asialo-gm1-ganglioside and Severe-Combined-Immunodeficiency

Topics: Anemia, Hemolytic; Animals; Chemical and Drug Induced Liver Injury; Female; G(M1) Ganglioside; Hepatomegaly; Immunization, Passive; Inflammation; Interleukin-2; Killer Cells, Lymphokine-Activated; Macaca fascicularis; Male; Mice; Mice, Inbred C57BL; Mice, SCID; Pulmonary Edema; Pulmonary Eosinophilia; Rats; Receptors, Interleukin-1; Recombinant Proteins; Severe Combined Immunodeficiency; Splenomegaly; Tumor Necrosis Factor-alpha; Viscera

T cells with antidonor specificities have been isolated from human recipients experiencing graft rejection after allogeneic bone marrow transplantation (BMT). Partial T-cell depletion of unrelated BM grafts with an anti- T-cell receptor (TCR) monoclonal antibody (MoAb) directed against the TCR alpha/beta heterodimer have shown that the incidence of graft-versus-host disease is low and that the incidence of durable engraftment is high. These studies suggest either that the number of residual TCR alpha/beta+ cells was sufficient to permit alloengraftment or that the preservation of cells other than TCR alpha/beta+ cells was beneficial for engraftment. With respect to the latter, one such candidate cell is the TCR gamma/delta+ T cell. Because no studies have specifically examined whether TCR gamma/delta+ cells might be capable of eliminating BM-derived hematopoietic cells, we established a new graft rejection model system in which transgenic (Tg) H-2d mice (termed G8), known to express gamma/delta heterodimers on high proportion of peripheral T cells, were used as BMT recipients. These Tg TCR gamma/delta+ cells respond vigorously to target cells that express the nonclassical major histocompatibility complex (MHC) class lb region gene products encoded in H-2T region of H-2T(b)+ strains. G8 Tg mice were used as recipients for C57BL/6 (B6: H-2(b); H-2T(b)) T-cell-depleted (TCD) donor BM. We show that G8 Tg (H-2(d), H-2T(d)) mice are potent mediators of B6 BM graft rejection and that the rejection process was inhibited by anti-TCR gamma/delta MoAbs. In contrast, BM from a B6 congenic strain that expresses the H-2T(a) allele, B6.A-Tl(a)/BoyEg, was readily accepted, suggesting that H-2T antigens on repopulating donor BM cells are the targets of host graft rejecting T cells that express the TCR gamma/delta heterodimer. PB chimerism studies were performed at > or = 1.5 months post-BMT using TCD BM from severe combined immunodeficient allogeneic donors, which is highly susceptible to rejection by the host. The addition of donor G8 TCR gamma/delta+ cells to TCD donor BM was shown to significantly increase alloengraftment in B6 recipients. These results show that (1) host TCR gamma/delta+ cells can reject repopulating donor cells, presumably by responding to nonclassical MHC class lb gene products expressed on BM-derived hematopoietic progenitor cells; and (2) donor TCR gamma/delta+ cells can facilitate the alloengraftment of rigorously TCD donor BM.

Topics: Animals; Antibodies, Monoclonal; Bone Marrow Transplantation; CD3 Complex; Female; G(M1) Ganglioside; Graft Rejection; H-2 Antigens; Histocompatibility Antigens Class I; Lymphocyte Activation; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, SCID; Mice, Transgenic; Radiation Chimera; Receptors, Antigen, T-Cell, gamma-delta; Severe Combined Immunodeficiency; T-Lymphocyte Subsets; Transplantation, Homologous

This study addresses the role of MHC class I molecules in the rejection of tumor grafts by SCID mice. Tumor cell lines, their corresponding MHC class I transfectants, and MHC class I-deficient mutants were inoculated to SCID mice. This allowed a study of tumor rejection responses in an environment with normal numbers of natural killer cells but largely devoid of functional T and B cells. C.B-17 (H-2d) SCID mice were found to reject low (10(2)) but not high (10(4)) doses of allogeneic (H-2b) tumor cells. The introduction of H-2Dd into such allogeneic tumor cells abrogated the rejection response with progressive tumor growth as a consequence. Introduction of H-2Kd or Ld had no or only marginal effects. The protective ability of H-2Dd was mapped to the alpha 1/alpha 2 domains of the molecule. H-2Dd protected allogeneic tumors from rejection also in C3H SCID mice of the H-2k haplotype, demonstrating that this ability was not dependent on H-2Dd expression in the host. Expression of endogenous H-2Kb and/or Db molecules partially protected wild-type allogeneic tumor cells from rejection since mutant allogeneic cells, devoid of class I expression, were rejected even after high-dose inoculation. Introduction of either allogeneic or xenogeneic class I molecules did not lead to rejection of otherwise MHC class I syngeneic (H-2d) tumor cells. The observed tumor cell rejection in SCID mice was dependent on natural killer cells. After depletion of asialo-GM1+ cells, all inoculated tumor cell lines grew progressively, independently of MHC class I expression. These results are compatible with a model where expression of certain, but not all, class I molecules protect from natural killer cell-mediated rejection. There was no evidence for rejection occurring as a consequence of the expression of allogeneic or xenogeneic class I molecules on the grafted cells. MHC class I expression may thus influence tumor cell recognition in mice lacking T-cell receptor expression.

Topics: Animals; Antigens, Neoplasm; B-Lymphocytes; G(M1) Ganglioside; Graft Rejection; Histocompatibility Antigens Class I; HLA-A2 Antigen; Killer Cells, Natural; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, SCID; Mutation; Neoplasm Transplantation; Neoplasms, Experimental; Severe Combined Immunodeficiency; T-Lymphocytes; Transfection; Tumor Cells, Cultured

Mice lacking functional T and B lymphocytes offer an in vivo animal model for the study of human immune functions. We have attempted to optimize the reconstitution of severe combined immunodeficiency (SCID) mice with human peripheral blood lymphocytes (PBL) using radiation, anti-asialo GM1 antibody or cyclophosphamide (Cy) treatment of the mice and in vitro stimulation of human PBL with interleukin (IL)-2 prior to their transfer to the mice. Total human IgG and tetanus-toxoid (TT)-specific human IgG responses of the mice were used as parameters of successful reconstitution. Treatment of the mice with anti-asialo GM1 antibody significantly enhanced total human IgG levels, but not TT-specific antibody responses, whereas irradiation or Cy treatment of the mice had no effect on human antibody production. In vitro treatment of human PBL with IL-2 prior to engraftment significantly decreased total human IgG responses of human PBL-grafted SCID mice. The immune responses of individual mice within a group were highly variable, which constitutes a major disadvantage of this model.

Topics: Animals; Antibodies; B-Lymphocytes; Cyclophosphamide; Disease Models, Animal; G(M1) Ganglioside; Humans; Immunoglobulin G; Interleukin-2; Mice; Mice, SCID; Recombinant Proteins; Severe Combined Immunodeficiency; Tetanus Toxoid; Whole-Body Irradiation

The severe combined immunodeficient (SCID) mouse engrafted with human peripheral blood lymphocytes (PBLs) is a potentially useful model for the study of cancer immunotherapy. For this application, rapid, consistent, and high level engraftment of SCID mice with functional human cytotoxic effector cells is necessary. To date, short term human lymphoid cell engraftment in SCID mice has generally been low and variable. Further, most of the human cells detected within the first 30 days are found in the peritoneal cavity. The purpose of the present study was to improve short term reconstitution of human PBLs in the SCID mouse. When untreated SCID mice were injected with human PBLs, the mean level of CD3+ cells in the spleens was < 5% on days 6-32 after injection, as determined by flow cytometry (FCM). Depletion of SCID mouse natural killer (NK) cells with anti-asialo GM1 only marginally improved short term reconstitution with human CD3+ cells. Preirradiation of SCID mice with 3 Gy improved reconstitution to over 16% CD3+ cells on days 12-14 following engraftment. However, the combination of pretreatment with anti-asialo GM1 plus radiation, significantly increased the mean percentage of human CD3+ cells in the spleen to 40% within 2 weeks following injection of PBLs. Human T cells positive for CD4, CD8, TcR alpha beta, and TcR gamma delta, and human NK and B cells were detected in the spleens of irradiated plus anti-asialo GM1 pretreated SCID mice. The presence of human lymphoid cells was confirmed by immunohistologic staining. The human immune cells in these mice were shown to be functional by the in vivo demonstration of an appropriate secondary immune response to the injection of tetanus toxoid and by an in vivo proliferative response to phytohemagglutinin. Human NK cells could be found in the spleens and peripheral blood of irradiated plus anti-asialo GM1 pretreated mice. These cells were also shown to be competent by their ability to lyse the human NK sensitive tumor targets K562 and MOLT-4 in 51Cr release assays. Thus, pretreatment of SCID mice with radiation plus anti-asialo GM1 significantly improves short term human PBL engraftment and provides a potentially useful model for the study of cancer immunotherapy.

Topics: Animals; CD4-Positive T-Lymphocytes; G(M1) Ganglioside; Graft vs Host Disease; Humans; Immunoglobulin G; Killer Cells, Natural; Lymphocyte Transfusion; Mice; Mice, SCID; Severe Combined Immunodeficiency; Spleen; T-Lymphocytes, Regulatory; Transplantation, Heterologous

In order to meet the present increased demands, we have tried to improve the methods to produce human monoclonal antibodies by using a SCID mouse grafted with human mononuclear cells. Initially, we gave an anti-asialo GM1 antibody to a SCID mouse to suppress the NK activity, as a pretreatment. Then, fifty million human mononuclear cells (MNC) from a healthy volunteer were injected intraperitoneally to the SCID mouse so as to construct a human immune system in the mouse (PBL-SCID mouse). We immunized the mouse with a synthetic peptide (pep 190) conjugated with KLH four times. The spleen cells taken from the immunized PBL-SCID mouse were fused with (mouse x human) heteromyeloma cells. The hybridoma cells were selected in GIT medium containing HAT and IL-6. Among 68 hybridoma-growing wells, we obtained one hybridoma clone (#41-1-4) which secreted a specific antibody to pep 190. The reactivity of this monoclonal antibody was tested by ELISA and the specificity of this antibody was confirmed by an absorption test with different kinds of proteins. This paper is the first report of the successful production of peptide-specific human monoclonal antibody by active in vivo immunization using a PBL-SCID mouse. By this active in vivo immunization system using a PBL-SCID mouse, human monoclonal antibodies for any sort of peptide antigens may easily be made available.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Epitopes; ErbB Receptors; Female; G(M1) Ganglioside; Humans; Hybridomas; Lymphocyte Transfusion; Mice; Mice, SCID; Molecular Sequence Data; Peptides; Proto-Oncogene Proteins; Receptor, ErbB-2; Severe Combined Immunodeficiency; Tumor Cells, Cultured; Vaccination

Mice with immunodeficiency provide an excellent in vivo model for cell transfer experiments. In this study, we compare the extent of immune deficiency of the original CB17 severe combined immune-deficient (SCID) mice with that of two other strains of immune-deficient mice, the recently developed C3H SCID mice and the beige/nude/X-linked immune-deficient (BNX) mice. Detectable levels of serum lg (higher than 0.4 microgram/ml) were found in 79% of CB17 SCID mice studied (n = 24) and in all BNX mice (n = 12); some leaky CB17 SCID mice had normal levels of Ig. In contrast, only 15% of C3H SCID mice (n = 61) had detectable serum lg; the highest Ig level in this strain was 9.6 micrograms/ml. Age had no effect on serum Ig concentrations of C3H SCID mice; in contrast, all old (> 1-year-old) CB17 SCID mice studied had detectable levels of serum Ig. Transfer of syngeneic, normal, neonatal thymocytes increased serum Ig of SCID mouse origin to near-normal levels in all CB17 SCID mice but had no effect on serum lg concentrations in C3H SCID mice. Treatment with anti-asialo-GM-1 antiserum to abrogate NK cell activity increased serum Ig levels in 37% of CB17 SCID mice but had no effect on Ig production in C3H SCID mice. Flow cytometric analysis failed to identify mature T or B cells in C3H SCID mice; in contrast, some leaky CB17 SCID mice had detectable numbers of T and B cells in the peritoneal cavity. After immunization with bacteriophage phi X 174, neither C3H nor CB17 SCID mice, including leaky mice, produced specific antibody to phage. In contrast, BNX mice produced small but significant amounts of anti-phage antibody. These results indicate that, of the three strains of immune-deficient mice, C3H SCID mice have the most severe immune defect. We predict that C3H SCID mice will be best suited for cell transfer experiments.

Topics: Animals; Antibodies, Viral; Antigens, Surface; Bacteriophage phi X 174; G(M1) Ganglioside; Immune Sera; Immunoglobulins; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, SCID; Severe Combined Immunodeficiency; Species Specificity; T-Lymphocytes