ALBERT

Description: Author(s): Yuanhui Wen, Yujie Chen, Yanfeng Zhang, Hui Chen, and Siyuan Yu Conventional caustic methods in real or Fourier space produced accelerating optical beams only with convex trajectories. We developed a superposition caustic method capable of winding light beams along nonconvex trajectories. We ascertain this method by constructing a one-dimensional (1D) accelerati… [Phys. Rev. A 94, 013829] Published Fri Jul 15, 2016

Description: Author(s): Yuanhui Wen, Yujie Chen, Yanfeng Zhang, Hui Chen, and Siyuan Yu An appropriate wave-front design will enable light fields that propagate along arbitrary trajectories, thus forming accelerating beams in free space. Previous strategies for designing such accelerating beams rely mainly on caustic methods, which start from diffraction integrals and deal only with tw… [Phys. Rev. A 95, 023825] Published Tue Feb 14, 2017

Description: CD8+TCR- bone marrow facilitating cells (FCs) facilitate engraftment of hematopoietic stem cells (HSCs) in both allogeneic and syngeneic recipients. We recently reported that co-administration of FCs with HSCs established chimerism and induced tolerance to renal allografts without graft-versus-host disease and engraftment syndrome in HLA-mismatched living donor renal transplant recipients. Homing of HSCs to the bone marrow niche is believed to be a crucial prerequisite for engraftment. Therefore, we evaluated whether FCs enhance functional HSC homing and retention in the hematopoietic niche using the in vivo syngeneic homing model followed by colony-forming cell (CFC) assay. B6 mice were conditioned with a supralethal dose (1200 cGy) of total body irradiation (TBI) and transplanted with 75,000 B6 FCs, 25,000 B6 HSCs or FCs plus HSCs 24 hours after TBI. At 18 hours after transplantation, bone marrow cells were harvested from the recipient’s femurs and tibias and placed in CFC assay using methylcellulose-based media. Bone marrow cells harvested from mice transplanted with FCs without HSCs or those receiving conditioning alone did not generate colonies, confirming that FCs themselves do not have repopulating capacity in vivo and post-TBI bone marrow was free of recipient HSCs under the supralethal dose of TBI. FCs significantly enhanced the efficiency of HSC homing as reflected by the bone marrow cells harvested from the mice transplanted with HSCs and FCs formed significantly higher numbers of colonies compared to that of the mice transplanted with HSCs alone (24.7 ± 2.9 vs. 15.6 ± 2.7; p 〈 0.05). The chemokine receptor CXCR4 plays a pivotal role in HSC homing. We therefore determined whether the effect of FCs on HSC homing was mediated by FCs increasing CXCR4 expression in HSC. CXCR4 expression in HSCs was measured by flow cytometry 18 hours after co-culture of HSCs and FCs in vitro. Incubation of FCs with HSCs did not lead to increased CXCR4 expression on HSCs compared with HSCs cultured alone. We next tested whether FCs enhance the responsiveness of HSCs to a low concentration of stromal cell-derived factor 1 (SDF-1, also known as CXCL12) gradient in Transwell chemotaxis assays in vitro. 500 HSCs were placed in the upper chamber and tested for migration of cells to the lower chamber of the Transwell in response to a 30 ng/ml SDF-1 gradient for 3 hours. The cells harvested from the lower chamber were placed in CFC assay. The migrated cells harvested from the lower chamber containing FCs formed 6-fold more colonies than HSCs obtained from the lower chamber that did not contain FC (22.3 ± 3.9 vs. 4.0 ± 1.2; p 〈 0.05). Furthermore, we found that small factions of total FCs were CXCL12-producing cells (2% of total FCs) and CD169+ cells (5% of total FCs). The migration ability of FCs was characterized by following patterns of homing after transplantation of 300,000 CellTracker Green labeled FCs or 300,000 FCs bearing different congenic marker. In 8,000,000 bone marrow cells harvested from femurs and tibias of the transplanted mice 18 hours post-transplantation, we detected 100 to 300 of transplanted CellTracker Green labeled FCs. On the 10 serial bone sections from femurs and tibias of the transplanted mice, we enumerated 15 to 20 of CellTracker Green labeled FCs. These data suggested that FCs might home to bone marrow within 18 hours post-transplantation. In summary, our results suggest that FCs enhance functional HSC homing and retention in the hematopoietic niche in vivo. This is not mediated by increased CXCR4 expression in HSCs. FCs also prime HSC migration to a low concentration SDF-1 gradient in vitro, possibly through production of priming factors. Disclosures Ildstad: Regenerex, LLC: Other.

Description: Abstract 3526 Poster Board III-463 Research on transplantation rejection, tolerance, and immunosuppressive agents has been directed toward the adaptive immune response as, until now, it has been believed to be the dominant mechanism for alloreactivity. The role of innate immunity in transplantation has not been fully defined. Despite the sentinel role of innate immunity in driving and shaping adaptive immunity, the contribution of innate immunity to bone narrow cells has only recently been identified. TLR4-driven MyD88-dependent immunity has been demonstrated to be critical for allogeneic innate immune responses to solid organ transplants. Whether innate immunity plays a role in BMC rejection has not been defined. In the present studies we tested whether the absence of MyD88 would enhance allogeneic engraftment in bone marrow transplantation (BMT). The core component of TLR signaling is activation of an IL-1-like pathway dependent upon the adapter MyD88. MyD88−/− B6 mice (n=6) were used to study the role of MyD88 signaling in allogeneic BMC rejection. As TRIF is another crucial adapter for TLR3 signaling and responsible for induction of signaling via type 1 IFNs, B6 TRIF−/− mice (n=6) were also investigated. Wild-type B6 (H2b, n=6) mice were used as controls. In the present study, MHC plus minor antigen-disparate BALB/c (H2d) mice served as allogeneic bone marrow donors. Recipients were nonmyeloablatively conditioned with anti-CD154 mAb (day0 and +3) to block CD40:CD154 co-stimulatory pathway and sirolimus (day0 to +4) to block late-stage T cell activation by inhibiting IL-2 responsiveness. Recipients were transplanted with 15 × 106 allogeneic (BALB/c; H2d) marrow cells 4-6 hours following conditioning with 100 cGy total body irradiation (TBI). Allogeneic engraftment was achieved in 33.3% of MyD88−/− recipients at 1 mo after BMT, a percentage similar to that for wild-type B6 mice. Surprisingly, 100% of TRIF−/− mice engrafted. The level of donor chimerism in TRIF−/− mice was 5.1 ± 0.6% at one month after BMT, which was significantly higher than in MyD88−/− (1.8 ± 1.0%; P 〈 0.0001) or in wild-type B6 mice (1.3 ± 0.8%; P 〈 0.005) and thereafter through all time points tested (2, 4 and 5 months, P 〈 0.05). The levels of donor chimerism increased with time and the level of donor chimerism reached 14.7 ± 7.1% TRIF−/− at five months. These results suggest that allogeneic BMC rejection is uniquely independent of the MyD88 pathway but rather involves TRIF signaling. These data suggest that the innate immune response to BMC differs from that for solid organs and may reflect the specificity of BMT compared with other transplants. These findings demonstrate for the first time that BMC elicit an innate immune response via the TRIF pathway and may open the door to novel approaches for immune-based conditioning to promote engraftment. Disclosures: Ildstad: Regenerex: Equity Ownership.

Description: Abstract 142 CD8+/TCR− graft facilitating cells (FC) have a potent capability to facilitate relatively small numbers of highly purified hematopoietic stem cells (HSC) in both allogeneic and syngeneic recipients. The mechanisms by which FC promote HSC engraftment have not been fully elucidated. We previously found that FC from non-obese diabetic mice (NOD) were compromised in enhancing engraftment of syngeneic and allogeneic HSC compared with FC from diabetes free congenic NOR mice. We therefore compared gene expression profiling between NOD FC and control NOR FC by microarray analysis. Among 18 most significant differentially expressed genes revealed from 45101 genes by false discovery rate control analysis with a cut-off value at level 0.05, dedicator of cytokinesis 2 (DOCK2) was identified as the gene with the most significant difference (P = 1.07 × 10−7). DOCK2 is a hematopoietic cell-specific member of the Caenorhabditis elegans Ced-5, mammalian DOCK180 and Drosophila melanogaster myoblast city (CDM) family of guanine nucleotide exchange factors. DOCK2 activates the small GTPase Rac and is indispensable for migration of plasmacytoid dendritic cells (pDC). FC is a heterogeneous cell population, with a predominant subpopulation resembling plasmacytoid precursor DC (p-preDC FC). In vitro studies demonstrated that FC increased clonogenicity of HSC and improved the ability of the impaired stroma to support late cobblestone area formation by HSC, which suggests that FC homing to hematopoietic niche as a potential component might be a perquisite for FC to enhance HSC engraftment. Therefore, we hypothesized that signaling pathways controlling cell migration via DOCK2 are critical for FC to enhance HSC engraftment. To test our hypothesis, DOCK2 expression data from microarray analysis was further confirmed using relative quantitative real-time PCR and high content image analysis. The expression level of DOCK2 in FC was significantly lower in NOD mice compared with NOR mice (p 〈 0.01 vs. NOR FC). Functional phenotypes of DOCK2-deficient FC were determined by Transwell migration assay and colony-forming cell assay in vitro, and by co-transplantation of HSC with FC in vivo as well as enumeration of CellTrack Green labeled FC by flow cytometry in spleen, thymus, and bone marrow of femurs and tibias 18 hours post-transplantation. Deficiency of DOCK2 in FC did not affect the ability of FC in promoting HSC colony formation when the two were cultured together. However, DOCK2-deficient FC were compromised in migration to the α-cheemokine, stromal derived factor-1 (SDF-1) at dose 200 ng/ml (Fig. A, P 〈 0.01 vs. wild-type FC). Homing of FC to spleen and bone marrow of femurs and tibias was also significantly impaired in DOCK2-deficient FC. Moreover, deficiency of DOCK2 in FC abrogated enhancement of HSC engraftment by FC in the syngeneic and allogeneic in vivo assays (Fig. B, syngeneic model: 500 B6 HSC plus 30K B6 or DOCK2−/−FC into lethally irradiated B6 recipients; Fig. C, allogeneic model: 10K B6 HSC plus 30K B6 or DOCK2−/−FC into lethally irradiated B10.BR recipients, P 〈 0.05 vs. B6 HSC plus wild-type FC). Taken together, our results indicate that deficiency of DOCK2 in FC leads to the dysfunction in migration, and suggest that the signaling pathways involved in FC migration are crucial for FC to enhance HSC engraftment. Disclosures: Ildstad: Regenerex LLC: Equity Ownership.

Description: Abstract 1885 CD8+/TCR− graft facilitating cells (FC) significantly enhance engraftment of hematopoietic stem cells (HSC) in both allogeneic and syngeneic recipients. We have shown that DOCK2−/− FC are compromised in cell migration in vitro and facilitating HSC engraftment in vivo. DOCK2 activates the small GTPase Rac and is indispensable for migration of neutrophils, lymphocytes and plasmacytoid dendritic cells (DC). A recent study demonstrated that DOCK2−/− DC were defective in antigen uptake and presentation. FC are a heterogeneous cell population, with a predominant subpopulation resembling plasmacytoid precursor DC. We previously found that FC induced antigen-specific regulatory T cells (Treg) in vitro and in vivo. In the present study, we evaluated whether DOCK2−/− FC could induce Treg. 5×105 wild-type (WT) sorted CD4+CD25− splenocyte T cells were incubated with 1×105 WT FC or DOCK2−/− FC at a 5:1 ratio in U bottom 96-well plates with long-term culture medium (IMEM with 20% horse serum, 10−6M hydrocortisone and 50μM β-mercaptoethanol) for up to 6 days. The co-cultures were harvested and CD4+CD25+ T cells were analyzed for FoxP3 by flow cytometry intracellular staining. WT CD4+CD25− splenocytes were cultured as negative control. Phagocytosis of APC-labeled polystyrene beads by WT and DOCK2−/− FC was assessed after incubating FC with the beads for 3 hours. We found that FC induced the generation of CD4+CD25+FoxP3+ Tregin vitro as expected, while DOCK2 deficiency completely abrogated the ability of FC to induce Treg generation. Moreover, the uptake of beads by DOCK2−/− FC was significantly impaired compared to their uptake by WT FC. In summary, DOCK2−/− FC were unable to induce the generation of CD4+CD25+FoxP3+ Tregin vitro and were compromised in phagocytosis. Disclosures: Ildstad: Regenerex, a biotech start-up company: Equity Ownership.

Description: Abstract 2424 Poster Board II-401 We previously reported that CD8+TCR- facilitating cells (FC) induce the generation of chimeric regulatory T cells (Treg) in vivo. Transplantation of a mixture of CD8+/TCR- FC and hematopoietic stem cells (HSC) into ablated recipients results in chimerism and tolerance. Treg harvested from the spleen of chimeras (chimeric Treg) potently increase long-term donor chimerism in secondary NOD recipient mice. Here, we evaluated whether chimeric Treg enhance engraftment of hematopoietic stem cells (HSC) in an antigen-specific manner. To prepare mixed chimeras (B6 → NOD), NOD recipients were conditioned with 950 cGy TBI and transplanted with 10,000 B6 HSC and 1,000 NOD HSC plus 45,000 CD8+TCR- B6 FC. At 5 weeks, CD8-CD4+CD25bright chimeric Treg were sorted from spleens of the mixed chimeras (B6 → NOD). 100,000 chimeric Treg were then mixed with 10,000 B6 HSC (donor-specific) + 10,000 B10.BR HSC (third-party) and transplanted into conditioned NOD recipients in competitive repopulation assays. NOD mice given HSC plus nonchimeric naïve B6 Treg or HSC alone served as controls. Two of the four animals that received HSC alone engrafted and exhibited an average of 6.7% donor B6 chimerism at 30 days, 11.2% at 60 days, and 10.6% at 90 days. Three of five animals given HSC plus naïve B6 Treg engrafted with 21.3% donor B6 chimerism at 30 days, 28.8% at 60 days, and 28.9% at 90 days. In contrast, eight of nine recipients of HSC + chimeric Treg engrafted. These animals exhibited a significantly higher level of donor B6 chimerism, ranging from 56.3% at 30 days, 75.4% at 60 days to 85% at 90 days (P = 0.034). None of the recipients engrafted with the MHC-disparate third-party B10.BR HSC. We then assessed the suppressive function of chimeric Tregin vitro by using MLR suppressor cell assays. CD8-/CD4+/CD25bright Treg were sorted from chimeric spleens 5 wks to 12 wks after HSC + FC transplantation. As shown in the Figure 1, Treg from naïve B6 mice resulted in 1.9 fold; 1.3 fold and 1.1 fold inhibition of proliferation at 1:1, 1:0.25, 1:0.125 responder/Treg ratios (n = 3). In contrast, chimeric Treg potently suppressed T cell proliferation by 10.5 fold; 3.2 fold; and 1.7 fold at responder/Treg ratios of 1:1, 1:0.25, 1:0.125 (n = 4). Chimeric Treg significantly suppressed T cell proliferation at responder/Treg ratios of 1:1 and 1:0.25 compared with naïve B6 Treg (P 〈 0.05). NOD responder splenocytes remained hypoproliferative in response to B6 stimulator and chimeric Treg compared with stimulator plus B6 Treg, suggesting that chimeric Treg are significantly more potent than naïve B6 Treg in suppressing effector T cell proliferation in vitro. These data show that chimeric Treg enhance donor B6 HSC engraftment but not third-party B10.BR HSC, demonstrating that chimeric Treg function in vivo in an antigen-specific fashion. These data also show that the mechanism of FC function in vivo is associated with the establishment of an antigen-specific regulatory feedback loop. Figure 1 Figure 1. Disclosures: Bozulic: Regenerex: Employment. Ildstad:Regenerex: Equity Ownership.

Description: Hematopoietic stem/progenitor cell (HSPC) transplantation is increasingly applied for treatment of hematologic malignancies and autoimmune diseases. Despite significant clinical advances of HSPC transplantation, the morbidity and mortality of ablative conditioning and graft-versus-host disease (GVHD) remain a challenge. These risks can be overcome through less toxic nonmyeloablative conditioning and approaches that selectively remove GVHD-causing cells while retaining engraftment-enhancing, tolerogenic cells. We were the first to discover CD8+ T cell receptor (TCR)- graft facilitating cells (FCs) in mouse bone marrow (BM). The addition of as few as 30,000 FCs to 10,000 HSPCs significantly enhances engraftment of hematopoietic stem cells (HSCs) in allogeneic recipients without causing GVHD. FCs also potently enhance engraftment of limiting numbers of syngeneic HSPCs. FCs are distinct from HSPCs since they do not generate multilineage engraftment when infused alone, nor do they function as a stem cell in vitro. The phenotype and mechanism of action of human FCs remains to be defined. We report here for the first time that two distinct FC subpopulations are critical to overall human FC function: CD8+TCR-CD56bright (CD56bright FCs) and CD8+TCR-CD56dim (CD56dim FCs). FACS analysis showed that the majority of CD56bright FCs are CD11c+CD11b+ and C-X-C motif chemokine receptor 4 (CXCR4)bright and keep a dendritic morphology after stimulation with CpG oligodeoxynucleotides (CpG ODN) (TLR9). The majority of CD56dim FCs express CD3ε and exhibit a lymphoid morphology. The CD56dim FC subpopulation significantly promotes homing of HSPCs to BM in non-obese diabetic/severe combined immunodeficiency/interleukin-2 receptor gnull (NSG) recipients and enhances hematopoietic colony formation. The CD56dim FC subpopulation produces rapid reconstitution of donor hematopoietic cells without causing GVHD. In contrast, recipients of CD56bright FCs plus HSPCs show low donor chimerism early after transplantation. However, the level of donor chimerism significantly increases with time. Both recipients of HSPCs plus CD56dim or CD56bright FC subpopulations showed durable donor chimerism at significantly higher levels in BM compared to recipients of HSPCs alone. Co-culture of CD56bright FCs with HSPCs up-regulates small innate immunity-related peptides such as cathelicidin or beta defensin-2, factors that prime responsiveness of HSCs to a low stromal cell-derived factor-1 (SDF-1) gradient. Both CD56bright and CD56dim FC subpopulations highly up-regulate mRNA expression of the HSC growth factors, stem cell factors and fms-related tyrosine kinase 3 (FLT3) Ligand (3,000 fold and 14 fold respectively). Taken together, our data indicate that: 1) human CD8+TCR- FCs are two distinct, complementary and synergistic subpopulations with multifactorial effects on HSCs; 2) CD56bright FCs significantly enhance durable chimerism in the BM of NSG mice and produce significantly increased levels of factors that prime HSC homing; 3) CD56dim FCs significantly enhance homing of human HSC to the BM in NSG recipients and promote HSC clonogenicity; and 4) recipients of HSCs plus CD56dim FCs or CD56bright FCs show durable donor human chimerism in peripheral blood, BM and spleen. Disclosures Bozulic: Regenerex, LLC, a start-up biotech company: Employment. Ildstad:Regenerex, LLC, a start-up biotech company: CEO Other.