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Biomedical subjects

M T Lotze

Publications and source records attributed to M T Lotze.

At least 19 recordsLinked to original sources
PubMed1996

Suppression of prostaglandin H synthase-2 induction in human monocytes by in vitro or in vivo administration of interleukin 4.

IL-4 is a potent modulator of monocyte function. Our previous studies demonstrated that the suppression of monocyte matrix metalloproteinase production by IL-4 is a result of its inhibition of PGE2 synthesis, which was attributed to an effect on prostaglandin synthase. Here we report on the in vitro and in vivo effects of IL-4 on monocyte prostaglandin H synthase-2 (PGHS-2) and its regulation by second messengers. Stimulation of monocytes with either LPS or Con A resulted in the induction of PGHS-2 which was significantly inhibited by IL-4. Inhibition of PGHS-2 mRNA and protein was detected at 0.05 to 0.1 ng/ml of IL-4 with substantial suppression at 10 to 20 ng/ml. If added later than 2 hr after LPS, IL-4 failed to suppress PGHS-2, indicating that IL-4 acted early in the signaling cascade. Moreover, the ability of exogenously added PGE2 or Bt2cAMP to restore PGHS-2 production in IL-4-treated monocytes further suggested early disruption of the pathway. The early event inhibited by IL-4 did not involve suppression of phospholipase activity, because LPS-induced arachidonic acid release was relatively unaffected by IL-4. Unlike PGHS-2, PGHS-1, the constitutively expressed PGHS, was not modulated by IL-4. Thus, IL-4 appears to selectively block PGHS-2 synthesis, thereby blocking subsequent steps in the pathway leading to the production of matrix metalloproteinases. In an extension of these findings, we examined peripheral blood monocytes from cancer patients undergoing IL-4 therapy. In these cells the induction of PGHS-2 expression by LPS was significantly reduced compared to that of monocytes obtained prior to IL-4 therapy. Although perhaps not relevant as an antitumor mechanism, these findings have important implications in defining the potent anti-inflammatory activities of IL-4 in vitro and in vivo.

Amino Acid Sequence↗
PubMed1996

Murine models of cancer cytokine gene therapy using interleukin-12.

Cytokine gene therapy, in particular IL-12 gene therapy, is one of the more novel and promising approaches in cancer therapy based on significant preclinical data derived mainly from murine tumor models. IL-12 is a heterodimeric cytokine that requires the simultaneous expression of both the p35 and p40 chains from the same cell for production of biologically active IL-12. A variety of biological functions have been attributed to IL-12 including the induction of IFN-gamma production and the promotion of predominantly Th1-type immune responses to antigens. Our previous studies using systemic administration of recombinant murine IL-12 have demonstrated profound antitumor efficacy against all tumors tested with the concomitant long-lived specific antitumor immunity in some cases. To determine whether the local secretion of IL-12 achieved by gene transduction has significant antitumor effects, fibroblast cell lines or murine tumor cell lines were transduced with expression plasmids or the retroviral vector TFG-mIL-12-Neo and inoculated intradermally (i.d.). Our first study using IL-12-transfected NIH3T3 cells admixed with the murine melanoma, BL-6, showed that local IL-12 expression suppresses tumor growth and promotes the acquisition of specific antitumor immunity. Subsequent studies showed that IL-12 gene therapy is also effective in treating established day 3 tumors. CD4+ and CD8+ T cells, as well as NK cells, appear to play important roles in the observed antitumor effects resulting from IL-12 paracrine secretion. Administration of neutralizing antibody specific for IFN-gamma also abrogated some of the IL-12-associated antitumor effects. Finally, this IL-12 gene therapy strategy to elicit an antitumor immune response was more effective when used in combination with the transduction of tumor cells with B7.1. Based on these promising results, a clinical protocol for the treating patients with cancer using genetically engineered fibroblasts to express IL-12 has been initiated at our institution.

Animals↗
PubMed1996

IL-12-engineered dendritic cells serve as effective tumor vaccine adjuvants in vivo.

The recent identification of tumor-associated antigens (TAA) and TAA-derived peptides presented by MHC molecules to T cells has provided the tools to design and test clinical vaccines for treating human malignancies, such as melanoma. While the most effective adjuvant for use in vaccine formulation remains unclear, autologous dendritic cells (DC) appear to be good candidate adjuvants. We have previously shown that syngeneic bone marrow-derived DC when pulsed ex vivo with relevant TAA-derived peptides can effectively vaccinate mice against a subsequent challenge with tumor or can effectively treat animals bearing established tumors. In this report, we have engineered murine interleukin-12 (mIL-12), a potent stimulator of cell-mediated immunity, into murine DC using retroviral-mediated or plasmid-based transfection procedures. Transfectants produced up to 25 ng rIL-12/10(6) cells/48 hours. These engineered cells are capable of promoting enhanced anti-tumor, antigen-specific CTL responses compared to nontransduced DC.

Adjuvants, Immunologic↗
PubMed1996

Tumor escape from immune recognition: lethal recurrent melanoma in a patient associated with downregulation of the peptide transporter protein TAP-1 and loss of expression of the immunodominant MART-1/Melan-A antigen.

In the last few years, mutiple protein target antigens for immunorecognition by T cells have been identified on human melanoma. How melanoma lesions escape from functional antigen-specific immune recognition remains poorly understood. We have identified the concomitant loss of the immunodominant T cell-defined MART-1/Melan-A antigen and downregulation of the TAP-1 gene in a recurrent metastatic melanoma that was resected in 1993. This phenotype was not observed for an earlier autologous melanoma lesion resected in 1987. The "antigen loss" could be restored in the variant tumor cell line by simultaneously providing both the MART-1/Melan-A gene (by retroviral transfer) and the TAP-1 gene (by a bioballistic approach) resulting in tumor cell sensitivity to MART-1/Melan-A-specific cytotoxic T lymphocytes. This suggests that tumor escape from immune surveillance may have occurred in vivo as a sequential result of (a) antigen loss, and (b) downregulation of the peptide-transporter protein TAP-1 expression by this patient's tumor over a 6-yr period from 1987 to 1993. These results suggest that the characterization of the T cell response to melanoma in individual patients and definition of the immunologically relevant genetic defects in tumors may be required to select the most effective therapeutic strategies for a given patient.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Systemic administration of cellular IL-10 induces an effective, specific, and long-lived immune response against established tumors in mice.

Cellular IL-10 (cIL-10), the collective term for human and murine IL-10, has both stimulatory and inhibitory effects on diverse cell types, including costimulation of T cell proliferation, chemoattraction of CD8+ T cells, and stimulation of lymphokine-activated killer cell activity. Human IL-10 (hIL-10) differs from its EBV homolog viral IL-10 (vIL-10) by only 16% at the amino acid level; however, vIL-10 shares with cIL-10 predominantly inhibitory effects, such as macrophage deactivation. We administered cIL-10 systemically to mice bearing established (day 7) sarcomas, melanomas, or colorectal carcinomas. At high doses (20 to 60 micrograms/day x 7 days), cIL-10 induced rejection of tumors, delaying tumor outgrowth or resulting in complete cure. Sublethal irradiation (500 rad) of mice prior to tumor inoculation abrogated the IL-10 effect. Cured mice were immune to subsequent rechallenge with 10-fold higher inoculation with the same, but not a different, tumor. IL-12 also has potent antitumor activity and interacts with IL-10 in both complementary and antagonistic ways; co-administration of both cytokines resulted in additive antitumor activity. To compare cIL-10 vs vIL-10 effects in vivo, we engineered CL8-1 melanoma transfectants bearing the vIL-10 or the murine IL-10 (mIL-10) gene. Local secretion of mIL-10 induced rejection of tumors, while vIL-10 resulted in accelerated outgrowth. Subsequent systemic administration of cIL-10 to mice bearing vIL-10-transduced tumors completely reversed the local suppressive effects, leading to rejection, suggesting distinct pathways for cIL-10 and vIL-10 effects. That cIL-10 can stimulate the acquisition of an effective, specific, and long-lived antitumor immune response in murine models and can reverse the local immunosuppressive effects of vIL-10 indicates a potential role for cIL-10 administration in the biologic therapy of cancer and suggests a broader interpretation of IL-10 biology.

Adenocarcinoma↗
PubMed1996

Therapy of murine tumors with p53 wild-type and mutant sequence peptide-based vaccines.

The BALB/c Meth A sarcoma carries a p53 missense mutation at codon 234, which occurs in a peptide, termed 234CM, capable of being presented to cytotoxic T lymphocytes (CTL) by H-2Kd molecules (Noguchi, Y., E.C. Richards, Y.-T. Chen, and L.J. Old. 1994. Proc. Natl. Acad. Sci. USA. 91:3171-3175). Immunization of BALB/c mice with bone marrow-derived dendritic cells (DC), generated in the presence of granulocyte macrophage colony-stimulating factor and interleukin 4, and prepulsed with the Meth A p53 mutant peptide, induced CTL that specifically recognized peptide-pulsed P815 cells, as well as Meth A cells naturally expressing this epitope. Immunization with this vaccine also protected naive mice from a subsequent tumor challenge, and it inhibited tumor growth in mice bearing day 7 subcutaneous Meth A tumors. We additionally determined that immunization of BALB/c mice with DC pulsed with the p53 peptide containing the wild-type residue at position 234, 234CW, induced peptide-specific CTL that reacted against several methylcholanthrene-induced BALB/c sarcomas, including CMS4 sarcoma, and rejection of CMS4 sarcoma in vaccination and therapy (day 7) protocols. These results support the efficacy of DC-based, p53-derived peptide vaccines for the immunotherapy of cancer. The translational potential of this strategy is enhanced by previous reports showing that DC can readily be generated from human peripheral blood lymphocytes.

Amino Acid Sequence↗
PubMed1996

Human intestinal Vdelta1+ lymphocytes recognize tumor cells of epithelial origin.

gammadelta T cells can be grouped into discrete subsets based upon their expression of T cell receptor (TCR) variable (V) region families, their tissue distribution, and their specificity. Vdelta2+ T cells constitute the majority of gammadelta T cells in peripheral blood whereas Vdelta1+T cells reside preferentially in skin epithelium and in the intestine. gammadelta T cells are envisioned as first line host defense mechanisms capable of providing a source of immune effector T cells and immunomodulating cytokines such as interleukin (IL) 4 or interferon (IFN) gamma. We describe here the fine specificity of three distinct gammadelta+ tumor-infiltrating lymphocytes (TIL) obtained from patients with primary or metastatic colorectal cancer, that could be readily expanded in vitro in the presence of IL-1beta and IL-7. Irrespective of donor, these individual gammadelta T cells exhibited a similar pattern of reactivity defined by recognition of autologous and allogeneic colorectal cancer cells, renal cell cancer, pancreatic cancer, and a freshly isolated explant from human intestine as measured by cytolytic T cell responses and by IFN-gamma release. In contrast, tumors of alternate histologies were not lysed, including lung cancer, squamous cell cancer, as well as the natural/lymphocyte-activated killer cell-sensitive hematopoietic cell lines T2, C1R, or Daudi. The cell line K562 was only poorly lysed when compared with colorectal cancer targets. Target cell reactivity mediated by Vdelta1+ T cells was partially blocked with Abs directed against the TCR, the beta2 or beta7 integrin chains, or fibronectin receptor. Marker analysis using flow cytometry revealed that all three gammadelta T cell lines exhibit a similar phenotype. Analysis of the gammadelta TCR junctional suggested exclusive usage of the Vdelta1/Ddelta3/Jdelta1 TCR segments with extensive (< or = 29 bp) N/P region diversity. T cell recognition of target cells did not appear to be a major histocompatibility complex restricted or to be correlated with target cell expression of heat-shock proteins. Based on the ability of some epithelial tumors, including colorectal, pancreatic, and renal cell cancers to effectively cold target inhibit the lysis of colorectal cancer cell lines by these Vdelta1+ T cell lines, we suggest that intestinal Vdelta1+ T cell lines, we suggest that intestinal Vdelta1+ T cells are capable of recognizing cell surface Ag(s) shared by tumors of epithelial origin.

Amino Acid Sequence↗
PubMed1996

Retrovirus-mediated transfer of viral IL-10 gene prolongs murine cardiac allograft survival.

A murine heterotopic, nonvascularized cardiac allograft model was used to examine the effects of the immunosuppressive cytokine, viral IL-10 (vIL-10), delivered by gene transfer on graft rejection. Retroviral-mediated gene transfer and expression of vIL-10 significantly prolonged allograft survival, without conventional systemic immunosuppression, from 12.1 +/- 0.8 days to 39.4 +/- 2.5 days (p < 0.0001). The effect was specific, dose dependent, and restricted to the site of transplantation. PCR analysis demonstrated specific expression of the transferred gene within the allograft. Analysis of the cellular infiltrate in the allografts showed a reduction in T cells and alloantigen-specific cytotoxic T cells and IL-2 producing helper T cells. Thus, the transient local expression of a gene encoding an immunosuppressive protein within a graft can generate local immunosuppression, making gene therapy a viable approach for facilitating transplantation.

Animals↗
PubMed1996

Successful culture and selection of cytokine gene-modified human dermal fibroblasts for the biologic therapy of patients with cancer.

Human autologous dermal fibroblasts have been cultured, transduced with the interleukin-4 (IL-4) gene and used as a vaccine together with irradiated autologous tumor cells in patients with cancer participating in a phase I/II clinical trial at the University of Pittsburgh Cancer Institute. In support of this clinical trial, methods have been devised to facilitate isolation of fibroblasts from freshly harvested skin specimens, to enhance their outgrowth in large-scale cultures, and to assay cytokine (IL-4) production following transduction with the cytokine gene +/- irradiation. Fibroblasts were isolated from skin specimens by enzymatic digestion, grown in primary cultures, and transduced with a retroviral vector containing the gene for human IL-4 and the NeoR gene as a selectable marker. Following selection in G418, the irradiated, IL-4-producing fibroblasts were administered to patients in a vaccine containing irradiated autologous tumor cells. Seventy-eight specimens of human skin were processed to obtain fibroblast suspensions. Cultures of fibroblasts were established from 68 of the 78 specimens (87%). Of 33 transduced and selected fibroblast cultures, 21 produced at least 1,000 units of IL-4/24 hours per 10(6) cells, as determined by ELISA, and 17/33 or 51% were used for therapy. The primary cultures were typically maintained for up to seven or eight passages. The mean +/- SD overall time for obtaining a required number of transduced, selected cells was 53 +/- 4 days. The fibroblasts continued to produce IL-4 in culture for 3 weeks even after irradiation. Similar results have been obtained with a retroviral vector encoding IL-12. This study shows that human dermal fibroblasts can be consistently and reproducibly expanded and genetically modified to serve as a source of cytokines or other gene products for gene therapy trials.

Adult↗
PubMed1996

Peptide-pulsed dendritic cells induce antigen-specific CTL-mediated protective tumor immunity.

Cytotoxic T lymphocytes (CTLs) are a critical component of the immune response to tumors. Tumor-derived peptide antigens targeted by CTLs are being defined for several human tumors and are potential immunogens for the induction of specific antitumor immunity. Dendritic cells (DC) are potent antigen-presenting cells (APCs) capable of priming CTL responses in vivo. Here we show that major histocompatibility complex class I-presented peptide antigen pulsed onto dendritic APCs induces protective immunity to lethal challenge by a tumor transfected with the antigen gene. The immunity is antigen specific, requiring expression of the antigen gene by the tumor target, and is eliminated by in vivo depletion of CD8+ T cells. Furthermore, mice that have rejected the transfected tumor are protected from subsequent challenge with the untransfected parent tumor. These results suggest that immunization strategies using antigen-pulsed DC may be useful for inducing tumor-specific immune responses.

Amino Acid Sequence↗
PubMed1996

Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines.

Antigen presentation by host dendritic cells (DC) is critical for the initiation of adaptive immune responses. We have previously demonstrated in immunogenic murine tumor models that bone marrow (BM)-derived DC pulsed ex vivo with synthetic tumor-associated peptides, naturally expressed by tumor cells, serve as effective antitumor vaccines, protecting animals against an otherwise lethal tumor challenge (Mayordomo, J.I., T. Zorina, W.J. Storkus, C. Celluzzi, L.D. Falo, C.J. Melief, T. Ildstad, W.M. Kast, A.B. DeLeo, and M.T. Lotze. 1995. Nature Med. 1:1297-1302). However, T cell-defined epitopes have not been identified for most human cancers. To explore the utility of this approach in the treatment of tumors expressing as yet uncharacterized epitopes, syngeneic granulocyte/macrophage colony-stimulating factor-stimulated and BM-derived DC, pulsed with unfractionated acid-eluted tumor peptides (Storkus, W.J., H.J. Zeh III, R.D. Salter, and M.T. Lotze. 1993. J. Immunother. 14:94-103) were used to treat mice bearing spontaneous, established tumors. The adoptive transfer of 5 x 10(5) tumor peptide-pulsed DC dramatically suppressed the growth of weakly immunogenic tumors in day 4 to day 8 established MCA205 (H-2b) and TS/A (H-2d) tumor models, when applied in three biweekly intravenous injections. Using the immunogenic C3 (H-2b) tumor model in B6 mice, tumor peptide-pulsed DC therapy resulted in the erradication of established d14 tumors and long-term survival in 100% of treated animals. The DC-driven antitumor immune response was primarily cell mediated since the transfer of spleen cells, but not sera, from immunized mice efficiently protected sublethally irradiated naive mice against a subsequent tumor challenge. Furthermore, depletion of either CD4+ or CD8+ T cells from tumor-bearing mice before therapy totally suppressed the therapeutic efficacy of DC pulsed with tumor-derived peptides. Costimulation of the host cell-mediated antitumor immunity was critical since inoculation of the chimeric fusion protein CTLA4-Ig virtually abrogated the therapeutic effects of peptide-pulsed DC in vivo. The analysis of the cytokine pattern in the draining lymph nodes and spleens of tumor-bearing mice immunized with DC pulsed with tumor-eluted peptides revealed a marked upregulation of interleukin (IL) 4 and interferon (IFN) gamma production, as compared with mice immunized with DC alone or DC pulsed with irrelevant peptides. DC-induced antitumor effects were completely blocked by coadministration of neutralizing monoclonal antibody directed against T helper cell 1-associated cytokines (such as IL-12, tumor necrosis factor alpha, IFN-gamma), and eventually, but not initially, blocked by anti-mIL-4 mAb. Based on these results, we believe that DC pulsed with acid-eluted peptides derived from autologous tumors represents a novel approach to the treatment of established, weakly immunogenic tumors, and serves as a basis for designing clinical trials in cancer patients.

Animals↗
PubMed1996

Interleukin-12 and B7.1 co-stimulation cooperate in the induction of effective antitumor immunity and therapy of established tumors.

Interleukin-12 (IL-12) promotes specific and long-lasting anti-tumor immunity mediated by T cells in a variety of murine tumor models. IL-12 also synergizes with B7.1 (CD80) co-stimulation to induce proliferation and cytokine production by both human and murine T cells in vitro. We evaluated the combined anti-tumor efficacy of IL-12 and B7.1 gene delivery in two apparently poorly immunogenic tumor models (TS/A and MCA207). In both of these models, expression of B7.1 and production of IL-12 in the inoculum led to improved anti-tumor immunity, with up to 80% long-term tumor-free animals (vs 0-20% of mice remaining tumor free when inoculated with either B7.1- or IL-12-transfected tumors alone). Tumor-free mice were capable of rejecting a subsequent rechallenge with the wild-type tumor in 66% of the cases. Cooperativity was dependent upon the level of IL-12 secreted by engineered cells. IL-12 delivery required B7 expression of therapeutic effects to be observed in these models. Vaccines provided at a site distal to a control, non-transfected tumor slowed (TS/A) or abrogated (MCA207) the progression of wild-type tumors. The synergistic anti-tumor effects associated with combined application of B7.1- and IL-12-transfected tumors were partially negated by systemic administration of the CD28-B7.1/B7.2 antagonist CTLA4-Ig or by inoculation with neutralizing antibodies directed against murine interferon-gamma or tumor necrosis factor-alpha, two cytokines elicited in response to IL-12 stimulation. These data support the potential clinical utility of combined gene therapy using IL-12- and B7.1-engineered autologous cells (tumor or fibroblasts) as a vaccine to elicit specific anti-tumor immunity.

Abatacept↗
PubMed1996

Amino acid substitutions at position 97 in HLA-A2 segregate cytolysis from cytokine release in MART-1/Melan-A peptide AAGIGILTV-specific cytotoxic T lymphocytes.

CD8+ T lymphocytes recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. Individual peptide termini appear to be fixed at the C- and N-terminal ends. In contrast, central peptide side chains residues may point in different directions and exhibit limited flexibility, dependent on the MHC class I structural variation. For instance, position 97 in HLA-A201 has been shown to shift individual peptide species into different coordinations, one oriented towards the peptide N terminus, or more towards the C-terminal end. The conformational shape of such non-anchor peptide residues may affect the affinity of MHC/peptide/TCR interaction, resulting in quantitative, or qualitative different T cell effector functions. To characterize the impact of different amino acid residues occupying position 97 in HLA-A2 on peptide binding and presentation to CTL, we generated a panel of mutated HLA-A2 molecules containing either M, K, T, V, G, Q, W, P or H at position 97. The HLA-A0201 presented melanoma-associated MART-1/Melan-A derived peptide AAGIGILTV was employed to assess the impact of such position-97 mutations on HLA-A2 in peptide binding measured in an HLA-A2 reconstitution assay and presentation to AAGIGILTV-specific polyclonal or clonal T lymphocytes as measured by cytotoxicity, or interferon (IFN)-gamma and granulocyte/ macrophage colony-stimulating factor (GM-CSF) secretion. The high-affinity AAGIGILTV peptide bound to all position-97 mutants, albeit with differential efficiencies, and elicited specific release of IFN-gamma and GM-CSF by CTL. CTL responses were triggered only by the HLA-A2 wild type, by HLA-A2-H97 (histidine position 97 mutant), and HLA-A2-W97. The HLA-A2-M97 presenting molecule elicited enhanced cytokine release and CTL effector functions by polyclonal and by clonal effector T cells. These results indicate that MHC class I-bound peptides can trigger specific cytokine release by effector T cells independently of their ability to induce cytolysis. We conclude that relatively minor changes in the MHC class I peptide binding groove, including substitutions at position 97, can affect recognition by antigen-specific T cells. Mutant MHC class I molecules, such as those described here, may act as partial peptide antagonists and could be useful for inducing T lymphocytes with qualitatively different effector functions.

Amino Acid Sequence↗