The influence of microenvironment on the course of CD8 + T cell responses in vivo was investigated by injecting H-2Kb-specific T cells from donor TCR transgenic (TCR-Tg) mice into H-2kb-Tg mice. H-2Kb expression in recipients was either ubiquitous (CBK mice) or restricted to myeloid and erythroid cells (K beta mice). Donor T cells proliferated as extensively and acquired similar surface phenotypes in spleen of both recipient types. Thus, neither the restricted pattern of H-2Kb expression nor the significantly reduced level of H-2Kb expression by myeloid cells in Kbeta recipients affects the ability of the splenic microenvironment to prime T cell proliferation in vivo. However, an unsustained burst of cytolytic activity was generated rapidly in spleen of CBK recipients, whereas relatively little cytolytic activity was generated in K beta spleen. This indicates that effector T cells were not generated efficiently in spleen of Kbeta recipients even though extensive T cell proliferation was taking place in this microenvironment. Furthermore, activated donor T cells dispersed rapidly throughout primary and secondary lymphoid organs of Kbeta recipients, whereas few T cells migrated from spleen in CBK recipients. Consequently, the course of CD8+ T cell responses and the anatomical distribution of activated T cells are profoundly influenced by the nature of the antigenic microenvironment encountered in vivo. We conclude that T cells rapidly proliferate and acquire new tissue-homing characteristics but do not differentiate into cytolytic effector cells at the site of priming when they encounter myeloid cells expressing low levels of antigen in vivo.
In vitro and in vivo activation of T cells was investigated T hybridoma cells. Nonprofessional APC expressing with invariant chain-antigen fusion protein. The CD4 T cell recombinant Ii HEL and H2-A k are also able to activate epitope amino acid 52-61 of hen egg lysozyme (HEL) was naive T cells from 3A9 TCR transgenic mice, a result not attached to the C-terminal end of invariant chain (Ii).achieved with peptide pulsed APC. To elicit an in vivo Expression of this recombinant Ii HEL directs the T cell immune response dendritic cells (DC) were transfected epitope to the class II processing pathway. Class II molwith rIi HEL cDNA: following immunization of CBA mice ecules of transfected antigen presenting cells (APC) are with transfected DC, a primary T cell response against the charged with this HEL epitope. The endogenously provided HEL epitope was induced. Thus the procedure described epitope competes with processing and presentation of here could be used to introduce antigens into the class II exogenously added antigen. APC expressing recombinant processing pathway and to elicit T cell activation both in Ii HEL stimulate a maximal IL-2 response of HEL-specific vitro and in vivo.
The invariant chain (Ii) gene encodes two differentially spliced variants Ii31 and Ii41. The Ii31 isotype is the dominant form expressed in all antigen-presenting cells (APC). Ii41 is differentially expressed and can be found in large quantities in Langerhans and dendritic cells. While a functional role of Ii in class II antigen presentation is now well established, a distinct role of the Ii isotypes remains controversial. We tested Ii31 and Ii41 L cell transfectants for antigen presentation of hen egg lysozyme (HEL) to T cell hybridomas. The result indicates that both Ii chains promote antigen presentation equally well. To test other APC than transfected L cells, we introduced a recombinant Ii41 gene into anti-deficient mouse line. There the transgene induces about one-third of total li expression of wild-type mice. Surface expression of class II molecules and the CD4 compartment which are deficient in Ii knock-out mice are restored in Ii41 transgenic mice. B lymphocytes from Ii41 transgenic mice and Ii31-expressing B lymphocytes from wild-type mice were used as APC for presentation of keyhole limpet hemacyanin and ovalbumin to T cell hybridomas. The results show that both Ii chains facilitate antigen presentation equally well.
Allelic exclusion of T cell receptor (TCR) genes is incomplete: a significant percentage (10-30%) of normal human and mouse peripheral T cells express two surface TCR alpha chains, and a small percentage of peripheral human T cells have been reported to express two surface TCR beta chains. A proportion of thymocytes in TCR transgenic mice rearrange endogenous T cell receptor genes, and peripheral T cells with two TCR alpha chains, transgenic and endogenous, have been reported. T cell clones with more than a single TCR heterodimer on their surface might be expected to show specificity for more than one cognate antigen: we report here a T cell clone with dual antigen specificity, isolated from an F5 TCR influenza nucleoprotein (NP 366-374/Db)-specific transgenic female mouse which had rejected an H-2-matched male skin graft. It was selected in vitro by stimulation with male H-2b spleen cells in the absence of the NP366-374 peptide but has specificity for both H-Y/Db and NP366-374. This contrasted with the single NP366-374/Db specificity shown by a control clone isolated from a Rag1-/- F5 mouse. The dual antigen specificity was associated with the rearrangement of endogenous TCR genes and cell surface expression of these as well as the TCR transgene.
Improved chemical inhibitors are required to dissect the role of specific antigen processing enzymes and to complement genetic models. In this study we explore the in vitro and in vivo properties of a novel class of targeted inhibitor of aspartic proteinases, in which pepstatin is coupled to mannosylated albumin (MPC6), creating an inhibitor with improved solubility and the potential for selective cell tropism. Using these compounds, we have demonstrated that MPC6 is taken up via mannose receptor facilitated endocytosis, leading to a slow but continuous accumulation of inhibitor within large endocytic vesicles within dendritic cells, and a parallel inhibition of intracellular aspartic proteinase activity. Inhibition of intracellular proteinase activity is associated with reduction in antigen processing activity, but this is epitope specific, preferentially inhibiting processing of T cell epitopes buried within compact proteinase-resistant protein domains. Unexpectedly, we have also demonstrated, using quenched fluorescent substrates, that little or no cleavage of the disulfide linker takes place within dendritic cells, but this does not appear to affect the activity of MPC6 as an inhibitor of cathepsins D and E in vitro and in vivo. Finally, we have shown that MPC6 selectively targets dendritic cells and macrophages in spleen in vivo. Access to non-lymphoid tissues is very limited in the steady state, but is strongly enhanced at local sites of inflammation. The strategy adopted for MPC6 synthesis may therefore represent a more general way to deliver chemical inhibitors to cells of the innate immune system, especially at sites of inflammation.Antigen presenting cells (including dendritic cells (DC) and macrophages) are equipped with an array of membrane and cytoplasmic receptors known as Pattern Recognition Receptors (PRR), with which they bind to microbial components (Pathogen Associated Molecular Patterns, PAMPs). Once internalised, any PAMP-associated proteins are subject to regulated proteolysis (the exogenous antigen processing pathway), producing peptides which bind to Class II Major Histocompatibility Complex (MHC) receptors, and hence stimulate T cell adaptive immunity 1 . The interaction between antigen presenting cell and T cell is widely recognised as being one of the key steps regulating both the magnitude and the A number of studies have used antibodies to deliver antigens to antigen presenting cells in vitro or in vivo 2 . This has achieved some significant successes. However, a wealth of experience from the field of tumour biology has shown that delivery of drugs via antibody conjugates poses formidable technical problems. An alternative approach is to target DC using ligands of lectins such as mannose receptors, themselves a family of PRRs 3,4 . We have explored this targeting strategy in the context of using the selective inhibitor pepstatin 1 to identify the role of aspartic proteinases cathepsins D and E in the proteolysis of antigen 5,6 . Pepstatin itself is a very potent inhibitor (IC...
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