Non-eosinophilic airway hyper-reactivity in mice, induced by IFN-γ producing CD4(+) and CD8(+) lung T cells, is responsive to steroid treatment.

Non-eosinophilic asthma is characterized by infiltration of neutrophils into the lung and variable responsiveness to glucocorticoids. The pathophysiological mechanisms have not been characterized in detail. Here, we present an experimental asthma model in mice associated with non-eosinophilic airway inflammation and airway hyper-responsiveness (AHR). For this, BALB/c mice were sensitized by biolistic DNA immunization with a plasmid encoding the model antigen ß-galactosidase (pFascin-ßGal mice). For comparison, eosinophilic airway inflammation was induced by subcutaneous injection of ßGal protein (ßGal mice). Intranasal challenge of mice in both groups induced AHR to a comparable extent as well as recruitment of inflammatory cells into the airways. In contrast to ßGal mice, which exhibited extensive eosinophilic infiltration in the lung, goblet cell hyperplasia and polarization of CD4(+) T cells into Th2 and Th17 cells, pFascin-ßGal mice showed considerable neutrophilia, but no goblet cell hyperplasia and a predominance of Th1 and Tc1 cells in the airways. Depletion studies in pFascin-ßGal mice revealed that CD4(+) and CD8(+) cells cooperated to induce maximum inflammation, but that neutrophilic infiltration was not a prerequisite for AHR induction. Treatment of pFascin-ßGal mice with dexamethasone before intranasal challenge did not affect neutrophilic infiltration, but significantly reduced AHR, infiltration of monocytes and lymphocytes as well as content of IFN-γ in the bronchoalveolar fluid. Our results suggest that non-eosinophilic asthma associated predominantly with Th1/Tc1 cells is susceptible to glucocorticoid treatment. pFascin-ßGal mice might represent a mouse model to study pathophysiological mechanisms proceeding in the subgroup of asthmatics with non-eosinophilic asthma that respond to inhaled steroids.

Disproportion in T-cell subpopulations in immunodeficient mutant hr/hr mice.

Mice of the HRS strain, which carry the mutant gene hr, were examined for abnormalities in representation of the three T-cell sets Ly1, Ly23, and Ly123 in the spleen. The salient feature of hr/hr mice, which are immunologically deficient, in comparison with +/hr segregants, was a gross disproportion in numbers of cells belonging to the Ly1 and Ly123 sets, at the age of 3--3.5 mo. At this age, Ly123 cells of hr/hr spleen outnumbered Ly1 cells by 2:1, whereas in +/hr spleens Ly123 cells were outnumbered by approximately 1:2. Cells from pooled lymph nodes of hr/hr mice did not show a correspondingly gross disporprotion of Ly1 and Ly123 cells. Total counts of splenic T cells, and of B cells, were not significantly different in hr/hr and +/hr mice.

CD4-mediated enhancement or inhibition of T cell activation does not require the CD4:p56lck association.

CD4 is the coreceptor molecule expressed on the surface of T cells specific for or restricted by class II molecules of the major histocompatibility complex (MHC). Its expression on T cells is required for an optimal response to antigen (Ag). Three mechanisms have been invoked for the involvement of CD4 in T cell activation. First, it was shown that CD4 binds to MHC class II molecules on antigen presenting cells (APCs) thereby favoring an adhesion between effector cells and APCs. Association of CD4 to the T cell receptor and to the tyrosine kinase p56lck have also been shown to be critically involved in the positive function of CD4. Here, we demonstrate that the interaction of CD4 with p56lck is not required to enhance the response of two CD4-dependent, Ag-specific T cell hybridomas. Mutant forms of CD4 (TCD4), which lose association to p56lck, were expressed in these T cells and were shown to enhance the Ag-specific response as efficiently as the wild-type CD4. Moreover both CD4-dependent and independent T cell responses were inhibited by CD4-specific mAbs even when CD4 was not associated with p56lck. These results indicate that mechanisms distinct from sequestration of p56lck and/or negative signaling operate in these inhibitions. Results demonstrating enhancement of TCR-mediated signaling by the coaggregation of TCD4 mutant to the TCR further confirm that the association of p56lck to CD4 is not absolutely required for the regulatory functions of CD4. Our results suggest that the mechanisms implicated in the enhancement of T cell stimulation via CD4 depend solely on the extracellular and transmembrane domains of CD4.

Acquisition of an additional antigen specificity after mouse CD4 gene transfer into a T helper hybridoma.

We have transfected the mouse CD4 gene into a beef insulin (BI)-specific murine T helper hybridoma that lacks CD4 surface expression. The CD4-expressing transfectants have acquired an additional reactivity for pork insulin (PI), which was not detectable in the original recipient cell. The transfectants' response to PI can be completely abrogated by anti-CD4 antibodies. The transfected clone showed a 50-fold increased sensitivity towards BI in comparison to the same CD4- hybridoma. These experiments suggest that CD4 may be important in determining the antigen fine specificity and, therefore, may also play a role in altering the T cell repertoire.

Antigen dose-dependent suppression of murine IgE responses is mediated by CD4(-)CD8(-) double-negative T cells.

BACKGROUND: The IgE response against protein antigens is profoundly influenced by the dose used for sensitization. OBJECTIVE: The aim of the study was to identify immune cells that are involved in antigen dose-dependent regulation of IgE formation. METHODS: Wild-type mice as well as T helper (Th)1-deficient IL-12p40(-/-) and IFN-gamma(-/-) mice were immunized by repeated intraperitoneal injection of either low doses (K01 mice) or high doses (K100 mice) of keyhole limpet haemocyanin adsorbed to aluminium hydroxide. Splenocytes of immunized mice were restimulated in vitro and antigen-dependent T cell proliferation and cytokine production were measured. The frequency of regulatory T cell subsets among splenocytes from K01 and K100 mice was compared using fluorocytometry and RT-PCR analysis. Splenocytes or T cell subpopulations were transferred into naïve mice and the effect of lymphocyte transfer on IgE production after priming of recipients with low antigen doses was determined. RESULTS: Specific IgE production was considerably impaired in K100 mice. Antigenic restimulation revealed hypoproliferation of K100 splenocytes and reduced production of Th2 cytokines IL-4, IL-5 and IL-13, but no induction of IFN-gamma production. Moreover, lymphocytes from K01 and K100 mice did not show significant differences in the expression of molecules associated with the phenotype or activity of conventional regulatory T cells. Transfer of splenocytes or purified T cells from K100 mice substantially suppressed the induction of IgE production in the recipients in an antigen- and isotype-specific manner. Neither CD4(+) nor CD8(+) T cells from K100 mice were able to inhibit IgE formation; instead, we identified CD4(-)CD8(-) double-negative T cells (dnT cells) as the principal T cell population, which potently suppressed IgE production. CONCLUSION: Our data demonstrate that CD4(-)CD8(-) dnT cells play a major role in the regulation of IgE responses induced by high antigen doses.

The specificity of the interaction between the agretope of an antigen and an Ia-molecule can depend on the T cell clonotype.

A series of T cell clones was developed from (B10 x B10.BR)F1 mice immunized with the isolated A chain of pig insulin. The T cell clones show considerable diversity as defined by their distinct reactivities to pig, beef, sheep and horse insulins in combination with the same syngeneic Ab alpha Ak beta molecules. These species variants of insulin differ from each other only in amino acid residues in position A8, A9 or A10 within the so-called A chain loop and responsiveness of mice to these variants is under Ir gene control. A detailed analysis of the stimulatory capacity of various insulin/Ia combinations including inhibition experiments with anti-Ia- and -L3T4 antibodies led to the following interpretation: the amino acid residues A8-A10 are involved in the interaction of the insulin A chain with the Ia molecules. This region can, therefore, be regarded as part of the agretope. Structural variations within this region can modify the stimulatory potency of the insulin variants. However, whether a particular amino acid substitution results in an enhancement or a reduction of the response depends on the fine specificity of the T cell clone involved. Thus, an interaction of Ia molecules with antigen cannot solely account for the functional specificity of an agretope, rather this also depends on the structure of the particular T cell receptor that participates in recognition.

Suppression of MHC class I antigens in oncogenic transformants: association with decreased recognition by cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) recognize peptide fragments derived from endogenous proteins, processed internally, and presented at the cell surface by major histocompatibility complex (MHC) class I molecules. The use of specific CTL for cancer therapy is limited because of their dependence on effective processing and presentation of appropriate antigenic peptides. Structural alterations, like point mutation or somatic loss, or dysregulation of key elements in the processing or presentation pathway, may allow cells to escape the immune surveillance. Indeed, the expression of MHC class I antigens on the surface of virus- and oncogene-transformed cells is low and correlates with tumorigenicity. Transformation of murine fibroblasts with the ras oncogene results in the suppression of cell surface expression of all H-2 loci as determined by FACScan analysis using a panel of monoclonal antibodies. We then examined whether the oncogene-mediated suppression of MHC class I surface expression was associated with reduced recognition of transformants by CD8+ T lymphocytes. Murine T lymphoma cells were stably transfected by the Ha-ras oncogene. The transfectants expressed distinct levels of the Ha-ras specific protein p21. Again, immunofluorescence analysis demonstrated an inverse correlation between oncogene and MHC class I surface expression in RMAras transformants. Allogeneic H-2Kb-restricted cytotoxic T lymphocytes were able to efficiently lyse the parental T lymphoma cells. In contrast, the CTL-mediated lysis of ras transformants was significantly downregulated compared with untransfected RMA cells. The efficiency of CTL-mediated lysis of RMAras cells was directly associated with reduced MHC class I membrane and high p21ras protein expression. Thus, the oncogene-mediated downregulation of MHC class I surface expression resulted in a reduced CTL response. Attempts are in progress to revert the defects in MHC class I surface expression of oncogenic transformants by introducing the different elements of the antigen presentation pathway. Such studies will not only provide improved understanding of the mechanisms of tumor escape, but also will suggest strategies to repair cellular defects in cancer patients having impaired expression of MHC class I antigens.

Maturation of epidermal Langerhans cells in vitro is accompanied by downregulation of 4F2 (CD98) as determined by differential display.

Following short-term culture, Langerhans cells mature morphologically and functionally into potent immunostimulatory cells. As regulation of gene expression accompanies this maturation process, it is likely that differentially expressed genes are involved in the maturation events. Using the recently described method of differential display, we generated cDNA expression patterns starting with mRNA of murine epidermal Langerhans cells isolated either directly (fLC) or following 3 d cultivation (cLC). Five hundred putative differentially expressed cDNA fragments were recovered from the gel. For a part of the fragments differential expression was confirmed by dot blot and Southern hybridization procedures. These cDNA fragments were subcloned and sequenced following the verification step. Database searches revealed that unknown genes as well as already characterized genes were identified. A cDNA fragment preferentially hybridizing with fLC was identified as the murine surface marker 4F2 (CD98). Downregulation of the activation marker 4F2/CD98 was confirmed by additional analysis at the mRNA and protein level. The downregulation of 4F2 surface expression on cLC is compatible with the notion that the committed, terminally differentiated cLC downregulate proteins involved in proliferation and cell survival.

Mouse langerhans cells differentially express an activated T cell-attracting CC chemokine.

Epidermal Langerhans cells represent an immature population of dendritic cells, not yet able to prime naïve T cells. Following in vitro culture Langerhans cells mature into potent immunostimulatory cells. We constructed a representative cDNA library of in vitro matured murine Langerhans cells. Applying a differential screening procedure 112 differentially expressed cDNA clones were isolated. Thirty-six clones represented cDNA fragments of the same gene, identifying it to be the most actively expressed gene induced in maturing Langerhans cells. A full-length cDNA was sequenced completely. The open reading frame codes for a protein of 92 amino acids containing a leader peptide of 24 amino acids, yielding a mature protein of 7.8 kDa molecular weight. Database searches revealed 99.4% sequence identity on the nucleotide level to the recently described mouse CC chemokine ABCD-1, as well as 74% sequence identity to the human CC chemokine, the macrophage-derived chemokine/stimulated T cell chemotactic protein. Expression was analyzed by reverse transcriptase-polymerase chain reaction on a large panel of cell types. Unlike the macrophage-derived chemokine, expression was not detected in macrophages stimulated by various cytokines. Expression is restricted to cultured Langerhans cells, in vitro cultured dendritic cells, and lipopolysaccharide-activated B cells. Recombinant protein was expressed in the yeast Pichia pastoris and purified to homogeneity. Whereas no chemotactic activity was observed in chemotaxis assays for naïve T cells, B cells, cultured dendritic cells, and Langerhans cells, a strong chemoattractant activity was exerted on activated T cells. Thus, production of this chemokine by dendritic cells may be essential for the establishment and amplification of T cell responses.

Expression of the actin-bundling protein fascin in cultured human dendritic cells correlates with dendritic morphology and cell differentiation.

Dendritic cells are key players of the immune system as they efficiently induce primary immune responses by activating naive T cells. We generated human dendritic cells from CD14+ blood precursors and investigated expression of the actin-bundling protein fascin during maturation by western blotting, immunofluorescence, and cytofluorometry. Cells obtained by culture of CD14+ blood precursors in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, which were only weakly positive for the maturation marker CD83, expressed low amounts of fascin. Addition of a cytokine cocktail including tumor necrosis factor alpha, interleukin-1beta, interleukin-6, and prostaglandin E2 induced maturation of the cells and enhanced fascin expression in parallel with CD83 expression. Isolated mature CD83+ cells displayed especially high fascin levels on western blots, as did gated CD83+ dendritic cells in cytofluorometry. Dendritic cells generated from CD34+ blood precursors expressed high levels of fascin as well. Confocal microscopy revealed that location of fascin within the cell was restricted to the area of the submembranous actin cytoskeleton and to the dendritic processes. Suppression experiments using antisense constructs of fascin hint at a retarded morphologic maturation of dendritic cells, supporting the view that fascin expression is pivotal for dendrite formation. Our data suggest that fascin could serve as a marker molecule to monitor the maturation state of in vitro generated dendritic cells for use in clinical trials.

Action of complement in the lysis of mouse sarcoma cells sensitized with H-2 alloantibody.

A modified cytotoxicity assay was adapted from classical erythrocytolytic assays, in which complement components were added in sequence to antibody-sensitized cells. This assay was applied to a model system in which mouse sarcoma cells were sensitized with H-2 alloantibody. The stepwise presentation of complement components combined with the stabilization of C2 by iodine treatment considerably augmented the lytic efficiency of human complement. More generally, the techniques adopted for this study provide a new model for obtaining basic information about selective reaction steps concerned in lysis of nucleated cells by alloantibody and complement. Comparisons of the lysis of sheep erythrocytes by xenoantibody with the lysis of mouse sarcoma cells by H-2 alloantibody, in our assay system with oxidized or untreated human complement, disclosed a difference in the kinetics of C142 formation, manifest in a lag phase and a protracted tmax for the sarcoma cells. These data may suggest that multiple complement-mediated functional lesions are necessary for immune lysis of nucleated cells.

PCR-amplified cDNA probes for verification of differentially expressed genes.

Differential display has proven to be a powerful technique for the detection and isolation of differentially expressed genes. By generating reproducible cDNA expression patterns, it is possible to compare gene expression by two or more cell types, developmental stages or tissues and to isolate as yet unknown differentially expressed genes. A sensitive method is necessary to verify the differential expression of the isolated cDNAs. Here we describe the use of adaptor-ligated. PCR-amplified total cDNA of the two cell types compared as a probe for Southern hybridizations with the isolated cDNAs.

Nonradioactive detection of differentially expressed genes using complex RNA or DNA hybridization probes.

The analysis of differential gene expression has become increasingly important in recent years. Typically, differentially expressed genes are identified in a primary screening procedure, yielding candidate genes whose differential expression has to be verified. We provide a highly sensitive, efficient and nonradioactive differential screening procedure to analyze numerous candidate genes in a single step. This comprises labeling of poly(A)+ RNA of the cell types analyzed with DIG Chem-Link and differential hybridization to the candidate genes fixed on dot blots. DIG Chem-Link allows, to our knowledge, for the first time efficient and direct nonradioactive labeling of RNA in vitro. Advantages of this method include extremely short exposure times and the feasibility to re-use the probes after prolonged storage. Using this procedure, we isolated several genes that are differentially expressed in maturing Langerhans cells.

Perpetual proliferation of LYT-1 cells requires repetitive signals for IL-2 receptor induction by antigen-presenting cells.

T cell lines with specificity for bovine insulin and ovalbumin were maintained by serial stimulation with antigen presented on irradiated syngeneic spleen cells, alternating 3 days later with subculture in IL-2 containing medium (CM). When the cultures were repetitively split in CM, with concomitant dilution of antigen-presenting cells, a gradual loss of proliferative capacity of the cells in the presence of CM was observed. Absorption studies revealed a 20-fold reduction of IL-2 receptors on the surface of T blasts assayed 12 days after antigenic stimulation as compared with day 5 blasts. This decrement in the number of IL-2 acceptor sites reflected an actual decrease in cell surface density of IL-2 receptors. Restimulation of the T blasts with antigen and spleen cells induced both a substantial increase in IL-2 receptor density and responsiveness to CM. Furthermore, the permanent presence of antigen and spleen cells during splitting of the T blasts in CM prevented the loss of responsiveness to IL-2. As an interpretation we propose that the Lyt-1 cells studied here clear their IL-2 receptors from the cell surface after interaction with IL-2. Thus, each new round of replication of the daughter cells would be dependent on induction of IL-2 receptors by activating signals provided by antigen/Ia structures on accessory cells as well as possibly accessory cell products such as IL-1, rendering Lyt-1 cells sensitive to regulatory influences.

Characterization of lymphokine-mediated activation of macrophages for antigen presentation: studies with long-term cultured bone marrow-derived macrophages and cloned T cells.

In cultures of bone marrow (BM) supplemented with L cell-derived colony-stimulating factor a pure population of macrophages (M phi) differentiates, which can be further propagated with a doubling time of 3.8 days. "Young" BMM phi obtained on day 8 of culture were shown to act as antigen-presenting cells inducing the antigen-specific proliferation of the cloned T cell line ST2/K.9, whereas "old" M phi had lost this ability. However, at any time tested (up to 132 days) the presentation function of old BMM phi could be completely restored by pulsing the cells with lymphokines (LK). A duration of 11 hr for the LK-pulse was sufficient to trigger the M phi to exert an optimal presentation function. This activity could be maintained when the LK-treatment was prolonged (tested up to 17 days). Activation was accompanied by a deceleration of growth. The LK effective in M phi activation were found to be contained in the supernatants of T cell lines stimulated by antigen or mitogen, and could be substituted by a low dose (5-10 units/ml) of recombinant interferon-gamma. In direct comparison LK-triggered BMM phi presented antigen as efficiently as peritoneal exudate M phi activated in vivo by ConA. Moreover, primed lymph node T cells responded to antigen-presenting BMM phi in a similar way as ST2/K.9 T cells. Therefore, these findings obtained with long-term cultured cells can be expected to reflect a physiological mechanism for the amplification of the immune response.

Consequences of antigen self-presentation by tumor-specific cytotoxic T cells.

CD8-positive cytotoxic T cells (CTL) recognize antigenic peptides in combination with major histocompatibility complex (MHC) class I molecules on the surface of syngeneic antigen presenting cells (APC). In the present paper we show that cells from tumor antigen-specific CTL clones present their cognate antigenic peptide to other CTL from the same clone. Inter-CTL peptide presentation resulted in activation of the cells of one CTL clone to MHC-unrestricted lysis of bystander cells. In contrast to the behaviour of this clone, another CTL clone did not lyse bystander cells after incubation with the cognate peptide, but was activated to self-destruction. The human herpes virus Epstein-Barr virus is involved in the pathogenesis of a broad spectrum of human neoplasias. Using freshly established non-clonal T cells with specificity for a peptide derived from an Epstein-Barr virus encoded antigen we found again lysis of MHC mismatched bystander cells as a consequence of inter-CTL peptide presentation, indicating that bystander lysis following antigen self-presentation is not a phenomenon restricted to long-term in vitro cultured T cell clones. The potential implications for immunosurveillance against cancer and for tumor escape mechanisms are discussed.

The role of NO in contact hypersensitivity.

Contact dermatitis or contact hypersensitivity (CHS) is a common T lymphocyte-mediated allergic disease characterized by local inflammatory skin reactions following contact with small reactive compounds called haptens. In common with other allergic processes, the development of contact dermatitis proceeds in two phases: a sensitization phase which occurs on first exposure to allergen, and an elicitation phase which occurs on subsequent exposure when the clinical manifestations of the disease are observed. This process is hapten-specific. While the pathophysiology of the sensitization phase is well characterized, our understanding of the elicitation phase is still incomplete, including the relative contribution of the different effector cells and mediators involved. Here we summarize current knowledge of the contribution of nitric oxide (NO) to skin inflammation with special focus on CHS. A number of inflammatory stimuli trigger expression of NO in human and animal skin, and topical application of an NO-releasing cream results in inflammation. Moreover, expression of the inducible isoform of nitric oxide synthase (iNOS) is induced in CHS and iNOS inhibitors injected intradermally suppress CHS responses. However, iNOS-deficient mice develop an aggravated CHS response late in the elicitation phase, suggesting that NO is involved in downregulation of CHS. Based on these data, we propose a comprehensive model of the role of NO in CHS.

Fine specificity of a T cell line reactive to bovine insulin.

A continuous T cell line was established from lymph node T cells of (high responder X low responder) hybrid mice ((B10 X B10.BR)F1) immune to bovine insulin (BI). T cells were expanded by restimulation with BI presented on irradiated syngeneic spleen cells and by culture in interleukin 2-containing medium. The proliferative response, measured in a 3H-thymidine incorporation assay, was specific for the selecting antigen BI; unrelated proteins did not induce DNA synthesis. However, in addition to BI the T blasts were efficiently restimulated by pig and sheep insulin, which are identical to BI except for the amino acid sequence in the A chain loop. This suggests that the T blasts recognized an antigenic determinant(s) different from the A chain loop epitope of BI, the essential antigenic moiety for cells of high responder parental H-2b mice. Induction of the blasts to DNA synthesis by the three insulins required the presence of F1 spleen cells. Parental strain B10 or B10.BR splenocytes failed to support a proliferative response, even when present as a mixture. The antigen-presenting determinants unique to F1 cells were mapped to the I-Ab/Kb and I-Ak loci. These data demonstrate that, although at least the majority of responding T cells in H-2b high responder mice recognize the A chain loop determinant of BI in the context of restriction elements of the b haplotype, T cells can be isolated from (high responder X low responder)F1 mice, which require recognition of other determinants on the insulin molecule in the context of F1 unique restriction structures for stimulation to occur.