Joint inflammations and exacerbations in mice by cloned helper T cells

Briefly, the aim of the study described in this thesis was to investigate whether the joint inflammations that occur in AlA can also be induced by cloned helper T cells and the antigen they recognize, and whether these joint inflammations can also show flare-up phenomena after repeated administration of the antigen. In Chapter 2 it is shown that cloned T cells with the helper phenotype can induce delayed type hypersensitivity (DTH) reactions and that these reactions can give rise to flare-up reactions after local, iv or oral administration of the antigen. We hypothesized that these DTH reactions underly the development of joint inflammations in the AlA model, and presumably in human rheumatoid diseases. In Chapter 3 we present the model of induction of joint inflammation by similar cloned T cells. Dose response curves of the antigen and the cloned T cells injected into the joints are shown, as are the kinetics of the joint inflammations induced. Furthermore we show that it is possible to evoke flare-up reactions of these joint inflammations, but also of joint inflammations induced by systemically administered T cells and local injection of the antigen. The induction of joint inflammation by the cloned T cells was found to be dependent on H-2 restricted interactions with recipient cells or tissues. In Chapter 4 we show that both the joint inflammation and the flareup reactions can be evoked in T cell deficient nude mice as well. Furthermore, this paper pays attention to the role of antigen in the retention of the cloned T cells in the joint. In our model, the induction of an inflammatory reaction is dependent on H-2 restricted interactions between T cells and recipient cells and tissues, presumably antigen presenting cells. In Chapter 5 the characterization of a macrophage cell line, AP284, is described that is able to present antigen efficiently in vivo to the cloned helper T cells used in our studies. Injection of AP284 and syngeneic cloned helper T cells into the hind foot of allogeneic mice induces a full blown DTH reaction. This model, in which both the antigen presenting cells and the T cells are monoclonal, is attractive to investigate the cellular interactions in the induction of T cell dependent inflammations. Therefore we applied this approach in our studies on AlA to investigate whether antigen activated helper T cells and macrophages are sufficient for the induction of joint inflammation in allogeneic recipients. The data presented in Chapter 6 show that this was indeed the case. Finally in Chapter 7 we studied in detail the histological and immunohistochemical characteristics of the inflammations and flare-up reactions in our model. The data emerging from our studies are discussed in the perspective of literature data in Chapter

Histological and immunohistochemical characterization of joint inflammation and flare-up reactions induced by cloned MT4+, Lyt-2-T cells

Abstract This report describes the histological and immunohistochemical characterization of joint inflammations and flare-up reactions in mice induced by cloned MT4+, Lyt-2− T cells. The T-cell clone used was specific for the antigen methylated bonne serum albumin (mBSA) and was inoculated locally into a joint together with the antigen. The histological examination was performed in methylmethacrylate sections, and the various cell types were quantified m distinct regions of the knee joint. The infiltrates consisted predominantly or granulocytes admixed with small numbers of histiocytes. Few lymphocytes were present, while plasma cells were not found. Fibrosis was prominent in the later stages of the inflammation. Immunohistochemical analysis of total unfixed, non-decalcified sections using monoclonal antibodies revealed the presence of T cells which were predominantly of the helper phenotype. sporadic B cells, and a considerable number of la-positive cells. Macrophages were scattered throughout the infiltrate. The synovial lining was shown to express Ia antigens and to contain cells that stained with macrophage markers Cell clusters were found including helper T (Th) cells, some B cells, and Ia-positive cells. These results are in line with immunohistological examinations in other arthritis models and resemble the early events in human rheumatoid arthritis. The data indicate that activated helper T cells are required and sufficient to give rise to the inflammatory infiltrates that are characteristic of the inflammations and exacerbations in human rheumatoid arthritis

Plasmacytoid dendritic cells of melanoma patients present exogenous proteins to CD4+ T cells after FcγRII-mediated uptake

Plasmacytoid dendritic cells (pDCs) contribute to innate antiviral immune responses by producing type I interferons. Although human pDCs can induce T cell responses upon viral infection, it remains unclear if pDCs can present exogenous antigens. Here, we show that human pDCs exploit FcγRII (CD32) to internalize antigen–antibody complexes, resulting in the presentation of exogenous antigen to T cells. pDCs isolated from melanoma patients vaccinated with autologous monocyte-derived peptide- and keyhold limpet hemocyanin (KLH)–loaded dendritic cells, but not from nonvaccinated patients or patients that lack a humoral response against KLH, were able to stimulate KLH-specific T cell proliferation. Interestingly, we observed that internalization of KLH by pDCs depended on the presence of serum from vaccinated patients that developed an anti-KLH antibody response. Anti-CD32 antibodies inhibited antigen uptake and presentation, demonstrating that circulating anti-KLH antibodies binding to CD32 mediate KLH internalization. We conclude that CD32 is an antigen uptake receptor on pDCs and that antigen presentation by pDCs is of particular relevance when circulating antibodies are present. Antigen presentation by pDCs may thus modulate the strength and quality of the secondary phase of an immune response

Arthritis by autoreactive T cell lines obtained from rats after injection of intestinal bacterial cell wall fragments

__Abstract__ T cell lines (B13, B19) were isolated from the lymph nodes of Lewis rats 12 days after an arthritogenic injection of cell wall fragments of Eubacterium aerofaciens (ECW), a major resident of the human intestinal flora. These cell wall fragments consist of peptidoglycan polysaccharide complexes (PPC). The cell lines that bear the helper phenotype were arthritogenic in knee or ankle joints upon intravenous injection into irradiated Lewis recipients. B13 was, however, not arthritogenic in irradiated F344 recipients that are largely RT1 identical. The arthritis induced in the knee joints of the irradiated Lewis rats was clearly shown by a 99mtechnetium-pertechnetate scanning technique and was confirmed histologically. In vitro the cell lines showed a proliferative response after stimulation with syngeneic spleen cells alone. The proliferation was significantly higher when bacterial PPC, isolated in soluble form from normal feces or ileostomy fluid were added. Recognition by B13 appeared to be MHC class II restricted. These results show that autoreactive T cell lines can be isolated from rats after injection of bacterial cell wall antigens and that these cell lines can be arthritogenic. This suggests a role for autoreactive T cells in the induction of bacterial cell wall arthritis and might give a clue for the arthritogenic properties of the normal human intestinal flora

Construction and Application of R Prime Plasmids, Carrying Different Segments of an Octopine Ti Plasmid from Agrobacterium tumefaciens, for Complementation of vir Genes

Several R prime plasmids have been obtained with high efficiency, by enclosing the R plasmid replicator, in an R::Ti cointegrate plasmid, between two copies of the transposon Tn1831, in the same orientation. These R primes carry different segments of an octopine Ti plasmid, and are compatible with Ti plasmids. They were used to study genetic complementation of Ti plasmid insertion mutants, outside the T-DNA region, which affected oncogenicity. Complementation was observed in both recombination-proficient and -deficient strains. The complementation in trans indicates that certain functions essential for tumor formation outside the T-DNA region are probably expressed in the bacterium. Therefore, the authors proposed to make a distinction between virulence (Vir) functions and oncogenic (Onc) functions of the octopine Ti plasmid of Agrobacterium tumefaciens. A large R prime was obtained, carrying the whole Ti plasmid, except a 7-Mdalton segment, containing the Ti plasmid replicator region. Strains harboring this plasmid induced normal tumors, showing that the replicator region of the octopine Ti plasmid is dispensible for tumor induction.

Site-Directed Mutagenesis in Escherichia coli of a Stable R772::Ti Cointegrate Plasmid from Agrobacterium tumefaciens

The host range of an octopine Ti plasmid is limited to Rhizobiaceae. This has been extended also to Escherichia coli in the form of a stable cointegrate with the wide-host-range plasmid R772. Its structure was studied by constructing a physical map of R772 and of the R772::pTiB6 cointegrate. An insertion sequence present in R772, called IS70, turned out to be involved in cointegrate formation. We found one intact copy of IS70 and a small segment of IS70, respectively, at the junctions of R772 and Ti DNA. The absence of a complete second copy of IS70 is a likely explanation for the stability of the cointegrate plasmid. A procedure for site-directed mutagenesis of this cointegrate plasmid in E. coli is described. The effect of mutations in the Ti plasmid part can be studied subsequently by transferring the cointegrate into Agrobacterium tumefaciens. The advantage of this procedure for Ti plasmids over other methods used at present is discussed.

Murine Macrophage Cell Line AP284 Presents Antigen to Cloned MT4+, Lyt-2− T Cells in vitro and in vivo

A murine macrophage cell line AP284 that appeared to be mature in phenotype was isolated. After repeated cloning, the cell line expressed the markers Mac-1, Mac-2, Mac-3, 2.4G2, F4/80 as well as Ia antigens. Moreover, it was positive for the enzymes nonspecific esterase and acid phosphatase, negative for alkaline phosphatase and was able to phagocytize latex beads. We studied whether this cell line was able to present antigen to cloned MT4+, Lyt-2− T cells specific for methylated bovine serum albumin (mBSA) or ovalbumin (OVA). The in vitro proliferative response of the cloned T cells specific for mBSA or OVA was found to be effectively supported by AP284. This proliferation could be blocked by monoclonal antibodies against Ia determinants. AP284 also effectively presented antigen in vivo as was shown in a foot swelling assay measuring delayed type hypersensitivity (DTH) to mBSA caused by specific cloned T cells with the helper phenotype. This offers a unique model system for studying the process of antigen presentation in which both the antigen presenting cells and the T cells are monoclonal