Conjoint Professor Margaret Dunkley

Pseudomonas aeruginosa is an environmentally ubiquitous, extracellular opportunistic gram-negative bacteria that causes significant morbidity and mortality to a disproportionately high degree for infections with this bacteria compared with other gram-negative bacteria. Patients at particular risk of infection are those with compromised respiratory function, in intensive-care support and taking immunocompromising pharmaceutical agents. Once acquired, infection is difficult to eradicate with chemotherapy and attempts to vaccinate against infection have been of little success. Over the past five years, we have pursued the concept of mucosal immunisation against respiratory infection with P. aeruginosa. Initial studies in an acute animal model clearly demonstrated that mucosal immunisation with a killed whole bacterial cell preparation could induce protective immune responses in the lung. Subsequent studies have shown that the protective immune mechanisms were dependent on antigen specific CD4+ T cells, the activation of alveolar macrophages, the recruitment and activation of polymorphs, predominantly neutrophils, the controlled secretion of TNF-alpha, IL-1 and IFN gamma and the presence of antibody. We have hypothesised that the protective response is under the control of T cells. A pre-clinical human trial of an oral whole killed cell preparation has been completed with no adverse side effects. A limited open trial in patients with bronchiectasis has also been completed. Preliminary analysis of the results has demonstrated that after oral vaccination, specific lymphocyte responses were observed to P. aeruginosa.

Pseudomonas aeruginosa, an oportunistic bacterial pathogen, is a major course of morbidity and mortality in subjects with compromised respiratory function despite the significant advances in therapeutic practices. The bacteria produces an armoury of products which modify its infective niche to ensure bacterial survival. The role of antibody in protection against pulmonary infection remains poorly defined. Protection appears to be associated with opsonizing antibody whilst some other antibody responses may be deleterious and promote further lung damage. Cell mediated responses are clearly important in protection against infection. This review proposes a vaccine strategy aimed at enhancing specific T cell responses in the lung which, through T cell-derived cytokines, drive the recruitment of neutrophils to the lung and the subsequent activation of these cells results in the clearance of bacteria from the lung. Copyright © 1995, Wiley Blackwell. All rights reserved

This paper examines the histology of rat lungs following intestinal immunization with killed mucoid Pseudomonas aerugtnosa and subsequent pulmonary challenge with live P. aeruginosa. The lungs of non-immune challenged rats developed a confluent haemorrhagic pneumonitis with degeneration and sloughing of the mucosa of the airways; perivascular infiltration with mononuclear cells was apparent 1¿2 h post-challenge; some neutrophils were present by 2 h post-challenge; by 12h post-challenge oedema and intra-alveolar haemorrhage were prominent and Gram-negative organisms were seen in large quantities. In contrast, immunized challenged animals showed a pronounced neutrophilic response 1¿2 h post-challenge; by 12 h post-challenge patchy abscesses were apparent with resolving inflammation and no organisms visible. The findings suggest that intestinal immunization prevents the development of fatal P. aeruginosa infections in the lung by accelerating the recruitment of polymorphonuclear neutrophils Copyright © 1995, Wiley Blackwell. All rights reserved

Studies of the migration of antigen-specific regulatory T cell subsets responding to gut immunization were undertaken to clarify their migratory potential and the role of antigen in their localization. In initial experiments, lymphocytes collected from the thoracic duct of rats after immunization of Peyer's patches (PP) with keyhole limpet hemocyanin (KLH), were enriched for T helper (Th) cells and labelled with the fluorochrome H33342. In other experiments, a higher frequency of antigen-specific T cells was achieved by short-term culture of the enriched Th cells in the presence of KLH and the blast cells labelled with 3H-thymidine. The distribution of both populations was determined after injection into immunized and unimmunized syngeneic recipients. Whereas the uncultured population (predominantly small Th cells) localized almost exclusively in follicular lymphoid tissues, the cells expanded by secondary culture (predominantly Th blasts) appeared in the gut lamina propria (LP) initially, then in PP and mesenteric lymph nodes. The Th blasts in the LP were almost always seen in close proximity to the gut epithelium. However, the migration of neither population appeared to be influenced significantly by antigen, in contrast to previous findings with regard to IgA-committed B cells. The initial subepithelial location of Th blasts in the gut LP and their subsequent appearance in PP may provide a mechanism by which antigen presented by epithelial cells could influence B cell differentiation in PP through modulation of signals expressed by these T cells.

The course of haemolytic anaemia in NZB mice has been altered by injection of spleen cells from diseased mice into younger ones before the onset of clinical disease. Recipients greater than 6 weeks of age developed early-onset autoimmune disease; recipients less than 6 weeks of age recovered from early induced disease and showed a delay in the onset of spontaneous disease as compared with untreated NZB mice. This delay was due to the induction in the young mice of splenic suppressor cells. These cells were non-adherent to nylon wool and suppressed autoantibody formation on transfer to old Coombs-positive recipients. Suppressor cells active against autoantibody-producing cells may be present in young untreated NZB mice, but not in sufficient numbers to suppress autoantibody production on adoptive transfer to Coombs-positive recipients; however, when Ig-negative cells from the spleens of very young NZB mice were transferred together with Ig-positive cells from Coombs-positive donor mice to irradiated NZB recipients, the autoantibody production of the transferred B cells was suppressed in some cases. Suppressor cell activity could also be induced by co-culture of spleen cells from old Coombs-positive and young Coombs-negative NZB mice in vitro.

To develop a precise assay for the T-cell progenitors of cytotoxic lymphocytes (CL-progenitors), lymphoid cells were cultured under optimal conditions in Marbrook vessels with mitomycin-treated allogeneic stimulator cells, and the total level of CL produced 5 days later estimated by a modified 51Cr release assay. Conditions were adjusted so an arithmetically linear cell dose response relationship was obtained. Three aspects of the cell dose response curve required attention. (1) At low responding cell inputs a macrophage-like cell became limiting (despite the presence of allogeneic macrophages in the stimulating cell population), leading to a lag in the response. This limitation was overcome by adding a low level of irradiated syngeneic macrophages, or by using irradiated syngeneic spleen 'filler' cells. (2) The slope of the resultant linear dose response region could be reduced if desired by changing from cellophane dialysis membranes to 0.1 µ pore size nuclepore membranes, suggesting a stimulatory role for some higher molecular weight soluble factor produced in the cultures. (3) At higher responding cell inputs a marked and extensive plateu was obtained. CL developing early in the response appeared to be destroying the allogeneic stimulator cells causing the response to be self-limiting. This problem was overcome by using a responding cell concentration lower than commonly employed. Assays using mixed leukocyte cultures in the lag or plateau regions could give misleading values for CL-progenitor activity. It is suggested that some examples of apparent synergism in CL generation may have resulted from these effects, rather than T-cell helper T-cell progenitor interactions. © 1978.

The proliferative response of various separated populations of mouse spleen and thymus lymphocytes to the mitogen phytohaemagglutinin (PHA) was not a direct function of the level of responsive T cells, but was governed by other regulatory effects. These included a stimulation by adherent macrophages, an inhibition by a separate population of adherent cells and an adherent cell independent restriction of proliferation at high cell concentration. In contrast, the proliferative response to Concanavalin A (Con A) was more closely related to the level of responsive T cells. All density and electrophoretically isolated sub-sets of splenic T cells appeared capable of a proliferative response to PHA and Con A, although under some conditions the PHA responsiveness of certain fractions was suppressed. In the thymus, the minor low T subpopulation appeared capable of response to both mitogens, and accounted for all the activity of the unfractioned thymus cells. No response to either mitogen could be obtained from the major, high T thymocyte population.

AMONG thymus-derived (T) lymphocytes of the mouse, several functionally distinct subsets may be identified (for review, see ref. 1). Early studies have shown synergy in graft-versus-host reactions of different populations of lymphoid cells2, and in vitro studies have demonstrated interactions between cells from anti-lymphocyte serum-treated mice (T1 cells) and adult thymectomised mice (T2 cells)3. A similar synergy has been observed in the generation of T helper and T suppressor cells in vitro4. Clearer definition of the subsets of T cells interacting in the induction of immune responses has come from the use of allo-antisera recognising differentiation antigens on T cells. In particular, the use of antisera to the Ly series of antigens5 has enabled the separation of amplifying (MLR) cells from killer cells6. Similarly in the humoral response helper T cells can be distinguished from suppressor T cells 7. In this report, we show that killer and suppressor T cells, both generated in vitro and carrying the Ly-2 and 3 antigens can also be separated using antisera to surface antigenic markers. We also present data suggesting that suppressor cells of similar phenotype may have an important function in vivo in the immune response. © 1976 Nature Publishing Group.

THYMUS-derived (T) cells have a major role in immune systems. They mediate various functions, such as T-B cooperation ("helper cells") 1 and the mixed lymphocyte reaction2, become killer cells3, are active in graft versus host responses (reviewed in ref. 4) and act as suppressor cells5. The identification of the cells responsible for these functions as T cells was facilitated by the use of antisera against the T-cell-specific alloantigen, Thy-1 (ref. 6) (formerly known as ¿). It has been reported that Ly allo-antigens, present exclusively on T cells7, identify functionally distinct subpopulations of T cells8. For example, T helper cells were lysed by anti-Ly-1, but not by anti-Ly-2, so that their phenotype is Ly-1+2-, whereas T killer cells were a distinct subpopulation, being Ly-1-2 +. We report here that specific T suppressor cells induced in vitro have a different Ly alloantigen phenotype from T helper cells induced in vitro from the same spleen cell pool. © 1975 Nature Publishing Group.