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Biomedical subjects

B A Pulaski

Publications and source records attributed to B A Pulaski.

15 recordsLinked to original sources
PubMed2000

Cooperativity of Staphylococcal aureus enterotoxin B superantigen, major histocompatibility complex class II, and CD80 for immunotherapy of advanced spontaneous metastases in a clinically relevant postoperative mouse breast cancer model.

One of the leading causes of death for women is metastatic breast cancer. Because most animal tumors do not accurately model clinical metastatic disease, the development of effective therapies has progressed slowly. In this study, we establish the poorly immunogenic mouse 4T1 mammary carcinoma as a postsurgical animal model. 4T1 growth characteristics parallel highly invasive human metastatic mammary carcinoma and, at the time of surgery, the extent of disease is comparable with human stage IV breast cancer. Progress in understanding the immune response has led to innovative immune-based anticancer therapies. Here, we test in this postsurgical model, a novel cell-based vaccine, combining MHC class II, CD80(B7.1), and SEB superantigen. Effective treatment of tumor-bearing mice with this immunotherapy requires expression of all three molecules. Mean survival time is extended from 5-7.5 weeks for control-treated mice to 6-10.5 weeks for therapy-treated mice. Increased survival is accompanied by a maximum of 100-fold decrease in clonogenic lung metastases. These therapeutic effects are particularly noteworthy because: (a) the postoperative model demonstrates that early metastases responsible for morbidity are established by 2 weeks after tumor inoculation with 7 x 10(3) parental 4T1 cells into the mammary gland; (b) the immunotherapy is started 4 weeks after tumor inoculation when the mice contain extensive, pre-established, disseminated metastases; and (c) CD4+ and CD8+ T cells are required for the effect.

Animals↗
PubMed2000

Immunotherapy with vaccines combining MHC class II/CD80+ tumor cells with interleukin-12 reduces established metastatic disease and stimulates immune effectors and monokine induced by interferon gamma.

Because they are difficult to treat, animal models of widespread, established metastatic cancer are rarely used to test novel immunotherapies. Two such mouse models are used in this report to demonstrate the therapeutic efficacy and to probe the mechanisms of a novel combination immunotherapy consisting of the cytokine interleukin-12 (IL-12) combined with a previously described vaccine based on MHC class II, CD80-expressing cells. BALB/c mice with 3-week established primary 4T1 mammary carcinomas up to 6 mm in diameter and with extensive, spontaneous lung metastases show a significant reduction in lung metastases following a 3-week course of immunotherapy consisting of weekly injections of the cell-based vaccine plus injections of IL-12 three times per week. C57BL/6 mice with 7-day established intravenous B16 melF10 lung metastases show a similar response following immunotherapy with IL-12 plus a vaccine based on B16 MHC class II, CD80-expressing cells. In both systems the combination therapy of cells plus IL-12 is more effective than IL-12 or the cellular vaccine alone, although, in the 4T1 system, optimal activity does not require MHC class II and CD80 expression in the vaccine cells. The cell-based vaccines were originally designed to activate tumor-specific CD4+ T lymphocytes specifically and thereby provide helper activity to tumor-cytotoxic CD8+ T cells, and IL-12 was added to the therapy to facilitate T helper type 1 lymphocyte (Th1) differentiation. In vivo depletion experiments for CD4+ and CD8+ T cells and natural killer (NK) cells and tumor challenge experiments in beige/nude/XID immunodeficient mice demonstrate that the therapeutic effect is not exclusively dependent on a single cell population, suggesting that T and NK cells are acting together to optimize the response. IL-12 may also be enhancing the immunotherapy via induction of the chemokine Mig (monokine induced by interferon gamma), because reverse PCR experiments demonstrate that Mig is present in the lungs of mice receiving therapy and is most likely synthesized by the tumor cells. These results demonstrate that the combination therapy of systemic IL-12 and a cell-based vaccine is an effective agent for the treatment of advanced, disseminated metastatic cancers in experimental mouse models and that multiple effector cell populations and anti-angiostatic factors are likely to mediate the effect.

Animals↗
PubMed1999

Cell-based vaccines for the stimulation of immunity to metastatic cancers.

We are developing vaccines for inducing immunity to metastatic cancers. Although primary tumors are frequently cured by surgery, chemotherapy, or radiation therapy, metastatic lesions often do not respond to these treatments or proliferate after conventional therapy is terminated. Vaccine therapy for established metastases as well as prophylactic vaccine treatment to prevent outgrowth of latent metastatic tumor cells would therefore be beneficial. Our goal is to activate CD4+ and CD8+ T lymphocytes; however, we have focused on activating tumor-specific CD4+ T-helper lymphocytes because of their pivotal role as regulatory cells and in the generation of long-term immunological memory. The vaccines are based on the premise that tumor cells express potentially immunogenic antigens that could be targeted for T-cell activation, and that if appropriately genetically modified, tumor cells could be antigen presenting cells for these antigens. To facilitate direct antigen presentation to CD4+ T cells, tumor cells have been transfected with syngeneic major histocompatibility complex class II, co-stimulatory molecule, and/or superantigen genes. In vivo studies in three mouse tumor models demonstrate that vaccination protects against future challenge with wild-type tumor, cures some solid primary tumors, reduces established metastatic disease, and extends mean survival time. Antigen presentation studies demonstrate that in vivo vaccine efficacy is directly related to in vitro antigen presentation activity. The relevance of antigen presentation activity of the vaccines is further confirmed by in vivo studies demonstrating that during the immunization process, the vaccines directly present tumor-encoded antigens to CD4+ T lymphocytes. Adaptation of these vaccines for the treatment of human metastatic cancers is discussed.

Animals↗
PubMed1998

IL-3 enhances both presentation of exogenous particulate antigen in association with class I major histocompatibility antigen and generation of primary tumor-specific cytolytic T lymphocytes.

Recent studies have reported that APC can present particulate exogenous Ag in the context of class I MHC to CD8+ CTL, and our laboratory demonstrated that IL-3 could enhance CTL generation to exogenous Ag. In this paper, we wished to determine whether presentation of particulate Ag could be enhanced by IL-3. A T cell hybridoma, B3Z86/90.14 (B3Z) restricted to Ova/Kb, was used as an indicator for presentation of particulate Ag with class I MHC. When activated, this hybridoma expresses lacZ, allowing a simple colorimetric measurement of Ag-specific T cell stimulation. We demonstrated that bone marrow cells stimulated by IL-3 in vivo and in vitro exhibited significantly increased presentation of exogenous OVA linked to beads. Lysate from OVA-transfected line 1 murine lung adenocarcinoma cells (line 1/OVA) was also presented by IL-3-stimulated bone marrow cells, suggesting that these APC can process tumor fragments or debris. Studies using TAP1/2-deficient mice and Ag presentation inhibitors indicate that this exogenous Ag presentation is mediated via the conventional class I MHC pathway. Adoptive transfer of IL-3-stimulated bone marrow cells pulsed with lysate from line 1/OVA tumor cells into naive recipient mice led to the generation of a potent CTL response. These observations indicate that use of such cells may provide a new avenue for development of tumor vaccines.

Animals↗
PubMed1998

Reduction of established spontaneous mammary carcinoma metastases following immunotherapy with major histocompatibility complex class II and B7.1 cell-based tumor vaccines.

For many cancer patients, removal of primary tumor is curative; however, if metastatic lesions exist and are not responsive to treatment, survival is limited. Although immunotherapy is actively being tested in animal models against primary tumors and experimental metastases (i.v. induced), very few studies have examined immunotherapy of spontaneous, established metastatic disease. The shortage of such studies can be attributed to the paucity of adequate animal models and to the concern that multiple metastatic lesions may be more resistant to immunotherapy than a localized primary tumor. Here, we use the BALB/c-derived mouse mammary carcinoma, 4T1, and show that this tumor very closely models human breast cancer in its immunogenicity, metastatic properties, and growth characteristics. Therapy studies demonstrate that treatment of mice with established primary and metastatic disease with MHC class II and B7.1-transfected tumor cells reduces or eliminates established spontaneous metastases but has no impact on primary tumor growth. These studies indicate that cell-based vaccines targeting the activation of CD4+ and CD8+ T cells may be effective agents for the treatment of malignancies, such as breast cancer, where the primary tumor is curable by conventional methods, but metastatic lesions remain refractile to current treatment modalities.

Animals↗
PubMed1998

Tumor antigen presentation: changing the rules.

Cell-based tumor vaccines have been developed on the basis of the hypothesis that tumor cells can be genetically modified to present antigen to T lymphocytes directly. Contrary to expectations, cross-priming is the predominant pathway for activation of tumor-specific CD8+ T cells, while direct presentation of antigen dominates activation of tumor-specific CD4+ T cells. These results pose interesting paradoxes for the generation of immune responses, and have definite implications for the development of anti-cancer vaccines.

Animals↗
PubMed1996

Interleukin 3 enhances cytotoxic T lymphocyte development and class I major histocompatibility complex "re-presentation" of exogenous antigen by tumor-infiltrating antigen-presenting cells.

We show that interleukin 3 (IL-3) enhances the generation of tumor-specific cytotoxic T lymphocytes (CTLs) through the stimulation of host antigen-presenting cells (APCs). The BALB/c (H-2d) spontaneous lung carcinoma line 1 was modified by gene transfection to express ovalbumin as a nominal "tumor antigen" and to secrete IL-3, a cytokine enhancing myeloid development. IL-3-transfected tumor cells are less tumorigenic than the parental cell line, and tumor-infiltrating lymphocytes isolated from these tumors contain increased numbers of tumor-specific CTLs. By using B3Z86/90.14 (B3Z), a unique T-cell hybridoma system restricted to ovalbumin/H-2b and implanting the tumors in (BALB/c x C57BL/6)F1 (H-2d/b) mice, we demonstrate that the IL-3-transfected tumors contain an increased number of a rare population of host cells that can process and "re-present" tumor antigen to CTLs. Electron microscopy allowed direct visualization of these host APCs, and these studies, along with surface marker phenotyping, indicate that these APCs are macrophage-like. The identification of these cells and their enhancement by IL-3 offers a new opportunity for tumor immunotherapy.

Animals↗
PubMed1995

Analysis of the effect of cytokines (interleukins 2, 3, 4, and 6, granulocyte-monocyte colony-stimulating factor, and interferon-gamma) on generation of primary cytotoxic T lymphocytes against a weakly immunogenic tumor.

We have investigated the role of cytokines (IL-2, IL-3, IL-4, IL-6, IFN-gamma, and GM-CSF) in the generation of primary cytotoxic T lymphocytes (CTL), within a single tumor system. The murine carcinoma line 1 was transfected with expression vectors with cDNA for these cytokines. Line 1 expresses low levels of class I MHC molecules, but can be induced with dimethyl sulfoxide or IFN-gamma to express high levels of class I. Class I low line 1 cells are not susceptible to CTL lysis, while class I high line 1 are lysed by CTL, which allows us to assay for CTL activity. To isolate primary CTL, tumor-infiltrating lymphocytes were isolated from cytokine-expressing or control tumors, growing in vivo. Most cytokines stimulated nonspecific killers, but IL-2 and IL-3 stimulated primary CTL. While IFN-gamma alone did not generate primary CTL, coexpression of IFN-gamma with IL-2 resulted in CTL generation. This is the first comparison of the effects of a series of cytokines on primary CTL development, and has important implications for vaccine development and immunotherapy.

Animals↗
PubMed1995

B7-1 enhances natural killer cell-mediated cytotoxicity and inhibits tumor growth of a poorly immunogenic murine carcinoma.

The B7-1 molecule expressed on antigen presenting cells is an important costimulatory molecule for T cell activation. It has been demonstrated that murine B7-1 can enhance host immunity and lead to tumor rejection via its costimulatory function. Here, we investigate how transfection of B7-1 into line 1, a poorly immunogenic murine lung carcinoma, affects the generation and function of different immune effector cells. Line 1 cells expressing B7-1 form tumors that grow at a slower rate than the parental line 1. Our studies have shown that tumor infiltrating lymphocytes present within the B7-1 expressing tumors are primarily composed of nonspecific killer cells with no specific cytotoxic T cells present. To determine if increased nonspecific killer cells could inhibit the tumor growth of line 1 in the presence of B7-1, we examined the cytotoxicity of natural killer (NK) cells and lymphokine-activated killer (LAK) cells on the B7-1-transfected line 1 and the parental line 1. We found that B7-1 augments the NK- but not LAK-mediated killing against line 1 as measured in an in vitro 51Cr-release cytotoxicity assay. This enhancement could be blocked by CTLA-4 Ig. In vivo depletion of NK cells led to growth of the B7-1-transfected line 1 at the same rate as the parental line 1. These results suggest that in addition to its costimulatory role for T cell activation B7-1 could be an accessory molecule that intensifies NK-mediated cytotoxicity. This novel finding may provide a mechanism for the effect of B7-1 on tumors of low immunogenicity.

Abatacept↗
PubMed1995

Synergistic effects of co-expression of the TH1 cytokines IL-2 and IFN-gamma on generation of murine tumor-reactive cytotoxic cells.

While the effect of cytokines on the generation of tumor-reactive cytotoxic cells has been a topic of active investigation, the effect of physiological cytokine combinations has not been determined. We have investigated the effect of co-expression of IL-2 and IFN-gamma on the generation of cytotoxic cells against the murine line 1 tumor in vivo. These cytokines were selected because they are normally produced in concert by a subset of T-helper cells called T-helper 1 (Th1). We transfected the line 1 murine carcinoma with cDNA for IL-2 and IFN-gamma, alone or combined. IFN-gamma alone does not elicit rejection of the transfectant, but IL-2 increases the tumorigenic dose by 10,000-fold above the parental cells. Co-expression of IFN-gamma and IL-2 increases this rejection to at least 100,000-fold above parental line 1. Unlike IL-2 transfectants, tumor cells expressing both IFN-gamma and IL-2 can also elicit rejection of admixed parental tumor cells. Finally, the IFN-gamma/IL-2 transfectants are more effective at generating memory cells that are cytolytic for the parental tumor. Our results show that synergistic interactions of Th1 cytokines can remarkably enhance the cytotoxic response to tumors.

Animals↗
PubMed1994

Transfection of transforming growth factor-beta producing tumor EMT6 with interleukin-2 elicits tumor rejection and tumor reactive cytotoxic T-lymphocytes.

One mechanism by which potentially immunogenic tumors evade the immune response is production of immunosuppressive factors. The murine mammary sarcoma EMT6 has previously been demonstrated to inhibit the proliferation of B-cells, suggesting that this tumor produces immunosuppressive factors. Here, we show that supernatant from EMT6 inhibits the development of cytotoxic T-lymphocytes (CTLs) and that this inhibition can be reversed by addition of recombinant interleukin (IL)-2. Furthermore, we show that EMT6 produces high levels of the immunosuppressant factor transforming growth factor (TGF)-beta. To determine if the T-cell growth factor IL-2 within the tumor microenvironment could reverse the immunosuppressive effect of EMT6, we transfected EMT6 with an expression vector containing the cDNA for murine IL-2 under the control of the beta-actin promoter. These transfectants produce significant levels of IL-2 (26 U/ml). EMT6/IL-2 is rejected by mice at 100-fold higher challenge than are parental cells or control transfectants (neomycin resistance only). Thy-1-expressing cells purified from EMT6/IL-2 tumors show greater cytotoxicity against the parental EMT6 cells than do those from the control transfectant. Thus, IL-2 can reverse the effects of TGF-beta on development and/or proliferation of CTL reactive with EMT6, allowing the establishment of mature effectors in vivo. This has significant implications for the development of CTL and immunotherapy for immunosuppressive tumors.

Animals↗
PubMed1993

Interleukin 3 enhances development of tumor-reactive cytotoxic cells by a CD4-dependent mechanism.

We investigated the effects that mouse interleukin 3 (IL-3), in comparison to mouse IL-2, has on the generation of cytotoxic effectors capable of killing line 1 tumor cells. These potent immunological mediators were delivered locally using gene transfection, rather than systemically, to the tumor site. We created line 1 transfectants that express high levels of IL-3 (3750 units/ml) or IL-2 (200 units/ml) by driving transcription from the beta-actin promoter. These levels of expression significantly enhanced tumor rejection in syngeneic mice. Tumor-infiltrating lymphocytes purified from IL-3 or IL-2 transfected tumors showed a dramatically enhanced cytotoxic response to parental line 1 targets. Also, IL-2, but not IL-3, expression enhanced the nonspecific lysis of YAC-1 cells. In vivo depletion of CD8+ cells with monoclonal antibody 2.43 abrogated the generation of cytotoxic effectors in both cases. Interestingly, depletion of CD4+ cells with monoclonal antibody GK1.5 abrogated the IL-3-mediated cytotoxic response but not the IL-2-mediated response. In vivo depletion of CD4+ or CD8+ cells abrogated the effect IL-3 had on reducing tumorigenicity. Reverse polymerase chain reaction analysis demonstrates that IL-3 transfected tumors, when compared to untransfected tumors, express increased levels of IL-2 and IL-4 mRNA. These results strongly suggest that IL-3, unlike IL-2, works to generate cytotoxic effectors by a mechanism that requires CD4+ cells.

Animals↗
PubMed1993

Interleukin-3 inhibits the generation of nonspecific killers by interleukin-2.

Interleukin (IL)-3 has effects on a wide variety of cell types, including immature cells of the immune system, as well as mature cells such as granulocytes. We have investigated the effects of IL-3 on generation of cytolytic cells that can kill tumor cells. Previously, we have shown that IL-3 can enhance the development of cytolytic T cells (CTL) reactive with the line 1 tumor in a CD4-dependent manner. It is of interest that we found the development of CTL in response to IL-3 was not accompanied by increased development of nonspecific killer cells. This was in contrast to IL-2, which enhanced development of both CTL and nonspecific cells. To determine if IL-3 could inhibit the development of nonspecific killers, we added IL-3 to cultures of fresh spleen cells stimulated with high levels of IL-2. In this assay, IL-3 showed very potent inhibitory activity against the generation of nonspecific killers. To determine if IL-3 could inhibit the IL-2-driven generation of non-specific killers in vivo, we injected a mixture of IL-2- and IL-3-producing line 1 cells. Each cytokine-producing transfectant was also injected alone. The cytotoxicity of tumor-infiltrating lymphocytes (TIL) from these tumors confirmed that the presence of IL-3 inhibits the generation of nonspecific killer cells in vivo.

Animals↗
PubMed1991

Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16.

Temperature-sensitive mutants defective in 60S ribosomal subunit protein L16 of Saccharomyces cerevisiae were isolated through hydroxylamine mutagenesis of the RPL16B gene and plasmid shuffling. Two heat-sensitive and two cold-sensitive isolates were characterized. The growth of the four mutants is inhibited at their restrictive temperatures. However, many of the cells remain viable if returned to their permissive temperatures. All of the mutants are deficient in 60S ribosomal subunits and therefore accumulate translational preinitiation complexes. Three of the mutants exhibit a shortage of mature 25S rRNA, and one accumulates rRNA precursors. The accumulation of rRNA precursors suggests that ribosome assembly may be slowed in this mutant. These phenotypes lead us to propose that mutants containing the rpl16b alleles are defective for 60S subunit assembly rather than function. In the mutant carrying the rpl16b-1 allele, ribosomes initiate translation at the noncanonical codon AUA, at least on the rpl16b-1 mRNA, bringing to light a possible connection between the rate and the fidelity of translation initiation.

Amino Acid Sequence↗