Showing papers in "Journal of Experimental Medicine in 1996"

B lymphocytes secrete antigen-presenting vesicles.

Abstract: Antigen-presenting cells contain a specialized late endocytic compartment, MIIC (major histocompatibility complex [MHC] class II-enriched compartment), that harbors newly synthesized MHC class II molecules in transit to the plasma membrane. MIICs have a limiting membrane enclosing characteristic internal membrane vesicles. Both the limiting membrane and the internal vesicles contain MHC class II. In this study on B lymphoblastoid cells, we demonstrate by immunoelectron microscopy that the limiting membrane of MIICs can fuse directly with the plasma membrane, resulting in release from the cells of internal MHC class II-containing vesicles. These secreted vesicles, named exosomes, were isolated from the cell culture media by differential centrifugation followed by flotation on sucrose density gradients. The overall surface protein composition of exosomes differed significantly from that of the plasma membrane. Exosome-bound MHC class II was in a compact, peptide-bound conformation. Metabolically labeled MHC class II was released into the extracellular medium with relatively slow kinetics, 10 +/- 4% in 24 h, indicating that direct fusion of MIICs with the plasma membrane is not the major pathway by which MHC class II reaches the plasma membrane. Exosomes derived from both human and murine B lymphocytes induced antigen-specific MHC class II-restricted T cell responses. These data suggest a role for exosomes in antigen presentation in vivo.

Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.

Abstract: Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic lineages throughout the lifetime of the animal. While experimenting with staining of murine bone marrow cells with the vital dye, Hoechst 33342, we discovered that display of Hoechst fluorescence simultaneously at two emission wavelengths revealed a small and distinct subset of whole bone marrow cells that had phenotypic markers of multipotential HSC. These cells were shown in competitive repopulation experiments to contain the vast majority of HSC activity from murine bone marrow and to be enriched at least 1,000-fold for in vivo reconstitution activity. Further, these Hoechst-stained side population (SP) cells were shown to protect recipients from lethal irradiation at low cell doses, and to contribute to both lymphoid and myeloid lineages. The formation of the Hoechst SP profile was blocked when staining was performed in the presence of verapamil, indicating that the distinctly low staining pattern of the SP cells is due to a multidrug resistance protein (mdr) or mdr-like mediated efflux of the dye from HSC. The ability to block the Hoechst efflux activity also allowed us to use Hoechst to determine the DNA content of the SP cells. Between 1 and 3% of the HSC were shown to be in S-G2M. This also enabled the purification of the G0-G1 and S-G2M HSC had a reconstitution capacity equivalent to quiescent stem cells. These findings have implications for models of hematopoietic cell development and for the development of genetic therapies for diseases involving hematopoietic cells.

Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation.

Abstract: We investigated the possibility that T helper cells might enhance the stimulatory function of dendritic cells (DCs). We found that ligation of CD40 by CD40L triggers the production of extremely high levels of bioactive IL-12. Other stimuli such as microbial agents, TNF-alpha or LPS are much less effective or not at all. In addition, CD40L is the most potent stimulus in upregulating the expression of ICAM-1, CD80, and CD86 molecules on DCs. These effects of CD40 ligation result in an increased capacity of DCs to trigger proliferative responses and IFN-gamma production by T cells. These findings reveal a new role for CD40-CD40L interaction in regulating DC function and are relevant to design therapeutic strategies using cultured DCs.

T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines.

Abstract: Analysis of the cDNA encoding murine interleukin (IL) 17 (cytotoxic T lymphocyte associated antigen 8) predicted a secreted protein sharing 57% amino acid identity with the protein predicted from ORF13, an open reading frame of Herpesvirus saimiri. Here we report on the cloning of human IL-17 (hIL-17), the human counterpart of murine IL-17. hIL-17 is a glycoprotein of 155 amino acids secreted as an homodimer by activated memory CD4+ T cells. Although devoid of direct effects on cells of hematopoietic origin, hIL-17 and the product of its viral counterpart, ORF13, stimulate epithelial, endothelial, and fibroblastic cells to secrete cytokines such as IL-6, IL-8, and granulocyte-colony-stimulating factor, as well as prostaglandin E2. Furthermore, when cultured in the presence of hIL-17, fibroblasts could sustain the proliferation of CD34+ hematopoietic progenitors and their preferential maturation into neutrophils. These observations suggest that hIL-17 may constitute (a) an early initiator of the T cell-dependent inflammmatory reaction; and (b) an element of the cytokine network that bridges the immune system to hematopoiesis.

A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1)

Abstract: Chemotactic factors are postulated to direct emigration of lymphocytes from the blood stream into sites of inflammation. Members of a family of chemotactic cytokines, termed chemokines, have been shown to attract lymphocytes but efficacy, i.e., the maximal percentage of attracted cells, has been low. We have identified a highly efficacious lymphocyte chemotactic activity in the supernatants of the murine bone marrow stroma cell line MS-5 which attracts 10-fold more lymphocytes in vitro than currently described lymphocyte chemoattractants. Purification of this chemotactic activity revealed identity to stromal cell-derived factor 1 (SDF-1). SDF-1 acts on lymphocytes and monocytes but not neutrophils in vitro and is both a highly efficacious and highly potent mononuclear cell attractant in vivo. In addition, SDF-1 induces intracellular actin polymerization in lymphocytes, a process that is thought to be a prerequisite for cell motility. Since SDF-1 is expressed constitutively in a broad range of tissues it may have a role in immune surveillance and in basal extravasation of lymphocytes and monocytes rather than in inflammation.

Mitochondrial control of nuclear apoptosis

Abstract: Anucleate cells can be induced to undergo programmed cell death (PCD), indicating the existence of a cytoplasmic PCD pathway that functions independently from the nucleus. Cytoplasmic structures including mitochondria have been shown to participate in the control of apoptotic nuclear disintegration. Before cells exhibit common signs of nuclear apoptosis (chromatin condensation and endonuclease-mediated DNA fragmentation), they undergo a reduction of the mitochondrial transmembrane potential (delta psi m) that may be due to the opening of mitochondrial permeability transition (PT) pores. Here, we present direct evidence indicating that mitochondrial PT constitutes a critical early event of the apoptotic process. In a cell-free system combining purified mitochondria and nuclei, mitochondria undergoing PT suffice to induce chromatin condensation and DNA fragmentation. Induction of PT by pharmacological agents augments the apoptosis-inducing potential of mitochondria. In contrast, prevention of PT by pharmacological agents impedes nuclear apoptosis, both in vitro and in vivo. Mitochondria from hepatocytes or lymphoid cells undergoing apoptosis, but not those from normal cells, induce disintegration of isolated Hela nuclei. A specific ligand of the mitochondrial adenine nucleotide translocator (ANT), bongkreik acid, inhibits PT and reduces apoptosis induction by mitochondria in a cell-free system. Moreover, it inhibits the induction of apoptosis in intact cells. Several pieces of evidence suggest that the proto-oncogene product Bcl-2 inhibits apoptosis by preventing mitochondrial PT. First, to inhibit nuclear apoptosis, Bcl-2 must be localized in mitochondrial but not nuclear membranes. Second, transfection-enforced hyperexpression of Bcl-2 directly abolishes the induction of mitochondrial PT in response to a protonophore, a pro-oxidant, as well as to the ANT ligand atractyloside, correlating with its apoptosis-inhibitory effect. In conclusion, mitochondrial PT appears to be a critical step of the apoptotic cascade.

Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model.

Abstract: Airways inflammation is thought to play a central role in the pathogenesis of asthma. However, the precise role that individual inflammatory cells and mediators play in the development of airways hyperreactivity and the morphological changes of the lung during allergic pulmonary inflammation is unknown. In this investigation we have used a mouse model of allergic pulmonary inflammation and interleukin (IL) 5-deficient mice to establish the essential role of this cytokine and eosinophils in the initiation of aeroallergen-induced lung damage and the development of airways hyperreactivity. Sensitization and aerosol challenge of mice with ovalbumin results in airways eosinophilia and extensive lung damage analogous to that seen in asthma. Aeroallergen-challenged mice also display airways hyperreactivity to beta-methacholine. In IL-5-deficient mice, the eosinophilia, lung damage, and airways hyperreactivity normally resulting from aeroallergen challenge were abolished. Reconstitution of IL-5 production with recombinant vaccinia viruses engineered to express this factor completely restored aeroallergen-induced eosinophilia and airways dysfunction. These results indicate that IL-5 and eosinophils are central mediators in the pathogenesis of allergic lung disease.

Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation.

Abstract: Neonatal thymectomy (NTx), especially around day 3 after birth, causes various organ-specific autoimmune diseases in mice. This report shows that: (a) T cells expressing the interleukin 2 receptor alpha chains (CD25) ontogenically begin to appear in the normal periphery immediately after day 3, rapidly increasing within 2 wk to nearly adult levels (approximately 10% of CD3+ cells, especially of CD4+ cells); (b) NTx on day 3 eliminates CD25+ T cells from the periphery for several days; inoculation immediately after NTx of CD25+ splenic T cells from syngeneic non-Tx adult mice prevents autoimmune development, whereas inoculation of CD25- T cells even at a larger dose does not; and furthermore, (c) similar autoimmune diseases can be produced in adult athymic nu/nu mice by inoculating either spleen cell suspensions from 3-d-old euthymic nu/+ mice or CD25+ cell-depleted spleen cell suspensions from older, even 1-yr-old, nu/+ mice. The CD25- populations from neonates or adults are also similar in the profile of cytokine formation. These results, taken together, indicate that one aspect of peripheral self-tolerance is maintained by CD25+ T cells that sustain potentially pathogenic self-reactive T cells in a CD25- dormant state; the thymic production of the former is developmentally programmed to begin on day 3 after birth in mice. Thus, NTx on day 3 can, at least transiently, eliminate/reduce the autoimmune-preventive CD25+ T cells, thereby leading to activation of the self-reactive T cells that have been produced before NTx.

Chemokine receptor specific for IP10 and mig: structure, function, and expression in activated T-lymphocytes.

Abstract: A human receptor that is selective for the CXC chemokines IP10 and Mig was cloned and characterized. The receptor cDNA has an open reading frame of 1104-bp encoding a protein of 368 amino acids with a molecular mass of 40,659 dalton. The sequence includes seven putative transmembrane segments characteristic of G-protein coupled receptors. It shares 40.9 and 40.3% identical amino acids with the two IL-8 receptors, and 34.2-36.9% identity with the five known CC chemokine receptors. The IP10/Mig receptor is highly expressed in IL-2-activated T lymphocytes, but is not detectable in resting T lymphocytes. B lymphocytes, monocytes and granulocytes. It mediates Ca2+ mobilization and chemotaxis in response to IP10 and Mig, but does not recognize the CXC-chemokines IL-8, GRO alpha, NAP-2, GCP-2. ENA78, PF4, the CC-chemokines MCP-1, MCP-2, MCP-3, MCP-4, MIP-1 alpha, MIP-1 beta. RANTES, 1309, eotaxin, nor lymphotactin. The exclusive expression in activated T-lymphocytes is of high interest since the receptors for chemokines which have been shown so far to attract lymphocytes, e.g., MCP-1, MCP-2, MCP-3, MIP-1 alpha, MIP-1 beta, and RANTES, are also found in monocytes and granulocytes. The present observations suggest that the IP10/Mig receptor is involved in the selective recruitment of effector T cells.

Bcl-2 inhibits the mitochondrial release of an apoptogenic protease.

Abstract: Bcl-2 belongs to a family of apoptosis-regulatory proteins which incorporate into the outer mitochondrial as well as nuclear membranes. The mechanism by which the proto-oncogene product Bcl-2 inhibits apoptosis is thus far elusive. We and others have shown previously that the first biochemical alteration detectable in cells undergoing apoptosis, well before nuclear changes become manifest, is a collapse of the mitochondrial inner membrane potential (delta psi m), suggesting the involvement of mitochondrial products in the apoptotic cascade. Here we show that mitochondria contain a pre-formed approximately 50-kD protein which is released upon delta psi m disruption and which, in a cell-free in vitro system, causes isolated nuclei to undergo apoptotic changes such as chromatin condensation and internucleosomal DNA fragmentation. This apoptosis-inducing factor (AIF) is blocked by N-benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (Z-VAD.fmk), an antagonist of interleukin-1 beta-converting enzyme (ICE)-like proteases that is also an efficient inhibitor of apoptosis in cells. We have tested the effect of Bcl-2 on the formation, release, and action of AIF. When preventing mitochondrial permeability transition (which accounts for the pre-apoptotic delta psi m disruption in cells), Bcl-2 hyperexpressed in the outer mitochondrial membrane also impedes the release of AIF from isolated mitochondria in vitro. In contrast, Bcl-2 does not affect the formation of AIF, which is contained in comparable quantities in control mitochondria and in mitochondria from Bcl-2-hyperexpressing cells. Furthermore, the presence of Bcl-2 in the nuclear membrane does not interfere with the action of AIF on the nucleus, nor does Bcl-2 hyperexpression protect cells against AIF. It thus appears that Bcl-2 prevents apoptosis by favoring the retention of an apoptogenic protease in mitochondria.

Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response.

Abstract: To investigate the role of TNF alpha in the development of in vivo immune response we have generated TNF alpha-deficient mice by gene targeting. Homozygous mutant mice are viable and fertile, develop lymph nodes and Peyer's patches and show no apparent phenotypic abnormalities, indicating that TNF alpha is not required for normal mouse development. In the absence of TNF alpha mice readily succumb to L. monocytogenes infections and show reduced contact hypersensitivity responses. Furthermore, TNF alpha knockout mice are resistant to the systemic toxicity of LPS upon D-galactosamine sensitization, yet they remain sensitive to high doses of LPS alone. Most interestingly, TNF alpha knockout mice completely lack splenic primary B cell follicles and cannot form organized follicular dendritic cell (FDC) networks and germinal centers. However, despite the absence of B cell follicles, Ig class-switching can still occur, yet deregulated humoral immune responses against either thymus-dependent (TD) or thymus-independent (TI) antigens are observed. Complementation of TNF alpha functioning by the expression of either human or murine TNF alpha transgenes is sufficient to reconstitute these defects, establishing a physiological role for TNF alpha in regulating the development and organization of splenic follicular architecture and in the maturation of the humoral immune response.

Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified.

Abstract: Dendritic cells (DC) are the most efficient APC for T cells. The clinical use of DC as vectors for anti-tumor and infectious disease immunotherapy has been limited by their trace levels and accessibility in normal tissue and terminal state of differentiation. In the present study, daily injection of human Flt3 ligand (Flt3L) into mice results in a dramatic numerical increase in cells co-expressing the characteristic DC markers-class II MHC, CD11c, DEC205, and CD86. In contrast, in mice treated with either GM-CSF, GM-CSF plus IL-4, c-kit ligand (c-kitL), or G-CSF, class II+ CD11c+ cells were not significantly increased. Five distinct DC subpopulations were identified in the spleen of Flt3L-treated mice using CD8 alpha and CD11b expression. These cells exhibited veiled and dendritic processes and were as efficient as rare, mature DC isolated from the spleens of untreated mice at presenting allo-Ag or soluble Ag to T cells, or in priming an Ag-specific T cell response in vivo. Dramatic numerical increases in DC were detected in the bone marrow, gastro-intestinal lymphoid tissue (GALT), liver, lymph nodes, lung, peripheral blood, peritoneal cavity, spleen, and thymus. These results suggest that Flt3L could be used to expand the numbers of functionally mature DC in vivo for use in clinical immunotherapy.

High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10.

Abstract: We have shown previously that dendritic cells (DC) produce IL-12 upon interaction with CD4+ T cells. Here we ask how this IL-12 production is induced and regulated. Quantitative PCR and in situ hybridization for IL-12 p40 and an ELISA specific for the p70 heterodimer were used to determine IL-12 production. We demonstrate that ligation of either CD40 or MHC class II molecules independently trigger IL-12 production in DC, and that IL-12 production is downregulated by IL-4 and IL-10. The levels of bioactive IL-12 that can be released by triggering with an anti-CD40 mAb or with a T cell hybridoma are high (range 260-4700 pg/ml from 1 X 10(6) DC in 72 h). The CD40-mediated pathway indicates that IL-12 production is induced in DC upon interaction with activated, CD40 ligand-expressing helper T cells, even in the absence of cognate antigen recognition. Side-by-side comparison of IL-12 production, and blocking experiments employing an anti-CD40 ligand mAb, suggest that the CD40-mediated pathway is quantitatively more significant than induction via the MHC class II molecule. The importance of the CD40/CD40 ligand interaction for IL-12 induction in DC likely contributes to the recent finding that mice lacking the CD40 ligand are impaired in mounting Th1 type cell-mediated immune responses.

Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo.

Abstract: Immunization with defined tumor antigens is currently limited to a small number of cancers where candidates for tumor rejection antigens have been identified. In this study we investigated whether pulsing dendritic cells (DC) with tumor-derived RNA is an effective way to induce CTL and tumor immunity. DC pulsed with in vitro synthesized chicken ovalbumin (OVA) RNA were more effective than OVA peptide-pulsed DC in stimulating primary, OVA-specific CTL responses in vitro. DC pulsed with unfractionated RNA (total or polyA+) from OVA-expressing tumor cells were as effective as DC pulsed with OVA peptide at stimulating CTL responses. Induction of OVA-specific CTL was abrogated when polyA+ RNA from OVA-expressing cells was treated with an OVA-specific antisense oligodeoxynucleotide and RNase H, showing that sensitization of DC was indeed mediated by OVA RNA. Mice vaccinated with DC pulsed with RNA from OVA-expressing tumor cells were protected against a challenge with OVA-expressing tumor cells. In the poorly immunogenic, highly metastatic, B16/F10.9 tumor model a dramatic reduction in lung metastases was observed in mice vaccinated with DC pulsed with tumor-derived RNA (total or polyA+, but not polyA- RNA). The finding that RNA transcribed in vitro from cDNA cloned in a bacterial plasmid was highly effective in sensitizing DC shows that amplification of the antigenic content from a small number of tumor cells is feasible, thus expanding the potential use of RNA-pulsed DC-based vaccines for patients bearing very small, possibly microscopic, tumors.

CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNF alpha.

Abstract: Human dendritic cells (DC) can now be generated in vitro in large numbers by culturing CD34+ hematopoietic progenitors in presence of GM-CSF+TNF alpha for 12 d. The present study demonstrates that cord blood CD34+ HPC indeed differentiate along two independent DC pathways. At early time points (day 5-7) during the culture, two subsets of DC precursors identified by the exclusive expression of CD1a and CD14 emerge independently. Both precursor subsets mature at day 12-14 into DC with typical morphology and phenotype (CD80, CD83, CD86, CD58, high HLA class II). CD1a+ precursors give rise to cells characterized by the expression of Birbeck granules, the Lag antigen and E-cadherin, three markers specifically expressed on Langerhans cells in the epidermis. In contrast, the CD14+ progenitors mature into CD1a+ DC lacking Birbeck granules, E-cadherin, and Lag antigen but expressing CD2, CD9, CD68, and the coagulation factor XIIIa described in dermal dendritic cells. The two mature DC were equally potent in stimulating allogeneic CD45RA+ naive T cells. Interestingly, the CD14+ precursors, but not the CD1a+ precursors, represent bipotent cells that can be induced to differentiate, in response to M-CSF, into macrophage-like cells, lacking accessory function for T cells. Altogether, these results demonstrate that different pathways of DC development exist: the Langerhans cells and the CD14(+)-derived DC related to dermal DC or circulating blood DC. The physiological relevance of these two pathways of DC development is discussed with regard to their potential in vivo counterparts.

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

Abstract: 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.

The fas counterattack : fas-mediated T cell killing by colon cancer cells expressing fas ligand

Abstract: Tumors escape immunological rejection by a diversity of mechanisms. In this report, we demonstrate that the colon cancer cell SW620 expresses functional Fas ligand (FasL), the triggering agent of Fas receptor (FasR)-mediated apoptosis within the immune system. FasL mRNA and cell surface FasL were detected in SW620 cells using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining, respectively. We show that SW620 kills Jurkat T cells in a Fas-mediated manner. FasR-specific antisense oligonucleotide treatment, which transiently inhibited FasR expression, completely protected Jurkat cells from killing by SW620. FasL-specific antisense oligonucleotide treatment of SW620 inhibited its Jurkat-killing activity. FasL has recently been established as a mediator of immune privilege in mouse retina and testis. Our finding that colon cancer cells express functional FasL suggests it may play an analogous role in bestowing immune privilege on human tumors. HT29 and SW620 colon cancer cells were found to express FasR mRNA and cell surface FasR using RT-PCR and immunofluorescence flow cytometry, respectively. However, neither of these cells underwent apoptosis after treatment by the anti-FasR agonistic monoclonal antibody CH11. Our results therefore suggest a Fas counterattack model for immune escape in colon cancer, whereby the cancer cells resist Fas-mediated T cell cytotoxicity but express functional FasL, an apoptotic death signal to which activated T cells are inherently sensitive.

Human tumor antigens recognized by t lymphocytes

Abstract: W e have come a long way since the identification of the first human tumor antigen recognized by autologous CTL. This report provides a brief appraisal of these antigens and their potential for cancer immunotherapy. Our comments will be restricted to nonviral antigens. The initial work carried out on mouse tumors revealed two possible mechanisms for generating new antigens that might be suflficiendy tumor-specific to be of relevance to immunotherapy. The first mechanism involved a point mutation, and the second involved the transcriptional activation o fa gene not expressed in normal tissues (1-3). Subsequent work on mouse tumors provided two interesting examples of tumor antigens resulting from point mutations (4, 5). Tumor-specific Shared Antigens. Three families of genes that appear to code for highly specific tumor antigens have been identified so far, namely, the MAGE, BAGE, and GAGE genes (6-9). These genes are frequently expressed in a wide range of tumor types such as melanoma, lung carcinoma, sarcoma, and bladder carcinoma, but very rarely in other tumor types such as brain tumors, renal carcinoma, and leukemia (7, 10-12). The only normal tissues where expression of these genes has been observed are testis and placenta (7). Starting from CTL clones obtained by stimulating lymphocytes with an autologous melanoma cell line, six antigens encoded by MAGE-1, MAGE-3, BAGE, and GAGE have been identified (8, 9, 13-15). For these six antigens, both the presenting HLA molecule and the antigenic peptide have been completely defined. Remarkably, all the relevant CTL were derived from the same melanoma patient, a patient with metastatic disease who enjoyed an extraordinarily favorable chnical course. Blood samples from several patients with a tumor expressing some or all of these genes were tested, and no such CTL were obtained by stimulating the lymphocytes with autologous tumor cells. More than 60% of Caucasian melanoma patients bear one of the presently defined antigens encoded by MAGE, BAGE, and GAGE. For other cancers such as head and neck tumors and bladder cancer, the frequencies range from 40% to 28%. For several reasons, it appears increasingly unlikely that immunization of patients against one of these antigens will cause harmful immunological side effects caused by the expression of the relevant gene in the testis. First, this expression appears to occur in germline cells, more precisely spermatocytes and spermatogonia (16). A similar observation has been made with the mouse equivalent of a MAGE gene by in situ hybridization (17). Because these germline cells do not express classical M H C class I molecules, gene expression should not result in antigen expression (18). These conclusions are further strengthened by immunization studies carried out with mouse tumor antigen P815A, which is encoded by a gene that is also expressed only in the testis. After immunization with P815 tumor cells, which carry this antigen, male mice produced a strong CTL response. No inflammation of the testis was observed in the following months, and the fertihty of these mice was normal (Uyttenhove, C., manuscript in preparation). A new mode of origin for antigens that are also tumorspecific shared antigens is described in this issue (19). Here, it seems that a gene that is ubiquitously expressed, namely, N-acetyl-glucosaminyltransferase V, contains an intron that appears to carry near its end a promoter that is activated only in melanoma cells. This atypical activation occurs in >50% of melanomas. This produces a message containing a new open reading frame, which codes for the antigenic peptide in its intronic part. Some CTL directed against breast, ovarian, and pancreatic carcinomas recognize an epitope of mucin, a surface protein composed of multiple tandem repeats of 20 amino acids (20-23). Whereas in normal cells mucin is heavily glycosylated, in these tumors the peptide repeats are unmasked by underglycosylation, resulting in CTL recognition. Remarkably, this recognition, which depends on the presence of multiple repeats, occurs in the absence of HLA restriction. The presence of this epitope was recently reported on myeloma cells, and mucin-specific CTL were isolated from the blood of a myeloma patient (24). These mucin antigens appear to be very specific for tumor cells, and the lack of HLA restriction should facilitate therapeutic vaccination trials. Differentiation Antigens. The observation that autolognus CTL can be generated readily against differentiation antigens present on normal melanocytes as well as melanoma cells was unexpected. Four genes encoding melanoma differentiation antigens have been identified: tyrosinase, Melan-A/Mart-1, gpl00, and gp75 (25-30). Most of the identified antigenic peptides are presented by HLA-A2, but other HLA-peptide combinations have been found (29-38). One tyrosinase peptide is presented by HLA-DR4 to CD4 T cells (34). The pattern of CTL precursors directed against these dif-

CTLA-4 engagement inhibits IL-2 accumulation and cell cycle progression upon activation of resting T cells.

Abstract: While interactions between CD28 and members of the B7 family costimulate and enhance T cell responses, recent evidence indicates that the CD28 homologue CTLA-4 plays a downregulatory role. The mechanism by which this occurs is not clear, but it has been suggested that CTLA-4 terminates ongoing responses of activated T cells, perhaps by induction of apoptosis. Here we demonstrate that CTLA-4 engagement by antibody cross-linking or binding to B7 inhibits proliferation and accumulation of the primary T cell growth factor, IL-2, by cells stimulated with anti-CD3 and anti-CD28. This inhibition is not a result of enhanced cell death. Rather it appears to result from restriction of transition from the G1 to the S phase of the cell cycle. Our observation that upregulation of both the IL-2R alpha chain and the CD69 activation antigen are inhibited by CTLA-4 engagement supplies further evidence that CTLA-4 restricts the progression of T cells to an activated state. Together this data demonstrates that CTLA-4 can regulate T cell activation in the absence of induction of apoptotic cell death.

A critical role for transforming growth factor-beta but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RB(low) CD4+ T cells.

Abstract: A T helper type 1 (Th1)-mediated colitis with similarities to inflammatory bowel disease in humans developed in severe combined immunodeficiency mice reconstituted with CD45RB(high) CD4+ splenic T cells and could be prevented by cotransfer of CD45RB(low) CD4+ T cells. Inhibition of this Th1 response by the CD45RB(low) T cell population could be reversed in vivo by an anti-transforming growth factor (TGF) beta antibody. Interleukin (IL) 4 was not required for either the differentiation of function of protective cells as CD45RB(low) CD4+ cells from IL-4-deficient mice were fully effective. These results identify a subpopulation of peripheral CD4+ cells and TGF-beta as critical components of the natural immune regulatory mechanism, which prevents the development of pathogenic Th1 responses in the gut, and suggests that this immunoregulatory population is distinct from Th2 cells.

Mitochondrial permeability transition is a central coordinating event of apoptosis

Abstract: In a number of experimental systems, the early stage of the apoptotic process, i.e., the stage that precedes nuclear disintegration, is characterized by the breakdown of the inner mitochondrial transmembrane potential (delta psi m). This delta psi m disruption is mediated by the opening of permeability transition (PT) pores and appears to be critical for the apoptotic cascade, since it is directly regulated by Bcl-2 and since mitochondria induced to undergo PT in vitro become capable of inducing nuclear chromatinolysis in a cell-free system of apoptosis. Here, we addressed the question of which apoptotic events are secondary to mitochondrial PT. We tested the effect of a specific inhibitor of PT, bongkrekic acid (BA), a ligand of the mitochondrial adenine nucleotide translocator, on a prototypic model of apoptosis glucocorticoid-induced thymocyte death. In addition to abolishing the apoptotic delta psi m disruption, BA prevents a number of phenomena linked to apoptosis: depletion of nonoxidized glutathione, generation of reactive oxygen species, translocation of NF kappa B, exposure of phosphatidylserine residues on the outer plasma membrane, cytoplasmic vacuolization, chromatin condensation, and oligonucleosomal DNA fragmentation. BA is also an efficient inhibitor of p53-dependent thymocyte apoptosis induced by DNA damage. These data suggest that a number of apoptotic phenomena are secondary to PT. In addition, we present data indicating that apoptotic delta psi m disruption is secondary to transcriptional events. These data connect the PT control point to the p53- and ICE/ Ced 3-regulated control points of apoptosis and place PT upstream of nuclear and plasma membrane features of PCD.

Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells.

Abstract: Human CD4+ T cells, activated by allogeneic monocytes in a primary mixed lymphocyte reaction in the presence of exogenous interleukin (IL) 10, specifically failed to proliferate after restimulation with the same alloantigens. A comparable state of T cell unresponsiveness could be induced by activation of CD4+ T cells by cross-linked anti-CD3 monoclonal antibodies (mAbs) in the presence of exogenous IL-10. The anergic T cells failed to produce IL-2, IL-5, IL-10, interferon gamma, tumor necrosis factor alpha, and granulocyte/macrophage colony-stimulating factor. The IL-10-induced anergic state was long-lasting. T cell anergy could not be reversed after restimulation of the cells with anti-CD3 and anti-CD28 mAbs, although CD3 and CD28 expression was normal. In addition, restimulation of anergized T cells with anti-CD3 mAbs induced normal Ca2+ fluxes and resulted in increased CD3, CD28, and class II major histocompatibility complex expression, indicating that calcineurin-mediated signaling occurs in these anergic cells. However, the expression of the IL-2 receptor alpha chain was not upregulated, which may account for the failure of exogenous IL-2 to reverse the anergic state. Interestingly, anergic T cells and their nonanergic counterparts showed comparable levels of proliferation and cytokine production after activation with phorbol myristate acetate and Ca2+ ionophore, indicating that a direct activation of a protein kinase C-dependent pathway can overcome the tolerizing effect of IL-10. Taken together, these data demonstrate that IL-10 induces T cell anergy and therefore may play an important role in the induction and maintenance of antigen-specific T cell tolerance.

Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo.

Abstract: Dendritic cells (DC) are described as "nature's adjuvant," since they have the capacity to sensitize T cells in vivo upon first encounter with the antigen. The potent accessory properties of DC appear to develop sequentially. In particular, the ability to process antigens and to sensitize native T cells develops in sequence, a process termed "maturation" that is well described in vitro. Here, we obtain evidence for maturation in vivo in response to the bacterial product lipopolysaccharide (LPS). Before LPS treatment, many DC are found at the margin between the red and white pulp. These cells lack the M342 and DEC-205 markers, but process soluble proteins effectively. 6 h after LPS, DC with the M342 and DEC-205 markers are found in increased numbers in the T cell areas. These cells have a reduced capacity to process proteins, but show increases in the B7 costimulator and T cell stimulatory capacity. 48 h after LPS, the number of DC in the spleen is reduced markedly. We interpret these findings to mean that LPS can cause DC in the marginal zone to mature and to migrate into and then out of the T cell areas.

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

Abstract: 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.

CTLA-4 ligation blocks CD28-dependent T cell activation.

Abstract: CTLA-4 is a CD28 homologue believed to be a negative regulator of T cell function. However, the mechanism of this downregulatory activity is not well understood. The present study was designed to examine the effect of CTLA-4 ligation on cytokine production, cell survival, and cell cycle progression. The results demonstrate that the primary effect of CTLA-4 ligation is not the induction of apoptosis. Instead, CTLA-4 signaling blocks IL-2 production, IL-2 receptor expression, and cell cycle progression of activated T cells. Moreover, the effect of CTLA-4 signaling was manifested after initial T cell activation. Inhibition of IL-2 receptor expression and cell cycle progression was more pronounced at late (72 h) time points after initial activation. The effects of anti-CTLA-4 mAbs were most apparent in the presence of optimal CD28-mediated costimulation consistent with the finding that CTLA-4 upregulation was CD28-dependent. Finally, the addition of exogenous IL-2 to the cultures restored IL-2 receptor expression and T cell proliferation. These results suggest that CTLA-4 signaling does not regulate cell survival or responsiveness to IL-2, but does inhibit CD28-dependent IL-2 production.

Interleukin 4, but not interleukin 5 or eosinophils, is required in a murine model of acute airway hyperreactivity.

Abstract: Reversible airway hyperreactivity underlies the pathophysiology of asthma, yet the precise mediators of the response remain unclear. Human studies have correlated aberrant activation of T helper (Th) 2-like effector systems in the airways with disease. A murine model of airway hyperreactivity in response to acetylcholine was established using mice immunized with ovalbumin and challenged with aerosolized antigen. No airway hyperractivity occurred in severe combined immunodeficient mice. Identically immunized BALB/c mice developed an influx of cells, with a predominance of eosinophils and CD4+ T cells, into the lungs and bronchoalveolar lavage fluid at the time that substantial changes in airway pressure and resistance were quantitated. Challenged animals developed marked increases in Th2 cytokine production, eosinophil influx, and serum immunoglobulin E levels. Neutralization of interleukin (IL) 4 using monoclonal antibodies administered during the period of systemic immunization abrogated airway hyperractivity but had little effect on the influx of eosinophils. Administration of anti-IL-4 only during the period of the aerosol challenge did not affect the subsequent response to acetylcholine. Finally, administration of anti-IL-5 antibodies at levels that suppressed eosinophils to < 1% of recruited cells had no effect on the subsequent airway responses. BALB/c mice had significantly greater airway responses than C57BL/6 mice, consistent with enhanced IL-4 responses to antigen in BALB/c mice. Taken together, these data implicate IL-4 generated during the period of lymphocyte priming with antigen in establishing the cascade of responses required to generate airway hyperractivity to inhaled antigen. No role for IL-5 or eosinophils could be demonstrated.

Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles.

Abstract: Notch is a highly conserved transmembrane protein that is involved in cell fate decisions and is found in organisms ranging from Drosophila to humans. A human homologue of Notch, TAN1, was initially identified at the chromosomal breakpoint of a subset of T-cell lymphoblastic leukemias/lymphomas containing a t(7;9) chromosomal translocation; however, its role in oncogenesis has been unclear. Using a bone marrow reconstitution assay with cells containing retrovirally transduced TAN1 alleles, we analyzed the oncogenic potential of both nuclear and extranuclear forms of truncated TAN1 in hematopoietic cells. Although the Moloney leukemia virus long terminal repeat drives expression in most hematopoietic cell types, retroviruses encoding either form of the TAN1 protein induced clonal leukemias of exclusively immature T cell phenotypes in approximately 50% of transplanted animals. All tumors overexpressed truncated TAN1 of the size and subcellular localization predicted from the structure of the gene. These results show that TAN1 is an oncoprotein and suggest that truncation and overexpression are important determinants of transforming activity. Moreover, the murine tumors caused by TAN1 in the bone marrow transplant model are very similar to the TAN1-associated human tumors and suggest that TAN1 may be specifically oncotropic for T cells.

Hodgkin and Reed-Sternberg cells in Hodgkin's disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells.

Abstract: In Hodgkin's disease (HD), the Hodgkin and Reed-Sternberg (HRS) cells represent only a minute population in the diseased tissue. The investigation of lineage derivation and clonal origin of these cells has yielded conflicting results. We have analyzed HRS cells micromanipulated from infiltrated tissue sections of 10 primary HD patients for rearranged V genes, extending a previous study. Clonally related rearrangements were found in nine cases, indicating that HRS cells represent a dominant clone of B lineage-derived cells in at least a large fraction of cases of HD. Rearranged VH genes from HRS cells carried a high load of somatic mutation, indicating that HRS cells are derived from germinal center (GC) cells or their progeny. Stop codons in some in-frame V gene rearrangements suggest that the HRS cell precursors reside inside GCs, have acquired crippling mutations that prevent antigenic selection, but escape apoptosis through some transforming event.

Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques.

Abstract: We used the simian immunodeficiency virus (SIV)/rhesus macaque model to study events that underlie sexual transmission of human immunodeficiency virus type 1 (HIV-1). Four female rhesus macaques were inoculated intravaginally with SIVmac251, and then killed 2, 5, 7, and 9 d later. A technique that detected polymerase chain reaction-amplified SIV in situ showed that the first cellular targets for SIV were in the lamina propria of the cervicovaginal mucosa, immediately subjacent to the epithelium. Phenotypic and localization studies demonstrated that many of the infected cells were likely to be dendritic cells. Within 2 d of inoculation, infected cells were identified in the paracortex and subcapsular sinus of the draining internal iliac lymph nodes. Subsequently, systemic dissemination of SIV was rapid, since culturable virus was detectable in the blood by day 5. From these results, we present a model for mucosal transmission of SIV and HIV-1.

A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis.

Abstract: Dendritic cells (DC), the most efficient antigen-presenting cells, are well equipped for activation of naive CD4+ T cells by their expression of high levels of major histocompatibility complex and costimulator molecules. We now demonstrate that some DC are equally well equipped for killing these same T cells. Murine splenic DC consist of both conventional CD8alpha- DC and a major population of CD8alpha+ DC. Whereas CD8- DC induce a vigorous proliferative response in CD4 T cells, CD8+ DC induce a lesser response that is associated with marked T cell apoptosis. By using various mixtures of T cells and DC from Fas-mutant lpr/lpr mice and Fas-ligand (FasL) mutant gld/gld mice, we show this death is due to interaction of Fas on activated T cells with FasL on CD8+ DC. Furthermore, we show by direct surface staining that CD8+ DC, but not CD8- DC, express FasL at high levels. These findings indicate that FasL+ CD8+ DC are a specialized subgroup of DC with a role in the regulation of the response of primary peripheral T cells.