Two distinct cytotoxic T lymphocyte subpopulations in patients with Vogt-Koyanagi-Harada disease that recognize human melanoma cells

The functional properties of cytotoxic lymphocytes from patients with Vogt-Koyanagi-Harada disease ( VKH ) specific for human melanoma cells (P-36 melanoma cell line established from a patient with malignant melanoma) were investigated by using monoclonal antibodies specific for human T cell subsets. Peripheral blood lymphocytes (PBL) from patients with VKH showed significant cytotoxic activity against the P-36 (SK-MEL-28) human melanoma cell line, but not against a human cervical carcinoma of the uterus cell line (HeLa-S3 cell line) or against a mouse melanoma cell line (B-16 cell line) originating from a C57BL/6 strain mouse or against the EL-4 mouse lymphoma cell line from a C57BL/6 mouse. The cytotoxic activity of the patients' PBL against the P-36 melanoma cell line was markedly reduced by pretreatment of the PBL with monoclonal anti-human Leu-1 antibody plus rabbit complement, but it was reduced to much less extent by pretreatment with either monoclonal anti-human Leu-2a or Leu-3a antibody plus rabbit complement. The specific cytotoxic activity of the patients' PBL against the P-36 human melanoma cell line is, therefore, mediated by T cells bearing Leu-1+ Leu-2a+ or Leu-1+ Leu-3a+ antigens. Furthermore, the cytotoxic activity was shown to be blocked not only by anti-Leu-2a antibody specific to human cytotoxic/suppressor T cells but also unexpectedly by anti-Leu-3a antibody which has previously been considered to be specific to human inducer/helper T cells. The results of this study suggest that at least two distinct subpopulations of cytotoxic T cells specific for P-36 human melanoma cells are present in the peripheral blood of VKH patients. These cytotoxic T cells have different surface antigens, Leu-2a and Leu-3a.     

Phenotypic characterization of cynomolgus monkey natural killer cells

Natural killer (NK) activity of cynomolgus monkey peripheral blood lymphocytes (PBL) was determined using B95-8 cells as target cells. Examination for the reactivity of human NK-related monoclonal antibodies (mAbs), anti-Leu-7, anti-Leu-11b, anti-NKH1A, and NC-1, with cynomolgus PBL revealed that Leu-11b (CD16) was the only antigen expressed on cynomolgus PBL. The percentage of Leu-11b-positive (Leu-11b+) cells correlated well with the level of NK activity when PBL taken from 21 monkeys were tested. After depletion of Fc receptor-positive (FcR+) cells, NK activity was lost concomitantly with the disappearance of Leu-11b+ cells. These results show that cynomolgus NK cells are mainly FcR+ which can be detected by mAb directed to Leu-11b. Cynomolgus PBL were separated by Ficoll-Hypaque centrifugation after E rosette formation with 2-aminoethylisothiouronium bromide-treated sheep red blood cells, and NK activities of both E rosette-forming (E+) and nonforming (E-) fractions were determined. The high level of killing was observed in the E- fraction, suggesting that the majority of cynomolgus NK cells was contained in the E- fraction. The separation of PBL by Percoll discontinuous density gradient showed cynomolgus NK cells were enriched in the low density fractions.     

Electron microscopic studies of saprobic and parasitic forms of Coccidioides immitis

During studies of both saprobic and parasitic cycles of Coccidioides immitis, we found that the hyphae contained septa with simple pores, Woronin bodies, pinocytotic vesicles and/or lomasomes. The alternating thallic arthroconidia were released by fracturing of the adjacent sterile cells. The endospores were formed by progressive cleavage of the spherules. The taxonomic classification of C. immitis still remains obscure.     

In vitro hemolysis of autologous erythrocytes caused by immune murine spleen cells and spherules of the fungus Coccidioides immitis.

This communication describes an in vitro system wherein mouse erythrocytes are lysed in the presence of spherules of the fungus Coccidioides immitis and spleen cells from syngeneic mice immunized with a variety of antigens. The antigens include: tobacco mosaic virus in complete Freund's adjuvant (CFA), CFA alone, separate components of CFA, sheep erythrocytes, and allogeneic tumor. Spleen cells from mice sublethally infected with C. immitis are also capable of participating in this response. The lytic phenomenon, which does not require complement, is dependent upon the number of spleen cells per culture, the number of spherules per culture, the time of culture incubation, the amount of antigen injected into the animal and the time after immunization at which spleen cells are recovered. Live spherules or spherules killed with heat, with dimethylsulfoxide, or with formalin were effective participants, together with immune spleen cells, in the lytic reaction.     

Depletion of NK cells with the lysosomotropic agent L-leucine methyl ester and the in vitro generation of NK activity from NK precursor cells

Human natural killer (NK) cell activity in peripheral blood lymphocytes (PBL) is totally inhibited by pretreatment of the effector cells with a lysosomotropic agent, L-leucine methyl ester (LeuOMe). This treatment specifically eliminates cells expressing the NK cell markers HNK-1, OKM1, B73.1, or Leu-11b, but has minimal effect on viability of cells with T cell markers Leu-1, OKT3, Leu-2a, or Leu-3a. LeuOMe also drastically decreased the proportion of K562 target-binding lymphocytes among PBL. PBL pretreated with LeuOMe respond normally in thymidine uptake to stimulation by phytohemagglutinin or allogeneic lymphocytes as long as irradiated autologous accessory cells are provided, indicating that the treatment is not toxic to T cells. NK activity can be regenerated in the NK cell-depleted PBL population by incubation with IL 2 or by mixed lymphocyte cultures, but not by alpha-interferon. Cells responsible for regeneration of such NK activity are probably large agranular lymphocytes, because they are resistant to LeuOMe treatment but have the same low buoyant density as NK cells in Percoll density gradient separation. The in vitro-generated NK is still sensitive to LeuOMe inhibition, but a higher concentration of the reagent is required to achieve total inhibition of the activity.     

Ultrastructure of Coccidioides immitis after exposure to the imidazole antifungals miconazole and ketoconazole

Scanning and transmission electron microscopy was performed on the various phases of Coccidioides immitis, exposed for different periods of time to the imidazole antifungals miconazole and ketoconazole. The development of spherules into endospores, which takes place in cultures under normal growth conditions, was suppressed in the drug treated cultures. Typical ultrastructural changes were localized at the cell periphery and in the vacuolar system. The drugs did induce changes in mature, resting endospore cultures and in cultures incubated statically at room temperature. Aerobically growing endospores were not susceptible to either drug. The transformation of arthroconidia into mycelium was fully prevented after treatment. Mycelial cells were most susceptible to the antifungals for necrosis was induced in a substantial part of the hyphae after exposure for 24 h.     

Spontaneous and lymphokine-induced cytotoxic activity of monkey intestinal mucosal lymphocytes

We determined the capacity of primate (macaque monkey) intestinal mucosal lymphocytes to mediate natural killer cytotoxicity, and characterized the nature of cells mediating this form of cytotoxicity in the intestine. Isolated macaque monkey intestinal mucosal lymphocytes were found to have intermediate levels of spontaneous cytotoxicity against K562 target cells compared to higher values found in lymphocytes of peripheral blood (PBL) and spleen and low values for mesenteric lymph node (MLN) lymphocytes. Intestinal lymphocytes were similar to PBL in having the same range of target cell specificites, in having augmentation of activity by interferon or interleukin 2, and in demonstrating specificity in cold target inhibition studies. Both intestinal and PBL spontaneous cytotoxic function in primates was mediated predominantly by cells bearing antigens cross-reactive with the anti-human monoclonal antibody Leu-11. The percentage of Leu-11+ lymphocytes was significantly lower in isolated intestinal, spleen, and MLN lymphocytes compared to peripheral blood. Furthermore, isolated intestinal lymphocytes differed from PBL in that intestinal Leu-11+ were predominantly Leu-15-, while Leu-11+ PBL were predominantly Leu-15+. These studies demonstrate that the lower spontaneous cytotoxic function of intestinal mucosal lymphocytes compared to PBL is associated with a lower number of effector cells and with effector cells which differ qualitatively in expression of the Leu-15 antigen.     

Salivary gland lymphocytes in primary Sjogren's syndrome lack lymphocyte subsets defined by Leu-7 and Leu-11 antigens

Primary Sjogren's Syndrome (SS) is an autoimmune disease characterized by dry eyes and dry mouth due to lymphocytic infiltration of lacrimal and salivary glands. Biopsies of their salivary glands provided an opportunity to characterize the phenotypic and functional properties of inflammatory site lymphocytes. We found that the salivary gland lymphocytes (SGL) of SS patients differed from the peripheral blood lymphocytes of the same patients because: a) SGL lacked lymphocytes reactive with anti-Leu-7 and anti-Leu-11 monoclonal antibodies; b) SGL lacked natural killer (NK) activity; and c) SGL lacked the ability to suppress polyclonal B cell responses in the presence of complement fragment C3a, a function that requires the presence of Leu-7+ cells. These studies also showed that the SGL of SS patients differed from tonsillar lymph node (LN) lymphocytes of immunologically normal individuals because tonsillar LN contained Leu-7+ T cells, and tonsillar LN could suppress polyclonal B cell responses in the presence of the complement fragment C3a. The absence of this regulatory subset in the salivary glands of SS patients may contribute to pathogenesis, because these cells may be important in the suppression of polyclonal antibody synthesis and in the elimination of neoplastic or viral infected cells.     

Mode of in vitro augmentation of natural killer cell activity by recombinant human interleukin 2: a comparative study of Leu-11+ and Leu-11- cell populations in cord blood and adult peripheral blood

Human newborn natural killer (NK) cell activity against K562 target cells was observed to be low compared with adult controls. Although Leu-7 (HNK-1)+ cells were negligible in cord blood, the proportions of Leu-11+ cells were equal to those of adult peripheral blood. Leu-11+ cells sorted from cord blood lymphocytes, as well as from adult lymphocytes exhibited the morphology of granular lymphocytes. In this study, we have investigated the phenotypic characterization of recombinant interleukin 2 (rIL 2)-induced cytotoxic lymphocytes against K562 cells by using anti-Leu-11 monoclonal antibody. Spontaneous cytotoxicity of lymphocytes was restricted to Leu-11+ cells in cord blood, as well as in adult blood, but this activity was low in cord blood Leu-11+ cells as compared with that of adult ones. NK cell activity of adult Leu-11+ cells could not be additionally enhanced after an 18-hr incubation with rIL 2(25 U/ml), whereas rIL 2 could potentiate the cytotoxicity of cord blood Leu-11+ cells approximately to the adult levels. It should be noted that cytotoxic activity of both Leu-11- cells from cord blood and adult blood that had no basal NK cells activity could be significantly potentiated by rIL 2. On the other hand, lymphokine-activated killer cells cytotoxic for HL-60 cell line could not be generated, and no proliferation of the lymphocytes was detected after an 18-hr incubation with rIL 2. It was shown that rIL 2 could not enhance the ability to bind to target cells in Leu-11+ and Leu-11- cells by means of a single cell conjugate assay, but the rate of target lysis of Leu-11+ cells from cord blood was significantly enhanced by rIL 2. These results suggested that rIL 2-induced cytotoxic effector cells were heterogeneous, and rIL 2 might potentiate the cytotoxicity of functionally immature NK cells or NK precursor cells.     

C-reactive protein antigenicity on the surface of human peripheral blood lymphocytes. Characterization of lymphocytes reactive with anti-neo-CRP

Previously, we have shown that antibodies specific for C-reactive protein determinants, not present on the native molecule, termed neo-CRP, also react with a significant percentage of PBL. In the present study, cells were evaluated by flow cytometry using alpha-neo-CRP antisera and mAb specific for lymphocyte subsets. With use of either monocyte-depleted PBL or Percoll-enriched large granular lymphocytes, we observed an overlap between cells reactive with alpha-neo-CRP and cells bearing the surface markers CD16, CD11b, Leu-7, and/or Leu-19, which are expressed on NK cells. In addition, we showed co-expression of the neo-CRP antigen with CD19, CD20, and HLA-DR, cell surface markers which are expressed on B lymphocytes. The major proportion of CD3+ cells failed to exhibit co-expression of neo-CRP. Single parameter flow cytometric analyses demonstrated that cells reactive with alpha-neo-CRP exhibited a bimodal staining pattern based on fluorescence intensity: high intensity neo-CRPbright and low intensity neo-CRPdim. Two-color analysis revealed that neo-CRPbright cells co-expressed CD19, CD20, and HLA-DR, whereas neo-CRPdim cells co-expressed CD16, CD11b, Leu-7, and Leu-19. Anti-neo-CRP also reacted with PBL obtained from patients with CD16+ lymphoproliferative disorders and from patients with chronic lymphocytic leukemia of B cell origin, but not with cells from patients with T cell or myeloid leukemias. The alpha-neo-CRP cells from patients with NK cell expansions showed dim fluorescence, whereas patients with B cell expansions showed bright fluorescence, consistent with the staining patterns observed with normal PBL. In addition, cell lines of T cell, B cell, NK cell, myeloid, and erythroid origin were evaluated for reactivity with alpha-neo-CRP. The cloned NK cell line NK 3.3 reacted as neo-CRPdim, but the B cell lines BL41, BL41/95, T1, T2, and CESS all reacted as neo-CRPbright. The cell lines K562, Molt-4, Hut-78, HL-60, U-937, and THP-1-0, which lack characteristic NK and B cell markers, did not react with alpha-neo-CRP. Additional study of the two-color histograms revealed a distinct diagonal staining pattern that was observed only when cells were co-stained with alpha-neo-CRP and either alpha-CD16 (alpha-Fc gamma RIII) or antibody IV3 (alpha-CDw32; alpha-Fc gamma RII). This finding suggests a 1:1 relationship between Fc gamma R on both NK and B cells and determinants recognized by alpha-neo-CRP.(ABSTRACT TRUNCATED AT 400 WORDS)     

HLA-linked nonresponsiveness to Cryptomeria japonica pollen antigen. I. Nonresponsiveness is mediated by antigen-specific suppressor T cell

The objective of this study was to elucidate the cellular mechanism of IgE nonresponse to the Cryptomeria japonica (Japanese cedar) pollen antigen (CPAg), which was shown in our previous study to be HLA-linked (1). We established an assay system for the measurement of small amounts of anti-CPAg IgE antibody, both in an antigen-specific and isotype-specific manner, and a culture system to induce antigen-driven IgE antibody synthesis in vitro. By using these methods, we clarified that the function of the HLA-DR molecule in the CPAg-driven IgE response is similar to that of I-A or I-E molecule in mice, namely the product of immune response genes (Ir-genes), because anti-HLA-DR monoclonal antibody blocked the response, and the interaction between monocyte and monocyte-depleted peripheral blood lymphocytes (PBL) to respond to CPAg was restricted by HLA-DR. Furthermore, PBL from nonresponders revealed a specific IgE response to CPAg when the Leu-2+3- T cell fraction was depleted, thereby suggesting that even nonresponders have Leu-2-3+ T cell and B cell clones specific for CPAg, and they apparently show no response due to the presence of CPAg-specific Leu-2+3- suppressor T cells. This suppressor T cell fraction abolished the IgE response of the autologous B + monocyte + Leu-2-3+ T cell in a CPAg-specific manner. The current cellular analysis together with our previous genetic analysis strongly suggest that the HLA-linked IgE nonresponse to CPAg is mediated by CPAg-specific suppressor T cells. The HLA-linked gene controlling the nonresponsiveness to CPAg is thus designated as the immune suppression gene for CPAg (Is-CPAg). Mapping of Is-CPAg within HLA-DQ subregion is discussed.     

Interleukin 2-dependent natural killer (NK) cell lines from patients with primary T cell immunodeficiencies

Bulk cultured cell lines with natural killer (NK) activity were derived by in vitro culture with interleukin 2-containing conditioned medium (IL 2-CM) of peripheral blood mononuclear leukocytes (PBL) from patients with primary T cell deficiencies. Lines were developed from three patients with severe combined immunodeficiency (SCID) and one patient with Nezelof's syndrome and contained several populations of cells with distinct phenotypes. All lines contained a cell population expressing the Leu-5 (50K) (sheep red blood cell receptor), 3A1 (40K), and OKT10 antigens, but lacking the pan T cell antigens Leu-1 (67K) and Leu-4 (19K) as well as the markers of T cell subsets Leu-2a (32K) and Leu-3a (56K). These cells failed to express the Leu-7 antigen and only weakly expressed OKM1. In addition, one line contained a population of Leu-5+, 3A1+, OKT10+, Leu-2a+, Leu 1-, and Leu 4- cells. Three of the lines also contained populations with classic T cell (Leu-1 and-Leu 4+) phenotypes. The lines were enriched in NK activity compared with the PBL from which they were derived. Their growth was strictly dependent on IL 2-CM. Highly purified IL 2, lacking any other detectable protein contaminants or lymphokine activities, was capable of supporting the growth of the Leu-5+, 3A1+ "null" cell populations from these lines without alteration in their functional activity or phenotype. Thus, studies of in vitro expanded cell lines from patients with severe disorders of T cell function and thymic involution indicate that this "null" cell population does not require thymic maturation to develop its effector function. This "null" cell population can be maintained in vitro in the presence of IL 2. This finding is analogous to the data obtained from study of NK cells in athymic (nude) mice.     

Natural cytotoxic effector cell activity against Shigella flexneri-infected HeLa cells

Virus and facultative intracellular bacteria both replicate within a host cell. The recognition and killing of virus-infected cells by natural killer (NK) cells is thought to be an important host immune function. However, little is known about immune recognition of bacteria-infected cells. In this report, we show for the first time that human peripheral blood lymphocytes (PBL) and large granular lymphocytes (LGL) purified from PBL have significant levels of cytotoxic activity against Shigella flexneri-infected HeLa cells. This cytotoxic activity was dependent on bacterial invasion of the HeLa cells, because HeLa cells pretreated with a noninvasive isogenic variant of S. flexneri or soluble bacterial products were not killed. Pretreatment of PBL with interleukin 2 (IL 2) or interferon-alpha greatly enhanced the cytotoxic activity of PBL against Shigella-infected HeLa cells. Cytotoxic activity present in PBL or in PBL pretreated with IL 2 was shown to be associated with both Leu-11+ and Leu-11- cell populations. These results suggest that NK cell killing of bacteria-infected cells may play an important role in host defense against facultative intracellular bacterial infections.     

Anti-lymphocyte antibody Leu-7 (HNK-1) recognizes a constituent of neuroendocrine granule matrix

Anti-lymphocyte monoclonal antibody HNK-1 (Leu-7) reacts with the cell surfaces of natural killer (NK) lymphocytes and with myelin-associated glycoprotein (MAG). This antibody reacts intensely with normal and neoplastic adrenal medullary cells. A small proportion of normal pancreatic islet cells, anterior pituitary, and gastroenteropancreatic endocrine cells also show Leu-7 immunoreactivity. In adrenal medulla, ultrastructural immunocytochemical studies and immunoblot analyses reveal that Leu-7 reacts with an intracellular protein of MW 75 KD which is localized within the matrices of the chromaffin granules. The MW of this protein differs from those of MAG and chromogranin A. The findings suggest that Leu-7 immunoreactivity might be a new marker for specific subsets of secretory granules.     

Sugars and Amino Acids as Carbon, Nitrogen, or Energy Sources for Coccidioides immitis Spherules and Endospores

Of various carbohydrates and amino acids tested, glucose, mannose, fructose, and glutamate were the most efficient substrates metabolized by the endospores and spherules of Coccidioides immitis.     

K562 killing by K, IL 2-responsive NK, and T cells involves different effector cell post-binding trigger mechanisms

The monoclonal antibody 13.3 specifically blocks the trigger process of the NK-K562 cytolytic sequence at a post-binding effector cell level. This antibody was used to define differences in the lytic trigger processes of NK and other mechanisms of K562 lysis. Monoclonal antibody 13.3 inhibited lysis of K562 target cells by freshly isolated peripheral blood lymphocytes (PBL) and purified large granular lymphocytes (LGL), but had no inhibitory effect on antibody-dependent cell-mediated cytotoxicity to K562 by these effectors. Lectin-dependent cellular cytotoxicity (LDCC) to this target cell was also unresponsive to 13.3. The 13.3-induced inhibition of NK-K562 lytic activity persisted when PBL were activated in culture with interleukin 2 (IL 2) for periods up to 48 hr. After 48 hr of culture, the degree of inhibition diminished progressively in medium containing fetal calf serum but not in medium containing autologous serum. This 13.3-unresponsive lytic activity in cultured PBL could be attributed to more than one cell type and was present in both the LGL and Fc gamma receptor-depleted T cell fraction. Thus, K562 lysis by freshly isolated human lymphocytes via NK, K, and LDCC mechanisms is characterized by heterogeneity of the post-binding effector cell trigger mechanism. K562 lysis by lymphocytes cultured with IL 2 is similarly heterogeneous.     

Tissue distribution of human NK cells studied with anti-Leu-7 monoclonal antibody

The distribution and numbers of human natural killer (NK) cells in different lymphoid tissues and several tumors were determined with the use of monoclonal antibody to the Leu-7 antigen and immunohistologic methods. Anti-Leu-7 reacts with large granular lymphocytes containing most of NK activity in the peripheral blood. The Leu-7+ cells were found mainly in the germinal centers of secondary follicles in lymph nodes, spleens, and tonsils. The mean number of Leu-7+ cells in 40 germinal centers was 223 +/- 103 SD. Only rare cells were found in the thymus. In the follicles studied in serial sections, the distribution of the Leu-7+ cells was distinctly different from that of T cells, B cells and Ia+ cells. The Leu-7+ cells did not react with OKT 6 antibody specific for immature (control) thymocytes and Langerhans' cells. By double staining techniques, two populations of Leu-7+ cells were identified in lymphoid tissues: those that did and did not express the Ia-like antigen. The numbers and localization of the Leu-7+ cells varied in different tumors, but often the Leu-7+ cells surrounded the neoplastic nodules or were seen in contact with individual cancer cells. Anti-Leu-7 may be a useful reagent for monitoring the presence and localization of NK cells in normal and malignant human tissues.     

Production of B cell growth factor by a Leu-7+, OKM1+ non-T cell with the features of large granular lymphocytes (LGL)

The present study reports the characterization of a non-T cell from human peripheral blood which is capable of releasing BCGF. This BCGF-producing non-T cell had a T3-, T8-, Leu-7+, OKM1+, HLA-DR-, Leu-11- surface phenotype and was likely to belong to the so-called large granular lymphocyte (LGL) subset because: after fractionation of non-T cells according to the expression of Leu-7 or HLA-DR markers, it was found in the Leu-7+, HLA-DR- fractions that were particularly enriched in LGL; it co-purified with LGL on Percoll density gradients; and it expressed Leu-7 and OKM1 markers that are shared by a large fraction of LGL. Although co-purified with cells with potent NK capacities, the BCGF-producing cell was not cytotoxic, because treatment of Leu-7+ cells with Leu-11 monoclonal antibody and complement abolished the NK activity but left the BCGF activity unaltered. The factor released by this LGL subset was not IL 1 or IL 2 mistakenly interpreted as BCGF, because: a) cell supernatants particularly rich in BCGF activity contained very little or no IL 1 or IL 2; b) BCGF-induced B cell proliferation was not inhibitable by anti-Tac antibodies (this in spite of the expression of IL 2 receptor by a proportion of activated B cells); and c) BCGF activity was absorbed by B but not T blasts.     

Leu-11+ lymphocytes with natural killer (NK) activity are precursors of recombinant interleukin 2 (rIL 2)-induced activated killer (AK) cells

Precursors of activated killer (AK) cells cytotoxic for human noncultured metastatic melanoma and colon carcinoma were characterized. These cells required 3 days incubation with recombinant interleukin 2 (rIL 2) and DNA synthesis for the induction of AK activity. Both negative and positive cell purification methods were used to identify the subpopulation of cells containing AK precursors. By complement-mediated cell depletion studies, AK precursors were largely present in the Leu-11+ fraction, and to a much lesser extent in the Leu-7+ and Leu-2a+ fractions; they were absent in Leu-3a+ and Leu-4+ cells. Lymphocyte subpopulations were then purified with a cell sorter to positively select for the subset containing AK precursors. Leu-11+ cells had the highest level of AK activity and proliferative response when cultured for 3 days with rIL 2 as well as the highest level of NK activity before culture. Leu-7+ cells had neither AK activity nor a proliferative response when cultured with rIL 2, although they still possessed high NK activity. The same levels of AK and NK activity were found in Leu-2a+ and Leu-2a- fractions, but both activities were absent among Leu-4+ and Leu-3a+ cells. Further fractionation with a two-step sorting technique showed that the highest AK activity resided in the Leu-7-Leu-11+ cell fraction. Morphologically, this subfraction was granular lymphocytes. Titration experiments or rIL 2-responsive cells showed that the number of cells required to achieve a comparable level of rIL 2 proliferative response were as follows: 35 X 10(3) cells from unseparated PBL, 10 X 10(3) cells from Leu-11+ cells, 3.3 X 10(3) from Leu-7-Leu-11+ cells, and 640 X 10(3) cells from Leu-7+ cells. These results indicate that the lymphocyte subpopulation that proliferates in the presence of rIL 2 and then develops AK activity was a subpopulation of Leu-11+ granular lymphocytes, which also possessed the highest NK activity. These Leu-11+ cells lacked the antigens defined by the Leu-7, Leu-3a, or Leu-4 antibodies. Although Leu-7+ cells did not respond to rIL 2 by themselves, they may play a role in the induction of AK activity.     

Role of the production of natural killer cell stimulatory factor (NKSF/IL-12) in the ability of B cell lines to stimulate T and NK cell proliferation.

We have previously used human Epstein Barr virus (EBV)-transformed B lymphoblastoid cell lines for the identification and purification of a novel cytokine, natural killer cell stimulatory factor (NKSF/IL-12), that has pleiotropic effects on human lymphocytes. B cell lines are also routinely employed as feeder cells for the culture of T and natural killer (NK) cells. In this report we describe the ability of two NKSF/IL-12 producing B cell lines (RPMI-8866 and Cess) and two nonproducing lines (Raji and Daudi) to stimulate the proliferation of T and NK cells in 8-day PBL cultures. We demonstrate, using an anti-NKSF/IL-12 neutralizing monoclonal antibody, that the endogenous production of NKSF/IL-12 in these cultures can significantly enhance the proliferation and cytotoxic activity of T and NK cells. We also report that the addition of exogenous rNKSF/IL-12 can greatly increase the number of T and NK cells obtained from the cultures following stimulation by the B cell lines. Aside from the possible practical applications, the enhanced proliferation of T and NK cells consistently observed in the presence of endogenously produced NKSF/IL-12 or exogenously added rNKSF/IL-12 in this system may further our understanding of the role of this cytokine during an in vivo immune response.