Biomedical subjects
Publications and source records attributed to S Man.
The power of the World Wide Web (WWW) lies in its ability to disseminate up-to-date information that may not be available by other means. Increasing exposure of research scientists and clinicians, amongst others, to the Internet has, in turn, stimulated the available resources to multiply and become more accessible and useful. Here, we review WWW sites of particular interest to immunologists.
Expression of resistance to cis-diamminedichloroplatinum(II) (CDDP), one of the most effective chemotherapeutic drugs used to treat a variety of malignancies, remains a serious obstacle for improving cancer treatment. To study possible genetic mechanisms underlying the development of CDDP resistance, we have adopted the approach of retroviral insertional mutagenesis. An early-stage CDDP-sensitive human melanoma cell line, WM35, was infected with a defective amphotropic murine retrovirus (murine stem cell virus), and the pooled cells were subsequently selected for CDDP-resistant variants. Nine CDDP-resistant clones independently derived from murine stem cell virus-infected WM35 cells were analyzed and it was found that five of these clones acquired an identical retroviral integration site, designated as CDDP resistance locus 1 (CRL-1), as revealed by isolation of retroviral flanking sequences. Furthermore, using the flanking sequence as probe, we have detected a 3.5-4.0-kilobase message, the expression of which is strongly increased in clones carrying a rearranged CRL-1 locus. These results strongly suggest that overexpression of CRL-1 confers resistance to CDDP in these clones. In addition, the present study indicates that retroviral insertional mutagenesis represents a potential strategy to identify genes responsible for CDDP resistance and possibly other chemotherapeutic drugs as well.
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Previous results from this laboratory demonstrated that the dominant influenza A epitope recognized by HLA-A2.1-restricted cytotoxic T lymphocytes (CTL) from HLA-A2.1 transgenic mice was the matrix protein 1 (M1) peptide epitope that is immunodominant in human CTL responses. However, analysis of a large number of CTL lines revealed a subset of influenza A/PR/8/34-specific murine CTL that recognized an HLA-A2.1-restricted epitope distinct from M1. Using recombinant vaccinia viruses encoding different influenza gene segments, the epitope recognized by these CTL was shown to be derived from A/PR/8 non-structural protein 1 (NS1). Because these CTL did not recognize targets infected with the A/Alaska/6/77 strain of influenza, candidate peptide epitopes were synthesized based on sequences that included an HLA-A2.1-specific binding motif, and that differed between A/PR/8 and A/Alaska. All of these CTL recognized a nonamer and a decamer peptide which contained a common eight amino acid sequence and two distinct sets of binding motif residues. However, the nonamer peptide was able to sensitize CTL for half-maximal lysis at 80- to 2500-fold lower doses than either the octamer or decamer. The homologous peptide derived from A/Alaska NS1 contained conservative amino acid changes at positions 4 and 8, and was not recognized at any tested concentration, although it bound with higher affinity to HLA-A2.1 than the peptide from A/PR/8. The A/PR/8 NS1 nonamer epitope was also recognized by human influenza A-specific CTL derived from two individuals. These results substantiate the general utility of HLA class I transgenic mice for the identification of human CTL epitopes for other pathogens.
The TCR structures of CTL derived from HLA-A2.1 transgenic mice were analyzed to determine features important in the interaction of murine TCR with the HLA-A2.1 + influenza M1(57-68) peptide complex. V beta 8.1 was dominant in 9 of 11 murine CTL lines, although three other V beta segments were also represented. Sequencing of TCR cDNA from a group of six independently derived CTLs that were V beta 8.1-positive demonstrated a restricted set of D-N-J beta sequences and an apparently restricted set of alpha-chains. However, at least five other distinct alpha beta pairs were found among HLA-A2.1 + M1 peptide-specific CTL in the absence of these chains. Consideration of all TCR sequences obtained demonstrated diverse beta-chain CDR3 regions with some restriction in V alpha segment usage and bias in amino acid sequence of alpha-chain CDR3 regions. Nevertheless, the strongest correlation with HLA-A2.1 + M1 specificity was clearly V beta 8.1 usage. Comparison with previously identified human TCR sequences specific for the same Ag-MHC complex revealed that the dominant murine V alpha and V beta segments used were not the homologues of the dominant human V beta and V alpha segments used. These results together with the lack of interspecies conservation in the alpha- and beta-chain CDR3 regions demonstrate that the dominant TCR structures recognizing HLA-A2.1 + M1(57-68) are substantially different between mouse and humans. Different factors may influence Ag-driven selection of the dominant TCRs used in each species.
BACKGROUND: Clinical drug resistance is either intrinsic (de novo) or often acquired rapidly in conjunction with chemotherapy. By contrast, the selection of drug-resistant mutant cell lines in monolayer culture systems is usually a more protracted process. Sublines of mouse EMT-6 mammary tumor cells selected for resistance to various alkylating agents in vivo after serial passage into syngeneic mice manifest their resistance in vitro only when cultured as three-dimensional multicellular aggregates or spheroids. PURPOSE: We examined whether a single exposure of mouse EMT-6 or human MDA-MB-231 breast cancer cells to alkylating agents in vitro is sufficient for the induction of a resistance phenotype, which may be detected by re-applying the drugs to cells grown as three-dimensional aggregates. METHODS: Mouse EMT-6 and human MDA-MB-231 breast cancer cells cultured as three-dimensional aggregates were exposed to a single dose of alkylating agent for 1-5 days. Aggregates were dispersed, and cells were plated as monolayer cultures for up to 8 weeks to allow for recovery. Colony-forming ability was assessed after a subsequent alkylating-agent exposure of cells cultured as monolayers or three-dimensional aggregates. RESULTS: A single in vitro exposure to 12.5-microM cisplatin (CDDP) for 5 days or 25 microM 4-hydroperoxycyclophosphamide (4-O2H CTX) for 1 or 3 days without changing the medium was sufficient to induce transient but substantial resistance in EMT-6 cells as determined by clonogenic assays. Such resistance was not detected when monolayer cell cultures were used. The concentration of 4-O2H-CTX and the length of time the cells remained in three-dimensional culture after initial exposure to this drug was associated with the degree of subsequent drug resistance of cells grown as three-dimensional cultures. Furthermore, this acquired resistance after a single drug exposure was accompanied by changes in the three-dimensional architecture of the cell aggregates, which now formed much more compact multicellular spheroids. Similarly, a single exposure to 4-O2H-CTX was enough to bring about resistance in MDA-MB-231 cells detectable only in three-dimensional cultures, as well as the change in three-dimensional architecture. CONCLUSIONS: Rapid acquisition of resistance likely represents a physiologic mechanism of adaptation operative at the multicellular level rather than a stable genetic change and may be one of the reasons for the rapid development of drug resistance acquired by tumors in vivo. IMPLICATIONS: In vivo drug exposure may result in transient and low levels of drug resistance that may nevertheless be clinically relevant.
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Surgical removal of early-stage radial growth phase or vertical growth phase primary cutaneous human melanomas usually results in cure of the disease. Hence there are few examples of genetically-related paired human melanoma cell lines for study in which one member of the pair is from a curable early-stage lesion and the partner is a more aggressive malignant variant. A rapid method of obtaining such variants is described. It consists of injecting cells from established early-stage radial growth phase or vertical growth phase melanoma cell lines--which are normally non- or poorly tumorigenic in nude mice--into such hosts, where the cell inoculum is co-mixed with Matrigel, a reconstituted basement membrane extract. This resulted in the rapid formation of progressively growing solid tumors from which permanent cell lines were established. Subsequently, the sublines were found to be frankly tumorigenic upon retransplantation into new nude mouse hosts in the absence of Matrigel co-injection. This process was repeated a second time, resulting in the isolation of secondary sublines, manifesting a stepwise increase in tumorigenic properties. The tumorigenic variant sublines were examined for their relative sensitivity to a panel of different cytokines that are normally growth inhibitory for melanoma cells from early-stage primary lesions. All the sublines were found to express an increased resistance to the cytokines transforming growth factor-beta, interleukin-6, interleukin-1 and tumor necrosis factor-alpha, and did so in a stable manner. Thus the results support the hypothesis that a progressive multicytokine resistance accompanies the progression of human melanoma. The availability of such related sublines should provide a valuable resource to help study the changes associated with, and perhaps causative of, disease progression in human malignant melanomas.
EMT-6 murine mammary tumor sublines highly resistant to cyclophosphamide, cis-diamminedichloro-platinum(II), or N,N',N"-triethylenethiophosphoramide were generated in vivo by sequential treatment of tumor-bearing mice with the respective drugs. Previous studies demonstrated the drug-resistant phenotypes of the sublines were not expressed in vitro when the cells were grown as monolayer cultures. We now show that expression of drug resistance--including patterns of cross-drug resistance observed in vivo--can be fully recapitulated in vitro when the cells are grown under in vivo-like, three-dimensional conditions--namely, as multicellular tumor spheroids. Moreover, the spheroids generated from all of the drug-resistant sublines manifested a much more compact structure. Immediate drug-sensitivity testing of single cells released by trypsin treatment from compact drug-resistant spheroids revealed that such cells lost much of their drug-resistant properties. The results suggest a possible mechanism of acquired drug resistance in tumors based on the response of a cell population (i.e., multicellular or tissue resistance) as opposed to classic (uni)cellular resistance mechanisms.
The T x B hybrid 174 x CEM.T2 (T2) has been shown to be defective in the processing of proteins for presentation by MHC class I molecules. It continues, however, to express significant quantities of HLA-A2.1, suggesting that this class I molecule is expressed either in a largely peptide-free form or in association with a small subset of peptides. In this paper T2 was used in conjunction with limiting dilution analysis to provide a direct estimate of the fraction of alloreactive cytotoxic T lymphocytes (CTLs) that were dependent upon the presence of peptides for their recognition of HLA-A2.1. Alloreactive cytotoxic T cell lines generated by stimulation with HLA-A2.1 expressing peripheral blood lymphocytes recognized T2 poorly. Split-well analysis of 240 clonal limiting dilution cultures demonstrated that this reflected the existence of two subpopulations. An average 85% of HLA-A2.1 specific CTLs recognized HLA-A2.1 on normal cells but not on T2. The remainder recognized HLA-A2.1 on both T2 and normal targets. CTL lines with the latter specificity could be generated by using T2 as a stimulator cell. Using target cells that either expressed a lower density of HLA-A2.1 or that expressed HLA-A2 molecules that had been mutated to affect CD8 binding, no significant differences in avidity between T2-reactive and T2-unreactive CTLs were seen. Thus the failure of the majority of alloreactive CTLs to recognize T2 is not a consequence of the lower level of HLA-A2.1 surface expression on this cell, but is instead due to the absence of appropriate epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)
It is known from a variety of experimental systems that the ability of tumor cells to grow locally and metastasize can be affected by the presence of adjacent normal tissues and cells, particularly mesenchymally derived stromal cells such as fibroblasts. However, the comparative influence of such normal cell-tumor cell interactions on tumor behavior has not been thoroughly investigated from the perspective of different stages of tumor progression. To address this question we assessed the influence of normal dermal fibroblasts on the growth of human melanoma cells obtained from different stages of tumor progression. We found that the in vitro growth of most (4 out of 5) melanoma cell lines derived from early-stage radial growth phase or vertical growth phase metastatically incompetent primary lesions is repressed by coculture with normal dermal fibroblasts, suggesting that negative homeostatic growth controls are still operative on melanoma cells from early stages of disease. On the other hand, 9 out of 11 melanoma cell lines derived from advanced metastatically competent vertical growth phase primary lesions, or from distant metastases, were found to be consistently stimulated to grow in the presence of dermal fibroblasts. Evidence was obtained to show that this discriminatory fibroblastic influence is mediated by soluble inhibitory and stimulatory growth factor(s). Taken together, these results indicate that fibroblast-derived signals can have antithetical growth effects on metastatic versus metastatically incompetent tumor subpopulations. This resultant conversion in responsiveness to host tissue environmental factors may confer upon small numbers of metastatically competent cells a growth advantage, allowing them to escape local growth constraints both in the primary tumor site and at distant ectopic tissue sites.
We have previously shown, using tumor cell populations genetically tagged by random integrations of plasmid DNA, that metastatically-competent clonal cell variants have a strong growth advantage within primary tumors over their non-metastatic counterparts. As a result, primary tumors can become overgrown by the progeny of such cells, a process referred to as "clonal dominance" of primary tumors by metastatically-competent cells. Because of the well-known "metastatic inefficiency" of the multi-step cascade process of spread and growth, clonal dominance within primary tumors may be necessary for distant metastatic spread or increase the probability of its occurrence. To examine this hypothesis mice were inoculated s.c. with mixture of non-metastatic and genetically tagged, metastatically-competent mouse mammary carcinoma cells in defined ratios, but always containing an excess of the unmarked non-metastatic population. Progressive overgrowth of the metastatic subpopulation was monitored as a function of time by Southern analysis of DNA obtained from mixed primary tumors. This allowed us to evaluate the effects that surgical removal of the primary tumor had before, during and after effective clonal dominance, and what influence this had on the subsequent formation of distant metastases. Surgical removal of primary tumors before metastatic clonal dominance resulted in a low (0.25%) frequency of lung metastases, whereas removal just 1 or 2 weeks later during or after clonal dominance was achieved resulted in a high (75-100%) frequency of such metastases. Our results support the hypothesis that dominance of primary tumors by metastatically competent cells may be necessary for distant metastatic spread, and also suggest that clonal interactions play a significant role in modulating the metastatic ability of tumor cells in vivo.
The frequencies of HLA class II-specific cytotoxic T lymphocyte precursors (CTLp) were studied in number of unrelated individuals using a limiting dilution analysis system optimized for the detection of CD4+ CTLp. Peripheral blood mononuclear cells (PBMC) were enriched for CD4+ T cells by immunomagnetic depletion of CD8+ T cells. In some allogeneic combinations high CTLp frequencies were obtained with no significant difference between PBMC and CD4-enriched PBMC populations. In other combinations CTLp frequencies in CD4-enriched PBMC were found to be at least twentyfold lower than in the starting, unfractionated PBMC, suggesting a predominance in these pairs of CD8+ CTLp. In addition there was variation in CTLp frequencies against the same set of HLA class II gene products between individuals, and variation in CTLp frequencies against different HLA class II gene products within individuals. The HLA class II specificity of the assay system was demonstrated unequivocally with detection of CTLp against HLA-DR1 expressed on a murine L cell transfectant.
Limiting dilution analysis (LDA) provides a practical and simple method for determining the frequency of defined clones of lymphocytes responding to a specific antigen or with a particular effector function. The importance of the technique stems from the fact that it is the only way to assess the immune response in humans, at the level of the cell, in a quantitative manner. In this article Claire Sharrock, Edward Kaminski and Stephen Man review the current status of the technique and its applications in human immunology.
Epstein-Barr virus (EBV)-immortalised B cell lines were established from patients receiving multiple administrations (two or more) of radiolabelled murine monoclonal antibodies for the treatment of ovarian cancer. Cells secreting anti-id2 Ig, an immunoglobulin with binding specificities comparable to the administered murine monoclonal antibody, were isolated by using magnetic beads coated with tumour-associated antigen, incubated with the cells and concentrated with a magnetic particle concentrator. Cross-linking of the immunoglobulin receptors by the antigen-coated beads appears to stimulate proliferation, resulting in increased secretion of the human anti-tumour-associated antigen antibodies. The T cell responses were studied and it was found that monoclonal antibody therapy appears to lead to an increase in the population of T cells committed to proliferate in response to both specific antigen and non-specific mitogens. Multiple administrations of monoclonal antibody induce the generation of T cells which proliferate in vitro following stimulation with murine antibodies. The relevant (administered) monoclonal antibody induces higher proliferation rates than an idiotypically unrelated antibody of the same isotype, indicating the generation of idiotypically restricted T cell responses in these patients.
Transplantation of human tumors into the organ or tissue of their origin (orthotopic transplantation) in nude mice can result in significant enhancement of tumor growth and metastases, compared with sc (ectopic) transplantation. Because melanocytes are normally found in the epidermal-dermal junction, intradermal inoculation of melanoma cells might be expected to improve their potential for malignant growth as xenografts. The purpose of our study was to examine this possibility. We found that because mouse epidermis and dermis are so thin, it was not possible to inject a bolus of tumor cells intradermally; instead the cells were actually deposited in the most superficial layer of the subcutis (i.e., subdermally). We evaluated the behavior of cells from a human melanoma cell line after sc or subdermal inoculation into National Institutes of Health Swiss athymic nude mice. The cells used were from (1) the predominantly amelanotic human malignant melanoma cell line MeWo, originally established from a melanotic lymph node metastasis, and (2) two MeWo variants resistant to wheat germ agglutinin (WGAr), which were selected for altered malignant capacities. Whereas 5 X 10(5) MeWo cells were required to achieve 100% tumor take with sc injection, only 2 x 10(4) cells were required with subdermal inoculation. Subdermal injection of the MeWo cells resulted in the development of highly melanotic and nonencapsulated primary tumors, which grew quickly into the dermis and epidermis and metastasized at high frequency to draining lymph nodes. In contrast, the tumors that developed after sc injection were found in the deepest layer of the subcutis and were predominantly amelanotic and encapsulated; they rarely metastasized to lymph nodes.(ABSTRACT TRUNCATED AT 250 WORDS)
HLA "matched" unrelated donor bone marrow transplants are associated with an increased incidence and severity of graft-versus-host disease in comparison with HLA-identical sibling transplants. This is presumably due to HLA and non-HLA histocompatibility differences between donor and recipient. Using a limiting dilution assay, we have previously demonstrated a relationship between cytotoxic T lymphocyte precursor frequency and HLA disparity. In this study we have compared CTL-p frequencies with clinical GVHD, and demonstrate for the first time a significant correlation (P less than 0.005) between high CTL precursor frequency prior to BMT and severity of acute GVHD after HLA A, B, DR "matched" unrelated donor transplants using T cell depleted marrow. This assay system may be of value in the final selection of HLA "matched" unrelated donors for BMT.
Spontaneous wheat germ agglutinin (WGA)-resistant mutants of the MeWo human malignant melanoma line were isolated after sequential selection in increasingly toxic concentrations of WGA, without prior mutagenesis. They were isolated in an attempt to obtain "membrane glycosylation mutants" having significantly altered metastatic properties when grown in nude mice, and to characterize the biochemical (oligosaccharide) changes associated with altered metastatic behavior. The lines were assessed for their sensitivity to other lectins, membrane glycoprotein profiles, ploidy levels, and their ability to produce "artificial" metastases in nude mice after i.v. inoculation. One mutant, called 70-W, manifested a 3- to 4-fold resistance to WGA compared with wild-type cells. When inoculated into NIH Swiss nude mice, 70-W cells not only produced extensive lung colony formation but also showed an extraordinary ability to disseminate widely and extensively in a clinical fashion to many extrapulmonary sites such as the subcutis, mesentery, muscle, and brain. Moreover the majority of these metastases were deeply pigmented facilitating visual identification of very small visceral metastases. A second mutant called 3S5 was isolated and found to be highly resistant to WGA (greater than 20-fold resistance). This line was virtually devoid of metastatic ability and was found by biochemical analysis to be phenotypically similar to the class I WGA resistant non-metastatic mutants previously isolated from the highly metastatic murine tumor MDAY-D2 which are known to be deficient in sialic acid and galactose. The similarity between these and earlier results using lectin resistant mutant rodent cell lines strongly suggests that sialylated glycoconjugates contribute to the metastasis of both animal and human tumors of different tissue origin. These new spontaneously derived WGA resistant MeWo mutants should be valuable new tools for the study of human tumor progression in vivo and factors involved in metastasis, especially the contribution of oligosaccharide moieties of cell surface glycoconjugates.