CD4+CD62L−, 74% vs. 50%, P<0.001; CD8+CD62L−, 77% vs. 41%, P<0.001); 3) Decreased proinflammatory cytokine production (CD4+IFNg+, 37% vs. 24%, P=0.04; CD8+IFNg+, 30% vs. 15%, P=0.007); 4) Decreased expression of intracellular CD3z chain (CD4+CD3z+, 86% vs. 56%, P<0.001; CD8+CD3z+, 85% vs. 68%, P<0.001). On day 14, donor MDSC-IL13 cells, distinguishable from donor BM and the host by a CD45 congenic marker, were re-isolated from the spleen of murine GVHD recipients. MDSC-IL13 cells retained their initial phenotype and were potent ex vivo suppressors of an allogeneic T cell response. Arginase-1 knockout MDSC-IL13 did not have a significant effect on reducing GVHD, indicating that arginase-1 expression was vital to the in vivo suppressive effect of MDSCs. MDSC-IL13 did not abrogate the graft-versus-tumor effect of donor T-cells. In MDSC-IL13 treated mice, donor T-cells retained their ability to eliminate A20 lymphoma cells, and at the same time, had improved survival when compared to mice receiving A20 cells plus T-cells all of which succumbed to GVHD (BM+T+A20 vs. BM+T+A20+MDSC-IL13, P<0.001). Since arginase-1 expression was critical for suppression, we exploited this mechanism of action by administering a pegylated form of human arginase-1 (PEG-arg1) to systemically deplete L-arginine. We find that GVHD survival is significantly improved (BM+T vs. BM+T+PEG-arg1, P=0.003). Conclusions:We can conclude that BM-derived MDSC-IL13 have the ability to dampen GVHD and enhance survival when using a fully-mismatched murine model of BMT. Arginase-1 expression was found to play a critical role in MDSC-mediated suppression. A GVT effect was not ablated by MDSC-IL13. In vivo administration of PEG-arg1 resulted in L-arginine depletion and significant GVHD reduction. Both MDSC infusion and PEG-arg1 administration are promising strategies that warrant further preclinical studies to prevent GVHD that may be readily translatable in the clinic. Disclosures:Highfill:UIniversity of Minnesota: Patents & Royalties; Louisiana State University: Patents & Royalties; Medical College of Georgia: Patents & Royalties. Rodriguez:University of Minnesota: Patents & Royalties; Louisiana State University: Patents & Royalties; Medical College of Georgia: Patents & Royalties. Ochoa:University of Minnesota: Patents & Royalties; Louisiana State University: Patents & Royalties; Medical College of Georgia: Patents & Royalties. Blazar:University of Minnesota: Patents & Royalties; Louisiana State University: Patents & Royalties; Medical College of Georgia: Patents & Royalties.

Potent Strategy for Treatmet of Gvhd and Autoimmune-Diseased by Infusion of Donor-Typed or Even Third-Party Tolerogenic Dendritic Cells by Psoralen Plus UVA Treatment

AbstractAbstract 2777We have reported that bone marrow derived dendritic cells with psoralen and UVA (PUVA-DCs) treatment acquired tolerogenicity in mice. With the purpose of potential application of PUVA-DCs in a clinical hematopoietic stem cell transplantations (HSCT) for graft-versus-host disease (GVHD), we showed that mixed lymphocyte reaction (MLR) was strongly inhibited when PUVA-DCs from the stimulator strain were added to the coculture (Stimulator (S): conventional DCs obtained from C57BL/6, Responder (R): splenocytes obtained from Balb/c, PUVA-DCs: C57BL/6). This suggests that infusion of host-typed PUVA-DCs would become a novel therapeutic approach for GVHD. However utilizing host-typed DCs has problems because of leukemic cell contaminations or low efficiency of cell culture from the patients receiving repetitive chemotherapy. Therefore next concern is whether PUVA-DCs generated from BM donor or even strangers would have same tolerogenicity as host-typed PUVA-DCs do. To test this, we performed MLR by adding PUVA-DC generated from the same strain of responder or third party strain (S: conventional DCs obtained from C57BL/6, R: splenocytes obtained from Balb/c, PUVA-DC: C57BL/6 or C3H). Proliferation was significantly inhibited when PUVA-DC generated from the stimulator strain were added to the coculture (p<0.05). Also significant inhibition was observed (p<0.05) when adding PUVA-DCs generated from third party, suggesting that PUVA-DCs have tolerogenicity in a MHC-independent manner.To clarify the mechanisms of how PUVA-DCs induce tolerogenicity, we performed MLR as mentioned above with the addition of neutralizing antibodies against IL-10 or TGF-beta1 or both, which have immunosuppressive effects. Neutralization of immunosuppressive cytokines had no effects on MLR. We then hypothesized that cell-to-cell contact between PUVA-DCs and alloreactive T-cells was needed to mediate the regulatory effect. To this end, we performed MLR using transwell to prevent cell-to cell contact. MLR was not suppressed when transwell was used, suggesting that PUVA-DCs dominantly regulates the alloreaction in a cell contact-dependent manner.This is the first report that PUVA-DCs prepared not only from host-typed but from donor-typed or even third-party could induce strong inhibition of alloreaction. Tolerogenic DCs prepared previously by several ways could not induce inhibition of alloreaction in vitro when these cells were prepared from donor-typed or third-party strains in mice. To apply tolerogenic dendritic cells for GVHD in clinical settings, it is necessary to obtain sufficient doses of PUVA-DCs with ease and safety guaranteed. Therefore in the future PUVA-DCs generated even from HLA mismatched iPS cells would be a promising approach. In conclusion, infusion of PUVA-DCs from donor-typed or even third party strain could have a potent strategy for treatment of lethal GVHD and autoimmune diseases. [Display omitted] Disclosure:No relevant conflicts of interest to declare.

Regulation of Acute Graft-Versus-Host Disease by MicroRNA-155

AbstractAbstract 245Acute Graft-versus-host disease (aGVHD) is a frequent and lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT) in which donor T cells destroy HLA mismatched host tissues by secreting soluble inflammatory cytokines (TNF-α, IFN-γ) and/or inducing direct cytotoxic cellular responses. Despite recent advances, GVHD still remains a major clinical problem, underscoring the need to elucidate further its mechanisms to then develop novel therapeutic strategies. Recent studies indicate that microRNAs (miR) play critical roles in the development and function of the immune system. In particular, miR-155 is required for normal function of B and T lymphocytes. MiR-155 has been shown to be up-regulated upon B and T cell activation and mice deficient for miR-155 are immunodeficient, exhibiting T cells with attenuated INF-γ and TNF-α release in response to antigen stimulation. Based on these observations, we hypothesized that miR-155 is up-regulated in donor T cells during aGVHD and is involved in the modulation of this process. To test this hypothesis, we initially measured miR-155 expression from activated CD8 T cells obtained from an experimental animal model of aGVHD. For this, a major histocompatibility complex (MHC) mismatched HSCT model was used in which spleen cells and BM from C57BL/6 (B6) donors were transferred i.v. into lethally irradiated B6D2F1 recipient mice (n=4). Two additional groups were included as controls with one group receiving no cell infusion (radiation only, n=4); and a second group receiving T cell depleted bone marrow only (t-BM) (5×106) (n=4). GVHD scores were performed daily according to Cooke et al, Blood 1996;8:3230-9. Recipient mice were sacrificed and tissues harvested when GVHD scores were ≥7 or at the end of the experiment. After an average of 3–4 weeks, mice receiving donor spleen cells but not BM alone developed severe GVHD (scores >7) which was confirmed by histology. Activated CD8 T cells from the spleen were isolated using CD8+/CD44+ antibodies and after obtaining total RNA, miR-155 expression was measured by RT-PCR. Mice receiving donor BM plus spleen cells developed severe aGVHD and exhibited increased miR-155 expression with respect to the BM only group (fold change 5.2, t-test p<0.001). To confirm that a causal relationship exists between miR-155 and aGVHD severity, we repeated the above MHC mismatched murine experiment using BL/6 mice deficient for miR-155 expression as donors (Rodriguez et al, Science 2007;316;608-611). The groups were as follows; radiation alone (n=4), t-BM (n=8), BM + wild type (WT) spleen cells (n=8), and t-BM+ miR-155 KO spleen cells (n=8). We found that mice receiving donor spleen cells from miR-155 KO mice exhibited dramatically lower mean GVHD scores (3 Vs. 5.5, t-test p=0.0007) and improved survival compared to those receiving WT spleen cells(87% vs. 13% of mice alive at 70 days, respectively; log-rank test p<0.001). Our results showed that recipients from miR-155 KO spleen cells did not exhibit high GVHD histological scores (III-IV) in the spleen, liver or gut, while 80%, 60% and 40% of the WT did. Overall survival, GVHD scores and histological GHVD findings were similar between miR-155 KO and WT t-BM only group. Since high levels of soluble TNF-α is characteristic for aGVHD, we measured serum TNF-α by ELISA assay in mice at the time of harvest. Mice receiving miR-155 KO spleen cells had significantly lower TNF-α levels (mean 14 pg/ml) than WT controls (mean 48 pg/ml, t-test, p=0.005). Finally, to establish the relevance of this finding to the human system, we measured miR-155 expression using Locked Nucleic Acid in situ hybridization from histologically confirmed gut biopsies of aGVHD patients (n=5), and healthy controls (n=3). We found a strong up-regulation of miR-155 expression in all patients with gut aGVHD while miR-155 expression was absent in normal gut. In summary, we have shown that miR-155 expression is up-regulated in CD8 T cells from mice with aGVHD and showed that mice receiving donor lymphocyte cells deficient for miR-155 exhibited less GVHD and improved survival as compared to mice receiving WT donor lymphocytes. Finally, up-regulation of miR-155 was also found in clinical specimens from patients with gut aGVHD. Altogether our data indicate a role for miR-155 in the modulation of aGVHD, and thus point to miR-155 as a novel target for therapeutic intervention in aGVHD. Disclosures:Off Label Use: Decitabine and bortezomib in AML.

Haploidentical Allogeneic Hematopoietic Cell Transplantation as Salvage Therapy for Engraftment Failure After Unrelated and Autologous Cell Transplantation.

AbstractAbstract 4529Engraftment failure is a rare but life-threatening complication of hematopoietic stem cell transplantation. The treatment of this condition is often challenging. We firstly reported haploidentical donor stem cells transplantation resulted in hematological reconstitution and long-time disease-free survival in a patient who developed engraftment failure after unrelated donor allogeneic stem cell transplantation and failure rescue treatment by re-infusion of autologous “back-up” stem cells. The 39-years-old male patient with acute myeloid leukemia(AML)-M2a achieved complete remission (CR) after one course of induction chemotherapy. He entered a lasting CR with 7 courses post-remission consolidation therapy and decided to receive unrelated donor allogeniec stem cell transplantation. In case of engraftment failure after allo-HSCT, autologous “back-up” cells were harvested after the last course chemotherapy (IDA 15 mg on days 1–2, 10 mg on day 3, Ara-c 300 mg on days 1–3, 200 mg on day 4), and mobilized with G-CSF 5 mg/kg/day for 5 days. The “back-up” cells consisting of 9.94×106/kg CD34+ cells were cryopreservated in liquid nitrogen. Fifteen months after de novo AML, the patient received a myeloablative conditioning regimen of busulfan and cyclophosphamide (busulfan 3.2 mg/kg/day on days -7 to -4, and cyclophosphamide 60 mg/kg/day on days -3 to -2), and an infusion of unrelated allogeneic peripheral stem cells from the Chinese Marrow Donor Program with a HLA-Cw allele mismatch on day 0. The graft contained 11.07×108/kg nucleated cells and 6.35×106/kg CD34+ cells. Pancytopenia was continuously observed during 28 days after transplantation and short tandem repeat-polymerase chain reaction (STR-PCR) analysis showed no donor chimera. As a rescue attempt for graft failure, cryopreservated autologous cells were re-infused on day +28. Unfortunately, pancytopenia was still continuously observed during 23 days after re-infused of “back-up” cells(51 days after unrelated transplantation), and bone marrow examination revealed severe bone marrow hypoplasia. On day +57 and +58 after unrelated transplantation, bone marrow cells containing 2.1×106/kg CD34+ stem cells and peripheral blood cells containing 2.81×106/kg CD34+ stem cells from a haploidentical donor sister (HLA matched in 5/10 alleles by high-resolution genotyping) were infused respectively after reduced-intensity conditioning with fludarabine and ATG (fludarabine 30mg/m2/d on day -5 to -1, ATG 100mg/d on day -4 to -1). Absolute neutrophil count >0.5×109/L was documented on day 12 after haploidentical transplantation. He achieved platelets count >20×109/L on day 28 after haploidentical transplantation. Twenty-nine days after haploidentical transplantation, bone marrow examination showed reconstitution and STR-PCR analysis indicated complete donor chimera. No grades III -IV aGVHD, extensive chronic GVHD, and severe infection after transplantation were observed. Recurrent bone marrow aspiration examinations showed the patient had been in CR. The patient remained alive during a 18-month follow-up after haploidentical transplantation. Our experience suggests that combined haploidentical donor BM and PBSC transplantation after Flu- and ATG-based conditioning could provide an effective therapeutic strategy for engraftment failure after unrelated allo-HSCT in adult patients. Considering the accessibility of haploidentical donors, haploidentical transplantation has the potential to act as a first-line choice for salvage therapy. Disclosures:No relevant conflicts of interest to declare.

The Novel Combination of Sirolimus and Bortezomib Prevents Graft-Versus-Host Disease but Mantains the Graft-Versus-Leukemia Effect After Allogeneic Transplantation.

AbstractAbstract 3738Graft-versus-host disease (GVHD) represents a major challenge and the main cause of morbidity and mortality after allogeneic transplantation. Using the standard GVHD prophylaxis based on a calcineurin inhibitor plus methotrexate (MTX). The incidence of acute GVHD is in the range of 30–60%, so that new strategies are required in order to decrease GVHD without hampering GVL. Sirolimus, an mTOR inhibitor, allows to decrease the risk of GVHD and increases the number of Treg after transplantation. Unfortunately, its use may increase the risk of microangiopathy and, moreover, its combination with calcineurin inhibitors blocks the development of Treg. Bortezomib is a proteosome inhibitor and blocks the nuclear translocation and activation of NF-kB. It induces depletion of alloreactive T and allows the expansion of T-cells with suppressive properties. Accordingly, both drugs could favour the development of a tolerogeneic immune response after transplantation. In the current study we have analyzed the synergistic effect of sirolimus together with bortezomib. Bortezomib 100 nM plus sirolimus 5nM synergistically inhibited T-cell activation as assessed by the expression of CD25, production of IFNg and expression of CD40L as well as proliferation assessed as expression of PKH. Remain vibility, these effects could not be attributed to a decreased viability of T-cells, as assessed by 7-AAD, at the concentrations evaluated. As compared to each drug alone, the combination significantly decreased the production of Th1 cytokines (IFNg, IL-2 and TNF) while regarding TH2 cytokines, only IL-6 significantly decreased upon combining both drugs. Concerning the mechanisms involved in this synergistic effect, the combination of both drugs resulted in an inhibition of the Akt and Erk ½ phophorylation, thus indicating that sirolimus inhibit pathways which could allow T-cells to escape from the effect of bortezomib at the doses used in the current experiment. In order to confirm in vivo the synergistic effect of sirolimus and bortezomib, a GVHD mouse model (C57/BL6-Balb/c) was carried out. Mice receiving both drugs (Bortezomib 1μg/day intravenous on days 0, +1, +2 postransplantat and sirolimus 0.25mg/Kg intra-peritoneal on days 0 to 12) has a significantly lower incidence of GVHD and longer survival as compared to each drug alone. We also wanted to evaluate whether the immunosuppressive effect of the combination was unspecific or, by contrast, it allowed induce specific immune tolerance against host but maintaining the immune response against other antigens. For this purpose a haematopoietic cells of Balb/c mice which were C57/BL6 complete chimeras were infused to NOD-SCID mice. While none of the donors had developed GVHD after transplantation plus sirolimus and bortezomib postransplant, the NOD-SCID mice succumbed due to GVHD. Furthermore, we infused WEHI cells to BALB/c after total body irradiation and we observed that, while all BALB/c mice receiving WEHI plus C57BL/6 donor BM cells died due to leukemic infiltration, none of those receiving WEHI cells plus C57BL/6 donor BM cells plus splenocytes (and GVHD prophylaxis with sirolimus and bortezomib) did develop leukemic infiltration, thus confirming that, using this approach, we were able to separate GVHD and GVL effect. In conclusion, the current study shows a potent synergistic effect between sirolimus and bortezomib in vitro and in vivo which prevent GVHD while maintaining GVL. Disclosures:Cañizo:CELGENE: Membership on an entity's Board of Directors or advisory committees. San Miguel:JANSSEN-CILAG: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; NOVARTIS: Membership on an entity's Board of Directors or advisory committees; MILLENNIUM: Membership on an entity's Board of Directors or advisory committees. Off Label Use: sirolimus and bortezomib are not approved for use in prophylaxis of graft versus host disease.

High Alpha-1 Antitrypsin Clearance Predicts Severity of Gut Graft Versus Host Disease (GVHD) In Children

AbstractAbstract 1242The clinical evaluation and management of gut GVHD is a significant challenge in pediatric hematopoietic stem cell transplantation (HSCT). It is often difficult to obtain pathological evidence to confirm the GVHD diagnosis and/or determine response to treatment. The severity of the disease itself may not just be related to the associated classic symptoms. Our objective herein was to prospectively study the effect of gut GVHD on protein losing enteropathy (PLE) as measured by alpha-1 antitrypsin clearance (AATC) in stools. Thirteen patients who were diagnosed with gut GVHD by clinical criteria were recruited; 5 patients were excluded as 4 had gut GVHD ruled out by biopsy and 1 was unable to complete study due to stool collection issues. Therefore, 8 patients; 6 males and 2 females, were studied. The median age was 9.5 years (range 6–17). Diagnoses included ALL (4), AML (2), Lymphoma (1) and Adrenoleukodystrophy (1). Donor types were; 7 unrelated (BM n=4 and cord n=3) one matched related BM donor. All patients received cyclosporine for GVHD prophylaxis, in addition 4 patients also received methotrexate and 3 others received prednisone as the second agent. All patients had negative stool EM for viruses and cultures for C.difficile on their first collection. Two 24 hour stool collections were taken from each patient at a mean of 8.5d (range 7–13d) apart. Patients' gut GVHD staging is summarized in Table 1.Table 1:Summary of Stage gut GVHD and PLEPatientsAATC Median(range)normal = <22Stool outputml/kg/d Median(range)Stage of gutGVHD (n=)# with PLECollection #1831 (3-339)28 (10-109)Stg 1 (2)4/8Stg 2 (3)Stg 3 (1)Stg 4 (2)Collection #2856 (8-561)19 (0-85)Stg 1 (0)5/8Stg 2 (0)Stg 3 (3)Stg 4 (1)At the time of 1st collection, 6 patients had ≥ stage II gut GVHD and at 2nd collection 4 patients had ≥stage II gut GVHD and 4 collections were of non-diarrheal stool. A total of 7 stool collections were taken at a diagnosis of ≥stage III gut GVHD. Mean alpha-1 antitrypsin clearance (AATC) from all 16 collections was 143 mls/day (range 3–561), levels >22 mls/day indicate the diagnosis of protein-losing enteropathy (PLE). Nine of the collections (56%) showed PLE with a mean AATC of 247mls/day (range 31–561). Six out of the 7 samples from patients with ≥stage III gut GVHD were positive for PLE. Five collections with stool volumes >30 ml/kg/day were positive for PLE. Stool volumes were significantly higher on second collection (Wilcoxon signed ranks test, p = 0.003) consistent with the second stool collections being significantly more likely to have an elevated AATC, therefore more severe PLE over time (p<0.001). We conclude that a highly significant positive correlation exists between the severity of PLE and the stage of gut GVHD, particularly obvious in patients with stage 3–4 GVHD (ANOVA, p=0.015). See Table 2.Table 2:Stage of gut GVHD and AATCStage ofGVHDNumber ofcollectionsMeanAATCStd.Deviation0426.0020.801217.0019.802384.67136.2934171.50176.2143406.33134.32When patients were grouped as stage 0–2 GVHD vs. stage 3–4 GVHD, those with stage 3–4 GVHD had significantly higher AATC values (p =0.02). Despite the small number of patients recruited, this study emphasizes the need to consider PLE as a useful aspect of the clinical picture. We suggest that in order to see a response to therapy and therefore a decrease in AATC, clinicians should not repeat stool collections before 2 weeks from the initiation of therapy. In light of the significant morbidity and mortality associated with grade 3 and 4 gut GVHD, and as an important therapeutic decision for these patients, one may consider escalating GVHD therapy if a patient's AATC is rising. Disclosures:No relevant conflicts of interest to declare.

Intra-Bone Marrow Transplantation of CD34+ Enriched Grafts Following a Non-Myeloablative Conditioning Allows Successful Engraftment In DLA-Identical Canine Litters.

AbstractAbstract 3711 Introduction:Successful engraftment following transplantation of hematopoietic stem cells (HSCT) depends mainly on pre- and posttransplant immunosuppression, graft type and composition as well as on the HSC numbers infused. Whereas some of the aforementioned parameters can be influenced in the clinical setting, the latter one is more difficult to address. HSCTs of grafts with limited HSC numbers are accompanied by increased graft failure rates, longer cytopenias and increased morbidity. Current concepts to overcome low HSC numbers include the combination of two unrelated grafts, expansion techniques, modification of the graft composition or the site of graft infusion. In preliminary rodent studies intra-bone marrow (IBM) compared to intravenous (IV) HSCT led to faster engraftment which might be explained by closer location of the HSC to the stem cell niches. Aims: To investigate the feasibility and efficiency of IBM-HSCT following a non-myeloablative conditioning regimen in a dog-leukocyte antigen (DLA) identical canine HSCT model. Method:DLA-identical siblings were used as donor/recipient pairs for HSCTs. Recipients received a single dose of 2 Gy total body irradiation before HSCT (day 0). Pre- and postgrafting immunosuppression consisted of CSA (d-1 to d+35) and MMF (d0 to d+27). Two IBM-HSCT cohorts were investigated and data compared to IV controls (CON). BM-grafts of the respective donors were infused unmodified IV (CON, n=9) or IBM after HSC enrichment using a buffy coat followed by ficoll density centrifugation (IBM-I, n=6; 5ml total volume) or IBM after HSC enrichment using buffy coat centrifugation only (IBM-II, n=6; 25 ml total volume). In the CON group the graft was infused in the cubital vein. In the IBM-groups the grafts were infused through a BM aspiration needle into the BM of the left humerus and femur over a period of 5 minutes. In 4 IBM animals graft migration analyses were performed using technecium99 marking. Chimerism and BM cellularity were determined at injection and opposite sides. Analyses of chimerism were performed via polymorphic nucleotide repeat analyses weekly. BM cellularity was determined biweekly. Complete blood count was performed daily. Result:Infusion of grafts directly into the BM was feasible: both volumes (5ml, 25ml) could be infused without any leakage at the injection sites. Tc99-marked BM cells stayed predominately at the injection site for the first 24 hours. All animals engrafted. Mean TNC numbers infused were 2.6 ×108/kg (range: 1.6–11.4; CON), 1.6 ×108/kg (range: 1–2.4; IBM-I), 3.7 ×108/kg (range: 2.1–5.8; IBM-II) (IBM-I vs CON: p=0.08, IBM-II vs CON: p=0.9, IBM-I vs II: p<0.02,). Mean CD34+ numbers infused were 0.6 ×106/kg (range: 0.3–2.2; CON), 2.5 ×106/kg (range: 0.3–6.4; IBM-I), 4.3 × 106/kg (range: 1.3–6.5; IBM-II) (IBM-I vs CON: p=0.06, IBM-II vs CON: p=0.01, IBM-I vs II: p=0.4). PBMC chimerisms at d+14, d+28 and d+56 were 25% (range: 8–46), 36% (range: 5–63), 28% (range: 4–54) (CON), 12% (range: 6–18), 23% (range: 14–39), 18% (range: 6–64) (IBM-I) and 40% (range: 23–60), 61% (range: 31–84), 45% (range: 25–70) (IBM-II) (IBM-I vs CON: p=0.03, p=0.05, p=0.1, IBM-II vs CON: p=n.s. (all), IBM-I vs II: p=0.06, p=0.04, p=0.1). Recovery of hematopoiesis occurred significantly slower in both IBM-BMT groups compared to CON (p<0.002): mean leukopenia (<1.0 gpt/l) durations were 4 days (range: 0–15; IBM-I), 0.5 days (range: 0–2, IBM-II) versus 0 days (CON); mean thrombocytopenia durations (<50Gpt/l) were 19 days (range: 2–49, IBM-I), 10 days (range: 8–16, IBM-II) versus 4 days (range: 0–6, CON). However, if only grafts with <2.0 ×106 CD34+/kg were analysed differences concerning leukocytes recovery times diminished between IBM and the IV groups, whereas they persisted in regards to thrombocytopenia. Analyses of cellularity and chimerism within a HSCT recipient (injection vs opposite site) revealed higher initial cellularities and significantly higher BM donor chimerisms up to day +56, both in favour of the injection site (p<0.03). Conclusion:Infusion of HSC grafts up to volumes of 25ml directly into the BM is feasible and allows successful donor engraftment following non-myeloablative conditioning. Duration of cytopenias following IBM-HSCT is still significant, perhaps due to the loss of precursor cells during graft preparation. Further studies are warranted to determine optimal graft preparation and IBM application techniques. Disclosures:No relevant conflicts of interest to declare.

Efficacy of pretreatment of allografts with methoxypolyethylene glycol-succinimidyl-propionic acid ester in combination with an anti-OX40L monoclonal antibody in relieving graft-versus-host disease in mice

We designed to determine whether pretreatment of allografts with methoxypolyethylene glycol-succinimidyl-propionic acid ester (mPEG-SPA) and an anti-OX40L monoclonal antibody (McAb) can relieve acute graft-versus-host disease in allogeneic bone marrow transplantation recipients. Responder splenocytes from C57BL/6 donor mice were incubated with stimulator splenocytes from BALB/c recipient mice for 7days in the presence or absence of anti-OX40L McAb followed by mPEG-SPA modification. Donor BM cells plus mixed culture T cells were then transplanted into myeloablatively irradiated BALB/c mice. The signs of GVHD were less evident in mice of groups B (mPEG-SPA modification group), C (anti-OX40L McAb pretreated group) and D (dual-treated group), with average survival durations all longer than those in group A (non-treated BMT group) (P<0.05). The survival rates on day 60 post-BMT in groups B, C and D were 50, 41.7 and 66.7%, respectively. After BMT, serum IL-4 and IL-10 levels elevated in groups B, C (P<0.05) and even more significantly increased in group D (P<0.01), while serum IFN-gamma levels decreased in these three groups (P<0.01). In conclusion, the combination of mPEG-SPA and anti-OX40L McAb can block T cell-activated antigens, co-stimulatory pathways and induce the immune shift of Th cells toward Th2 cells; their effects in ameliorating GVHD are synergistic.

Abstract 4326: High-resolution imaging and antitumor effects of GFP(+) bone marrow-derived cells homing to syngeneic mouse colon tumors

Abstract Bone marrow-derived cells (BMDCs) are believed to play a significant role in the growth and spread of certain tumors, namely cancers of the breast, brain, lung, and stomach. To date there are limited reports of bone marrow involvement in colon cancer pathogenesis, but such findings have the potential to generate novel treatments for patients with colon cancer. We have established two mouse models (Balb/c and C57/BL6 backgrounds) for imaging BMDCs from whole tumor to single-cell resolution, whereby the bone marrow of lethally irradiated host animals is reconstituted with EGFP-expressing bone marrow cells from matched donors. The bone marrow transplants (BMTs) yield mice with fluorescently labeled bone marrow and BMDCs can subsequently be monitored within a tumor through optical imaging, as well as ex vivo techniques. We have confirmed successful BM reconstitution at six or more weeks following transplantation. At least six weeks after transplantation, surviving Balb/c and C57/BL6 transplanted mice were injected with CT26 mouse colon cancer cells and p53-deficient Ras/Myc-transformed colonocytes, respectively. We have found up to 45% of cells dissociated from the tumors are GFP-positive and we are currently working to identify the various BM lineages represented within these populations. Interestingly, we have found that tumor growth is reduced in BMT animals compared with untransplanted host mice, as well as EGFP-expressing BM donor mice. We are currently working to determine whether an immune response associated with the BMT might account for these apparent anti-tumor effects. It will clearly be important to separate the molecular and cellular (T cells, NK cells, macrophages, etc) basis of the anti-tumor effect of the BMDCs from any pro-tumorigenic effect that could be subverted for therapeutic gain. Additionally, as an extension of our model we are performing orthotopic cecal wall injections in order to determine whether microenvironmental nuances alter BM homing to the colon versus subcutaneous space. It is expected that the identified BMDCs will aid in the development of novel cancer therapeutic and delivery strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4326.

Alloreactive CD8 T cells require cell-intrinsic PD-1 and upregulate Nr4a and Egr family members during deletional tolerance in vivo (145.43)

Abstract Objective: To characterize deletional tolerance of peripheral alloreactive CD8 T cells induced by anti-CD40L and allogeneic BMT. Methods: B6 mice received 3Gy TBI on Day -1 and 2mg anti-CD40L ip prior to B10.A BMT on Day 0. For CD8 transfer, KbDb KO mice were given 6-9x10^6 purified, recipient-type CD8 cells on Day 0. For deletion and microarray studies, synchimeras containing 10-20% 2C TCR Tg (donor-reactive) cells among CD8 T cells were used. Results: Using adoptive transfer, we determined that the inhibitory PD-1 receptor is required on CD8 T cells for tolerance (6/7 vs. 0/8 chimeras with WT vs. KO CD8s). 2C cells in animals treated with blocking anti-PD-1 initially disappeared at a similar rate to those in tolerized mice. On Day 6 the frequency of apoptotic 2C cells in tolerant controls was equivalent to that in anti-PD-1-treated mice (p = 0.2) and greater than that in mice with no mAb (p = 0.03). Following complete rejection of donor BM, 2C cells in anti-PD-1-treated mice expanded (p = 0.004 vs. tolerant at Day 42). Microarray analysis on sorted 2C+ CD8 T cells isolated on Day 6 post-BMT revealed significantly higher transcript levels for the apoptosis-associated Nr4a family members Nur77 (7.3 fold, p = 0.01), Nurr1 (23 fold, p = 0.01), and Nor1 (9 fold, p = 0.007) and the tolerance-associated Egr family members Egr-1 (4 fold, p = 0.01), Egr-2 (15 fold, p = 0.001), and Egr-3 (8 fold, p = 0.04) in tolerant compared to rejecting mice.

Donor APCs Promote Gvhd in MHC-Mismatched Transplants by Indirectly Presenting Host Minor Histocompatibility Antigens.

Abstract 689In MHC-mismatched allogeneic bone marrow/stem cell transplantation (alloBMT), T cells that at least in-part recognize the allogeneic MHC are likely the dominant mediators of acute GVHD. This would explain why far fewer T cells induce acute GVHD in MHC-mismatched as opposed to MHC-matched alloBMT. However, despite this difference, in human recipients of haploidentical allografts, the organ distribution and histologic appearance of GVHD, especially chronic GVHD, are similar. Moreover, late-onset GVHD in MHC-mismatched alloBMT occurs when recipient antigen presenting cells (APCs) have been replaced by donor APCs. We therefore investigated how indirect presentation of host minor histocompatibility antigens (miHAs) by donor APCs contributes to GVHD. To test this, we employed the MHC/miHA mismatched B6 (H-2b) (right arrow) BALB/c (H-2d) strain pairing. We compared GVHD in irradiated BALB/c mice that were reconstituted with B6 CD4 cells and either B6 BM or B6 MHCII-/- BM. We reasoned that if BALB/c miHAs presented by donor B6 APCs were important, GVHD would be reduced in recipients of MHCII-/- BM. As measured by weight loss and survival, this was indeed the case. However, it was possible that receiving MHCII-/- donor BM reduced GVHD for reasons unrelated to the presentation of BALB/c miHAs. To address this we compared GVHD in irradiated B6.C (H-2d) recipients of B6 CD4 cells and either B6 or B6 MHCII-/- BM. This strain pairing shares the same MHC mismatch as does the B6(right arrow)BALB/c model, but lacks miHAs except those encoded by the congenic H-2d region. In contrast to the prior experiments, B6.C recipients of MHCII-/- or wild type BM developed similar GVHD. To further define indirect-presentation of miHAs in MHC/miHA-mismatched alloBMT, we used B6 Marilyn CD4+ T cell receptor transgenic T cells that recognize an IAb-restricted peptide derived from the male DBY protein. Male or female BALB/c mice were irradiated and reconstituted with female B6 BM and female polyclonal B6 CD4 cells along with graded numbers of Marilyn T cells. At day 7 and 14 post transplant Marilyn cells expanded at least 1000-fold greater in male than in female recipients. Marilyn T cell expansion was similar when male antigen was restricted to hematopoietic or nonhematopoietic cells. There was little expansion of Marilyn cells in male BALB/c mice if donor BM was MHCII-/-, confirming that expansion of Marilyn T cells depended on donor APCs presenting DBY. Indirectly primed Marilyn cells were functional as measured by IFN-γ production after restimulation by the DBY peptide in vitro. As we saw reduced GVHD in the B6 (right arrow) BALB/c model in recipients of MHCII-/- BM, we reasoned we should be able to detect polyclonal CD4 cells responding to host miHA presented by donor APCs. That was the case as we found an increase in DBY-reactive polyclonal CD4 cells by ELISPOT in male recipients as compared to female recipients at day 7 (1.5 fold for IFN-γ and TNF-αa). This difference was lost when donor BM was MHCII-/-. In summary, indirect presentation of host miHAs by donor APCs in MHC-mismatched alloBMT is surprisingly efficient and alloreactive CD4 cells that target host miHAs presented by donor APCs contribute to GVHD in MHC/miHA-disparate alloBMT. The generation of T cells that target host miHAs presented by donor APCs allows GVHD to persist even when host APCs that prime T cells that recognize the allogeneic MHC have been eradicated. This provides a mechanistic explanation for why late onset GVHD, including chronic GVHD, in MHC-mismatched alloBMT resembles that in MHC-matched alloBMT as both may target host miHAs indirectly presented by donor-derived APCs. These data also provide further rationale for targeting donor-derived APCs as a method for preventing and treating GVHD. Disclosures:No relevant conflicts of interest to declare.

Innate Immunity Is Responsible for Early Allorejection of Donor Bone Marrow Cells.

Abstract 3523Poster Board III-460Adaptive immunity, especially T cells, has long been believed to be the dominant immune response in allogeneic transplantation. However, innate immunity has been recently shown to pose a significant barrier to the induction of tolerance to solid organ transplants. The role of the innate immune system in bone marrow cell alloreactivity has not been addressed. The innate immune system provides the first line of defense in the removal of pathogens because of the delay in generation of adaptive immune responses. Our present findings show that innate immunity is a significant first line barrier in bone marrow cell (BMC) rejection. To determine the effect of innate immune cell populations on rejection of donor BMCs, T cell deficient mice (TCR β/δ−/−) were used as BMC recipients in in vivo cytotoxicity assays (Figure A). TCRβ/δ−/− mice have normal innate immune cell populations but do not initiate adaptive cell-mediated cytotoxic or humoral responses. 20 × 106 CFSE labeled donor (high CFSE fluorescence intensity) and recipient control splenocytes (low CFSE fluorescence intensity) were injected into TCRβ/δ−/− and wildtype control B6 recipient mice. Donor cell survival was compared over time. Donor BALB/c splenocytes were eliminated in wildtype B6, with rejection complete by day 3. The kinetics of elimination of donor cells in TCRβ/δ−/− mice was similar to that for wildtype B6 controls, with donor cells eliminated by day 3. These results indicate that early rejection of the splenocytes in wildtype mice was T cell-independent. The acute rejection of BMC in wildtype B6 recipients occurred within 3 days, which is prior to the time required for T cell activation. Thus the effectors mediating BMC rejection would be the innate immune cells: macrophages, neutrophils, or NK cells. To rule out potential involvement of natural Abs in the cytotoxicity we observed in the TCRβ/δ−/− mice, Rag−/− mice were used as recipients (Figure B). Rag−/− mice do not produce mature T cells or B cells. 20 × 106 CFSE labeled donor (high CFSE intensity) and recipient control BM cells (low CFSE intensity) were injected into Rag−/−mice. Rag−/− and wildtype B6 control mice exhibited similar kinetics of donor cell cytolysis. The rapid elimination of allogeneic cells from immunocompetent mice is comparable with that observed in T cell- (TCRβ/δ−/−) or T and B cell- (Rag−/−) deficient mice indicates that allogeneic cells are subject to T cell-independent rejection at the early time period after cell infusion (≤ 3 days). As the kinetics of cytotoxicity were similar in experiments using either splenocytes or BMCs as target cells in our later experiments, our data suggest that the innate immune system is responsible for early allorejection of donor BMC at the early inductive period for adaptive immunity. These findings may have significant impact on the development of immune-based nonmyeloablative conditioning strategies and show for the first time that a dominant factor in BMC rejection is contributed by innate immune responses. [Display omitted] Disclosures:Bozulic:Regenerex: Employment. Ildstad:Regenerex: Equity Ownership.

Donor Dendritic Cells Home to Lymphoid Organs in Allogeneic Transplant Recipients, Enhance Donor T-Cell Chimerism, and Polarize Donor T-Cell Activation.

Abstract 62 Background:The fate of donor dendritic cells (DC) contained in the allograft, and their role in regulating immunity following allogeneic transplant has not been fully explored. We have previously demonstrated that donor BM CD11b− DC added to purified hematopoietic stem cells (HSC) and T-cells polarize donor T-cells to Th1 immunity, and improve leukemia survival among tumor-bearing transplant recipients compared with recipients of allogeneic CD11b+ donor antigen presenting cells (APC) or grafts with no donor APC (Li, et al Blood 2008, 112:1252). Objective: In this study, we studied the homing of donor DC in the primary and secondary lymphoid organs of transplant recipients in murine allogeneic HSC transplantation. Methods: In this study we transplanted between 5 × 104 and 1 × 106 FACS-sorted lineage− CD11c+ CD11b− DC (predominately plasmacytoid DC (pDC) and pDC precursors) and lineage− CD11c+ CD11b+ APC and APC precursors (CD11b+ APC) purified from GFP+ or luciferase+ transgenic donors in combination with 3 × 103 lineage− c-kit+ Sca-1+ HSC and 1 × 106 CFSE stained donor T-cells in B6□B10BR, B6□BALB/C, and FVB□B10BR allogeneic transplant models to explore homing, maturation, and persistence of donor DC in the primary and secondary lymphoid organs of transplant recipients. Homing and short-term maturation of donor DC was assessed 3 days and 10 days post-transplant by FACS analysis gating on GFP+ donor DC. In vivo maturation of DC was assessed by FACS gating on GFP+ donor APC progeny in transplant recipients and compared with DC subsets cultured with CD40L (1μg/mL) or LPS (10ng/mL). Proliferation and IFN-ψ production of CFSE-labeled donor T-cells isolated from recipients' peripheral blood, lymph nodes, and spleen was assessed by flow cytometric analysis of CFSE and intracellular cytokine staining. Persistence of donor DC was assessed by bioluminescence imaging 7, 14, 21, and 28 days post-transplant in FvB□B10BR allogeneic transplant model using 5 × 104 pDC and CD11b+ APC from FvB luciferase+ transgenic donors transplanted with 3 × 103 HSC and 3 × 105 T-cells from FvB wild-type donors. Results: The frequencies of GFP+ pDC and CD11b+ APC recovered from transplant recipients at day 10 post-transplant were 2.2 ± 0.2% and 1.8 ± 0.3%, respectively in BM and 4.9 ± 1.5% and 1.9 ± 0.5%, respectively in recipient spleen. Absolute numbers of GFP+ donor DC were measured in the sections of spleen by confocal microscopy and were similar comparing recipients of pDC and CD11b+ APC (9.0/μL and 7.3/μL spleen, respectively, at day 10). The GFP+ CD11b+ APC had a scatter profile consistent with myelo-monocytic differentiation while GFP+ pDC retained mononuclear properties. The expression of MHC-II and CD80/CD86 on GFP+ CD11b+ APC recovered from transplant recipients was minimal, indicating predominant differentiation to myeloid-derived suppressor cells rather than to DC. FACS analysis of lineage expression patterns in donor GFP+ pDC and CD11b+ APC recovered from recipient BM were similar compared with sorted APC cultured with CD40L or LPS. PDC up-regulated MHC-II, CD80 and CD86 expression, while CD11b+ APC expressed high levels of PDL1 and PDL2 and up-regulated F4/80 and Gr-1-associated markers. In contrast, the same donor APC populations recovered from the spleen expressed lower levels of DC-associated markers. Bioluminescent imaging showed that luciferase+ donor DC persisted and expanded in the lymph nodes and spleen of transplant recipients for up to one month. The immunological effect of transplanting pDC led to increased donor T-cell proliferation, IFN-ψ production and CD25 expression compared with transplanting grafts containing HSC, T-cells and CD11b+ APC, or grafts containing only HSC and T-cells in both MHC-mismatched murine HSCT models. In conclusion, the results indicate that different subsets of donor APC and DC home to lymphoid tissues and up-regulate a pattern of co-stimulatory and co-inhibitory molecules consistent with their observed immunological immune activity in vivo. Maturation of pDC in vivo was slower than that in vitro, and pDC maturation was slower in the recipients' BM compared with recipient's spleen suggesting that the post-transplant microenvironment may impair DC differentiation. Less mature pDC in the stem cell graft that home to the BM may facilitate donor engraftment and immune reconstitution, while more mature pDC that home to the spleen may polarize donor T-cell Th1 responses. Disclosures:Waller:NIH-National Heart, Lung, and Blood Institute, NHLBIP01H1086773: Research Funding.