NK Cell Reactivity Research Articles

Transgenic expression of Ly49A on T cells impairs a specific antitumor response.

Inhibitory MHC receptors determine the reactivity and specificity of NK cells. These receptors can also regulate T cells by modulating TCR-induced effector functions such as cytotoxicity, cytokine production, and proliferation. Here we have assessed the capacity of mouse T cells expressing the inhibitory MHC class I receptor Ly49A to respond to a well-defined tumor Ag in vivo using Ly49A transgenic mice. We find that the presence of Ly49A on the vast majority of lymphocytes prevents the development of a significant Ag-specific CD8+ T cell response and, consequently, the rejection of the tumor. Despite minor alterations in the TCR repertoire of CD8+ T cells in the transgenic lines, precursors of functional tumor-specific CD8+ T cells exist but could not be activated most likely due to a lack of appropriate CD4+ T cell help. Surprisingly, all of these effects are observed in the absence of a known ligand for the Ly49A receptor as defined by its ability to regulate NK cell function. Indeed, we found that the above effects on T cells may be based on a weak interaction of Ly49A with Kb or Db class I molecules. Thus, our data demonstrate that enforced expression of a Ly49A receptor on conventional T cells prevents a specific immune response in vivo and suggest that the functions of T and NK cells are differentially sensitive to the presence of inhibitory MHC class I receptors.

Impaired Anti-Viral T Cell Responses Due to Expression of the LY49A Inhibitory Receptor

Inhibitory receptors specific for alleles of MHC class I proteins play an important role in determining the reactivity and specificity of NK cells. To determine whether these receptors are also able to regulate T cell functions, we have studied anti-viral immune responses in mice transgenic for a class I-specific inhibitory receptor, Ly49A. Although nontransgenic mice express Ly49A primarily on NK cells and some T cells, the Ly49A transgenic mice express Ly49A on all lymphocytes, including T cells. We have assessed the activation, expansion, cytokine production, and cytotoxic activity of CD8 T cells in both transgenic and nontransgenic mice following infection with lymphocytic choriomeningitis virus. As expected, nontransgenic mice made a potent virus-specific CD8 T cell response following virus infection. However, as measured in cytolysis assays and by cytokine production, virus-specific CD8 T cell activity was reduced in Ly49A transgenic mice. This inhibition was largely, but not always exclusively, dependent upon the presence, either in vivo or in vitro, of the Ly49A ligand, H-2Dd. Strikingly Ly49A transgenic mice have reduced capacity to control infection with the virulent lymphocytic choriomeningitis virus variant clone 13. Overall, these studies demonstrate that expression of killer inhibitory receptors can modulate anti-viral T cell responses in vivo and in vitro.

Maintenance and reversibility of natural killer cell- and T cell-independent B lymphocyte xenotolerance in athymic nude rats.

We previously described that a tolerogeneic regimen (TR) including (1) the infusion of a minced hamster heart suspension (MHH), (2) a single injection of an anti-natural killer (NK) cell serum (rabbit anti-asialo GM1 serum), and (3) a 4-week course of the B cell immunosuppressant leflunomide (20 mg/kg/ day) induced T cell-independent (T-I) B lymphocyte and NK cell tolerance for hamster xenoantigens in T-deficient athymic nude rats. In addition, the TR allowed for long-term hamster cardiac xenograft (Xg) survival when Xgs were transplanted 2 weeks (Day 0) after the initiation of the TR (started on Day - 14). The present study was undertaken to investigate some of the characteristics of this T-I xenotolerance in more detail. To investigate the duration of the effect of the TR on the T-I xenotolerance, hamster Xgs were transplanted at various times after initiation of the TR. To investigate whether the maintenance of the T-I xenotolerance depended on the presence of the graft, tolerated Xgs were removed on Day +28, and the subsequent evolution of the T-I xenotolerance as well as of second hamster Xg was followed. In addition, the reversibility of NK cell nonresponsiveness by recombinant interleukin-2 was investigated in vitro. Xgs transplanted on day 0 or Day +7 showed long-term survival. However, all Xgs transplanted on Day +15, +30, and +60 were rapidly rejected. The latter rejection occurred in the absence of formation of anti-hamster immunoglobulin (Ig)M xenoreactive antibodies (xAbs) but correlated with the recovery of anti-hamster NK cell reactivity from day +14 on. Rejected Xgs showed infiltration of NK cells but absence of IgM xAbs or complement factor deposition. When tolerated first Xgs (transplanted on Day 0) were removed on Day +28, second hamster Xgs survived without treatment when transplanted 1 or 2 weeks later. However, second hamster Xgs transplanted 3 weeks after removal of the first Xgs were all rapidly rejected. Again, the latter rejection was characterized by the infiltration of the Xgs with NK cells and by the absence of anti-hamster IgM xAbs formation. Xenoreactive NK cell nonresponsiveness was not only shorter than xenoreactive B cell nonresponsiveness, but was also more fragile. This was evident from the fact that after addition of recombinant interleukin-2 in vitro, specific anti-hamster NK nonresponsiveness was easily broken. NK cell and T-I B cell xenotolerance can be induced in T-deficient rats. Compared with B cell xenotolerance, the maintenance of NK cell xenotolerance is much shorter, more dependent on the presence of the graft, and easily reversible in vitro.

Duration of stressor and natural killer cell activity

To examine whether duration of an acute stressor affected the time line of natural killer cell (NK) activity in response to acute stress, this study collected NK measures from 31 males and 24 females at six times (before, during and after either a 5-min or M-min computerized Stroop task). Both stress periods were associated with increased self-report of distress and anxiety, and participants in the 30-min task group had increased systolic and diastolic blood pressures and heart rate. Regardless of task length, NK activity was elevated during all in-task samples, and activity returned to baseline levels following the task, where it remained through the duration of the study. However, computation of NK activity per cell in a subset of subjects suggested that increases in activity were due to a redistribution of NK cells to the periphery. No gender differences in NK cell reactivity were found.

Human NK cell and ADCC reactivity against xenogeneic porcine target cells including fetal porcine islet cells.

In vitro studies of human NK cell-mediated cytotoxicity and ADCC against porcine target cells were performed. Stimulation of human PBMC responder cells with either allogeneic or xenogeneic porcine cells led to a marked increase in NK cell reactivity. Maximum reactivity was reached following 3-6 days of in vitro culture. The sensitivity of target cells ranked as follows: K562 > porcine PHA-induced lymphoblasts > resting porcine PBMC. Limiting dilution analysis showed that allo- and xeno-stimulation in vitro led to differentiation of similar frequencies of effector NK cells. Split culture experiments showed that single NK effector cells were cytotoxic against both K562 and porcine lymphoblasts, demonstrating that individual NK cells lack species specificity. NK effector cell generation stimulated by xenogeneic cells was cyclosporin A (CsA) sensitive and dependent on the presence of autologous responder T lymphocytes, a dependence that was completely reconstituted by the sole addition of human IL-2. Xenostimulation of enriched CD3+ cells also led to a preferential appearance of CD16+ or CD56+ lymphoblasts. Natural xenoreactive human anti-porcine antibodies are mainly of IgM and IgG2 subclasses, but antibodies in xenoimmunised patients reactive against porcine lymphocytes and fetal porcine islet cells were also of IgG1 and IgG3 subclasses. The same subclass distribution was found among antibodies specific for gal(alpha)1,3 gal epitopes as shown by tests performed with alpha1,3 galactosyltransferase-transfected Raji cells (human Burkitt lymphoma cells). Natural antibodies did not mediate ADCC, whereas gal(alpha)1,3 gal-specific antibodies in sera from xenoimmunised patients did. Fetal porcine islet cells were sensitive to human NK cell-mediated cytotoxicity and to ADCC mediated by xenoimmune sera.

Murine NK cell allospecificity-1 is defined by inhibitory ligands.

Hemopoietic allografts of normal and neoplastic origin are subject to NK cell-mediated resistance in mice. Susceptibility to this resistance is controlled by MHC-linked genes in a recessive manner. Several distinct specificities could be postulated to explain the strain-dependent pattern of resistance. These presumptive specificities for recognition are H-2 haplotype dependent, but the correspondence is not one-to-one. For example, resistance of H-2d or H-2b/d host to H-2 b graft operationally defines specificity-1, establishing its link with haplotype H-2b. To examine the molecular basis of specificity-1, spontaneous Dd-loss mutant clones were isolated from H-2b/d and H-2d hemopoietic cell lines, i.e., 416B of (C57BL/6 x DBA/2)F1 (B6D2F1) origin and L1210 of DBA/2 origin, both of which lack specificity-1. The expression of specificity-1 in the mutant clones was examined in vivo and in vitro. The results indicate that Dd-loss clones of 416B and L1210 lines express specificity-1. These data suggest that murine NK cell allospecificity-1 is defined primarily by the absence of the Dd molecule or other class I molecules sharing the protective motifs; no H-2b-associated genes play a relevant role. This conclusion is consistent with the missing self hypothesis of NK cell reactivity, and is in agreement with the observation that lysis of B6 targets by B6D2F1 NK cells is mediated mostly by cells that express Ly-49A and/or Ly-49G2.

The effect of specific and nonspecific immunotherapy on natural killer cell activity in patients with non-small-cell lung cancer.

Fourteen patients with stages I and II non-small-cell lung carcinoma receiving adjuvant immunotherapy after surgery were studied serially for natural killer (NK) cell cytotoxicity assays. Seven patients received active nonspecific immunotherapy with Freund's complete adjuvant alone and seven patients received active specific immunotherapy with tumor-associated antigen plus Freund's complete adjuvant. Both groups were analyzed as a single group in view of the lack of any difference in the results. The results indicated that NK-cell cytotoxicity v K562 was lower in untreated patients than in normal controls, but the difference was not significant. There was no evidence of specific cytotoxicity v the lung cell line A549 in untreated patients when compared with normal controls. Compared with pretreatment values, elevated cytotoxicity v both K562 and A549 was observed at every post-treatment time point tested, and this was statistically significant at several time points by a paired t test. No specific effect was noted after immunization with lung antigen on cytotoxicity against the lung cell line A549. Other correlations studied did not show any relationship between delayed hypersensitivity skin testing or serum carcinoembryonic antigen levels and NK-cell cytotoxicity. It is concluded that NK-cell reactivity was increased nonspecifically by active specific and nonspecific immunotherapy. This increase seems to peak at the 12-month period and was significant when compared with pretreatment levels.

Induction of autologous reactivity of human NK cells.

'Mature' human natural killer cell activity could be enriched by adsorption-elution with fetal fibroblast as adsorbents against normal allogeneic skin fibroblasts, but not against autologous targets. However, when the natural killer activity was augmented by contact with tumour cells (HeLa), autologous and allogeneic skin fibroblast targets were killed without preference. Adsorption-elution with augmenting target cells as adsorbents resulted in an efficient enrichment of NK activity showing non-discriminate cytotoxicity. Morphologically, this was associated with an enrichment of large granular lymphocytes previously shown to be responsible for human NK activity. We conclude that the NK activity of human 'mature' NK cells shows a relative autologous exemption, whereas 'pre-NK' cells augmented to full activity in vitro are non-discriminative.

Expression of Human T Lymphocyte Antigens by Killer Cells

K cells, the effectors of antibody-dependent cell-mediated cytotoxicity, were found to express human T but not B lymphocyte antigens detected by rabbit anti-HTLA and anti-HBLA. Pretreatment of effector cells with anti-HTLA+C inhibited ADCC by specifically lysing K cells: no inhibition of ADCC by anti-HTLA occurred when deltaC was substituted for C. By contrast, pretreatment of effector cells with anti-HBLA nonspecifically inhibited ADCC, probably for forming antigen-antibody complexes with HBLA+ cells in effector suspensions: a) treatment with anti-HBLA deltaC was more inhibitory of ADCC than treatment with anti-HBLA+C, and b) the inhibitory effect of anti-HBLA on ADCC was either eliminated or markedly reduced if effector suspensions were first passed through a nylon fiber column, a procedure that removed most HBLA+ cells without affecting K cell activity. HTLA antigens expressed by K cells and NK cells are the same as HTLA antigens expressed by thymocytes since thymocytes completely absorb the anti-K cell and NK cell reactivity of anti-HTLA.