Sylvia B. Pollack

Prevention of rejection of allogeneic bone marrow transplants by NK 1.1 antiserum.

We have studied the effect of in vivo administration of NK 1.1 antiserum on two functions of natural killer (NK) cells: (A) in vitro cytotoxicity of B6 mice to T cell lymphoma, YAC-1, and (B) potential of B6 mice to reject allogeneic BALB/c bone marrow transplants. We demonstrated that a single i.v. injection of 0.4 ml of NK 1.1 antiserum significantly reduced in vitro NK cell cytotoxic potential and concomitantly prevented rejection of allogeneic bone marrow transplants. NK 1.1 antiserum was effective in diminishing both of these functions when injected 2, 6, or 24 hr before transplantation and NK cell assay, but it was ineffective when given 48 hr before transplantation or the NK cell test. As a specificity control, we investigated the effect of specific anti-T-cell-directed monoclonal antibodies, Thy 1.2 and Lyt 2.2, in the same systems. Neither of these antibodies exerted any effect on NK cell cytotoxicity to YAC-1 or rejection of allogeneic bone marrow transplants. These studies indicate that NK cells represent one of the major components of the mechanism of bone marrow graft rejection.

Self-generating density gradients of Percoll provide a simple and rapid method that consistently enriches natural killer cells

The separation or enrichment of natural killer (NK) cells from the heterogeneous cell populations in murine spleen or bone marrow is a vital step for the study of NK cells. We report in this study a simple and rapid method for the enrichment of NK cells from B cell-depleted spleen cells, using a self-generating density gradient of polyvinyl pyrrolidone-coated silica (Percoll). Nylon wool-passed spleen cells are suspended in Percoll that is isotonic and isosmotic with mouse blood at a density of 1.087 g/ml and ultracentrifuged at 30,000 x g for 10 min. This method consistently enriches for NK cell cytotoxic activity, in spleen cells of both unstimulated and interferon-stimulated mice, as measured in the chromium release assay. There is a concomitant enrichment for cells bearing the NK marker asialo GM-1 and depletion of L3T4 or Lyt-2-bearing T cells. In contrast to discontinuous, step-wise gradients, the self-generating Percoll gradient, which relies on the intrinsic property of Percoll to form a continuous density gradient, appears to provide the cells with a physiological environment both before and during the centrifugation step.

In vivo functions of natural killer cells

This review focuses on recent experiments in which the natural killed (NK) compartment has been directly manipulated in vivo either by passive transfer of NK-enriched cell populations or by selection depletion of NK cells. These data have provided direct evidence for the role of NK cells in vivo. It is evident that even these experiments have inherent limitations due to the complexity of in vivo interactions. In the aggregate, however, these data build a compelling case for the in vivo activity of NK cells and for their biologic importance. Most of the experiments were carried out in mice. Although there is heterogeneity among NK cells, these studies deal mainly with classical NK cells defined as bone marrow-derived, non-B (Ig/sup -/), non-T (Lyt 1/sup -/2/sup -/) lymphocytes that are nonadherent and bear the NK-associated antigens NK-1 and asialo-GMl. A natural model which has been exploited to study NK cells in the intact host is also discussed.

DIRECT EVIDENCE FOR ANTI-TUMOR ACTIVITY BY NK CELLS IN VIVO: GROWTH OF B16 MELANOMA IN ANTI-NK 1.1-TREATED MICE

Publisher Summary This chapter examines evidence for anti-tumor activity by natural killer (NK) cells in vivo. A model was developed to directly test the role of NK cells in vivo by specific depletion of cells: C57B1/6 (B6) mice were depleted of NK cells by in vivo treatment with antisera to the NK-associated alloantigen NK 1.1. When tested in vitro in the presence of complement, anti-NK 1.1 kills fewer than 5% of B6 spleen cells (SC), has no effect on cytolytic T cells or plaque forming cells. Anti-NK 1.1 alone does not affect NK activity. Injection of B6 mice with 25 μl of anti-NK 1.1 reduced the ability of their SC to lyse YAC-1 in vitro by 70% within 2 h. NK activity gradually returned to control levels but still was significantly depressed at 48 h. Decreased NK activity was comparable when the antiserum was injected i.p. or i.v. In a series of four experiments, tumor cell clearance was reduced 2–4-fold in the anti-NK 1.1 treated mice compared to injection controls. These results provided direct evidence that NK cells play an in vivo role in the elimination of circulating tumor cells.

Inhibition by autologous lymphoid cells of antibody-dependent cellular cytotoxicity (ADCC) to a tumor target

Abstract Murine lymph node cells (LNC), which we showed previously to noncompetitively inhibit antibody-dependent cellular cytotoxicity (ADCC) to an erythrocyte target, were tested for their ability to inhibit ADCC to a tumor target, EL-4. Both a 4-hr 51 Cr-release cytotoxicity assay and an overnight 125 IUdR (iododeoxyuridine) postlabeling cytostasis assay were used. Normal autologous lymph node cells inhibited spleen cell-mediated ADCC in both assays. Inhibition by LNC was dose dependent, but comparable numbers of sheep erythrocytes did not inhibit, indicating that LNC-mediated inhibition was not simply a matter of crowding. Inhibitory activity was enriched in LNC after removal of Fc receptor-bearing cells on EA monolayers.

Characterization of natural killer cell activity in Macaca nemestrina

Natural killer (NK) cell activity was evaluated in three groups of Macaca nemestrina that varied with respect to SAIDS D retrovirus serotype 2 (SRV‐2/W) and viremic status. Target cells used were Raji and K562 cells. No significant differences (ANOVA) in mean NK activity were detected among the three groups of animals studied. Using Raji targets, mean LU30/106 ± SEM was 6.3 ± 1.6 for seronegative (V‐Ab−) animals, 7.3 ± 1.5 for seropositive (V‐Ab+) animals, and 10.2 ± 3.5 for persistently viremic (V + Ab−) animals. Using K562 targets, mean LU30/106 was 7.6 ± 1.7 for seronegative (V‐Ab−) animals, 6.5 ± 2.5 for seropositive (V‐Ab+) animals, and 5.1 ± 1.9 for persistently viremic (V+Ab−) animals. Percentage blood CD16+ and CD8+cells also were not different in the three groups of animals. NK activity did not always correlate with percentage of CD16+ or CD8+ cells in peripheral blood at the time the assays were done. In persistently viremic animals, there was a strong positive correlation between percent CD16+ and CD8+ cells and NK activity using K562 cells but not Raji cells. Depletion experiments indicated that lysis was mediated by both CD8+ and CD16+ cells with both Raji and K562 cells. However, Raji targets were a better indicator of killing mediated by CD16+ cells. Our studies indicate that M. nemestrina may be classified as high or low responders with regard to NK activity, and there was no correlation with SRV‐2/W viral or antibody status. Additionally, our results suggested that group housing of M. nemestrina was usually associated with increased NK activity. In conclusion, studies of NK activity in M. nemestrina should consider target cells used, phenotype of effectors, endogenous (high or low) levels of NK activity in individual animals, and housing conditions.

ANTI-NK 2.1: AN ACTIVITY OF NZB ANTI-BALB/c SERUM

Publisher Summary This chapter presents a study focusing on anti-NK 2.1. Anti-NK 1.1 was raised by immunizing BALB/c × C3H F-1 mice with CE spleen cells (SC). The same immunization schedule was used to raise NZB anti-BALB/c, that is, NZB/B1NJ mice were injected intraperitoneally with progressively greater doses of BALB/c SC at 2-week intervals. After the fourth immunization, the NZB mice were bled individually and their sera tested for C dependent depletion of NK activity. BALB/c SC were treated with antiserum and C or antiserum alone in a one-step lysis and then tested at 3 effector:target ratios on 51Cr-labeled YAC-1 target cells. The sera were also screened for autoantibody by dye exclusion assay on BALB/c SC. Sera with autoantibody or low titers of anti-NK activity were discarded and the remaining sera pooled. Sera were subsequently obtained on days 4 and 7 after further immunizations. Analysis of NZB anti-BALB/c serum revealed that antibodies to NK-associated antigens were indeed present but that the activity was not to an allele of NK1. The data suggests that this antiserum detects an NK-associated antigen provisionally designated NK 2.1, which is neither an allele of nor linked to NK 1.