asialo-gm1-ganglioside and Immunologic-Deficiency-Syndromes

Despite major deficits in their immune system, SCID, Nude, and NIH III mice reject allo- and xenografts, particularly leukemic cell lines, albeit less readily than immunologically intact mice. Since variation among these immunodeficient mouse strains in rejection of a human lymphoid cell line (CCRF-CEM) parallels splenic non-MHC-mediated cytolytic activity, non-MHC-restricted cytolytic activity may be responsible for retained resistance to leukemic cell transplantation. SCID mice that had the least cytolytic activity accepted 100% of their grafts. The converse was true for NIH III mice that showed the greatest cytolytic activity and were relatively resistant to CEM cell engraftment. Different approaches to ablate NK activity and thus enhance engraftment led to variable results for each strain. A single dose (500 micrograms) of anti-asialoGM1 (AsGM1) markedly reduced NK activity in SCID and NIH III mice by 60 and 40%, respectively. A moderate 20% decrease was seen in Nude mice at this dose. In contrast, gamma irradiation suppressed NK activity by greater than 80% of baseline levels in all three strains. Of importance, total cytolytic activity in immunosuppressed Nude and NIH III mice, although significantly depressed compared to untreated mice of the same strain, still remained higher than that seen in nonimmunosuppressed SCID mice. Enhanced engraftment and systemic dissemination of CEM cells in immunosuppressed mice correlated directly with decreased total splenic cytolytic activity in all three strains. These results have implications for the use of immunodeficient models for transplantation, tumor immunobiology, and engraftment of a human immune system.

Topics: Animals; Cytotoxicity, Immunologic; Female; G(M1) Ganglioside; Humans; Immunologic Deficiency Syndromes; Immunosuppression Therapy; Lymphocyte Transfusion; Major Histocompatibility Complex; Mice; Mice, Inbred BALB C; Mice, SCID; Transplantation, Heterologous

To develop a model of human thymus growth in vivo, we have implanted postnatal human thymus under the renal capsule of severe combined immune deficient (SCID) mice and assayed for graft survival and graft characteristics 1-3 mo after engraftment. Three groups of SCID mice were engrafted with postnatal human thymus: untreated SCID mice, SCID mice pretreated with 400 cGy of gamma irradiation 1-5 d before engraftment, and SCID mice treated with intraperitoneal anti-asialo GM-1 antiserum every 4-5 d during engraftment. In the untreated group of SCID mice, only 37% of grafts survived and consisted of human thymic microenvironment components and human immature thymocytes. Irradiation of SCID mice before engraftment improved survival of human thymic grafts to 83%, but these grafts were largely devoid of thymocytes and contained only thymic microenvironment components with large numbers of thymic macrophages. Treatment of SCID mice with anti-asialo GM-1 antiserum throughout the engraftment period also promoted human thymus engraftment (70%) and induced SCID B cell Ig production (SCID[Ig+]) in 38% of animals. In SCID(Ig-) anti-asialo GM-1-treated mice, the human thymic grafts were similar in content to those in untreated SCID mice. However, in anti-asialo GM-1-treated animals with grafts that became SCID(Ig+), all animals were found to have mouse-human chimeric grafts in that the human thymic microenvironment (human fibroblasts, thymic epithelium, vessels) was colonized by murine T cells. These data demonstrate that human postnatal thymus will grow as xenografts in SCID mice, and that the components of human thymus that engraft are dependent on the immunosuppressive regimen used in recipient mice. A striking finding in this study was the induction of T and B lymphopoiesis in SCID mice by abrogation of NK cell activity with in vivo anti-asialo GM-1 treatment. These data strongly suggest that asialo GM-1+ NK cells and/or macrophages play a role in mediation of suppression of lymphopoiesis in SCID mice.

Topics: Animals; Female; G(M1) Ganglioside; Glycosphingolipids; Humans; Immunoglobulins; Immunologic Deficiency Syndromes; Immunophenotyping; Immunosuppression Therapy; Infant; Infant, Newborn; Male; Mice; T-Lymphocytes; Thymus Gland; Transplantation, Heterologous; Whole-Body Irradiation

The relationship between NK cell and T cell progenitors was investigated by using mice with severe combined immune deficiency (scid). Scid mice are devoid of mature T and B cells because they cannot rearrange their Ig and TCR genes. However, they have normal splenic NK cells. Thymus of scid mice, although markedly hypocellular, contains cells that lyse YAC-1, an NK-sensitive tumor cell. By flow cytometry, two populations of cells were identified in the scid thymus. Eighty percent of the cells were Thy-1+, IL-2R(7D4)+, J11d+, CD3-, CD4-, CD8- whereas the remaining were IL-2R-, J11d-, CD3-, CD4-, and CD8-. By cell sorting, all NK activity was found in the latter population, which is phenotypically similar to splenic NK cells. To determine if the thymus contains a bipotential NK/T progenitor cell, J11d+, IL-2R+ cells were cultured and analyzed for the generation of NK cells in vitro. These cells were used because they resemble 15-day fetal and adult CD4- CD8- thymocytes that are capable of giving rise to mature T cells. Cultured J11d+ thymocytes acquired non-MHC-restricted cytotoxicity, but in contrast to mature NK cells, the resulting cells contained mRNA for the gamma, delta, and epsilon-chains of CD3. This suggests that J11d+ cells are early T cells that can acquire the ability to kill in a non-MHC-restricted manner, but which do not give rise to NK cells in vitro. The differentiative potential of scid thymocytes was also tested in vivo. Unlike bone marrow cells, scid thymocytes containing 80% J11d+ cells failed to give rise to NK cells when transferred into irradiated recipients. Together these results suggest that mature NK cells reside in the thymus of scid mice but are not derived from a common NK/T progenitor.

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; Blotting, Northern; CD3 Complex; Cytotoxicity, Immunologic; G(M1) Ganglioside; Glycosphingolipids; Immunologic Deficiency Syndromes; Interferon Type I; Interleukin-2; Ionomycin; Killer Cells, Natural; Mice; Mice, Mutant Strains; Receptors, Antigen, T-Cell; Recombinant Proteins; RNA, Messenger; Spleen; Tetradecanoylphorbol Acetate; Thymus Gland

Splenocytes from mice of genotype scid/scid.bg/bg were tested in vitro to characterize the nature of the immunological deficit in these doubly mutant animals. The cells were unresponsive to the mitogens LPS and Con A and to alloantigens, as predicted for scid/scid genotype. Splenocytes from scid/scid.bg/bg lysed the NK cell-sensitive target cell line YAC at levels approximately 50% lower than those observed for scid/scid splenocytes. Splenocytes from SCID-beige mice failed to lyse the NK-resistant, LAK-sensitive cell line P815 but showed high levels of activity against the murine placental cell line Be6. Lytic activity was found in both nonadherent and plastic adherent cells and was eliminated by pretreatment of the effectors with anti-asialo-GM1 and complement. Incubation of 1 x 10(5) splenocytes with hrIL-2 failed to induce blastogenesis in scid/scid.bg/bg cells but produced a response in cultures of scid/scid or bg/bg spleen cells. However, blastogenesis and elevated levels of LAK-type killing were observed following incubation of higher numbers of scid/scid.bg/bg splenocytes in hrIL-2. Thus, doubly mutant scid/scid.bg/bg mice have reduced NK cell activity, in comparison to scid/scid mice, and appear to possess LAK-like effector cells and LAK cell precursors.

Topics: Animals; Cell Adhesion; Concanavalin A; Cytotoxicity, Immunologic; G(M1) Ganglioside; Genotype; Glycosphingolipids; Immunity, Cellular; Immunologic Deficiency Syndromes; Interleukin-2; Killer Cells, Lymphokine-Activated; Killer Cells, Natural; Lipopolysaccharides; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Mice; Mice, Mutant Strains; Recombinant Proteins; Spleen

The lineage of lymphokine-activated killer (LAK) cells is poorly understood. To examine the relationship between LAK and natural killer (NK) cells we utilized two congenitally immunodeficient mice, namely severe combined immunodeficient (scid) and athymic (nude) mice that lack T cells but have normal NK cells. LAK activity was evaluated by the ability to lyze NK-resistant P815 cells. When cultured with human recombinant interleukin 2, splenocytes of scid and nude mice could generate LAK activity at levels comparable to or more than those of normal C.B-17 mice. LAK effector cells in these immunodeficient mice as well as normal mice had the phenotype resembling that of NK cells with asialo-GM1 (aGM1) expression. In vivo treatment with anti-aGM1 antiserum completely abolished the induction of LAK activity from splenocytes of normal mice. In contrast, LAK activity in splenocytes of scid and nude mice was still demonstrable even after this treatment, indicating that most LAK precursors in both mice were cells without aGM1 antigen. The aGM1- progenitors for LAK activity, probably in common with NK progenitors, appeared to be more expanded in scid and nude mice than in normal mice. The use of such congenitally immunodeficient mice should be helpful in studying the differentiation step of LAK as well as NK cells from their precursors.

Topics: Animals; Antigens, Differentiation; Cell Differentiation; Cytotoxicity, Immunologic; G(M1) Ganglioside; Glycosphingolipids; Immunity, Cellular; Immunologic Deficiency Syndromes; Interleukin-2; Killer Cells, Natural; Mice; Mice, Mutant Strains; Mice, Nude; Spleen

We have examined the ability of in vivo treatment of mice with recombinant interleukin 2 (rIL-2) to affect natural immunity measured against tumor (YAC-1) or virally infected (herpes simplex type 1) target cells. rIL-2 treatment leads to significant increases in natural killer/lymphocyte-activated killer (NK/LAK) function and spleen cells recovered. This effect is dose dependent and strain related. The latter parameter correlated with the pretreatment NK activity level of the strain. The rIL-2 induced NK/LAK augmentation is also kinetically restricted as treatment must have occurred within 48-72 h of assay to be effective. The rIL-2 therapy effectively enhances both antitumor and antiviral NK/LAK activity and results in a noticeable increase in asialo-GM1-positive cells in the spleens of treated mice as well as a significant increase in IL-2 receptor expression as monitored by either cytometry or radioligand binding. In vivo treatment of mice with an antibody directed to the ASGM1 determinant effectively reduces the rIL-2 augmentation of both antitumor and antiviral activity even though this treatment does not affect the pretreatment level of antiviral activity. Various natural and induced immunodeficiency states (immunotherapy, irradiation, immunosuppressive drugs, cytoreductive drugs) have been examined for the ability of in vivo treatment with rIL-2 to enhance NK/LAK activity. In vivo rIL-2 administration is differentially effective in enhancing NK/LAK activity in these situations. Notably, in these induced immunodeficiency states, although NK/LAK activity is commonly enhanced, the number of spleen cells recovered often is only marginally affected. Thus, as expected, a limiting aspect in this use of a natural immunomodulator is the number of potentially responsive cells present in the immunodeficiency condition. In addition, correlations between rIL-2 effect, several of the immunodeficiency states, and vascular leak syndrome are briefly discussed.

Topics: Animals; Antineoplastic Agents; Dose-Response Relationship, Drug; Female; G(M1) Ganglioside; Glycosphingolipids; Immunologic Deficiency Syndromes; Immunosuppressive Agents; Interleukin-2; Killer Cells, Natural; Male; Mice; Mice, Inbred Strains; Neoplasms, Experimental; Receptors, Interleukin-2; Recombinant Proteins; Species Specificity; Virus Diseases; Whole-Body Irradiation

We have studied the influence of interferons (IFNs) on the metastatic potential of mouse colon adenocarcinoma, COLON 26, cells. Pre-treatment of the cells in vitro for 24 hr with recombinant murine IFN-gamma (rMuIFN-gamma) significantly increased the number of lung tumour nodules when cells were injected i.v. into immunocompetent BALB/c mice and BALB/c nude mice. However, when MuIFN-gamma-pre-treated cells were injected into beige (NK-deficient) nude mice or anti-asialoGM 1 (asGM 1)-serum-treated BALB/c mice (NK-depleted) no enhancement of metastatic potential was seen. Pre-treatment of COLON 26 cells with recombinant human IFN-alpha A/D (Bg1 I), an IFN with equal activity on human and mouse cells, did not significantly enhance their subsequent metastases in immunocompetent or immunodeficient mice. In fact, there was a small but significant decrease in the number of tumour nodules in the lungs of beige nude and asGM 1-treated mice. The effects of rMuIFN-gamma on COLON 26 cells did not appear to be related to an alteration in MHC expression. COLON 26 cells constitutively express H-2D and H-2K antigens and both IFNs had equal enhancing (approx. 2-fold) activity on the expression of these antigens at the doses used in this experiment (10(3)U/ml). We conclude that pre-treatment with rMuIFN-gamma renders COLON 26 cells resistant to in vivo NK-cell lysis via a mechanism that does not involve changes in MHC expression.

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Cytotoxicity, Immunologic; G(M1) Ganglioside; Glycosphingolipids; H-2 Antigens; Immunologic Deficiency Syndromes; Interferon-gamma; Killer Cells, Natural; Lung Neoplasms; Macrophages; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Tumor Cells, Cultured

The ability of in vitro addition of recombinant interleukin 2 (rIL-2) to differentially enhance natural cytotoxicity was assessed using cells from mice with natural and induced cellular defects. In vivo treatment with most immunosuppressive or cytoreductive agents, anti-asialo-GM1 antibody, or gamma irradiation dramatically reduced in vitro cytotoxicity against natural killer (NK) sensitive targets by direct reduction in either percentage specific lysis or lytic units per spleen. In most cases, in vitro addition of rIL-2 (at concentrations causing augmented NK function in cells from naive Balb/C mice) enhanced cytotoxic activity of cells from treatment groups to a normal value but not within the rIL-2-enhanced range of nontreated animals. Additionally, cytotoxic activity of cells from animals treated with certain drugs or gamma irradiation could be augmented by rIL-2 when measured by percentage lysis but not lytic units per spleen. In vivo treatment with cyclosporin A did not affect natural cytotoxic activity and addition of rIL-2 augmented the NK activity in a similar fashion to the profile of naive cells. In experiments using cells from beige (C57Bl/6-bg) mice which have a natural defect in NK activity against YAC-1 targets, addition of rIL-2 (at concentrations causing augmented natural cytotoxic function in cells from C57Bl/6 mice) could not effectively enhance in vitro natural cytotoxic function.

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Agents; Cytotoxicity, Immunologic; G(M1) Ganglioside; Glycosphingolipids; Immune Sera; Immunity, Innate; Immunologic Deficiency Syndromes; Immunosuppressive Agents; Interleukin-2; Mice; Mice, Inbred AKR; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Mutant Strains; Recombinant Proteins; Whole-Body Irradiation

H-2+ and H-2- cells of B16 melanoma were established by repeated fluorescence-activated cell sorting. The H-2- line formed no metastasis in untreated C57BL/6 mice, whereas the H-2+ cells showed evidence of metastatic development. This difference was ascribed mainly to the increased susceptibility of H-2- cells to attack by natural effector mechanisms, particularly asialo GM1+ NK cells. After treatment with both anti-asialo GM1 serum and whole body irradiation (400 rad), numerous colonies of H-2- cells formed in the lung, whereas the metastasis was only marginally enhanced by irradiation and moderately by treatment with anti-asialo GM1 serum. With the H-2+ cells, treatment with each modality significantly increased the number of metastatic colonies. Therefore collaboration of asialo GM1+ NK cells and radiosensitive natural effectors seems to be the main mechanism involved in the synergistic effects on defense against H-2- cell metastasis, and to a lesser extent against H-2+ cell metastasis. Irradiation (1000 rad) to the right lung to abrogate the organ-associated defense increased the colonies, particularly in the H-2+ cells. On the other hand, treatment with anti-asialo GM1 serum increased colonization in the early phase of metastasis with H-2- cells and may have abolished asialo GM1+ NK cells capable of recognizing the reduced expression of H-2 antigens and eliminating H-2- cells in the blood-born phase. Natural defense mechanisms probably exert suppressive effects on the metastasis of H-2+ cells, mainly in the organ-associated phase after extravasation.

Topics: Animals; Cell Line; Cell Survival; Female; G(M1) Ganglioside; Glycosphingolipids; H-2 Antigens; Immune Sera; Immunization, Passive; Immunologic Deficiency Syndromes; Killer Cells, Natural; Lung Neoplasms; Lymphatic Metastasis; Lymphocyte Cooperation; Melanoma; Mice; Mice, Inbred C57BL; Phenotype; Spleen

Mice with severe combined immunodeficiency syndrome (SCID) exhibit an impairment in both T and B cell maturation, whereas myelopoiesis remains unaffected. We report here that spleens from SCID mice have undergone phenotypic expansion of cells bearing the NK-2 and asialo GM1 markers (70-80%) characteristic of NK cells and this expansion is accompanied by a 3-4-fold enrichment in NK cytolytic activity over their normal C.B-17 littermates. Furthermore, the NK cells from SCID mice do not rearrange or express T cell receptor alpha or beta genes, or a third T cell rearranging gene, gamma. These findings suggest that (a) T cell receptors are not necessary for NK-mediated cytolysis, and (b) either NK cells constitute an entirely distinct lineage or NK cell function is acquired in pre-T cells prior to the expression of T cell receptor genes.

Topics: Animals; Female; G(M1) Ganglioside; Glycosphingolipids; Immunologic Deficiency Syndromes; Killer Cells, Natural; Male; Mice; Phenotype; Receptors, Antigen, T-Cell; Recombination, Genetic; Transcription, Genetic

Spleen cells from C.B- 17 scid mice with severe combined immunodeficiency disease exhibit natural killer cell (NK) activity against YAC lymphoma targets in a standard 4-hr 51Cr release assay. The cytolytic activity is demonstrable only at high effector to target ratios but can be augmented at least sevenfold by the interferon inducer poly I:C. The pattern of target lysis is specific, because splenocytes from poly I:C-primed C.B-17 scid mice lyse NK-sensitive YAC cells and not the insensitive P815 mastocytoma. The presence of several NK-associated antigens on C.B-17 scid splenocytes was tested by pretreating cells with the appropriate antiserum plus complement before testing for NK activity. The results indicate that a proportion of NK effectors in C.B-17 scid mice bear surface NK 2.1 and Asialo GM1 but are negative for Thy-1.

Topics: Animals; Antibodies, Monoclonal; Cytotoxicity, Immunologic; G(M1) Ganglioside; Glycosphingolipids; Immunologic Deficiency Syndromes; Isoantibodies; Killer Cells, Natural; Lymphoma; Mice; Mice, Mutant Strains; Phenotype; Spleen