Vaccination with Leukemia Cell Expression Membrane Bound GM-CSF Overcomes the Host Immunosuppression Induced by Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5435-5435
Author(s):  
Di Ling ◽  
Ralph B. Arlinghaus ◽  
Xiaoyang Ling
Keyword(s):  
Cell Surface ◽  
Treg Cells ◽  
T Cell Response ◽  
Leukemia Cells ◽  
Cell Mediated Immunity ◽  
Immune Recognition ◽  
Gm Csf ◽  
Treg Population ◽  
Membrane Bound ◽  
Murine Leukemia

Abstract Immunotherapy of leukemia involves stimulating host-cell mediated immunity by facilitating immune recognition of leukemia cells, which are normally weakly immunogenic. We previously showed that vaccination with membrane bound GM-CSF leukemic cells protects mice from leukemia challenge (Ling et al., Oncogene, 2007). In these studies, after addition of a transmembrane domain to the original GM-CSF coding sequence (tmGM-CSF), the construct was transduced into murine leukemia cells (WEHI-3B), which was shown to be more than 98% on the cell surface. Vaccination with lethally irradiated tmGM-CSF expressing murine leukemia cells prevents leukemia in immunocompetent mice (BALB/c), as 100% of vaccinated BALB/c mice were protected from leukemia (Ling et al, Oncogene 2007). No protection was observed by vaccination of nude mice, indicating that adaptive immunity is involved in the protective response. In the present studies, we extended our original observation and provided evidence to show that leukemic mice undergo immunosuppression and that vaccination with leukemia cells expressing cell surface tmGM-CSF overcomes immunosuppression. Vaccination with lethally irradiated leukemia cells expressing cell surface tmGM-CSF overcame the immunosuppression induced by leukemia development, as normal levels of CD4+/CD25+/Foxp3+ T-regulatory (Treg) cells were maintained in spleens and thymus after challenge with leukemia cells. In contrast, the Treg population was significantly increased in leukemic mice vaccinated with leukemia cells lacking cell surface tmGM-CSF (p<0.001) after leukemia challenge, and these mice had a lower CD8+/Treg cell ratio (p<0.01). The ratio of CD8+/Treg cells was higher in tmGM-CSF/GFP vaccinated mice than in GFP vaccinated mice (p<0.001), which in-turn leads to a more effective CD8+ T-cell response. DC levels were also increased from normal levels in mice vaccinated with tmGM-CSF+ leukemia cells compared to control vaccinations. These results suggest that vaccination with leukemia cells expressing GM-CSF on their cell surface leads to an effective cell-mediated immune response in the vaccinated host by overcoming an impaired host cellular immunity induced up-regulation of Treg cells caused by the leukemia process. This strategy has potential for use in the treatment of various human leukemias.

Vaccination with Leukemia Cells Expressing Cell-Surface-Associated GM-CSF Blocks Leukemia Induction in Immunocompetent Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2531-2531
Author(s):  
Xiaoyang Ling ◽  
Yan Wang ◽  
Ralph B. Arlinghaus
Keyword(s):  
Immune System ◽  
Cell Surface ◽  
Immune Tolerance ◽  
Leukemia Cells ◽  
Host Immune System ◽  
Over Expression ◽  
Gm Csf ◽  
Significant Difference ◽  
Specific Antigens ◽  
Immunocompetent Mice

Abstract The fundamental basis for immunotherapy of leukemia is that leukemia cells express specific antigens that are not expressed by normal hematopoietic cells. However, the host immune system appears to be tolerant of leukemia cells. To overcome this immune tolerance, we transduced WEHI-3B mouse monocyte leukemia cells (1) with a transmembrane form of GM-CSF (tmGM-CSF). The tmGM-CSF was constructed using the pDisplay vector for cell-surface targeting (Invitrogen) into the pLOX lentivirus gene transfer vector (2). After infection of WEHI-3B cells with a recombinant lentivirus encoding tmGM-CSF, nearly all the transduced cells expressed tmGM-CSF on the cell-surface, as determined by flow cytometry analysis using anti-GM-CSF. To determine whether vaccination with tmGM-CSF expressing WEHI-3B cells would prevent leukemia formation, immunocompetent BALB/c mice were immunized with lethally-irradiated WEHI-3B cells (106, 3 times 7 day intervals), which express tmGM-CSF, prior to challenging vaccinated mice with WEHI-3B cells (5x104) that express GFP as a marker. 100% of vaccinated mice were protected from leukemia. Non-vaccinated mice succumbed to leukemia within 50–55 days. Vaccination of mice with lethally-irradiated WEHI-3B cells expressing CD40L protected 80% of the mice from leukemia. In contrast, mice immunized with lethally-irradiated WEHI-3B/GFP cells lacking tmGM-CSF were not protected. Mice vaccinated three times at 5,12, 19 days after challenge with WEHI-3B/GFP cells had a significant increase in survival in that 60% of mice were alive and healthy at 16 days (to this date) after all control non-vaccinated mice had died. Similar vaccine studies were performed with BCR-ABL (b3a2)+ 32D cells (106) in immunocompetent C3H/HeJ mice (3). These mice die of leukemia within 35 days. After infection of BCR-ABL+ 32D cells with the lentivirus encoding tmGM-CSF/GFP, tmGM-CSF was expressed on the cell-surface. The C3H/HeJ mice challenged with BCR-ABL+32D/GFP cells (106) showed a significant level of protection by vaccination with lethally-irradiated tmGM-CSF+ 32D BCR-ABL cells (106, 2 times at 7 day intervals); 40% of the vaccinated mice remained healthy; all non-vaccinated mice died of leukemia. There was a significant difference in survival (P=0.03) between the vaccinated and non-vaccinated groups. Interestingly, the spleens of vaccinated C3H/HeJ mice that died of leukemia at the same time as non-vaccinated mice approached normal size whereas non-vaccinated mice had enlarged spleens. Our findings suggest that over-expression of cell-surface tmGM-CSF in leukemia cells can overcome immune tolerance, allowing the immune system to efficiently recognize and destroy the leukemia cells, providing extended survival of vaccinated mice. Because significant protection from death was achieved by vaccination after challenge with leukemia cells, tmGM-CSF expression in leukemia cells has potential as a therapeutic strategy for treatment of leukemia.

scholarly journals In vitro generation of tumor-specific cytotoxic lymphocytes. Secondary allogeneic mixed tumor lymphocyte culture of normal murine spleen cells

1977 ◽  
Vol 146 (2) ◽  
pp. 468-482 ◽  
Author(s):  
S Gillis ◽  
KA Smith
Keyword(s):  
Cell Surface ◽  
Lymphocyte Culture ◽  
Leukemia Cells ◽  
Cytotoxic Lymphocytes ◽  
Mixed Tumor ◽  
Spleen Cells ◽  
Syngeneic Tumor ◽  
Murine Spleen ◽  
Murine Leukemia

In vivo or in vitro immunity to murine leukemia virus (MuLV)-induced leukemia cells which do not effectively produce virus, has been difficult to demonstrate. Because immunizations with allogeneic murine leukemia cells have been used to confer syngeneic tumor immunity to virus- producing cells, we attempted to generate lymphocytes, cytotoxic to syngeneic nonproducer leukemia cells, by stimulating normal murine spleen cells with allogeneic nonproducer leukemia cells in mixed tumor lymphocyte culture (MTLC) reactions in vitro. Secondary allogeneic MTLC of normal C57BL/6 or DBA/2 spleen cells effectively produced syngeneic tumor-specific cytotoxic lymphocytes. Target cells lysed in lymphocyte- mediated cytolysis (LMC) assays, included both Friend and Rauscher virus- induced syngeneic murine leukemia cells and chemically-induced hematopoietic tumor cells. Syngeneic tumor cells were lysed regardless of whether they produced infectious MuLV or expressed viral antigens gp-71, p-30, or p-12 at the cell surface. Syngeneic normal cells (thymus, lymph node, or Concanavalin A-stimulated spleen cells) used as targets in LMC assays were uneffected by lymphocytes harvested from secondary allogeneic MTLC. Several other in vitro culture treatments including secondary syngeneic MTLC and repetitive mixed lymphocyte culture stimulations were incapable of generating tumor-specific cytotoxic lymphocytes. Based upon these results, we propose that secondary MTLC stimulation of normal spleen cells with allogeneic nonproducer leukemia cells selects for the proliferation of two subpopulations of antigen-specific cytotoxic lymphocytes. The population capable of effecting syngeneic tumor cell lysis is directed against tumor-associated cell surface antigens which may be distinct from viral structural proteins or glycoproteins. The growth of these tumor-specific cytotoxic lymphocytes may be enhanced by a soluble allogeneic effect factor produced by the proliferation of the second subpopulation of lymphocytes generated in repetitive allogeneic MTLC, namely those lymphocytes with specificities directed against differing histocompatibility antigens.

scholarly journals Vaccination with leukemia cells expressing cell-surface-associated GM-CSF blocks leukemia induction in immunocompetent mice

Oncogene ◽  
2006 ◽  
Vol 25 (32) ◽  
pp. 4483-4490 ◽  
Author(s):  
X Ling ◽  
Y Wang ◽  
M F Dietrich ◽  
M Andreeff ◽  
R B Arlinghaus
Keyword(s):  
Cell Surface ◽  
Leukemia Cells ◽  
Gm Csf ◽  
Immunocompetent Mice

In vitro induction of cell-mediated immunity to murine leukemia cells

1978 ◽  
Vol 4 (3) ◽  
pp. 161-169 ◽  
Author(s):  
Eli Kedar ◽  
Ziva Raanan ◽  
Maya Schwartzbach
Keyword(s):  
Leukemia Cells ◽  
Cell Mediated Immunity ◽  
In Vitro Induction ◽  
Murine Leukemia

scholarly journals A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition

2001 ◽  
Vol 155 (7) ◽  
pp. 1265-1274 ◽  
Author(s):  
Laurent Coscoy ◽  
David Jesse Sanchez ◽  
Don Ganem
Keyword(s):  
Cell Surface ◽  
Immune Recognition ◽  
Glutathione S Transferase ◽  
Host Cell Membrane ◽  
Mhc I ◽  
Target Proteins ◽  
Cell Membrane Proteins ◽  
Membrane Bound ◽  
E2 Enzymes

Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells. This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins. Here, we show that expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface. MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity. In vitro, addition of a MIR2–glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion; this reaction is abolished by mutation of the Zn-coordinating residues of the PHD domain. Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

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