Involvement of human membrane-associated complement components in the rosette formation between marmoset red blood cells and human leukocytes

The majority of human monocytes, a subpopulation of B lymphocytes, and all B lymphoblastoid cell lines (B-LCL) tested but one form rosettes with Marmoset red blood cells (MaRBC). None of the human peripheral T cells, T-LCL, and B chronic lymphoid leukemia cells (B-CLL) used bind to MaRBC. The binding could not be correlated with any membrane markers or antigens present on cultured cells or peripheral blood leukocytes (PBL). Blocking of the rosette formation by preincubation of MaRBC with purified human complement (C) components and cobra venom or by pretreatment of leukocytes and cultured cells with antisera to human C components suggested that membrane-associated C components present on PBL or B-LCL are involved in the binding to MaRBC.     

Cellular activities during a mixed leucocyte reaction in the teleost sea bass Dicentrarchus labrax

In this investigation a number of "in vitro" activities of sea bass peripheral blood leucocytes (PBL) against allogeneic PBL inactivated by irradiation were studied. Stimulator PBL were cultured with inactivated allogeneic PBL, and direct counting of lymphocytes was done after 2 weeks by immunofluorescence and flow cytometry using mAbs DLT15 and DLIg3 specific for T-cells and B-cells, respectively. In a one-way mixed leucocyte reaction (MLR), results showed an increase of T lymphocytes, whereas B lymphocytes had values similar to those in control PBL. The increase of T-cells in MLR cultures was also confirmed using RT-PCR by analyzing the expression of the T-cell receptor (beta-subunit) mRNA. The addition of 5 microg/ml of cyclosporin A (CsA) to the MLR caused a significant decrease in T-cell proliferation. Leucocytes from MLR cultures displayed an enhanced cytotoxic activity against xenogeneic target cells with respect to control PBL, raising the possibility of the presence of cytotoxic-like T lymphocytes. Cellular activation of PBL was confirmed in 2 weeks MLR by measuring antibody-induced intracellular Ca(++) mobilization with Fura-2 AM. This work represents the first direct quantitative determination of an "in vitro" T-cell activity in a teleost species.     

Low density lipoprotein receptor degradation is influenced by a mediator protein(s) with a rapid turnover rate, but is unaffected by receptor up- or down-regulation

Treatment of cultured human skin fibroblasts with cycloheximide retarded the down-regulation of low density lipoprotein (LDL) receptor activity caused by 25-hydroxycholesterol. The rate of LDL receptor degradation, measured directly by means of [35S]methionine pulse-chase experiments, was also markedly inhibited by cycloheximide (or puromycin), suggesting that continuous synthesis of a short-lived mediator protein(s) was necessary for normal LDL receptor turnover. In the absence of cycloheximide, both the up- and down-regulation of LDL receptor activity took place with a half-time of approximately 12 hr. Pulse-chase measurements with [35S]methionine yielded a receptor half-life (t1/2) of 11.7 +/- 2.2 hr (n = 10) in up-regulated cells; the t1/2 in the partially down-regulated state was similar. The presence of LDL or 25-hydroxycholesterol did not alter this degradation rate. Regulation of LDL receptor activity under these various culture conditions therefore probably occurred solely as a result of changes in the rate of receptor synthesis. The cycloheximide-sensitive factor(s) that influences receptor turnover apparently did not play a regulatory role in the up- or down-regulation of the LDL receptor.     

Epstein Barr virus binding induces internalization of the C3d receptor: a novel immunotoxin delivery system

Epstein Barr virus (EBV) infection of human B lymphocytes is initiated by selective binding of the virus to the C3d receptor (EBV/C3d receptor) on the cell surface and results in polyclonal proliferation of infected cells. In these studies we examined the fate of the EBV/C3d receptor during viral infection by using an immunotoxin made from a monoclonal antibody (HB5) reactive with the receptor and the potent toxin, gelonin. Binding of the HB5-gelonin conjugate to the EBV/C3d receptor before EBV infection (at concentrations as low as 10(-11) M) significantly inhibited the subsequent polyclonal proliferation of virus-infected B lymphocytes. HB5 antibody and gelonin alone did not inhibit proliferation. Because internalization of gelonin-antibody conjugates is required to cause cytotoxicity, these results indicate that infection of B lymphocytes with EBV selectively induced endocytosis of the EBV/C3d receptor with concomitant internalization of the immunotoxin. Proliferation of B lymphocytes that were activated by prior infection with EBV, or activated by cross-linking of their surface immunoglobulin molecules, was not inhibited by the antibody-toxin conjugate even at concentrations as high as 10(-7) M. Also, the growth of B lymphoblastoid cell lines cultured in the presence or absence of infectious EBV was not inhibited by HB5-gelonin. Thus, our results suggest that the EBV/C3d receptor is internalized only during the infection of normal B lymphocytes by EBV, with co-internalization of immunotoxin, and indicate that internalization of the EBV/C3d receptor-immunotoxin complex does not occur simply as a consequence of activation and proliferation of B lymphocytes. The use of a ligand to induce endocytosis of its receptor offers a new strategy for the selective delivery of immunotoxins to cells and may be more generally applicable.     

Regulation of C5a and formyl peptide receptor expression on human polymorphonuclear leukocytes

Fluorescein conjugates of C5a (FL-C5a) and formyl methionine-leucine-phenylalanine-lysine (FL-FMLPL) have been used to determine how the expression of receptors for these peptides is regulated on human polymorphonuclear leukocytes (PMN). Video intensification microscopy showed that receptors for FL-C5a were homogeneously distributed on the surface of the PMN, but within minutes were mobilized into patches and internalized by the PMN. Internalization of C5a receptors was confirmed in studies in which external FL-C5a fluorescence was quenched by reducing the pH. A similar rapid internalization was observed with FL-FMLPL. This process was inhibited for both fluorescent ligands by monensin. Reexpression of C5a and formyl peptide receptors after internalization occurred with both receptors. By comparison, the rate of reexpression of formyl peptide receptors was much faster than that observed with C5a receptors with the half maximal reexpression time for each being 5 to 10 min and 18 to 60 min, respectively. C5a receptor reexpression was completely blocked by monensin suggesting receptor recycling, whereas monensin had little effect on FMLPL receptor reexpression. The reexpression of both receptors occurred in the presence of cycloheximide indicating that this process occurred independent of protein synthesis. Additional studies on formyl peptide receptor showed that when PMN were treated with ionomycin to fully mobilize the intracellular pool of FMLPL receptors, receptor reexpression failed to occur. These studies show that both C5a and formyl peptide receptors are internalized after binding ligand, but that their reexpression occurs through different mechanisms. C5a receptors appear to be recycled to the cell surface whereas formyl peptide receptors are reexpressed predominantly by translocation from an intracellular pool.     

Theileria parva: Early events in the development of bovine lymphoblastoid cell lines persistently infected with macroschizonts

The cellular origin and development of bovine lymphoblastoid cell lines persistently infected with macroschizonts of Theileria parva was studied. Cultures of lymphoblastoid cells isolated from cattle with patent East Coast fever were compared with those obtained by infecting normal lymphocytes in vitro with sporozoites. The young lines were contrasted with a continuous line which had been isolated earlier. The mononuclear cells were separated from the blood and the inoculum enriched for lymphoblastoid cells and/or lymphocytes by removing the monocytes. The lines arose directly from lymphoblastoid cells transplanted into culture or from lymphocytes infected by sporozoites. In primary cultures of lymphoblastoid cells from the peripheral blood, there was an increase in the proportion of infected cells without the eclipse of the parasite, the macroschizonts were larger than those observed in the inoculum or the continuous line, and there was concurrent microschizont differentiation. In lymphocyte cultures challenged with sporozoites, small mononucleated trophozoites were observed after 2 days which differentiated into typical macroschizonts but microschizonts were rare. In all cultures, the infected cells had mitotic indices of 4 to 5%. As the young lines were passaged, the parasites came to resemble those of the continuous line. The macroschizont size in the continuous line was stable and most had six to eight nuclei but when cultured at high cell concentrations the number of parasite nuclei increased. Minicultures of lymphocytes were used to quantitate the infectivity of sporozoites obtained from organ cultures of Rhipicephalus appendiculatus savliary glands. Sporozoites from ticks fed on rabbits for 5 days were approximately six times more infective than those from glands of ticks fed for 2 days and then cultured at 32 °C for 3 days. Glands from unfed ticks cultured for 5 days failed to yield infective sporozoites.     

Monoclonal anti-actin antibody recognizes a surface molecule on normal and transformed human B lymphocytes: expression varies with phase of cell cycle

A monoclonal anti-actin antibody, 2C9, was used to study the distribution of an actin-like cell-surface antigen (hereafter termed actin) on a lymphoblastoid cell line LA350 and on human peripheral blood lymphocytes. It was determined that 8-40% of LA350 cells and 3-15% of peripheral blood lymphocytes from healthy donors stain specifically with 2C9, almost exclusively on IgM-positive cells. Treatment of cells with 2C9 prior to incubation caused cell-surface actin to first patch and then to cap. Treatment of cells with nonspecific protease caused a loss of surface actin, with reexpression of the marker after 8-12 hr. The expression of LA350 surface actin also increased with DNA synthesis and was demonstrated to be maximal during late G1/early S phase. Thus, this antigen may be a sensitive marker for activated lymphocytes. These studies contribute to our understanding of the expression and distribution of actin-like membrane proteins that may participate in regulatory signals mediated by anti-actin antibody.     

Target specificity and cell surface structures involved in the human cytolytic T lymphocyte response to endothelial cells

Two long-term cytolytic T lymphocyte (CTL) lines derived from the peripheral blood lymphocytes (PBL) of a single donor were analyzed for target specificity and involvement of cell surface molecules in CTL-target interactions. One line, AH2, was generated after stimulation with B lymphoblastoid cells. Cytolysis by these cells was restricted to targets expressing the appropriate HLA-A2 specificity and was blocked by mAb recognizing CD2, CD3, CD8, LFA-1, and LFA-3. The second line, AE1, was generated after stimulation with cultured endothelial cells derived from human newborn preputial microvessels. These CTL lysed all human target cells tested, except autologous cells and the Class I negative cell line Daudi. In addition, mAb specific for CD2, CD3, and CD8 did not affect cytolysis. Anti-LFA-1 and -LFA-3 mAb blocked cytolysis of B lymphoblastoid targets but not endothelial targets. These results indicate that some CTL utilize as yet uncharacterized cell surface structures for CTL-target interactions.     

Increased proliferation, lytic activity, and purity of human natural killer cells cocultured with mitogen-activated feeder cells.

The addition of mitogen-prestimulated periferal blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) cultures to enriched populations of natural killer (NK) cells obtained from PBL of normal donors in the presence of rIL-2 resulted in highly significant increases in proliferation, purity, and cytolytic activity of cultured NK cells. Two sources of enriched NK cell preparations were used: (i) Adherent-lymphokine activated killer (A-LAK) cells obtained by adherence to plastic during 24 hr activation with 10(3) Cetus U/ml rIL-2; and (ii) NK cells negatively selected from PBL by removal of high-affinity rosette-forming cells and CD3+ lymphocytes. Coculture of A-LAK cells for 14 days with autologous or allogeneic Con A-activated PBL (10(6) cells/ml) or selected EBV-transformed LCL (2 x 10(5) cells/ml) as feeder cells increased fold expansion by a mean +/- SEM of 629 fold +/- 275 (P less than 0.019) and 267 fold +/- 54 (P less than 0.0001), respectively, compared to 55 +/- 20 in A-LAK cultures without feeder cells. The addition of either activated PBL or EBV lines to A-LAK cultures also led to a significant increase in the percentage of NK cells (CD3- CD56+) (84 +/- 2.4 and 84 +/- 2.6%, respectively, P less than 0.0001 for both), compared to 53 +/- 7.2% in cultures without feeders. The presence of feeder cells in cultures of A-LAK cells also led to significantly higher anti-tumor cytolytic activity compared to control cultures, as measured against NK-sensitive (K562) and NK-resistant (Daudi) target cells. Mitogen-stimulated CD4+ PBL purified by positive selection on antibody-coated flasks were better feeders than CD8+ or unseparated PBL. In the presence of feeder cells, it was possible to generate up to 6 x 10(9) activated NK cells from 2 x 10(8) fresh PBL by Day 13 of culture. Enhanced NK cell proliferation in the presence of feeder cells was not attributable to a detectable soluble factor. The improved method for generating A-LAK or activated-NK cells should facilitate cellular adoptive immunotherapy by providing sufficient numbers of highly enriched CD3- CD56+ effector cells with high anti-tumor activity.     

Evidence for active synthesis of two allelic b locus markers by single lymphocytes from heterozygous rabbits.

The double expression on single cells of both b4 and b6 allotypic markers by the mixed antiglobulin technique was observed on single peripheral blood lymphocytes (PBL) from heterozygous rabbits which were treated with pronase and incubated overnight to allow for regrowth of surface immunoglobulin (Ig). The percent of double staining rosettes observed after lymphocyte processing prior to pronase stripping ranged from 10–30%. Following pronase treatment at concentrations allowing for maximum stripping but assuring high cellular viability, the total rosetting population was reduced to 1–3% in the majority of cases. Occasionally, pronase stripping was less effective. Overnight incubation in serum free medium resulted in the reappearance of surface Ig with double staining rosettes ranging from 8–30%. Exposure of b6 or b4 normal rabbit serum to b4 or b6 cells, respectively, resulted in uptake of b6 in which most of the passively acquired Ig eluted off during overnight incubation, while negligible uptake of b4 was seen. Coincubation of normal b4, 4 PBL which presumably are capable of donating Ig with pronase treated b6, 6 PBL and vice versa also resulted in the absence of mixed rosettes. That simultaneous active synthesis of both allelic markers results in double allotype expression was demonstrated by kinetics data. Supporting this was the observation that only after washing of cycloheximide inhibited cells did allotype reexpression resume.     

Expression of IFN-alpha and IFN-gamma receptors on normal human small resting T lymphocytes and large granular lymphocytes

The expression of interferon (IFN) receptors was studied on freshly isolated human lymphocytes from normal donors. Highly enriched populations of small resting T lymphocytes and large granular lymphocytes (LGL) were found to constitutively express high-affinity receptors for IFN-alpha and IFN-gamma. Both types of lymphocytes also had lower-affinity IFN-alpha binding sites. T lymphocytes had a mean of 230 IFN-alpha and 520 IFN-gamma high-affinity receptors per cell, whereas LGL had 520 IFN-alpha and 760 IFN-gamma receptors. However, because LGL were larger than the T lymphocytes, the IFN receptor density was similar on the two types of lymphocytes. The affinity of binding was similar on the two types of normal lymphocytes and on the cultured lymphoblastoid cell line Daudi. The number of IFN receptors per cell and the affinities of the IFN-receptor interactions varied little among the normal donors. Both the freshly isolated normal lymphocytes and the cultured cell line Daudi had separate receptors for type I (alpha and beta) and type II (gamma) IFN. Taken together, our data indicate that two types of freshly isolated normal lymphocytes constitutively express IFN receptors that are similar to those present on the lymphoblastoid cell line Daudi derived from a patient with Burkitt's lymphoma.     

Suppression of DNA synthesis in normal, but not neoplastic, nuclei by cytoplasmic extracts from resting cells

We have previously shown that a heat-stable protein in cytoplasmic extracts from human quiescent peripheral blood lymphocytes (PBL) is capable of inhibiting the induction of DNA synthesis in isolated resting nuclei. We now report that these cytoplasmic extracts are also capable of suppressing DNA synthetic activity in replicative nuclei isolated from mitogen-activated PBL. PBL extracts had little or no inhibitory effect, however, on replicative nuclei derived from several transformed lymphoblastoid cell lines. These results suggest that the growth of normal lymphocytes may be negatively controlled by cytoplasmic inhibitory factors. Furthermore, the relative resistance of tumor cell nuclei to these inhibitory signals provides a possible explanation for the loss of growth control in neoplastic cells.     

Binding of human recombinant 125I-interferon gamma to receptors on human cells

Recombinant human interferon gamma (rIFN-gamma) produced in Escherichia coli was labeled with 125I to study its binding to receptors of HeLa and lymphoblastoid cells. All the cell lines examined had receptors for rIFN-gamma, although the binding varied considerably among different cell lines. The binding of 125I-rIFN-gamma was competed up to 90% by the addition of unlabeled rIFN-gamma, although not by the addition of IFN-alpha or -beta. By adding increasing concentrations of unlabeled rIFN-gamma to binding assays containing a constant amount of 125I-rIFN-gamma, we determined a KD of 3.7 and 6.3 X 10(-10) M for its binding to Daudi and HeLa cells, respectively. About 13,000 receptors per cell were present in Daudi and 5,000 in HeLa cells. The Mr of the IFN-gamma/receptor complex was determined by cross-linking experiments to be about 125,000. This complex is smaller than the IFN-alpha/receptor complex that has a Mr of about 140,000. The rIFN-gamma receptor was down-regulated when HeLa cells were treated with this interferon, but not when these cells were treated with IFN-beta. These findings suggest that the receptors for IFN-alpha and -gamma differ in several characteristics. The turnover of the rIFN-gamma receptor was measured by inhibiting protein synthesis with cycloheximide and the half-life of this receptor was found to be 2 h. The unglycosylated rIFN-gamma was bound to cellular receptors with an affinity similar to that previously reported for natural IFN-gamma. The lymphoblastoid cell lines examined had high affinity receptors for rIFN-gamma, but did not respond to treatment with this IFN with an induction of the synthesis of the enzyme (2'-5')oligo(A) synthetase, whereas HeLa cells responded to rIFN-gamma. The reason for the lack of response of lymphoblastoid cells is presently unknown.     

Leucoagglutinin induces differential proliferation of lymphocyte subsets

In this study we have established culture conditions that allow the preferential and rapid expansion of either T cell receptor (TCR)+/CD3+16- T lymphocytes or TCR-/CD3-16+ natural killer (NK) cells, or the non-selective outgrowth of both subsets. Optimal proliferation of lymphocytes was obtained using a combination of irradiated allogeneic peripheral blood lymphocytes (PBL) and irradiated Epstein Barr virus (EBV) transformed lymphoblastoid B cell lines (B-LCL). Addition of 1 microgram/ml leucoagglutinin to the culture medium induced a preferential outgrowth of TCR+/CD3+16- T lymphocytes. The proportion of TCR-/CD3-16+ NK cells was decreased to 5% or less, although still a 2000-fold multiplication of TCR-/CD3-16+ NK cells was obtained at day 13. Without leucoagglutinin a 1000-fold increase of about 70% pure TCR-/CD3-16+ NK cells was obtained at day 13. Intermediate concentrations of leucoagglutinin (0.1-0.3 micrograms/ml) resulted in a non-selective expansion of both NK cells and T cells. Irrespective whether leucoagglutinin was added or not, the number of TCR+/CD3+8+ lymphocytes increased more rapidly relative to the TCR+/CD3+4+ lymphocytes resulting in an increased TCR+/CD3+8+ population size. Also under limiting dilution conditions leucoagglutinin increased the frequency of proliferating cells. In contrast to the preferential outgrowth of TCR+/CD3+8+ lymphocytes in bulk cultures, approximately 80% of the clones generated was TCR+/CD3+4+, demonstrating a growth promoting effect of TCR+/CD3+4+ lymphocytes on TCR+/CD3+8+ lymphocytes in PBL bulk cultures.     

Application of flow cytometry to studies on the distribution, persistence, and regeneration rate of Epstein-Barr virus receptors

Flow cytometry has been applied to study the persistence and regeneration rate of Epstein-Barr virus (EBV) receptors and other membrane proteins in normal and tumor cells. EBV receptors were detected in a binding assay utilizing fluorescein isothiocyanate-conjugated virions (FITC-EBV). After stripping receptors from the cell surface with trypsin, binding of FITC-EBV was rapidly regenerated in the lymphoma cell line Loukes, reaching 75% of the control levels by 3 h. Fresh human lymphocytes regenerated receptors at a much slower rate, reaching 50% of control levels by 24 h. The regenerating receptors did not reappear simultaneously on all the cells. Subpopulation of new receptor-positive cells increased gradually during the incubation at 37 degrees C. Reappearance of receptors was inhibited in the absence of protein synthesis. Conversely, receptors persisted in the nontrypsinized cells in the absence of protein synthesis. These results show that certain parameters of membrane receptor turnover, such as the change in receptor distribution within a cell population, receptor persistance, and regeneration rate, can be measured at the single-cell level by flow cytometry.     

Lysis of autologous human melanoma cells by in vitro allosensitized peripheral blood lymphocytes

Summary Peripheral blood lymphocytes (PBL) of melanoma patients were sensitized in vitro with lymphocytes of a single donor or with a pool of lymphocytes of 5–20 different donors. After 6–7 days, the cytotoxic activity of the sensitized PBL was tested against cultured autologous tumor cells and lymphocytes in a ⁵¹Cr-release assay. Tumor lysis was observed in 13 of 16 cases in which patients' PBL (Pt-PBL) were stimulated by a pool of allogeneic lymphocytes and in five out of seven cases when single sensitization was performed. In no case was lysis of autologous normal lymphocytes or blasts seen. Cultivation of Pt-PBL with irradiated autologous tumor cells never led to the induction of lymphocytes cytotoxic to melanoma cells. Lysability by pool-activated autologous Pt-PBL of fresh cryopreserved tumor cells was compared to that of short-term cultured tumor cells, and no significant differences were observed. Cold-target inhibition experiments indicated that the cytotoxicity of Pt-PBL was tumor-restricted since only autologous melanoma cells but not lymphocytes were able to inhibit the reaction. These results indicate that activation of Pt-PBL is necessary in order to elicit or amplify their antitumor activity.     

Regulation of nuclear antigen expression on the cell surface of human monocytes.

The expression of cell surface nuclear Ag was studied by examining the binding of anti-histone mAb to viable human peripheral blood cells. Freshly isolated cells showed no binding of these mAb. However, in vitro culture in the presence of LPS induced a dose-dependent expression of cell surface nuclear Ag on monocytes (M3+ cells). The addition of IL-1 beta to cultures also induced expression of cell surface nuclear Ag, whereas IFN-gamma was without effect. Release of nuclear material into the supernatants over time was demonstrated by using a chromatin-specific sandwich ELISA. Analysis of the DNA in the released nuclear material demonstrated banding at multiples of 190 bp, suggesting the release of polynucleosomes. Although LPS was required for cell surface nuclear Ag expression, it did not affect the release of nuclear material into the supernatants. The ability of monocytes to bind exogenous chromatin was studied by adding biotinylated-chromatin to PBL and detection with FITC-avidin. Freshly isolated PBL bound no chromatin, but when PBL were cultured in the presence of LPS, monocytes bound chromatin in a saturable manner. The LPS induction of the capacity to bind exogenous chromatin was blocked by cycloheximide. These data suggest that monocyte activation is associated with the expression of a chromatin (?nucleosome)-binding receptor and that this receptor is capable of binding nuclear material released into the cellular milieu. Monocytes may thus provide an important mechanism for the removal of extracellular nuclear material at sites of cell death and/or inflammation. The binding of nuclear Ag to cell surfaces and potential abnormalities of this binding may play a role in the induction of antinuclear antibodies and/or tissue damage in diseases such as SLE.     

Comparison of the Fc receptors for IgE on human lymphocytes and monocytes

Fc receptors for IgE (Fc epsilon R) on human peripheral blood lymphocytes and monocytes and cultured lymphoblastoid and macrophage-like cell lines were compared with respect to: 1) binding affinity for radiolabeled IgE, 2) inhibition of IgE-specific rosette formation and inhibition of binding of radiolabeled IgE by an antiserum raised against Fc epsilon R isolated from a lymphoblastoid cell line, and 3) m.w. of radiolabeled cell surface proteins precipitated with the anti-Fc epsilon R serum. Scatchard analysis of 125I-IgE binding to lymphocytes, monocytes, and their corresponding cell lines showed biphasic binding curves with all cell types, from which 2 binding affinities were calculated to be KA = 6.2 +/- 1.1 and 2.0 +/- 0.5 x 10(7) M-1. The anti-Fc epsilon R serum inhibited both IgE rosette formation and binding of radiolabeled IgE by lymphocytes and monocytes but did not inhibit IgE rosettes formed by basophils. The inhibitory activity of the anti-Fc epsilon R serum could be absorbed with Fc epsilon R(+) but not with Fc epsilon R(-) cell lines. The anti-Fc epsilon R serum precipitated 2 peptides having m.w. of approximately 47,000 and 23,000 daltons from lysates of both cell surface-labeled lymphocyte and macrophage cell lines. These data indicate that Fc epsilon R on normal lymphocytes and monocytes, as well as on cultured lymphoblastoid and macrophage-like cells, are related structurally, since they share antigenic determinants, bind IgE with a similar affinity, and have similar m.w. However, they differ in all 3 parameters from Fc epsilon R on basophilic granulocytes.     

Natural killer cells in the blood and bone marrow of the rhesus monkey

Natural killer (NK) cells in peripheral blood lymphocytes (PBL) and bone marrow (BM) cells of the rhesus monkey were detected by their functional activity against K562 cells. Animals could be grouped into "high" or "low" NK responders, a trait found to be consistent over a period of 2 years. NK active cells in PBL were in the nonadherent population, with the majority bearing Fc receptors and a further subdivision of these into CR+ (complement receptor) and CR- NK cells. Of 10 monoclonal antibodies directed against different epitopes of human lymphocytes, OKT11, OKT10, and Leu 11 showed reactivity with rhesus NK cells. Only OKT10 was reactive with the effector site of the cell, as shown by its capability to block NK function. Of the Leu 11 monoclonal antibodies (a, b, c), Leu 11c was nonreactive while Leu 11a and Leu 11b were shown by immunofluorescence to bind to 7 to 21% of PBL; Leu 11b was also cytotoxic to the NK cells. Leu 11b did not prevent binding of Leu 11a to PBL, suggesting reactivity of these antibodies with different epitopes. Percoll fractionation of PBL and BM revealed a greater enrichment of NK activity with BM; also, with PBL peak NK activity occurred in fractions 4 and 5 while this occurred in fraction 5 with BM. Although Percoll PBL fractions contained a higher percentage of Leu 11b cells, the NK activity of the BM fractions was proportionately greater. The majority of PBL cells with NK activity were FcR+ while significant activity could be attributed to FcR- cells of BM, in both the unseparated and Percoll fractions of each tissue. The data suggest NK active cells of BM may be distinct from those found in PBL.     

In vitro induction of HLA-restricted cytotoxic T lymphocytes against autologous Epstein-Barr Virus transformed B lymphoblastoid cell line

Cytotoxic T lymphocytes (CTL) against autologous EBV-transformed B lymphoblastoid cell line (LCL) were induced in vitro by culturing peripheral blood lymphocytes (PBL) of healthy donors together with mitomycin C-treated autologous LCL for 6 days. The cytotoxic cells developed only from the E-rosette-positive fraction but not from the negative fraction of PBL. These CTL killed autologous LCL but not PWM-stimulated autologous PBL. In addition, the CTL killed allogeneic LCL when at least 1 of the HLA-A antigens was identical with that of the LCL of CTL donor. However, identity of HLA-B and HLA-C antigens was not enough for a significant killing of allogeneic LCL. The specificity of the CTL was also confirmed by a cold target inhibition test. These results indicated that the CTL induced specifically recognized EBV-transformed cells with HLA restriction.