Cells In Peripheral Blood Leukocytes Research Articles

Orelabrutinib, a Selective Bruton's Tyrosine Kinase (BTK) Inhibitor in the Treatment of Primary Immune Thrombocytopenia (ITP)

Background:Primary immune thrombocytopenia (ITP) is the most common autoimmune hemorrhagic disorder characterized by decreased platelet count. increased risk of bleeding, and poor quality of life. Only about 70% patients response to first-line treatments, some patients are still refractory or relapsed after combined therapies, therefore it is necessary to explore new therapeutic targets. Bruton's tytosine kinase (BTK) is a non-receptor tyrosine kinase of Tec family, which is widely expressed in hematopoietic cells including B cells, monocytes/macrophages, and others. BTK participates in a variety of signaling pathways of innate and adaptive immunity, and plays an important role in cell survival and maturation. Platelet destruction mediated by anti-platelet glycoprotein antibodies is considered to be the main cause of ITP. B cells differentiate into plasma cells and produce autoantibodies due to the intolerance to autoantigens, which are important effectors in the pathogenesis of ITP. We speculated that inhibition of BTK may reduce platelet destruction by inhibiting B cell activation and autoantibody production. Orelabrutinib is a new generation of BTK inhibitor which has been used in hematological malignancies, this is the first study to explore the mechanisms of BTK inhibitor in the treatment of ITP.Methods:The concentrations of orelabrutinib were set at 1 nM, 10 nM, 100 nM and 1 μM in the in vitro study. Peripheral blood mononuclear cells (PBMCs) were isolated from active ITP patients and healthy controls and cultured for 72 hours, the apoptosis rate of PBMCs in each group was measured by Annexin V/PI double staining. CD19 + B cells of ITP patients were sorted by magnetic beads and stimulated with anti-human IgM to evaluate the activation of B-cell receptor (BCR) pathway and differentiation of plasma cells, respectively. Further, we transfused the splenocytes of immunized CD61-KO mice (C57BL/6) into the severe combined immunodeficient (SCID) mice to establish the active ITP murine models. Orelabrutinib was administered intragastrically at 10mg/kg, once a day. The control group was treated with 0.5% CMC at the same volume and frequency. Platelet count was measured weekly, the peripheral blood was collected and the B cell subsets in spleen were detected by flow cytometry at days 28 after splenocyte transfusion.Results:The proportion of early apoptotic cells (Annexin V +PI -) in PBMCs from both ITP patients and healthy controls was increased by orelabrutinib at 1μM,but there was no statistical difference. Orelabrutinib significantly inhibited the expression of CD69 in a dose-dependent manner at the concentrations of 10nM, 100nM and 1μM. Another early activation marker of BCR signaling pathway, CD86, was also found to be inhibited by orelabrutinib at 100nM and 1μM. The number of CD138 + plasma cells was decreased after treated with orelabrutinib at 10nM, 100nM and 1μM without dose-dependent manner. In the murine models, mice administered with orelabrutinib had significantly higher platelet count than the control mice at days 7, 14, 28 after splenocyte transfusion. The frequency of total B cells in peripheral blood leukocytes, the proportion of GL-7 + germinal center B cells and plasma cells in splenocytes were all determined to be lower in mice treated with orelabrutinib than the control group, though did not reach the statistical significance.Conculsion:Orelabrutinib could effectively suppress the activation and differentiation of B cells in vitro and in vivo, thus alleviate the thrombocytopenia in active ITP murine models. It could be the new treatment strategy for refractory/relapsed ITP patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immune gene expression in cyprinid herpesvirus-2 (CyHV-2)-sensitized peripheral blood leukocytes (PBLs) co-cultured with CyHV-2-infected goldfish fin cell line.

Goldfish is one of the preferred ornamental fish which is highly susceptible to cyprinid herpesvirus-2 (CyHV-2) infection. The present study aimed to analyse immune gene expression in a co-culture of CyHV-2-sensitized goldfish peripheral blood leukocytes (PBLs) with CyHV-2-infected fantail goldfish fin cell lines (FtGF). Goldfish were sensitized with intraperitoneal TCID50 dose (107.8±0.26/mL) of CyHV-2. After 2 weeks, PBLs were collected and co-cultured with CyHV-2-infected FtGF cells keeping both uninfected FtGF cells and PBL control groups. After 2 days of co-culture, WST-1 assay for cell proliferation was performed at 450 nm during the 2nd, 4th and 6th days of co-culture. The results showed a significant increase (p < 0.05) in cell density in CyHV-2-infected PBL and virus-infected FtGF cells during the 4th day post co-culture which confirmed effector cell generation. Expressions of few immune genes were checked taking RNA samples of CyHV-2-induced PBLs post co-culture with infected FtGF cells along with uninfected FtGF cells as control group at different time periods (2nd, 4th and 6th days) in triplicate. The results indicated increased expression of CD8α, IFNγ, b2m, MHC I, LMP 7, IL-10, IL-12 and GATA3 except Tapasin. From the above study, we concluded that goldfish showed both Th1- and Th2-mediated immune responses to CyHV-2. The current findings support the scope for further vaccine development against CyHV-2 for goldfish.

Assessment of Glycogen and Lipid Contents in Neutrophil Leukocytes of Peripheral Blood during Prenosological Diagnosis of Flight Personnel

The flight factors in highly maneuverable aircrafts lead to a significant and early progress of maladaptive shifts in pilot’s body as compared with non-flying professions. The aim of this study was to find out whether glycogen and lipid contents in segmented neutrophils of pilot peripheral blood depend on age and total flight hours, and to use findings for prenosological diagnostics of maladaptive shifts in body. The results of cytochemical studies of the content of glycogen and lipids in neutrophils from peripheral blood of pilots during their training flights are given. It has been established that a persistent increase in lipid content in neutrophils with an mean cytochemical coefficient higher than 2.60, along with a decrease in glycogen content in these cells at less than 2.00, indicates a decrease in the energy reserves of phagocytes, and peripheral blood leukocyte cell maladaptation. This condition develops as a response of the pilot’s body to the complex effect of flight factors on highly maneuverable aircraft with a total flight time of more than 1500 h and a pilot age of 36 years or more. The revealed patterns can be used in prenosological diagnostics of flight personnel.

Inhibition of CD83 Alleviates Systemic Inflammation in Herpes Simplex Virus Type 1-Induced Behçet’s Disease Model Mouse

Behçet's disease (BD) is an autoinflammatory disease that can lead to life- and sight-threating complications. Dendritic cells (DCs) are the most potent antigen-presenting cells that can regulate multiple inflammatory pathways. The objective of this study was to investigate the association of the DC stimulatory molecule CD83 with BD. Frequencies of costimulatory molecules expressing DCs in peripheral blood leukocytes (PBL) were measured by flow cytometry (FACS). The severity of symptoms in HSV-1-induced BD symptomatic mice was also assessed. Frequencies of CD83-positive cells were significantly increased in mice exhibiting BD symptoms, compared to those in asymptomatic mice. Abatacept, a CD80/86 blocker, significantly decreased the frequencies of CD83-positive cells in a time- and dose-dependent manner. BD symptomatic mice treated with Abatacept showed gradual reduction in the severity score of symptoms. Intraperitoneal injection of CD83 siRNA significantly reduced the frequencies of CD83-positive cells in PBL and peritoneal macrophages. After CD83 siRNA injection, BD symptoms of mice were improved and disease severity was decreased. Discontinuation of CD83 siRNA deteriorated symptoms while readministration of CD83 siRNA again improved BD symptoms of mice. These results clearly indicate the involvement of CD83-expressing cells in the inflammatory symptoms of BD. Therefore, CD83 might be useful as a therapeutic target for BD.

Macropinocytosis-dependent endocytosis of Japanese flounder IgM+ B cells and its regulation by CD22

B cells in fish are proven to be endocytic and have a great contribution to innate immunity like phagocytosis. In this study, the endocytic capacity and the corresponding internalization pathways of IgM+ B cells in Japanese flounder (Paralichthys olivaceus) were investigated. The results showed that IgM+ B cells in peripheral blood leukocytes (PBL) and splenic leukocytes (SL) exhibited different abilities to ingest 0.5 μm and 1 μm latex beads through macropinocytosis-dependent endocytic pathway. Japanese flounder CD22 (PoCD22) co-stimulatory signals were identified to be essential for the innate immune responses in B cells. Most of IgM+ B cells and some IgM− cells were demonstrated to be PoCD22 positive. When PoCD22 was blocked by antibody, the endocytic activities and reactive oxygen species (ROS) activities of SL IgM+ B cells were significantly increased, while the endocytic and ROS activities of PBL IgM+ B cells were significant decreased. These results collectively suggest that Japanese flounder IgM+ B cells are able to employ macropinocytosis-dependent endocytic pathway, which is under the regulation of CD22.

Characterization of UDP-Activated Purinergic Receptor P2Y6 Involved in Japanese Flounder Paralichthys olivaceus Innate Immunity

Uridine 5’-diphosphate (UDP)-activated purinergic receptor P2Y6 is a member of a G-protein-coupled purinergic receptor family that plays an important role in mammalian innate immunity. However, the role of the P2Y6 receptor (P2Y6R) in fish immunity has not been investigated. In this report, we characterized a P2Y6R gene from Japanese flounder (Paralichthys olivaceus) and examined its role in fish innate immunity. Sequence analysis reveals that the Japanese flounder P2Y6R protein is conserved and possesses four potential glycosylation sites. Quantitative real-time RT-PCR analysis shows that P2Y6R is broadly distributed in all examined Japanese flounder tissues with dominant expression in the liver. In addition, P2Y6R gene expression was up-regulated in head kidney macrophages (HKMs) upon lipopolysaccharides (LPS) and poly(I:C) stimulations but down-regulated by LPS challenge in peripheral blood leukocytes (PBLs). Furthermore, pharmacological inhibition of the endogenous P2Y6 receptor activity by the potently selective P2Y6R antagonist, MRS 2578, greatly up-regulated pro-inflammatory cytokine interleukin (IL)-1β, IL-6 and TNF-α gene expression in PBL cells treated with UDP. Moreover, LPS- and poly(I:C)-induced gene expression of IL-1β and TNF-α in Japanese flounder PBL cells was attenuated significantly by inhibition of P2Y6R activity with antagonist MRS 2578. Collectively, we, for the first time, showed the involvement of functional purinergic P2Y6R in fish innate immunity.

Frequencies of IL-15Rα+ cells in patients with Behçet’s disease and the effects of overexpressing IL-15Rα+ on disease symptoms in mice

It has been suggested higher serum levels of IL-15 and lower expression levels of IL-15 receptor alpha (IL-15Rα) are correlated with pathogenesis of Behçet's disease (BD). However, whether overexpressing IL-15Rα could be used as a therapeutic candidate for BD is currently unclear. Therefore, the purpose of this study was to determine whether overexpressing IL-15Rα could affect BD symptoms in a mouse model. IL-15/IL-15Rα complex expressing vector or protein complex of IL-15/IL-15Rα-Fc was used to treat BD mice. Frequencies of IL-15Rα+ cells in peripheral blood leukocytes (PBL) and lymph node cells were determined using a flow cytometer. BD symptoms in mice improved after treatment with IL-15/15Rα expression vector or IL-15/IL-15Rα-Fc protein complex. In addition, treatment with pIL-15/15Rα significantly (p = .016) decreased disease severity score of BD mice compared to treatment with control vector. Frequencies of IL-15Rα+ cells were also significantly (p = .01) higher in peritoneal macrophages of pIL-15/15Rα treated BD mice than those of mice treated with control vector. Frequencies of IL-15Rα+ PBL were also significantly higher in BD mice treated with IL-15/IL-15Rα-Fc protein complex than those in the control group. These results suggest up-regulating IL-15Rα+ cells could be used as novel therapeutic strategies to control BD in the future.

Characteristic patterns of HLA presentation and T cell differentiation in adult-onset Still's disease.

We examined the expression of human leukocyte antigen (HLA) and composition of differentiated T cells in the peripheral blood to understand the characteristics of the immune changes in patients with adult-onset Still’s disease (AOSD). This study enrolled patients with AOSD (n = 14), patients with rheumatoid arthritis (RA, n = 20), and healthy controls (HC, n = 20). The percentage of surface-stained cells with HLA-DP, DQ, and DR alleles and the composition of differentiated T cells in peripheral blood leukocytes (PBLs) were evaluated by flow cytometry. AOSD patients exhibited significantly higher percentages of lymphocytes presenting HLA-DP and HLA-DR, and lower percentages of cells presenting HLA-DQ, than RA patients or HC. The proportions of CD4+, CD4+CCR7+, CD4+CD62L–, and CD8+CD62L– cells from PBLs were decreased in AOSD patients relative to RA patients or HCs. By contrast, AOSD patients had higher proportions of CD8+naïve T cells in whole blood relative to RA patients or HC. The proportions of CD4+ effector memory T cells, CD8+ naïve T cells, and CD8+ effector memory T cells in whole blood cells and CD4+ effector memory T cell in lymphocytes were significantly associated with the systemic score. While the proportions of CD4+, CD8+, CCR7+, CD4+CCR7+, CD4+CD62L–, and CD8+CD62L– cells were significantly decreased in AOSD patients, and the proportion of CD8+naïve T cells was elevated in AOSD and correlated with the systemic score. Further studies of a large cohort of AOSD patients will be necessary to evaluate these markers in the pathogenesis of AOSD.

Immune responses of flounder Paralichthys olivaceus vaccinated by immersion of formalin-inactivated Edwardsiella tarda following hyperosmotic treatment.

The aim of the present study was to evaluate the effects of hyperosmotic immersion (HI) vaccination and determine the optimum hyperosmotic salinity for flounder Paralichthys olivaceus by investigating its immune responses following vaccination. Flounder were immersed in 1 of 3 hyperosmotic solutions at 50, 60 and 70‰ salinity, then transferred into 30‰ salinity normal seawater containing formalin-inactivated Edwardsiella tarda for vaccination (3 HI groups), or were immersed in normal seawater as direct immersion (DI group). The results showed that the percentages of surface membrane immunoglobulin-positive (sIg+) cells in peripheral blood leukocytes and spleen leukocytes induced by HI were significantly higher than that with DI (p < 0.05), and the 50‰ salinity group showed the strongest response among the HI groups, which reached peaks at Week 4. ELISA assay showed that the specific serum antibodies gradually increased after vaccination and reached peak at Day 32, and the fish treated with HI showed stronger antibody responses; among the HI groups, a significantly higher specific antibody level was detected in the 50‰ salinity group at Day 32 (p < 0.05). Similarly, the fish treated with HI showed higher specific mucosal antibody levels compared to the DI group, and the mucosal antibody showed a faster response, with peak time arriving 1 wk earlier than for the serum antibody. The relative percent survival (RPS) of flounder treated with HI at 50, 60 and 70‰ salinities were 79, 71 and 57% respectively, while this was 43% in the DI group. These results demonstrated that HI, especially the 50‰ salinity, could efficiently enhance the immune response of flounder and show higher RPS. This has significant value for immunological prevention of edwardsiellosis in flounder.

Cytomegalovirus in Bone Marrow Cells Correlates with Cytomegalovirus in Peripheral Blood Leukocytes

In allogeneic hematopoietic stem cell transplant recipients with bone marrow (BM) suppression, cytomegalovirus (CMV) pp65 antigenemia and DNA were detectable in peripheral blood leukocytes (PBL) and BM cells. A relationship between CMV infection of PBL and BM cells has been found.

FTY720 Markedly Increases Alloengraftment but Does Not Eliminate Host Anti-Donor T Cells that Cause Graft Rejection on Its Withdrawal

The immunomodulator FTY720 (FTY) is beneficial in models of graft-versus-host disease, solid organ transplantation, and autoimmunity and has been approved for use in patients with multiple sclerosis. FTY modifies the homing and migration of many cell types. We report that FTY has profound positive and negative effects on allogeneic bone marrow (BM) engraftment in sublethally irradiated recipients. FTY increased donor hematopoietic progenitors in the BM, resulting in high donor engraftment in the B cell, myeloid cell, and natural killer cell, but not T cell, lineages. Donor T cell progenitors within the thymus of FTY-treated recipients were dramatically reduced, resulting in a lack of donor T cell reconstitution. In addition to preventing the ingress of donor (and host) T cell progenitors, FTY prevented the egress of fully functional host CD4+CD8- and CD4-CD8+ thymocytes that on cessation of FTY administration were able to exit from the thymus and contribute to a rapid and complete rejection of a well-established donor BM graft. When used in combination with anti-CD40L mAbs to block the CD40L:CD40 costimulatory pathway, FTY markedly enhanced anti-CD40L mAb-mediated alloengraftment promotion. In contrast to FTY alone, the combination of anti-CD40L mAb and FTY resulted in a surprisingly stable, multilineage, long-term donor chimerism. These data illustrate FTY's profound migration modulating effects and suggest a use in combinatorial therapy in achieving stable alloengraftment under nonmyeloablative conditions.

Peripheral blood lymphocytes from low-grade squamous intraepithelial lesions patients recognize vaccine antigens in the presence of activated dendritic cells, and produced high levels of CD8 + IFNγ + T cells and low levels of IL-2 when induced to proliferate

BackgroundMost infections with human papillomavirus (HPV) are resolved without clinical intervention, but a minority evolves into chronic lesions of distinct grades, including cervical-uterine cancer. It is known that in most cases the immune system mediates elimination of HPV infection. However, the mechanism of immune evasion leading to HPV persistence and development of early cervical lesions is not fully understood. The aim of the present work was to evaluate the potential of peripheral blood leukocytes (PBL) from low-grade squamous intraepithelial lesions (LSIL) patients to be activated ex-vivo by vaccine antigens, the participation of cytotoxic lymphocytes and regulatory T cells, and to determine the secretion of Th1 and Th2 cytokines mediated by stimulation of T cell receptors.ResultsWe found that PBL from LSIL patients showed a significantly lower proliferation rate to vaccine antigens as compared to that of healthy donors, even though there was not a difference in the presence of antibodies to those antigens in sera from both groups. We did not find differences in either the frequency of CD4 + CD25 + FoxP3+ in PBL, or the levels of IL-4, IL-5 and IL-10 in plasma or conditioned media from PBL incubated with TcR agonists in vitro, between the two groups. However, we detected a lower production of IL-2 and a higher proportion of CD8 + IFNγ + cells in PBL from LSIL patients as compared with PBL from normal donors. We also observed that PBL from patients infected by HPV-16 and −18 were not able to proliferate in the presence of soluble HPV antigens added to the culture; however, a high level of proliferation was attained when these antigens were presented by activated dendritic cells.ConclusionsOur results suggest that the immunodeficiency reported in LSIL patients could be due to the inability of specific cytotoxic T lymphocytes that for some unknown reason are present but unable to mount a response when challenged with their antigens, probably related to an in situ IL-2 production deficiency.

Sex differences in response to chronic administration of Tacrolimus in spontaneously hypertensive rats

Hypertension in male spontaneously hypertensive rats (SHR) is thought to be mediated in part by enhanced immune function. Tacrolimus (FK‐506) is a potent immunosuppressive used to decrease occurrence of allograft rejection. The present study tested the hypothesis that there are sex differences in response to tacrolimus in SHR. Young male (YM, age 3 mos; n= 6) and young female (YF, aged 3 mos; n= 4–5) SHR received tacrolimus (0.25 mg/kg/day i.p.) for 14 d. Rats were implanted with radiotelemeters and mean arterial pressure (MAP) was measured chronically. Tacrolimus increased MAP in males (Control: 143 ±3 mmHg vs. Tacrolimus‐treated: 163 ±4 mmHg, p=S), but had no effect in females (Control: 132 ±3 mmHg vs. Tacrolimus‐treated: 133 ±2 mmHg). After the experiment, blood was taken and the numbers of CD4+ and CD8+ T cells in peripheral blood leukocytes (PBL) were measured by flow cytometry. The number of CD4+ T cells decreased by similar percentage in females (Control: 42 ±7 vs. Treated: 30 ±4% gated) and males (Control: 49 ±2 vs. Treated: 32 ±3% gated) treated with tacrolimus. However, tacrolimus treatment decreased the number of CD8+ by 72% in females (Control: 18 ±6 vs. Treated: 5 ±2% gated) and 40% in males (Control: 33 ±2 vs. Treated: 20 ±1% gated). The data show that in spite of similar reductions in CD4+ cells and greater reduction in CD8+ T cells, tacrolimus had no effect on MAP in females. In males, tacrolimus increased MAP despite decline in CD4+ and CD8+ T cells. Thus the contribution of CD4+ and CD8+ T cells in mediating the hypertension in male and female SHR is not clear.

Presence of Bcr-Abl Positive Cells in Peripheral Blood Leukocytes of Immunosuppressed Solid Organ Transplant (SOT) Recipients

In chronic myeloid leukemia (CML) the p210 BCR-ABL protein, generated by a t(9;22)(q34;q11) translocation, is the underlying mechanism of leukemogenesis. Although the presence of p210 BCR-ABL is normally restricted to CML patients (pts), previous reports demonstrated low levels of bcr-abl transcripts in healthy individuals that may be controlled by an intact immune system (Bose et al. Blood 92:3362, 1998). Interestingly, several articles reported cases of CML occurring in pts after SOT (Pelloso et al. Leukemia Res 29:353, 2005). Therefore, immunosuppressed SOT recipients should represent an optimal population to investigate the frequency of bcr-abl transcripts in non-leukemic individuals in order to address a potential impact of immunosuppression on the presence of bcr-abl transcripts. This possibility was investigated by studying peripheral blood leukocytes for the presence of bcr-abl transcripts in a total of 201 individuals of whom 100 were SOT recipients (n=50 kidney, n=46 liver, n=3 heart, n=1 heart-lung) and 101 were control individuals (n=87 patients with renal failure, n=14 healthy individuals). The male to female ratio in the study group was 54:46 (median age: 55.38 years, range: 22–83) and matched the control group (median age: 59.8 years, range: 32–96). Included in the study group were 13 pts with post transplant malignancies who were previously treated with standard chemotherapeutic regimens. Immune suppressive drugs given to all SOT recipients included steroids (90%), cyclosporine A (69%), azathioprine (22%), tacrolimus (69%), sirolimus (44%), mycophenolic acid (63%) and others (37%). For the detection of the bcr-abl transcript we used a nested reverse transcriptase-polymerase chain reaction (RT-PCR) assay that is routinely used in our institution. All samples were tested at least twice. In 5/100 immunosuppressed pts at least 1 of 2 RT-PCR products was bcr-abl positive in the second round amplification. This rate significantly exceeded the control group that was completely bcr-abl negative (0/101 p = 0.0242). Of the 5 bcr-abl positive pts, 3 were liver transplant and 2 were kidney transplant recipients. The latency in this group (interval between transplantation and bcr-abl PCR) was at 58.4 months (range: 24-135 months) and shorter when compared to the latency in the remaining study group (104.8 months; range 1 – 369). Additionally, all samples were tested for both the pml-rara and aml1-eto transcripts that are detectable in subsets of pts with acute myeloid leukaemia but none of the samples tested positive for these transcripts. Our findings are extended by three case reports of SOT recipients (2 kidney, 1 liver) who developed CML in a total of 2088 transplantations in our centre in 9 years. In these three pts (2 males, 1 female), CML occurred at 84.3 months (range: 32–183) following SOT. In summary our data show: 1. The presence of bcr-abl transcripts in immunosuppressed non-leukemic SOT pts and the absence of bcr-abl transcripts in healthy normals when tested with routine PCR protocols. 2. The absence of pml-rara and aml1-eto transcripts in both the study and control group. And 3. the occurrence of CML in three patients following SOT suggesting a higher frequency of CML in this population.

Upregulation of carp GDNF mRNA by the immunomodulator AS101

Although the glia derived neurotrophic factor (GDNF) is defined as a molecule that maintains neuronal cells, it possesses a range of functions outside the nervous system. For example, it is essential for uretric branching in kidney morphogenesis and for regulating the differentiation of stem cells during spermatogenesis, cardiac, hair follicle and vascular differentiation and the maintenance of immune cells. In the present work, the presence of GDNF in carp peripheral blood leukocytes (PBL) and head kidney cells (HK) was evidenced and its evolutionary importance in both neural and immune systems development was suggested. Using the northern-blot technique, we could observe the expression of two different transcripts of this gene. GDNF upregulation was detected using semi-quantitative PCR, following ex vivo treatment of PBL and HK cells with the immunomodulator AS101 which was previously shown to inhibit IL-10 and to up-regulate GDNF protein levels in human SVG astrocyte cell line, in 6-OHDA hemi-parkinsonian mice in vivo and in rat glomerular mesengial cells in vitro.

CTLA-4 blockade augments human T lymphocyte-mediated suppression of lung tumor xenografts in SCID mice.

Previous studies by others using transplantable murine tumor models have demonstrated that the administration of antibodies that block CTLA-4 interaction with B7 can provoke the elimination of established tumors, and that the tumor suppression is mediated by T-cells and/or cells expressing NK1.1. Studies from our lab have established in a human/severe combined immunodeficient (SCID) mouse chimeric model that autologous peripheral blood leukocytes (PBL) can suppress the growth of tumor xenografts in a PBL dose-dependent fashion, and that this suppression is dependent upon the patient's T and NK cells. Using this human/mouse chimeric model, we sought to determine whether an antibody blockade of CTLA-4 would enhance the anti-tumor response of a patient's PBL. It was first important to determine whether the tumor suppression observed in the SCID model was dependent upon CD28/B7 co-stimulation. Blockade of B7 with a human CTLA-4-Ig fusion protein completely abrogated the lymphocyte-mediated tumor suppression, confirming in this model that tumor suppression is dependent upon a CD28/B7 co-stimulation. Using two different CTLA-4 specific monoclonal antibodies, we observed that CTLA-4 blockade significantly enhanced the human lymphocyte-mediated tumor suppression in mice co-engrafted with PBL and tumor cells. This enhancement was observed in both an allogeneic setting (in which the PBL were allogeneic with respect to the tumor) and an autologous setting (in which the PBL and tumor were from the same patient). These results sustain the notion that human anti-tumor immune response can be augmented (in vivo) by blocking the interaction between CTLA-4 and B7.

In Vivo Bone Marrow Stem Cell Gene Transfer in Mice by In Situ Delivery of a 3rd-Generation Lentiviral Vector Using Intrafemoral Injection.

HIV-based lentiviral vectors (LV) have become powerful tools for in vivo gene transfer and gene expression in non-dividing cells after local injection. However, the potential of in vivo bone marrow stem cell gene transfer by “in situ” bone cavity injection has not been well unexplored. This in vivo approach could take full advantage of any source of stem cells present in bone cavity and avoid many of the difficulties encountered by ex vivo hematopoietic stem cell (HSC) gene transfer. To determine if intra bone marrow (iBM) injection of LV can efficiently transduce BM stem cells, a 3rd-generation self-inactivating LV, containing eGFP regulated transcriptionally by a CMV promoter, was used to inject intra-femorally into adult Boy/J Ly45.1 mice (at 2 x106 IU/mouse). Blood counts were normal in all LV-injected mice (n=4) and buffer-injected mice (n=4). GFP-expressing peripheral blood leukocytes (PBL) were observed in both myeloid (up to 4%) and lymphoid subpopulations (up to 2%) 3-month post injection. To evaluate gene transfer and transgene expression in multi-potential progenitors, the colony-forming cell (CFC) assay was performed using low-density bone marrow 4-months post injection, resulting in 4–5% GFP+ CFU-granulocyte macrophage (CFU-GM) and CFU-granulocyte, erythroid, macrophage and megakaryocyte (CFU-GEMM). This was consistent with the 4-color FACS analysis data demonstrating that up to 3.7% of HSC/progenitors (Lin−c-kit+Sca1+ cells) from LV-injected mice expressed GFP transgene 4-months post injection. To further evaluate HSC gene transfer, BM from injected mice was transplanted into lethally irradiated adult C57/Bl6 (Ly45.2) mice. Four months later, successful engraftment was demonstrated in all BM recipients with 97–99% donor-derived cells in PBL and 91–93.3% in BM. Moreover, significant levels of GFP+ CFU were observed in all recipients ranging from 8.4 ± 2.3% to 17.7 ± 4.2%. To further study the LV-mediated gene transfer profile in HSC, mesenchymal progenitors and systemic distribution by intra-femoral injection, molecular analyses are in progress including real-time QPCR and clonality analysis by LAM-PCR. Our results indicate successful LV-mediated gene transfer into HSC by iBM injection. These data would potentially open a door to a novel approach for treatment of human diseases, but also provide a new tool to study adult stem cell plasticity and the nature of hematopoiesis.

Genetic Line Effect on Peripheral Blood Leukocyte Cell Surface Marker Expression in Chickens

To determine the role of genetics in baseline lymphocyte parameters, several distinct lines of chickens were examined for differences in peripheral blood leukocyte (PBL) populations. Four highly inbred chicken lines (MHC congenic Fayoumi lines M15.2 and M5.1, and MHC congenic Leghorn lines G-B1 and G-B2), two advanced intercrosses [F5 (Broiler × G-B2) and F5 (Broiler × M15.2)], and an outbred population of broilers were used. Leukocytes isolated from healthy adult birds were labeled with monoclonal antibodies: chCD3, chCD4, chCD8, chBu-1, and hCD14. Flow cytometry was used to determine the total percentage of positively labeled cells for each surface marker in a sample, as well as the mean fluorescent intensity, or surface marker density, of a labeled subset. Significant line differences for percentage positive CD3 T cells and the ratio of B cells:T cells (represented by the Bu-1:CD3 ratio) were found. The effect of line was also significant for CD3 and CD8 T cell receptor density. Effects of sex and MHC on PBL cell surface marker expression were not significant in the lines examined. This study demonstrates the effect of genetic line on resting leukocyte composition of peripheral blood in the chicken lines examined. Observed PBL differences add to our growing knowledge of the varied roles that immune system status (defined by specific cell populations) and genetic background have in determining susceptibility and disease progression in chickens.

630. Tamoxifen-Inducible Expansion of Murine Hematopoietic Stem Cells by Retroviral-Mediated Transfer of a HOXB4-ERT2 Fusion Protein

Top of pageAbstract Overexpression of the homeobox transcription factor HOXB4 can increase self-renewal divisions in hematopoietic stem cells (HSCs). As a result, HOXB4 expression is being explored as a gene therapy strategy for increasing the number of genetically modified HSCs. While vectors expressing HOXB4 have not led to any cases of hematopoietic malignancy in transplanted mice, several other HOX family members do cause transformation in this context. An additional concern is that HOXB4 expression can interfere with hematopoietic differentiation in some systems. Therefore, to circumvent any potential adverse effects associated with exogenous HOXB4 expression, we developed an inducible HOXB4 system (HOXB4-ERT2) consisting of a mutated ligand binding domain of the estrogen receptor fused to the carboxyl terminus of HOXB4 via a 10 amino acid linker. Secondary replating assays were used to evaluate the self-renewal capacity of myeloid progenitor cells, and showed that cells transduced with the HOXB4-ERT2 vector generated 3, 5 and 8 fold more secondary colonies than GFP transduced cells in the presence of 100, 200 and 300 nM tamoxifen (TAM) respectively. We next examined whether HOXB4-ERT2 transduced HSCs could be expanded in vivo by treating transplanted mice with TAM. Murine bone marrow (BM) cells expressing the CD45.1 leukocyte antigen were transduced with MSCV-based vectors expressing either GFP, HOXB4 or HOXB4-ERT2. Recipient mice (CD45.2) were lethally irradiated and then transplanted with 2.5105 CD45.1 BM cells, 10% of which were GFP positive, together with 1105 fresh CD45.2 BM cells, giving a total of 7% GFP positive cells in each graft. For the HOXB4-ERT2 group, half of the recipients received TAM treatment daily for 3 weeks starting immediately after the transplant, while the other half received no treatment. Secondary transplants were performed at week 21. The percentages of GFP positive cells in peripheral blood leukocytes (PBLs) are summarized in the table. Our results showed that HSCs transduced with the HOXB4-ERT2 vector expanded in vivo with TAM treatment. Comparing TAM treated with untreated mice, GFP positive cells in PBLs increased 7 and 27 fold in primary and secondary recipients respectively. Hematological parameters were within normal range in all transplanted animals. In summary, these results show that HOXB4 function can be controlled both in vivo and in vitro using our HOXB4-ERT2 system. This may allow for increased safety by allowing inactivation of HOXB4 function after HSC expansion has occurred. In addition, we are using this system to study the kinetics of the HOXB4 effect by inducing HOXB4 function at different time points post-transplant and early results indicated that there was a restricted time frame for HOXB4-induced expansion.

In vitro transmission of HCMV between fibroblasts and peripheral blood leukocytes in the presence of IL-4.

We demonstrated transmission of human cytomegalovirus (HCMV) from the human lung fibroblast MRC-5 to peripheral blood leukocytes (PBLs). mRNA of the HCMV immediately-early (IE) antigen was detected in PBLs cultured with IL-2 or IL-2 + IL-4 that made direct contact with HCMV-infected MRC-5, whereas it was not detected in PBLs prevented from making cell-to-cell contact. However, mRNA of HCMV IE was not detected in PBLs cultured with IL-2 and IFN-gamma that made direct contact with HCMV-infected MRC-5. Transmission of the pp65 antigen was increased in culture medium containing IL-4. At a higher viral infection titer, cell-free HCMV infected adherent PBLs cells. The subset, which did not adhere, did not infect cell-free viruses even at a very high multiplicity of infection. Moreover, the adhered subset of PBLs infected with HCMV was able to transmit HCMV to non-infected fibroblasts. Our results suggest that cell-to-cell contact (when PBLs make direct contact with HCMV-infected cells) is important in the mechanism of HCMV transmission and that the adherent cells of PBLs are one of the most important vehicles for HCMV infection. Moreover, we suggest that type 2 cytokines such as IL-4 enhance the transmission of HCMV to PBLs.