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The role of the T lymphocyte growth factor, interleukin 2 (IL2), on human lymphocyte proliferation was investigated. The results define the extent and the chronological sequence of IL2 effects on unstimulated mononuclear cells and on an activated population enriched in IL2 receptor positive cells. Important differences were observed in the proliferative response to the mitogen PHA when the IL2 concentration in the culture medium was varied. The data suggest that IL2 concentration is critical in the responsiveness to a mitogenic stimulus.

Apoptosis allows the clearance of unwanted cells from living tissues without causing inflammation. Processing of phagocytosed apoptotic cells yields Ags that access the cytosol and the MHC class I pathway of engulfing cells and are recognized by Ag-specific CTL. We show here that injection of apoptotic RMA cells, a syngeneic T cell lymphoma, into C57BL/6 mice results in priming of a functional and long-lasting tumor-specific immune response. Cross-priming of CTLs by apoptotic cells requires CD4+ T cell help. Apoptotic cells, however, are at least 20-fold less immunogenic than nonreplicating live cells. Immunogenicity of apoptotic cells is proportional to the number of cells injected, correlates with the serum concentration of IL-10 and IL-1β cytokines, and is enhanced in IL-10 knockout mice. Moreover, immunization with dendritic cells (DCs), but not macrophages (Mφ), pulsed with apoptotic cells primes tumor-specific CTLs and confers protection against a tumor challenge. Our findings demonstrate that tumor cells undergoing apoptosis are, though scarcely, immunogenic in vivo, outline the different roles of Mφ and DCs in the physiologic clearance of unwanted cells, and have implications in designing immunomodulating vaccines.

Programmed death via apoptosis is the metazoan physiologic mode of cell death. Apoptotic cells are recognized by scavenger phagocytes via a number of membrane receptors and engulfed. Thereafter, little is known of their fate, or that of phagocytes. Here, we have traced apoptotic cells upon their engulfment by macrophages. After 3 h, apoptotic cells were contained in discrete well-defined vacuoles. Upon overnight chase, several small vesicles, possibly originating from the fragmentation of original vacuoles, were evident all over the macrophage body. Furthermore, Ags were diffused in the cytosol of some cells, which raises the possibility that epitopes from engulfed apoptotic cells may associate with macrophage MHC class I molecules and be recognized by T lymphocytes. Indeed, Ag-specific CTLs recognize and specifically lyse syngeneic macrophages upon phagocytosis of MHC class I-positive or -negative apoptotic cells, provided that they contain the relevant Ags. Synthesis and membrane expression of class I molecules by macrophages, together with functional transporters associated with Ag presentation, were necessary for recognition and lysis. The indirect presentation of epitopes from engulfed apoptotic cells by scavenger Ag-presenting phagocytes may, in the absence of "danger" signals, have implications for the establishment of central and peripheral self-tolerance.

For an efficient antitumor cytotoxic response, tumor antigenic peptides need to be presented by professional antigen-presenting cells in association with MHC class I molecules. We established in vitro short-term human CTL lines from healthy and melanoma-bearing subjects, using as antigen-presenting cells autologous adherent cells after phagocytosis of latex beads coated with melanoma peptides. Melanoma peptides were obtained by acid extraction of melanoma cells that matched with donor peripheral blood mononuclear cells, at least for one HLA-A allele. The cytotoxic activity of the lines was specific for the melanoma from which peptides were obtained and for melanoma sharing HLA alleles. These results demonstrate that a complex mixture of naturally processed melanoma peptides conjugated to a phagocytic substrate that targets them into the MHC class I pathway of adherent cells can prime a CTL response in healthy subjects in vitro, and that peptides from allogeneic tumors may be used to propagate CTL in melanoma patients. Our data support the feasibility of active and passive vaccination procedures with nonliving vaccines in cancer patients.

Pentraxins are acute-phase proteins produced in vivo during inflammatory reactions. Classical short pentraxins, C-reactive protein, and serum amyloid P component are generated in the liver in response to interleukin (IL)-6. The long pentraxin PTX3 is produced in tissues under the control of primary proinflammatory signals, such as lipopolysaccharide, IL-1 beta, and tumor necrosis factor-alpha, which also promote maturation of dendritic cells (DCs). Cell death commonly occurs during inflammatory reactions. In this study, it is shown that PTX3 specifically binds to dying cells. The binding was dose dependent and saturable. Recognition was restricted to extranuclear membrane domains and to a chronological window after UV irradiation or after CD95 cross-linking-induced or spontaneous cell death in vitro. PTX3 bound to necrotic cells to a lesser extent. Human DCs failed to internalize dying cells in the presence of PTX3, while they took up normally soluble or inert particulate substrates. These results suggest that PTX3 sequesters cell remnants from antigen-presenting cells, possibly contributing to preventing the onset of autoimmune reactions in inflamed tissues. (Blood. 2000;96:4300-4306)

Tumor cells undergoing programmed death are an attractive source of tumor-associated antigens, and evidences are available for their therapeutic efficacy in vivo when used either alone or in association with dendritic cells. However, little is known about the specificity of the immune response induced by such antigen formulation. Indeed, activation of specific proteases during apoptosis may influence the cytoplasmic degradation of proteins and the generation of CTL epitopes. We show here that on injection of C57BL/6 mice either with RMA lymphoma cells induced to apoptosis or bone marrow-derived dendritic cells pulsed with apoptotic RMA cells, a specific and protective CTL response is induced, which, however, is not directed against the immunodominant CTL epitope gag(85-93). Lack of in vivo expansion of gag(85-93)-specific CTL in vaccinated mice is attributable to the apoptosis-dependent loss of gag(85-93) in dying tumor cells. Indeed, we found loss of gag(85-93) in RMA, MBL-2, and EL-4G+ lymphoma cells, which share gag(85-93) as an immunodominant CTL epitope, induced to apoptosis by UV irradiation, mitomycin C, doxorubicin, or daunorubicin. This phenomenon appears to be caspase-dependent, because caspase inhibition by N-benzyloxycarbonyl-Val-Ala-asp-fluoromethylketone prevents apoptosis of lymphoma cells and loss of gag(85-93). Therefore, subversion of the epitope hierarchy in apoptotic tumor cells might be relevant in the induction of tumor-specific T-lymphocyte responses.

A 57-year-old man with a history of recurrent infections from the age of 50 was hospitalized with a diagnosis of common variable hypogammaglobulinemia (CVH). Immunological studies revealed a severe reduction of circulating immunoglobulins of all classes. Immunophenotyping of peripheral blood mononuclear cells (PBMC) with monoclonal antibodies, revealed normal values of total B and T cells with CD4/CD8 ratio sharply reduced (0.35) as compared to normal (1.6) because of an increase of CD8 and a decrease of CD4 cells. The surface expression of IL-2 receptor was normal. Natural cytotoxic and phagocytic system presented several abnormalities: a deep impairment of NK activity was found in spite of a normal number of NK cells, as ascertained by Leu 19 and B73.1 monoclonal antibodies. The defective NK activity was not restored by interferon alfa, but was normalized by recombinant IL-2. Phagocytic function, as defined by zymosan-stimulated O2- production was almost absent. The involvement of natural cytotoxic and phagocytic systems in CVH has been rarely reported; the possible causative role of a chronic viral infection (Epstein-Barr virus?) is discussed, on the base of anamnesis.

The behaviour of human lymphocyte surface markers was investigated after in vitro alloactivation. Evidence was obtained that the expression of surface receptors is modified during mixed lymphocyte culture. In vitro allogeneic stimuli are able to induce a very significant increase of Fc receptors bearing cells: the biological significance of these data is discussed.

When a group of 104 aged subjects was screened for autoimmune reactions, positive reactions for the rheumatoid factor and/or autoantibodies (ANA, anti-thyroid, PCA, anti-smooth muscle, anti-mitochondria) were recorded in 40.4%. Immunological functions were studied in 32 positive aged subjects, 32 age- and sex-matched negative controls, and 32 young subjects. Some differences attributable to the process of aging were quite evident, such as a depression in the percentage of E rosette forming peripheral lymphocytes and in their response to PHA, and an increase in the percentage of IgG-bearing peripheral lymphocytes and in the serum levels of IgA and three complement fractions (C'3, C'4, and C'3-PA). No clear-cut picture was noted when autoimmunity-positive and -negative, aged subjects were compared. However, some differences between sexes suggest that autoimmune reactions are linked to a depressed T cell function mainly in males, whereas the reverse is true for females.

Human one-way mixed lymphocyte culture induces a significant increase of EA(7S) rosette-forming cells. Using fractionation procedures, an increased number of Fc receptor-bearing cells in the T high-enriched populations was found from alloactivated lymphocytes compared with similar fractions obtained after autologous control cultures. Additional experiments showed a parallel increase of E-rosette-forming cells in the Fc receptor-enriched alloactivated fractions. The results indicate that an increase of T.G lymphocytes occurs during in vitro alloactivation in man.

Dendritic cells (DCs) and natural killer (NK) cells are key players at the interface between innate resistance and acquired immunity. NK cells can induce DC maturation, a differentiation process whereby DCs respond to a environmental stimulus and acquire the ability of eliciting adaptive immunity. Conversely, maturing DCs promote NK functions in vivo and in vitro. This interplay has important consequences on the immune response to pathogens and possibly to neoplastic cells. Here, we show that B16 melanoma cells actively modulate the interaction between DCs derived from bone marrow precursors and NK/LAK cells propagated from the spleen of C57BL/6 mice. DCs increased in a dose-dependent manner the ability of NK/LAK cells to kill melanoma cells and to produce cytokines. This activatory cross-talk entailed the production of IL-18 by DCs and of IFN-gamma by NK/LAK cells. Melanoma cells were not a passive target of NK activity; they regulated the outcome of the interaction between DCs and NK/LAK cells, inhibiting the in vitro production of cytokines as effectively as the genetic deletion of IL-18 or IFN-gamma. Interference with the NK/DC interaction possibly represents a mechanism used by growing tumors to evade the immune response.

We have investigated the role of p55 and p75 tumor necrosis factor receptors 1 and 2 (TNFR1 and TNFR2, respectively) in TNF-induced alteration of endothelial permeability in vitro and in vivo. Stimulation of TNFR1 with an agonist antibody or a receptor-selective TNF mutein increased the flux of125I-albumin through endothelial cell monolayers. An antagonist anti-TNFR1 antibody, but not antagonist anti-TNFR2 antibodies, blocked the activity of TNF in vitro. Stimulation of TNFR1, but not TNFR2, induced cytoskeletal reorganization associated with increased permeability. SB-203580, a p38 mitogen-activated protein kinase inhibitor, blocked TNFR1-induced cytoskeletal reorganization and permeability. A selective mouse TNFR1 agonist and human TNF, which binds to murine TNFR1, increased the leakage of trypan blue-albumin from liver vessels in mice. These results indicate that stimulation of TNFR1 is necessary and sufficient to increase endothelial permeability in vitro and in vivo. However, an antagonist anti-murine TNFR2 antibody partially inhibited the effect of murine TNF on liver vessels, suggesting that TNFR2 also plays a role in the regulation of TNF-induced vascular permeability in vivo.

Fas and Fas ligand (FasL) have been found both in lymphoid and in non-lymphoid malignancies, and are thought to play a role in the interplay between tumors and the immune system. Here we investigated Fas/FasL expression, function and intracellular signalling pathways in human melanomas. Of 5 melanoma cell lines, 3 expressed Fas at their surface, and all of them expressed FasL. FasL was functional, since it triggered Fas-induced apoptosis of human T lymphocytes clones. Conversely, cross-linking of Fas molecule with a specific monoclonal antibody failed to induce apoptosis in any of the melanomas tested, or ceramide intracellular accumulation or caspase-3 activation, pointing to an early alteration in the Fas-triggered signaling cascade. All melanomas retained the ability to undergo apoptosis induced by cytotoxic lymphocytes, which was mediated by the granule exocytosis mechanism. This suggests that melanoma cells evade immune-mediated Fas-triggered apoptosis via a selective blockade of the Fas apoptotic pathway. Cytotoxic lymphocytes, however, may circumvent tumor resistance to Fas-induced death via granzyme-mediated apoptosis, further supporting the development of immunotherapeutic strategies in the treatment of cancer.

Objective: To verify whether features of CNS involvement can be detected in SLE patients without overt neuropsychiatric manifestations.Methods: 114 SLE patients who had never received a diagnosis of neuropsychiatric lupus (never NPSLE) were studied and compared to 65 SLE patients with known neuropsychiatric involvement (NPSLE). The study relied on evaluation of neurocognitive functions by means of a battery of neuropsychological tests, on psychiatric and neuropsychological assessments and on neuroimaging studies (computed tomography, magnetic resonance, single photon emission computed tomography (SPECT)).Results: Clinical features, including disease duration/activity and pharmacological therapy, of never-NPSLE and NPSLE patients were similar. Short-term and long-term memory, visuo-spatial and verbal information processing were similarly compromised in never-NPSLE and in NPSLE patients; only attention was significantly more compromised in NPSLE patients. Psychiatric morbidity was higher than expected in never-NPSLE patients, although less than in the control neuropsychiatric group. Ischemic lesions, multiple small high intensity lesions and cortical atrophy, detected by CT and MR scans, as well as abnormal SPECT were also frequently detected in never NPSLE patients. Interestingly, left parietal and occipital area hypoperfusion by SPECT was significantly more frequent in the patients with impaired visuo-spatial intelligence and short-term memory.Conclusions: Most abnormalities detected by available diagnostic tools and characteristics of neuropsychiatric SLE are also present in non-symptomatic patients. They may derive from an unexpected widespread involvement of the CNS and are not per se sufficient, in the absence of clinical manifestations, for a diagnosis of neuropsychiatric SLE.

Cytotoxic T lymphocytes (CTL) recognize antigenic peptides presented by major histocompatibility complex class I (MHC-I) molecules on the surface of target cells. Optimal induction of CD8+ CTL depends on the amount of relevant peptide/MHC-I complexes and the presence of co-stimulatory molecules on antigen-presenting cells (APC). The antigen-processing defective mutant cell line RMA-S, when cultured at low temperature, expresses high amounts of MHC-I molecules that do not contain endogenously derived peptides. These "empty" MHC-I molecules can be stabilized by addition of MHC-binding peptides. RMA-S cultured at low temperatures with selected peptides have been used for in vitro CTL induction with conflicting results. RMA-S cells do not express detectable amounts of B7 co-stimulatory molecule. This could explain their unpredictable efficiency as APC. We have evaluated whether RMA-S cells, stably transfected with cDNA encoding for the human B7.1 molecule could provide effective co-stimulation for CD8+ T lymphocytes. RMA-S/B7 cells, loaded with different synthetic peptides, demonstrated a high and sometimes unique efficiency for in vitro primary CTL induction, even when "sub-optimal" antigen peptides were used. Most importantly, RMA-S/B7 cells pulsed with naturally processed peptides extracted from the poorly immunogenic B16 melanoma cells were able to prime CD8+ cells against B16 melanoma. We conclude that the use of RMA-S/B7 cells as APC represents an ideal strategy for in vitro CTL immunization without prior in vivo priming. This system may also help to address the issue of the different contributions of co-stimulation and relative occupancy of MHC-I by single peptide epitopes in CTL priming.

Dendritic cells (DCs) are promising tools for tumor immunotherapy. Their efficacy in the tumor environment increases when tumor cells die as a consequence of chemo/radiotherapy or when local stimuli promoting DC maturation and function are available. Dying tumor cells could represent a source of tumor antigens, which DCs cross-present to tumor-specific T cells. The outcome of cross presentation is in turn determined by the maturation state of DCs. Natural killer (NK)/lymphokine-activated killer (LAK) cells injected into growing tumors could both provide a source of dying cells for cross-presentation and deliver stimuli for DC maturation. Here, we report that NK/LAK cells recognized and killed in vivo major histocompatibility complex class I(low) highly tumorigenic, nonimmunogenic B16F1 melanoma cells when injected into exponentially growing neoplastic lesions. The simultaneous injection of immature DCs was required to heal animals. Similar results were obtained injecting NK/LAK cells and DC into growing Raucher leukaemia virus induced cell line lymphomas. Cured mice failed to reject other implantable tumors, and developed a specific cytotoxic response against the original neoplasm; moreover, they developed a long-lasting memory, and were protected against further challenges with living tumor cells only when both cell populations were introduced. The response associated to the preferential recruitment within tumors of eosinophils. The simultaneous injection in solid tumors of DCs and NK/LAK cells represents an attractive approach for antineoplastic immunotherapeutic strategies.