Heterogeneity and Mechanism of Action of Human Natural Killer Lymphocytes: Differential Distribution of Receptors for Helix pomatia Haemagglutinin (HP Receptors)

Neuraminidase-treated lymphocytes from the peripheral blood of normal human donors were fractionated on columns charged with Helix pomatia haemagglutinin (HP) coupled to Sepharose 4B. While lymphocytes lacking HP receptors (HP-) passed directly through the column (fraction I), lymphocytes with HP receptors (HP+) were subsequently eluted in two distinct fractions with two different concentrations of the competitive hapten N-acetyl-D-galactosamine (fraction II and III, respectively). The natural cytotoxicity of these lymphocytes to various tumour target cells (K562, T24, MANO, HCV29) was tested in a 51Cr release assay. Natural cytotoxicity was found in all three fractions recovered from the HP columns. In general, the cytotoxicity of the lymphocytes in fractions I and II was significantly enhanced over that of the unfractionated lymphocytes. Surface marker analysis and fractionation studies indicated that natural cytotoxicity in these target systems is exerted by both HP+ and HP- lymphocytes bearing Fc receptors for IgG. Since the HP receptor is considered to be a marker to T lymphocytes, the findings suggest that a significant fraction of these NK cells may be of T-cell lineage. The surface marker profiles of these NK cells are very similar to those of antibody-dependent K cells. Addition of Fab fragments of immunoadsorbent-purified rabbit antibodies to human immunoglobulin inhibited the natural cytotoxicity of HP-column-fractionated lymphocytes to various degrees, indicating that part but not all of it reflects antibody-dependent K-cell reactions. Since cytotoxicity in all three HP fractions was inhibitable in this way, the results suggest that immunoglobulin-dependent natural cytotoxicity may be displayed by both HP+ and HP- effector cells.     

Expression of perforin in murine natural killer cells and cytotoxic T lymphocytes in vivo.

We have previously detected perforin expression in a subpopulation of asialo GM1+ natural killer (NK) cells and CD8+ T lymphocytes in murine spleen cells by immunocytochemical staining with an anti-perforin monoclonal antibody. In the present study, more detailed analyses of perforin expression in murine cytotoxic lymphocyte subpopulations were performed. The expression of perforin in asialo GM1+ spleen cells was predominantly confined to the NK1.1+ subset, where all NK activity also resided. Perforin expression was also studied on alloreactive cytotoxic T lymphocyte (CTL) induced in vivo. The cells expressing perforin in peritoneal exudate lymphocytes predominantly resided in the CD8+ T cell subpopulation co-expressing asialo GM1 where an allospecific CTL activity also resided. Furthermore, the percentage of perforin-positive cells in this population was greatly reduced after stimulation with anti-CD3 or anti-T cell receptors antibodies, which induce serine esterase release from the cytoplasmic granules. These findings highly suggest that perforin is involved in in vivo NK cell- and CTL-mediated cytolysis.     

Natural cytotoxic T cells responsible for anti-CD3-induced cytotoxicity in mice.

T cells obtained from normal mouse spleen cells showed significant cytotoxic activity against Fc receptor positive tumor cells in the presence of anti-CD3 monoclonal antibody (mAb). This activity was designated as natural cytotoxic T cell (NCT) activity and compared with natural killer (NK) activity. Considerable levels of NCT activity were detected in mouse strains with both high and low NK activity. NCT cells were distributed in both lower and higher density fractions of Percoll discontinuous density gradients, while NK cells were enriched in the lower density fraction of Percoll gradients. Moreover, NCT activity was resistant to in vivo anti-asialo GM1 treatment, in contrast to NK cells. These results indicate that NCT cells, which have different characteristics from NK cells, are present in normal, nonimmunized mouse spleen cells. Unexpectedly, CD4+ T cells sorted from normal mouse spleen T cells revealed significant NCT activity, as did CD8+ T cells. It was also demonstrated that NCT cells require the LFA-1 molecule to lyse tumor cells in the presence of anti-CD3 mAb.     

Enrichment of mouse splenic natural killer cells using discontinuous polyvinylpyrrolidone silica (Percoll) gradients.

A simple and rapid method is reported here for enriching murine spleen cells with natural killer (NK) function as assessed by short-term cytolysis assay of 51Cr-labelled YAC-1 lymphoma target cells. The established method used for the enrichment of NK reactive cells, including large granular lymphocytes (LGL) from human and rat peripheral blood lymphocytes, does not substantially enrich for mouse splenic NK cell activity. A reproducible procedure for enriching mouse splenic NK cells has been developed using a four- or five-step discontinuous Percoll gradient in the density range of 1.062 g/ml (top) to 1.092 g/ml (bottom) and osmolarity (310-340 mOsm/kg) nearer to that of mouse blood and tissue. A four- to 25-fold (usually about nine-fold) increase in NK cell activity, consisting of 50-100% of the recovered lytic unit activity, is found in which are the cells forming band 3, approximately 10% of the recovered cell number. This cell population with enriched NK cell activity has a characteristic density less than or equal to 1.077 g/ml, but more than 1.070 g/ml when centrifuged under appropriate conditions. Similar enrichment was obtained with a four-step gradient at an uniform osmolarity of 320 mOsm/kg throughout. Although the lymphocytes in band 3 show relatively little heterogeneity in appearance, only a minor population of the cells contain granules.     

Localization of an immunorecessive epitope on SV40 T antigen by H-2Db-restricted cytotoxic T-lymphocyte clones and a synthetic peptide

SV40 tumor (T) antigen possesses four distinct antigenic determinants, sites I, II, III, and IV, recognized by SV40-specific H-2b-restricted cytotoxic T-lymphocytes (CTL) clones. SV40-transformed C57BL/6 (B6) mouse kidney cells, designated K-3, 1, 4, K-1, 4, and K-4, 1, have been isolated by immunological selection with SV40 T antigen site-specific CTL clones in vitro. The cells have lost the expression of all four antigenic sites and cannot be lysed by the CTL clones specific for antigenic sites I, II, III, and IV. To search for additional SV40-specific antigenic sites on SV40 T antigen, B6 mice were immunized with K-3,1,4 cells and stimulated spleen cells with K-3,1,4 cells in vitro. Repeated stimulation of the spleen cell culture with gamma-irradiated K-3,1,4 cells in the presence of IL-2 was necessary to generate CTL activity against K-3,1,4 cells. A new group of H-2Db-restricted CTL clones designated as Y-5 was isolated which were cytotoxic to K-3,1,4 cells. The antigenic site recognized by CTL clone Y-5, site V, was localized in the carboxy terminal half of the SV40 T antigen. By the use of a synthetic peptide corresponding to SV40 T antigen in the carboxy region, the antigenic site V was localized between amino acids 489 and 503.     

Perforin, a pore-forming protein detectable by monoclonal antibodies, is a functional marker for killer cells

Perforin is one of the important cytolytic factors in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this paper, we report rat mAbs against mouse perforin established by immunization with a recombinant mouse perforin fragment. These mAbs reacted with purified mouse perforin prepared from cytoplasmic granules of an NK-like cell line in ELISA and Western blot analysis. However, none of these mAbs blocked the hemolytic activity of mouse perforin or absorbed it when fixed in the solid phase. These results indicate that all of these mAbs react with denatured but not with native mouse perforin. By using a combination of the mAbs, we established a sandwich ELISA, for quantitating the cellular contents of perforin. These mAbs were also useful for immunohistochemical staining analysis, and perforin was detected in the cytoplasmic granules of CTL and NK cell lines. Perforin was also detected in a minor population of lymphocytes of the spleen, liver, and lymph node. In normal spleen cells of 5- to 8-week-old mice, 12-15% of asialo GM1+ cells and 7-21% of CD8+ T cells were perforin-positive, but CD4+ T cells, B cells, and macrophages were totally negative. These data clearly show that perforin is expressed in cells of a cytotoxic character in normal mice, in the same way as in primed mice.     

Natural Killer Cells Do Not Belong to the Recirculating Lymphocyte Population

Natural Killer (NK) cells constitute a cell type with an as yet undefined lineage, although certain similarities with T lymphocytes have been found in the mouse. Our present results show that NK cells have a significant difference compared with T and B cells in their capacity to traverse the blood-lymph barrier. Thoracic duct lymphocytes from mice or rats are thus devoid of NK activity, when at the same time potential, cytotoxic T lymphocyte (CTL) activity or antibody-dependent cellular cytotoxicity function can be demonstrated. Chronic thoracic duct drainage in the rat also leads to an increase in NK activity per unit cell number in the other lymphoid organs. Thoracic duct lymphocytes from mice and rats may thus serve as convenient sources of CTL and/or killer cells in situations in which it is important to minimize NK cell involvement.     

Expression of Helix pomatia (HP) haemagglutinin receptors on cytolytic lymphocytes activated in mixed cultures

The expression of receptors for Helix pomatia (HP) haemagglutinin, a T cell marker, was assayed on human lymphocytes cultivated with K562 or allogeneic lymphocytes. The receptor was detected on the cells after neuraminidase treatment by reactivity with FITC conjugated HP. By affinity chromatography the lymphocyte populations were separated into 3 subsets: (1) a subset which did not attach to the lectin column; (2) and (3) two subsets attaching with different avidities, and therefore eluting with different concentrations of the lectin-binding sugar hapten. The subsets were characterized for T cell markers, HP and E receptors, the B cell marker SIg and also for Fc receptors, and were tested for lytic potential against K562 and allogeneic blasts. A high proportion of HP receptor positive T blasts did not attach to the lectin column, and thus had low avidity HP receptors, confirming that activation of T lymphocytes is accompanied by decreased expression of T markers. The passed fraction which was enriched in blasts had the strongest cytotoxic function, while the fraction rich in cells with high avidity HP receptors and containing mainly small cells, had weak activity. This was true for both the anti-K562 and allospecific activities. Thus the phenotypic characteristics of the cells exerting the two types of lytic function were similar. The distribution of the lytic potential in the three subsets correlated with the presence of E and EA receptor positive blasts.     

Resistance of mouse cytolytic cells to pore-forming protein-mediated cytolysis

Pore-forming protein (perforin, PFP) was isolated from a mouse large granular lymphocyte (LGL) [natural killer (NK-like)] cell line. Purified PFP lysed a variety of mouse tumor cell lines and helper T lymphocyte cell lines. However, LGL and cytotoxic T lymphocyte cell lines were resistant to PFP-mediated cell lysis. The presence of hemolytic activity in the granule was examined in these resistant cell lines. Four out of five of these resistant cell lines had hemolytically active granules. We determined whether NK cells freshly isolated from BALB/c nude mouse spleens were resistant to PFP-mediated cytolysis. Nylon column-passed spleen cells with an enriched content of NK cells exhibited more resistance than whole spleen cells. Moreover, when spleen cells were treated with PFP the remaining live cells showed enriched NK activity suggesting that normal peripheral cells with NK activity are resistant to PFP. These results indicate that cytolytic cells containing PFP have developed defense mechanisms to inhibit PEP-mediated cell lysis.     

Monoclonal antibodies against a rat leucocyte antigen block antigen-induced T-cell responses via an effect on accessory cells.

The MRC OX-45 and OX-46 mouse monoclonal antibodies recognize a rat cell surface glycoprotein of 45,000 MW that is present on a wide variety of haematopoietic cells and on endothelial cells. MRC OX-45 IgG or F(ab')2 blocked the primary mixed lymphocyte response (MLR) and the secondary response of T lymphocytes to the soluble antigen DNP-BGG. In contrast, the antibodies had no effect on the cytotoxic activity of specific (CTL) or non-specific (NK) killer cells or on proliferative responses stimulated by lectins or oxidative mitogenesis. The inhibitory effect was at the level of stimulator cells rather than responders since mouse anti-rat xenogeneic MLRs were inhibited but rat anti-mouse responses were unaffected. However, the effect was not a direct one because inhibition was seen when irradiated spleen cells were used as stimulators but not when cell populations highly enriched for dendritic cells were used. In the latter case, inhibition potentiated by antibody could be restored if a peritoneal cell population enriched for macrophages was added back to the cultures. The inhibitory effects of these monoclonal antibodies seem most likely to be due to potentiation of nonspecific suppression by macrophages.     

Difference between antigen-binding receptor repertoires in effector cytotoxic T lymphocytes and their secondary precursors (memory cells) specific to H-2Kb.

The fine specificity of antigen-binding receptors was compared in pCTL-2 and secondary effector CTL (cytotoxic T lymphocytes) induced in vivo with the H-2Kb alloantigen in recombinant inbred mice. The lymphocyte preparations were enriched by elution from macrophage monolayers of various origins, including the donor (B6 strain), the H-2Kb mutant bm1, the H-2Kk allele B10.A(4R) and the recipient strain B10.D2(R101) as a control. Anti-Kb pCTL-2 eluted from third-party bm1 or B10.A(4R) monolayers gave rise to CTL progeny that lysed, equally well, both donor TC and those third-party TC from whose monolayer the pCTL-2 had been eluted, but which were unable to lyse irrelevant third-party TC. The lytic activities of secondary CTL whose precursors had been eluted from bm1 or B10.A(4R) monolayers were 6 and 12 times lower, respectively, than pCTL-2 eluted from the donor monolayer. Opposite results were shown for receptors of enriched secondary anti-Kb effector CTL. Irrespective of their elution source, whether donor, mutant or allele variant, the eluted effector CTL were able to lyse the donor TC to a similar degree and much more than the given third-party TC; moreover, they retained cross-reactivity in all cases. It is suggested that CTL receptors are homogeneous in specificity for a whole composite immunodominant epitope and differ from each other only in the affinity/lability of the combining site. In contrast, pCTL-2 can be separated into fractions: receptors of each fraction are strictly specific (with high affinity) to a particular portion of the same composite CTL epitope. It seems likely that the pCTL-2 receptor antigen-binding site is modified during pCTL-2 in vivo differentiation into effector CTL.     

Generalized toxicity of L-leucyl-leucine-methyl ester for lymphocyte functions

This study investigated the hypothesis that sensitivity to L-leucyl-leucine-methyl ester (Leu-leu-O-Me) can be used to identify a role for cytotoxic lymphocytes in alloreactivity. Treatment of donor lymphocytes with Leu-leu-O-Me profoundly inhibited their ability to induce three separate models of murine graft-versus-host reaction (GVHR). This was observed for all aspects of GVHR examined, including weight loss, splenomegaly and activation of natural killer (NK) cells. These effects were not influenced by whether specific cytotoxic T lymphocytes (CTL) were involved in individual models of GVHR. Leu-leu-O-Me not only eliminated NK and CTL activity in vitro, but had identical effects on the proliferation of T and B lymphocytes in response to a variety of mitogenic stimuli. Finally, Leu-leu-O-Me prevented immune lymphocytes from mediating both class I and class II major histocompatibility complex (MHC)-restricted delayed-type hypersensitivity (DTH) responses in vivo. It is concluded that Leu-leu-O-Me has non-specific toxicity for most lymphocyte function and that it cannot be used as a specific means of predicting the functional roles of CTL.     

Cytolytic T lymphocytes and antibodies to myocytes in adriamycin-treated BALB/c mice. Evidence for immunity to drug-induced antigens.

Female BALB/c mice were given a single intravenous injection of between 0.1 and 10 mg adriamycin/kg body weight and were killed between 2 and 16 days later. Natural killer (NK) cell activity in the spleen was measured using YAC cell targets. Natural killer cell activity was slightly elevated 2 to 5 days after drug injection and significantly depressed by day 9 compared with spleen cells from untreated animals. Adriamycin-treated mice developed both cytolytic T lymphocytes (CTL) and antibodies to drug-treated myocytes. Peak CTL response occurred between days 9 and 13, whereas antibody reactivity continued to increase throughout the observation period. The effector cell belonged to the CD8+ T lymphocyte subpopulation, because cytolytic activity could be reduced by treating the cells with anti-Lyt 2 antibody and complement, whereas anti-L3T4 (CD4+ cell-specific) treatment either had no effect or increased cytotoxicity. Both CTL and antibody reactivity could be absorbed with adriamycin-treated myocyte monolayers but not by non-drug-treated myocytes. Furthermore CTL reactivity could be only partly removed by adriamycin-treated skin fibroblasts. Adriamycin concentrations in the heart were measured by flourometry and demonstrated only a gradual decrease in the drug over the 16-day period. Immunofluorescent staining of myocardial sections demonstrated increased numbers of both T lymphocytes and macrophages in the hearts of adriamycin-treated mice compared with untreated controls.     

Natural Killer Cells Contribute to Inflammation but Do Not Appear to be Essential for the Induction of Clinical Lymphocytic Choriomeningitis

The inflammatory exudate found in cerebrospinal fluid (CSF) of mice 6 days after intracerebral infection with lymphocytic choriomeningitis virus (LCMV) contains substantial populations of both cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Removal of NK cell activity by in vivo treatment with antibody to the asialo GM1 ganglioside and studies with NK-deficient bg/bg mice did not clearly determine whether NK cells contribute in any way to the development of clinical LCM. However, the LCM disease process induced in cyclophosphamide-suppressed, LCMV-infected recipients by the adoptive transfer of LCMV-immune spleen cells occurs in the absence of NK cell effector function in spleen, lymph nodes, or CSF of the recipients, though potent CTL populations are present in all of these sites. In this situation, NK cells are apparently not required for the induction of neurological symptoms that are indistinguishable from those of classical LCM.     

Lactosamine type asialooligosaccharide recognition in NK cytotoxicity

Inhibition of pig NK cell activity by asialooligosaccharides (aOS) isolated from human serum glycoproteins was investigated. Train-tennary aOS (aOSIII) of ceruloplasmin was found to be the most potent inhibitor up to the concentration 0.1 micrograms/ml, which is in agreement with its highly specific binding to NK-activity-enriched pig lymphocytes (with a morphology similar to human large granular lymphocytes (LGL]. Only lectins with the specificity to Gal(beta 1----4)GlcNAc or Gal(beta 1----3)GalNAc structures exhibited inhibition of NK cytotoxicity. F(ab)2 fragments of rabbit antibodies against pig spleen membrane lectin cross-reacting with the pig liver membrane lectin completely inhibited NK activity when preincubated with the effectors or present in the incubation mixture during the assay. These data suggest that lectin receptors on cells of pig NK-activity-enriched fraction specific for aOSIII and antigenically related to membrane lectins isolated from pig spleen and liver, are involved in the NK recognition of several xenogeneic targets.     

Effect of Irradiation on Rat Renal Transplant Rejection

Leucocytes were selectively eliminated either from a DA renal allograft or from a WF host by irradiation of either the host or the graft on different days after the transplantation. The recovery of inflammatory leucocytes and the generation of lymphoid killer cells--that is, the natural killer (NK) cells and the cytotoxic T lymphocytes (CTL)--were analysed separately in the two compartments. Early irradiation of the graft did not affect the recovery of leucocytes in either compartment. The NK activity was only slightly reduced in the graft but was distinctly reduced in the spleen. A delay in the generation of the CTL activity was observed in the spleen. Late irradiation of the graft reduced the recovery of leucocytes in both compartments. The disappearance of the NK activity increased in the graft but not in the spleen. The CTL activity in the spleen developed normally up to day 6, whereafter it declined. After selective irradiation of the host a fair number of leucocytes remained in the graft, compared with a nearly complete disappearance of leucocytes from the graft and blood. The NK and CTL activity declined rapidly in both compartments. The data demonstrate a bidirectional interdependence between the graft and the host during the rejection.     

Natural killer cells in murine muscular dystrophy. IV. Characterization of Percoll fractionated splenic and thymic natural killer cells and natural killer-sensitive thymocyte targets

The Natural Killer (NK) activity in the thymus and NK-sensitive thymocyte targets of dystrophic mice was investigated. Dystrophic and normal mouse thymocytes or spleen cells were layered on discontinuous Percoll gradients (5 or 10% increments, respectively) between 40 and 70% and centrifuged at 1700 g for 30 min. All fractions were tested for either NK activity or used a 51Cr-labeled NK-sensitive targets in a 6-hr 51Cr release assay. The density interface between the 50% (1.060 g/ml) and 60% (1.075 g/ml) Percoll fractions of either dystrophic or normal mouse spleen cells and the 40% (1.050 g/ml) and 50% (1.060 g/ml) Percoll fractions of either dystrophic or normal mouse thymocytes were found to contain the largest proportion of NK activity using YAC-1 lymphoma tumor cells as targets. In addition, the NK activity in dystrophic mouse spleen cells and thymocytes was significantly greater when compared with normal mouse controls. Target binding cell studies revealed that these Percoll fractions of dystrophic mouse spleen cells and thymocytes had greater numbers of conjugate-forming cells when compared with normal control groups. Cell depletion experiments using either anti-Thy 1.2, anti-asialo-GM 1 or anti-NK-1 plus complement treatment revealed that the cell responsible for NK activity in the 50% Percoll fraction interface of dystrophic mouse spleen cells was asialo-GM 1 positive. NK-1 positive, and partially Thy 1.2 positive. However, the cells displaying NK-activity in the thymus of normal or dystrophic mice were found to be highly Thy-1.2 positive and peanut agglutinin (PNA) negative. The density interface between the 60% (1.075 g/ml) and 65% (1.081 g/ml) Percoll fractions of either normal or dystrophic mouse thymocytes contained the largest proportion of NK-sensitive target cells. Interestingly, the 60% Percoll fraction of dystrophic mouse thymocyte targets was significantly more susceptible to NK-mediated lysis than that of the normal mouse thymocyte population. Cell depletion experiments revealed that the NK-sensitive thymocyte population was similar in both mice, that is, Thy-1.2 positive, cortisone sensitive, PNA positive, Dolichos biflorus (DBA) negative and asialo GM-1 negative. The results indicate that there are density differences between splenic and thymic NK cells. In addition, there are density and phenotypic differences between thymic NK cells and thymic NK-sensitive target cells. The findings support the hypothesis that there are different populations of NK cells.     

A method of increasing EA positive cells in mouse spleen lymphocytes

Treatment of mouse spleen lymphocytes with a high concentration of trypsin (1 mg/ml) increases the number of EA positive cells to include nearly all the B lymphocytes. It seems that all B lymphocytes express Fc receptors but in some cells these are hidden by other proteins present on the cell surfaces.     

A New Rat Lymphocyte Surface Marker: Characterization and Separation of Cells with Receptors for Helix pomatia Hemagglutinin

Thirty-two percent of neuraminidase-treated DA rat spleen lymphocytes and 48% of lymph node lymphocytes possess receptors for Helix pomatia hemagglutinin (HP). Moreover, these HP-receptor-bearing cells can be separated from B cells by affinity chromatography on HP-Sepharose columns. The virtual absence of immunoglobulin (Ig) receptors and the close correlation with reported T-cell content of these lymphoid tissues suggest that HP-receptor lymphocytes are probably T cells and that HP may provide a convenient marker, for both the identification and the purification of rat T lymphocytes.     

Locally superantigen-activated peritoneal cytolytic T lymphocytes belong to the CD8+ CD45RC- subset and lyse MHC class II+ tumor cells.

Bacterial encoded superantigens (SA) are capable of activating and targeting cytolytic human and mouse T lymphocytes (CTL) to lyse major histocompatibility complex class II positive (MHC class II+) target cells. In this study both in vitro and in vivo activated rat CTL were directed against MHC II+ tumor targets by bacterial encoded SA. Polyclonal in vitro activation of rat peripheral blood T lymphocytes generated CTL capable of killing MHC class II+ human BSM cells coated by staphylococcal enterotoxin (SE) -A, -E, -D, and TSST-1 but not by SEB or SEC1-3. Allo selective peritoneal CTL generated by intraperitoneal stimulation with allogeneic spleen cells were directed against BSM cells by SEA, -D, and -E but not by SEB, SEC1-3 or TSST-1. Based on the above observations, and in order to locally activate CTL, SEA was chosen for in vivo priming of rats by intraperitoneal inoculation of the toxin. SEA injection generated highly cytolytic CTL, and maximum cytolytic responses were seen at 50-250 micrograms SEA per animal with a peak in response 48-72 hours after injection of the toxin. The cytolytic activity of peritoneal SEA reactive effector cells was confined to the TCR alpha beta+ CD4- CD8+ CD45RC- cell population. MHC class II- colon carcinoma cells were insensitive to lysis by SEA reactive CTL but colon carcinoma cells induced to express MHC class II by interferon-gamma (IFN-gamma) treatment were efficiently lysed in the presence of SEA. Comparison of rat and human MHC II+ colon carcinomas revealed a peak in sensitivity to lysis at 10-100 ng SEA/ml for both tumor targets. These findings suggest that superantigens can be used in local immunotherapy of peritoneal tumors such as ovarian and colorectal carcinomatosis, with inducible or constitutive expression of MHC class II.