CB6F1 Mice Research Articles

Suppression of tumor growth in renal cancer treatment with tumor vaccination after haploidentical bone marrow cell reconstitution

To investigate whether whole tumor cell vaccination strategies in combination with bone marrow transplantation (BMT) can stimulate graft-versus-tumor effect (GVT). Twenty-six BALB/c mice were randomly divided into 3 groups: BMT group (group A, n = 10), BMT + vaccination group (group B, n = 10), control group (group C, n = 6). (BALB/c × C57BL/6) F1 mice [CB6F1, H-2K(b/d)] were used as donors. BALB/c mice of group C were only inoculated with Renca cell (2.6 × 10(6)). Mice of group A and B were conditioned with 8 Gy irradiation, followed by infusion by bone marrow cell of CB6F1 mice on day 1, then inoculated with Renca cell (2.6 × 10(6)) on day 8. All mice of group B were immunized subcutaneous on the back with 5 × 10(5) irradiated Renca tumor cells on day 9 and day 16. All mice of group C were inoculated with Renca cell (2.6 × 10(6)) on day 8. In group A and B, all mice were analyzed by fluorescence activated cell sorter (FACS) on day 14, and 28 day after BMT. Mice were killed on day 32 after inoculation with tumor cell and collected blood sample. All tumors were taken out to be weighed and then fixed in 10% buffered formalin, embedded in paraffin, and cut into 5 µm slices. The slices were stained with HE and examined by TdT mediated-dUTP nick end labeling (TUNEL). Liver, skin, intestine, and spleen were biopsied for histopathological examination. The results of chimera showed that engraftments of group A, B were full donor chimerism, and the chimerism of those remained above 90% and preserved even after 28 days. The tumor weight, tumor volume increment in the group B was lower than group A and C (P < 0.05). The tumor suppressing rates of the group A and B were 54%, 60% respectively. The area ratio of tumor necrosis and apoptosis index (AI) of the tumor in the group B were higher than group A and C (P < 0.05). Graft-versus-host disease was not observed in each group. The mechanism of GVT after haploidentical allogeneic bone marrow transplantation with tumor vaccination may be the promotion of tumor necrosis and apoptosis.

Alloreactive NK-Cells Cure Tumor Stem Cell Containing Breast Cancer.

Abstract 4082Poster Board III-1017 BackgroundPatients with metastasized solid tumors can not be cured by current standard treatment options. One hypothesis is that slow cycling chemo-resistant tumor stem cells give rise to new tumors after cytoreductive treatment, ultimately leading to chemoresistant tumors. Acute myeloid leukemia tumor stem cells can be killed by alloreactive T- or NK-cells, as patients have a higher survival chance after allogeneic transplantation. In the past we published the curative effectiveness of allogeneic spleen and bone marrow transplantation in breast cancer bearing mice. Our current aim was to study if either alloreactive T- or NK-cells are able to cure these mice. In addition we wanted to know if the tumors contain slow cycling tumor stem cells. MethodsThe 4T1 breast cancer cell line was cultured under standard conditions. Fifty thousand 4T1 cells were injected s.c. in the flank of CB6F1 (H-2b/d) mice. At day 12 mice were treated with 2× 2Gy total body irradiation and 200 mg/kg cyclophosphamide (CY), followed by transplantation of spleen and bone marrow cells. B6CBAF1 (H-2b/k) mice were used as allogeneic donors. These donors harbour alloreactive NK-cells towards CB6F1 recipients as determined by an in vivo NK-cell alloreactivity assay. NK-cell or T-cell depleted grafts were obtained from donors injected with anti-AsialoGM1 or a combination of anti-CD4 and anti-CD8 respectively some days before spleen and bone marrow harvest. Tumor stem cells were defined as slow cycling (i.e. label retaining) Hoechst extruding cells. 4T1 tumor cells were labelled with Vybrant CM-Dil (Dil) and then injected s.c. in CB6F1 mice. Fourteen days later tumors were excised. Part of the tumor was prepared as single cell suspension. Subsequently the presence of Dil retaining tumor stem cells was determined by fluorescence microscopy. The other part of the tumor was snap frozen and studied by histochemistry and fluorescence microscopy. For in vitro cytotoxicity testing 4T1 cells were again labelled with Dil and then seeded in 96 well plates at 30 cells/well. Chemotherapeutics were added in the different plates: 4-OH-CY (the active metabolite), cisplatine and doxorubicin, in concentrations far above the lowest maximal lethal dose as determined in MTT assays. Wells were checked weekly for growth and presence of tumor stem cells. Hoechst staining was used in these cultures to underscore that Dil retaining chemorefractory cells are indeed stem cells. [Display omitted] ResultsNinety percent of CB6F1 mice injected with 4T1 breast cancer cells developed a tumor growing progressively to a size that made it necessary to sacrifice them. Growth in mice treated with radio- and chemotherapy only or additionally with CB6F1 spleen and bone marrow cells was delayed for 10 days compared with tumor growth in untreated mice, but the incidence was equally high (90% and 80% respectively). In contrast, but in concord with our previous published results, only 10% of 4T1 breast cancer bearing mice transplanted with haploidentical spleen and bone marrow cells did show progressive tumor growth (follow-up 99 days at the time of abstract submission, p<0.004 log-rank test)). Similarly, mice transplanted with spleen and BM cells from T-cell depleted donors did not develop tumors at all (p<0.004 log-rank test), in contrast to recipients of in vivo NK-cell depleted grafts (90% tumor incidence).Label retaining tumor stem cells are unmistakably present in tumors at the day of transplantation, evidenced by the presence of a minute number of Dil retaining cells in single cell suspension of the tumor and in tumor tissue sections. More direct evidence for chemo-resistance of 4T1 tumor stem cells is derived by results of in vitro co-culture experiments. When Dil labelled 4T1 cells are plated in 30 cells/well concentration, approx. 25% of the wells contain one or two label retaining cells while the other wells comprise non-label retaining cells only. After incubation with chemotherapeuticals growth is observed only in wells containing a label retaining cell. These label retaining cells are also Hoechst negative. ConclusionsThis report provides the first evidence that tumor stem cells can be eliminated in vivo by alloreactive NK cells resulting in cure of chemorefractory breast cancer. It is also shown in vitro that only tumor stem cell containing tumor colonies resist the effect of various chemotherapeuticals commonly used in oncology as they do in vivo. These results strongly encourage the exploration of clinical alloreactive NK therapy in patients with metastasized solid tumors. Disclosures:No relevant conflicts of interest to declare.

Allospecific Effector Memory T Cells Are Able to Mediate Second-Set Skin Graft Rejection but Unable to Induce Graft-Versus-Host Disease.

Abstract 232Several different groups have independently demonstrated that non-allospecific effector memory T cells do not induce graft-versus-host disease (GVHD). Limited data are available regarding the ability of allospecific effector memory T cells to induce GVHD. We first studied this question in the C57BL/6 into BALB/c model. Similar to the data previously published by other groups, purified CD62L- effector memory T cells isolated from donors, who were primed with the host antigens 8 weeks earlier, had decreased ability to induce acute GVHD compared with unseparated and CD62L+ T cells. Similar results were observed when the parous female mice, who were sensitized to the host antigens during pregnancy, were used as memory T cell donors. In order to study this question more definitely and to understand the mechanisms underlying these surprising observations, we further studied the ability of allospecific effector memory T cells to induce GVHD using a novel GVHD model mediated by transgenic TEa T cells. All TEa T cells are CD4+ and recognize the same peptide in the context of I-Ab. This peptide corresponds to positions 52-68 from the alpha-chain of I-E class II molecules and is expressed in all antigen presenting cells from H-2b/I-E+ strains such as CB6F1 mice. To generate memory T cells, naïve TEa cells were first parked in Rag1−/− mice and then immunized with irradiated CB6F1 spleen cells. More than eight weeks later, ∼98% of TEa cells obtained a memory phenotype (CD44high, CD45RB-.CD127+,CD11a bright, FasL bright, Ki67-, CD28-, KLRG-). Of them, about 93% were CD62L-CD44high effector memory T cells and 7% were CD62L+CD44high central memory T cells. These Rag1−/− mice that contained memory phenotype TEa cells rejected CB6F1 skin grafts much faster than naïve TEa mice did (median survival time: 6.5 vs. 13 days, P=0.01), suggesting that the memory phenotype T cells contained in these mice are functional. Moreover, CD62L-CD44high TEa cells purified from these mice mediated faster and stronger in vitro proliferative responses against alloantigens than naïve TEa cells did, further demonstrating that they are true functional effector memory T cells. We next tested the ability of these effector memory TEa cells to induce GVHD. Effector memory TEa cells were obtained after depletion of CD62L+ cells using magnetic beads and the purity was more than 99%. Transplantation of 1′105 TEa naïve T cells together with 1′107 T cell depleted bone marrow cells into lethally irradiated CB6F1 recipients induced lethal GVHD in all recipients and all animals in this group died within 35 days after transplantation. In contrast, none of the effector memory TEa cell recipients developed GVHD and all of them survived more than 100 days post transplantation (P<0.01, compared with naïve T cell control). To understand the mechanisms underlying these observations, we studied the kinetics of TEa proliferative responses upon challenge with alloantigens. The data indicated that effector memory TEa cells reached the peak responses faster than naïve TEa cells did. CFSE tracking experiment further confirmed this observation. Simultaneous staining with anti-Anexin V antibody and 7-AAD demonstrated that effector memory TEa cells undergone apoptosis and died faster than naïve T cells did. In conclusion, these data underscore the fundamental difference of alloresponses mediated by antigen-specific effector memory T cells in graft rejection and GVHD settings. The TEa transgenic T cell skin graft and GVHD models would allow further understanding of the unique alloresponses mediated by allospecific memory T lymphocytes in GVHD. Disclosures:No relevant conflicts of interest to declare.

Parasitaemia levels inPlasmodium chabaudiinfected-mice modify IFN-γ and IL-10 expression after a homologous or heterologous challenge

CB6F1 mice infected with the nonlethal Plasmodium chabaudi chabaudi AS suffer parasitaemia levels up to 40% (full parasitaemia, FP) and develop both homologous and heterologous (against the lethal Plasmodium yoelii 17XL) protective immunity. However, if mice are treated with anti-malarial drug when parasitaemia is below 10% (low parasitaemia, LP), they only develop homologous immunity. For the better understanding of this interesting dissociation related to the degree of parasitaemia, in this work, we studied the genetic expression of some cytokines. We found that during primary parasitaemia both FP and LP mice showed at first a TNF-alpha, IL-2 and IFN-gamma response which is followed by an IL-4 and IL-10 response. When FP and LP mice were challenged with either the homologous (FP + AS and LP + AS mice) or the heterologous parasite (FP + 17XL and LP + 17XL mice), we observed that LP + 17XL mice, which failed to develop heterologous immunity and succumbed to the challenge, showed a stronger IFN-gamma and a weaker IL-10 expression than FP + 17XL mice, which developed heterologous immunity and survived the challenge. The importance and the possible implications of these findings are discussed.

Mesenchymal stem cells protecting multiple organ failure

Objective The patients with lethal irradiation after sucessful hematopoietic stem cells transplan-tation had blood recovery, but did not avoid to died of multiple organ failure(MOF). To overcome the block, the article investigated mechanisms of mesenchymal stem cells (MSCs) protecting lethal radiated mice from multiple organ failure after haploid bone marrow cells transplantation. Method BALB/c mice irradiated with 8Gy60COγ-rays were randomly divided into two groups: MSCs group, infused MSCs labeled with cm-DiI and bone marrow monocytes of CB6F1 mice; Control group, only infused bone marrow monocytes; normal group, mice were infused cm-DiI marked MSCs without irradiation. The distribution of MSCs and the serous densities of Il-2, Il-10 and TNF-α in the recipients were observed after transplantation. Results MSCs collected in the bone marrow and the intes-tine in normal group at 15 d,in MSCs group MSCs enriched the different organs at 3,15 and 30 d. MSCs regulated down the secretion of IL-2 and TNF-α,and up the IL-10 density. Conclusions MSCs protected mice from multiple organ failure through above effects and may be open a new treatment strategy on acute radiation syndrome by stem cells. Key words: Acute radiation syndrome; Mesencbymal stem cells; Cytokine; Multiple organ failure

Epitope-Specific Regulatory T cells Differentially Modulate the CD8 T Cell Response to Respiratory Syncytial Virus in Mice (45.29)

Abstract RSV infection is the leading cause of lower respiratory disease in infants and young children, accounting for &amp;gt;120,000 hospitalizations each year in the US alone. Although CD8 T cell effector function is key to virus clearance, CD4 T cells coordinate the immune response and can lead to immunopathology. Therefore, they play a central role in the immunity and immunopathogenesis of RSV infection. To facilitate in depth evaluation of RSV-induced CD4 T cell responses we identified two new I-Ab- restricted dominant CD4 T cell epitopes, M213-223 and M227-37, in RSV M and M2 that also contain well characterized CD8 T cell epitopes. CD4 T cells from lung and spleen of RSV-infected CB6F1 mice produce different patterns of IL-2, IFN-γ and other cytokines after peptide stimulation. Novel tetrameric peptide-I-Ab complexes identified 0.5% and 1.6% RSV M- and M2-specific CD4 T cells in lungs of the infected mice. Peptide-stimulated CD4 T cells produced different patterns of regulation on CD8 T cell responses to RSV M and M2 epitopes in vitro. The regulatory function of the CD4 T cell correlated with the level of FoxP3 expression in epitope-specific CD4 T cells Our study demonstrates that the biophysical properties of the epitope may dictate the functional properties of the CD4 T cell response. Understanding how epitope structure influences T cell function will provide new insight into viral pathogenesis and may improve vaccine design.

A bioassay to evaluate oocyte vitrification procedures

OBJECTIVE: Vitrified human oocytes display a high potential for survival, fertilization and cleavage, but their developmental potential to blastocyst is low. In contrast, similarly vitrified mouse oocytes develop to blastocyst at a comparable rate to fresh oocytes. In this study we assess the viability of these mouse blastocysts by monitoring their re-expansion and morphology following vitrification-thaw. DESIGN: Blastocysts developing from fresh or vitrified mouse oocytes were evaluated after vitrification to assess their ability for re-expansion and morphology following thaw. MATERIALS AND METHODS: Metaphase II oocytes were collected from PMSG+hCG-treated CB6F1 mice and denuded with hyaluronidase. Mature oocytes were equilibrated and vitrified using a DMSO+ethylene glycol+sucrose procedure. After thaw and cryoprotectant removal in sucrose solutions, the surviving oocytes were inseminated by capacitated sperm. The resultant zygotes were cultured for 4 days at 37C in Quinn's Advantage sequential cleavage/blastocyst media (Sage) supplemented with 6% PlasmanateR. All good quality blastocysts (a well-defined inner cell mass and trophectoderm) were punctured with a micro glass needle to release the blastocoel fluid and vitrified. They were examined for survival immediately post-thaw and for blastocoels re-expansion and morphology 3h later. RESULTS: In response to vitrification-thaw, 98% of 90 mouse oocytes survived; their cleavage and blastocyst formation rates were comparable to those of non-vitrified eggs (78% vs 80%; 71% vs 66%, respectively) as was the formation rate of good quality blastocysts (60% vs 63%). All blastocysts in both groups survived the vitrification-thaw procedure. However, when examined at 3h post-thaw, the number of re-expanded blastocysts derived from vitrified oocytes was significantly lower (62% vs 98%, p<0.001). Moreover, good quality morphology was observed in fewer of the expanded blastocysts that developed from vitrified eggs (51% vs 73%, p<0.05). CONCLUSIONS: The decreased ability of blastocysts derived from vitrified eggs to re-expand and display good quality morphology post-vitrification suggests that they are less viable. However, not all blastocysts are similarly affected. Such results suggest that monitoring blastocyst recovery post-vitrification is a reasonable bioassay to evaluate oocyte vitrification or cryopreservation protocols. Such bioassays will optimize these procedures for fertility preservation.

366: Allospecific Effector Memory T cells have Decreased Ability to Induce Acute GVHD

Several different groups have independently demonstrated that non-allospecific memory T cells do not induce GVHD. The data related to the ability of allospecific memory T cells to induce GVHD have not been conclusive. In order to study this question more definitely, we have developed a novel GVHD model using TEa TCR transgenic mice as donors. TEa CD4+ TCR transgenic mice (C57BL/6 background) express a TCR that recognizes the peptide ASFEAQGLANIAVDKA in the context of I-Ab. This peptide corresponds to positions 52–68 from the alpha-chain of I-E class II molecules and is expressed in all APCs from H-2b/I-E+ strains such as CB6F1 mice.

Evaluation of the Friend Virus model for the development of improved adenovirus-vectored anti-retroviral vaccination strategies

We evaluated the suitability of the Friend Virus (FV) model for the development of improved adenovirus vectors for anti-retroviral vaccination using two types of adenovirus vectors, encoding F-MuLV Env and Gag, which differed only in their fiber genes (Ad5 and Ad5F35). Genetically FV-resistant C57BL/6 mice and highly susceptible CB6F1 hybrid mice were vaccinated by either homologous or heterologous prime-boost regimen. After FV challenge, viral loads in the spleens of C57BL/6 mice were reduced approximately 250-fold and were below the detection threshold in >50% of the mice. Vaccination outcome was critically influenced by the route of vector administration. In CB6F1 mice, vaccination resulted in reduced viremia, delayed onset of splenomegaly, and induction of FV-specific T cells as assessed by tetramer staining. Heterologous prime-boost vaccination resulted in significantly higher neutralizing antibody titers, translating into improved immune protection, in contrast to coexpression of cytokines. Our results suggest that the FV model can provide insight into the development of improved adenovirus vectors for HIV-1 vaccination.

Mechanisms of Migration and Generation of Allo-Reactive Donor T Cells That Cause Organ Specific Acute GvHD in Allogeneic BMT Recipients.

Background: Allo-reactive donor T cells are primarily responsible for GvHD in allogeneic BMT. A number of studies have shown that increased allo-reactivity is found among the CD62L+ subset of donor T-cells, but the mechanisms for organ specific allo-reactivity are poorly defined. Our hypothesis is that rapid proliferation and migration of CD62L+ naive donor CD4+ and CD8+ T cells to specific organs leads to acute GvHD. Methods: We used a parent (C57BL/6) to (C57BL/6 × BALB/c) CB6F1 allogeneic BMT model with a combination of T cell depleted BM (TCD BM) and splenocytes. 30 × 106 congeneic donor splenocytes labeled with CFSE were transplanted with 5 × 106 TCD congeneic BM into lethally irradiated (11Gy) CB6F1 mice. Recipients were sacrificed within 3.5 days of transplant and FACS was used to measure proliferation of CFSE-labeled donor T-cells isolated from blood, spleen, liver, lungs, thymus, BM, lymph nodes, and peritoneal exudates cells (PEC). Syngeneic C57BL/6 recipients served as controls. At least 5 mice per group were used in each experiment. Results: There was increased homing of CFSE-labeled donor T-cells to most organs in allogeneic compared to syngeneic BMT recipients. CD45.1+ donor cells were 4-fold higher in spleen, p=0.01; 9-fold higher in liver, p=0.002; 14-fold higher in PEC, p=0.017; 136-fold higher in lung, p=0.0006; 126-fold higher in BM, P=0.002, 1482-fold higher in thymus p=0.002 compared to syngeneic recipients. Allogeneic and syngeneic recipients had equivalent numbers of donor CFSE-labeled lymphocytes in PBMC and lymph nodes. The tissue specific homing of CD4+ and CD8+ donor T-cells was also found significantly higher in most organs except the PBMC and LNs. Donor splenocytes were 80% CD62L+ before transplant, but the frequency of CD62L+ donor T-cells had declined to 15–16% in BM, 4–10% in liver, 17–30% in spleen and 10 to 25% in the thymus within 3.5 days post-transplant. In syngeneic recipients, 80% of donor T-cells remained CD62L+ within 3.5 days post-transplant. Most donor T-cells that divided rapidly lost expression of CD62L, while non-replicating donor CD4+ and CD8+ T cells remained predominately CD62L+. The expression of CD44 on donor T-cells were the opposite, with CD44+ cells undergoing less, and CD44− cells dividing more in allogeneic transplant recipients. In syngeneic BMT, donor CD4+ and CD8+ T-cells underwent minimal proliferation within the first 3.5 days post-transplant. Intracellular cytokine staining showed that high levels of IFN-g and TNF-a synthesis was seen among CD62L+ CD4+ and CD8+ T cells that had yet to divide (and had un-diluted CFSE staining). Conclusion: Migration of allogeneic donor T cells to tissues and local proliferation occurs rapidly after allogeneic BMT compared to recipients of syngeneic transplants. The dissociation of CD62L expression from lymph node homing suggests lack of the CD62L-receptor expression in lymph node HEV following irradiation, or a dominant effect of other chemokine receptors in directing donor T-cell preferentially to other organs. The marked and preferential homing of donor T-cells to the recipient thymus and bone marrow may play a role in achieving donor hematopoietic and T-cell chimerism in recipients of allogeneic BMT. Tissue specific homing of naive CD62L+ donor T-cells, with a high proliferative capacity, is likely responsible for the initiation of acute GvHD at these sites.

IL10 -Transduced Mesenchymal Stem Cells Improve the Acute Graft-Versus-Host Disease Protection in a Murine Model.

Recent research has suggested that mesenchymal stem cells(MSCs) have some immunosuppressive properties which may be of interest to lessen GVHD after transplant. Although encouraging, very limited systematic study has been performed to assess the ability of MSCs to suppress GVHD in vivo. In the early study, we failed to demonstrate murine MSCs attenuate the acute GVHD in an established murine model of acute GVHD (C57BL/6 mice → CB6F1 mice). Because of its anti-inflammatory properties and its association with dendritic cells and regulatory T cells, IL10 may has substantial benefit on the induction of immune tolerance and graft survival. In this study, we investigated whether the co-transplantation of genetically transduced MSCs expressing IL10 could improve the GVHD protection.Methods: 8–9 week old male CB6F1 mice (C57BL/6×Balb/c) were given 12Gy (100cGy/min) total body irradiation (6mv,X-ray). 4 hours after the lethal irradiation, the mice received one of four groups of donor mice cells (8–10 week old female C57BL/6 mice) via tail vein injection:1.1×107 bone marrow MNC+3×107 splenic MNC (n=10) (aGVHD inoculum, control);2.1×107 bone marrow MNC+3×107 splenic MNC+1×105 murine marrow-derived MSCs (n=15);3.1×107 bone marrow MNC+3×107 splenic MNC+1×105 IL10-transduced MSCs (transfected with IL10- recombinant adenoviral vector)(n=15);4.1×107 bone marrow MNC+3×107 splenic MNC+5×105 IL10-transduced MSCs (n=15).The weight, survival time, WBC count and chimerism of recipient mice were observed, the severity of aGVHD was assessed with both an aGVHD scoring system for physical signs and a semi-quantitative score for histologic examination. The serum of recipient mice was collected on the day 14, 28, 35,or the brink of death after transplant, then be used to detect the levels of cytokines (IL10,IL4,INF-γ and TNF-α) by ELISA.Results: The injection of 1×107 bone marrow MNCs plus 3×107 splenic MNCs from C57BL/6 donor into CB6F1 recipient following 12Gy irradiation resulted in a reliable model of aGVHD: the clinical signs and histologic degree was relatively coincident, the incidence of aGVHD were 100% and all mice died within +27d∼+33d. The co-transplant of 1×105 MSCs failed to decrease the incidence and severity of aGVHD significantly(81.8% vs 100%, score 6.0±2.83 vs 9.0±0.38, P>0.05).Compared with the group of 1×105 MSCs or control, 1×105 IL10-transduced MSCs demonstrated significantly reduced the incidence and severity of aGVHD(0%,score 0.5±0.05,P<0.01), while increased cell dose(5×105) failed to enhance the effect significantly. Serum levels of cytokines showed the significant increase of IL-10 (46.7±3.4 pg/ml vs 17.5 ±2.6 pg/ml, P<0.05), the corresponding increase of IL-4 (20.7±2.5 pg/ml vs 17.7±3.8 pg/ml, P=0.13),and more dramatic decrease of INF-γ (90.4±16.1 pg/ml vs 234.0 ±83.2 pg/ml, P<0.01) in the group of IL10-transduced MSCs.Conclusion: Benefical effects on aGVHD protection could be observed when MSCs were engineered to expressed IL10. The significant increase of Th2 type cytokines and decrease of INF-γ may be one of the mechanisms for the aGVHD protection in this murine model.

Suppression of Ewing's Sarcoma Tumor Growth, Tumor Vessel Formation, and Vasculogenesis Following Anti–Vascular Endothelial Growth Factor Receptor-2 Therapy

We previously showed that bone marrow cells participate in new tumor vessel formation in Ewing's sarcoma, and that vascular endothelial growth factor 165 (VEGF(165)) is critical to this process. The purpose of this study was to determine whether blocking VEGF receptor 2 (VEGFR-2) with DC101 antibody suppresses tumor growth, reduces tumor vessel formation, and inhibits the migration of bone marrow cells into the tumor. An H-2 MHC-mismatched bone marrow transplant Ewing's sarcoma mouse model was used. Bone marrow cells from CB6F1 (MHC H-2(b/d)) mice were injected into irradiated BALB/cAnN mice (MHC H-2(d)). TC71 Ewing's sarcoma cells were s.c. injected 4 weeks after the bone marrow transplantation. Mice were then treated i.p. with DC101 antibody or immunoglobulin G (control) twice a week for 3 weeks starting 3 days after tumor cell injection. DC101 antibody therapy significantly reduced tumor growth and tumor mean vessel density (P < 0.05) and increased tumor cell apoptosis. Decreased bone marrow cell migration into the tumor was also shown after DC101 therapy as assessed by the colocalization of H-2K(b) and CD31 using immunohistochemistry. DC101 inhibited the migration of both human and mouse vessel endothelial cells in vitro. These results indicated that blocking VEGFR-2 with DC101 antibodies may be a useful therapeutic approach for treating patients with Ewing's sarcoma.

Pharmacologic Levels of Dietary Arginine in CB6F1 Mice Increase Serum Ammonia in the Healthy State and Serum Nitrite in Endotoxemia

Therapeutic or pharmacologic doses of arginine are used to enhance blood flow and immune function despite the lack of dose-response studies and the potential for adverse effects. This study determined the optimal level of oral arginine supplementation required to elevate serum arginine concentrations yet limit adverse effects in healthy and endotoxemic mice. Male CB6F1 mice were fed one of the following diets: The standard AIN93G (3 g arginine/100 g of protein) or this diet modified to provide 10 g, 20 g, or 30 g arginine/100 g of protein. On day 14, mice were injected with lipopolysaccharide (endotoxemic) or saline (healthy) and 4 hours later were exsanguinated. Weight gain was reduced 50% in the group fed the 30 g arginine vs standard diet. Serum arginine, ornithine, citrulline, histidine, lysine, serine, threonine, tyrosine, and phenylalanine were greater and glutamate levels were lower in healthy supplemented mice; lipopolysaccharide treatment negated these changes. Serum ammonia concentration was 52% greater in healthy mice fed the 30 g arginine vs standard diet. Serum nitrite and urea were unaffected by supplementation in healthy mice. Serum nitrite was 37% greater in endotoxemic mice fed 30 g vs 10 g arginine, and serum urea was 27% greater in mice fed 20 g or 30 g vs 10 g arginine. Changes in serum arginine or its metabolites were observed with all of the modified diets; however, a 30-g arginine diet was associated with an initial impairment of growth and potential adverse effects.

226: Differential requirement for NKG2D in the rejection of haplomismatched and MHC-I deficient bone marrow

Mouse Natural Killer (NK) cells mediate rejection of MHC-mismatched or MHC-deficient bone marrow allografts. Counteracting activating and inhibitory signals regulate NK cell activity. The ability of F1 mice to reject parental bone marrow (BM) cells (hybrid resistance) has been attributed to the presence in the recipient of a subset of NK cells that lack inhibitory receptors for the MHC class I antigens expressed by the donor cells. Evidence supporting the “missing self” hypothesis of hybrid resistance was provided by demonstration that MHC class-I deficient donor BM is rejected by otherwise syngeneic recipients. We have previously demonstrated a role for NKG2D, an activating NK cell receptor, in rejection of Balb/c (H-2d) parental BM by (Balb/c x C57Bl/6; H-2b/d) F1 (CB6F1) recipient mice. NKG2D ligands are expressed on the regenerating myeloid BM cells in Balb/c and NOD mice but not substantially on the regenerating C57Bl/6 BM cells. Moreover, the rejection of Balb/c BM by CB6F1 recipients was blocked by a neutralizing anti-NKG2D monoclonal antibody. The purpose of this study was to determine whether NKG2D blockade was sufficient to prevent rejection of MHC-I deficient BM. Beta-2 microglobulin deficient (B2M-/-) Balb/c or NOD bone marrow was transplanted into irradiated CB6F1 mice that had been pretreated with neutralizing antibodies to NKG2D. In contrast to results with wild-type Balb/c or NOD BM whose rejection was prevented by anti-NKG2D, B2M-/- donor cells were rejected despite NKG2D blockade. Furthermore, syngeneic MHC class I+ Balb/c recipients were able to reject B2M-/- Balb/c donors despite NKG2D blockade. These results suggest that rejection of MHC class I-bearing BM requires activation via NKG2D, whereas MHC class I-deficient BM elicit a sufficiently strong NK response that augmentation by NKG2D signaling is not essential for the rejection. Therefore, the hybrid resistance model in which MHC class I-bearing BM are used for transplantation may better reflect the situation in human hematopoietic stem cell transplantation. Mouse Natural Killer (NK) cells mediate rejection of MHC-mismatched or MHC-deficient bone marrow allografts. Counteracting activating and inhibitory signals regulate NK cell activity. The ability of F1 mice to reject parental bone marrow (BM) cells (hybrid resistance) has been attributed to the presence in the recipient of a subset of NK cells that lack inhibitory receptors for the MHC class I antigens expressed by the donor cells. Evidence supporting the “missing self” hypothesis of hybrid resistance was provided by demonstration that MHC class-I deficient donor BM is rejected by otherwise syngeneic recipients. We have previously demonstrated a role for NKG2D, an activating NK cell receptor, in rejection of Balb/c (H-2d) parental BM by (Balb/c x C57Bl/6; H-2b/d) F1 (CB6F1) recipient mice. NKG2D ligands are expressed on the regenerating myeloid BM cells in Balb/c and NOD mice but not substantially on the regenerating C57Bl/6 BM cells. Moreover, the rejection of Balb/c BM by CB6F1 recipients was blocked by a neutralizing anti-NKG2D monoclonal antibody. The purpose of this study was to determine whether NKG2D blockade was sufficient to prevent rejection of MHC-I deficient BM. Beta-2 microglobulin deficient (B2M-/-) Balb/c or NOD bone marrow was transplanted into irradiated CB6F1 mice that had been pretreated with neutralizing antibodies to NKG2D. In contrast to results with wild-type Balb/c or NOD BM whose rejection was prevented by anti-NKG2D, B2M-/- donor cells were rejected despite NKG2D blockade. Furthermore, syngeneic MHC class I+ Balb/c recipients were able to reject B2M-/- Balb/c donors despite NKG2D blockade. These results suggest that rejection of MHC class I-bearing BM requires activation via NKG2D, whereas MHC class I-deficient BM elicit a sufficiently strong NK response that augmentation by NKG2D signaling is not essential for the rejection. Therefore, the hybrid resistance model in which MHC class I-bearing BM are used for transplantation may better reflect the situation in human hematopoietic stem cell transplantation.

Homeostatic Expansion of Donor FoxP3+ CD25+CD4+ T Cells in Recipients of CD4+ Cells Depleted Allogeneic Hematopoietic Graft.

Background: In allogeneic BMT patients, the presence of allo-reactive donor CD4+ T cells in the graft were reported to be the primary cause of GvHD. Moreover, donor T-cells are required to promote the stem cell engraftment and to decrease the disease relapse. A number of studies also reported that a subset of CD4+CD25+ T cells usually generated de novo from the thymus that expressed FoxP3 regulate the T cells allo-reactivity in vivo. Thus, to establish a therapeutically useful adoptive T-cells immunotherapy, we depleted the CD4+ T cells from the graft and transplanted along with T cell depleted (TCD) BM cells in clinically relevant parent to F1 experimental allogeneic BMT model. Our hypothesis is that CD4-depleted graft will not cause GvHD, preserve the thymic function, homeostatically produce donor BM-derived CD4+ T cells along with FoxP3+CD4+CD25+ regulatory T cells with beneficial anti-opportunistic infection and anti-tumor effects.Methods: We used a parent (C57BL/6) to (C57BL/6 X BALB/c)CB6F1 allogeneic BMT model with a combination of TCD BM and splenocytes as the hematopoietic graft. CD4+ or CD8+ cells were selectively depleted from the splenocytes of C57BL/6 donor mice using MACS column. 1×106 CD4-depleted splenocytes or a mixture of 2×106 CD8-depleted and 1×106 CD4-depleted splenocytes and/or grafts containing 10×106 unfractionated splenocytes along with 5×106 TCD BM cells harvested from the congeneic C57BL/6 donor mice, were adoptively transferred to lethally irradiated (11Gy) CB6F1 mice. GvHD was monitored twice weekly by weight loss and other clinical signs. After 50 days post transplant recipients mice were bled or sacrificed and lymphocytes isolated from blood and different organs were analyzed by multicolor FACS.Results: Within 50 days of transplant the recipients of CD4-depleted splenocytes had 100% survival without GvHD whereas recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes suffered from severe GvHD (%weight loss below 20%) with 50% survival. Surprisingly, very significantly expansion of total CD4+ T cells (37% ± 7% of lymphocytes, CD4:CD8 ratio 6:1) occurred in the blood of recipients of CD4-depleted splenocytes. In contrast the recipients of mixture of CD4- and CD8-depleted splenocytes DLI or whole splenocytes had only few CD4+ T cells (~2% ± 2% of lymphocytes, CD4:CD8 ratio 1:2). Over 90% of the CD4+ T cells in the blood of recipients of CD4-depleted splenocytes were from the donor BM and included significantly higher number of CD25+CD4+ T cells compared with the recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes. Similarly, significantly increased numbers of FoxP3+CD25+CD4+ regularity T cells were also found in the spleen and thymus of recipients of CD4-depleted splenocytes compared with the recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes (p<0.005).Conclusion: Adoptive immunotherapy with the CD4-depleted hematopoietic graft results better immune reconstitution, caused extensive homeostatic expansion of donor stem cell-derived CD4+ T cells including significantly increased levels of FoxP3+CD25+ CD4+ regulatory T cells derived from de novo thymopoiesis without GvHD. The presence of donor FoxP3+CD25+ CD4+ regulatory T cells in the hematopoietic graft are not necessary for post-transplant expansion of donor stem-cell-derived regulatory T-cells via thymopoiesis.

P-192: The viability of mouse oocytes in response to vitrification is dependent on the time of retrieval after hCG stimulation

P-192: The viability of mouse oocytes in response to vitrification is dependent on the time of retrieval after hCG stimulation

O-154: The effect of chelating cytosolic calcium on the viability of vitrified mouse oocytes

Vitrification is an alternative to cryopreserve MII oocytes, however, the embryonic potential of oocytes vitrified by choline chloride DMSO-EG-sucrose vitrification system is still low. Recently, it has been reported that DMSO and EG may induce the increase of intracellular calcium. This increase in calcium may have detrimental effects: premature cortical granule release, zona pellucida hardening, metaphase-to-anaphase transition, and/or egg activation. Preventing the increase of intracellular calcium by calcium-free media combined with a cell membrane permeable calcium chelator was carried out to assess the viability of mouse oocytes exposed to a choline chloride DMSO-EG-sucrose based vitrification system. Prospective study using mouse oocytes at a University-based research laboratory. MII oocytes were collected from 6-8 weeks old CB6F1 mice by hyperstimulation of 5IU PMSG and 5IU hCG. MII oocytes were denuded by brief exposure to 100iu/ml hyaluronidase, and then cultured in vitro less than 1hour prior to exposure of choline chloride DMSO-EG-sucrose vitrification system. Oocytes were loaded in pulled straw and heat-sealed just before plunging directly into liquid nitrogen. The experimental groups include presence or absence of calcium in the vitrification system, and concentration of BAPTA AM, a cell permeant calcium chelator. Group 1, calcium-based PBS; Group 2: calcium-free PBS; Groups 3 - 7: calcium-free PBS + varying concentrations (0.1μM, 0.5μM, 5μM, 50μM and 500μM) of BAPTA AM. After thawing, eggs were allowed to recover for one hour. Surviving oocytes were inseminated by capacitated sperm. Embryos were cultured in sequential Quinn’s Media (Sage) supplemented with 6% plasmanate, and monitored at day 1 and day 5(Table 1). Day 5 blastocysts were graded and corroborated by double staining of Oct 3/4 (detect functional inner cell mass) and of Hoechst 33342 (detect total embryonic cell number). Mouse oocytes, in response to 0.1μM BAPTA based vitrification system, display high rate of survival (97%) and cleavage (71%), comparable to that response to calcium based and calcium-free based vitrification system. However, raising the concentration of BAPTA to 0.5μM and above, the rate of survival (<82%) and cleavage (<55%) declined dramatically. Furthermore, more embryos in group of 0.1μM BAPTA develop into good (45% ≥3BB) and excellent (32% ≥4AA) blastocyst than in other groups. The group of 0.5μM BAPTA yielded less blastocysts, but still generated a number of good and excellent blastocysts comparable to the group of 0.1μM BAPTA. BAPTA influences the viability of mouse oocytes in a concentration dependant manner. BAPTA, at a concentration of 0.1μM (equivalent to cytosolic Ca2+ concentration -10-7 M = 0.1μM), exhibits the maximum benefit to improve the viability of vitrified oocytes. The effect of BAPTA on post-implantation embryos and full-term offspring needs to be addressed in further study.

Graft-versus-tumor effects on murine mammary carcinoma in a model of nonmyeloablative haploidentical stem cell transplantation

Despite a slight decrease in mortality over the last decade, breast cancer still remains a leading cause of cancer-related death in women. Although anti-tumor effects have been observed after allogeneic stem cell transplantation (SCT), this treatment is not standard care owing to graft-versus-host disease (GVHD) and scarcity of suitable donors. With the aim of reducing treatment-related mortality and increasing donor availability in clinical situations, we developed a preclinical mouse model that combines nonmyeloablative conditioning with the use of haploidentical donor-recipient pairs. To mimic active disease, CB6F1 mice were inoculated with 5 x 10(4) 4T1 mammary carcinoma cells 10 days before transplantation. Keratinocyte growth factor (KGF) was used as GVHD prophylaxis. Syngeneic (CB6F1) SCT did not cure any of the mice and KGF treatment did not influence tumor development. After transplantation with haploidentical (B6CBAF1) bone marrow and splenocytes, however, tumor outgrowth was reduced and long-term disease-free survival (>3 months) was observed in 9/18 (50%) (P=0.0011) of the animals. We conclude that in a model of murine breast cancer, a graft-versus-tumor effect can be induced by a nonmyeloablative haploidentical SCT procedure.

Evaluation of human immunodeficiency virus type 1 subtype C gag, pol, and gagpol DNA and alphavirus replicon vaccines

The worldwide HIV-1 vaccine research endeavor is focused increasingly on subtype C, which is now the predominant strain of the present HIV/AIDS epidemic. Expression cassettes of HIV-1 subtype C gag, pol and versions of gagpol fusion cassettes were constructed and evaluated for their relative abilities to induce cellular immune responses in mice. Animals were vaccinated with DNA or alphavirus replicon particle-based vaccines and cellular immune responses were measured by flow cytometry. Five new major histocompatibility complex (MHC) class I-restricted T cell epitopes in subtype C Gag and Pol were identified. Although two CD8 + T cell epitopes within Gag were immunodominant in BALB/c and CB6F1 mice, the overall breadth of the T cell responses in mice immunized with plasmids or recombinant alphavirus replicon particles encoding gagpol fusion genes was improved over single antigen genes (i.e. gag or pol alone). The patterns of epitope dominance were consistent among mice although there were variations observed between different animals in the relative contributions of the various epitopes to the total response. These data are consistent with observations in non-human primates (Otten GR, Schaefer M, Doe B, Liu H, Magede JZ, Donnelly J, et al. Potent immunogenicity of an HIV-1 gag-pol fusion DNA vaccine delivered by in vivo electroporation. Vaccine 2005, in press [1]) and support a subtype C in-frame gagpol fusion gene vaccine.

Antioxidants do not explain the disparate longevity between mice and the longest-living rodent, the naked mole-rat

The maximum lifespan of naked mole-rats (NMRs; Heterocephalus glaber) is greater than that of any other rodent. These hystricognaths survive in captivity >28 years, eight-times longer than similar-sized mice. The present study tested if NMRs possess superior antioxidant defenses compared to mice and if age-related interspecies changes in antioxidants were evident. Activities of Cu/Zn superoxide dismutase (Cu/Zn, SOD), Mn SOD, catalase and cellular glutathione peroxidase (cGPx) were measured in livers of physiologically equivalent age-matched NMRs (30, 75 and 130 months) and CB6F1 mice (4, 12 and 18 months). In mice, Mn SOD activity increased with age, while the activity of catalase and cGPx declined. None of the antioxidants changed with age in mole-rats. cGPx activity of NMRs was 70-times lower ( p < 0.0001) than in mice, and resembled that of cGPx knock-out animals. NMRs may partially compensate for the lower cGPx when compared to mice, by having moderately higher activities of the other antioxidants. It is nonetheless unlikely that antioxidant defenses are responsible for the eight-fold longevity difference between these two species. Maintenance of constant antioxidant defenses with age in NMRs concurs with previous physiological data, suggesting delayed aging in this species.