P815 Tumor Cells Research Articles

Phytochemical profile of Anacardium occidentale L. (cashew tree) and the cytotoxic and toxicological evaluation of its bark and leaf extracts

Anacardium occidentale (Anacardiaceae), popularly known as cashews, is a vegetable native to South America, consumed worldwide in the food sector. Its leaves and bark are used in the preparation of teas for health maintenance, being indicated in ethnobotanical studies for the treatment of cancer, which is considered a serious public health problem worldwide. The objective of this study was to evaluate the cytotoxicity of the A. occidentale leaf and bark extracts over tumor and normal cells, as well as their in vivo toxicity using the Drosophila melanogaster fruit fly. Cytotoxicity was evaluated using the MTT method [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide] in five cell lines (HL-60, NCI-H292, HCT-116, P815 and L929). Toxicity was evaluated by fly mortality rate and negative geotaxis, as well as by oxidative stress markers (Fe2+ quantification, protein and non-protein thiols) in the D. melanogaster model. The chromatographic study was performed by UPLC-QTOF/MS. As a result, 11 compounds present in the A. occidentale leaf extract (LEAo) and 6 compounds in the bark extract (BEAo) were identified, with gallic acid and galloyl hexoside being present in both samples. The bark extract presented a greater cytotoxic activity for HCT-116, HL60 and P815 tumor cells. The extracts also presented a low cytotoxicity with the normal cell line, L929. No changes in the levels of protein and non-protein thiols (NPSH) were observed, whereas a considerable increase in Fe2+ levels was observed in the Fe2+ quantification assays from homogenized flies exposed to the leaf extract in the D. melanogaster assays. However, further in-depth studies need to be carried out to elucidate the mechanisms of action of the extracts and to assess their pharmacological safety.

PRMT5 promotes STAT-1 mediated T cell inflammatory response and is a therapeutic target for acute graft-versus-host disease

Abstract Introduction T cell mediated acute graft-versus-host disease (aGVHD) is the main cause of non-relapse mortality in allogeneic hematopoietic cell transplant recipients. Protein arginine methyltransferase 5 (PRMT5) is a post-translational modifier and promotes activation of memory TH cells. We investigate mechanisms by which PRMT5 regulates T cell function and propose PRMT5 inhibition as a therapeutic strategy for aGVHD. Materials and Methods PRMT5 expression and function was evaluated in T cells of healthy and aGVHD mice and humans. We assessed T cell proliferation and effector function, using C220, a novel PRMT5 inhibitor. We tested effects of PRMT5 inhibition using in vivo mouse models of aGVHD, where mice received T cell depleted bone marrow + allogeneic splenocytes. In graft-versus-leukemia (GVL) experiments, P815 tumor cells were administered. Mice were treated with C220 or vehicle and monitored for survival and clinical aGVHD scores. Results PRMT5 expression and function is upregulated in T cells of mice and humans with aGVHD. Inhibition of PRMT5 reduces T cell proliferation, perturbs cell cycle and downregulates STAT-1 mediated interferon response. Administration of C220 improved survival in aGVHD mouse models and maintained tumor specific CTL response contributing to retention of Graft versus leukemia (GVL) effect. Conclusions Inhibition of PRMT5 using C220, down-regulates T cell proliferative and effector response, induces cell-cycle arrest, reduces interferon response and perturbs signaling pathways. C220 shows potent biological activity in vivo by reducing aGVHD clinical severity and prolonging survival in mouse models without compromising GVL. Therefore, PRMT5 is a novel and druggable target for aGVHD.

Influence of the alkylating function of aldo-Ifosfamide on the anti-tumor activity.

The present work investigates the influence of different DNA damages caused by different isophosphoramide mustards on the 3-hydroxypropanal-assisted apoptotic antitumor activity of oxazaphosphorine cytostatics using I-aldophosphamide-perhydrothiazine (IAP) and mesyl-I-aldophosphamide-perhydrothiazine (SUM-IAP) for in-vitro and in-vivo experiments. IAP and SUM-IAP hydrolyze spontaneously to the corresponding I-aldophosphamide derivatives. They differ in the chemical structure of the alkylating moiety, whereas IAP has two chlorethyl groups in the SUM-IAP molecule, one chlorethyl group is substituted by a mesylethyl group. With both substances, cytotoxicity studies on P388 tumor cells in vitro and therapy experiments in mice bearing advanced growing P388 tumors were carried out. IAP was significantly more cytotoxic in-vitro than SUM-IAP, but the antitumor activity of SUM-IAP was by order of magnitude higher than the antitumor activity of IAP. The reason for these findings is discussed with respect to the enzymatic cleavage of the various I-aldophosphamide derivatives to the corresponding isophosphoramide mustards and 3-hydroxypropanal. Overall, the findings indicate that antitumor activity of ifosfamide and derivatives of ifosfamide can be improved considerably by altering the alkylating moiety of the molecule, but retaining the aldophosphamide structure.

Cytotoxic and Antiviral Triterpenoids from the Mangrove Plant Sonneratia paracaseolaris.

A chemical investigation was conducted on the aerial parts of the mangrove plant Sonneratia paracaseolaris, yielding five new triterpenoid paracaseolins A–E (1–4, and 11) together with twelve known analogues (5–10, 12–17). Their structures were established by extensive spectroscopic methods and comparisons their spectroscopic data with those of the known related compounds. The cytotoxicities against P388, HeLa, A549, and K562 tumor cell lines and anti-H1N1 (Influenza A virus) activities for the isolates were evaluated. Compound 4 showed potent cytotoxicity against the A549 cell line with an IC50 value of 1.89 µM, and compound 1 exhibited significant anti-H1N1 virus activity with an IC50 value of 28.4 µg/mL. A preliminary structure activity relationship was discussed.

Differential Cytotoxic Activity of Essential Oil of Lippia citriodora from Different Regions in Morocco

The aim of this work was to investigate the cytotoxic effect of the essential oil of dried leaves of Lippia citriodora (H.B. & K.) harvested in different regions of Morocco. This effect was evaluated against the P815 murine mastocytoma cell line using the MTT assay. Interestingly, this work demonstrated for the first time that these essential oils exhibited a strong cytotoxic activity against the P815 cell line, with IC50 values ranging from 7.75 to 13.25 μg/ml. This cytotoxicity began early and increased in a dose- and time-dependent manner. The chemical profile of these essential oils was analyzed by gas chromatography coupled to mass spectrometry. Importantly, the difference in terms of major components' contents was not significant suggesting probably that the differential cytotoxicity between these essential oils could be attributed to the difference in the content of these essential oils in minor compounds, which could interact with each other or with the main molecules. Finally, this study demonstrated for the first time that essential oils of L. citriodora from different regions in Morocco induced apoptosis against P815 tumor cell line.

The MEK inhibitor trametinib separates murine graft-versus-host disease from graft-versus-tumor effects.

The efficacy of allogeneic hematopoietic stem cell transplantation for hematologic malignancies is limited by the difficulty in suppressing graft-versus-host disease (GVHD) without compromising graft-versus-tumor (GVT) effects. We previously showed that RAS/MEK/ERK signaling depends on memory differentiation in human T cells, which confers susceptibility to selective inhibition of naive T cells. Actually, antineoplastic MEK inhibitors selectively suppress alloreactive T cells, sparing virus-specific T cells in vitro. Here, we show that trametinib, a MEK inhibitor clinically approved for melanoma, suppresses GVHD safely without affecting GVT effects in vivo. Trametinib prolonged survival of GVHD mice and attenuated GVHD symptoms and pathology in the gut and skin. It inhibited ERK1/2 phosphorylation and expansion of donor T cells, sparing Tregs and B cells. Although high-dose trametinib inhibited myeloid cell engraftment, low-dose trametinib suppressed GVHD without severe adverse events. Notably, trametinib facilitated the survival of mice transplanted with allogeneic T cells and P815 tumor cells with no residual P815 cells observed in the livers and spleens, whereas tacrolimus resulted in P815 expansion. These results confirm that trametinib selectively suppresses GVHD-inducing T cells while sparing antitumor T cells in vivo, which makes it a promising candidate for translational studies aimed at preventing or treating GVHD.

Absence of Coronin 1B in Donor T Cells Diminishes Acute Gvhd By Impairing T Cell Accumulation in Secondary Lymphoid Tissue

Allogeneic stem cell transplant is a standard treatment for patients with high-risk and relapsed myeloid and lymphoid malignancies. However, donor T cells from the stem cell graft mediate graft-versus-host disease (GVHD), which is a common cause of morbidity and mortality for transplant recipients. Our group and others have shown that migration of donor T cells into secondary lymphoid tissue (SLT) and subsequent migration to target organs is critical to the pathogenesis of acute GVHD.The Coronin family of proteins consists of actin-binding proteins, which regulate filament formation by interacting with the Arp2/3 complex. Coronin 1B, a ubiquitously expressed member of the Coronin family, is required for lamellipodial protrusion and effective cell migration. Previous work has not evaluated a role for this protein in the function of T lymphocytes or during acute GVHD.To evaluate the effect of Coronin 1B in acute GVHD pathogenesis, we transplanted B6 T cell depleted bone marrow cells with wild type or Coronin 1B-/- T cells to lethally irradiated B6D2 and BALB/c recipient mice and evaluated clinical score of GVHD and overall survival. B6D2 recipients of Coronin 1B-/- T cells demonstrated 100% survival (Figure 1A. p< .001 as determined by Log-rank (Mantel-Cox) test) and significantly decreased clinical scores after transplant. This was confirmed with improvement in survival in BALB/c recipients of Coronin 1B-/- T cells. Additionally, Coronin 1B-/- T cells were capable of eliminating P815 tumor cells, indicating that loss of Coronin 1B does not inhibit graft-versus-tumor activity. By day 12 post- transplant, all mice receiving bone marrow alone developed tumor compared to none of the mice receiving Coronin 1B-/- T Cells. However, protection was not complete as 40% of Coronin 1B-/- T cell recipients developed tumor by day 23.To determine the effect of Coronin 1B on T cell migration during GVHD, B6D2 recipients were given GFP-expressing wild type or Coronin 1B-/- T cells along with T cell depleted bone marrow. Lymphoid tissue and target organs were harvested and analyzed by flow cytometry or GFP ELISA. We observed decreased accumulation of Coronin 1B-/- CD4+ (Figure 1B. p< .01 as determined by Student's t -test) and CD8+ T cells in the inguinal lymph node, mesenteric lymph node, and the spleen 4 days after transplant with no difference in accumulation in lymphoid tissue on days 7 and 14 after transplant. Additionally, we found decreased accumulation of Coronin 1B-/- donor T cells in the lung, colon and spleen 14 days after transplant (Figure 1C. p< .05 by Student's t -test). We also quantified the amount of cytokine in target organs by ELISA, and observed a decrease in IFN-γ and TNF-α in the colon 14 days after transplant.Our data demonstrate that Coronin 1B-/- T cells elicit reduced GVHD compared to wild type T cells. This was correlated with decreased accumulation of Coronin 1B-/- T cells in SLT early after transplant. These data indicate that targeting the migration of T cells to SLT is a viable approach to prevent acute GVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Synthesis and tumor cell growth inhibitory activity of biotinylated annonaceous acetogenins

Nineteen biotinylated squamocin/bullatacin derivatives have been synthesized for targeted delivery to biotin receptor overexpressed tumor cells. Most biotinylated squamocin and bullatacin derivatives show similar in vitro cytotoxicity against the biotin receptor non-overexpressed L1210 cells as squamocin and bullatacin, respectively, while against biotin receptor overexpressed 4T1 and P815 tumor cells, several derivatives show significantly higher potency and better selectivity. Among all the synthesized compounds, 15,28-di-O-(6-biotinylamidohexanoyl)squamocin (16) is the most potent, which is 10 and 26 times more active than squamocin against 4T1 and P815 cells, respectively. Compound 16 also appears to be six and fifteen times more selective than squamocin towards 4T1 and P815 cells, respectively, against L1210 cells. The structure activity relationship analysis has revealed that the preferred site for biotinylation is different for squamocin and bullatacin, and it also depends on whether a linking spacer is present.

Interleukin-15 administration increases graft-versus-tumor activity in recipients of haploidentical hematopoietic SCT

Utilizing a clinically relevant haploidentical (HI) murine transplant model, lethally irradiated B6D2F1 (H2K(b/d)) mice were transplanted with T cell-depleted (TCD) BM from B6CBAF1 (H2K(b/k)) mice. We found that administration of IL-15 significantly increases the numbers of CD8+ T and natural killer (NK) cells in spleen and BM after transplantion without GVHD. Graft-versus-tumor (GVT) potency of the graft was evaluated upon tumor challenge using P815 tumor cells (H2(d)). IL-15 administration without T-cell infusion did not result in any survival improvement. However, IL-15 in combination with very low-dose T-cell infusion (1 × 10(4)) significantly increased GVT activity and improved survival in recipients of HI hematopoietic SCT (HSCT). This effect was observed when IL-15 was given at a later time point, rather than immediately following transplantation. IL-15 administration also specifically increased slow-proliferative CD8+ T-cell proliferation and IFN-γ secretion in CD8+ T cells in recipients of CFSE (carboxyfluorescein succinimidyl ester)-labeled HI T-cell infusion, whereas there was no effect on CD4+ T-cell proliferation, suggesting the critical effect of IL-15 on CD8+ T-cell homeostasis in HI host. We conclude that IL-15 can be used for enhancing antileukemia effect of HI-HSCT, which requires presence of donor-derived T cells.

Computational QSAR models with high-dimensional descriptor selection improve antitumor activity design of ARC-111 analogues

ARC-111 has potent topoisomerase I-targeting activity and pronounced antitumor activity. To design ARC-111 analogues with improved efficiency, we performed analyses on the quantitative structure–activity relationship of 22 ARC-111 analogues assessed in P388 tumor cells. First, the support vector regression (SVR) models were constructed and optimized based on literature descriptors (the low-dimensional descriptor space) and the worst descriptor elimination multi-round (WDEM) method. The optimized SVR model had greater generalization ability than multiple linear regression (MLR) and stepwise linear regression (SLR) in the independence test, which indicated that our nonlinear WDEM method could remove redundant descriptors more effectively, and our optimized SVR was a more powerful modeling technique. Second, to identify more accessible and effective descriptors, our modeling descriptors with clear meanings were selected from a large number of descriptors calculated by the software PCLIENT. Through the high-dimensional descriptor selection nonlinear method and the WDEM method, seven independent variable combinations with tens of descriptors were selected out of 2,923 descriptors. The seven corresponding SVR models performed better in the independent test, compared to MLR and SLR. The evaluation measures supported the excellent predictive power of the new models. According to the interpretability analysis of the SVR model, the regression significance of the model and the importance of single indicator were evaluated based on F tests. Our study offers some useful theories for understanding the function mechanism and finds parameters for designing ARC-111 analogues with enhanced antitumor activity.

IL-15 Administration Increases Graft Versus Leukemia Activity in Recipients of Haploidentical Hematopoietic Stem Cell Transplantation,

Abstract 4027The success of haploidentical (HI) hematopoietic stem cell transplantation (HSCT), suggests that graft-versus-leukemia (GVL) effect might have a substantial role in this transplant modality. Rigorous T-cell depletion (TCD) of the graft decreases the occurrence of graft-versus-host disease (GVHD) in HI-HSCT, however this results in immunodeficiency and high disease relapse rate, especially in patients with resistant or residual leukemia. Therefore, enhancing GVL activity of HSCT without increasing GVHD is crucial for improving the outcome of haploidentical transplant. Post-transplant IL-15 administration is shown to enhance immune reconstitution, particularly donor-derived NK and CD8+ T cell populations in murine models. We evaluated the efficacy of IL-15 for enhancing GVL effect in recipients of HI-HSCT.For developing clinically relevant haploidentical transplant models, different hybrid mice with B6 background that share the same haplotype (H2Kb) are used for our murine haploindentical transplant experiments. Lethally irradiated B6D2F1/J (H2Kb/d) mice are transplanted with B6CBAF1/J (H2Kb/k) TCD bone marrow (BM) and T cells at varying doses. Some animals were also given P815 tumor cells on the day of transplant. Administration of IL-15 significantly increased the numbers of CD8+ T and NK cells in the spleen and BM in the T cell depleted model at post-transplant day 28. Infusion of very low dose haploidentical T cells (1×104) with TCD-BM resulted in a conflicting effect on immune reconstitution, i.e. increased T cell numbers, and decreased NK cell population. Post-transplant IL-15 administration also changed this immune reconstitution pattern and significantly increased both T and NK cell numbers in recipients of HI-HSCT. In P815 challenged mice that were transplanted with very low dose T cell added TCD-BM, IL-15 administration significantly increased anti-tumor activity of the graft and improved survival (Figure 1) without increasing GVHD. This effect was observed when IL-15 administration was given at a later time point rather than immediately following transplantation, possibly allowing for more donor cell engraftment and T cell proliferation to take place. IL-15 administration without T cell infusion did not result in any survival improvement. We conclude that in our experimental HI transplant models, IL-15 administration augments anti-tumor effect of the HI-HSCT without increasing GVHD risk, and this effect requires presence of donor derived T cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Human Mesenchymal Stem Cells Attenuate Graft-Versus-Host Disease and Maintain Graft-Versus-Leukemia in Murine Allogeneic Bone Marrow Transplantation

Abstract 1907Defining in vivo effects and biodistribution of human bone marrow-derived mesenchymal stem cell (hMSCs) following allogeneic bone marrow transplantation (alloBMT) could impact the clinical utility of MSC therapy for the prevention and treatment of graft-versus-host disease (GvHD). Using an established model of murine alloBMT, we defined hMSC effects on GvHD and graft-versus-leukemia (GvL) activity. We first studied whether hMSC could modulate in vitro murine T-cell (TC) alloreactivity in mixed leukocyte cultures (MLCs). Specifically, hMSCs added to MLCs significantly reduced TC proliferation in a concentration-dependent manner distinct from human fibroblasts. In contrast to MLC cultures alone, MLCs containing hMSCs had significant reduction in TNFα, IFNγ, and IL-10 levels and higher levels of PGE2 and TGFβ1. Modulation in the inflammatory milieu was associated with changes in TC phenotypes, including more naïve and less activated TC surface marker expression (CD62L+CD69−) and the induction of CD4+CD25+FoxP3+ T-regulatory cells. To determine whether hMSCs could modulate in vivo mTC alloreactivity, irradiated recipient B6D2F1 (H-2bxd) mice were transplanted with allogeneic C57BL/6 (H-2b) BM and purified splenic TCs (B6→B6D2F1) and then were tail-vein injected with hMSC infusions (1 million per injection) on days one and four post-transplant. Syngeneic transplant recipients (B6D2F1→B6D2F1) were used as controls. hMSC-treated alloBMT mice had significantly prolonged survival and improved clinical GvHD scores, reduced splenic TC expansion and TNFα and IFNγ-producing TCs, and lower circulating TNFα and IFNγ levels versus untreated alloBMT mice. Bioluminescence imaging showed redistribution of labeled hMSCs from the lungs to abdominal organs within 72 hours following infusion. Importantly, GvHD target tissues (small and large bowel and liver) harvested from hMSC-treated alloBMT mice had significantly lower GvHD pathology scores than untreated alloBMT mice. We next determined the effects of hMSCs on GvL activity using the murine mastocytoma cell line, P815 (H-2d). TCs co-cultured with hMSCs maintained potent in vitro cytotoxic T-lymphocyte (CTL) activity comparable to untreated control CTLs. After challenge with P815 tumor cells, hMSCs-treated alloBMT mice had less severe GvHD, eradication of tumor burden, and improved leukemia-free survival compared to alloBMT control mice. Lastly, indomethacin (IM) added to MLC-hMSC co-cultures significantly reversed attenuation in both murine TC alloreactivity and surface activation expression. In addition, IM administered to hMSC-treated alloBMT mice reversed hMSC-associated survival advantage, suggesting that PGE2 in part mediates hMSC immunomodulatory effects. Together, our results show that hMSC infusions effectively attenuate GvHD and maintain GvL potency in alloBMT mice and reveal potential biomarkers and mechanisms of action underlying hMSC effects. Disclosures:Solchaga:Bimemetic Therapeutics: Employment. Cooke:Amgen: Provides experimental drug and central pharmacy support for 2 trials for which I am Co-PI.

The influence of pretransplantation conditioning on graft-vs.-leukemia effect in mice

The therapeutic potential of allogeneic hematopoietic stem cell transplantation relies on the graft-vs.-leukemia (GVL) effect to eradicate residual tumor cells immunologically. The relationship of conditioning intensity to GVL effect was investigated. Lethally irradiated (either 900 or 1300 cGy) B6D2F1 (H-2(b/d)) recipients were transplanted from B6 (H-2(b)) donors. P815 or L1214 (H-2(d)) tumor cells were injected intravenously or subcutaneously on day 1 post-transplantation to generate a GVL model. Tumors in allogeneic mice treated with 1300 cGy exhibited markedly delayed subcutaneous tumor growth invivo as compared with mice treated with 900 cGy, while intravenous tumor growths were comparable between the two radiation doses. Serum levels of tumor necrosis factor-α or interferon-γ were similar and the percentages of donor T-cell proliferation or apoptosis early after hematopoietic stem cell transplantation were comparable. The numbers of CD8(+) T cells from recipients that received 1300 cGy were significantly elevated in skin and tumor tissues. CD62L(low) and CD44(high) CD8(+) T-cell fractions were also elevated in 1300 cGy. After the higher radiation dose, P815-specific interferon-γ responses of splenic CD8(+) T cells were markedly enhanced and the fractions of T cells of interferon-γ-producing T cells in tumor tissues but not in spleen were increased. The protein concentrations of chemokines in skin and tumor tissues were substantially elevated in 1300 cGy compared to 900 cGy. The higher radiation dose (1300 vs. 900 cGy) resulted in significantly enhanced GVL effect against an extramedullary tumor and the alterations in effector T-cell trafficking into tumor tissue are the most likely mechanism.

The protective effect of schisandra lignans on stress-evoked hepatic metastases of P815 tumor cells in restraint mice

The present study was conducted to investigate the effects of schisandra lignans extract (SLE) on stress-evoked hepatic metastases of mastocytoma P815 tumor cells, which was closely related with immune function. The high-performance liquid chromatography (HPLC) fingerprint of SLE was recorded and the percentage composition of schisandra lignans was determined as 82.63%. The contributions of the immunomodulatory properties of SLE to the protective effects on stress-induced hepatic metastases were studied. Our results found that restraint stress significantly promoted hepatic metastases of P815 tumor cells. However, oral administration of SLE (100 and 200mg/kg/d, 14d) significantly reduced the number of metastatic colonies in liver of restrained mice. SLE was further found to be significantly improving T lymphocyte proportions and increasing cytotoxic T lymphocyte (CTL) activity of immunized spleen cells in stressed mice. These results indicated that the protective effects of SLE on hepatic metastases were related to its alleviation of the adverse effects of stressors for bio-homeostasis and immunoprotection. The obtained data provided evidences to elucidate the traditional use of Fructus schisandrae as a tonic or sedative.

Double Haploidentical Hematopoietic Stem Cell Transplantation (HSCT) Results In Successful Engraftment of Bone Marrow From Both Donors without Graft Versus Host or Graft Versus Graft Effects.

AbstractAbstract 1453The use of haploidentical donors extends the potential clinical application of HSCT. However, relapse of resistant malignancy contributes to low success rates in high risk patients. Relapse may be due to the ability of leukemic cells to immunologically escape a single donor's GVL effects. We hypothesized that the use of two haploidentical donors, each targeting a different recipient haplotype, will increase anti- leukemia activity after double haploidentical SCT. We performed murine studies to establish new single haploidentical (SH) and double-haploidentical (DH) murine models that mimic the possible scenarios which might be encountered clinically rather that using more traditional Parent → F1 models.We first established a haploidentical transplant model using two different hybrid mouse strains as donor and recipient in the experiments. Lethally irradiated B6CBAF1 (H2Kb/k) recipients were transplanted with T cell depleted (TCD) bone marrow (BM) from B6D2F1 (H2Kb/d) donors. Recipient mice harvested at days 28, 42 and 56, showed more than 90% donor cell engraftment, including donor derived lymphopoiesis and myelopoiesis, without evidence of graft versus host disease. Subsequently, lethally irradiated B6CBAF1 (H2Kb/k) recipients were transplanted with TCD-BM from two haploidentical donors (DH model) including B6SJF1 (H2Kb/s) (donor 1 - D1) and B6D2F1 (H2Kb/d) (donor 2 - D2). We observed recipients for 90 days and all mice survived without evidence of GVHD or weight loss. Analyses of blood collected retro-orbitally at day 90 revealed that recipients of DH transplants had significantly higher WBC and neutrophil counts than recipients of SH HSCT from either D1 or D2 respectively. DH recipients consistently showed successful engraftment with mixed chimerism in both bone marrow and spleen. There was no difference in thymopoiesis, B cell and myeloid cell reconstitution compared to SH transplants. In contrast, the number of splenic T cells was higher in SH recipients of D1 marrow (B6SJF1). We then explored the effects of low dose T cell infusions (1×105) on chimerism of donor cells. Low dose T cell infusion from either D1 or D2 did not affect the BM cellularity, but did increase the degree of dominance of that donor's cells in the BM and spleen.A similar outcome was observed when this study was extended to other models such as B6C3 + C3D2F1 → B6D2F1 and B6CBAF1 + B6SJF1 → CB6F1 models where all recipients of DH transplants survived without evidence of GVHD.Recipients of TCD DH transplants were challenged with P815 tumor cells. We used B6SJF1 (D1) + B6CBAF1 (D2) → B6D2F1 model in tumor experiments. Interestingly, recipients of TCD-DH transplants exhibited a significantly better survival than recipients of D1 SH or D2 SH transplants. However, after a low dose T cell infusion (1 ×105), recipients of D2 BM survived significantly better than recipients of D1 BM. In contrast to the TCD model, recipients of DH BM + DH T cells show similar probability of survival with recipients of D2 SH BM + D2 SH T cells.We conclude that TCD DH HSCT results in successful engraftment of both types of BM cells. Additionally, infusion of low dose haploidentical T cells improves the anti-tumor effect without stimulating GVHD. Double haploidentical HSCT may be an ideal platform to enhance GVL effects after transplantation. Disclosures:No relevant conflicts of interest to declare.

Synthesis, crystal structures and anticancer activities of two decavanadate compounds

Two ammonium decavanadate(V) compounds, (H2tmen)3V10O28·6H2O (1) and (H2en)3V10O28·2H2O (2), where H2tmen and H2en represent the doubly protonated N,N,N′,N′-tetramethylethylenediammonium ion and doubly protonated ethylenediammonium ion, respectively, have been synthesized and structurally characterized by elemental analyses, spectroscopic (i.r., e.s.r.) studies, and single-crystal X-ray diffraction. Both crystal structures reveal the presence of the [V10O28]6− cluster anions, doubly protonated diammonium cations, and lattice water molecules. Hydrogen bonds assemble these two compounds to form three-dimensional networks. The in vitro anticancer activity against A549 and P388 tumor cells lines has also been determined by the MTT-based assay, and the results suggest that both compounds can inhibit proliferation of these two kinds of tumor cells.

Cytotoxic mechanisms of IL-4 polarized CTLs (101.28)

Abstract IL-4 down-regulates perforin and granzymes and depresses rapid (4 hr) cytotoxic T lymphocyte (CTL) killing. Curiously, IL-4 can increase anti-tumor immunity in animal models. We wanted to know how IL-4 polarized CTLs kill and found that they have potent delayed cytotoxicity. We cultured BALB/c mouse splenocytes with conA and 500 units/ml mouse IL-4 or IL-2 for 3 days and then re-cultured the cells for 3 days with cytokines without conA. The IL-4 CTLs were polarized as indicated by expression of the IL-4 inducible lipase PLRP2. By flow cytometry, IL-4 CTLs expressed Grz B at lower levels than IL-2 CTLs. We assayed cytotoxicity using anti-CD3-redirected death of P815 tumor cells. Death was measured at 48 hrs by loss of EGFP+ P815 cells using flow cytometry with cell count beads to account for dead cell disintegration. Cytotoxicity by the IL-4 CTLs was 80% at effector lymphocyte to P815 (E:T) ratios of 1:10. IL-4 CTLs had perforin-independent mechanisms, with similar activity for WT and Prf1-/- CTLs. We monitored T cells for death receptor ligands. Quantitative RT-PCR indicated that IL-4 cells maintained comparable levels of mRNA for LTβ, LIGHT, TRAIL and FasL, but had lower levels of TNFα and TNFβ, than IL-2 CTLs. We conclude that IL-4 polarized CTLs have potent cytotoxicity which could be mediated by death receptor ligands and/or by novel IL-4 inducible potential cytotoxins. Supported in part by NIH grants R01CA38942, T32CA09563, P20 RR-016464 &amp; P20-RR015581.

IL-12 Enhances Tumor Immunity Via Differential Effects On Regulatory T Cells and Cytotoxic Lymphocytes.

Abstract 1656Poster Board I-682To study the roles of IL-12 and Interferon-gamma (IFNg) in tumor immunity, we used RMAS lymphoma cells to challenge IL-12 receptor beta 2-deficient (IL-12Rb2-/-) and IFNg receptor 1-deficient (IFNgR1-/-) mice that are in the syngeneic C57BL/6J background. We intravenously injected mice with a dose of 1 × 104 RMAS cells that caused death in about 50% of wild-type (WT) mice. As shown in the Figure below, all of the WT mice treated with exogenous IL-12 were rescued from death caused by tumor growth; endogenous IL-12 was not sufficient to impact tumor growth since IL-12Rb2-/- mice showed a survival rate similar to that of WT mice. However, all of the IFNgR1-/- mice succumbed to tumor growth, indicating that endogenous IFNg is required for tumor immunity in this system. Furthermore, IL-12 treatment did not improve the survival of the IFNgR1-/- mice, suggesting that IFNg signaling is required for IL-12's anti-tumor effect. [Display omitted] We previously showed that an IL-12/IFNg axis can inhibit tumor-induced regulatory T cell (Treg) proliferation in vitro (Cao et al, 2008 ASH Annual Meeting). We have subsequently examined their effects on Treg cells in vivo. Compared to naive mice, significant Treg expansion (4.9 ± 2.1 fold, n=5, p=0.025) was observed in the peritoneal cavity of WT mice within 2 weeks after an intraperitoneal injection of 1 × 104 RMAS cells. This expansion was completely blocked by treatment with exogenous IL-12. Treg cells in the IL-12Rb2-/- mice expanded to levels comparable to that in WT animals, suggesting that endogenous IL-12 was not sufficient to control Treg expansion. In contrast, significantly higher Treg expansion was observed in IFNgR1-/- mice (36.8 ± 11.8 fold, n=5, p=0.002), which was partially inhibited by IL-12 treatment (13.2 ± 3.5 fold, n=5, p=0.002), suggesting that an IFNg-independent mechanism may also account for IL-12's anti-Treg effect.To further study the effects of IL-12 and IFNg on cytotoxic T lymphocyte (CTL) function, we performed mixed lymphocyte reactions (MLR) and used flow-based killing assays (FloKA) to measure cell contact-dependent killing of allogeneic P815 tumor cells. MLR-activated CTLs were found to kill tumor targets via perforin/granzyme-mediated cytotoxicity. At a 10:1 (effector:target) ratio, granzyme AxB-deficient CTLs and perforin-deficient CTLs displayed significantly reduced killing (8.6 ± 1.2% and 4.5 ± 0.9%, respectively) compared to WT CTLs (36.1 ± 3.5%). IL-12 supplement (2ng/ml) to the MLR significantly increased the killing activity of WT CTLs (65.3 ± 4.2%), but had no significant effect on granzyme AxB-deficient CTLs or perforin-deficient CTLs. In contrast, IFNg supplement (10ng/ml) to the MLR had no significant effect on the killing activity of CTLs. Conversely, MLR-activated IFNgR1-/- CTLs killed P815 cells as efficiently as WT CTLs and responded to IL-12 treatment as efficiently as WT CTLs.Taken together, these data suggest that IL-12 treatment inhibits tumor-induced Treg expansion and stimulates IFNg-dependent anti-tumor immune responses. In addition, IL-12 also activates perforin/granzyme-dependent function of cytotoxic T lymphocytes. These differential effects on diverse immune components may collectively result in enhanced tumor immunity. DisclosuresNo relevant conflicts of interest to declare.

Lipid‐dependent cytotoxicity by the lipase PLRP2 and by PLRP2‐positive cytotoxic T lymphocytes (CTLs)

IL-4 induces a lipase, pancreatic lipase related protein 2 (PLRP2), in cytotoxic T lymphocytes (CTLs). Because PLRP2 in semen can mediate lipid-dependent toxicity to sperm, we questioned whether CTL-derived PLRP2 could support similar cytotoxicity toward tumor cells. Recombinant PLRP2 was toxic to P815 tumor cells in 48 h when lipid and another protein, colipase, were present. However, PLRP2-positive CTLs (induced with many lots of IL-4) were unable to mediate lipid-dependent cytotoxicity. Notably, CTLs induced with only one lot of IL-4 had lipid-dependent cytotoxicity. The exceptional lot of IL-4 was effective in multiple experiments at inducing lipid-dependent cytotoxicity. The lipid-dependent cytotoxicity it induced was determined to be perforin-independent. CTLs induced with IL-4 that was unable to induce lipid-dependent cytotoxicity had mRNA for PLRP2 but not mRNA for colipase. Therefore, we added exogenous colipase to the CTL assays but still cytotoxicity was unchanged. We conclude (1) that lipid-dependent cytotoxicity, promoted by the lipase PLRP2 and colipase, will kill tumor cells and (2) that more than PLRP2 alone is required for lipid-dependent cytotoxicity mediated by CTLs.

Hydrolysis of tumor cell lipids after CTL-mediated death

Contributions of lipases to CTL function have been debated, including if T cell lipases damage target cells. Expression of the lipase pancreatic lipase-related protein 2 (PLRP2) was previously found in IL-4 cultured lymphocyte cell lines but absent from IL-2 cultured lymphocytes. Here, we evaluated IL-2 and IL-4 induced CTLs for hydrolysis of target cell lipids and killing. Using anti-CD3 redirected lysis of [(3)H]-oleic acid-labeled P815 tumor cells, we detected the release of the radioactive fatty acid (FA). When PLRP2(+/+) and PLRP2(-/-) CTLs were compared, there was more killing by the PLRP2(+/+) CTLs. However, [(3)H]-oleic acid release was similar per dead P815, suggesting that lipid hydrolysis was produced by the dead P815s rather than by PLRP2. The FA release and death were completely dependent on perforin and also occurred when P815s were killed by perforin-containing T cell granule extracts that lacked lipase activity. Death by the cytotoxic granules extracts was unaffected by the addition of lipases. A lipase inhibitor, tetrahydrolipstatin, blocked FA release without affecting CTL-mediated cytotoxicity. Also, CTL-mediated death caused as much FA release as death by disruption of cells by freeze-thawing. The released oleic acid may be sufficient to promote secondary apoptotic responses after CTL-induced trauma.