Interleukin-2 effects on human B cells activatedin vivo

While activated B cells, as well as T cells, can express functional interleukin-2 receptors (IL-2R), the physiologic role of IL-2 in B-cell responses is still in doubt. Accordingly, we have examined the role of recombinant IL-2 (rIL-2) in the proliferative response, IL-2R expression, and the terminal differentiation of tonsillar B cells in comparative studies with T cells from the same source. For these analyses of physiologically activated lymphocytes, the B and T cells were first separated, then divided into subpopulations enriched for either resting or preactivated cells on the basis of relative cell density or activation antigen expression. As expected, the relatively low-density fraction of T cells was enriched for IL-2R-positive (Tac⁺) cells and displayed vigorous proliferative responses to IL-2 along with heightened Tac antigen expression. Moreover, limiting dilution analysis revealed that growth of these activated T cells could be perpetuated with the addition of IL-2. By comparison, relatively few tonsillar B cells expressed the Tac antigen. The addition of rIL-2 to cultured B cells of relatively low density resulted in an increase in Tac⁺ cells but only a minimal proliferative response. The subpopulation of tonsillar B cells expressing the Bac-1 activation antigen contained most of the IL-2 inducible Tac⁺ B cells, and rIL-2 induced an efficient but transient proliferative response by these activated B cells. The rIL-2-induced Tac⁺ B cells were noted to be relatively large. A fraction of these Tac⁺ cells, but not the Tac^– cells, produced and secreted immunoglobulins. Incubation with rIL-2 enhanced the Ig secretion, and the anti-Tac antibody blocked this enhancement. Time-course analysis revealed that rIL-2 induced transient Tac expression, whereas mature plasma cells in 6-day cultures no longer expressed detectable Tac antigen. In conclusion, these observations suggest that IL-2 transiently upregulates expression of IL-2R, via which it induces the terminal growth and differentiation of activated B cells into plasma cells.     

Histological evidence of natural killer cell aggregation against malignant melanoma induced by adoptive immunotherapy with lymphokine-activated killer cells

Adoptive immunotherapy with lymphokine-activated killer (LAK) cells and systemic administration of recombinant Interleukin-2 (RIL-2) was carried out in a case of malignant melanoma with lung metastases. Histological specimens from the lung showed a metastatic melanoma heavily invaded by atypical lymphoid cells with convoluted nuclei of varying size. Immunohistochemistry revealed that these cells had the characteristic exclusively of natural killer cell (Leu-7+). Nodules of these cells mimicked the appearance of non-Hodgkin's lymphoma of pleomorphic type. Molecular cytogenetic analysis, however, showed the absence of rearranged bands for the T-cell receptor beta-chain gene, indicating the absence of T-cell clones. At autopsy, 1 month after the LAK therapy, the heavy invasion of convoluted cells had disappeared. These findings clearly indicate that the LAK cell plus RIL-2 therapy induced Leu-7+ lymphoid cells, phenotypically suggestive of natural killer cell aggregation in the tumours.     

健康人外周血单个核细胞诱导的NK细胞对脑胶质瘤杀伤作用的探讨

目的 对体外培养的NK细胞杀伤脑胶质瘤细胞进行探讨,为脑胶质瘤的免疫细胞疗法提供理论基础.方法 从4个健康人外周血提取单个核细胞,在KRATM培养环境下,诱导产生NK细胞和LAK细胞.分别用细胞计数法和流式细胞仪检测NK细胞增殖和细胞表面特异性标志CD3/CDl6/CD56,并用MTr法检测NK细胞及LAK细胞对脑胶质瘤细胞的杀伤.结果 经过体外培养,平均可得到7.93×109个以上细胞.和LAK细胞相比,NK细胞对脑胶质瘤细胞LNI8、LN229、T98G和U87MG的敏感性更高.结论 NK细胞能够对脑胶质瘤细胞产生有效免疫应答,为过继性免疫细胞治疗脑胶质瘤提供可能.     

Invariant natural killer T cells balance B cell immunity

Invariant natural killer T (iNKT) cells mediate rapid immune responses which bridge the gap between innate and adaptive responses to pathogens while also providing key regulation to maintain immune homeostasis. Both types of important iNKT immune responses are mediated through interactions with innate and adaptive B cells. As such, iNKT cells sit at the decision‐making fulcrum between regulating inflammatory or autoreactive B cells and supporting protective or regulatory B cell populations. iNKT cells interpret the signals in their environment to set the tone for subsequent adaptive responses, with outcomes ranging from getting licensed to maintain homeostasis as an iNKT regulatory cell (iNKT_reg) or being activated to become an iNKT follicular helper (iNKT_FH) cell supporting pathogen‐specific effector B cells. Here we review iNKT and B cell cooperation across the spectrum of immune outcomes, including during allergy and autoimmune disease, tumor surveillance and immunotherapy, or pathogen defense and vaccine responses. Because of their key role as influencers, iNKT cells provide a valuable target for therapeutic interventions. Understanding the nature of the interactions between iNKT and B cells will enable the development of clinical interventions to strategically target regulatory iNKT and B cell populations or inflammatory ones, depending on the circumstance.     

Interleukin-2 (r-IL-2)-induced lymphokine-activated killer (LAK) cells and their precursors express the VGO1 antigen

Precursor and effector cells of recombinant interleukin-2 (r-IL-2)-induced lymphokine-activated killer (LAK) activity were investigated for their expression of VGO1. Peripheral blood lymphocytes (PBL) from normal donors were purified and separated in a FACS 420 into VGO1⁺-and VGO1^–-cell fractions before and after culture for 96 hr with 100 U/ml of r-IL-2. Their lytic activity against K 562 and Daudi cells was measured in a⁵¹Cr release assay. The majority, if not all, of the LAK effector and precursor cells was VGO1⁺ lymphocytes. The expression of VGO1 by LAK precursor cells remained stable under the culture conditions used in our experiments. VGO1^– lymphocytes cultured with r-IL-2 demonstrated neither LAK-induced activity nor expression of VGO1 antigen.     

In vivo treatment with interferon causes augmentation of IL-2 induced lymphokine-activated killer cells in the organs of mice.

Interferon-alpha (IFN-alpha) has been shown to synergize with IL-2 in the regression of a variety of established murine tumours and studies are underway to explore this combination in patients with advanced cancers as well. To understand the mechanism of synergy we have studied lymphokine-activated killer (LAK) cell activity in various compartments of mice in response to IFN-alpha and IL-2 administration. The effects of IFN-gamma, TNF-alpha and IL-4 were also examined. C57BL/6 mice were injected intraperitoneally with HBSS, IL-2 alone, IFN-alpha alone or both, two times a day for 7 days. On days 4 and 8, LAK activity was tested in a 4-h chromium release in cells obtained from lungs, spleen, and liver using fresh MCA-102 tumour cells as targets. The cells from control mice failed to lyse the MCA-102 target. IL-2 caused the generation of LAK activity and an increase in total cell yield in all the organs after 3 days of injection. IFN-alpha failed to generate LAK activity but when administered along with IL-2, caused synergistic enhancement of LAK lysis of MCA-102 target cells. Cell yield in this group was lower as compared with the IL-2-treated group. LAK activity tested after 7 days of IL-2 therapy was significantly decreased compared with that observed after 3 days. However, activity remained at as high a level after 7 days of therapy as after 3 days of therapy in animals treated with IFN-alpha and IL-2. FACS analysis revealed that asialo GM-1+ (ASGM-1) and NK1.1+ cells were increased in number in IL-2 and IL-2 plus IFN-alpha-treated spleen; however, the number of these cells was similar in both groups. In the liver, ASGM-1+ cells were higher in the IL-2 plus IFN-alpha group than in the group treated with IL-2 alone. By in vitro depletion utilizing antibody and Rbc' experiments, it was clear that both ASGM-1+ and NK1.1+ cells from the spleen mediated most of the cytotoxicity of MCA-102 targets. Pre-treatment irradiation (5 Gy) of mice completely abrogated the capability of IL-2 or IL-2 plus IFN-alpha to generate LAK activity. IFN-gamma also had a stimulatory effect on IL-2 induction of LAK activity. Tumour necrosis factor-alpha (TNF-alpha) and IL-4 failed to generate LAK activity and, in combination with IL-2, no additional stimulatory effect was observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phorbol ester enhances both interleukin 2 receptor expression and immunoglobulin secretion in human Epstein-Barr virus-immortalized B cells

The enhanced expression of interleukin 2 (IL2) receptors by 12-O-tetradecanoylphorbol 13-acetate (TPA) on sublines of an Epstein-Barr virus-immortalized human B17 B cell line (M. Steinitz et al., Immunobiology 1979. 156: 41.) was studied by immunofluorescence using the anti-Tac monoclonal antibody and by binding studies with purified radiolabeled IL2. These studies show that TPA at a final concentration of 5 ng/ml greatly increased Tac antigen expression on a number of sublines of B17. IL2-binding studies revealed that TPA induced an increase in not only the number of IL2 receptors per cell but also the affinity of the receptors for IL2. The number and affinity of IL2 receptors on the C76 subline treated with TPA appear to be similar to those of activated normal human peripheral T cells. Furthermore, TPA-induced differentiation of these B cell lines was measured by induction of immunoglobulin secretion using an enzyme-linked immunosorbent assay. The capacity of TPA to induce differentiation in human B cells and the biological significance of IL2 receptor expression by activated B cells are discussed.     

Lymphokine-activated killer cytotoxicity against pancreas adenocarcinoma cell lines and vascular endothelial cells

Eight pancreas carcinoma cell lines of duct cell origin (PCI-6, 10, 19, 24, 35, 43, 55, and 64) were established. Using one of these lines, PCI-24, human umbilical vein endothelial cells (HUVEC), and several recombinant cytokines, conditions and specificity of antl-PCI LAK induction were Investigated, with the focus on a search for lymphokine-activated killer (LAK) activity that differentiates neoplastic (PCI) from non-neoplastic (HUVEC) cells. Interferon-γ (IFN-γ), IFN-α, IL-4, 11–6, and IL-7, but not tumor necrosis factor-α (TNF-α) or IL-1β, induced a weak LAK activity against PCI-24, whereas IL-2-induced (1000U/mL) LAK exhibited a far more potent cytotoxicity. When these cytokines were added at the suboptimal dose IL-2 (100U/mL), no significant augmentation in LAK activity was induced. Staphylococcal protein A (SpA) induced LAK activity as potent as that seen with IL-2 (1000 U/mL). Both IL-2-induced and SpA-induced LAK had a potent, dose-dependent cytotoxicity against HUVEC. HUVEC inhibited both IL-2– and SpA-induced LAK cytotoxicity against PCI-24 to almost the same extent as seen with PCI-24. Thus, two potent LAK-inducers did not generate LAK activity that differentiates neoplastic from non-neoplastic cells. Thus, in vitro cytotoxicity of LAK agalnst non-neoplastic endothelial cells is unavoidable when handling cytokines in LAK induction.     

Production of human monoclonal antibodies against Epstein-Barr virus-specific antigens by the virus-immortalized lymphoblastoid cell lines

The possible production of human monoclonal antibodies against Epstein-Barr virus (EBV) was assessed through the EBV immortalization technique. When individual lymphocyte samples from 50 clinical patients and healthy donors were immortalized by EBV, 4 lymphoblastoid lines yielded antibodies to EBV antigens. These positive lines were cloned and each line yielded cultures that secreted monoclonal antibodies against either viral capsid antigen (VCA) or membrane antigen (MA) component. Above all, a clonal line TAKA-SP-8 produced 5 micrograms MA antibody/10(6) cells/ml for more than 12 months. The culture fluid specifically immunoprecipitated a single polypeptide with a size of 93K from both P3HR-1 and B95-8 cell extracts. FUKA-SP-3, on the other hand, secreted 5 micrograms VCA antibody/10(6) cells/ml for at least 8 months. This antibody recognized two polypeptides with sizes of 123K and 120K, from P3HR-1 and B95-8 cell extracts, respectively. When B95-8 and P3HR-1 EBV were treated with the human MA monoclonal, both nuclear antigen (EBNA) synthesis and early antigen (EA) induction were strongly inhibited. All EBV antibody-producing cultures were exclusively achieved from splenic lymphocytes of patients with autoimmune diseases, but not from other donors.     

Phenotype characteristics of human B cells studied by Epstein-Barr virus infection II. C3 receptor switching during human B cell differentiation

Our experiments showed that two C3 receptors (C3-R) appeared on membrane surfaces in a distinct sequence during the differentiation of human B cells. The experimental results supporting this conclusion were (1) the lymphoblastoid cells established from immature B cells expressed more EAC^m-R (C3bi and C3d receptors) than EAC^h-R (C3b receptor), (2) the lymphoblastoid cells originated from mature B cells showed more EAC^h-R than EAC^m-R, (3) the above characteristic expression of C3-R was demonstrated on every clone isolated in soft agar from cells in B cell lineage at various differentiation stages, indicating that a cell line consists of homogeneous population in terms of C3-R expression. From these results, it was concluded that C3-R switched from EAC^m-R to EAC^h-R during B cell differentiation.     

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. IX. The quantitation of natural killer cell activity

On analysis ofin vitro assays of human natural killer (NK) cell function the inadequacy of commonly used methods of expressing lytic activity was apparent. A comparison was made of the data obtained using modifications of two equations—the simple exponential fit and the von Krogh equations. Both of these equations were found to satisfy the following essential criteria for use in these assays. First, the majority of the results obtained in the chromium-release assay could be used in data reduction; second, the resultant dose-response curve was reduced to linearity; and third, a single numerical expression was obtained which was directly proportional to the cytotoxic activity. Of the two methods the more conventional exponential fit was found to be the simpler to use. The closeness of fit of the experimentally derived data to the ideal curves did not support the possibility that normal lymphocyte preparations contain suppressor cells capable of inhibiting NK activity. Data have also been presented showing that NK-sensitive targets could be categorized with respect to their susceptibility by comparing the slopes of the target cell survival curves obtained using the exponential fit equation. These observations are relevant to the accurate assessment of NK activity in patient populations and to the determination of the effects of disease and its treatment on this activity.     

Combinational IL-2/IL-15 induction does not further enhance IL-15-induced lymphokine-activated killer cell cytotoxicity against human leukemia/lymphoma cells

In vitro comparisons of induction of perforin (PFP), granzyme B (GRB), production of cytokines, and cell-mediated cytotoxicity by interleukin-2 (IL-2), interleukin-15 (IL-15), or combinational IL-2/IL-15-induced lymphokine-activated killer cells were studied in this study. Whereas IL-2-induction was associated with a decrease in cultured cell population over a 7-day period, IL-15 alone or in combination with IL-2 resulted in significant increase including cytotoxic T lymphocytes and subsets of CD56+ lymphocytes, particularly cytokine-induced killer and cytolytic natural killer-T lymphocytes. The overall PFP, GRB, and tumor necrosis factor-alpha expression in different subtypes were also significantly higher with IL-15 alone or in combination with IL-2 induction with resultant superior cytotoxicity compared to IL-2 treatment. There was no significant advantage of addition of IL-2 over IL-15 induction. These results offer further information on the cytotoxic potency of these cytokines and their mechanisms of action implicating potential use of IL-15 as part of cytokine adoptive immunotherapy.     

人外周血树突状细胞对LAK细胞杀伤活性的影响

从人外周血中分离出树突状细胞，体外观察了其对ＬＡＫ细胞活性的影响。发现：５×１０２～１×１０４／ｍｌ树突状细胞对ＬＡＫ细胞活性起增强作用，而５×１０４～１×１０５／ｍｌ树突状细胞却抑制ＬＡＫ杀伤活性。这说明人外周血树突状细胞对ＬＡＫ细胞活性起双相性调节作用。     

Impaired T cell activation and cytokine production by calcitriol-primed human B cells

The biologically active form of vitamin D₃, 1, 25-dihydroxyvitamin D₃ (calcitriol), is a potent modulator of the immune response. We have shown previously that calcitriol modulates the immunoglobulin response in vitro and in vivo in mice and humans. To analyse the underlying molecular mechanisms we studied whether calcitriol-primed B cells modulate T cell activation and function. Human B cells were stimulated with anti-CD40 and interleukin (IL)-4 in the presence of increasing concentrations of calcitriol. After removal of calcitriol, primed B cells were co-cultured with autologous CD4⁺ T cells; the B cell phenotype T cell activation and their consecutive cytokine production were also assessed. Naive T cells co-cultured with calcitriol-primed naive B cells showed a reduced expansion, nuclear factor of activated T cells, cytoplasmic 2 (NFATc2) expression and cytokine production upon restimulation. CD86 expression on B cells after calcitriol priming was identified as an underlying mechanism, as T cell activation and expansion was rescued by activating anti-CD28 antibodies. Our data indicate that calcitriol-primed B cells display an impaired capacity to activate T cells. Taken together, we identified a novel B cell-dependent vitamin D immune regulatory mechanism, namely by decreased co-stimulation of calcitriol-primed B cells.     

Biological response modifier enhances the activity of natural killer cell against human cytomegalovirus-infected cells

Peripheral blood lymphocytes (PBL) from two human cytomegalovirus (CMV)-seronegative donors and eight CMV-seropositive donors were cultured for 3 days with or without the biological response modifier OK-432 and examined for lysis of K562 cells and CMV-infected MRC-5 cells. OK-432-stimulated PBL exhibited significantly greater natural killer (NK) activity than did unstimulated PBL. There was no difference in activity of NK cells in PBL prepared from CMV-seronegative and -seropositive donors. Antibody-complement depletion studies suggested that OK-432-stimulated NK activity was associated with Leu-7-positive cells. The ability of OK-432 to sustain the NK activity in PBL was decreased when the CD4-positive population of lymphocytes was eliminated by antibody-complement depletion prior to OK-432 stimulation. The ability of OK-432 to sustain the NK activity of PBL was also significantly decreased in the presence of monoclonal antibody against recombinant human interleukin-2. The results suggest that the activity of human NK cells against K562 and CMV-infected MRC-5 target cells can be sustained in vitro by OK-432-stimulated T-helper cells and that the effect of the T-helper cells is mediated, at least in part, by interleukin-2.     

Activity of human natural killer cells against target cells differing in sensitivity to interferon

Department of Interferons, N. F. Gameleya Research Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110. No. 10, pp. 406–409, October, 1990.     

An improved method for the generation of human lymphokine activated killer cells

In an effort to make interleukin-2/lymphokine-activated killer cell (IL-2/LAK) therapy safer for cancer patients, we examined the efficacy of using Fenwal PL732 bags as tissue culture flasks. These bags can be sterilly connected using tubing kits thus reducing the risk of contamination to the cells. Peripheral blood mononuclear cells were obtained from normal donors or cancer patients undergoing IL-2/LAK cell therapy. Following Ficoll-Hypaque purification, these cells were incubated in the presence of IL-2 in either PL732 plastic bags or standard tissue culture flasks. Our results showed that LAK cells could be generated from either normal donors or cancer patients in the bags as well as in the flasks. Comparisons were made of the LAK cell populations obtained from the two sources and showed that each was similar in terms of morphology as determined by Wright stain differentials. The populations of cells were also similar in regard to cell surface phenotype as determined by flow cytometric analysis. In addition, recoveries from either tissue culture vessel as well as cell viability of the LAK cells were comparable. Finally, the LAK cells obtained from both sources were assessed for cytolytic activity against the tumor cell lines K562 and Daudi. These results showed that the cytolytic activity of the LAK cells against these target cells was the same whether the cells were obtained from the flasks or the bags.