The effect of interferon on lymphocyte-mediated effector cell functions: selective enhancement of natural killer cells.
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Animals↗
Biomedical subjects
M Hokland
Publications and source records attributed to M Hokland.
Enhanced expression of beta2-microglobulin and HLA antigens on human lymphoid cells by interferon.
Mononuclear cells from the blood of healthy normal humans were kept in cultures under nonstimulating conditions for 16 hr in the presence or absence of human interferon. The relative quantities of HLA antigens and beta(2)-microglobulin on the cultured cells were determined by quantitative immunofluorescence (fluorescence-activated cell sorter) and by the capacity of cells to absorb out cytotoxic antibodies against the relevant antigens. Interferons of different origin and purities enhanced the expression of HLA antigens and beta(2)-microglobulins, whereas membrane immunoglobulins and antigens recognized by antiserum raised against human brain and T cells were the same on interferon-treated and control cells. Similar interferon effects were observed on an Epstein-Barrvirus-negative Burkitt lymphoma cell line. The enhanced expression of histocompatibility antigen subsequent to intereferon treatment was observed on B- and T-enriched lymphocyte populations and was found to be dose dependent with the optimum with "physiological" concentrations of interferon. Pretreatment of lymphocytes with interferon for 2 hr was found to be as effective as having interferon present during the total culture period. The interferon-induced enhancement of antigen expression on cells was dependent on active protein synthesis.
Adolescent↗
Two small lymphocyte subpopulations in human peripheral blood. I. Purification and surface marker profiles.
By means of simple rosette sedimentation methods two subsets from human peripheral blood lymphocytes have been isolated: (1) (E, Fc)- and (2) (E, Ig)-. The first subset was obtained by centrifuging suspensions of macrophage-depleted PBL in which E and EA rosettes had been allowed to form simultaneously. The dominant marker of these E- Fc- cells was surface Ig, and during 4 days of culture this population did not alter its surface markers. Subset 2 was obtained in two ways following rosette centrifugation with AET-treated SRBC and rabbit anti-human Ig-coated autologous RBC. This 'Null cell population' was shown to be highly variable as judged by the surface markers applied after 4 days of culture, and it is suggested that Null cells contain a number of immature lymphoid cells that may acquire their surface marker during culture. It is concluded that the methods described for purification of small subsets of human lymphocytes are effective and easy to perform and might be used to purify cells for functional studies.
B-Lymphocytes↗
Two small lymphocyte subpopulations in human peripheral blood. II. Functional characterization in vitro.
Two small lymphocyte subpopulations from human peripheral blood were produced by rosette sedimentation procedures, and tested for in vitro functions by stimulation with mitogens, PPD and allogeneic lymphocytes, and for effector capacities in ADCC. The non-T, non-Fc subpopulation had previously been found to be a SmIg+ fraction which did not aquire new surface markers during unstimulated culture, did not respond to mitogens, PPD or allogeneic cells but were potent MLC stimulators. In addition, they were poor ADCC effector cells. The non-T, non-B ('Null') cells, which showed varying surface marker profiles from donor to donor, were able to respond to mitogens and PPD, but were weak MLC responders. Potent ADCC effector capacity was, however, found to be exhibited by this subpopulation.
Antibody-Dependent Cell Cytotoxicity↗
An improved technique for obtaining E rosettes with human lymphocytes and its use for B cell purification.
The standard E rosette method and two previously described methods claimed to give improved E rosetting for enumeration of human T lymphocytes have been compared with respect to the speed of rosette formation, and the mechanical stability of the rosettes formed. Following rosette formation with the three methods, the lymphocyte-erythrocyte suspensions were sedimented on Ficoll-Isopaque to deplete them of rosette-forming cells and red cells. The purified lymphocyte preparations were tested for B and T cell markers to determine the degree of contamination with T cells. One of the improved rosetting methods was clearly better than the others, and led to the recovery of B lymphocytes with a contamination of only 2.0+/-1.9 per cent of T lymphocytes.
B-Lymphocytes↗
Endogenous and adoptively transferred A-NK and T-LAK cells continuously accumulate within murine metastases up to 48 h after inoculation.
In murine models, therapeutic efficacy of adoptive immunotherapy (AIT) of cancer with lymphokine activated killer (LAK) cells is seen only when applied together with substantial doses of interleukin-2 (IL-2), probably because this cytokine is imperative for both motility and viability of the LAK cells. We wanted to investigate whether IL-2 in addition mediates an immunostimulatory activation and expansion of endogenous effector cells contributing to tumor regression. Using an immunoperoxidase technique, we have been able to longitudinally analyze the accumulation of tumor infiltrating lymphocytes expressing the pan-T cell/activated lymphocyte phenotype (Thy1.2), the natural killer (NK) cell phenotype (AsGM,) as well as the cytotoxic T (CD8) cell phenotype within experimental established B16 pulmonary melanoma metastases in C57BL/6 mice during the first 48 h after high dose IL-2 monotherapy. Whereas a substantial and selective infiltration of AsGM1+ lymphocytes in tumor tissue was seen (262 and 937 cells per sq.mm malignant tissue at 0 and 48 h, respectively), only a minor increase in accumulation of CD8+ cells was seen (106 and 171 cells per sq.mm tumor tissue at 0 and 48 h, respectively). The addition of adoptive transfer with lymphokine-activated adherent NK (A-NK) cells to the high-dose IL-2 treatment resulted in more than a 1.5 fold increase in infiltrating AsGM1+ cells compared to IL-2 therapy alone (1520 compared to 937 AsGM1+ cells per sq.mm malignant tissue). No substantial accumulation of CD8+ cells was observed in this setting either. In contrast, the treatment with high dose IL-2 together with adoptive transfer of mitogen-stimulated, lymphokine-activated T killer (T-LAK) cells increased the infiltration of CD8+ cells 10-fold compared to IL-2 monotherapy (2078 compared to 171 CD8+ cells per sq.mm malignant tissue, respectively). Interestingly, infiltration of both endogenous and exogenous cells continued over time, since the effector-to-tumor cell ratio in metastatic tissue dramatically increased from 1:8 and 1:6 at 16 h to 1:3 and 1:2 at 48 h after adoptive transfer of A-NK and T-LAK cells, respectively. These data underline the longevity of LAK cells in vivo and highlight the importance of IL-2 treatment in recruiting endogenous immune cells to tumor areas.
Adjuvants, Immunologic↗
Expression and function of LFA-1 on A-NK and T-LAK cells: role in tumor target killing and migration into tumor tissue.
To study the role of the adhesion molecule lymphocyte function-associated antigen 1 (LFA-1) in lymphokine-activated natural killer (A-NK) and mitogen-stimulated lymphokine-activated T killer (T-LAK) cell function, we evaluated the correlation between LFA-1 expression, as well as conjugate formation and cytotoxicity. Furthermore, the role of LFA-1 in the extravascular phase of migration of A-NK and T-LAK cells into B16 tumor tissue in vivo was studied. We demonstrate an 8-fold increased LFA-1 expression on 4-day-old A-NK and T-LAK cells compared to unstimulated spleen cells. Moreover, we demonstrate that while LFA-1 expression on T-LAK cells was strictly correlated to conjugate formation and cytotoxic capacity, A-NK cells showed a less clear correlation between LFA-1 expression and these functions. Interestingly, anti-CD11a alone did not inhibit cytotoxicity, anti-CD18 alone did only inhibit cytotoxicity moderately (25%) whereas anti-CD11a and anti-CD18 in combination almost abrogated cytotoxicity against tumor targets P815 and YAC-1. Finally we demonstrate that pre-incubation of T-LAK cells with anti-bodies to CD11a and CD18 in combination inhibited their migration into the B16 tumors by more than 50-60%. In contrast, this pretreatment did not inhibit A-NK cell migration. This difference could not be explained by differences between A-NK and T-LAK cells with respect to LFA-1 expression, turnover of antibody-receptor complexes or complement-mediated lysis induced by the anti-LFA-1 antibodies. We conclude that T-LAK cell binding to and lysis of tumor target cells and migration into the intercellular space of tumors depend strongly on the expression of LFA-1. In contrast, LFA-1 expression of A-NK cells showed a less clear correlation to these functions.
Animals↗
Morphological studies of effector cell-microvessel interactions in adoptive immunotherapy in tumor-bearing animals.
Murine lymphokine-activated natural killer (A-NK) cells are able to migrate to and accumulate in tumor metastases. However, the exact migratory pattern is as yet unknown. In the present study, we have investigated the migration from the vasculature towards malignant tissues of various effector cells. Our results indicate that murine A-NK cells seem to be arrested for an extended period of time in the microvasculature and also, although infrequently, to adhere to the endothelial lining of larger vessels close to tumor tissues before extravasation. While murine T lymphokine-activated killer and rat A-NK cells accumulate significantly in the subendothelial areas of larger venules in normal tissues, no such accumulation is observed with respect to murine A-NK cells. Electron microscopy reveals that the murine A-NK cells undergo an extreme deformation during extravasation and tumor infiltration. Furthermore, the cells are shown to be in an activated stage probably facilitating their migration, and hence, the elimination of tumor cells.
Adenocarcinoma↗
Infiltration and lysis of tumour cell aggregates by adherent interleukin-2-activated natural killer cells is distinct from specific cytolysis.
Adoptively transferred activated natural killer (A-NK) cells infiltrate tumours in vivo. Two in vitro B16-F10 melanoma tumour models were used to study with fluorescence and electron microscopy the infiltration of adherent interleukin 2 (IL-2) A-NK cells: (1) substratum-bound sessile microtumours (MTs), and (2) three-dimensional cell growth on macroporous gelatinous microcarriers (Cultispheres). From 2 h and on increasing numbers of A-NK cells infiltrated the MTs regularly surrounded by a widened intercellular space. An IL-2-dependent disintegration of MTs began at 6-8 h resulting in a release of vital and dead cells. A-NK cell invasion into Cultispheres effectively displaced the melanoma cells from the highly convoluted substratum. Thus, A-NK cell infiltration had a protease-like effect on the tumour cell aggregates which might have a bearing on the interpretation of their cytolytic effect on target cells. Ultrastructural evidence was not obtained of specific A-NK/target conjugate formation or of granule-mediated target cell destruction in either model tumour.
Adoptive Transfer↗