Enhancement of antitumor immunity against B16 melanoma tumor using genetically modified dendritic cells to produce cytokines

Dendritic cells (DC) that have been genetically modified to express cytokine genes may be novel tools for inducing antitumor immune responses. In the present study, the pMX retroviral vector was modified to express the mouse IL-2 (mIL-2pMX) and mouse IL-12 (mIL-12pMX) genes. Supernatants from 293 cells transfected with pMX retroviral vectors were harvested and used to transduce mouse lin- bone marrow (BM) progenitor cells. After 48 h co-culture with pseudotype retrovirus, BM cells were cultured for 12 days in the presence of mGM-CSF, mSCF and mTNF-alpha to obtain a DC-enriched fraction. Flow cytometric analysis showed that GFP protein expression in these cultures was 20-40% and that 40-50% of the cultured BM cells were positive for the DC marker, DEC205. About 60% of cells sorted for DEC205 also expressed GFP. The supernatants of DC-mIL-2 and DC-mIL-12 cultured for 48 h contained 5.2 +/- 0.15 and 33.9 +/- 2.6 ng cytokine protein per milliliter, respectively. Intratumoral injection of DC-mIL-2 or DC-mIL-12 on days 8 and 15 after the intradermal injection of 1 x 105 B16F10 cells, resulted in a significant reduction in tumor size by day 21, as compared with mice treated with unmodified DC or DC-GFP. Longer term analysis as assessed at day 42 revealed that B16 tumor-bearing mice treated with cytokine gene-modified DC survived significantly longer than mice from other groups. Spleen cells obtained from DC-treated mice were specifically sensitized for the generation of CTL by subsequent restimulation with gene-modified DC. These results suggested that DC genetically modified to express IL-2 or IL-12 can induce potent antitumor responses against well-established, poorly immunogenic B16F10 tumors. Gene Therapy (2000) 7, 2113-2121.     

Transforming growth factor beta selectively inhibits normal and leukemic human bone marrow cell growth in vitro

The effects of transforming growth factor beta 1 or beta 2 (TGF-beta 1 or -beta 2) on the in vitro proliferation and differentiation of normal and malignant human hematopoietic cells were studied. Both forms of TGF- beta suppressed both the normal cellular proliferation and colony formation induced by recombinant human interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). In the presence of GM-CSF or IL-3, optimal concentrations of TGF-beta (400 pmol/L) inhibited colony formation by erythroid (BFU-E), multipotential (CFU-GEMM), and granulocyte-macrophage (CFU-GM) progenitor cells by 90% to 100%, whereas granulocyte or monocyte cluster formation was not inhibited. In contrast, neither form of TGF-beta had any effect on G- CSF-induced hematopoiesis. The suppressive action appeared to be mediated directly by TGF-beta since antiproliferative responses were also observed in accessory cell-depleted bone marrow cells. In contrast to normal bone marrow cells, both GM- and G-CSF-induced proliferation of cells from patients with chronic myelogenous leukemia were suppressed in a dose-dependent manner by TGF-beta. Differential effects of TGF-beta on the proliferation of established leukemic lines were also observed since most cell lines of myelomonocytic nature studied were strongly inhibited where erythroid cell lines were either insensitive or poorly inhibited by TGF-beta. These results suggest that TGF-beta is an important modulator of human hematopoiesis that selectively regulates the growth of less mature hematopoietic cell populations with a high proliferative capacity as opposed to more differentiated cells, which are not affected by TGF-beta.     

Detection of human T-cell leukemia/lymphoma virus, type II, in a patient with large granular lymphocyte leukemia.

We studied a patient with large granular lymphocyte (LGL) leukemia for evidence of human T-cell leukemia/lymphoma virus (HTLV) infection. Serum from this patient was positive for HTLV-I/II antibodies by enzyme-linked immunosorbent assay (ELISA) and was confirmed positive in Western blot and radioimmunoprecipitation assays. Results of a synthetic peptide-based ELISA showed that the seropositivity was caused by HTLV-II and not HTLV-I infection. Analyses of enzymatic amplification of DNA from bone marrow sections using the polymerase chain reaction (PCR) were positive for HTLV-II specific gag, pol, env, and pX gene sequences. Cloning and sequencing of amplified products showed that the HTLV-II pol and pX sequences in patient DNA differed from the sequences of 17 other HTLV-II isolates examined in our laboratory. HTLV infection may have a role in some patients in the pathogenesis of LGL leukemia.     

The synthetic peptide WKYMVm attenuates the function of the chemokine receptors CCR5 and CXCR4 through activation of formyl peptide receptor-like 1

The G protein-coupled 7 transmembrane (STM) chemoattractant receptors can be inactivated by heterologous desensitization. Earlier work showed that formly peptide receptor-like 1 (FPRL1), an STM receptor with low affinity for the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalamine (fMLF), is activated by peptide domains derived from the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp120 and its activation results in desensitization and down-regulation of the chemokine receptors CCR5 and CXCR4 from monocyte surfaces. This study investigated the possibility of interfering with the function of CCR5 or CXCR4 as HIV-1 coreceptors by activating FPRL1. Cell lines were established expressing FPRL1 in combination with CD4/CXCR4 or CD4/CCR5 and the effect of a synthetic peptide, WKYMVm, a potent activator of formyl peptide receptors with preference for FPRL1 was determined. Both CXCR4 and CCR5 were desensitized by activation of the cells with WKYMVm via a staurosporine-sensitive pathway. This desensitization of CXCR4 and CCR5 also attenuated their capacity as the fusion cofactors for HIV-1 envelope glycoprotein and resulted in a significant inhibition of p24 production by cell lines infected with HIV-1 that use CCR5 or CXCR4 as coreceptors. Furthermore, WKYMVm inhibited the infection of human peripheral monocyte-derived macrophages and CD4(+) T lymphocytes by R5 or X4 strains of HIV-1, respectively. These results indicate that heterologous desensitization of CCR5 and CXCR4 by an FPRL1 agonist attenuates their major biologic functions and suggest an approach to the development of additional anti-HIV-1 agents. (Blood. 2001;97:2941-2947)     

Transient reversal of bone marrow aplasia associated with lymphocyte depleted Hodgkin's disease after combination chemotherapy

This report describes a patient with lymphocyte depleted Hodgkin's disease who presented with bone marrow aplasia. The aplastic marrow reverted to normal after initiation of MOPP chemotherapy; however, 4 months after completion of therapy, bone marrow aplasia recurred in the absence of recurrent Hodgkin's disease. The patient remains free of Hodgkin's disease 34 months after completion of chemotherapy. Bone marrow abnormalities in Hodgkin's disease are reviewed and the current understanding of the pathological mechanisms leading to aplastic anemia is discussed.     

Administration of recombinant human interleukin-7 alters the frequency and number of myeloid progenitor cells in the bone marrow and spleen of mice.

The administration of greater than or equal to 5 micrograms interleukin-7 (IL-7) twice a day to mice for 4 to 7 days increased by twofold to fivefold the total number of splenic and peripheral blood leukocytes, but did not appreciably increase bone marrow (BM) cellularity. This regimen of IL-7 administration also resulted in a greater than 90% reduction in the frequency and total number of single lineage colony-forming unit-culture (CFU-c) and multilineage CFU-granulocyte, erythroid, monocyte, megakaryocyte colonies that could be cultured from the BM, but a fivefold to 15-fold increase in the number of these progenitors that could be cultured from the spleen. All of these effects were reversible with progenitor and white blood cell numbers returning to near normal by day 6. Morphologic analysis of cells obtained from the BM of IL-7-treated mice showed an increase in lymphoid cells. Surface phenotype analysis showed that most of this IL-7-induced increase in lymphocytes was attributable to an increase in immature B cells (B220+, sIg-), while cells expressing the myelomonocytic markers 8C5 and MAC-1 decreased by twofold to threefold. Further studies showed that the administration of IL-7 to mice that had been rendered leukopenic by the injection of cyclophosphamide (Cy) or 5-fluorouracil (5FU) exhibited a more rapid recovery and/or overshoot in their peripheral blood lymphocytes when compared with mice treated with Cy or 5FU alone. These results show that IL-7 can differentially regulate myelopoiesis in the BM and spleen, while stimulating lymphopoiesis.     

Differential effect of transforming growth factor-{beta}1 on the activation of human naive and memory CD4+ T lymphocytes

Transforming growth factor-ß1 (TGF-ß1) canhave stimulatory or inhibitory effects on cell growth. For severalcell types, the effect of TGF-ß1 was found to correlatewith the differentiation stage of the cells and the presenceof other cytoklnes. We have studied here the influence of TGF-ß1on CD4⁺ T cell activation in relation to the differentiationstage of the cells by evaluating the effect of TGF-ß1on the prollferatlve responses of purified CD4⁺CD45RA⁺ (unprfmed)and CD4⁺CD45RO⁺ (primed) lymphocytes. Under certain conditions,TGF-ß1 exerted a co-stlmulatory effect on peripheralblood CD4⁺CD45RA⁺ T cells whereas the outgrowth of CD4⁺CD45RO⁺T cells was suppressed in any activation system tested. Theenhancement of prollferatlve responses by TGF-ß1 inTCR/CD3 or CD2 stimulated cultures of CD45RA⁺ cells involvedup-regulatlon of CD25 expression and was dependent on the presenceof exogenous IL-2 or CD28 mAbs; IL-7 driven proliferatlve responseswere suppressed by TGF-ß1. These observations wereconfirmed in experiments with purified cord blood (CB) CD4⁺T cells inasmuch as addition of TGF-ß1 caused a 2-to 7-fold increase in IL-2 driven proliferatlve responses ofthese cells. Finally we show that, in contrast to the effectof TGF-ß1 during primary stimulation of CB CD4⁺ Tcells, TGF-ß1 suppressed T cell proliferation for40% in secondary cultures of these cell. Our findings indicatethat TGF-ß1 Is a blfunctlonal regulator of CD4⁺ Tcell growth in vitro, with co-stimulatory capacities duringCD45RA⁺ T cell mediated primary responses and growth suppresslveeffects during secondary responses of CD45RO⁺ T cells.     

Induction of the autonomous stage of transformation in erythroid cells infected with SFFV: helper virus is not required

The erythroleukemia induced by the Friend spleen focus-forming virus (SFFV) in mice exemplifies a multistep oncogenic process. Its sequential steps include a rapid polyclonal hyperplastic stage and a more slowly developing malignant stage characterized by autonomous erythroid cells. We report here that the helper virus normally present in mice infected by SFFV is not required for development of the second stage of transformation. In this study, mice were infected with a polycythemia-inducing variant of SFFV which was prepared as a helper-free stock (L. Wolff and S. Ruscetti, 1985, Science 228, 1549). Highly malignant cells could be detected in helper-free SFFV-infected mice by their transplantability into the omentum of sublethally irradiated mice, and erythroleukemia cell lines, typical of previously isolated Friend murine erythroleukemia cell lines, could be established from diseased spleens. Like their helper virus-containing counterparts, the lines established with helper-free SFFV are inducible for hemoglobin synthesis with a variety of chemicals, but not erythropoietin, and express p53, a marker of malignant transformation. Although the cells expressed SFFV encoded proteins, none expressed gene products of replication competent murine leukemia viruses.