ovalbumin and Precursor-Cell-Lymphoblastic-Leukemia-Lymphoma

Patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are threatened by potentially lethal viral manifestations like cytomegalovirus (CMV) reactivation. Because the success of today's virostatic treatment is limited by side effects and resistance development, adoptive transfer of virus-specific memory T cells derived from the stem cell donor has been proposed as an alternative therapeutic strategy. In this context, dose minimization of adoptively transferred T cells might be warranted for the avoidance of graft-versus-host disease (GVHD), in particular in prophylactic settings after T-cell-depleting allo-HSCT protocols. To establish a lower limit for successful adoptive T-cell therapy, we conducted low-dose CD8(+) T-cell transfers in the well-established murine Listeria monocytogenes (L.m.) infection model. Major histocompatibility complex-Streptamer-enriched antigen-specific CD62L(hi) but not CD62L(lo) CD8(+) memory T cells proliferated, differentiated, and protected against L.m. infections after prophylactic application. Even progenies derived from a single CD62L(hi) L.m.-specific CD8(+) T cell could be protective against bacterial challenge. In analogy, low-dose transfers of Streptamer-enriched human CMV-specific CD8(+) T cells into allo-HSCT recipients led to strong pathogen-specific T-cell expansion in a compassionate-use setting. In summary, low-dose adoptive T-cell transfer (ACT) could be a promising strategy, particularly for prophylactic treatment of infectious complications after allo-HSCT.

Topics: Adolescent; Animals; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Proliferation; Child; Cytomegalovirus; Cytomegalovirus Infections; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Homeodomain Proteins; Humans; Immunization; Immunotherapy, Adoptive; Male; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Ovalbumin; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Severe Combined Immunodeficiency; Transplantation, Homologous; Virus Activation

The recognition and neutralization of tumour cells is one of the big challenges in immunity. The immune system has to recognize syngeneic tumour cells and has to be primed and respond in an adequate manner. Priming of a leukaemia-specific immune response is a crucial step in tumour immunology that can mislead to tumour tolerance either by T cell ignorance, deletion or Treg induction. To resemble the situation of acute lymphoblastic leukaemia (ALL) in patients, we used the murine BALB/c model with syngeneic BM185 tumour cells. We established a tumour cell line that expresses the neo-antigen ovalbumin (BM185-OVA/GFP) to allow the application of T cell receptor transgenic, antigen-specific CD4(+) T cells. Here, we demonstrate that effective anti-ALL immunity can be established by in vivo priming of CD4(+) T cells that is sufficient to differentiate into effector cells. Yet they failed to control tumour alone, but initiated a Th1 response. An efficient tumour clearance was dependent on both antigen-specific CD4(+) T cells and CD8(+) effector T cells from the endogenous repertoire. The tolerogeneic milieu was characterized by increased Tregs numbers and elevated IL-10 level. Tregs hamper effective antitumour immune response, but their depletion did not result in reduced tumour growth. In contrast, neutralization of IL-10 improved median mouse survival. Future therapies should focus on establishing a strong CD4+ T cells response, either by adjuvant or by adoptive transfer.

Topics: Animals; Bone Marrow Cells; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Cells, Cultured; Dendritic Cells; Female; Flow Cytometry; Green Fluorescent Proteins; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-10; Interleukin-12; Interleukin-2; Kaplan-Meier Estimate; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, SCID; Ovalbumin; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Time Factors

As a primary drug for the treatment of acute lymphoblastic leukemia (ALL), encapsulation of L-asparaginase (ASNase) into red blood cells (RBC) has been popular to circumvent immunogenicity from the exogenous protein. Unlike existing methods that perturbs RBC membranes, we introduce a novel method of RBC-incorporation of proteins using the membrane-translocating low molecular weight protamine (LMWP). Confocal study of fluorescence-labeled LMWP-ovalbumin, as a model protein conjugate, has shown significant fluorescence inside RBCs. Surface morphology by scanning electron microscopy of the RBCs loaded with LMWP-ASNase was indistinguishable with normal RBCs. These drug loaded RBCs also closely resembled the profile of the native erythrocytes in terms of osmotic fragility, oxygen dissociation and hematological parameters. The in vivo half-life of enzyme activity after administering 8 units of RBC/LMWP-ASNase in DBA/2 mice was prolonged to 4.5+/-0.5 days whereas that of RBCs loaded with ASNase via a hypotonic method was 2.4+/-0.7 days. Furthermore, the mean survival time of DBA/2 mice bearing mouse lymphoma cell L5178Y was improved by approximately 44% compared to the saline control group after treatment with the RBC loaded enzymes. From these data, an innovative, novel method for encapsulating proteins into intact and fully functional erythrocytes was established for potential treatment of ALL.

Topics: Animals; Antineoplastic Agents; Asparaginase; Biological Transport; Cell Line, Tumor; Chemistry, Pharmaceutical; Drug Carriers; Drug Compounding; Enzyme Stability; Erythrocyte Transfusion; Erythrocytes; Feasibility Studies; Hemolysis; Mice; Mice, Inbred DBA; Microscopy, Confocal; Microscopy, Electron, Scanning; Molecular Weight; Osmotic Fragility; Ovalbumin; Pilot Projects; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protamines; Sheep

Tumor escape mechanisms in leukemia are not well defined. To dissect immunological mechanisms responsible for immune tolerance toward leukemia, we established a murine model system allowing clonotypic analysis of leukemia-specific CD4 T cells recognizing ovalbumin (OVA). Upon i.v. injection of genetically engineered leukemia cells, dendritic cells (DCs) engulfed, processed and presented OVA to OVA-specific CD4 T cells. Consequently, leukemia-specific T cells were primed in vivo as shown by expression of activation markers and proliferative responses. However, in spite of detectable CD4 T cell responses in vitro and in vivo, no effective anti-leukemia immunity was established. In contrast, adoptively transferred DO11.10 T cells that were primed ex vivo mediated effective antitumor immunity. Furthermore, ex vivo primed DO11.10 T cells showed high expression of Th1 cytokines (interferon-gamma, tumor necrosis factor-alpha and interleukin-2) whereas in vivo primed OVA-specific CD4 T cells showed incomplete differentiation (proliferation without cytokine production). We conclude that activated T cells lacking effector function develop through incomplete differentiation in leukemia-bearing mice. Thus, priming conditions of leukemia-specific CD4 T cells critically determines the balance between immunity or tolerance toward leukemia.

Topics: Animals; CD4-Positive T-Lymphocytes; Cell Proliferation; Dendritic Cells; Disease Models, Animal; Female; Flow Cytometry; Fusion Proteins, bcr-abl; Humans; Immune Tolerance; Immunity; Interferon-gamma; Leukemia, Experimental; Mice; Mice, Inbred BALB C; Ovalbumin; Precursor Cell Lymphoblastic Leukemia-Lymphoma; T-Lymphocytes, Regulatory; Th1 Cells; Thymidine; Tumor Cells, Cultured