Th17-cell plasticity in Helicobacter hepaticus-induced intestinal inflammation.

Bacterial-induced intestinal inflammation is crucially dependent on interleukin (IL)-23 and is associated with CD4(+) T helper type 1 (Th1) and Th17 responses. However, the relative contributions of these subsets during the induction and resolution of colitis in T-cell-sufficient hosts remain unknown. We report that Helicobacter hepaticus-induced typhlocolitis in specific pathogen-free IL-10(-/-) mice is associated with elevated frequencies and numbers of large intestinal interferon (IFN)-γ(+) and IFN-γ(+)IL-17A(+) CD4(+) T cells. By assessing histone modifications and transcript levels in IFN-γ(+), IFN-γ(+)IL-17A(+), and IL-17A(+) CD4(+) T cells isolated from the inflamed intestine, we show that Th17 cells are predisposed to upregulate the Th1 program and that they express IL-23R but not IL-12R. Using IL-17A fate-reporter mice, we further demonstrate that H. hepaticus infection gives rise to Th17 cells that extinguish IL-17A secretion and turn on IFN-γ within 10 days post bacterial inoculation. Together, our results suggest that bacterial-induced Th17 cells arising in disease-susceptible hosts contribute to intestinal pathology by switching phenotype, transitioning via an IFN-γ(+)IL-17A(+) stage, to become IFN-γ(+) ex-Th17 cells.

Lineage fate alteration of thymocytes developing in an MHC environment containing MHC/peptide ligands with antagonist properties.

A18 TCR transgenic thymocytes which are H-2E(k) restricted and normally selected into the CD4 lineage, are exclusively selected into the CD8 lineage in an H-2(q) MHC background. CD8 T cell selection in the H-2(q) background is far more efficient than default selection of A18 CD8 cells on a CD4(-/-) H-2E(k +) background. This suggests the involvement of special selecting ligands. Analogues of the cognate peptide for A18 with antagonist properties for the A18 TCR have previously been shown to effect a lineage diversion from CD4 to CD8 in fetal thymic organ cultures and intriguingly the MHC(q) background contains unidentified natural MHC class II ligands which similarly show antagonist properties for the A18 TCR. Despite the presence of these unidentified MHC class II ligands in the H-2(q) background and their potential influence on developing A18 thymocytes, however, MHC class I molecules were essential for thymic selection of A18 CD8 T cells.

Low CD4(+) T cell responses to the C-terminal region of the malaria merozoite surface protein-1 may be attributed to processing within distinct MHC class II pathways.

The C-terminal fragment of merozoite surface protein-1 (MSP-1) of the mouse malaria parasite Plasmodium chabaudi chabaudi (AS) stimulates a weak CD4 T cell response when compared to the response to a more structurally simple region of the molecule. The tertiary structure of the C-terminal region of MSP-1 is maintained by five disulfide bonds. A peptide from this region could only be processed and loaded onto newly synthesized MHC class II molecules, whereas a peptide from the structurally simple region was available for loading onto recycling MHC class II. CD4(+) T cell hybridomas took longer to recognize an epitope derived from the disulfide-bonded region whether native parasite or recombinant MSP-1 antigen was used. Reduction of disulfide bonds in the C-terminal region subsequently allowed peptides to be loaded onto recycling MHC class II and greatly enhanced the rapidity of the T cell response. These data demonstrate that differential processing occurs intramolecularly in MSP-1, which may be responsible for the observed weak CD4 T cell responses against this region. The consequences of this in vivo may be that limited T cell help is available for protective antibody production which has important implications for designing vaccines based on MSP-1.

Differential survival of naive CD4 and CD8 T cells.

In this paper we compare survival characteristics of transgenic and polyclonal CD4 and CD8 T cells. Transgenic CD4 T cells have an intrinsically lower capacity for survival, reflected in their gradual disappearance in thymectomized hosts, their increased sensitivity to apoptosis in vitro, and fewer divisions during homeostatic proliferation upon transfer into syngeneic lymphopenic hosts compared with CD8 T cells. Homeostatic proliferation, however, does not generally result in phenotypic conversion of activation markers unless cognate or cross-reactive Ag is present. T cells from the A18 TCR transgenic strain normally selected into the CD4 lineage are fragile as CD4 T cells, yet display the typical robust survival pattern of CD8 T cells when diverted into the CD8 lineage in a CD4-deficient host. Polyclonal CD4 and CD8 T cells also show distinctive patterns of survival, emphasizing that survival signals are relayed differently in the two lymphocyte subpopulations. However, expression levels of Bcl-2 in either transgenic or polyclonal naive CD4 and CD8 T cells are similar, excluding a role for this molecule as a key factor in differential survival of CD4 vs CD8 T cells.

Following the development of a CD4 T cell response in vivo: from activation to memory formation.

The in vivo differentiation of CD4 T cells from naive to memory cells was followed after their adoptive transfer together with syngeneic dendritic cells into MHC mismatched adoptive hosts lacking lymphocytes and NK cells. Functional and molecular changes were measured as the antigenic stimulus, provided by the cotransferred dendritic cells, disappeared. Memory cells as opposed to effector cells show an inversion in the relative expression of Bcl-2 family members in favor of antiapoptotic molecules, and compared with naive cells they have an increased ratio of bcl-xL to bcl-2. They differ qualitatively from naive T cells, suggesting that accelerated CD4 memory responses can occur without the need for increased frequencies of specific T cells.

The HSC73 molecular chaperone: involvement in MHC class II antigen presentation.

Heat shock proteins (HSP) are conserved proteins, many of which share the ability for indiscriminate peptide binding and ATPase-coupled peptide release. In this paper, we show that heat shock cognate protein (HSC)73, a constitutively expressed member of the HSP70 family, could be a candidate for chaperone activity within the MHC class II presentation pathway. HSC73 expression in macrophages was shown to overlap with expression of MHC class II; overexpression of HSC73 in stable transfectants of a macrophage line markedly enhanced their presentation of exogenous Ag without affecting presentation of processing independent peptide. Ag from an exogenous source was demonstrated to associate with HSC73 in macrophages, and this association was sensitive to ATP treatment and inhibited by deoxyspergualin, an immunosuppressive agent that has previously been shown to bind specifically to HSC73. Furthermore, deoxyspergualin reduced Ag presentation by macrophages in relation to the amount of HSC73 expressed in these cells. The data are consistent with a potential role for HSC73 in binding and protecting peptides from extensive degradation and/or facilitating the kinetics of peptide transfer to MHC class II molecules.

Triggering a second T cell receptor on diabetogenic T cells can prevent induction of diabetes.

In this paper, we test the hypothesis that triggering of a second T cell receptor (TCR) expressed on diabetogenic T cells might initiate the onset of diabetes. A cross between two TCR-transgenic strains, the BDC2.5 strain that carries diabetogenic TCRs and the A18 strain that carries receptors specific for C5, was set up to monitor development of diabetes after activation through the C5 TCR. F1 BDC2. 5 x A18 mice developed diabetes spontaneously beyond 3-4 mo of age. Although their T cells express both TCRs constitutively, the A18 receptor is expressed at extremely low levels. In vitro activation of dual TCR T cells followed by adoptive transfer into neonatal or adult F1 mice resulted in diabetes onset and death within 10 d after transfer. In contrast, in vivo immunization of F1 mice with different forms of C5 antigen not only failed to induce diabetes but protected mice from the spontaneous onset of diabetes. We propose that antigenic stimulation of cells with low levels of TCR produces signals inadequate for full activation, resulting instead in anergy.

DNA vaccination: transfection and activation of dendritic cells as key events for immunity.

The mechanisms underlying initiation and maintenance of CD4 T cell responses after DNA vaccination were studied using a construct coding for nonsecreted fifth component of complement (C5) protein, thus restricting the availability of antigen. The only cell types to express C5 were keratinocytes at the site of DNA application and a small number of dendritic cells present in the draining lymph nodes. Antigen expression persisted for up to 12 wk in keratinocytes, but dendritic cells did not express C5 beyond 2 wk after vaccination. Cross-priming of dendritic cells by C5 expressed in keratinocytes did not occur unless keratinocyte death was induced by irradiation in vitro. CD4 T cells were activated in the draining lymph nodes only and subsequently migrated to the spleen, where memory T cells persisted for longer than 40 wk despite the absence of a source of persistent antigen. While DNA vaccination resulted in transfection of a small proportion of dendritic cells only, it led to general activation of all dendritic cells, thus providing optimal conditions for effective T cell activation and maintenance of memory.

The Ii41 isoform of invariant chain mediates both positive and negative selection events in T-cell receptor transgenic mice.

The functional role of invariant chain in T-cell selection events and antigen presentation is well established. The invariant chain gene encodes differentially spliced isoforms, Ii31 and Ii41. The Ii41 isoform has been described to increase the efficiency of antigen presentation. We have analysed the effect of the Ii41 isoform on positive and negative selection of transgenic CD4 T cells with specificity for a natural self antigen (C5) which are crucially dependent on invariant chain for their development and functional antigen recognition. The data show that Ii41 fully substitutes for wild-type invariant chain in both positive and negative selection events during functional maturation of T cells with specificity for a natural, blood-borne self antigen.

Normal thymic selection of TCR transgenic CD4 T cells, but impaired survival in the periphery despite the presence of selecting MHC molecules.

In this paper, we investigate selection in the thymus and survival in the periphery of CD4 T cells, which carry a major histocompatibility class II-restricted transgenic TCR (A18 TCRtg) specific for a natural self Ag, the fifth component of complement (C5). A18 TCRtg thymocytes develop normal numbers of CD4 single-positive (SP) thymocytes, but do not show pronounced overselection as do some other TCR transgenic strains. CD4 SP cells are mature as judged by termination of CD8 synthesis, resistance to cortisone, and functional competence. The kinetics of positive selection, determined by BrdU labeling, are very fast. CD4 SP thymocytes are demonstrable within 2 days of labeling, and within 8 days after labeling a large proportion (20%) of lymph node T cells are recent thymic emigrants. The high number of recent thymic emigrants suggests rapid turnover of CD4 T cells in the periphery, which was confirmed by thymectomy and determination of CD4 T cell life spans. A18 TCRtg T cells have a t(1/2) of approximately 6 wk, despite the presence of selecting MHC molecules. This explains the failure to accumulate high numbers of peripheral T cells and suggests that the MHC-bound ligand(s) responsible for initiating survival signals is limiting for the selection and maintenance of A18 transgenic CD4 T cells.

Antagonist peptide selects thymocytes expressing a class II major histocompatibility complex-restricted T cell receptor into the CD8 lineage.

CD4/CD8 lineage decision is an important event during T cell maturation in the thymus. CD8 T cell differentiation usually requires corecognition of major histocompatibility complex (MHC) class I by the T cell receptor (TCR) and CD8, whereas CD4 T cells differentiate as a consequence of MHC class II recognition by the TCR and CD4. The involvement of specific peptides in the selection of T cells expressing a particular TCR could be demonstrated so far for the CD8 lineage only. We used mice transgenic for an MHC class II-restricted TCR to investigate the role of antagonistic peptides in CD4 T cell differentiation. Interestingly, antagonists blocked the development of CD4(+) cells that normally differentiate in thymus organ culture from those mice, and they induced the generation of CD8(+) cells in thymus organ culture from mice impaired in CD4(+) cell development (invariant chain-deficient mice). These results are in line with recent observations that antagonistic signals direct differentiation into the CD8 lineage, regardless of MHC specificity.

Rejection of H-Y disparate skin grafts by monospecific CD4+ Th1 and Th2 cells: no requirement for CD8+ T cells or B cells.

We wished to determine whether CD4+ T cells could reject a skin graft that was discordant for a single minor transplantation Ag in the absence of CD8+ T cells or Ab. Transgenic A1(M) mice were constructed that express the rearranged V beta 8.2 and V alpha 10 TCR genes from a T cell clone that is specific for the male Ag (H-Y) in the context of H2-Ek. In addition, the RAG-1(-/-) background was bred onto these mice to eliminate any endogenous TCR rearrangements. As expected, clonal deletion was found to be complete in the thymus of male A1(M) x RAG-1(-/-) mice, while only CD4+ T cells were positively selected and found in the periphery of females. Female A1(M) x RAG-1(-/-) mice were able to rapidly reject (in <14 days) male (but not female) skin grafts in a CD4-dependent fashion. After multiple grafts, it was confirmed that no CD8+ T cells or surface Ig+ B cells were present. An immunofluorescent analysis of spleen cells after grafting showed that the majority of T cells expressed activation markers (CD44, CD25, and intracytoplasmic IL-2) and a significant proportion were making IFN-gamma and IL-4. Surprisingly, the transfer of either Th1 or Th2 CD4+ T cell lines from these mice into T cell-depleted recipients was sufficient to cause a specific rejection of male skin.

Antigen-specific T cell receptor antagonism by antigen-presenting cells treated with the hemolysin of Listeria monocytogenes: a novel type of immune escape.

We have examined the influence of listeriolysin O (LLO), the hemolysin secreted by the pathogenic bacterium Listeria monocytogenes, on major histocompatibility complex class II-dependent T cell activation. Stimulation of T cells by native antigens but not by peptides is inhibited upon pretreatment of antigen-presenting cells (APC) with LLO. Experiments presented here reveal that this inhibition is not due to a lack in processing of antigen by APC but is the result of an irreversible inactivation of T cells that recognize antigen on LLO-treated APC. Incubation of mixtures of two different T cells where only one antigen was presented on LLO-treated APC suggested that T cell inactivation is antigen specific. The inactivation was dominant and could be observed even in the presence of amounts of synthetic peptides that normally lead to T cell responses. This condition is reminiscent of the T cell inhibition observed when antagonistic and stimulatory peptides are added to APC at the same time. Our results thus reveal a novel type of interference by pathogens with antigen presentation and T cell stimulation that could give the pathogen a decisive advantage in dissemination and disease.

Modulation of Basophil Histamine Release and T Cell Reactivity by Chemically Modified Allergen.

Modification of a model allergen ovalbumin (OA) with succinylation (Suc-OA) led to inhibition of allergen histamine releasing activity as it was tested on basophils of OA-sensitive patients. A whole blood leukocyte histamine release was performed by glass fibre based histamine assay. IgE-binding activity of Suc-OA was significantly reduced as it was shown by RAST inhibition technique. Suc-OA stimulated OA-specific T cell hybrid 3DO-548 and ACP:LK35 to produce cytokine release at the same level as in the case of non-modified OA. Succinylation of OA was concluded to result in selective blockade of B cell and preservation of T cell epitopes of the allergen suggesting a new approach for allergen specific immunotherapy.

Excessive degradation of intracellular protein in macrophages prevents presentation in the context of major histocompatibility complex class II molecules.

The endogenous major histocompatibility complex (MHC) class II presentation pathway allows biosynthesized, intracellular antigens access for presentation to MHC class II-restricted T cells. This pathway has been well documented in B cells and fibroblasts, but may not be universally available in all antigen-presenting cell types. This study compares the ability of different antigen-presenting cells, expressing endogenous C5 protein (fifth component of mouse complement) as a result of transfection, to present their biosynthesized C5 to MHC class II-restricted T cells. B cells and fibroblasts expressing C5 were able to present several epitopes of this protein with MHC class II molecules, whereas macrophages were unable to do so, but readily presented C5 from an extracellular source. However, macrophage presentation of endogenous C5 could be achieved when they were treated with low doses of the lysosomotropic agent ammonium chloride. In the presence of an inhibitor of autophagy, presentation of endogenous C5 was abrogated, indicating that biosynthesized C5 is shuttled into lysosomal compartments for degradation before making contact with MHC class II molecules. Taken together, this suggests that proteolytic activity in lysosomes of macrophages may be excessive, compared with fibroblasts and B cells, and destroys epitopes of the C5 protein before they can gain access to MHC class II molecules. Thus, there are inherent differences in presentation pathways between antigen-presenting cell types; this could reflect their specialized functions within the immune system with macrophages focussing preferentially on internalization, degradation, and presentation of extracellular material.

T-cell anergy induced by antigen presenting cells treated with the hemolysin of Listeria monocytogenes.

Antigen presenting cells (APC) that are infected with listeriolysin (LLO) secreting Listeria lack the ability to stimulate MHC class II restricted T-cells by conventional antigens. Similarly, T-cell activation by native proteins but not by peptides was inhibited upon pretreatment of APC with purified listeriolysin. The inhibition is due to an irreversible inactivation of T-cells that recognize antigen on infected or LLO treated APC. Inhibition was found to dominate over stimulation by peptides. This condition is reminiscent of T-cells inactivation by antagonistic peptides and represents a novel type of immune escape.

Bone marrow-derived dendritic cells can process bacteria for MHC-I and MHC-II presentation to T cells.

Dendritic cells can engulf particulate Ags and induce T cell proliferative responses after pulsing with particulate Ag. However, their capacity to process viable Gram-negative bacteria for presentation by MHC-I and MHC-II has not been shown. We therefore characterized the ability of murine bone marrow-derived dendritic cells to process Escherichia coli and Salmonella typhimurium, expressing defined epitopes for presentation by MHC-I and MHC-II molecules. The I-Ak-restricted 46-61 epitope from hen egg white lysozyme (HEL(46-61)) or the Kb-restricted 257-264 epitope from chicken egg OVA (OVA(257-264)) was expressed as fusion proteins in the bacterial cytoplasm as the Crl-HEL and Crl-OVA fusion proteins, respectively. Bacteria expressing Crl-HEL or Crl-OVA, or beads coated with HEL or OVA, were coincubated with murine bone marrow-derived dendritic cells, and Ag processing and presentation were quantitated using T cell hybridomas. The data show that granulocyte-macrophage CSF-stimulated dendritic cells can process live intact Gram-negative bacteria for peptide presentation by MHC-I and MHC-II. Cytochalasin D inhibition studies revealed that processing for both MHC-I and MHC-II presentation required cytoskeletal rearrangements. Processing for MHC-I and MHC-II presentation was inhibited by ammonium chloride, suggesting that acidic compartments were required. Thus, granulocyte-macrophage CSF-stimulated murine bone marrow dendritic cells are capable of processing exogenous particulate Ags, including bacteria with no known mechanism for phagosomal escape, for peptide presentation by both MHC-I and MHC-II. These data suggest that dendritic cells may be important in priming both CD4+ and CD8+ T cells to bacterial Ags.