Presence Of Recombinant Interleukin Research Articles

Inhibition of Apoptosis Stages of Human Blood Lymphocytes after Exposure to Carbon Monoxide in the Presence of Recombinant Interleukin-2.

We studied the effect of carbon monoxide (60-, 75-, and 90-min exposure) on the expression of antiapoptotic proteins (survivin and Bcl-2) in human blood lymphocytes in the presence of recombinant IL-2 in an apoptosis-inducing dose (0.1 ng/ml). Incubation of cells in atmosphere with carbon monoxide in the presence of recombinant IL-2 was accompanied by accumulation of Bcl-2 protein with simultaneous decrease of survivin content. It was concluded that carbon monoxide plays a role in the dysregulation of apoptosis of human blood lymphocytes Bcl-2 (i.e. CO inhibits the proapoptotic effect of recombinant IL-2).

Characterisation of transcription factors in rainbow trout potentially involved in dendritic cell maturation and activation

Dendritic cells (DCs) are heterogeneous cell populations derived from the bone marrow in mammals and play essential roles in antigen presentation and activation of innate and adaptive immune responses after infection. Phenotyping DCs mainly relies on expression of multiple cell surface markers, making it difficult for comparative studies in different species. Recently several transcription factors (TFs) controlling DC differentiation and maturation have been characterised in humans and mice and are highly conserved. These include zinc finger and BTB domain containing 46 (Zbtb46), zinc finger protein 366 (Znf366) and basic leucine zipper transcription factor/ATF-like 3 (Batf3). To search for common molecular markers to assist identification of DC like cells in fish, in this study the gene homologues for these DC-TFs were sequenced in rainbow trout and their phylogeny was analysed. Gene expression was studied in tissues of healthy fish and in gills of Atlantic salmon after exposure to Paramoeba perurans. Furthermore, monocytes/macrophages were isolated from rainbow trout and cultured in the presence of recombinant interleukin 4/13 for 7 days and then examined for DC-TF expression after stimulation with Toll-like receptor ligands such as lipopolysaccharide, bacterial flagellin, polyI:C and imidazoquinoline.

Inhibition of B-Cell Activation by Retinal Pigment Epithelium

To determine whether retinal pigment epithelial (RPE) cells can inhibit B-cell activation in vitro. Primary cultured RPE cells were established from normal C57BL/6 mice. Activated target B cells were established from splenic B cells stimulated with anti-mouse CD40 antibody and lipopolysaccharide (LPS) in the presence of recombinant interleukin 4 (rIL-4). B-cell activation was assessed by examining proliferation through [(3)H]-thymidine incorporation or carboxyfluorescein succinimidyl ester dilution, and antibody production was determined by ELISA. Expression of costimulatory molecules and the receptors on B cells was evaluated by flow cytometry. Neutralizing anti-TGFβ antibodies were used in the assay. Addition of primary cultured RPE cells suppressed B-cell proliferation in response to anti-CD40, LPS, and rIL-4 stimulation. Similarly, antibody production by these activated B cells was suppressed. Suppression of B-cell activation was mediated by a soluble factor because supernatants from cultured RPE cells were sufficient to inhibit B-cell responses. Moreover, TGFβ was identified as the soluble mediator given that RPE-supernatants failed to suppress B-cell activation if pretreated with neutralizing anti-TGFβ antibodies. Cultured RPE cells suppress the activation of B cells in vitro. These data support the hypothesis that retinal pigment epithelium has immunosuppressive properties that are capable of suppressing B-cell activation.

The effect of enantiomers of beta-agonists on myofibroblast-derived vascular endothelial growth factor and other matrix components in the presence of dust-mite extract

Beta(2)-adrenergic receptor agonists have been shown to modulate airway epithelial cell and smooth muscle release of cytokines and growth factors transforming growth factor (TGF) beta, associated with remodeling, is known to up-regulate the synthesis of vascular endothelial growth factor (VEGF) and stimulate differentiation of fibroblasts to the myofibroblast phenotype. VEGF and fibronectin can promote angiogenesis and (S)-albuterol can induce VEGF secretion from normal human lung fibroblasts (NHLF). We hypothesize that (S)-albuterol could stimulate myofibroblast secretion and expression of VEGF and fibronectin in the presence of Dermatophagoides pteronyssinus extract. Cultured NHLFs were stimulated with IL-1beta, TGF-beta, D. pteronyssinus, and treated with (R)- and (S)-enantiomers of albuterol. VEGF and fibronectin and basic fibroblast growth factor (bFGF) were measured by ELISA and mRNA. VEGF secretion by fibroblasts was twofold higher with 10(-7) M of (R) relative to (S) (p < 0.05). Myofibroblast secretion of VEGF was increased twofold over fibroblasts, but there was no difference between enantiomers. (S)-albuterol at 10(-8)-10(-4) M caused an increase in VEGF mRNA that paralleled VEGF secretion relative to 10(-8)-10(-4) M. Fibronection secretion by myofibroblasts but not fibroblasts was increased by 10(-5) M of (S) relative to (R) in the presence of recombinant interleukin 1 (rhIL-1)beta and D. pteronyssinus (S)-albuterol at 10(-6) M increased bFGF. The 10(-6) M of (S)-albuterol, but not (R)-albuterol, may promote angiogenesis. Increased fibronectin or bFGF by (S)-albuterol could enhance matrix deposition and remodeling in a subset of asthmatic patients.

Persistent IL-10 production is required for glioma growth suppressive activity by Th1-directed effector cells after stimulation with tumor lysate-loaded dendritic cells

Injection of dendritic cells (DC) pulsed with tumor antigens is a novel treatment strategy against malignancies, and aims to elicit anti-tumoral cell-mediated immune responses. We studied the in vitro proliferative responses and cytokine production in T cell cultures after 2 stimulations with autologous DC loaded with tumor lysates derived from glioblastoma multiforme (GBM) cells in the presence of recombinant interleukin (rIL)-6/rIL-12 in the first, and rIL-2/rIL-7 in the second stimulation. After the second stimulation, T cells were co-cultured with glioblastoma (GBM) cells and tumor growth suppression by T cells was assessed using a MTT assay. Although loaded DC induced a significant shift towards T helper cell type 1 (Th1) cytokine production as compared to unloaded DC, persistent interleukin (IL)-10 production by T cells both at the end of 2 stimulations with loaded DC and during the effector phase was also required for their tumor suppressive activity. A stronger glioma growth suppressive activity by T cells stimulated with tumor lysate-loaded DC than by control T cells, cultured with unloaded DC, was seen only if the relative IL-10 production after two stimulations with loaded DC was at least 40% of the IL-10 production after two stimulations with unloaded DC. If less than 40% IL-10 was produced in the experimental condition compared to the control condition, T cells also lost their tumor growth suppressive activity. Addition of rIL-10 during stimulation increased the suppressive activity on tumor cell viability and interferon (IFN)-gamma production by T cells that showed Th1 response upon stimulation with loaded DC. The data point towards the production of both IFN-gamma and IL-10 by responding effector T cells, and towards an immune modulatory rather than immune suppressive role of IL-10 to generate anti-tumoral effector T cells against GBM.

Allele-dependent Similarity between Viral and Self-peptide Presentation by HLA-B27 Subtypes

Molecular mimicry is discussed as a possible mechanism that may contribute to the development of autoimmune diseases. It could also be involved in the differential association of the human major histocompatibility subtypes HLA-B(*)2705 and HLA-B(*)2709 with ankylosing spondylitis. These two subtypes differ only in residue 116 of the heavy chain (Asp in B(*)2705 and His in B(*)2709), but the reason for the differential disease association is not understood. Using x-ray crystallography, we show here that the viral peptide pLMP2 (RRRWRRLTV, derived from latent membrane protein 2 (residues 236-244) of Epstein-Barr virus) is presented by the B(*)2705 and B(*)2709 molecules in two drastically deviating conformations. Extensive structural similarity between pLMP2 and the self-peptide pVIPR (RRKWRRWHL, derived from vasoactive intestinal peptide type 1 receptor (residues 400-408)) is observed only when the peptides are presented by B(*)2705 because of a salt bridge between Arg(5) of both peptides and the subtype-specific heavy chain residue Asp(116). Combined with functional studies using pLMP2/pVIPR-cross-reactive cytotoxic T cell lines and clones, together with target cells presenting these peptides or a modified peptide analogue, our results reveal that a pathogen-derived peptide can exhibit major histocompatibility complex class I subtype-dependent, drastically distinct binding modes. Furthermore, the results demonstrate that molecular mimicry between pLMP2 and pVIPR in the HLA-B27 context is an allele-dependent property.

Enhanced extracellular growth of Staphylococcus aureus in the presence of selected linear peptide fragments of human interleukin (IL)-1beta and IL-1 receptor antagonist.

Replication of Staphylococcus aureus is significantly enhanced in the presence of recombinant interleukin (IL)-1beta. In this study, specific binding of IL-1beta to the surface of S. aureus significantly increased growth of S. aureus in the presence of IL-1beta and IL-1ra in a concentration-dependent manner. Although IL-1ra enhanced the growth of S. aureus, there was a significant reduction in IL-1beta-mediated growth enhancement of S. aureus when 25-fold excess amounts of IL-1ra (in comparison with the IL-1beta concentration) were present in the culture medium. Thus, IL-1beta may influence the growth of S. aureus through a receptor-mediated event. By using 5 linear peptides spanning limited regions of IL-1beta, the growth-promoting regions were localized to amino acid residues 118-147 and 208-240. These results build on the newly evolved concept of direct interactions between the soluble mediators of inflammation and infectious agents.

Observed localization of the long-term cultured rat adherent natural killer cells in mammary tumor tissues.

Adherent natural killer (A-NK) cells were isolated from splenic lymphocytes and treated with long-term culture in the presence of recombinant interleukin-2 (rIL-2). Immunocytochemical and flow-cytometric analysis revealed that most of the A-NK cells strongly expressed lymphocyte-function-associated antigen 1 (LFA-1alpha, and LFA-1beta) throughout the incubation. All A-NK cells from 8- to 150-day cultures, particularly those cultured for 8 days, showed significant cytolytic activity against all targets. Analysis of the tissue distribution of the injected [3H] uridine-labeled A-NK cells demonstrated that, in the first 3 h, most (over 60%) cells localized in the lungs, and that most cells remained temporally within the cavities of blood capillaries of the lungs and moved gradually into lymphoid and other tissues. Peritumoral injection of various kinds of adjuvant, particularly Freund's complete adjuvant (FCA) plus bacillus Calmetee-Guerin (BCG), resulted in a marked accumulation of [3H]A-NK cells in mammary tumor tissues 24 h after injection, and simultaneously in the formation of vessels resembling high-endothelium venules (HEV), infiltration of LFA-1+ lymphocytes and expression of the ICAM-1 molecule on the tumor cells in the sites of tumor tissues. When 30 x 10(6) A-NK cells were intravenously administered, significant retardation of tumor growth and prolongation of survival of tumor-bearing rats were observed in the groups that received the prior injection of adjuvants, especially FCA + BCG and Freund's incomplete adjuvant (FIA) + BCG. The suppressive effect of combination therapy on tumor growth was blocked effectively by the injection of anti-ICAM-1 mAb. These results indicate that the prior injection of proper adjuvant into the peritumoral region is effective for the selective accumulation or infiltration of A-NK cells into the sites of tumor tissues, and results in the marked retardation of tumor growth.

Efficient In Vivo Marking of Primary CD4+ T Lymphocytes in Nonhuman Primates Using a Gibbon Ape Leukemia Virus-Derived Retroviral Vector

AbstractHigh efficiency retroviral-mediated gene transfer to rhesus CD4+ peripheral blood lymphocytes (PBL) was accomplished using an optimized transduction protocol using a gibbon ape leukemia virus (GaLV) envelope-containing packaging cell line PG13. Engineered CD4+ PBL were administered to three nonmyeloablated animals in three or four separate infusions over 9 months. Polymerase chain reaction (PCR) demonstrated in vivo reconstitution of the genetically engineered CD4+ PBL at levels between 1% and 10% of the circulating leukocytes. This level of gene marking indicates that up to 30% of endogenous circulating CD4+ cells can be genetically engineered. The high levels of marked lymphocytes persist for the first 3 weeks following reinfusion then decline to ≤ 0.1% over the next 21 weeks. Lymph node (LN) biopsies were performed to determine if the engineered CD4+ lymphocytes could traffic to lymphoid tissues. Marked lymphocytes were detected in LN biopsies 100 days following reinfusion of the transduced cells. Expression of retroviral vector-derived sequences was detected by reverse transcriptase (RT)-PCR analysis from CD4-enriched lymphocytes that were activated by culturing in the presence of recombinant interleukin-2 (rIL-2). A humoral immune response to fetal bovine serum (FBS) was detected in all animals following the second administration of the culture expanded CD4+ lymphocytes. No antibody response was detected to the neomycin-resistance (NeoR) transgene, the murine retroviral group-specific antigen (gag), or GaLV envelope (env) proteins.

Isolation of Decidual Lymphocytes From Chorionic Villus Samples: Phenotypic Analysis and Growth in Vitro

Giemsa stained cell isolates prepared from chorionic villus samples (CVS) contain granulated cells morphologically similar to large granular lymphocytes. Phenotypic characterization of these cellular isolates by two-color immunofluorescence and subsequent in vitro culture in the presence of recombinant interleukin-2 (rIL-2) were done in order to determine whether CVS could serve as a source of decidual lymphocytes. A major fraction of the CVS-derived lymphocytes were characterized as decidual NK cells, exhibiting high levels of CD56 expression (CD56+bright), without concomitant expression of CD16. The T cell population present in CVS-derived lymphocytes contained both CD4+ and CD8+ cells in a ratio somewhat reduced compared to that found in peripheral blood. While both T cells and CD56+bright cells from CVS proliferate in vitro in response to rIL-2 alone, preferential growth of CD56+bright cells was accomplished using a selective culture technique wherein co-culture with an irradiated, B lymphoblastoid cell line promoted the growth of CD56+ cells. CVS contains decidual NK cells and T cells that proliferate in response to rIL-2 and/or third party stimulator cells. These culture techniques will allow investigations into the maturation and/or activation of decidual NK cells and T cells.

皮膚悪性腫ようのＢＲＭ療法のその後の展開 養子免疫療法 皮膚悪性腫ように対する臨床応用，悪性黒色腫を中心として

Treatment for metastatic melanoma has not yet been established. Use of biological response modifiers (BRM) have been found to enhance therapeutic benefits. Rosenberg reported the effects of adoptive immunotherapy with lymphokine-activated killer (LAK) cells and with tumor-infiltrating lymphocytes (TIL). We cultured patient's peripheral blood lymphocytes or TIL massively for a short time in the presence of recombinant interleukin 2 and immobilized anti-CD3 antibody. In clinical trials involving 10 cases, the mean number of harvested T cells after 14 days culture was 2.0×1010, and the mean expansion index was about 2000-fold. We called the acquired lymphocytes “immobilized anti-CD3 antibody-activated T lymphocytes (CD3-AT) ”and studied their clinical efficasy as a part of multidisciplinaly treatment. Of 10 malignant melanoma patients receiving CD3-AT, one had complete response and one had partial response. Moreover we administered CD3-AT to 2 patients of metastatic sweat gland carcinoma and one patient of progressive angiosarcoma, partial response of the angiosarcoma was observed. No side effects of CD3-AT have not yet been observed. Expanded T lymphocytes were infused to the patients with skin malignancies, and safety and feasibility was confirmed.

Proliferative, phenotypic and functional and molecular characteristics of tumour-infiltrating lymphocytes obtained from unselected patients with malignant melanomas and expanded in vitro in the presence of recombinant interleukin-2

Tumour-infiltrating lymphocytes (TIL) derived from several histotypes, including melanoma, can be expanded in vitro in the presence of recombinant interleukin-2 (rIL-2) and infused as part of experimental adoptive immunotherapy protocols. Several authors have described the isolation and the expansion of TIL, but little is known about the actual proportion of unselected melanoma biopsies that give rise to 'positive' TIL cultures. We evaluated the proliferative, phenotypic, functional and molecular characteristics of cultured TIL obtained from 26 patients with metastatic melanomas, eligible for inclusion in a protocol of adoptive immunotherapy. Sixteen proliferating cultures were obtained. All TIL belonged to the T cell lineage and were characterized by a wide range of cytolytic activity against autologous and, to a lesser extent, allogeneic melanoma cells. Molecular analysis of the constant region of T cell receptor-beta (TCR-beta) showed an oligoclonal population of TIL in the majority of expanded samples, indicating that a selected subset of lymphocytes present in the tumour site could be expanded in vitro. These features, together with the high number of proliferating samples, make these populations of TIL suitable for infusion into patients with advanced disease.

5] Granzyme B

Publisher Summary This chapter highlights purification and assay methods for granzyme B. The highest levels of granzyme B are found in the granules of cytotoxic T lymphocytes (CTLs). Lower levels of granzyme B are found in helper T cells and in thymocytes. The proposed physiological roles of granzyme B include the inhibition of colon cancer cell growth and the induction of chromatin DNA degradation during T cell-mediated apoptosis. Human and mouse granzyme B are isolated from cytoplasmic granules of activated CTL lines grown in the presence of recombinant interleukin 2. Granzyme B preferentially cleaves substrates with negatively charged side chains, and prefers aspartic over glutamic acid, as aspartic acid has a shorter side chain. Granzyme B also exhibits a weak esterase activity toward substrates containing a serine or an asparagine, as these two residues can form hydrogen bonds with the Arg-208. Granzyme B is inhibited by a variety of inhibitors, such as lima bean trypsin, soybean trypsin, and chymostatin.

The induction of murine tumor infiltrating lymphocytes (TIL) by interleukin-2 or T cell growth factor.

Mice were injected in the foot pad with either 5 x 10(5) syngeneic plasmacytoma (MOPC104E) or fibrosarcoma cells (Meth A). Lymph nodes containing tumor cells were harvested 14 days later and cultured. In the presence of recombinant interleukin-2 (r-IL-2) predominantly tumor cells proliferated. Culture with T cell growth factor (TCGF) resulted in the growth of lymphoid cells. Concanavalin A (Con A) had only a modest effect on elimination of tumor cells in the culture. Tumor-infiltrating lymphocytes (TIL) prepared from the lymph nodes showed specific tumor-neutralizing activity when grown in the presence of TCGF. In vitro examination revealed that Meth A cells could not be lysed by TIL, while TIL from MOPC tumors showed tumor specific activity. This study may explain negative results in human trials with TIL induced by IL-2 alone.

Generation of natural killer cells from both Fc gamma RII/III+ and Fc gamma RII/III- murine fetal liver progenitors

In vitro culture of day-15.5 murine fetal liver (FL) cells in the presence of recombinant interleukin-2 (IL-2) results in the expansion of Fc gamma RII/III+ CD3-Ti-NK1.1+ cells displaying both natural killer (NK) and antibody-dependent cell cytotoxicity (ADCC) cytolytic activities. These FL-derived NK cells express Fc gamma RIII (CD16) in association with an Fc epsilon RI gamma homodimer on their surface. In contrast, in vitro expansion of FL cells in the absence of IL-2 generates noncytotoxic cells belonging to the myelomonocytic lineage (Mac1+Gr1+NK1.1-). Hence, IL-2 appears to be critical for the proliferation and differentiation of NK cells from FL progenitors. Experiments in which FL cells were fractionated by density gradient centrifugation before in vitro expansion showed that NK progenitors are contained within a cell population with a density of 1.04 < d < 1.08 g/mL. Cells with d > 1.08 g/mL (representing > or = 40% of FL cells) have no such NK progenitor activity. In addition, after intrathymic injection into Ly5 congenic host animals, day-15.5 CD4-CD8- FL cells mature into CD4+CD8+ thymocytes within 12 days. Interestingly, this T-cell progenitor activity is restricted to subpopulations of FL cells that also contain NK progenitors, but is absent in high-density (d > 1.08 g/mL) FL cells. Finally, fractionation of FL cells according to surface expression of Fc gamma RII/III complexes shows that NK (and T-lymphocyte) progenitors are found in both Fc gamma RII/III+ and Fc gamma RII/III-FL subpopulations.

Generation of Natural Killer Cells From Both FcγRII/III+ and FCΓRII/III− Murine Fetal Liver Progenitors

AbstractIn vitro culture of day-15.5 murine fetal liver (FL) cells in the presence of recombinant interleukin-2 (IL-2) results in the expansion of FcγRII/III + CD3-Ti−NK1.1+ cells displaying both natural killer (NK) and antibody-dependent cell cytotoxicity (ADCC) cytolytic activities. These FL-derived NK cells express FcγRIII (CD16) in association with an Fc∊Rl7 homodimer on their surface. In contrast, in vitro expansion of FL cells in the absence of IL-2 generates noncyto-toxic cells belonging to the myelomonocytic lineage (Mac1 +Gr1 +NK1.1−). Hence, IL-2 appears to be critical for the proliferation and differentiation of NK cells from FL progenitors. Experiments in which FL cells were fractionated by density gradient centrifugation before in vitro expansion showed that NK progenitors are contained within a cell population with a density of 1.04 < d < 1.08g/mL. Cells with d > 1.08 g/mL (representing ≥;40% of FL cells) have no such NK progenitor activity. In addition, after in-trathymic injection into Ly5 congenic host animals, day-15.5CD4−CD8− FL cells mature into CD4+CD8+ thymocytes within 12 days. Interestingly, this T-cell progenitor activity is restricted to subpopulations of FL cells that also contain NK progenitors, but is absent in high-density (d > 1.08 g/mL) FL cells. Finally, fractionation of FL cells according to surface expression of FcγRII/III complexes shows that NK (and T-lymphocyte) progenitors are found in both FcγRII/III+ and FCΓRII/III−FL subpopulations.

Lymphocyte activation in HIV-1 infection. I. Predominant proliferative defects among CD45RO+ cells of the CD4 and CD8 lineages

The proliferative defects of CD4 and CD8 cells taken from 474 HIV-1-seropositive individuals during various stages of disease were quantitated. Phytohaemagglutinin (PHA) and soluble anti-CD3 were used in optimal mitogenic concentrations in the presence of recombinant interleukin-2 (rIL-2) and conditioned medium, and the proliferation of cells from HIV-1-seropositive donors was assessed in co-culture with HIV-1-seronegative cells in order to exclude effects of cytokine deficiency. Defects within the CD45RA+ ('unprimed') and CD45R0+ ('primed') T-cell populations were also investigated. Quantitative immunofluorescence and double and triple labelling in flow cytometry were performed for (1) CD25 (IL-2 receptor alpha chain) expression, (2) lymphocyte and T-cell survival, and (3) blast transformation and proliferation--in relation to the original input of cells for each subpopulation. T cells from normal and HIV-1-seropositive donors were CD25+ at day 1. In HIV-1-seropositive patients a variable number of CD4 and CD8 lymphocytes failed to further increase CD25, and died as a sign of activation-associated lymphocyte death (AALD). Forty-two per cent of asymptomatic subjects, including 32% of those with CD4 cell counts > 400 x 10(6)/l, showed a poor blast transformation (< 30% blasts). Cells from HIV-1-seropositive donors showed poor blast responses when co-cultured with HIV-1-seronegative cells; both CD4 and CD8 cells were handicapped. In asymptomatic HIV-1-seropositive people T cells with the CD45R0+ RA- ('primed') phenotype were three to five times more vulnerable to AALD than the CD45RA+ RO- ('unprimed') cells. In patients in Centers for Disease Control and Prevention (CDC) disease stage IV both CD45R0+ and -RA+ populations were severely affected. This is the first quantitative analysis to demonstrate that in HIV-1 infection mitogen-stimulated CD45R0+ ('primed') T cells preferentially die upon activation. Both the CD4 and CD8 lineages are affected, as seen in animal models of graft versus host disease. AALD may explain defects of immunological memory. The analysis of AALD may be a suitable assay for studying whether antiviral drugs influence the proliferative responses of lymphocytes.

Cultured human thymocytes lacking CD2 and CD11a/CD18 antigens are functional and adhere to endothelial cells via CD56 or CDw49d molecules

The preferential growth of CD3 −CD2 −CD1la/CD18 − thymocytes was obtained by stimulation of CD2 −CD3 − thymic cells with low doses of PMA (0.5 ng/ml) and subsequent culture in the presence of recombinant interleukin-2 (100 U/ml). After 2–3 weeks, CD3 −CD2 −CD11a/CD18 − thymocytes represented 40–60% of the total proliferating cells. Highly purified CD3 −CD2 −CD11a/CD18 − cell populations were obtained by depletion of the CD11a/CD18 + thymocytes by immunomagnetic beads. Moreover, these populations proliferated for 2–5 weeks and did not change their surface phenotype. It is of note that these cells, despite the lack of CD2 and CD11a/CD18 adhesion molecules, could bind to umbilical vein endothelial cells as efficiently as did CD3 +CD2 +CD11a/CD18 + thymocytes. Furthermore we demonstrate that (a) CD56 molecule is involved in the adhesion of CD3 −CD2 −CD11a/CD18 − thymic cells, but not of peripheral CD3 −CD56 + lymphocytes, to untreated or IFN-γ- and/or TNF-α-treated endothelium, (b) anti-CDw49d mAb could inhibit the adhesion of this thymus-derived population to either IFN-γ- or TNF-α-treated endothelial cells but not to untreated endothelium, and (c) CD56 antigen expressed by these cultured thymocytes has a sialic acid content different from that of peripheral lymphocytes. Indeed, isoelectrofocusing analysis showed that CD56 molecule expressed on CD3 −CD2 −CD11a/CD18 − thymocytes displayed an isoelectric point (p I5.0) different from that of CD56 antigen expressed by peripheral NK cells (p I4.7 and 5.4). Further, we noted that CD56 antigen showed the same p I 5.8 after desialylation obtained using neuraminidase treatment. Finally, CD3 −CD2 −CD11a/CD18 − thymocytes mobilized Ca 2+ and released TNF-α and IFN-γ after treatment with lectins.

TCR γδ Bearing T Lymphocytes Infiltrating Human Primary Cutaneous Melanomas

In situ immunophenotypic studies show that tumor infiltrating T lymphocytes from six of 11 fresh cutaneous primary melanomas tested contain T-cell receptor gamma delta expressing cells whereas those from 8 metastatic melanomas did not. In three cases these cells represent an unexpectedly high proportion of the lymphocytic infiltrate, ranging from 15 to 25%. This elevated percentage is maintained when tumor infiltrating lymphocytes are propagated in vitro for 2 or 3 weeks in the presence of recombinant interleukin 2 and autologous tumor cells. Functional studies reveal that gamma delta lines or clones developed from lymphocytes infiltrating primary melanoma display, in addition to their natural killer-like activity, a potent autologous tumor cell cytotoxic activity that does not seem to be restricted by major histocompatibility complex gene products.

Cytotoxicity of interleukin 2-induced lymphokine-activated killer (LAK) cells against human leukemia and augmentation of killing by interferons and tumor necrosis factor

Adoptive immunotherapy with interleukin 2-induced lymphokine-activated killer (LAK) cells or tumor-infiltrating lymphocytes (TIL) and the induction of anti-tumor responses by IL-2 alone have proven to be promising approaches in cancer therapy. The present study investigated the cytotoxicity of LAK cells towards human leukemia cells. LAK cells were generated by culturing peripheral blood mononuclear cells from normal donors for six days in the presence of recombinant interleukin 2 (IL-2). Cytotoxicity was evaluated using a standard 4-h chromium-release assay. A significant lysis of fresh uncultured leukemia cells by IL-2-activated killer cells could be detected in 77 of 150 leukemias examined. The mean Cr-release was 35.7 ± 12.9% in the LAK cell-sensitive vs 9.9 ± 5.9% in the resistant leukemias. With a view to the therapeutic utilization of the LAK-cell system, we attempted to improve the efficiency of its cytotoxic mechanisms. Combined application of IL-2 and interferon-α, interferon-γ, or tumor necrosis factor-α in cultures for generation of activated killer cells significantly improved the effectiveness of cytotoxic mechanisms. Our results suggest that the performance of adoptive immunotherapy with ex vivo-activated LAK cells and the in vivo induction of cytotoxic immune responses by IL-2 alone or combined with different lymphokines or cytokines may be of value in treating human leukemia, especially when the tumor burden is low, e.g. during maintenance therapy or after bone marrow transplantation to eliminate minimal residual disease or in early relapse.