Highly Reproducible Engraftment of Chronic Lymphocytic Leukemia (B-CLL) Cells in NOD/SCID Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 52-52 ◽  
Author(s):  
Peter Ebeling ◽  
Jan Duerig ◽  
Florian Grabellus ◽  
Ulrich Duehrsen ◽  
Siegfried Seeber ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cell Clone ◽  
Cell Types ◽  
Scid Mice ◽  
Lymphocytic Leukemia ◽  
In Vivo Model ◽  
Acute Leukemias ◽  
Human T Cells

Abstract In contrast to normal hematopoiesis and acute leukemias, research in CLL still is hampered by the lack of a reliable in vivo model for primary B-CLL. We here report highly reproducible engraftment of B-CLL cells, when 1x10^8 MNC derived from the peripheral blood of CLL patients were transplanted i.v and i.p. into NOD/SCID mice. So far, 14 different CLL samples were investigated in 41 mice. At weeks 4, 8 or 12 mice were sacrificed and bone marrow (BM), spleen, and peritoneal fluid (PF) were analyzed by FACS for human CD19/CD5/CD23/CD45 (B-CLL) cells and CD45/CD3/CD5 (T) cells. Additionally, HE- and immunostaining was performed on spleen sections. Analysis at week 4 revealed engraftment in NOD/SCID mice for 13/14 samples (spleen: 13/14, BM: 4/14, PF: 12/14). B-CLL cells were observed predominantly in the spleen (8.9±2.4% or 9.1±4.4x10^5 cells) and PF (19.0±4.4% or 3.4±1.8x10^5 cells) with much lower engraftment in BM (0.6±0.3% or 0.1±0.1x10^5 cells). Detection of B-CLL cells in peripheral blood could be obtained in 3/14 experiments. Also substantial engraftment of human T-cells was observed in 13/14 experiments (spleen: 13/14, BM: 8/14, PF: 11/14). T-cells engraftment was highest in the spleen (23.8±9.8% or 28.7±13.1x10^5 cells) and somewhat lower in PF (16.4±8.2% or 3.0±1.6x10^5 cells) and BM (7.3±3.8% or 2.9±1.1x10^5 cells). Subpopulation analysis revealed a CD4+ phenotype in 65, 59 and 72 % of T-cells within spleen, PF and BM, respectively. Noteworthy, immunohistological analysis of HE stained spleen sections of engrafted animals revealed a pseudofollicular infiltration with human CD45LCA+ cells along splenic arterioles. Within these pseudofollicles human B-CLL but also CD3+ T-cells were detected. Contribution of B-CLL and T-cells to individual follicles was highly variable ranging from 5–95% for both cell types. When engraftment was analysed separately for the i.p and the i.v. route, engraftment of transplanted cells in PF seemed to be depended on the i.p. route whereas splenic engraftment was obtained following i.v. as well as i.p. injection. Sustained B-CLL engraftment was seen after 8 weeks (spleen: 3.1±1.4% or 7.3±3.1x10^5 total cells; PF: 57.6±23.3% or 1.0±0.5x10^5 cells; n=3 mice) and 12 weeks (spleen: 1.4±1.3% or 0.3±0.3x10^5 cells; PF: 10.2±7.3% or 0.5±0.5x10^5 cells; n=2 mice). Thus, we have shown efficient engraftment of human B-CLL cells in the spleen and PF of NOD/SCID mice. This in vivo model should significantly help to understand B-CLL biology and to test novel therapeutic approaches. The observed pseudofolicular pattern of splenic infiltration supports the theory of T-cells creating a “microenvironment” sustaining the growth of the leukemic B cell clone.

scholarly journals Severe Combined Immunodeficiency Mice Engrafted With Human T Cells, B Cells, and Myeloid Cells After Transplantation With Human Fetal Bone Marrow or Liver Cells and Implanted With Human Fetal Thymus: A Model for Studying Human Gene Therapy

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1800-1810 ◽  
Author(s):  
Sergey Yurasov ◽  
Tobias R. Kollmann ◽  
Ana Kim ◽  
Christina A. Raker ◽  
Moshe Hachamovitch ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
T Cells ◽  
Peripheral Blood ◽  
Severe Combined Immunodeficiency ◽  
Precursor Cells ◽  
Scid Mice ◽  
Combined Immunodeficiency ◽  
Human T Cells

To develop an in vivo model wherein human hematopoiesis occurs, we transplanted severe combined immunodeficiency (SCID) mice with either human fetal bone marrow (HFBM) or human fetal liver (HFL). After transplantation of SCID mice with cultured HFBM (BM-SCID-hu mice) or HFL cells (Liv-SCID-hu mice), significant engraftment of the mouse bone marrow (BM) and population of the peripheral blood with human leukocytes was detected. Human colony-forming unit–granulocyte macrophage and burst forming unit-erythroid were detected in the BM of the BM-SCID-hu and Liv-SCID-hu mice up to 8 months after transplantation. When the HFBM or HFL cells were transduced with a retroviral vector before transplantation, integrated retroviral sequences were detected in human precursor cells present in the SCID mouse BM and in leukocytes circulating in the peripheral blood (PB) up to 7 months after transplantation. The PB of the BM-SCID-hu mice also became populated with human T cells after implantation with human thymic tissue, which provided a human microenvironment wherein human pre-T cells from the BM could mature. When the HFBM was retrovirally transduced before transplantation, integrated retrovirus was detected in sorted CD4+CD8+ double positive and CD4+ single positive cells from the thymic implant and CD4+ cells from the PB. Taken together, these data indicated that the BM of our BM-SCID-hu and Liv-SCID-hu mice became engrafted with retrovirally transduced human hematopoietic precursors that undergo the normal human hematopoietic program and populate the mouse PB with human cells containing integrated retroviral sequences. In addition to being a model for studying in vivo human hematopoiesis, these mice should also prove to be a useful model for investigating in vivo gene therapy using human stem/precursor cells.

scholarly journals Severe Combined Immunodeficiency Mice Engrafted With Human T Cells, B Cells, and Myeloid Cells After Transplantation With Human Fetal Bone Marrow or Liver Cells and Implanted With Human Fetal Thymus: A Model for Studying Human Gene Therapy

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1800-1810 ◽  
Author(s):  
Sergey Yurasov ◽  
Tobias R. Kollmann ◽  
Ana Kim ◽  
Christina A. Raker ◽  
Moshe Hachamovitch ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
T Cells ◽  
Peripheral Blood ◽  
Severe Combined Immunodeficiency ◽  
Precursor Cells ◽  
Scid Mice ◽  
Combined Immunodeficiency ◽  
Human T Cells

Abstract To develop an in vivo model wherein human hematopoiesis occurs, we transplanted severe combined immunodeficiency (SCID) mice with either human fetal bone marrow (HFBM) or human fetal liver (HFL). After transplantation of SCID mice with cultured HFBM (BM-SCID-hu mice) or HFL cells (Liv-SCID-hu mice), significant engraftment of the mouse bone marrow (BM) and population of the peripheral blood with human leukocytes was detected. Human colony-forming unit–granulocyte macrophage and burst forming unit-erythroid were detected in the BM of the BM-SCID-hu and Liv-SCID-hu mice up to 8 months after transplantation. When the HFBM or HFL cells were transduced with a retroviral vector before transplantation, integrated retroviral sequences were detected in human precursor cells present in the SCID mouse BM and in leukocytes circulating in the peripheral blood (PB) up to 7 months after transplantation. The PB of the BM-SCID-hu mice also became populated with human T cells after implantation with human thymic tissue, which provided a human microenvironment wherein human pre-T cells from the BM could mature. When the HFBM was retrovirally transduced before transplantation, integrated retrovirus was detected in sorted CD4+CD8+ double positive and CD4+ single positive cells from the thymic implant and CD4+ cells from the PB. Taken together, these data indicated that the BM of our BM-SCID-hu and Liv-SCID-hu mice became engrafted with retrovirally transduced human hematopoietic precursors that undergo the normal human hematopoietic program and populate the mouse PB with human cells containing integrated retroviral sequences. In addition to being a model for studying in vivo human hematopoiesis, these mice should also prove to be a useful model for investigating in vivo gene therapy using human stem/precursor cells.

Generation of primary antigen-specific human cytotoxic T lymphocytes in human/mouse radiation chimera

Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 721-730 ◽  
Author(s):  
H Segall ◽  
I Lubin ◽  
H Marcus ◽  
A Canaan ◽  
Y Reisner
Keyword(s):  
T Cells ◽  
T Cell ◽  
Human Lymphocytes ◽  
Scid Mice ◽  
Human Antibody ◽  
Adoptive Therapy ◽  
Activation Markers ◽  
Human T Cell ◽  
Human T Cells

Severe combined immunodeficient (SCID) mice are increasingly used as hosts for the adoptive transfer of human lymphocytes. Human antibody responses can be obtained in these xenogeneic chimeras, but information about the functionality of the human T cells in SCID mice is limited and controversial. Studies using human peripheral blood lymphocytes (PBL) injected intraperitoneally (IP) into SCID mice (hu-PBL-SCID mice) have shown that human T cells from these chimeras are anergic and have a defective signaling via the T-cell receptor. In addition, their antigenic repertoire is limited to xenoreactive clones. In the present study, we tested the functionality of human T cell in a recently described chimeric model. In this system, BALB/c mice are conditioned by irradiation and then transplanted with SCID bone marrow, followed by IP injection of human PBL. Our experiments demonstrated that human T cells, recovered from these hu-PBL-BALB mice within 1 month posttransplant, proliferated and expressed activation markers upon stimulation with anti-CD3 monoclonal antibody. A vigorous antiallogeneic human cytotoxic T-lymphocyte (CTL) response could be generated in these mice by immunizing them with irradiated allogeneic cells. Moreover, anti-human immunodeficiency virus type 1 (HIV-1) Net- specific human CTLs could be generated in vivo from naive lymphocytes by immunization of mouse-human chimeras with a recombinant vaccinia-nef virus. This model may be used to evaluate potential immunomodulatory drugs or cytokines, and could provide a relevant model for testing HIV vaccines, for production of antiviral T-cell clones for adoptive therapy, and for studying human T-cell responses in vivo.

scholarly journals Microarray Analysis of Host Cell Gene Transcription in Response to Varicella-Zoster Virus Infection of Human T Cells and Fibroblasts In Vitro and SCIDhu Skin Xenografts In Vivo

2003 ◽  
Vol 77 (2) ◽  
pp. 1268-1280 ◽  
Author(s):  
Jeremy O. Jones ◽  
Ann M. Arvin
Keyword(s):  
T Cells ◽  
Gene Regulation ◽  
Varicella Zoster Virus ◽  
Host Cell ◽  
Gene Transcription ◽  
Cell Types ◽  
Cellular Gene ◽  
Varicella Zoster ◽  
Human T Cells

ABSTRACT During primary infection, varicella-zoster virus (VZV) is spread via lymphocytes to skin, where it induces a rash and establishes latency in sensory ganglia. A live, attenuated varicella vaccine (vOka) was generated by using the VZV Oka strain (pOka), but the molecular basis for vOka attenuation remains unknown. Little is known concerning the effects of wild-type or attenuated VZV on cellular gene regulation in the host cells that are critical for pathogenesis. In this study, transcriptional profiles of primary human T cells and fibroblasts infected with VZV in cell culture were determined by using 40,000-spot human cDNA microarrays. Cellular gene transcription in human skin xenografts in SCID mice that were infected with VZV in vivo was also evaluated. The profiles of cellular gene transcripts that were induced or inhibited in infected human foreskin fibroblasts (HFFs), T cells, and skin in response to pOka and vOka infection were similar. However, significant alterations in cellular gene regulation were observed among the three differentiated human cell types that were examined, suggesting specific differences in the biological consequences of VZV infection related to the target cell. Changes in cellular gene transcription detected by microarray analysis were confirmed for selected genes by quantitative real-time reverse transcription-PCR analysis of VZV-infected cells. Interestingly, the transcription of caspase 8 was found to be decreased in infected T cells but not in HFFs or skin, which may signify a tissue-specific antiapoptosis mechanism. The use of microarrays to demonstrate differences in effects on host cell genes in primary, biologically relevant cell types provides background information for experiments to link these various response phenotypes with mechanisms of VZV pathogenesis that are important for the natural course of human infection.

scholarly journals Disseminated human immunodeficiency virus 1 (HIV-1) infection in SCID-hu mice after peripheral inoculation with HIV-1.

1994 ◽  
Vol 179 (2) ◽  
pp. 513-522 ◽  
Author(s):  
T R Kollmann ◽  
M Pettoello-Mantovani ◽  
X Zhuang ◽  
A Kim ◽  
M Hachamovitch ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Lymph Nodes ◽  
Peripheral Blood ◽  
Interleukin 2 ◽  
Human T Cells ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Human Immunodeficiency Virus 1

A small animal model that could be infected with human immunodeficiency virus 1 (HIV-1) after peripheral inoculation would greatly facilitate the study of the pathophysiology of acute HIV-1 infection. The utility of SCID mice implanted with human fetal thymus and liver (SCID-hu mice) for studying peripheral HIV-1 infection in vivo has been hampered by the requirement for direct intraimplant injection of HIV-1 and the continued restriction of the resultant HIV-1 infection to the human thymus and liver (hu-thy/liv) implant. This may have been due to the very low numbers of human T cells present in the SCID-hu mouse peripheral lymphoid compartment. Since the degree of the peripheral reconstitution of SCID-hu mice with human T cells may be a function of the hu-thy/liv implant size, we increased the quantity of hu-thy/liv tissue implanted under the renal capsule and implanted hu-thy/liv tissue under the capsules of both kidneys. This resulted in SCID-hu mice in which significant numbers of human T cells were detected in the peripheral blood, spleens, and lymph nodes. After intraimplant injection of HIV-1 into these modified SCID-hu mice, significant HIV-1 infection was detected by quantitative coculture not only in the hu-thy/liv implant, but also in the spleen and peripheral blood. This indicated that HIV-1 infection can spread from the thymus to the peripheral lymphoid compartment. More importantly, a similar degree of infection of the hu-thy/liv implant and peripheral lymphoid compartment occurred after peripheral intraperitoneal inoculation with HIV-1. Active viral replication was indicated by the detection of HIV-1 gag DNA, HIV-1 gag RNA, and spliced tat/rev RNA in the hu-thy/liv implants, peripheral blood mononuclear cells (PBMC), spleens, and lymph nodes of these HIV-1-infected SCID-hu mice. As a first step in using our modified SCID-hu mouse model to investigate the pathophysiological consequences of HIV-1 infection, the effect of HIV-1 infection on the expression of human cytokines shown to enhance HIV-1 replication was examined. Significantly more of the HIV-1-infected SCID-hu mice expressed mRNA for human tumor necrosis factors alpha and beta, and interleukin 2 in their spleens, lymph nodes, and PBMC than did uninfected SCID-hu mice. This suggested that HIV-1 infection in vivo can stimulate the expression of cytokine mRNA by human T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

In Vivo Eradication of Human Clonigenic Acute Lymphoblastic Leukemic Cells Expressing the Wilms Tumor Protein, WT-1, by HLA Restricted WT-1 Specific T-Cells in NOD/SCID Mice Monitored by Bioluminescent Imaging.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 581-581
Author(s):  
Ekaterina Doubrovina ◽  
Mikhail Doubrovin ◽  
Elena Kanaeva ◽  
Richard J. O’Reilly
Keyword(s):  
T Cells ◽  
Wilms Tumor ◽  
Scid Mice ◽  
Leukemic Cells ◽  
Luciferase Reporter ◽  
Bioluminescent Imaging ◽  
Acute Leukemias ◽  
Post Infusion

Abstract WT-1 is expressed in 60–80% of acute leukemias, CML and high risk forms of MDS. Its expression has been hypothesized to be critical to the growth or survival of leukemic stem cells. Previously, alloreactive HLAA0201− T-cells recognizing a complex of WT-1 peptide and HLA A0201 were reported to prevent growth of leukemic HLA A0201+ CD34+ Ph+CML progenitor cells in NOD/ SCID mice (Transplantation, vol 75, No9, 2003). In this study, we have assessed the capacity of HLA-restricted, WT-1 peptide specific CTL (WT1-CTL) lacking alloreactivity to prevent the outgrowth of a human acute preB-lymphocytic leukemia (B-ALL)in NOD/SCID mice. This leukemia contained 65% of the blasts expressed WT-1 as determined by FACS analysis. For these studies the leukemic cells were transduced to express a luciferase reporter gene, permitting sequential monitoring of growth in vivo by bioluminescent imaging. WT-1 specific T-cells were generated from normal HLA A0201+ donor PBMC by in vitro sensitization with autologous dendritic cells loaded with the immunogenic HLA A0201 binding WT-1 peptide, RMFPNAPYL, and shown to be selectively cytotoxic against HLA A0201+WT-1+ leukemias and peptide loaded PHA blasts. T-cells from the same donor sensitized with autologous EBV BLCL and exhibiting HLA A0201 restricted EBV-specific cytotoxic activity served as controls. WT-1-CTL or EBV CTL were co-incubated in vitro with the WT-1+ HLA A0201+ BALL-LUC at a 4:1 effector target ratio for 7 hours at 37°C. Thereafter, separate groups of 5 NOD/SCID mice received intravenous infusions of cells from each of the co-cultures, at doses providing 12 × 106 WT1 CTL or EBVCTL and 3 × 106 BALL-LUC cells/mouse. A third group received 3×106 BALL-LUC alone. Leukemia growth was monitored at 2–3 day intervals from day 1–45 post infusion. In all 3 groups, BALL-LUC could be detected in the thorax by imaging at day 1. In mice treated with BALL-LUC alone or together with EBV-CTL, signal accumulation in the thorax increased steadily through 45 days of observation. By day 17, BALL-LUC were also detected throughout the head, abdomen and pelvis, and thereafter also increased until sacrifice at day 45. Autopsy confirmed presence of leukemic nodules in the lung and leukemic cells in blood, spleen and marrow as well as other organs. In contrast, in mice treated with WT1-CTL+ BALL LUC, signal intensity in lung decreased by day 4. In 4/5 of these mice, BALL-LUC could not be detected thereafter. In one mouse from this group, BALL-LUC were first detected in the head 31 days post infusion. At autopsy on day 45, this mouse had detectable BALL in the skull but in no other sites. WT-1 expression of residual leukemic cells is being analyzed. The other mice treated with WT-1 CTL had no detectable residual disease. These results suggest that clonogenic BALL cells express WT-1 and are susceptible to eradication in vivo by WT-1 peptide specific cytotoxic T-cells. The elimination of such clonogenic leukemic cells is sufficient to prevent subsequent development of leukemia.

Cytotoxicity Is Not Required for the Anti-Leukemia Efficacy of Human H-Y-Specific CD4+ T Lymphocytes In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3553-3553
Author(s):  
Attilio Bondanza ◽  
Lothar Hambach ◽  
Zohara Aghai ◽  
Monica Casucci ◽  
Bart Nijmeijer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Clone ◽  
Scid Mice ◽  
Helper T Cell ◽  
T Cell Clone ◽  
Control Clone

Abstract Abstract 3553 Poster Board III-490 Introduction Minor histocompatibility antigens (mHag) play a major role in the graft-versus-leukemia (GvL) effect following HLA-matched allogeneic hemopoietic cell transplantation (allo-HCT). Clinically, the GvL effect coincides with the emergence of mHag-specific CD8+ cytotoxic T lymphocytes (CTL). Experimentally, targeting a single mHag with human CD8+ CTL has a major anti-leukemia effect in NOD/scid mice. Altogether, these observations suggest that mHag-specific cytotoxicity by CD8+ T cells is an important component of the GvL effect. In contrast, little is known on the contribution of mHag-specific CD4+ T cells. Female-to-male allo-HCT is characterized by a low rate of leukemia relapse, indicating that H-Y-encoded mHag are potent leukemia-regression antigens. Earlier, we described a DRB3*0301-restricted H-Y mHag epitope inducing CD4+ helper T-cell responses in H-Y-mismatched HLA-matched allo-HCT. Aim: The aim of this study is to elucidate the role of mHag-specific human CD4+ T lymphocytes on the GvL effect. Methods The ALL-CM leukemia cell line, derived from a male (i.e. H-Y+) HLA-A0201+, DRB30301+ patient, reproducibly engrafts in NOD/scid mice after administration of 10×106 cells. Both an HLA-A0201-restricted H-Y-specific CD8+ CTL clone and the DRB30301-restricted H-Y-specific CD4+ helper T-cell clone that we earlier described were used to investigate the anti-leukemia efficacy of CD8+ and CD4+ T cells in NOD/scid mice. Results In vitro, the CD8+ H-Y specific CTL clone was highly cytotoxic against the ALL-CM leukemia. The H-Y specific CD4+ helper T-cell clone did not lyse the leukemia, but produced IFN-γ upon recognition. Infusion of the H-Y-specific CD8+ CTL clone (25×106 cells/mouse) 3 days after ALL-CM leukemia challenge significantly delayed leukemia progression by 3 weeks compared to a CMV-specific CD8+ CTL control clone (p<0,001). Despite no measurable in vitro cytotoxicity, the H-Y-specific CD4+ helper T-cell clone (25×106 cells/mouse) delayed leukemia progression by 2 weeks compared to a leukemia non-reactive HLA-DR1-specific CD4+ helper T-cell control clone (p<0,001). In vitro co-incubation of the H-Y-specific CD4+ helper T-cell clone did not influence leukemia proliferation but induced up-regulation of MHC-class I and II, CD80, CD86 and CD40. In vitro, pre-incubation of leukemia cells with the H-Y-specific CD4+ helper T-cell clone irradiated did not improve the in vivo anti-leukemia efficacy of the H-Y-specific CD8+ CTL clone. Co-infusion of the H-Y specific CD4+ helper T-cell clone did not augment the in vivo persistence of the H-Y-specific CD8+ CTL T-cell clone. Nevertheless, the co-infusion resulted in a delay in leukemia progression of approximately 5 weeks, suggesting an additive, non overlapping anti-leukemia mechanism. Conclusions Minor Hag-specific human CD4+ T lymphocytes may contribute to the GvL effect through a direct, non cytotoxic mechanism, which could be additive to that of CD8+ CTL. The nature of this non cytotoxic GvL effect is currently under investigation. A.B. and L.H. equally contributed to this study. Disclosures: No relevant conflicts of interest to declare.

Different Pattern of CD154 and CD40 Expression On B-CLL and T Lymphocytes In Peripheral Blood, Bone Marrow and Lymph Node Microenvironment In B-Cell Chronic Lymphocytic Leukemia (B-CLL)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3607-3607
Author(s):  
Ozren Jaksic ◽  
Branimir Gizdic ◽  
Tajana Stoos Veic ◽  
Vlatka Pandzic Jaksic ◽  
Rajko Kusec ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
T Cells ◽  
Lymph Node ◽  
Peripheral Blood ◽  
Lymphocytic Leukemia ◽  
Significant Difference ◽  
Cd40 Expression ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Abstract 3607 Background: In B-cell chronic lymphocytic leukemia (B-CLL) there is a well documented intraclonal and interclonal variability of B-CLL cells in different lymphoid compartments with respect to the expression of a number of surface and intracellular molecules (for example CD38 and ZAP-70). This variability in part may reflect a number of interactions of malignant B-CLL clone with supporting microenviroment including cells (T-cells, nurse-like cells, etc.), cytokines, chemokines and stroma. One of the key interactions of B-CLL clone is with T-cells, through CD154/CD40 system. It is important pathway modulating survival, drug resistance and immunity. It is known that CD154 is transiently expressed on CD4+ T cells, as well as that CD154 can be coexpressed on B-CLL cells with CD40 in a subpopulation of B-CLL patients. Its expression on B-CLL cells can be induced by gene therapy and lenalidomide, being in part responsible for their therapeutic effects. Aim of this study was to determine the level of expression of CD154 and CD40 in vivo on B-CLL cells and T lymphocytes and to evaluate intra and interclonal differences due to different microenvironment, i.e. peripheral blood, bone marrow and lymph nodes. Methods: peripheral blood, (PB), bone marrow (BM) and lymph node (LN) samples were taken by conventional techniques (venepuncture and fine needle aspiration) on the same day. The expression level of CD154 and CD40 molecules on CD19+CD5+ B-CLL cells and CD19-CD5+ T cells was analyzed by flow cytometry. Results were expressed as mean fluorescence intensity (MFI) and analyzed by paired tests. Results: samples taken from 21 typical B-CLL patients with median age of 72 years were analyzed. There were 9 males and 12 females. Mean beta-2 microglobuin was 4.3mg/l, mean Total Tumor Mass size was 8.9 and mean Tumor Distribution pattern was 0.75. There were 2, 14 and 5 patients in Rai stage 0, I+II and III+IV, respectively. There were 6 previously treated patients (but off therapy 3 months before sampling). The expression level of CD154 was absent/low on T-cells and in 14/21 patients on B-CLL cells. However in 7/21 patients B-CLL cells had higher CD154 expression (“CD154 positive” patients). There was no detectible difference in CD154 expression on T cells between compartments, while on B-CLL cells there was highest expression in lymph nodes and lowest in peripheral blood (p<0.01). CD40 expression on B-CLL cells was significantly higher than CD154, i.e. all cases were positive, and there was no significant difference between lymphoid compartments. There was no significant difference between CD154 positive and negative patients in measured disease parameters. Conclusions: our results show that CD154 expression on T-cells is absent/low and not significantly different between lymphoid compartments regardless of different microenvironment milieu. CD40 expression on B-CLL cells is high and comparable through compartments. In subset of patients there is CD154 positivity on B-CLL cells and shows strong association with lymphoid compartments possibly indicating microenviroment influence on CD154/CD40 system in B-CLL in vivo. These results warrant further studies to indentify the role of CD154 expression on B-CLL cells in pathologic process and its regulation and may eventually uncover novel or modulate existing innovative therapeutic approaches (like gene therapy or immunomodulatory agents like lenalidomide). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Infection of Dendritic Cells by the Maedi-Visna Lentivirus

2000 ◽  
Vol 74 (21) ◽  
pp. 10096-10103 ◽  
Author(s):  
Susanna Ryan ◽  
Laurence Tiley ◽  
Ian McConnell ◽  
Barbara Blacklaws
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Cell Types ◽  
Infected Sheep ◽  
In Vivo Model ◽  
Visna Virus ◽  
Afferent Lymph ◽  
Infection Experiments

ABSTRACT The early stages of lentivirus infection of dendritic cells have been studied in an in vivo model. Maedi-visna virus (MVV) is a natural pathogen of sheep with a tropism for macrophages, but the infection of dendritic cells has not been proven, largely because of the difficulties of definitively distinguishing the two cell types. Afferent lymphatic dendritic cells from sheep have been phenotypically characterized and separated from macrophages. Dendritic cells purified from experimentally infected sheep have been demonstrated not only to carry infectious MVV but also to be hosts of the virus themselves. The results of the in vivo infection experiments are supported by infections of purified afferent lymph dendritic cells in vitro, in which late reverse transcriptase products are demonstrated by PCR. The significance of the infection of afferent lymph dendritic cells is discussed in relation to the initial spread of lentivirus infection and the requirement for CD4 T cells.

scholarly journals Disseminated and Sustained HIV-Infection in CD34+ Cord Blood Cell Transplanted Rag2−/−gc−/− Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 489-489
Author(s):  
Markus G. Manz ◽  
Stefan Baenziger ◽  
Roxane Tussiwand ◽  
Roberto F. Speck
Keyword(s):  
T Cells ◽  
Hiv Infection ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Receptor Expression ◽  
In Vivo Model ◽  
Immunodeficient Mice ◽  
Genetically Engineer

Abstract HIV research has been hampered due to the lack of assessable animal models that mirror infection in humans. HIV is a human-specific virus, and consequently laboratory rodents as mice or rats are not susceptible to infection. Although non-human primates as chimpanzees can be infected, they do not develop HIV-associated immunodeficiency, while sooty mangabeys, rhesus macaques, and baboons are only susceptible to HIV related simian-immunodeficiency virus. Efforts to genetically engineer rodents to become HIV targets have largely failed; even if infection in vitro was achieved, HIV replication in vivo was limited or absent. Thus, substitute xeno-chimeric models have been developed by transplanting immunodeficient mice with either human peripheral blood leukocytes (hu-PBL-SCID), or pieces of human fetal tissues containing hematopoietic cells (SCID-hu). Both hu-PBL-SCID and SCID-hu mice sustain HIV infection and replication in vivo. However, in hu-PBL-SCID mice xeno-reactivity and successive loss of human leukocytes limit infection to a relatively short time frame. In SCID-hu mice, HIV infection can be observed for extended times; however, availability of transplantable human fetal organs is restricted for practical and ethical reasons, and HIV pathology in these mice is mainly limited to tissue implants. Given these limitations hu-PBL-SCID and SCID-hu mice did not fully match the demand for a small animal model that closely mirrors infection in humans. Recently, we found that injection of human cord blood CD34+ cells into newborn Rag2−/−gc−/− mice leads to development of human T, B, and dendritic cells, successive formation of primary and secondary lymphoid organs in situ, and some in vivo immune responses. We here tested these mice as a model system for in vivo HIV-1 infection. Mice with a PB CD45+ and CD4+ cell chimerism of 29.4±18.2% and 2.7±3.0%, respectively, were infected i.p. with either CCR5-tropic YU-2 (n=15), or CXCR4-tropic NL4–3 (n=19) HIV-strains at 10–28 weeks of age. Independent of viral strains used, HIV-RNA levels peaked two to six weeks after infection, with up to about 2x10E6 copies/ml plasma, while thereafter viremia mostly stabilized at lower levels, and was maintained for up to 190 (YU-2) and 120 (NL4–3) days, the longest time followed. A marked relative CD4+ T cell depletion in peripheral blood occurred in CXCR4-tropic strain infected mice, while this was less pronounced in CCR5-tropic strain infected animals. Thymus infection, as determined by p24 immunohistochemistry, was almost exclusively observed in CXCR4-tropic strain infected mice, while spleen and lymph node HIV infection occurred irrespective of co-receptor selectivity, consistent with respective co-receptor expression on human CD4+ T-cells. P24 expressing, and thus productively HIV infected non-T cells such as CD68+ macrophages were only occasionally detected as expected in a non-inflammatory in vivo setting. In summary, the here presented data establishes newborn human CD34+ cell transplanted Rag2−/−gc−/− mice as a new tool to study HIV infection and pathogenesis in vivo, closely resembling HIV infection in humans. This straight-forward to generate, cost-effective, ethically unproblematic, and easy to monitor new in vivo model should thus be valuable to study virus-induced pathology, as well as pharmacologic or genetic approaches aiming to prevent or treat HIV infection.

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