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T L Darrow

Publications and source records attributed to T L Darrow.

34 records · Page 2Linked to original sources
PubMed1991

MHC-restricted recognition of autologous melanoma by tumor-specific cytotoxic T cells. Evidence for restriction by a dominant HLA-A allele.

Autologous melanoma-specific CTL recognize a common tumor-associated Ag (TAA) in the context of HLA class I antigens. We have demonstrated that HLA-A2 can be a restricting Ag and, in T cell lines homozygous for HLA-A2, that CTL can be generated by stimulation with HLA-A2 allogeneic melanomas. In the current study, we have investigated T cell lines from patients who are heterozygous at HLA-A region locus, to determine the relative importance of each A-region allele in this MHC-restricted recognition of tumor. We have shown that HLA-A1 can be a restricting Ag, and that allogeneic melanomas expressing HLA-A1 can substitute for the autologous tumor in the generation of HLA-A1-restricted CTL. However, when T cell lines express both HLA-A1 and HLA-A2, the HLA-A2 allele governed restriction of the melanoma TAA. Three autologous-stimulated HLA-A1, A2 CTL lines all demonstrated restriction by the HLA-A2 allele, when examined in cytotoxicity assays, cold-competition assays, and proliferation assays. There was no evidence of restriction by the second HLA-allele, HLA-A1. Although the autologous-stimulated CTL use a single A-region allele for tumor recognition, the autologous HLA-A1, A2 tumors are lysed by both HLA-A1-restricted and HLA-A2-restricted CTL. The dominance of restricting alleles was further demonstrated when HLA-matched allogeneic melanomas were used as the stimulating tumor to generate tumor-specific CTL. Stimulation of the heterozygous (HLA-A1, A2) lymphocytes with HLA-A2-matched allogeneic melanomas resulted in CTL specific for the autologous tumor, and restricted by the HLA-A2 Ag. However, stimulation with an HLA-A1-matched allogeneic melanoma failed to induce tumor-specific CTL restricted by the HLA-A1 Ag. The data suggest there is a dominance of HLA-A region Ag at the level of the T cell, such that only one is restricting in the recognition of the autologous melanoma. At the level of the tumor, however, the TAA is expressed in the context of both HLA-A region alleles. We can generate specific CTL from lymph node cells or PBL and HLA-A region matched allogeneic melanomas; however, because most patients are heterozygous at the HLA-A region locus, an understanding of the dominant restricting alleles must be obtained so that an appropriately matched allogeneic melanoma can be selected.

Alleles↗
PubMed1990

Generation of human autologous melanoma-specific cytotoxic T-cells using HLA-A2-matched allogeneic melanomas.

Autologous tumor-specific cytotoxic T-lymphocytes (CTLs), generated by repeated stimulation with autologous melanoma and expanded in interleukin 2, are major histocompatibility complex restricted. These CTLs recognize a common tumor-associated antigen in the presence of HLA class I determinants, suggesting that allogeneic melanomas which express the restricting HLA-A region antigen could substitute for the autologous tumor in the generation of CTLs. This was investigated in the HLA-A2 system. Four T-cell lines were established by stimulation of lymphocytes with either autologous tumor or an HLA-A2-matched allogeneic melanoma. Allogeneic stimulated CTLs specifically lysed the autologous tumor and demonstrated an identical pattern of HLA-A2 restriction, when compared to the autologous stimulated CTLs. Lysis by the allogeneic stimulated CTLs was blocked by a monoclonal antibody to HLA class I antigens; lysis was also inhibited by both autologous tumor or HLA-A2 allogeneic melanomas when evaluated in cold target competition studies. The allogeneic stimulated CTLs proliferated in response to both autologous tumor and HLA-A2 melanomas, but not in response to HLA-A2 nonmelanomas. By phenotypic analysis these CTLs were CD3+ and predominantly CD8+ cells. We conclude that autologous tumor-specific CTLs can be generated using HLA-A region-matched allogeneic melanomas for stimulation. Since established, HLA-typed melanoma tumor lines can be used in the absence of autologous tumor; this procedure can be applied clinically to a broad patient population and may prove useful in the adoptive immunotherapy of melanoma.

Autoimmunity↗
PubMed1990

Inhibition of the growth of human melanoma xenografts in nude mice by human tumor-specific cytotoxic T-cells.

Melanoma-specific T-cells (CTLs) are specifically cytotoxic for autologous tumor, when assayed in vitro. To examine their effectiveness in vivo, we tested the ability of these human T-cells to inhibit growth of human melanoma xenografts by using a Winn assay. Nude mice receiving specific CTLs (n = 10) demonstrated a dramatic inhibition of tumor growth. All treated mice were tumor-free at day 50 and nine remained tumor-free at day 65, vs. control mice (n = 10) with average tumor volumes of 321 mm3 and 808 mm3, respectively. To control for the possibility that a non-specific response to the human T-cells could inhibit tumor growth, an additional group received allospecific CTLs. There was no inhibition of tumor growth in this group (n = 8), with the average tumor volume of 2,768 mm3 at day 40 vs. 1,882 mm3 in the control group (n = 10). We conclude that these tumor-specific CTLs can inhibit tumor growth in vivo and may prove useful in the adoptive immunotherapy of melanoma.

Animals↗
PubMed1990

Modulation of in vitro autologous melanoma-specific cytotoxic T-cell responses by phorbol dibutyrate and ionomycin.

Human melanoma-specific, HLA restricted, cytotoxic T-cell lines can be generated by in vitro stimulation and culturing of peripheral lymphocytes, or lymph node cells, with autologous or HLA-A region matched melanomas in the presence of a low concentration (5 U/ml) of IL-2. Stimulation is followed by a period of clonal expansion and differentiation into cytotoxic T-cells specific for melanoma. We investigated the effect of the PKC modulating drug phorbol dibutyrate combined with the calcium ionophore Ionomycin on growth and differentiation of the cell lines. The growth of the T-cell lines was substantially augmented in the presence of the drugs with increases of 10-fold or more in clonal expansion by 3 weeks of culture. The cell lines were IL-2 dependent for growth in the presence or absence of the drugs and the phenotypic distribution remained predominantly CD3+ T-cells of mixed CD4 and CD8 phenotypes. In spite of the increased rate of growth in the presence of the drugs, autologous melanoma-specific cytotoxicity was almost completely abrogated in those cultures. The cells were, however, nonspecifically lytic in the presence of concanavalin A. The melanoma-specific cytotoxic response was completely restored following culture with IL-2 alone. The results suggest that the human tumor-specific cytotoxic T-cell response can be induced and amplified in the presence of immune modulating drugs.

Antigens, CD↗
PubMed1990

Implications of leukoregulin to autologous tumor-specific human T-cell populations.

Interactions between the cellular and humoral compartments of the human immune system are well documented. Leukoregulin (LR) is a hormone with the ability to upregulate the natural killer (NK) phenomenon by increasing target cell sensitivity to NK lymphocyte cytotoxicity. In the present report, the interactions between LR and human cytotoxic T-lymphocytes (CTLs) are described. Two cultured T-cell populations are specifically cytotoxic for autologous human melanoma and fail to lyse allogeneic melanoma, K562, or autologous peripheral blood lymphocytes (PBLs) in 4 h chromium release assays. Pretreating allogeneic melanoma or K562 with LR resulted in 19-67% lysis at an effector:target (E:T) ratio of 5:1, while no more than 10% lysis was observed without LR. Autologous PBLs were also subject to lysis when pretreated with LR (50% at an E:T of 40:1 with LR, and 2% without LR). The observed effect was dose-related and was most effectively inhibited by autologous cold targets, but persisted in the presence of antibody to human lymphocyte antigen class I antigens (w6/32). Lysis of autologous melanoma was not increased by pretreatment with LR. LR appears to mediate lysis of melanoma, NK targets, and normal PBLs by tumor specific CTLs. The effect is dose-related and non-major histocompatibility complex-restricted. The data do not support a significant role for LR in the normal physiology of human CTLs, but the striking effects in vitro may prove to be experimentally or therapeutically useful.

Antibodies, Monoclonal↗
PubMed1989

The role of HLA class I antigens in recognition of melanoma cells by tumor-specific cytotoxic T lymphocytes. Evidence for shared tumor antigens.

CTL lines were established in vitro by stimulating patient lymphocytes with autologous melanoma cells in the presence of IL-2. Resulting CTL lines lysed autologous melanoma and failed to lyse several allogeneic melanomas or K562. The mechanism of target cell recognition by autologous tumor-specific CTL was evaluated in this system, using several CTL lines: DT6, DT105, DT141, DT166, DT169, and DT179. Autologous melanoma lysis was inhibited by W6/32, mAb directed against HLA class I Ag, but not by L243, mAb directed against HLA class II Ag. CTL from DT6, DT141, DT166, DT169, and DT179 lysed fresh and cultured allogeneic melanomas, which shared the HLA-A2 Ag, but failed to lyse allogeneic melanomas, which shared B-region or C-region Ag, or shared no HLA class I Ag. CTL from DM141 lysed DM93, which shared A2 and Bw6, but failed to lyse DM105, which shared only Bw6. DM105 CTL failed to lyse allogeneic melanomas that shared HLA-A1, or that shared B or C region Ag, but they did lyse allogeneic melanoma DM49, which expressed an A region Ag that either was A10 or was serologically cross-reactive with A10. A T cell leukemia line, three EBV transformed B cell lines, and a pancreatic cancer line, all of which expressed HLA-A2, were not lysed by DM6 or DM179 CTL. Furthermore, HLA-matched nonmelanomas failed to inhibit autologous tumor lysis in cold target inhibition assays, whereas an HLA-A2+ allogeneic melanoma, DM93, inhibited autologous tumor lysis as effectively as the autologous tumor itself. HLA-A2, and possibly other HLA-A-region Ag, appear to function in HLA-restricted recognition of shared melanoma associated Ag by CTL.

Antibodies, Monoclonal↗
PubMed1989

Melanoma-specific cytotoxic T cells generated from peripheral blood lymphocytes. Implications of a renewable source of precursors for adoptive cellular immunotherapy.

Cytotoxic T lymphocytes (CTL) specific for autologous human melanoma have been generated in vitro from peripheral blood lymphocytes (PBL) of five patients with resectable stage II malignant melanoma. The PBL were cultured with 5u/ml recombinant IL-2 and were repeatedly stimulated with irradiated fresh or cultured autologous tumor cells. Cytotoxicity was determined by four-hour chromium release assays. Specific cytotoxicity developed in 30 to 40 days, after three or four stimulations with tumor. The PBL-derived CTL are CD3+ and are mixed for CD4+ and CD8+ phenotypes. They lysed autologous melanoma and failed to lyse allogeneic melanoma, K562, or autologous lymphocytes. The lysis of autologous tumor was maintained for more than 4 months. The cells proliferated in response to autologous, but not allogeneic melanoma cells, in a dose-dependent manner. Lysis of the autologous tumor target was inhibited with w6/32, a monoclonal antibody to HLA Class I antigens. It is concluded that PBL may serve as a plentiful and renewable source of precursor cells for the generation of autologous tumor-specific CTL, which may be useful in specific adoptive cellular immunotherapy of melanoma.

Antibodies, Monoclonal↗
PubMed1988

Autologous lymph node cell-derived tumor-specific cytotoxic T-cells for use in adoptive immunotherapy of human melanoma.

The in vitro development of tumor-specific cytotoxic T-cells from draining and tumor-involved lymph nodes obtained from melanoma patients were examined. Fresh draining or tumor-involved lymph node cells (LNC) demonstrate no significant cytotoxic activity against a variety of tumor targets including autologous melanoma. Natural killer cell (NK) activity is very low or absent in all of these specimens. Culture of the cells with irradiated autologous tumor and expansion in recombinant interleukin 2 (rIL-2) results in strong cytotoxicity for autologous tumor cells. The cultured cells are T-cells of mixed CD4 and CD8 phenotypes. Following restimulation with autologous tumor, these lines are capable of becoming specifically cytotoxic for autologous tumor as tested in direct killing and in cold target inhibition studies. The LNC yield from fresh specimens ranges from 1 X 10(7) to more than 1 X 10(9) cells averaging 5 X 10(8) cells. After the cells are cultured, we can achieve up to a 60-fold or more increase in cell numbers, that demonstrate strong cytotoxicity for melanomas. The potential for adoptive immunotherapy using such specifically sensitized cytotoxic T-cells of mixed phenotypes is discussed.

Cell Line↗
PubMed1987

Human T cells specifically activated against autologous malignant melanoma.

Lymphocytes from ten patients with melanoma were specifically stimulated in vitro with autologous melanoma cells and expanded in interleukin 2. Significant lysis of autologous melanoma cells was demonstrated in T cells derived from six of these patients. The mean percent of lysis of autologous tumor cells at an effector-target ratio of 20:1 was 46% among these six patients. The T cells derived from two patients developed specificity in lysing autologous melanoma cells. In both cases, specificity was enhanced by the in vitro stimulation with autologous tumor cells. Restimulation with autologous melanoma cells was associated with increasing specificity over time. Whether derived from peripheral blood lymphocytes or from lymph node cells, T cells from one patient lysed fresh autologous melanoma cells more potently than K562, allogeneic melanoma cells, and nonmelanoma cells. On day 38, at an effector-target ratio of 10:1, cell lysis of K562, an osteosarcoma, a pancreatic cancer, and three allogeneic melanomas was 3%, 4%, 7%, 8%, 7%, and 2%, respectively, while lysis of autologous melanoma cells was 47%. Specificity was maintained beyond day 60. The T cells could be expanded over 50-fold within one month.

Cell Separation↗
PubMed1987

Immune response of chimpanzee to purified melanoma 250 kilodalton tumor-associated antigen.

Melanoma high molecular weight tumor-associated antigen (TAA), having a molecular weight of 250 kilodaltons (Kd), was purified from a crude cell membrane extract through a combination of lectin affinity, immunoadsorption, and high performance liquid molecular filtration chromatography. Compared to the starting extract, purified TAA was 600-fold higher in TAA activity per microgram of protein. Purified TAA was used to immunize a chimpanzee and the resulting antiTAA immune response was evaluated. Postimmune chimpanzee serum reacted in solid phase radioimmunoassay against purified TAA with a titer in excess of 100,000. In contrast, preimmune serum had a titer of less than 100 in the same assay. By immunoprecipitation analysis, we were able to demonstrate reactivity of the chimpanzee immune serum with a 250 Kd TAA in spent culture medium from melanoma cells metabolically labeled with 35S-methionine and with iodinated purified 250 Kd TAA. Reactivity of the chimpanzee antiserum for the 250 Kd TAA was confirmed in blocking and reciprocal immunodepletion studies using murine monoclonal antibody 9.2.27. These studies suggest that the 250 Kd TAA defined by murine monoclonal antibodies may prove to be immunogenic in man and that manipulation of the immune response to this TAA might be used to the clinical benefit of the patient.

Animals↗
PubMed1983

Augmentation of syngeneic tumor-specific immunity by semiallogeneic cell hybrids.

Hybrid cell lines were established from fusions between lipopolysaccharide- (LPS) stimulated C57BL/6J spleen cells and MPC-11 tumor cells (45.6TG1.7, abbreviated M45), and were tested for their ability to immunize semiallogeneic mice against a parental tumor challenge. These hybrids were tumorigenic in syngeneic (BALB/c X C57BL/6J) F1 (CB6F1) mice but did not grow in semiallogeneic (BALB/c X A/J) F1 (CAF1) mice. All hybrids express both parental major histocompatibility antigens (H-2b and H-2d) as detected by indirect immunofluorescence and by their ability to function as either stimulators or targets for allogeneic cytotoxic lymphocytes (CTL). M45 tumor-associated antigens (TAA) were expressed on the hybrid surface as shown by their ability to act as either stimulators or targets for syngeneic CTL specific for M45 TAA. Immunization of semiallogeneic CAF1 mice with the hybrids i.p. followed by a challenge with M45 tumor cells resulted in extended survival when compared to untreated mice or animals immunized i.p. with M45 tumor cells. This immunity was specific and was not due to an allogeneic effect; immunization with an unrelated H-2bd tumor, 70Z/3, or H-2bd B6D2F1 spleen cells or with semiallogeneic spleen cells plus M45 did not protect mice from M45 challenge. Interestingly, prophylactic priming with semiallogeneic hybrid tumor cells or parental myeloma cells led to M45-specific CTL and "help" for an in vitro CTL response; however, the degree of CTL priming by hybrid tumors was not augmented when compared to the level of CTL achieved with parental tumor alone. Hence, stimulation of CTL activity per se by hybrid tumor cells cannot explain the protective effect of hybrid tumor immunization. These studies nevertheless confirm that semiallogeneic hybrids, which we show express TAA and alloantigens, can be used to immunize mice against a lethal syngeneic myeloma tumor challenge.

Animals↗
PubMedDate not supplied

Modulation of specific active immunization against murine melanoma using recombinant cytokines.

Specific active immunization with tumour cells and IL-1beta or IL-2 was examined in a murine model. Mice were treated with irradiated B16 melanoma, IL-1beta or IL-2 only, or with B16 plus cytokines prior to i.v. challenge with viable B16. Lung metastases were recorded after 28 days. Treatment with cytokine alone was not protective. Treatment with B16 alone afforded moderate protection. Treatment with B16 in combination with either cytokine resulted in a significant level of B16 specific protection which was dependent on the dose of cytokine used. Multiple immunizations with B16 provided limited protection which was significantly improved with IL-2. Immunization with B16 in combination with both cytokines at doses that alone failed to enhance immunity resulted in significant protection, suggesting that the two cytokines act at least additively. These studies demonstrate the significant benefit of specific active immunization with tumour cells in combination with low doses of IL-1beta or IL2.

Analysis of Variance↗