T cell functions in Plasmodium falciparum and other malarias.
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Animals↗
Biomedical subjects
M Troye-Blomberg
Publications and source records attributed to M Troye-Blomberg.
Regulation of the immune response in Plasmodium falciparum malaria: IV. T cell dependent production of immunoglobulin and anti-P. falciparum antibodies in vitro.
T cells from patients with acute Plasmodium falciparum malaria were investigated for induction of immunoglobulin- or anti-malaria antibody secretion in vitro. Stimulation of autologous T/B cell mixtures (2T:1B) with low concentrations of P. falciparum antigen and cultured for 12 days gave rise to a T-dependent IgG secretion which was significantly elevated over that in medium controls. This was achieved with both a crude P. falciparum antigen and a partially purified preparation enriched in Pf 155, a merozoite-derived antigen deposited in the red cell membrane at invasion (Perlmann et al., 1984). Control antigen (RBC ghosts) induced IgG secretion only when added at high concentrations (greater than 10 micrograms/ml). Neither of the antigens induced IgG secretion at concentrations of less than or equal to 10 micrograms/ml in control cultures of lymphocytes from patients with P. vivax malaria. Supernatants from cultures of P. falciparum patients frequently contained anti-P. falciparum antibodies when nanogram quantities (10-100 ng/ml) of either one of the two malaria antigen preparations was used for stimulation. No anti-P. falciparum antibodies were induced by the control antigen at any concentration. The induced anti-P. falciparum antibodies were directed to intracellular parasites and. at lower frequencies, to Pf 155 as detected on the surface of infected erythrocytes. The induction in vitro of anti-P. falciparum antibodies appeared to be correlated with the presence of such antibodies in the sera of the lymphocyte donors. The lymphocytes of only one out of eight P. vivax patients responded to antigen stimulation by secreting anti-P. falciparum antibodies. However, this donor (but not any of the others), was also P. falciparum seropositive. Taken together, these results indicate that the induction of anti-P. falciparum antibody secretion reflects a secondary response in vitro of cells primed in vivo. The present experimental system should be well suited to map parasite antigen for their capacity to induce T cell dependent responses in P. falciparum malaria.
Acute Disease↗
Human monoclonal antibodies to Pf 155, a major antigen of malaria parasite Plasmodium falciparum.
Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.
Animals↗
Pf 155, a candidate for a blood stage vaccine in Plasmodium falciparum malaria.
Pf 155 is a Mr 155,000 P. falciparum antigen, which is deposited in the erythrocyte membrane at merozoite invasion. The antigen is detected by a modified immunofluorescence assay giving staining of the surface of ring stage infected erythrocytes. Cell lines producing human monoclonal antibodies to Pf 155 were established and two different antibodies of IgM and IgG class, respectively, were characterized further. Both antibodies gave a strong surface immunofluorescence and in immunoblotting they react strongly with Pf 155, but also with some lower molecular weight material, including polypeptides of Mr 135,000 and 120,000. Both antibodies were efficient inhibitors of P. falciparum reinvasion in vitro. Pf 155 as well as the Mr 135,000 and 120,000 polypeptides were shown to bind with high affinity to human erythrocyte glycophorin. An octapeptide (GluGluAsnValGluHisAspAla) corresponding to a repeated sequence in Pf 155 was synthesized. Rabbit antibodies to the octapeptide gave a distinct surface immunofluorescence and reacted with Pf 155 and the Mr 135,000 and 120,000 polypeptides in immunoblotting. Human antibodies reacting with the octapeptide were isolated by affinity chromatography from the serum of a P. falciparum immune individual. These antibodies showed a strong reaction with Pf 155 as determined by immunoblotting and surface immunofluorescence. Pf 155 reactive antibodies affinity purified on monolayers of P. falciparum infected erythrocytes are very efficient inhibitors of parasite reinvasion. Such antibody preparations depleted of octapeptide reactive antibodies showed a markedly decreased reinvasion inhibitory capacity, while a high inhibitory activity was recovered in the octapeptide reactive antibodies. Pf 155 fulfills several criteria thought to be proper for antigens involved in anti-malarial protective immunity.
Amino Acid Sequence↗
Production of IL 2 and IFN-gamma by T cells from malaria patients in response to Plasmodium falciparum or erythrocyte antigens in vitro.
T cells from patients acutely infected with malaria exhibit a disease-related stimulation of DNA synthesis in response to Plasmodium falciparum antigen in vitro. This response is weak and short-lived, suggestive of induction of suppressor mechanisms. Exogenous T cell growth factor (IL 2) that was added to antigen-stimulated T cell cultures enhanced proliferation in antigen-responsive cultures, indicating that the lymphocytes expressed IL 2 receptors. In contrast, the addition of IL 2 to cultures that did not respond to antigen had no effect. Antigen-responsive cultures contained endogenous IL 2 as well, and the antigen-induced lymphocyte proliferation was correlated with IL 2 production. However, the results suggested that IL 2 production by the patients' T cells was insufficient or actively shut off, and that this was responsible for the premature cessation of their DNA synthesis. Supernatants from 60% of the T cell cultures treated with malaria antigen and from 30% treated with RBC ghost antigen contained interferon-gamma (IFN-gamma), as determined by a cytopathic effect inhibition assay combined with acid treatment and antibody neutralization or by an IFN-gamma-specific ELISA. There was no obvious correlation between antigen-induced lymphocyte proliferation and the presence of IFN-gamma in the culture supernatants. A high IFN-gamma activity was also seen in antigen-treated cultures from P. falciparum-immune donors living in highly endemic malaria areas. In contrast, no IFN-gamma was found in supernatants of antigen-treated T cells from healthy donors or patients with Plasmodium vivax malaria. Thus, the IFN-gamma activity of these cultures appears to reflect the presence of antigen-reactive T cells and may be useful as a sensitive indicator of cellular immunity in P. falciparum malaria.
ABO Blood-Group System↗
Regulation of the immune response in Plasmodium falciparum malaria. III. Proliferative response to antigen in vitro and subset composition of T cells from patients with acute infection or from immune donors.
T cells from patients with acute Plasmodium falciparum malaria were investigated for their proliferative responses in vitro to malarial antigen. Of 26 patients, 14 had acute and short lived (less than or equal to 8 days) disease episodes, most of them for the first time, while 12 had been ill for more than 8 days at the time of the blood samples were taken. The lymphocytes from the first group gave a weak, and apparently P. falciparum specific proliferation, peaking after 3-4 days, but waning within 5-6 days, suggesting the induction of suppression. No such responses were obtained with control antigen consisting of normal RBC membranes. The P. falciparum antigen-induced proliferative response was completely lacking in the second group of patients. Since both groups responded equally to T cell mitogen, the results are indicative of a malaria specific non-responsiveness. In contrast T cells from a group of apparently immune donors living in highly endemic P. falciparum malaria areas developed strong and long lasting proliferative responses to P. falciparum antigen with a peak on days 5-6. T cells from acutely infected P. vivax patients did not respond to either the P. falciparum antigen or to the control antigen. The cellular basis of the proliferative responses were investigated by surface marker studies with monoclonal antibodies. Within the T cell preparations, the T4+/T8+ cell ratio was close to normal for both the immune donors and for those with acute P. vivax infection. In contrast, this ratio was depressed for both groups of patients with acute P. falciparum infection. However, this was due to reduced number of circulating T4+ cells in the patients with short disease episodes whereas it was due to increased numbers of T8+ cells, probably including suppressor cells, in those who were ill for more than 8 days.
Antibodies, Monoclonal↗
Human spontaneous lymphocyte-mediated cytotoxicity (SLMC) against malignant and normal tissue-derived target cell lines tested in autologous and allogeneic combinations by the microcytotoxicity assay.
Effector cell types and effector mechanisms of human spontaneous lymphocyte-mediated cytotoxicity (SLMC) were studied in a 44-h microcytotoxicity titration assay. Peripheral blood lymphocytes from cancer patients and controls were used as effector cells either unfractionated or after fractionation by rosetting techniques or affinity chromatography. The possible immunoglobulin dependency of the reactions was studied by incorporation of specific Fab fragments of rabbit anti-human IgG antibodies in the incubation mixtures. Twelve different target cell lines of either high or low sensitivity to SLMC and with or without easily detectable HLA antigens were used. Most of the target cells were cell lines derived from transitional cell carcinoma of the urinary bladder (TCC). Both allogeneic and autologous lymphocyte target cell combinations were tested. Although high- and low-sensitivity target cells differed significantly in susceptibility to lysis, the predominating SLMC was displayed by Fc-receptor-positive lymphocytes in both allogeneic and autologous combinations. Addition of the Fab anti-immunoglobulin reagent to the incubation mixtures resulted in strong inhibition of cytotoxicity regardless of the type of target cells used and in allogeneic as well as in autologous lymphocyte target cell mixtures. However, in some combinations no inhibition was seen and inhibition was usually not complete, suggesting that both immunoglobulin-dependent (i.e., ADCC-like) and immunoglobulin-independent mechanisms were involved in the cytotoxicity reactions. The results of the microcytotoxicity assay were compared with those obtained with aliquots of the same lymphocytes and target cells in an 18-h 51Cr-release assay. While similar results were obtained with high-sensitivity target cells, with low-sensitivity targets and in some autologous combinations the two assay systems registered lymphocyte/target cell interactions which differed with regard to specificity, effector cell type, and immunoglobulin dependency.
Cell Line↗
Regulation of the immune response in Plasmodium falciparum malaria. II. Antigen specific proliferative responses in vitro.
The antigen-induced DNA synthesis in vitro in lymphocytes from patients with acute Plasmodium falciparum malaria was investigated. The patients and healthy controls from Sweden or Colombia were the same as those studied in the accompanying paper (Troye-Blomberg et al., 1983). The malarial antigens used were sonicated membrane preparations or purified and concentrated supernatants from in vitro cultures of P. falciparum; similar preparations derived from normal human erythrocytes served as control antigen. In the patients' lymphocytes P. falciparum antigens induced a weak or moderate but significant stimulation of DNA synthesis, peaking after 3-4 days of incubation. This early response was specific for P. falciparum since it was not obtained with lymphocytes from healthy donors nor with those from patients with acute P. vivax or P. ovale malaria. No antigen-induced response was seen in about half of the P. falciparum patients. However in a few negative cases, available for consecutive testing, positive reactions were seen with lymphocytes taken 2 weeks after infection when the blood of these patients was free of parasites. The early response induced in patients' lymphocytes to the P. falciparum antigens was not obtained with RBC antigen. However, these preparations frequently induced a response rising to significant levels later during incubation (day 5-6). Similar delayed responses were obtained when either patients' or control donors' lymphocytes were exposed to the P. falciparum antigens. This indicates that both the RBC and the parasite preparations contained mitogenic substances affecting human lymphocytes in general and easily obscuring the P. falciparum specific response seen only in the patients. This latter response was relatively low and short lived, suggesting that it reflected a secondary in vitro stimulation of in vivo primed lymphocytes and that it was regulated by suppressor mechanisms.
Acute Disease↗
Monoclonal anti-parasite and anti-RBC antibodies produced by stable EBV-transformed B cell lines from malaria patients.
To produce human monoclonal antibodies associated with infectious disease, peripheral blood lymphocytes (PBL) from patients with Plasmodium falciparum malaria were transformed with EB-virus in vitro. To enrich for malaria-specific B cells, PBL were incubated for 3 days with unsoluble P. falciparum antigen before EBV-transformation. Furthermore, cyclosporin A was added during and after transformation to eliminate T cell suppression of B cell growth. Microcultures were screened for antibodies against blood stage antigens of P. falciparum or of noninfected erythrocytes by ELISA and indirect immunofluorescence. Cultures producing anti-P. falciparum and/or anti-erythrocyte antibodies were developed from the lymphocytes of eight patients, including some individuals with their first infection. Positive cultures were cloned and propagated for several weeks. Seven of 15 clones producing antibody at a stable rate have now been kept in cultures for more than 1 yr. Of six cultures analyzed in detail, all produced IgM antibodies of either K or lambda isotype. Although three clones were monoclonal after one cloning, three were oligoclonal. Of the former, two produced P. falciparum-specific antibodies directed to an antigen associated with the surface of merozoites. One of the oligoclonal cultures produced anti-erythrocyte antibodies, and it was probably reacting with spectrin.
Antibodies, Monoclonal↗
Regulation of the immune response in Plasmodium falciparum malaria. I. Non-specific proliferative responses in vitro and characterization of lymphocytes.
The mitogen-induced DNA synthesis in vitro in lymphocytes from 20 patients acutely ill with Plasmodium falciparum malaria was compared with that of 16 healthy donors. Within both groups part of the donors were individuals who had only experienced short exposure or none at all to the parasite (Sweden) while the other part were donors living in a malaria endemic area (Colombia). The proliferative response to the T cell mitogen La (leucoagglutinin from PHA) of the patients was significantly reduced as compared with that of the controls. With pokeweed mitogen which stimulates T cells and induces a T cell-dependent activation of B cells, no difference between patients or controls was seen. The results were similar for the donors of different geographical origin and malaria background. Lymphocytes and monocytes from the peripheral blood of these donors were also studied for surface marker distribution by means of monoclonal antibodies. Both the absolute and the relative frequencies of T cells in the blood of the malaria patients were significantly reduced as compared with the controls. Furthermore, in almost all eight patients tested, the ratio between T4+ T cells (including the helper/inducer subsets) and T8+ T cells (including the suppressor and cytotoxic subsets) were below 1:1 while they were close to 2:1 in the controls. The results indicate that the relative frequency of T8+ T cells, expressed as percentage total T cells (T3+) was significantly elevated in the P. falciparum patients. The possible relationship between this imbalance and the irregular La response of the patients lymphocytes requires further investigation of lymphocyte function.
Acute Disease↗
Association between immune recognition of the malaria vaccine candidate antigen Pf155/RESA and resistance to clinical disease: a prospective study in a malaria-endemic region of west Africa.
We have measured cellular and humoral immune responses to short synthetic peptides representing epitopes of the malaria vaccine candidate antigen Pf155/RESA in a longitudinal, prospective study of clinical immunity to Plasmodium falciparum malaria in a cohort of 354 Gambian children aged 3-8 years. A significant association was observed between presence of antibodies to the 3' repeat region peptide (EENV)6 and resistance to clinical malaria. The prevalence of protective antipeptide antibodies varied significantly between different ethnic groups, suggesting that immune recognition of some Pf155/RESA epitopes may be genetically regulated. There was no obvious association between proliferative or interferon gamma responses to T cell epitopes of Pf155/RESA and resistance to malaria infection or disease. At an individual level, the presence of peptide-binding antibodies was associated with the induction of interleukin 4 messenger ribonucleic acid expression in T cells activated with the overlapping T cell epitope EENVEHDA(EENV)2. This suggests that measurement of interleukin 4 production by T cells may represent a functional assay for T helper activity.
Animals↗
Mapping of IgE binding regions in the major rat urinary protein, alpha 2u-globulin, using overlapping peptides.
Exposure to laboratory animals poses a hazard for development of occupational allergy. Identification of antigenic determinants of allergenic proteins may be valuable for immunotherapeutic purposes. Overlapping peptides of the major allergen in rat urine, Rat n 1.02, corresponding to the protein alpha2u-globulin were synthesised on solid support and screened simultaneously to locate IgE binding linear epitopes using a simple modified ELISA procedure. Thirty-nine peptides were synthesised, each 8 amino acids long with 4 amino acids overlaps. Sera from fifteen rat-sensitized subjects were analyzed and as controls sera from 7 non-rat-sensitized individuals were used. In general low binding and a great individual variation between sera from rat allergic individuals were seen. Some peptides were more frequently recognized by IgE antibodies in sera from rat allergics. These peptides were mainly clustered towards the N-terminal and C-terminal parts of the protein. Taken together our data suggest the existence of linear IgE binding epitopes in the rat urine allergen, Rat n 1.02. However, the role of these sequences in the allergic reaction needs further investigation.
Alpha-Globulins↗
Increase of circulating CD8+CD57+ lymphocytes after measles infection but not after measles vaccination.
Natural measles virus infection is recognised to induce immunosuppression, contributing to an increased susceptibility to other infections. A cell population that could be involved in this process is the CD8CD57 double-positive lymphocyte subset (CD8+CD57+), known to be significantly expanded in some viral infections, e.g. human immunodeficiency virus (HIV) infection. We therefore studied the level of CD8+CD57+ lymphocytes during measles infection and measles vaccination. Twenty-two measles patients were examined 5-57 days after the onset of fever and several months later. Healthy, age-matched controls were examined twice. Eleven children receiving measles-mumps-rubella (MMR) vaccination were examined before, 9-19 days and 5-9 months afterwards. Blood samples were analysed for the proportion of peripheral blood mononuclear cells carrying both CD8 and CD57, and for other cell surface markers (CD4, CD14, CD3, CD16(CD56) or CD20). Elevated proportions of CD8CD57 double-positive cells were found in the peripheral blood of children with natural measles early after infection (p < 0.05), whereas the proportion of other cell surface markers remained stable. No corresponding change in CD8+CD57+ lymphocytes was noted in MMR-vaccinated children or in healthy controls. Since CD8+CD57+ lymphocytes could be related to the immunosuppression seen in some viral infections, our finding of elevated CD8CD57 double-positive lymphocytes during acute measles infection would suggest that this population of lymphocytes is involved in measles-induced immunosuppression. The absence of an increase of CD8CD57 in children vaccinated with the conventional live attenuated measles vaccine, in contrast to children with natural measles infection, would thus indicate that the vaccine does not induce immunosuppression as measured in our in vitro system.
Adolescent↗