anti-NK Activity Research Articles

Heterogeneity in the effect of different ixodid tick species on human natural killer cell activity.

Tick saliva plays a vital role in blood-feeding, including manipulation of the host response to tick infestation. Furthermore, a diverse number of tick-borne pathogens are transmitted to vertebrate hosts via tick saliva, some of which exploit the immunomodulatory activities of their vector's saliva. We report that salivary gland extracts (SGE) derived from Dermacentor reticulatus adult ticks induce a decrease in the natural killer (NK) activity of effector cells obtained from healthy human blood donors. The decrease was observed with SGE from both female and male D. reticulatus fed for either 3 or 5 days on mice, but no significant effect was observed with SGE from unfed ticks or ticks that had fed for 1 day. These results indicate that the tick anti-NK factor(s) is only active after blood-feeding has commenced. Microscopic examination revealed that the first step of NK activity, namely effector/target cell conjugate formation, was affected by SGE. The observed reduction in conjugate formation occurred when effector (but not target) cells were treated with SGE for 30 min, and the effect persisted after 12 h of treatment. Similar but less potent anti-NK activity was detected for SGE from Amblyomma variegatum and Haemaphysalis inermis. By contrast, SGE derived from Ixodes ricinus and Rhipicephalus appendiculatus female ticks did not decrease NK activity. The apparent absence of such activity in these two important vectors of tick-borne viruses suggests that control of NK cells does not play an important role in promoting virus transmission, at least for these particular species.

Association of natural killer cell depletion with induction of mixed chimerism and allograft tolerance in non-human primates.

Nonmyeloablative T cell depletion followed by donor bone marrow infusion has proved to be an effective approach to induction of mixed chimerism and tolerance of organ allografts in non-human primates. To help define the mechanisms involved we have compared T cell depletion with ATG versus anti-CD2 monoclonal antibody with respect to establishment of mixed chimerism and induction of tolerance. Both nonmyeloablative regimens included low dose total body irradiation (1.5 Gy x 2), thymic irradiation (7 Gy), splenectomy and kidney plus donor bone marrow transplantation, followed by a 4-week posttransplant course of cyclosporine. In addition, the ATG group (13 recipients) received antithymocyte globulin, although the LOCD2b group (10 recipients) were treated with an anti-CD2 monoclonal antibody (LOCD2b). In the ATG group, 11 of 13 monkeys developed multilineage chimerism and 9 survived for more than 100 days without kidney allograft rejection. In contrast, 0/10 monkeys in the LOCD2b group developed chimerism, 5 died of infection and 5 suffered progressive rejection; only 1 recipient survived beyond 100 days. Sequential monitoring of peripheral blood mononuclear cells revealed greater T cell (CD3+) depletion in the LOCD2b-treated animals compared to those receiving ATG. However, NK cells (CD16+CD8+) were significantly more depleted in the ATG group and NK function remained abrogated longer after ATG than LOCD2b treatment (3 weeks vs. <5 days). Despite excellent T cell depletion by LoCD2b, ATG was more effective in inducing chimerism and tolerance. This difference correlated with anti-NK activity of the two reagents. These data suggest that NK cells may also resist engraftment of allogeneic bone marrow cells in this model.

Expression of natural killer (NK) cell-specific alloantigens on a mouse NK-like cell line.

As part of the strategy for screening for natural killer (NK) cell-specific monoclonal antibodies (MoAb) we have raised a number of murine NK-like cell lines in media containing interleukin-2 (IL-2). The detection of specific NK cell alloantigens on a C57BL/6 cell line in long-term culture in IL-2 is the subject of this paper. The C57BL/6 cell line has the morphology of large granular lymphocytes (LGL) and exhibits strong cytolytic activity against the archetype NK cell target, YAC-1. Absorption of three anti-NK antiserum, NZB anti-BALB/c (anti-NK-2.1), BALB/c anti-DBA/2 (anti-NK-3.1) and CE anti-CBA (anti-NK-4.1), with the C57BL/6 cell line removed the anti-NK activity from these antisera. Flow cytometric studies of the C57BL/6 cell line demonstrated significant binding of the anti-NK-1.1 MoAb produced by hybridoma PK136. Our results suggest that the C57BL/6 NK-like cell line exhibits some of the properties of naive NK cells and expresses all the known NK cell-specific alloantigens, NK-1.1, NK-2.1, NK-3.1 and NK-4.1 and therefore is potentially useful in selecting NK specific hybridomas and in studying the biology of NK cells.

Preparation of monoclonal antibodies against a glycolipid asialo GM1.

Five IgM monoclonal antibodies (MAbs), MW-1, MW-2, MW-3, MW-4, and MW-5, against a glycolipid asialo GM1 were prepared from hybridoma clones obtained by the fusion of mouse NS-1 myeloma cells with spleen cells from a mouse immunized with asialo GM1 adsorbed to naked Salmonella. All the MAbs reacted only with asialo GM1 when their reactivities were examined by enzyme-linked immunosorbent assay (ELISA) and thin-layer chromatography (TLC)-immunostaining using structurally related glycolipids. The MAbs showed a complement-dependent lysis of mouse natural killer (NK) cells, but the lytic activities were weaker than that of a rabbit polyclonal anti-asialo GM1 antibody. When they were mixed, the anti-NK activity was increased to a level almost comparable to that of the polyclonal antibody. These results suggest that all the MAbs obtained are specific for asialo GM1 and that they may be different in fine specificity for the glycolipid. Significance of the MAbs in immunological and neurochemical studies is discussed.

Role of natural killer (NK) cells in the production of the murine T lymphocyte allorecognition repertoire

Limiting dilution cultures of alloreactive (anti-H2K b) CTL were established from thymocyte or spleen cell pools of C3H/HeJ and their congenic bg/bg partner, or of SJL/J and their congenic bg/bg partner. CTL populations in these cultures were assayed for cross-reactive lysis of a panel of splenic Con A blasts of H2K bm mutant mice. There was some slight elevation of frequency of CTL p in the thymocyte lymphoid pool of bg/bg mice; more strikingly, the repertoire of anti-H2K b specificities was clearly altered in both strains in the bg/bg animals. There was apparently an increased diversity (more specificities represented at higher frequencies) in the thymocyte pool and a decreased diversity in the spleen cell CTL p pool in animals with the bg/bg marker. Similar shifts in the allorecognition repertoire of normal C3H/HeJ mice were produced by inoculation of neonatal mice with a rabbit anti-NK heteroantibody (antiasialo GM1). Preabsorption of this serum such that it lost anti-NK activity also abolished this effect of in vivo neonatal injection. Furthermore, injection of bg/bg bone marrow-reconstituted C3H/HeJ ( bg/+) mice with a C3H spleen cell-derived NK line also caused a shift in the allorecognition repertoire toward that seen in the normal littermate control animals.

Cytotoxic antibodies to natural killer cells in systemic lupus erythematosus

Natural killer (NK) cell function is frequently depressed in systemic lupus erythematosus (SLE) and an NK cell inhibitory activity is present in the serum. In an effort to determine whether autoantibodies to NK cells are made by patients with SLE, serum specimens from 11 patients with SLE were examined for the presence of anti-NK antibodies. Anti-NK cell antibody was detected in 7 of 11 specimens tested in a microcytotoxicity assay using highly enriched populations of NK cells from normals prepared by discontinuous Percoll density centrifugation. None of four control sera was positive. Both IgM and IgG antibodies were identified, but only a single class of immunoglobulin was observed in plasma from any one patient. There was no noticeable relationship between SLE clinical disease activity and the presence of anti-NK cell antibody. Specificity of the anti-NK cell antibody contained in three sera was examined following absorption with either T cells or NK cells. In two cases the cytotoxic activity against both NK cells and T cells was removed following absorption with T cells. However, in one case, the inability to absorb out the anti-NK activity with T cells, while eliminating the anti-T-cell activity, suggested that this patient had serum autoantibody recognizing antigenic determinants specific to the NK cell membrane. A heterogeneity of autoantibody formation exists with antibodies to NK cells representing a potential mechanism for the inhibition of NK cell activity in SLE.

In vivo effect of anti-asialo GM1 antibody on natural killer activity.

Mounting evidence suggests that natural killer cells (NK) play an important part in the destruction of incipient tumours. If that is so, the destruction of NK cells in vivo should allow tumour cells to proliferate. We have recently described an antiserum having anti-NK activity which was raised against mouse brain tissue, and its effective antibody was shown to be directed against the cell surface glycolipid GM1 with its sialic acid groups removed (asialo GM1)1. We show here that intravenous (i.v.) injection of microlitre amounts of anti-asialo GM1 into BALB/c nu/nu mice almost completely abolishes NK activity and enhances the local growth of syngeneic lymphoma, RL♂1 as well as the incidence of tumour take. Evidence suggests that this effect is due to membrane damage producing loss of NK function rather than to cytolytic membrane attack.

A glycolipid on the surface of mouse natural killer cells.

Cytotoxic treatment with rabbit antiserum raised against purified glycosphingolipid "asialo GM1" was capable of eliminating natural killer (NK) activity of spleen cells from different inbred mouse strains including CBA/J, C57BL/6, BALB/c, AKR, and athymic nude mice. The anti-asialo GM1 antiserum showed little cross-reactivity with structurally related glycolipids, e.g. GM), GD 1 b and asialo GM2 in the microflocculation test. The specific reactivity of this antiserum with NK cells was confirmed by the quantitative absorption of anti-NK activity with graded amounts of asialo GM1 but not with other glycosphingolipids. The absorption of anti-brain-associated T cell antigen (anti-BAT) with asialo GM1 also effectively diminished its anti-NK activity, leaving the ability to kill T cells intact. This suggests that the antibody to asialo GM1 is responsible for the anti-NK activity contained in the anti-BAT antiserum. In contrast to the extreme sensitivity of NK cells to anti-asialo GM1, alloreactive cytotoxic T killer cells generated in the mixed lymphocyte culture were not killed by anti-asialo GM1 and complement. These results indicate that asialo GM1 is expressed on mouse NK cells in a high concentration.

Identification of a cell-surface antigen selectively expressed on the natural killer cell.

We have studied the cell-surface phenotype of natural killer (NK) cells of NZB and B6 mice which react to an MuLV+ lymphoid tumor. (a) NK cells do not express Thy1, Ly2, or Ig surface markers. (b) NK cells express an antigen recognized by C3H anti-CE antiserum ('anti-Ly1.2 antiserum'). Inasmuch as NK activity of spleen cells from B6 and B6/Ly1.1 congenic strains were both equally sensitive to C3H anti-CE antiserum, the NK antigen is distinct from Ly1.2. This point was confirmed by the observation that alphaNK activity was removed by absorption of C3H anti-CE antiserum with spleen cells from either B6 or B6/Ly1.1 congenic strains. Absorption of C3H alphaCE serum with BALB/c thymocytes and spleen cells (which are Ly1.2+NK-) removed anti-Ly1.2 activity and left anti-NK activity intact. This absorption step could be circumvented by inserting the BALB/c genotype into the recipient immunized to CE cells (i.e., (C3H X BALB/c)F1 alphaCE spleen cells). This antiserum, provisionally termed 'anti-NK', defines a new subclass of lymphocytes which may play a central role in the immunosurveillance against tumors.