Genetic differences in the age-associated decrease in inducibility of natural killer cells by interferon-alpha/beta

Natural killer (NK) cells, which are important in viral infections and anti-tumor activity, show reduced cytotoxicity in aged mice. The mechanism(s) for this age-related decline in NK activity has not been clearly established. We assessed changes in NK cytotoxicity in splenocytes and peripheral blood mononuclear cells after interferon (IFN)-alpha/beta stimulation in adult (6 months) and aged (22-26 months) C57Bl/6, Balb/c, and (Balb/c x C57Bl/6)F1 mice. Aged C57Bl/6 and Balb/c mice had a significantly reduced IFN-alpha/beta-stimulated NK cytotoxicity compared to adult mice. In contrast, adult and aged F1 mice showed similar NK cytotoxicity after IFN-alpha/beta induction. The decreased ability of NK cells of aged mice to respond to induction by IFN-alpha/beta was not due to a requirement for an increased amount of IFN or for a longer period of treatment with IFN. Further, this decreased response did not appear to be the result of suppressive activity of adherent cells or T cells. While the percentage of NK cells (NK1.1+) was similar in adult and aged mice, the (CD8+ NK1.1+) subset of NK cells was significantly increased in aged mice. Importantly, the percentage of CD8+ NK1.1+ cells was inversely related to the cytotoxicity observed after IFN-alpha/beta treatment.     

Ly-49D transfected NK cells show reduced interleukin-10 production in response to H-2d and increased lytic activity: implication by interaction using class I MHC alloantigen+ target cells in pregnancy

PROBLEM: Mating of CBA/J (H-2k) with DBA/2 (H-2d) males leads to a high rate of spontaneous resorption (about 40%), which is not seen in other mating combinations, such as CBA/J X BALB/c. The activation of natural killer cells (NK cells) seems to be a key mechanism for the maternal-fetal intolerance in allogeneic pregnancy, and recurrent spontaneous abortion. The effect of expression of the NK cell activating receptor Ly49D recognizing BALB/c or DBA/2 class I MHC was investigated. METHOD OF STUDY: Intracellular interleukin (IL)-100 production was detected and target cell survival rates were calculated after 22 hr coincubation of rat NK cells transfected, or not. with a murine Ly49D receptor, with either male BALB/c or male DBA/2 splenocytes, by using flow cytometry. RESULTS: Ly49D negative rat NK cells produced 13.7% more IL-10 than Ly49D positive rat NK cells, and more splenocytes were killed by Ly49D transfected rat NK cells (survival rate 2.45%) than by Ly49D negative rat NK cells (survival rate 4.36%). CONCLUSION: After physiological stimulation with BALB/c or DBA/2 splenocytes, rat NK cells are able to synthesize IL-10. Recognition of mouse splenocyte major histocompatibility complex (MHC) by Ly49D mice receptor decreased IL-10 production. The observed increase in killing activity might be a result of this phenomenon. NK cell activation via the Ly49D receptor might play an important role in pregnancy failure, but cannot explain why CBA/J X DBA/2 matings are abortion prone, and CBA/J X BALB/c matings are abortion resistant.     

Alleviation of chronic GVHD in mice by oral immuneregulation toward recipient pretransplant splenocytes does not jeopardize the graft versus leukemia effect

Chronic graft versus host disease (cGVHD) is the result of an immune-mediated attack by transplanted donor lymphocytes, entailing inflammatory damage to host target organs. Clinically, the post-bone marrow transplantation (BMT) graft versus leukemia (GVL) effect may be associated with GVHD. Immune hyporesponsiveness induced by oral antigen administration has recently been shown to prevent the development of cGVHD in a murine model. To evaluate whether amelioration of cGVHD in mice by induction of oral immune regulation in donors toward recipient pretransplant lymphocyte antigens is associated with attenuation of the GVL effect donor B10.D2 mice were fed with Balb/c splenocytes, B10.D2 splenocytes, bovine serum albumin (BSA), or regular chow, every other day for 10 days. Subsequently, transplantation of 2 x 10(7) splenocytes from donor B10.D2 mice to recipient Balb/c mice was undertaken, followed by inoculation of 3 x 10(3) BCL-1 leukemia on the day of BMT. Control groups were fed identically without leukemia inoculation. Mice were followed for survival and leukemia progression. Induction of tolerance was assessed by a mixed lymphocyte reaction (MLR). Cutaneous GVHD was assessed macroscopically. To elucidate the mechanism of any observed effect, serum interferon (IFN), interleukin (IL-2), IL-12, IL-4, and IL-10 were determined by enzyme-linked immunosorbent assay and flow cytometry analysis for CD4+, CD8+, and NK1.1+ lymphocyte subpopulations was performed. There was no significant difference in leukemia progression manifested by survival or white blood cell counts of orally immune-regulated mice compared with control animals. Cutaneous cGVHD was significantly ameliorated in Balb/c mice transplanted from tolerized B10.D2 mice. This effect was associated with a significant reduction in the mixed lymphocyte response of effector splenocytes from tolerized B10.D2 mice against Balb/c target splenocytes; significantly decreased serum IFN-gamma and IL-2; increased serum IL-12 levels; increased peripheral NK1.1+ cells; and CD4+/CD8+ lymphocyte ratio. Oral tolerization of BMT donors toward recipient antigens ameliorates cGVHD without hampering the GVL effect.     

Changes of natural killer cell activity in different mouse lines by acute and asymptomatic flavivirus infections

Effect of certain flaviviruses on the activity of mouse natural killer (NK) cells was investigated using the classical mouse splenocyte system and YAC-1 cells for demonstration of NK cell cytotoxicity. Infection of mice with Langat and West Nile (WN) viruses was accompanied by temporary activation of NK cells. In mice infected with tick-borne encephalitis (TBE) virus the stimulation phase of NK cell cytotoxicity on days 2-4 post-infection (p.i.) was followed by suppression of their activity. As to the surface markers (sensitivity to antitheta and antiimmunoglobulin serum, respectively), the flavivirus-activated NK cells did not differ from the endogenous NK cells of intact mice. The stimulatory effect of flaviviruses on cytotoxicity of NK cells varied in different mouse lines. An increased NK cell activity at early stages of TBE virus infection was observed in mouse lines characterized by low (C57B1/6) and medium (BALB/c)--but not by high (CBA)-activity of their non-stimulated NK cells. Suppression of NK cell activity at later stages of TBE virus infection was not associated with virus multiplication in mouse splenocytes.     

Influence of NK cell magnetic bead isolation methods on phenotype and function of murine NK cells

Natural killer (NK) cells are a promising tool for cell therapy due to their capacity to lyse tumor cells without prior activation and their influence on the innate as well as the adaptive immunity. To characterize distinct NK cell populations, it is important to find a reliable isolation method. We investigated separation methods for NK cell isolation by magnetic bead labeling. There are three commonly used different MACS protocols to isolate NK cells from murine spleen: CD49b (DX5) MicroBeads, the NK Cell Isolation Kit and a separation method which is based on a positive selection for NKp46 expressing cells. Interestingly, we found a significant difference of NK cell purities depending on the mouse strains and the purification protocol used. We observed a significantly higher level of purity and yield of NK cells by preparations from Balb/c splenocytes. In this study, we modified the negative selection protocol and adapted it to C57Bl/6 mice to obtain equal purity, viability and yields of NK cells in the different mouse strains. Moreover, we optimized the NKp46 NK cell selection method by addition of a B cell depletion step. To our knowledge, this is the first report that has directly compared and essentially modified the different NK cell purification strategies in parallel, both in C57Bl/6 and Balb/c mouse strains. Altogether, these results are a basic prerequisite for the further development of NK cell therapy protocols in murine in vivo models.     

Peripheral blood lymphocytes resistant to Epstein-Barr virus immortalization manifest high natural killer (NK) type activity against NK-resistant target cells

Epstein-Barr virus (EBV) readily immortalizes human peripheral blood lymphocytes (PBL) in vitro. We found recently that PBL from two EBV-seropositive healthy adults were exceptionally resistant to immortalization by EBV. In contrast to PBL from other EBV-seropositive donors sensitive to immortalization by EBV (S-PBL), the "resistant" PBL (R-PBL) respond to EBV infection with an early interleukin-2 (IL-2) synthesis and high interferon gamma (IFN gamma) production. In order to determine whether these differences in cytokine responses between R-PBL and S-PBL could be associated with a detectable difference in lymphocyte cytotoxicity, we compared the natural killer (NK) activity of R-PBL and S-PBL effectors by using both NK-sensitive (i.e. K562) and NK-resistant (i.e. Raji) targets. We found that, while effectors from EBV-infected R-PBL and S-PBL cultures exhibited comparable NK activity against the K562 targets, they differed remarkably in their cytolytic activity against Raji cells. At days 3 and 5 of culture, effectors from EBV-infected R-PBL showed a significantly higher lytic activity against Raji targets, whereas S-PBL did not. Culture of EBV-infected R-PBL and S-PBL effectors in the presence of recombinant IL-2 (rIL-2) for 5 days resulted in increases of their lytic activity against Raji cells, whereas pretreatment of these effectors with recombinant IFN gamma (rIFN gamma) was found to increase only R-PBL cytotoxicity. These results suggest that the resistance of R-PBL to EBV immortalization could be associated with a lymphokine-mediated early cellular cytotoxic response of the NK/LAK (lymphokine-activated killer cell) type against EBV-infected cells.     

Ontogeny of natural killer cell activity and antibody dependent cell mediated cytotoxicity in pigs

Cells from pigs of various ages were collected from peripheral blood, mesenteric lymph node, spleen and thymus and their ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity was determined. ADCC against chicken red blood cell (CRBC) was present in cells from peripheral blood, lymph node and spleen, but was absent in thymic cells. There were no age-related differences in ADCC to CRBC and cells from fetal pigs had similar activities to cells from adult pigs. Maximal cytotoxicity against CRBC was found in the polymorphonuclear leukocyte (PMN) cell fraction. In contrast to the good response against CRBC, PMN cells were not lytic in the ADCC assay when PI3 virus-infected cells were used as target cells. Peripheral blood lymphocytes (PBL) had low but significant lytic activity against PI3 virus-infected cells in the presence of high concentrations of specific antiserum. NK cell activity against K562 target cells was readily detected in PBL of pigs older than 2 weeks but was not observed with cells from spleen, lymph node or thymus from pigs of any age. PBL of pigs younger than 2 weeks of age had low but detectable NK activity: however, fetal pigs had no NK activity against K562 target cells. In contrast, when PI3 virus infected Vero cells were used as target cells, NK cells were detected in spleen and PBL, but not lymph nodes or thymus, of pigs greater than one day of age. Similar to the absence of activity to K562, none of the lymphoid cells from fetal pigs had NK activity against PI3 virus-infected Vero cells. The present results suggest that the effector cells that mediate ADCC are distinct from those that mediate NK activity in that cells mediating ADCC develop earlier and are found in different organs than the NK cells. Additionally, the cells that mediate NK activity against viral infected cells may be different from those that mediate NK activity for K562 target cells; however, regardless of the target, NK cells are not present before birth in the pig.     

Natural killer cells in viral arthritis.

Changes in natural killer (NK) cell activity were studied in patients with polyarthritis associated with rubella or Ross River virus infections. In 30 of 32 Ross River virus patients, peripheral NK cell activity was depressed at some stage of the disease but returned to normal levels as patients recovered from arthritic symptoms. Similar changes did not occur in rubella patients and no difference was found between changes in peripheral NK activity and serum interferon (IFN) levels in rubella patients with arthritis and those without. Neither the peak of NK cell activity in peripheral blood lymphocytes (PBL) recovered early in Ross River virus and rubella infections, nor the depression of NK cell activity late in Ross River virus infections could be correlated with changes in serum IFN levels. The decrease in PBL-NK cell activity in epidemic polyarthritis (EPA) patients could not be attributed solely to loss of NK cells from the peripheral circulation because limiting-cell-dilution (LCD) analyses indicated changes in peripheral NK cell activity were due to changes in both the number and lytic activity of NK cells. Despite the association between HLA-DR7 and EPA no differences were found in levels of peripheral NK cell activity in DR7+ and DR7- EPA patients. The demonstration that peripheral NK cells could kill autologous synovial cells suggested that NK cells in joints of EPA patients may contribute to the arthritis associated with Ross River virus infection.     

Characterization of equine natural killer and IL-2 stimulated lymphokine activated killer cell populations.

Natural killer (NK) cells are an important component of the innate immune system. Though intensively studied in humans and rodents. NK cells remain less well characterized in other species. Studies are often limited by the lack of specific cell markers; however, the mAb NK-5C6 has been suggested to recognize an evolutionarily conserved molecule on NK cells and reacts with cells from several species. This mAb was used in the current investigation to identify and characterize equine NK cells, and was found to label approximately 10% of peripheral blood lymphocytes (PBL). Two-color flow cytometry analysis identified the NK-5C6+ cell population as being CD3-CD4- and CD8-, but positive for MHC class I and LFA-1 expression. Depletion of CD3+ T cells increased the percent NK-5C6+ cells in PBL; this enriched population demonstrated a specific cytotoxic response against a major histocompatibility complex (MHC) deficient NK target cell line (K-562), but not MHC+ target cells (EqT8888). These results provide evidence for an equine NK cell population, which exhibits endogenous lytic activity and a phenotype similar to that of human and mouse NK cells. Stimulation of peripheral blood mononuclear cells (PBMC) with IL-2 promoted the development of LAK cells. These cells were predominantly CD3+ T cells, demonstrated intracellular perforin expression, and effectively lysed both K-562 and EqT8888 target cells. Hence, equine NK cells can be identified by the NK-5C6 mAb and distinguished from IL-2 stimulated LAK cells by their cytotoxic response to specific target cell lines.     

Gamma interferon production is critical for protective immunity to infection with blood-stage Plasmodium berghei XAT but neither NO production nor NK cell activation is critical

We have examined the roles of gamma interferon (IFN-gamma), nitric oxide (NO), and natural killer (NK) cells in the host resistance to infection with the blood-stage malarial parasite Plasmodium berghei XAT, an irradiation-induced attenuated variant of the lethal strain P. berghei NK65. Although the infection with P. berghei XAT enhanced NK cell lytic activity of splenocytes, depletion of NK1.1(+) cells caused by the treatment of mice with anti-NK1.1 antibody affected neither parasitemia nor IFN-gamma production by their splenocytes. The P. berghei XAT infection induced a large amount of NO production by splenocytes during the first peak of parasitemia, while P. berghei NK65 infection induced a small amount. Unexpectedly, however, mice deficient in inducible nitric oxide synthase (iNOS-/-) cleared P. berghei XAT after two peaks of parasitemia were observed, as occurred for wild-type control mice. Although the infected iNOS-/- mouse splenocytes did not produce a detectable level of NO, they produced an amount of IFN-gamma comparable to that produced by wild-type control mouse splenocytes, and treatment of these mice with neutralizing anti-IFN-gamma antibody led to the progression of parasitemia and fatal outcome. CD4(-/-) mice infected with P. berghei XAT could not clear the parasite, and all these mice died with apparently reduced IFN-gamma production. Furthermore, treatment with carrageenan increased the susceptibility of mice to P. berghei XAT infection. These results suggest that neither NO production nor NK cell activation is critical for the resistance to P. berghei XAT infection and that IFN-gamma plays an important role in the elimination of malarial parasites, possibly by the enhancement of phagocytic activity of macrophages.     

Effects of triiodothyronine supplements on splenic natural killer cells in malnourished weanling mice

The main purpose of this investigation was to determine whether exogenous triiodothyronine (T₃) administered according to a protocol known to prevent depression in acquired immunity in weanling murine protein-energy malnutrition (PEM) would, likewise, influence the splenic natural killer (NK) cell in this disease. Weanling mice of disparate inbred strains, C57BL/6J and CBA/J, were subjected to wasting PEM produced by means of two low-protein diets (0.5% crude protein) identical in every respect except that one diet contained supplemental T₃ (0.2 μg/g diet). NK cell lytic activity toward YAC-I targets was assessed in vitro using suspensions containing 0.5 × 10⁶ mononuclear spleen cells. Lytic activity in this assay was low in mice fed the unsupplemented low-protein diet, but was not depressed in malnourished animals given exogenous T₃. Surface marker analysis using the NK cell-specific antigen, NK 1.1 (PK 136), revealed no effect of the low-protein diet or of exogenous T₃ on the proportion of splenic mononuclear cells exhibiting NK 1.1⁺ phenotype. Previous investigations have shown that acquired immune competence in PEM can be manipulated, by means of endocrine hormonal intervention, independently of continued wasting disease. The present results extend this fundamental new concept to include an innate immune function, namely NK cell lytic activity. In this system of experimental PEM, exogenous T₃ prevented depression in NK cell lytic activity expressed on a per cell basis. The malnourished weanling rodent is a particularly powerful experimental system with which to investigate the mechanisms whereby thyroid hormones influence NK cells.     

Loss of surface CD200 on stored allogeneic leukocytes may impair anti-abortive effect in vivo

PROBLEM: Prevention of spontaneous abortion by allogeneic mononuclear leukocyte immunotherapy has proven ineffective in the CBA x DBA/2 murine abortion model if the leukocytes are stored overnight before inoculation. The mechanism and generality of the phenomenon has not been elucidated. METHODS: As prevention of recurrent abortion in the CBA x DBA/2 model requires allogeneic BALB/c lymphoid cells bearing paternal antigens and the tolerance-signaling molecule CD200 (OX-2), we evaluated effects of cell storage on cell surface CD200 expression using flow cytometry of fresh or stored cells stained with monoclonal anti-CD200 antibody. Release of putative CD200 molecules into culture supernatant during storage was tested by the ability of supernatants to block binding of anti-CD200 to freshly isolated cells. Similar studies were done using human peripheral blood mononuclear leukocytes. Possible binding of soluble CD200 to immunoglobulin G (IgG) molecules in plasma as a basis for the anti-abortive effect of intravenous immunoglobulin G (IVIG) was tested using the standard peripheral blood lymphocyte (PBL) natural killer (NK) cell lysis of (51)Cr-labeled K562 cells and monoclonal anti-human CD200 antibodies. RESULTS: Loss of anti-abortive effect of BALB/c cells with overnight storage at 4 degrees C and blocking of protective effect of freshly isolated cells with anti-CD200 antibody was confirmed. Supernatants of stored cells acquired a low level of protective activity against abortion in the CBA x DBA/2 model. Cell surface CD200 was lost with overnight storage at 4 or 22 degrees C, and supernatants acquired the ability to block binding of anti-CD200 antibody to fresh cells. Similar results were obtained using human PBL. However, if cells were stored overnight in IgG containing plasma, binding was not blocked. Suppression of NK cell lysis by PBL was abrogated if anti-CD200 antibody was added to the assay. CONCLUSIONS: Loss of the tolerance signal CD200 from allogeneic cells occurs with storage overnight, and their ability to protect against abortion is lost. CD200 appears to be shed into the supernatant, and may associate with IgG molecules rendering IVIG suppressive.     

Effect of thymic hormone treatment on several immune functions of nude mice

Studies of the effect of short-term, intense treatment with thymic hormone on mitogen response, cytotoxicity to EL-4 lymphoma and natural killer cell (NK) activity was investigated Balb/c nude mice (about 12-16-week-old) were treated 5 times per week for 3 weeks with: Facteur Thymic Serique (FTS) and Thymopentin (TP5, Thymopoietin 32-36) at 1 microgram and 10 ng; TM4 1 ng (an enzyme resistant variant of FTS); Thymosin Fraction V (TF5), 10 and 1 microgram; and 0.1 ml saline, and killed 2 days after the last treatment. The animals were monitored for changes in weight, hematocrit, peripheral blood lymphocyte (PBL) and spleen mitogen response. Additional groups of nude mice were immunized with 1 x 10(7) 5000 R irradiated EL-4 cells 10 days before sacrifice and tested for the presence of cytotoxic T-lymphocytes (CTL). The results show that weight and hematocrit were similar among the groups. Treatment with FTS significantly elevated the number of PBL. Spleen stimulation in mice treated with 1 microgram TP5 was depressed to mitogen concanavalin A (ConA) and lipopolysaccharide (LPS) stimulation. The phytohemagglutinin (PHA) response was not different among the treatment groups. The PBL mitogen response to ConA and LPS was generally increased over saline control in the hormone treated groups but was not statistically significant. The PHA response was only slightly elevated. No CTL was generated in nude mice in any of the groups. However, there was a statistically significant general depression of NK activity in all of the hormone treated animals compared with saline. The results indicate that the basic differentiation defect of the T-cells of nude mice cannot be restored to full functional activity by short-term treatment.     

Impact of 90Sr on Mouse Natural Killer Cells and their Regulation by Alpha Interferon and Interleukin 2

Male CBA/SU mice were exposed to ionizing radiation by intraperitoneal injection of the bone-seeking beta-emitter 90Sr. NK-cell lytic activities in spleen, peripheral blood, and lymph nodes were severely depressed or completely abolished. In contrast, production of the NK regulatory proteins alpha interferon (IFN-alpha) and interleukin 2 (IL-2) was normal 5-8 weeks after 90Sr injection. IFN-alpha, produced in vivo or in vitro by cells from injected mice, was able to enhance strongly NK lytic activities. These data indicate that 90Sr acts on the bone marrow, where it interferes with the maturation and seeding of NK precursor cells. The mechanisms regulating NK activities in peripheral organs remained relatively unchanged. Finally, we did not detect any major organ redistribution of NK cells as a result of 90Sr irradiation.     

Immunomodulatory effect of Listeria monocytogenes infection. II. Natural cell cytotoxicity in infected mice

The natural cell cytotoxicity of blood leukocytes, splenocytes and peritoneal cells derived from Balb/c mice being on day 1, 3, 5, 7, 9 of listeriosis to allogeneic fibroblast L 929 was examined. The intensity of cytotoxic reaction was assessed by the per cent of specific 51Cr release from target cells. To check if this reaction correlates with the level of monocyte-macrophage lineage cells present in the effector cells of if it depends on the development of delayed type hypersensitivity to Listeria antigen, Balb/c mice were infected with Listeria monocytogenes and simultaneously treated with ATS. It was found that Listeria monocytogenes infection in mice enhanced the natural cell cytotoxicity of tested effectors cells, particularly of peritoneal cells in animals being on the 5th-7th day of listeriosis. The treatment of mice with ATS which is known to inhibit the development of delayed type hypersensitivity to Listeria antigen, completely suppressed the stimulatory effect of this infection on the natural cell cytotoxicity to allogeneic fibroblasts. This finding suggests that the enhancement of natural cytotoxic activity of tested effector cells results from the action of activated macrophages cooperating with Listeria sensitized T lymphocytes.     

A new surface marker on mouse natural killer cells: Receptors for Helix pomatia A hemagglutinin

Natural killer (NK) cells in normal mouse spleen have so far been found to lack conventional cell surface markers. Recently, neuraminidase treatment has been shown to uncover receptors for the A hemagglutinin of the snail Helix pomatia (HP) on populations (preferentially T cells) of human blood lymphocytes. We have used HP coupled to Sepharose in columns to fractionate mouse spleen cells. Fractions consisted of passed cells (fraction I) and cells eluted with the competitive hapten N-acetyl-D -galactosamine at either 0.1 mg/ml (fraction II) or 1.0 mg/ml (fraction III). NK activity and percentage of T and B cells in each fraction were determined. B cells were found enriched in fraction I. T lymphocytes, among them alloreactive cytotoxic T cells (CTL), were eluted in fraction III. However, NK activity of normal CBA spleens was highest in fraction II, fraction III was somewhat enriched, whereas fraction I was depleted of lytic activity. Thus, while NK cells possess HP receptors, their binding properties seem to differ from those of CTL. HP receptors represent the first simple and reliable marker detected on NK cells and might be useful for purification of this cell type.     

Human T cells in hu-PBL-SCID mice proliferate in response to Daudi lymphoma and confer anti-tumour immunity.

In vitro culture of human peripheral blood lymphocytes (PBL) with Daudi (Burkitt lymphoma) cells results in selective proliferation of V gamma 9/V delta 2 T cells with high cytotoxicity against Daudi cells. After adoptive transfer into severe combined immunodeficient (SCID) mice, these cells exert specific anti-tumour activity against Daudi lymphoma. To test whether cytotoxic V gamma 9/V delta 2 T cells are induced in SCID mice, human PBL injected intraperitoneally were stimulated with irradiated Daudi cells (PBL/Daudi-SCID). After 7-14 days, PBL/Daudi-SCID had a significantly higher percentage of human gamma delta T cells in their peritoneal cavity, lymph nodes and blood than controls (PBL-SCID). DNA content analysis of T cell subsets from PBL/Daudi-SCID showed a significantly higher percentage of cells in S + G2 + M phases of the cell cycle in the TCR-gamma delta-1+ than in CD3+ cell population. Human cells recovered from PBL/Daudi-SCID showed specific cytotoxicity against Daudi cells. PBL/Daudi-SCID inoculated with a lethal dose of Daudi lymphoma survived significantly longer than controls. This protection was specific for Daudi cells and was not mediated by murine natural killer (NK) cells. Thus human peripheral blood T cells grafted in SCID mice proliferate in response to antigen and confer specific immunity.     

Miniaturization of the standard 51Cr release assay for long term follow-up of NK activity of individual mice

Two modifications for miniaturizing the standard 51chromium release assay have been described. Using peripheral blood lymphocytes (PBL) as effectors and very few target cells, this permits longitudinal studies on NK cell activity of individual mice to be performed. The modifications are based on methods to increase the chromium uptake by the target cell. Firstly, chromium uptake by the YAC-1 target cells was enhanced to such an extent that appreciable activity could be detected against as little as 750 cells/well at an E/T ratio of 50:1. Secondly, the uptake was increased even further by labelling the target cells in the presence of isotonic sucrose, thus permitting the use of only 125 target cells/well at an E/T of 25:1. The long term pattern of NK activity in individual BALB/c mice was studied by repeated bleedings over a period of 8 months. Considerable fluctuations with time were observed in the pattern of NK activity of individual mice. However, the NK activity averaged over a large number of mice did not show a significant decrease with age. These methods may allow the study of the long term pattern of NK activity in individual mice and the response of this activity to physiological and environmental factors.     

Inhibition of natural killer activity by calcitonin gene-related peptide

The effect of calcitonin gene-related peptide (CGRP) on natural killer (NK) cell activity in spleen cells from Balb/c mice and nude mice was studied. CGRP dose-dependently (10(-9) to 10(-7) M) inhibited NK activity of spleen cells from both strains of mice. This inhibitory effect was observed at the effector to target ratios of 12.5:1 to 100:1. Maximum inhibition by 10(-7) M CGRP was about 60%. The inhibition of NK activity by CGRP was also observed in anti-Thy 1.2 plus complement treated Balb/c spleen cells. Furthermore, when cells were treated with 10(-9) to 10(-7) M CGRP the concentration of intracellular cyclic AMP increased in spleen cells of nude mice. The characteristics of these cells were similar to those of NK cells, (1) being petri dish and nylon wool nonadherent, (2) expressing asialo GM1 antigen, and (3) lacking readily detectable Thy 1 antigen and immunoglobulin. In addition, the intravenous injection of asialo GM1 completely abolished NK activity in spleen cells from nude mice and the increase in intracellular cyclic AMP in spleen cells by CGRP was less in spleen cells from mice given an anti-asialo GM1 injection. Our present study suggests that CGRP inhibits NK cell activity by increasing the intracellular cyclic AMP concentration. CGRP may be implicated in the regulation of NK function.     

Changes in Natural Killer Activities in Experimental Secondary Amyloidosis

Natural killer (NK) cell activity in experimental murine amyloidosis was studied. In CBA/J mice, which show a high incidence of amyloidosis, NK activity was significantly decreased after 1 week of casein treatment. In C3H mice, which show a low incidence of amyloidosis, NK activity was not changed by casein treatment. Pretreatment with lipopolysaccharide in vivo enhanced the NK activities in CBA/J and C3H mice. These increases were not observed after casein treatment. The lowered NK activity of cells from CBA/J mice after casein treatment was restored to the normal range by indomethacine in vitro. Depletion of adherent cells from the spleen cells treated with casein had no effect on NK activity. Single-cell assay showed that casein treatment impaired the killing but not the binding of NK cells to target cells. After casein treatment, the splenic serum amyloid A (SAA) level gradually increased in CBA/J mice but remained low in C3H mice. NK activity was suppressed by the addition of serum obtained from CBA/J mice treated with casein but not by normal control serum. And partially purified AA protein obtained from the spleen of CBA/J mice treated with casein also suppressed NK activity in vitro.