摘要:

The immunosuppressive activity of tumor cells may be mediated by tumor-derived cytokines such as transforming growth factor-β (TGF-β) and interleukin-10 (EL-10). A human breast cancer celt line derived from malignant ascites (BRC 173) secreted TGF-β, but not IL-10, into tissue culture supernatant (TCS). BRC 173 TCS suppressed natural killer (NK) and lymphokine-activated killer (LAK) cell activity and also blocked the generation of HLA-A*0201-restricted tumor-reactive cytotoxic T-lymphocyte (CTL) lines in vitro. Human α2-macroglobulin (α2M), a plasma protein and cytokine carrier that binds isoforms in the TGF-β family, was tested for its ability to neutralize the immunosuppressive activity in BRC 173 TCS. α2M was converted to its activated conformation by reaction with methylamine (α2M-MA) and then incubated with normal human peripheral blood lymphocytes (PBL) in the presence of IL-2 and BRC 173 TCS. Lysis of NK targets (K562) and LAK cell targets (DM6 melanoma) by the PBL was examined after 6 days of culture. PBL cultured in IL-2, without TCS or α2M-MA, were lytic for both target cells. BRC 173 TCS substantially suppressed the lytic activity of the PBL in the presence of IL-2. When TGF-β-neutralizing antibody was added to the PBL culture medium with IL-2 and TCS, a majority of the lytic activity was restored. α2M-MA (280 nM) neutralized almost all of the immunosuppressive activity in the TCS, restoring 80–100% of the lytic activity without any apparent effect on the activity of IL-2. The ability of α2M-MA to counteract immunosuppressive cytokines in breast cancer TCS was evident in serum-containing and serum-free medium. These studies demonstrate that activated α2M can function as a selective cytokine neutralizer to thereby promote the activation of NK, LAK, and tumor-specific CTL responses.

ISSN:1524-9557    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Crosslinking of CD28 receptors on resting T lymphocytes by B7 cost-imulatory molecules expressed by antigen-presenting cells (APCs) plays a critical role in T-cell activation. Human melanomas express major histocompatibility complex (MHC)-restricted tumor-associated antigens that can be recognized by cytotoxic T lymphocytes (CTL), yet they remain poorly immunogenic. One mechanism for the failure of T-cell response is the lack of expression of costimulatory molecules by human melanoma cells. We have transfected the B7–1 gene into three HLA-A2-expressing human melanoma cell lines, and studied their capacity to stimulate primary human T cells. B7-expressing melanoma cells were excellent inducers of T-cell proliferation, cytokine production, and cytolytic activity in allogeneic mixed lymphocyte cultures through a process dependent on the function of the T-cell receptor as well as interactions between B7:CD28, CD2:LFA-3, and LFA-1:ICAM-1. Subset analysis demonstrated that CD4*T cells or addition of exogenous interleukin-2 was required for the induction of CD8+CTL. Untransfected parental melanoma cells were inert as APCs in these cultures. Rotating stimulation of T cells with the three B7-expressing cell lines led to the generation of T-cell lines that were cytolytic for HLA-A2 melanoma cells and other HLA-A2+targets that were pulsed with HLA-A2-restricted MART-1 peptides: These data demonstrate that expression of B7–1 by human mela-noma cells converts them into effective APCs for the in vitro induction of MHC-restricted, melanoma-specific CTL.

ISSN:1524-9557    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

We have previously described the induction of murinc CD8+major his-tocompatibility complex class I restricted cytotoxic T cells to the 20 amino acid repeat region of human Mucinl (MUC1) variable number of tandem repeats regiona mucin greatly increased in expression in breast cancer and proposed as a target for immu-notherapy. Mannan-MUCl immunization protocol induces a high cytotoxic T lymphocyte (CTL) frequency, and some protection of mice against a tumor challenge. The CTL frequency can be substantially increased using cyclophosphamide (Cy), from 1/84,900 without Cy to 1/8,100 with Cy. Furthermore, in the presence of Cy, established tumors are rapidly eradicated, which does not happen in its absence. Cy clearly gives a major increase in the frequency of CTL precursors (CTLp) to MUC1 and could be of therapeutic value in patients.

ISSN:1524-9557    

出版商:OVID    

年代:1998

数据来源: OVID

ISSN:1524-9557    

出版商:OVID    

年代:1998

数据来源: OVID

ISSN:1524-9557    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

We have taken several approaches to investigate the capacity of the secretory pathway to liberate major histocompatibility complex (MHC) class I-restricted antigenic peptides from precursor porypeptides. Cells lacking the peptide transporter (TAP) are unable to deliver peptides from cytosolic antigens to class I molecules. TAP can be bypassed by targeting peptides directly to the endoplasmic reticulum (ER) using NH2-terminal signal sequences. This results in the generation of enormous numbers of MHC class I complexes (50,000 peptides/cell), and recombinant vaccinia viruses expressing such peptides are highly immunogenic. In contrast to signal sequence-targeted peptides, peptides are liberated very inefficiently from internal locations in ER-targeted full-length proteins, indicating that the secretory pathway has a limited capacity for generating antigenic peptides from most polypeptide contexts. We have, however, identified a location in proteins from which peptides can be liberated in numerous contexts in the secretory pathway. Placing a number of different peptides at the COOH termini of a secreted protein and two proteins with type II membrane anchors resulted in their TAP-independent presentation. These findings demonstrate that the secretory compartment possesses proteases able to liberate COOH-terminal antigenic peptides from virtually any context, entirely consistent with a role for these proteases in the processing of TAP-transported antigenic peptide precursors.

ISSN:1524-9557    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Lymphocytes and dendritic cells (DCs) are critical for immune responses, yet how they develop from pluripotenl hematopoietic stem cells is poorly defined. In humans and mice, it is possible to isolate phenotypically defined subsets of bone marrow (BM) cells that represent intermediate progenitors without long-term repopu-lating characteristics but with specific lineage differentiation properties. For instance, murine BM CD34+CD45RA+cells are progenitors for B and T lymphocytes with no in vivo repppulation activity. In human BM, a small subset (5%) of cells having the phenotype CD34+Lin-CD10+CD45RA+CD38+Thy-1-c-kit represents a new class of hematopoietic progenitor cells that gives rise to lymphocytes [T, B, and natural killer (NK) cells] and to DCs but does not produce myeloid or erythroid cells. The identification of such progenitor cells provides the opportunity to define the differentiation and growth requirements for the production of lymphocytes and DCs. Genes involved in lineage specification can also be studied. Altogether, these studies have fundamental implications for understanding the biology of pivotal lineages of immune cells. This understanding could be used to treat a variety of immunodeficiencies and to design novel immunotherapies particularly in the context of hematopoietic cell transplantation.

ISSN:1524-9557    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Dendritic cells differentiated in vitro from blood and other sources using cytokines hold particular promise as immunothcrapeulic agents in cancer. However, there are currently no data to show that human in vitro-derived dendritic cells are immunogenic in vivo. We have developed a primate model of immunotherapy using dendritic cells differentiated in vitro from blood monocytes by culturing with human granulocyte/ macrophage colony-stimulating factor and interieukin-4. We measured the immune response to antigen elicited by in vitro-derived and antigen-treated dendritic cells following a single intravenous inoculation and boost in chimpanzees. The antigens tested were ovalbumin, a complex foreign protein, and a peptide derived from the MUC-1 mucin tumor antigen, a relatively uncomplex self antigen. Four chimpanzees were immunized either with antigen-pulsed dendritic cells (two animals) or mock-treated dendritic cells (one animal) given intravenously or both antigens given in adjuvant subcutaneously (one animal). Each animal received a boost of both antigens in adjuvant 10 days later. All animals responded with an IgG-mediated humoral response to ovalbumin measured in the serum at day 24. This was associated with a proliferative cellular response to ovalbumin in the inguinal lymph node draining the boost injection. In contrast, antibody responses to mucin peptide were detected in one animal in response to the boost injection, and no T cell proliferative responses to mucin peptide were detected in the draining lymph node of any animal. To determine if the single inoculation of antigen-pulsed dendritic cells elicited any immunity, we measured the T cell response to ovalbumin in blood monoouclear cells harvested prior to the boost Ovalbumin-specific proliferative responses that were antigen dose dependent were detected in one of two treated animals. In contrast, ovalbumin given with adjuvant and mock-treated dendritic cells induced no response. The three animals inoculated with dendritic cells, either antigen or mock treated, had moderate T cell responses to bovine serum albumin, a constituent of the medium used to culture cells prior to injection. We conclude from these data that in vitro-derived dendritic cells can elicit T cell responses to a complex foreign antigen following a single intravenous injection in a large primate, ft is likely that immunity to a simple self antigen, MUC-1 mucin peptide, may require multiple inoculations. The results support the use of dendritic cells differentiated in vitro as vehicles for immunotherapy in humans.

ISSN:1524-9557    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

The recent identification of tumor-associated antigens and tumor-associated antigen-derived peptide epitopes recognized by cytolytic T lymphocytes (CTLs) in the context of major hislocompatibility complex (MHC) class I molecules has prompted the development of peptide-based vaccines for the treatment of human cancers, particularly melanoma. The design of such clinical protocols requires an understanding of the inherent immunogenicity of the peptide(s) and a choice of a facilitating adjuvant promoting cellular immunity against these peptidcs. We have evaluated the abilities of a series of defined synthetic peptide epitopes derived from MART-1/Melan-A, gp100, tyrosinase, and MAGE-3 or unfractionatcd peptides natu-rally presented by melanoma MHC molecules to elicit HLA-A2-restricted and melanoma-reactive CTLs from the peripheral blood of normal donors or patients with metastatic melanoma. Autologous peripheral blood dendritic cells (DCs), which were easily generated from all donors when cultured in the presence of recombinant human interleukin-4 and recombinant human granulocyte-macrophage colony-stimulating factor were pulsed with melanoma peptides and used to “prime” and/or “boost” CTL cultures in vitro. Our results suggest that antimelanoma CTLs may be reproducibly generated in short-term in vitro cultures in this manner using either a subset of the defined synthetic peptides (MART-1/Melan-A27–35, MART-1/Melan-A32–40, gp100280–288, tyrosinase368–376and MAGE-3271–279) or unfractionated peptides (containing both idiotypic and shared melanoma epitopes) derived from freshly isolated autologous melanoma lesions. These in vitro data support the use of autologous DCs prepulsed with such peptides as an appropriate antigen adjuvant delivery system in melanoma peptide-based vaccines.

ISSN:1524-9557    

出版商:OVID    

年代:1998

数据来源: OVID