Isolation and expansion of tumor-infiltrating lymphocytes.
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Biomedical subjects
E M Lord
Publications and source records attributed to E M Lord.
Two regions of the H-2 Dd promoter are responsive to dimethylsulfoxide in line 1 cells by a mechanism distinct from IFN-gamma.
The line 1 lung carcinoma is a spontaneous BALB/c tumor deficient in class I Ag expression at the protein and mRNA levels. Exposure of line 1 cells to 3% DMSO or IFN-gamma increases class I Ag protein and mRNA dramatically. We have examined the regulation of class I Ag induction by DMSO in line 1 cells. We found DMSO induces class I Ag expression in line 1 cells by a mechanism distinct from IFN, because the kinetics of class I Ag induction by these agents were dramatically different, 7 days vs 3 days, and DMSO did not act through an IFN second messenger. At the molecular level, class I H chain transcription in line 1 cells was low. Treatment with 3% DMSO or IFN-gamma increased H chain transcription four-fold and sevenfold, respectively, indicating that class I H chain expression is regulated at the level of transcription in line 1 cells. Using reporter gene constructs, we mapped the regions in the Dd H chain promoter that increase H chain expression after DMSO treatment of line 1 cells. Two regions of the Dd promoter, D1, from -210 to -133 bp, and D2, from -125 to -61 bp, were found to be independently responsive to DMSO. These regions were also responsive to IFN-gamma in line 1 cells. However, consistent with our cellular results, DMSO and IFN induction of class I H chain expression differed at the molecular level as determined by D1 point mutations that diminished IFN-gamma responsiveness but did not alter induction by DMSO. Thus, DMSO appears to regulate class I transcription through multiple regions of the class I H chain promoter in line 1 cells by a mechanism distinct from IFN-gamma.
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CD4-class II major histocompatibility complex interaction does not enhance killing by a class I-restricted CD4+CD8+ cytotoxic T cell clone.
As unusual tumor-specific cytotoxic T lymphocyte (CTL) clone was isolated which expressed both CD4 and CD8 molecules. The target cells for this CTL can be induced to express either class I major histocompatibility complex (MHC) alone (with dimethylsulfoxide) or both class I and class II MHC (with interferon-gamma). Lysis of the tumor target depends on expression of class I MHC molecules, but does not require expression of class II MHC molecules. Furthermore, the lysis of target cells expressing both class I and class II is inhibited only by antibodies to class I (Kd), and not by antibodies to class II, demonstrating that the T cell receptor is class I restricted. We have used this CTL to assess the role of the interaction between CD4 and class II MHC in the absence of a class II-restricted T cell receptor. Our data indicate that CD4-class II interaction does not contribute to recognition by T cells in the absence of binding of the T cell receptor to class II molecules.
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Roles for the extracellular matrix in plant development and pollination: a special case of cell movement in plants.
Pattern formation in plants is now thought to be primarily dependent on positional information during development. We discuss the prevalent theories on how position is deciphered by cells in an organism and highlight the recent advances implicating molecules of the cell wall or extracellular matrix (ECM) in this process. We compare the functions of the ECM in plants and animals and describe the various cell and substrate adhesion molecules of the animal ECM which play a role in morphogenesis and cell movement. We propose that analogous molecules may occur in plants and provide evidence for the presence of a substrate adhesion molecule like vitronectin in plants and algae. We provide a model for how substrate adhesion molecules may be involved in a special case of cell movement in plants, pollination.
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Immunological Characterization of a Tapetal Protein in Developing Anthers of Lilium longiflorum.
Antiserum was raised in rabbits against a lily (Lilium longiflorum) anther-specific protein (LLA-15). Monospecific anti-LLA-15 antibodies were prepared to investigate the distribution of LLA-15 during anther development in a variety of flowering plants. Immunoblot analyses of total protein from floral and vegetative organs confirmed that LLA-15 or LLA-15-like proteins accumulated to detectable levels only in a discrete stage of anther development. In situ localization using anti-rabbit immunoglobulin G conjugated with gold particles confirmed that LLA-15 was specifically localized in the tapetal tissue of lily anthers. The maximal level of LLA-15 was strictly coincident with the peak of tapetal secretory functions. Immunoblots of two-dimensional polyacrylamide gels of lily anther proteins indicated that the seven LLA-15 isoforms ranged from isoelectric point 5.6 to 6.1. In vitro translation of lily anther mRNAs showed that four of these isoforms were primary products, the additional three being a result of posttranslational processing of the primary translation products.
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Mechanism of cytolytic T lymphocyte killing of a low class I-expressing tumor.
Many tumors have been shown to express minimal levels of class I MHC Ag, which makes them more resistant to recognition and lysis by cytolytic T lymphocytes. Line 1, a BALB/c spontaneous lung carcinoma, normally expresses very low levels of class I Ag, but expression can be increased 50-fold by treatment with agents such as DMSO or IFN-gamma. Because class I Ag serve as restricting elements for cytolytic T cell recognition of tumor Ag, we wished to determine if cytotoxic T lymphocytes could play a role in the immune response to this type of class I low, but inducible, tumor. After immunization in vivo and restimulation of splenic cells in vitro we were able to generate T cell clones that lysed line 1 cells induced to express high levels of class I, but did not lyse uninduced, low class I expressing line 1 cells in short term (6-h) 51Cr release assays. Paradoxically, incubation of the T cells with uninduced class I low line 1 cells for a few days resulted in complete destruction of the tumor cells. We demonstrate that the T cells, stimulated by the tumor cells, produce IFN-gamma, which in turn induces class I expression on the line 1 cells making them susceptible to lysis by the T cell clone. This suggests that a positive feedback reaction can occur in generating a response to this and perhaps other inducible tumor cell lines.
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Class-I MHC expression in the mouse lung carcinoma, line 1: a model for class-I inducible tumors.
We have examined the expression and biological effects of class-I MHC molecules on the immune response to the line I lung carcinoma. The line I system is of interest because these tumor cells have very low constitutive levels of class-I molecules but can be induced to express levels found on spleen cells, by culturing the cells with agents such as dimethylsulfoxide (DMSO) or interferon gamma (IFN-gamma). This induction is significant immunologically, since induced cells can be lysed very effectively by cytotoxic T lymphocytes (CTL), whereas the uninduced cells cannot. CTL clones that are reactive with line I cells have been generated and used in vitro and in vivo, to examine the interactions of T cells with line I. We have shown that the expression of class I on tumor cells is induced in vivo by IFN-gamma, and that this induction is associated with the ability to reject the tumor. We will also introduce preliminary data concerning the mechanism of induction in which CTL appear to induce class-I MHC both in vitro and in vivo. The results are discussed in terms of a model which may be important generally for class-I inducible tumors.
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A kinematic analysis of gynoecial growth in Lilium longiflorum: surface growth patterns in all floral organs are triphasic.
Sequential marking experiments combined with histological and morphological examination indicate that growth of the gynoecium in Lilium longiflorum Thunb. (Easter lily) is triphasic. Phase I (gynoecia less than 10 mm in length) is characterized by spatial and temporal variation in local relative growth rate and mitotic index throughout the entire gynoecium. In Phase II (10-100 mm), a steady growth peak is restricted to the proximal 5 to 15 mm of the style, while the ovary shows uniform growth. In Phase III (100-135 mm), the ovary ceases growth and the maximal growth zone of the gynoecium migrates from the proximal to the distal end of the style. Comparison of surface growth patterns and differentiation events in the gynoecium to those previously described in the tepal and another demonstrates the triphasic nature of growth in the whole lily flower.
Cell Division↗
Lymphocytic infiltration and cytotoxicity under hypoxic conditions in the EMT6 mouse mammary tumor.
Infiltration of lymphocytes, neutrophils and macrophages was evaluated in hypoxic and well-oxygenated areas of the EMT6 mouse mammary adenocarcinoma, by in vivo staining with the fluorescent dye Hoechst 33342 followed by cell sorting on the basis of fluorescence intensity. Tumors were grouped by days post-injection (days 11-14, 15-17 and 20-27). As lymphocytes are the only host cell population in this tumor model to possess lytic activity against EMT6 tumor cells, the ability of sensitized T lymphocytes to lyse syngeneic EMT6 cells was examined under conditions of varying oxygen concentrations. Infiltrating lymphocytes were detected to the same extent in cell fractions from both areas in all tumors. In contrast, neutrophils were found in significantly higher percentages in the hypoxic population than in the well-oxygenated cell fraction of all but the largest tumors. Macrophages were present in significantly higher percentages in the well-oxygenated fraction than in the hypoxic fraction of day-11 to -14 tumors. Extreme radiobiological hypoxia (0% O2) resulted in a significant decrease in T-cell-mediated lysis of EMT6 tumor cells, compared to lysis in room air (20% O2), but lysis was not impaired under conditions of mild radiobiological hypoxia (1% O2). Our study indicates that host-cell infiltration into areas of differing oxygenation may be quantitated via in situ Hoechst staining followed by cell sorting; in the EMT6 tumor, lymphocytes appear to infiltrate hypoxic areas to the same extent as well-oxygenated areas, and T-lymphocyte killing of syngeneic tumor cells is significantly reduced, although still present, under these hypoxic conditions.
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Alteration of the metastatic potential of line 1 lung carcinoma cells: opposite effects of class I antigen induction by interferons versus DMSO or gene transfection.
Class I antigens are necessary for the recognition of tumor cells by cytotoxic T lymphocytes (CTL). The line 1 lung carcinoma is a spontaneous murine tumor deficient in class I antigen expression. Consistent with this, line 1 cells are highly metastatic in vivo. We investigated whether increasing class I antigen expression on line 1 cells could alter the metastatic potential of these tumor cells using an in vivo lung metastasis model. We used three methods to induce class I antigen expression on line 1 cells: gene transfection, treatment with dimethyl sulfoxide (DMSO), or treatment with interferon (IFN)-beta or -gamma. We found that line 1 cells expressing a transfected class I gene were significantly less metastatic than parental line 1 cells. DMSO-treated line 1 cells also formed significantly fewer metastases than parental line 1 cells. These results indicate that increased class I antigen expression decreases the metastatic potential of line 1 cells in vivo. However, we did not observe a significant decrease in the number of lung metastases in mice receiving line 1 cells treated with IFN-beta or -gamma, despite high levels of class I antigen expression. Thus, increasing class I antigen expression with IFN has an opposite effect on metastasis from class I antigen expression induced by transfection or DMSO. These results show that the method used to increase class I antigen expression is critical in terms of the in vivo effect observed. To investigate a possible mechanism for the differences observed in vivo between these class I expressing cells, we tested whether IFN alters or blocks susceptibility of line 1 cells to immune effector cells. We found IFN treatment increased the ability of line 1 cells to be recognized by CTL but concomitantly decreased the susceptibility of line 1 cells to NK cell lysis by a non-class I antigen-related mechanism. In contrast, transfected or DMSO-treated line 1 cells which were less metastatic in vivo were susceptible to both CTL and NK-mediated lysis. Taken together, these results suggest that immune intervention against metastasizing line 1 cells may involve NK cells and CTL.
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Tumor necrosis factor-mediated cytotoxicity by tumor-associated macrophages.
We have directly demonstrated that macrophages present within solid EMT6 mammary tumors (of BALB/c origin) produce TNF-alpha (TNF). These tumor-associated macrophages lysed WEHI-164, a TNF-sensitive cell line, very efficiently. This cytotoxicity was abrogated in the presence of anti-TNF antisera. In contrast, EMT6 cells, the tumor from which the macrophages were obtained, were not effectively lysed by the macrophages and were 100-fold less sensitive to lysis by recombinant mouse TNF. Thus, marked heterogeneity exists among tumors regarding sensitivity to TNF-mediated cytotoxicity. Similarly, macrophages which infiltrate into EMT6 multicellular spheroids implanted into the peritoneal cavity as well as free cells within the cavity exhibited TNF-mediated cytotoxicity of WEHI-164 cells, but failed to lyse EMT6 cells. The kinetics of lysis by these cells was similar to that of recombinant mouse TNF.
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Requirement for proliferating cell nuclear antigen expression during stages of the Chinese hamster ovary cell cycle.
Proliferating cell nuclear antigen (PCNA/cyclin) is a nuclear protein that can stimulate purified DNA polymerase delta in vitro, and its synthesis correlates with the proliferation rate of cells. We have attempted to determine whether synthesis of PCNA/cyclin in Chinese hamster ovary cells is necessary to regulate entry into S phase. We have measured cellular PCNA/cyclin concentration of the mRNA or protein throughout the cell cycle. Cells were separated by centrifugal elutriation into populations enriched for G-1, S, and G-2/M phases. Quantitative Northern hybridization analysis was performed on RNA isolated from each cell population by using a cDNA clone of PCNA/cyclin as a probe. Results demonstrated that although intact PCNA/cyclin mRNA is present during all phases of the cell cycle, an induction of about 3-fold occurs during S phase. Two-parameter staining for PCNA/cyclin and DNA, and analysis by flow cytometry, confirmed that the quantity of PCNA/cyclin protein in the cells increases severalfold in G-1 or early S phase but generally is invariant in S and G-2/M phases. This cell cycle dependence of PCNA/cyclin expression suggests that the observed synthesis is a prerequisite for initiation of DNA replication. Introduction of an antisense oligonucleotide complementary to the PCNA/cyclin mRNA to inhibit PCNA/cyclin synthesis effectively prevented entry of G-1 phase cells into S phase. A complementary sense oligonucleotide used as a control did not have an inhibitory effect. This result suggests that a threshold concentration of PCNA/cyclin is necessary for entry into S phase.
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Growth Patterns Inferred from Anatomical Records : Empirical Tests Using Longisections of Roots of Zea mays L.
Our objective was to test whether accurate growth analyses can be obtained from anatomical records and some mathematical formulas. Roots of Zea mays L. were grown at one of two temperatures (19 degrees C or 29 degrees C) and were prepared with standard techniques for light microscopy. Positions of cell walls were digitized from micrographs. The digitized data were averaged and smoothed and used in formulas to estimate growth trajectories, Z(t), velocities, v(z), and strain rates, r(z), where Z(t) is the location occupied by the cellular particle at time t; and v(z) and r(z) are, respectively, the fields of growth velocity and strain rate. The relationships tested are: for Z(t), t = n * c; v(z) = l(z) * f; and r(z) = f * ( partial differential/ partial differentialz (l(z))). In the formulas, n represents the number of cells between the origin and the position Z(t); l(z) is local cell length; the constant c, named the ;cellochron,' denotes the time for successive cells to pass a spatial point distal to the meristem; l(z) is local cell length, and f is cell flux. Growth trajectories and velocity fields from the anatomical method are in good agreement with earlier analyses based on marking experiments at the two different temperatures. Growth strain rate fields show an unexpected oscillation which may be due to numerical artifacts or to a real oscillation in cell production rate.
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Molecular analysis of deficient class I H-2 antigen expression by mouse lung carcinoma cells.
We have continued our investigations of line lung carcinoma cells to understand the molecular basis of decreased expression of class I H-2 Ag and class I Ag induction with DMSO. We show that line 1, a murine lung carcinoma cell line, has low levels of class I Ag (H-2K, D, and L) because it is deficient in both class I and beta 2-microglobulin (B2M) RNA, and that these mRNA can be coordinately induced with DMSO. Evidence presented herein also shows that IFN-gamma can induce surface expression of class I Ag and suggests that it may act through a different mechanism than DMSO in inducing class I Ag. To further evaluate the regulation of class I expression, H-2Dp genes were transfected into line 1 cells. The transfected H-2 genes appear to be constitutively expressed at much higher levels than are the endogenous class I genes because surface expression of the foreign Dp Ag on the transfectants is elevated relative to the endogenous H-2d haplotype class I Ag. Both Dp surface expression and Dp mRNA are induced after treatment with DMSO. In all the Dp transfectants, we observed higher constitutive levels of class I mRNA as well as increased constitutive levels of endogenous B2M mRNA when compared to control or untransfected line 1 cells, however, we could not correlate these constitutive levels with Dp copy number. These results suggest that the regulation of class I and B2M genes is linked and that expression of class I genes can affect the expression of B2M genes.
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Lysis of a lung carcinoma by poly I:C-induced natural killer cells is independent of the expression of class I histocompatibility antigens.
Cells from the line 1 murine carcinoma express little if any H-2d when grown in normal medium. These cells are susceptible to splenic cell populations with NK activity, stimulated by prior injection of poly I:C, but are not lysed by NK-deficient splenocytes from homozygous beige mice treated with anti-asialo GM1. Incubation of line 1 cells in medium containing DMSO leads to a dramatic stimulation of H-2d expression but no change in lytic susceptibility to splenic NK cells. Transfection of H-2Dp into line 1 leads to a constitutive and DMSO-inducible expression of H-2Dp at functionally significant levels, but this expression appears to have no influence on NK cytolytic susceptibility.
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Cell cycle dependent activities of DNA polymerases alpha and delta in Chinese hamster ovary cells.
The activities of DNA polymerases alpha and delta, in extracts from Chinese hamster ovary (CHO) cells, were assayed in order to determine whether these polymerases are regulated during the cell cycle. An exponential population of CHO cells was separated into enriched populations of G-1, S, and G-2/M phases of cell cycle by centrifugal elutriation. Total cell homogenates from each population were assayed for DNA polymerase activity by measuring labeled nucleotide incorporation into the exogenous templates oligo(dT).poly(dA) and DNase I activated calf thymus DNA. In these experiments, specific DNA polymerase inhibitors were added to assays of the cellular extracts to allow for the independent measurement of activities of DNA polymerases alpha and delta. Comparisons of total DNA polymerase activity from cellular extracts, sampled from each portion of the cell cycle, demonstrated no significant change with respect to the concentration of total protein. However, results indicate that the activity of DNA polymerase delta increases with respect to that of DNA polymerase alpha in the G-2/M portion of the cell cycle. This difference in relative activities of DNA polymerases alpha and delta suggests a coordinate regulation of a specific species of DNA polymerase during the cell cycle.
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Comparison of fluorescence intensity of Hoechst 33342-stained EMT6 tumour cells and tumour-infiltrating host cells.
Hoechst 33342 is a fluorescent dye used for cell selection from tumours based upon intratumour location. When the dye is administered i.v. to tumour-bearing animals, cellular fluorescence is directly related to the proximity of cells to blood vessels. The present study compared inherent Hoechst fluorescence between in vitro-stained EMT6/Ro (mouse mammary sarcoma) cells and host cells, to determine if these populations have different staining characteristics that may influence cell selection procedures. Tumour cell fluorescence exceeded host cell staining by 8-fold when pure cell populations (EMT6/Ro monolayer cells, mouse spleen and peritoneal cells) were compared, and 3-fold for tumour cell-enriched and host cell-enriched populations from solid tumours. Inherent uptake of HO 33342 appeared to be correlated with cell volume. These differences in inherent dye uptake between host and tumour cells were found to be minor in comparison to the fluorescence gradient between the 10% brightest and 10% dimmest (78-fold) cell populations from in vivo-stained tumours.
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Specific (EMT6) and non-specific (WEHI-164) cytolytic activity by host cells infiltrating tumour spheroids.
The development of a serum-free, low-protein culture medium has allowed the detection of tumour-specific cytolytic cells in EMT6 immunized mice bearing EMT6 multicellular tumour spheroids. Spheroid associated (SAC) and peritoneal (PC) effector cells were specific to EMT6 as the target cell, not killing line 1, P815 or RIF-1. The natural killer (NK) cell sensitive target YAC-1 was also not lysed by SAC or PC, indicating undetectable levels of NK cells present within infiltrated spheroids. In contrast, high levels of cytolytic activity were present in SAC, PC and spleen cells against WEHI-164, a line sensitive to natural cytotoxic (NC) and macrophage mediated killing. The EMT6 specific activity was mediated by Thyl+, Lyt2+ cells. The anti-WEHI-164 effector cell population was Thyl-, Lyt2-. The WEHI-164 killer cells were present in SAC and PC from unimmunized mice while the EMT6 specific effector cells were not. After separation of SAC and PC by size using centrifugal elutriation, anti-EMT6 activity was present only in the lymphocyte fraction while anti-WEHI-164 activity was enriched in the macrophage fraction.
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