Neutralizing antibody to interleukin 4 induces systemic protection and T helper type 1-associated immunity in murine candidiasis.

An interleukin 4 (IL-4)-specific monoclonal antibody (mAb) was administered to mice infected systemically with the yeast Candida albicans, and the animals were monitored for mortality, development of delayed-type hypersensitivity, production of antibodies of different isotypes, release of IL-2, IL-4, IL-6, and interferon gamma (IFN-gamma) in vitro by splenic CD4+ lymphocytes, and levels of IL-4 and IFN-gamma mRNA in these cells. Neutralization of IL-4 by three weekly injections of mAb in several independent experiments resulted in an overall cure rate of 81% versus 0% of controls. Cure was associated with efficient clearance of the yeast from infected organs and histologic evidence of disease resolution, detection of strong T helper type 1 (Th1) responses, and establishment of long-lasting protective immunity. Soon after infection, and as a result of the first or second injection of mAb, there was a decrease in IL-4 mRNA in CD4+ cells, which was accompanied by an increase in the levels of IFN-gamma-specific transcripts. Our data thus indicate that the production of IL-4 by Th2 cells may limit Th1-associated protective immunity in murine candidiasis.

Identification of a 2-propanol analogue modulating the non-enzymatic function of indoleamine 2,3-dioxygenase 1.

Indoleamine 2,3 dioxygenase 1 (IDO1) is a metabolic enzyme that catalyzes the conversion of the essential amino acid tryptophan (Trp) into a series of immunoactive catabolites, collectively known as kynurenines. Through the depletion of Trp and the generation of kynurenines, IDO1 represents a key regulator of the immune responses involved in physiologic homeostasis as well as in neoplastic and autoimmune pathologies. The IDO1 enzyme has been described as an important immune checkpoint to be targeted by catalytic inhibitors in the treatment of cancer. In contrast, a defective expression/activity of the enzyme has been demonstrated in autoimmune diseases. Beside its catalytic activity, the IDO1 protein is endowed with an additional function associated with the presence of two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which, once phosphorylated, bind SHP phosphatases and mediate a long-term immunoregulatory activity of IDO1. Herein, we report the screening of a focused library of molecules bearing a propanol core by a protocol combining microscale thermophoresis (MST) analysis and a cellular assay. As a result, the combined screening identified a 2-propanolol analogue, VIS351, as the first potent activator of the ITIM-mediated function of the IDO1 enzyme. VIS351 displayed a good dissociation constant (Kd = 1.90 µM) for IDO1 and a moderate cellular inhibitor activity (IC50 = 11.463 µM), although it did not show any catalytic inhibition of the recombinant IDO1 enzyme. Because we previously demonstrated that the enzymatic and non-enzymatic (i.e., ITIM-mediated) functions of IDO1 reside in different conformations of the protein, we hypothesized that in the cellular system VIS351 may shift the dynamic conformational balance towards the ITIM-favoring folding of IDO1, resulting in the activation of the signaling rather than catalytic activity of IDO1. We demonstrated that VIS351 activated the ITIM-mediated signaling of IDO1 also in mouse plasmacytoid dendritic cells, conferring those cells an immunosuppressive phenotype detectable in vivo. Thus the manuscript describes for the first time a small molecule as a positive modulator of IDO1 signaling function, paving the basis for an innovative approach to develop first-in-class drugs acting on the IDO1 target.

Tryptophan catabolism in IDO+ plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) represent a specialized cell population that produces large amounts of type I interferons, the so-called natural interferon-producing cells. Recently, murine and human pDCs have been credited with a unique ability to express indoleamine 2,3-dioxygenase (IDO) and to mediate immunosuppression in specific settings. This suggests an important role for IDO-expressing pDCs in controlling the balance of inflammation and tolerance. Here we review recent advances in our understanding of how these cells may be critical at the interface of inflammation and tolerance and discuss the potential for therapeutic IDO modulation as an immunoregulatory maneuver targeting pDC function.

O6-alkylguanine-DNA alkyltransferase attenuates triazene-induced cytotoxicity and tumor cell immunogenicity in murine L1210 leukemia.

Methylating and chloroethylating triazene compounds (TZCs) are effective antitumor agents in murine leukemias and can induce the appearance of novel antigens in leukemic cells (chemical xenogenization). Recently, it has been shown that TZCs might have a role in the treatment of patients affected by acute myelogenous leukemias that express low levels of the DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (OGAT). In this report, we have evaluated the role of this DNA repair enzyme in the leukemic cell response to the xenogenizing and cytotoxic properties of TZCs. OGAT-deficient murine leukemic L1210 cells were transfected with a recombinant ecotropic retrovirus containing the coding region for the human OGAT protein. Selected clones expressed the human OGAT transcript and had greatly increased OGAT activity. Compared to OGAT-deficient cells, OGAT-expressing cells were considerably more resistant to the xenogenizing properties of 1-(p-chlorophenyl)-3,3- dimethyl-triazene, measured in terms of leukemia graft rejection, and were less susceptible to the cytotoxic activity of the TZCs 8-carbamoyl-3-methyl-imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one and 8-carbamoyl-3-(2-chloroethyl)imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one. These data suggest that methylation of the O6 position of guanine is involved in the appearance of increased tumor immunogenicity after exposure to methylating TZC and that OGAT is able, at least in part, to counteract the cytotoxic effects of methylating and chloroethylating agents.

T cell apoptosis by tryptophan catabolism.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that, expressed by different cell types, has regulatory effects on T cells resulting from tryptophan depletion in specific local tissue microenvironments. Different mechanisms, however, might contribute to IDO-dependent immune regulation. We show here that tryptophan metabolites in the kynurenine pathway, such as 3-hydroxyanthranilic and quinolinic acids, will induce the selective apoptosis in vitro of murine thymocytes and of Th1 but not Th2 cells. T cell apoptosis was observed at relatively low concentrations of kynurenines, did not require Fas/Fas ligand interactions, and was associated with the activation of caspase-8 and the release of cytochrome c from mitochondria. When administered in vivo, the two kynurenines caused depletion of specific thymocyte subsets in a fashion qualitatively similar to dexamethasone. These data suggest that the selective deletion of T lymphocytes may be a major mechanism whereby tryptophan metabolism affects immunity under physiopathologic conditions.

Dendritic cells, interleukin 12, and CD4+ lymphocytes in the initiation of class I-restricted reactivity to a tumor/self peptide.

Cell-mediated immunity involving CD8+ lymphocytes is effective in mediating rejection of murine mastocytoma cells bearing P815AB, a tumor-associated and self antigen showing similarity to tumor-specific shared antigens in humans. Although this antigen may act as an efficient target for class I-restricted responses in immunized mice, neither P815AB expressed on tumor cells nor a related synthetic nonapeptide will activate unprimed CD8+ cells for in vivo reactivity, measured by skin test assay. We review evidence showing that the failure of P815AB to initiate CD8+ cell reactivity may be due to defective recruitment of accessory and Th1-like cells to the afferent phase of the response initiated by transfer of mice with dendritic cells pulsed in vitro with the P815AB peptide. Although the copresence of a T helper peptide in dendritic cell priming in vitro with P815AB may compensate for the poor generation of accessory and Th1 cells in the adoptively transferred mice, recombinant IL-12 can replace the helper peptide in both effects. Effective priming to P815AB in vivo is achieved by either exposing dendritic cells to IL-12 prior to P815AB priming or administering the recombinant cytokine in vivo. Different approaches suggest that IL-12 may act both on accessory cells to improve presentation of previously undescribed class II-restricted epitopes of P815AB and on CD4+ cells to improve recognition of such epitopes. In particular, at the CD4+ cell level, IL-12 apparently acts as an adjuvant and an inhibitor of anergy induction. These data offer useful information for developing vaccination strategies using dendritic cells and class I-restricted tumor peptides in humans.

Intrasplenic immunization for the induction of humoral and cell-mediated immunity to nitrocellulose-bound antigen.

Intrasplenic immunization of mice has been shown to induce both specific humoral and cell-mediated immunity to minute amounts of nitrocellulose-bound antigen, electroblotted from sodium dodecyl sulfate-polyacrylamide gels. The test antigens used were aberrantly expressed molecules immunoprecipitated from the lysate of highly immunogenic ('xenogenized') murine lymphoma cells, derived by mutagenesis from a parental, nonimmunogenic cell line. The stained bands of nitrocellulose blots corresponding to the appropriate molecular weights were cut out and the resulting strips deposited in the spleens of recipient mice on three occasions at 15 day intervals. 2 weeks later, the antibody response in the serum was analyzed using a standard ELISA procedure. Cell-mediated immunity was investigated in vitro in terms of cytotoxic T lymphocyte (CTL) activity to radiolabeled xenogenized tumor target cells. In vivo, the immunized mice were assayed for their ability to mount a delayed-type hypersensitivity (DTH) response following footpad challenge with the xenogenized tumor. Our results confirm previous data that the intrasplenic deposition of minute amounts of protein immobilized on a solid matrix effectively stimulates production of specific antibodies. In addition, our results demonstrate that this procedure may also result in the development of T cell-dependent responses detectable in in vitro and in vivo assays of cell-mediated immunity.

The immunosuppressive activity of proinflammatory cytokines in experimental models: potential for therapeutic intervention in autoimmunity.

The role of cytokines in the pathogenesis of T cell-mediated diseases, and in particular autoimmune responses, has been the subject of intense investigation in the past few years. Transgenic strains of mice have been generated, each expressing individual cytokines in organs targeted by autoimmunity. These animal models provide the most advanced tool available for analyzing the relationship between cytokines and T-dependent autoimmune responses. On the one hand, these experiments confirm that the local expression of proinflammatory cytokines is pivotal in initiating and maintaining pathogenic responses to self. On the other hand, and somewhat unexpectedly, these models have also revealed that cytokine factors controlling autoimmunity can act both as potentiating and inhibitory agents, depending upon the site and timing of exposure. As a result, one major concept emerging from different experimental models, including those originally established in our laboratory, is that proinflammatory cytokines may ameliorate autoimmunity. In this review, we analyze the mechanisms whereby cytokines that are considered as proinflammatory may in contrast suppress immune responses to self antigens. Besides emphasizing that the proinflammatory/immunogenic properties of a given cytokine may not be an intrinsic property, we review evidence that the regulation imposed by the cytokine network on autoimmunity is a finely tuned balance between activation and downmodulation of an individual autoreactive T cell repertoire. By emphasizing that factors such as the duration of cytokine exposure and the type of cell population involved strongly influence that balance, we underline the potential therapeutic implications of cytokine-mediated modulation of autoimmunity.

Endogenous retroviral gp70 genes of the murine lymphoma L5178Y: analysis of restriction fragment polymorphism upon induction of drug-mediated immunogenicity.

Highly immunogenic ("xenogenized") tumor variants appear after treatment of murine lymphoma L5178Y with the triazene derivative DTIC, this phenomenon being associated with the appearance of structurally abnormal gp70 env proteins in the cell variants. In the present study, we have isolated and sequenced several PCR-amplified gp70 cDNA genes from L5178Y cells. One of the resulting clones was used as a probe in Southern and Northern analysis of parental and xenogenized cells. The results indicated that xenogenization of tumor cells is associated with changes in retroviral env sequences detectable at the genomic level.

Evidence for tumor necrosis factor alpha as a mediator of the toxicity of a cyclooxygenase inhibitor in Gram-negative sepsis.

To investigate the effect of cyclooxygenase inhibition in experimental Gram-negative sepsis, indomethacin was administered to mice at different times (1 or 5 days, or 1 h) before sublethal infection with an intravenous inoculum of Pseudomonas aeruginosa Early indomethacin exposure did not alter the outcome of infection, yet treatment at the time of bacterial challenge resulted in a high mortality rate. Polymerase chain reaction-assisted mRNA amplification in the spleens of infected mice revealed that tumor necrosis factor alpha (TNF-alpha) messenger was selectively expressed by the drug-treated and infected mice during the 24 h preceding death. Higher TNF-alpha levels were found in sera from these mice, whose macrophages produced increased levels of nitric oxide in vitro. Both pentoxifylline, an inhibitor of TNF-alpha synthesis, and an inhibitor of nitric oxide production improved survival in the indomethacin-treated and infected mice, although no such effect followed the administration of TNF-neutralizing antibodies. These data support the notion that cyclooxygenase inhibitors may exert both positive and negative effects in Gram-negative sepsis, the latter presumably involving overproduction of TNF-alpha.

Dacarbazine-induced immunogenicity of a murine leukemia is attenuated in cells transfected with mutated K-ras gene.

Strong immunogenicity is induced by antitumor triazene compounds in tumor cells through a mutagenic mechanism. A highly immunogenic <> clone, isolated from a dacarbazine-treated L5178Y leukemia of DBA/2 mice, was transfected with K-ras mutated at codon 12 (i.e. ras(m12)). This transfected clone presents at least 2 mutations, one concerning K-ras gene, and the other affecting an unrelated gene, responsible for the generation of a highly immunogenic, MHC class I restricted non-self peptide. The results indicate that cells of <> clone transfected with ras(m12) were less immunogenic than cells of the same origin transfected with the vector alone. Moreover, ras(m12)-transfected cells showed lower levels of H-2K(d) gene expression with respect to those detectable in control cells. In addition, in vivo and in vitro sensitization against <> clone carrying mutated ras did not result in a strong cytotoxic T lymphocyte response against ras(m12)-transfected non immunogenic L5178y target cells. These preliminary results suggest that K-ras mutation could down-regulate the level of tumor immunogenicity, possibly acquired through a mutagenic process affecting other unrelated genes.

Loss of growth arrest DNA damage genes expression in oral melanomas.

29 oral melanomas were stained immunohistochemically for the GADD34, GADD45 and GADD153. GADD34 was found in 5/29 melanomas and its expression did not exceed 21%, averaging 4.1% of melanoma cells. GADD45 was observed in 8/29 oral melanomas and cell positivity averaged 2.8%. GADD153 was found in 2/29 melanomas and the percentage of positive cells ranged between 0 and 31%, averaging 1.2%. Not one significant correlation between GADD genes in oral melanomas was found. Loss of GADD gene expression and lack of correlation between them show advanced disturbances in their cooperation, leading to a high genetic instability of oral melanoma cells.

Using an ancient tool for igniting and propagating immune tolerance: IDO as an inducer and amplifier of regulatory T cell functions.

Although most CD4(+)CD25(+) regulatory T (T(reg)) cells develop in the thymus (i.e., natural T(reg) or nT(reg)), accumulating evidence suggests that they can also develop in the periphery (adaptive/induced T(reg) or iT(reg)). Both types of cells are functionally associated with the expression of Foxp3, a transcription factor that is constitutively expressed in nT(reg) cells and inducible during iT(reg) cell generation from CD4+CD25- T lymphocytes. Multiple factors are involved in the generation and function of T(reg) cells, but a major role seems to be played by indoleamine 2,3-dioxygenase (IDO). IDO can both deplete tryptophan in local tissue microenvironments and generate immunoregulatory catabolites, known as kynurenines. Tryptophan starvation and presence of kynurenines can induce the conversion of naïve CD4(+)CD25(-) T cells into highly suppressive CD4(+)CD25(+)Foxp3(+) T(reg) cells. In turn, T(reg) cells induce IDO in dendritic cells (DCs) and convert inflammatory into regulatory DCs, which can further expand the T(reg) cell compartment by tryptophan catabolism. Evidence suggests that the modulation of IDO activity favors the interconversion between T(reg) cells and T helper type 17 (T(H)17) inflammatory cells. Thus, in the periphery, tolerogenic immune responses mediated by T(reg) cells can be induced and amplified by IDO, a tryptophan catabolizing enzyme that also contributes to the plasticity of the T(reg) cell lineage.

Hemisection and vital treatment of a fused tooth--literature review and case report.

Fusion and gemination of permanent teeth are developmental anomalies of the dental hard tissues which may require endodontic and surgical treatment for functional, orthodontic or aesthetic reasons. Following a review of the dental literature on tooth fusion and gemination, a case of fusion of a maxillary central incisor and a supernumerary tooth and its endodontic and surgical treatment is presented.

Cell-mediated immunity to chemically xenogenized tumors--VI. The effect of cell treatment with retroviral env antisense oligonucleotides.

The occurrence of aberrant, retrovirus-related proteins is a common finding in immunogenic clones of the triazene-xenogenized L5178Y lymphoma line (L5178Y/DTIC). In clone D-cells, newly expressed 80 kDa antigens related to xenotropic murine leukemia virus env gene products induce specific humoral and cell-mediated responses and possess biologic activity in vivo. To further clarify the relationship between immunogenic properties of clone D and retroviral gene expression, tumor cells were treated in vitro with antisense oligonucleotides complementary to xenotropic and/or polytropic env sequences of murine leukemia virus. The cells were then assayed for expression of antigens recognized by humoral and cell-mediated responses with specificity for clone D. The results demonstrated that inhibition of env mRNA translation adversely affected the expression of immunogenic determinants in the xenogenized tumor cells.

Cell-mediated immunity to chemically xenogenized tumors. II. Evidence for accessory function and self-antigen presentation by a highly immunogenic tumor variant.

To determine whether antigen-presenting ability might be involved in the superior immunogenicity of chemically xenogenized tumors over that of parental cells, we tested a murine lymphoma line xenogenized by a triazene derivative for expression of Ia antigens, ability to present soluble antigen in vitro, and production of factor(s) active in a mouse thymocyte assay. Results showed that Ia antigens, absent on nonimmunogenic parental L5178Y cells, were expressed on a xenogenized, highly immunogenic tumor variant (clone D), as detected by immunofluorescence. While the ability of parental cells to stimulate lymphocyte proliferation in vitro was lost on removal of Ia+ cells from the responder population, considerable augmentation of reactivity was observed upon depletion of Ia+ cells from the population of splenocytes responding to the xenogenized cells. Under these conditions, stimulation was blocked by anti-Ia antibodies, or an anti-L3T4 reagent or antibodies to the novel antigenic determinants induced by xenogenization. In addition, no stimulating activity was observed following exposure of clone D cells to glutaraldehyde or lysosomotropic agents such as chloroquine and ammonia. When the ability of clone D cells to present ovalbumin in vitro was assayed, it was found that the xenogenized cells could present the soluble antigen to specifically primed lymphocytes. Moreover, clone D cells could substitute for splenic adherent cells in the proliferative reaction of splenocytes to concanavalin A. Finally, when the supernate from clone D-cell culture pulsed with phorbol myristic acetate was tested in a mouse thymocyte assay, considerable IL-1-like activity was disclosed.

Increase of natural killer activity of mouse lymphocytes following in vitro and in vivo treatment with lithium.

The in vivo and in vitro influence of lithium lactate on mouse natural killer activity was investigated. In vitro exposure of effector-target mixture to graded concentrations of lithium did not substantially modify the natural killer activity of mouse splenocytes, untreated or pretreated with cyclophosphamide. However in vitro treatment of effector splenocytes increased the frequency of NK-percursor cells. The in vivo treatment with lithium lactate greatly increased the natural killer activity in intact mice, whereas it did not improve this cytotoxic function in host immunodepressed by cyclophosphamide. These data suggest that lithium salts produce a modulation of natural killer activity of mouse spleen cells, probably through a mechanism involving the increase of the number of NK-precursors in hosts not subjected to cytotoxic chemotherapy.

Cell-mediated immunity to chemically xenogenized tumors--III. Generation of monoclonal antibodies interfering with reactivity to novel antigens.

To develop monoclonal antibodies (MAbs) recognizing drug-mediated tumor antigens on a chemically xenogenized murine lymphoma, hybridomas were constructed with splenocytes from histocompatible mice hyperimmunized with L5178Y cells antigenically altered by triazene treatment in vivo (clone D, derived from a polyclonal L5178Y/DTIC subline). Screening of supernatants with parental and xenogenized cells showed that nine MAbs displayed exclusive or preferential reactivity with clone D cells as detected by immunofluorescence, and failed, as a rule, to bind normal or unrelated malignant cells of the same or different haplotype. Moreover, no reactivity was displayed to the triazene-xenogenized variants of antigenically unrelated tumors. All nine MAbs, however, were capable of binding a panel of L5178Y/DTIC clones in addition to clone D. When the ability of these antibodies to interfere with the development of cell-mediated immunity to clone D cells in vitro was tested, it was found that the proliferative reaction and generation of cytolytic activity by syngeneic lymphocytes were inhibited by addition of several MAbs to the tumor--lymphocyte co-cultures.