ABSTRACT We investigated the effects of peripheral blood mononuclear cells expanded with irrelevant control and mycobacterial antigens on the intracellular growth of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in human macrophages. More than 90% of the cells present after 1 week of in vitro expansion were CD3 + . T cells were expanded from purified protein derivative-negative controls, persons with latent tuberculosis, and BCG-vaccinated individuals. T cells expanded with nonmycobacterial antigens enhanced the intracellular growth of BCG in suboptimal cultures of macrophages. T cells expanded with live BCG or lysates of Mycobacterium tuberculosis directly inhibited intracellular BCG. Recent intradermal BCG vaccination significantly enhanced the inhibitory activity of T cells expanded with mycobacterial antigens ( P < 0.02), consistent with the induction of memory-immune inhibitory T-cell responses. Selected mycobacterial antigens (Mtb41 > lipoarabinomannan > 38kd > Ag85B > Mtb39) expanded inhibitory T cells, demonstrating the involvement of antigen-specific T cells in intracellular BCG inhibition. We studied the T-cell subsets and molecular mechanisms involved in the memory-immune inhibition of intracellular BCG. Mycobacteria-specific γδ T cells were the most potent inhibitors of intracellular BCG growth. Direct contact between T cells and macrophages was necessary for the BCG growth-enhancing and inhibitory activities mediated by control and mycobacteria-specific T cells, respectively. Increases in tumor necrosis factor alpha, interleukin-6, transforming growth factor β, and vascular endothelial growth factor mRNA expression were associated with the enhancement of intracellular BCG growth. Increases in gamma interferon, FAS, FAS ligand, perforin, granzyme, and granulysin mRNA expression were associated with intracellular BCG inhibition. These culture systems provide in vitro models for studying the opposing T-cell mechanisms involved in mycobacterial survival and protective host immunity.

Tuberculosis (TB) vaccine development is hindered by the lack of clear surrogate markers of protective human immunity to Mycobacterium tuberculosis. This study evaluated the hypothesis that immune-mediated inhibition of mycobacterial growth would more directly correlate with protective TB immunity than other immunologic responses. Bacille Calmette-Guérin (BCG) vaccination, known to induce partial protection against TB, was used as a model system to investigate mechanistic relationships among different parameters of antigen-specific immunity. Effects of primary and booster intradermal BCG vaccinations were assessed in 3 distinct assays of mycobacterial inhibition. Correlations between vaccine-induced growth inhibition and other immune responses were analyzed. BCG significantly enhanced all antigen-specific responses. Peak responses occurred at 2 months after boosting. Statistical analyses suggested that each assay measured unique aspects of mycobacterial immunity. Despite previous evidence that type 1 immune responses are essential for TB immunity, interferon-gamma production did not correlate with mycobacterial inhibition. These results have important implications for TB vaccine development.

Induction of human gammadelta T cells was investigated in subjects who were vaccinated with live recombinant canarypox virus expressing human immunodeficiency virus (HIV) proteins or soluble MN rgp120. Both canarypox and rgp120 induced antigen-specific lymphoproliferative and interferon (IFN)-gamma responses. However, only canarypox vaccination induced increased gammadelta T cell responses detectable after secondary in vitro expansion (P<.02). These enhanced gammadelta T cell responses were specific for canarypox but not HIV antigens. Canarypox-specific gammadelta T cells were predominantly Vgamma9(+) and produced intracellular and secreted IFN-gamma. gammadelta T cell lines generated from canarypox vaccinees responded to canarypox antigens but not to mycobacterial antigens shown previously to induce bacille Calmette-Guérin-specific gammadelta T cells. Furthermore, canarypox vaccinations were associated with significantly higher NK cell expansions (P=.02). Increased IFN-gamma production by gammadelta T and NK cells could enhance the induction of protective type 1 memory immunity. Thus, stimulation of gammadelta T cells might be an important feature of live vaccines.

The expression of the proliferation‐associated nuclear antigen Ki‐67 in peripheral blood mononuclear cells was studied in 30 patients with acute malarial illness and 11 healthy controls from Addis Ababa or Nazareth in Ethiopia. Seventeen patients had Plasmodium falciparum infections and 13 had Plasmodium vivax. Two‐colour immunoenzymatic staining was developed in order to simultaneously detect the expression of the nuclear antigen Ki‐67 and determine the surface phenotype of the cell. The median percentage of proliferating, Ki‐67 positive lymphocytes was significantly higher in patients with acute P. falciparum (11.8%) and P. vivax (15.6%) illnesses compared to the controls (4.3%). The majority of Ki‐67 positive cells were T cells (CD3+) while the relative increase of Ki‐67 expressing cells was similar for both the CD4+ and CD8+ T‐cell subsets. Our data show an increased number of activated cells driven to proliferation in the peripheral blood of patients during acute malaria illness.

We investigated the ability of T cells expanded with mycobacterial antigens from healthy purified protein derivative—reactive donors and bacille Calmette-Guérin (BCG)—vaccinated volunteers to inhibit intracellular growth of BCG. Peripheral blood mononuclear cells were incubated for 7 days with mycobacterial whole lysate, live BCG, tetanus toxoid as control antigen, or medium alone. Autologous monocytes were separated by plastic adherence, allowed to mature for 6 days, and infected with BCG before serving as target cells. Expanded effector cells were cocultured with target cells for 72 h. Cocultures were then treated with 0.2% saponin to lyse infected monocytes and release intracellular BCG. Quantities of viable BCG present in these lysates were studied by colony-forming unit counting and radiometric labeling. We reproducibly found that lymphocytes expanded with mycobacterial whole lysate or live BCG significantly inhibited the intracellular growth of BCG, compared with lymphocytes expanded with tetanus toxoid or rested in medium. In addition, BCG vaccination enhanced the ability of T cells to inhibit intracellular mycobacterial growth in 3 of 5 volunteers. This assay may be useful for estimates of protective immunity induced by tuberculosis vaccines in human trials.

The functional properties, regarding parasite growth inhibition in vitro, the cytotoxic potential and cytokine profiles of human γδ+ and αβ+ T cells, T‐cell lines and clones stimulated with Plasmodium falciparum‐antigen‐or T‐cell mitogen in vitro were investigated. Using reverse transcriptase‐polymerase chain reaction (RT‐PCR) and specific primers, mRNA for the cytolytic molecules perforin, granzyme A and B, Fas and Fas ligand (FasL) were detected in both the γδ‐ and the αβT cells. Despite this fact, only γδT cells inhibited, both Vδ1+ and Vδ2+, the in vitro growth of the asexual blood stages in a dose dependent manner. The inhibition required cell‐to‐cell contact and was not observed until the second parasite replication implied that the likely γδT‐cell target was the extracellular merozoite or schizont. The failure of αβT cells to inhibit the growth of the parasite suggests requirement of additional cytolytic molecules/signals or different receptor specificities exhibited by the γδT cells. Both the γδ‐ and αβT cells expressed mRNA for a large number of cytokines. Interferon (IFN)‐γ, interleukin (IL) IL‐5, IL‐6, IL‐8, tumour necrosis factor alpha (TNFα), tumour necrosis factor beta (TNF‐β)/lymphotoxin (LT) and T‐cell growth factor beta‐1 (TGF‐β1) were observed in all activated clones tested. No IL‐3 was detected, while IL‐1β, IL‐2, IL‐4, IL‐10 and GM‐CSF were variably expressed. In conclusion, our data show that γδT cells in malaria nonimmune individuals inhibit the asexual blood stages of P. falciparum malaria, while similarly activated αβT cells do not. Thus, it is likely that the γδT cells could play a mandatory role in the elimination of parasites and/or the regulation of the early immune response to malaria infection.

Most children and adults living in areas where the endemicity of Plasmodium falciparum malaria is high have significantly elevated levels of both total immunoglobulin E (IgE) and IgE antimalarial antibodies in blood. This elevation is highest in patients with cerebral malaria, suggesting a pathogenic role for this immunoglobulin isotype. In this study, we show that IgE elevation may also be seen in severe malaria without cerebral involvement and parallels an elevation of tumor necrosis factor alpha (TNF). IgE-containing serum from malaria immune donors was added to tissue culture plates coated with rabbit anti-human IgE antibodies or with P. falciparum antigen. IgE-anti-IgE complexes as well as antigen-binding IgE antibodies induced TNF release from peripheral blood mononuclear cells (PBMC). Nonmalaria control sera with no IgE elevation induced significantly less of this cytokine, and the TNF-inducing capacity of malaria sera was also strongly reduced by passing them over anti-IgE Sepharose columns. The cells giving rise to TNF were adherent PBMC. The release of this cytokine probably reflects cross-linking of their low-affinity receptors for IgE (CD23) by IgE-containing immune complexes known to give rise to monocyte activation via the NO transduction pathway. In line with this, adherent monocytic cells exposed to IgE complexes displayed increased expression of CD23. As the malaria sera contained IgG anti-IgE antibodies, such complexes probably also play a role in the induction of TNF in vivo. Overproduction of TNF is considered a major pathogenic mechanism responsible for fever and tissue lesions in P. falciparum malaria. This overproduction is generally assumed to reflect a direct stimulation of effector cells by certain parasite-derived toxins. Our results suggest that IgE elevation constitutes yet another important mechanism involved in excessive TNF induction in this disease.

Lymphocyte subset distributions and activation in the peripheral blood were studied in 39 patients with acute malaria and 16 healthy controls from Addis Ababa and Nazareth, Ethiopia. As confirmed by polymerase chain reaction (PCR), 15 patients were infected with Plasmodium falciparum (Pf), 17 with P. vivax (Pv) and seven were double-infected (Di) with both Pf and Pv. Three-colour flow cytometry was used for phenotyping. Total leucocyte and lymphocyte counts were lower in malaria patients than in controls. The T cell count was reduced in Pf patients, while in the Pv and Di patients there was a reduction in the natural killer (NK) cell count. The CD4/CD8 ratio remained unchanged. gammadelta+ T cells were significantly elevated in Pf and Di patients, but not in Pv patients. The increase in gammadelta+ T cells was mostly due to an increase in Vdelta1+ cells. Analyses of cellular activation indicated by the expressions of CD25 and HLA-DR revealed significantly higher numbers of activated CD3+ cells, including gammadelta+ T cells, in all patient groups compared with controls. Our results thus indicate that in acute malaria illness there is a complex pattern of change in lymphocyte subset distribution and activation, including gammadelta+ T cells. These patterns in Pf infection seem to be distinct from those in Pv infection.

The phenotypic composition of peripheral blood lymphocytes in 45 healthy adults (15 each from Bangladesh, Ethiopia and Sweden) was analysed as an indicator of the influence of environment and/or ethnic background on the human immune response. The possible interference of technical factors was minimized by highly standardized handling of samples and by use of a similar simultaneous 3-colour flow cytometry analysis technique for all samples. The percentage of CD4+ cells was lower, and the percentage of CD8+ cells was higher, in Bangladeshi and Ethiopian subjects than in those from Sweden. A higher percentage of CD57+/CD8+ T cells was also found in these 2 groups than in Swedish subjects. The percentage of gamma delta T cells was higher in Bangladeshi subjects and a difference in T cell receptor V beta expression was also noted between Bangladeshi and Swedish subjects. The data suggest that environmental or genetic factors are important bias factors to be considered in immunophenotyping studies. Possibly differences in the pattern or level of microbial challenge, as well as nutritional factors, may lead to different adaptive changes in the immune response. The potential influence of such immune adaptation on the response to vaccination or pharmaceutical therapy may be important in the development of new strategies of medical intervention in different geographical regions or ethnic groups.