Adam S. Hey

Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis

The T cell response to antigens from Leishmania major promastigotes was investigated in peripheral blood mononuclear cells from Sudanese individuals with a history of cutaneous leishmaniasis (CL), Sudanese individuals with positive DTH reaction in the leishmanin skin test but with no history of skin lesions, and in Danes without known exposure to Leishmania parasites. Proliferation and production of interferon‐gamma (IFN‐γ) and IL‐4 in antigen‐stimulated cultures was measured. Lymphocytes from individuals with a history of CL proliferated vigorously and produced IFN‐γ after stimulation with either a crude preparation of L. major antigens or the major surface protease gp63. These cultures produced no or only little IL‐4. Also cells from leishmanin skin test‐positive donors with no history of CL produced IFN‐γ and no IL‐4 in response to major antigens. Cells from the unexposed Danes were not activated by gp63. The cells from Danish donors produced either IFN‐γ or IL‐4, but not both cytokines after incubation with the crude preparation of L. major antigens. The data show that the T cell response to Leishmania antigens in humans who have had uncomplicated CL or subclinical L. major infection is an IFN‐γ‐producing Th1‐like response.

Dichotomy of the human T cell response to Leishmania antigens. II. Absent or Th2-like response to gp63 and Th1-like response to lipophosphoglycan-associated protein in cells from cured visceral leishmaniasis patients.

The T cell response to different Leishmania donovani aniigens was investigated using peripheral blood mononuciear cells (PBMC) from Kenyans cured of visceral leishmaniasis and non‐exposed Danes. Crude promastigote and amastigote antigens both induced proliferation and interferon‐gamma (IFN‐γ) production in PBMC from cured patients, while cells from non‐exposed donors gave weak responses. A similar pattern was indticed by lipophosphoglycan‐associated protein (LPGAP). By contrast, the major surface protease of Leishmania. gp63, induced only a weak proliferative response without IFN‐γ production in five of 17 samples from cured patients. Four of the five responding cultures produced IL‐4, i.e. the response to this antigen was of the Th2 type. Furthermore, sera from acutely ill visceral leishmaniasis patients contained high levels of IgG antibodies to gp63. The Th2‐tike response to gp63 in patients cured of visceral leishmaniasis differs from the Thl‐like response to the same antigen observed in patients cured of cutaneous leishmaniasis.

Leishmania major surface protease Gp63 interferes with the function of human monocytes and neutrophils in vitro

In the present study the effect of Leishmania major surface protease Gp63 on the chemotaxis and oxidative burst response of human peripheral blood monocytes and neutrophils was investigated. It was shown that prior incubation of cells with Gp63 inhibited chemotaxis of neutrophils but not monocytes towards the chemotactic peptide f‐met‐leu‐phe. On the other hand, chemotaxis of both neutrophils and monocytes towards zymosan‐activated serum containing C5a was inhibited by Gp63. Monocyte and neutrophil chemiluminescence response to opsonized zymosan was reduced by preincubation of the cells with Gp63 in a concentration‐dependent manner. Notably, monocytes were inhibited to a much greater degree than neutrophils by a given concentration of Gp63, and they were also inhibited at much lower concentrations of the protease. The inhibitory effect of Gp63 on chemotaxis and chemiluminescence was completely abolished by heat inactivation of the protease at 70 °C for 15 min. Neither neutrophil nor monocyte chemiluminescence was inhibited by Gp63 when cells were stimulated with PMA. Our data suggest that the major surface protease Gp63 might play an important role in the initial stages of Leishmania/macrophage interactions and the intracellular survival of the parasite.

Dichotomy of the T cell response to Leishmania antigens in patients suffering from cutaneous leishmaniasis; absence or scarcity of Th1 activity is associated with severe infections

The T cell response was studied in 25 patients suffering from cutaneous leishmaniasis caused by Leishmania major with severe (n= 10) and mild (n= 15) disease manifestations. Peripheral blood mononuclear cells (PBMC) from the patients were activated by sonicates of Leishmania promastigotes (LMP) and amastigotes (LDA), and the surface protease gp63. The proliferative responses to Leishmania antigens were lower in patients with severe disease than in patients with mild disease (P= 0·01–0·05), and such a difference was not observed in the response to purified protein derivative of tuberculin (PPD) or tetanus toxoid (TT). LMP‐induced interferon‐gamma (IFN‐γ) production was lower in patients with severe than in patients with mild disease (P < 0·05). When the IL‐4 and IFN‐γ responses of each patient were considered, two response patterns were observed in the cultures activated by the Leishmania sonicates. One response pattern was characterized by high production of IFN‐γ without production of IL‐4 (a Th1‐like pattern), the other was characterized by low IFN‐γ levels which in most cases were associated with IL‐4 production (not a Th1‐like pattern). These patterns could not be distinguished when the cells from the same donors were stimulated by TT and PPD. The percentages of patients with a Th1‐like response pattern after stimulation by LMP in patients with severe and mild disease manifestations were 30% and 80%, respectively. This difference was statistically significant (P= 0·034).

Measurement of antigen-dependent interleukin-4 production by human peripheral blood mononuclear cells Introduction of an amplification step using ionomycin and phorbol myristate acetate

The study of T cell responses in parasitic disease and allergy in humans has been limited by difficulties in the measurement of interleukin-4 (IL-4) in supernatants from antigen-stimulated peripheral blood mononuclear cells (PBMC). To obtain measurable amounts of IL-4 in vitro, we have added an amplification step to the antigen-specific response. Human PBMC were stimulated by tetanus toxoid (TT) or tuberculin (PPD) for 6 days and then pulsed with ionomycin and phorbol myristate acetate (PMA) for 24 h. TT-stimulated cells from nine revaccinated donors but not from seven unvaccinated donors and four that had only received childhood vaccinations against tetanus produced high levels of IL-4 (median (range) 1500 (300-3800), 316 (0-1600), and 270 (100-410) pg/ml, respectively, as measured by an enzyme linked immunosorbent assay, P = 0.005). PPD did not increase IL-4 production above the background level, although the majority of PPD-stimulated PBMC proliferated and produced interferon-gamma (IFN-gamma) in cultures without ionomycin and PMA. TT-induced IL-4 production correlated positively with proliferation. Culture supernatants did not interfere with IL-4 immunoreactivity and failed to affect ionomycin and PMA induced IL-4 production. The findings suggest that proliferating antigen-specific T cells were the source of IL-4 in these experiments. The method should prove useful for comparing the IL-4 producing ability of antigen-specific T cells from different individuals.

Activation of Human T Lymphocytes by Leishmania Lipophosphoglycan

This study describes Leishmania antigen‐induced activation of lymphocytes isolated from Kenyan donors, previously treated for visceral leishmaniasis. and from Danish and Kenyan controls. Peripheral blood mononuclear cells (PBMC) from cured Kala‐Azar patients proliferated and produced Interferon‐γ in vitro in response lo lipophosphoglycan (LPG) isolated from Leishmania major. The proliferative response was mainly due lo activation of Cn2‐posilive T cells, PBMC from controls did not respond to LPG. hut to sonicates prepared from both L. major and L. donovani promastigotes. The surface glycoprotein GP 63 failed lo activate PBMC from any of the donors tested.

Measurement of serum antibodies against native Leishmania gp63 distinguishes between ongoing and previous L. donovani infection.

An enzyme‐linked immunosorbent assay (ELISA) using native gp63 for detection of serum antibodies to Leishmania was evaluated. The test identified antibodies in sera from 16 of 16 visceral leishmaniasis (VL) patients and 9 of 12 sera from patients with Trypanosoma brucei infection. In comparison, sera from 80 Danish controls and 40 control donors from a malaria endemic area of Ghana without known exposure to Leishmania were negative, as were sera from 12 Kenyan malaria patients and 9 schistosomiasis patients. After cure of VL, sera rapidly became negative. Only 1 of 7, 1 of 21, and 1 of 27 sera from cured VL patients 6–12 months, 1–2 years and >2 years after cure were positive. Thus, in contrast to other serological tests for VL, the gp63 ELISA seems to distinguish an ongoing from a past infection. This might prove useful both for diagnostic and epidemiological purposes.

Field application of an ELISA using redefined Leishmania antigens for the detection of visceral leishmaniasis

Two soluble antigens from Leishmania donovani of 116 kDa and 70 kDa molecular mass, and a soluble mixture of crude antigens, were used in an enzyme-linked immunosorbent assay (ELISA) for the detection of visceral leishmaniasis (VL) in the field, and compared with the direct agglutination test (DAT). The tests were carried out on 8 VL patients, 34 normal individuals from an area endemic for the disease, and 68 former visceral leishmaniasis patients 1-5 years after treatment. The 70 kDa ELISA and the DAT had a sensitivity and specificity of 100% (95% confidence interval 63-100%), while the 116 kDa ELISA and the soluble crude antigen ELISA were 37.5% (9-76%) and 50% (16-84%) sensitive, respectively. When using ELISA (116 kDa or 70 kDa), 68-69% of sera tested 1-2 years, and 92-94% of sera tested 5 years, after treatment were negative. In contrast, when DAT or ELISA with crude antigen were used, the negativity rate was 31% 1-2 years, and 53% 5 years, after treatment. DAT was therefore not an accurate test for diagnosis in the field. The use of the 70 kDa antigen in ELISA was an accurate alternative to DAT in the detection of VL.

Thl-Like Human T-Cell Clones Recognizing Leishmania gp63 Inhibit Leishmania major in Human Macrophages

The major surface protease of Leishmania major, gp63, has been suggested as a vaccine candidate for cutaneous leishmaniasis. In this study gp63 was purified from L. major promastigotes. A panel of human T‐cell clones recognizing this protein were generated from individuals who had previously had self‐healing cutaneous leishmaniasis. The T‐cell clones expressed CD4, and the alpha chain of the T‐cell antigen receptor. Gp63 reactive T‐cell clones activated by antigen or by immobilized anti‐CD3 antibody released relative large amounts of interferon‐gamma and no or little interleukin‐4, thereby resembling Th l cells. Autologous mononuclear cells and Epstein‐Barr virus‐transformed B cell lines were equally efficient in presenting the antigen to the T cells. The gp63 reactive T cells induced resistance to infection in cultured human macrophages by L. major. The data confirm that human CD4+ T cells recognizing gp63 can take part in the host defence against L. major infections.