Human suppressor T cell clones lack CD28.

Previously we showed that certain T cell lines and clones from a lepromatous leprosy patient displayed a dose-dependent suppression of the proliferation of autologous T cells to Mycobacterium leprae (M. leprae) but not mitogen or an unrelated antigen. The latter cells were also cloned and did not display this suppressive activity, were CD4+ and proliferated vigorously to M. leprae presented by autologous HLA-DR molecules. We shall refer to these cells as T helper (Th) cells. Most of the suppressive T cell clones (Ts) were also CD4+ and also proliferated to M. leprae presented by HLA-DR, but much less strongly than Th cells. In this study we report on our search for (a) the mechanism of this apparently antigen-specific suppression by T cells, and (b) a possible phenotypic difference between Th and Ts clones. The two main conclusions are that Ts clones possess a lytic machinery, but that M. leprae-specific suppression and cytotoxicity can be clearly dissociated, and that the only phenotypic difference between Th and Ts is the presence of the CD28 marker on Th and its absence on Ts clones.

Suppression of T-cell proliferation by Mycobacterium leprae and its products: the role of lipopolysaccharide.

Addition of soluble molecules obtained from sonicated Mycobacterium leprae markedly suppressed the proliferative response to the mitogen anti-CD3 of peripheral blood mononuclear cells and isolated T cells. Suppression was nonspecific and occurred with cells from lepromatous and tuberculoid leprosy patients as well as control donors. The purified lipoarabinomannans from M. leprae and Mycobacterium tuberculosis had a similar spectrum of inhibition whereas their deacylated derivatives were without effect. All mycobacterial preparations of either a crude or purified state, which suppressed cellular responses, contained appreciable quantities of bacterial lipopolysaccharide by the Limulus amebocyte assay. Contamination with lipopolysaccharide could account for the extent and nonselectivity of the T-cell suppression. Suppression was also monocyte-dependent and in part due to the release of arachidonate metabolites of the cyclooxygenase pathway.

CD2 expression and function in lepromatous leprosy.

Leprosy is a spectral disease in which clinical presentation is thought to be related to the host immune response. Previous investigations have suggested that selective unresponsiveness to Mycobacterium leprae in patients with lepromatous leprosy is due to the presence of M. leprae-specific T-suppressor cells. However, it has recently been suggested that CD2 modulation was the mechanism for the observed impaired immune response in lepromatous patients. Therefore, we studied the expression of CD2 and CD3 on lymphocytes in lepromatous skin lesions and peripheral blood mononuclear cells (PBMC). Using immunohistochemical techniques, we found that virtually all of the CD3+ cells in leprosy skin lesions expressed CD2. In addition, indirect immunofluorescence flow cytometry demonstrated that most CD3+ cells in the peripheral blood possessed the CD2 marker, suggesting that CD2 expression of T-lymphocytes is normal. T-cell activation using paired anti-T11(2) and anti-T11(3) or anti-CD3 monoclonal antibodies demonstrated similar 3H-thymidine incorporation and gamma interferon production in the PBMC of lepromatous patients in comparison with the PBMC of their contacts and tuberculoid patients. However, lepromatous PBMC did not proliferate or produce gamma interferon in response to M. leprae. Our data suggest not only that CD2 expression is normal on T lymphocytes in lepromatous leprosy skin lesions but also that CD2 expression in peripheral blood lymphocytes is functional in T-cell activation. Defective CD2 modulation does not appear to be the mechanism for specific unresponsiveness in lepromatous leprosy.

Reduced suppressor cell response to Mycobacterium leprae in lepromatous leprosy.

We have previously shown that concanavalin A (ConA) induction of suppressor cell activity is impaired in patients with lepromatous leprosy (LL). In this study, we demonstrated that the proportion of cells bearing the Leu8 antigen (associated with suppressor-inducer cells) is low in LL patients and tends to normalize during the erythema nodosum leprosum (ENL) episode. Antigen-induced suppressor cell function was evaluated by a two-stage assay. In the first stage, peripheral blood mononuclear cells (PBMC) were cultured for 5 days either in the presence of gamma-irradiated Mycobacterium leprae or in tissue culture medium as a control. In the second stage, mitomycin C-treated suppressor or control cells were added to phytohemagglutinin (PHA)- or ConA-stimulated autologous PBMC. The results indicate that the ability of M. leprae to induce suppressor activity was lower in LL patients than in patients with tuberculoid (TT) and intermediate clinical (BB, BL, BT) forms and Mycobacterium bovis BCG-immunized normal controls. In ENL patients, the percent suppression was between that of TT and normal individuals. M. leprae-induced suppression was more effective on ConA- than on PHA-triggered T-cell proliferation in all groups. In contrast, normal PBMC cultured for 5 days in RPMI 1640 medium (N-C) and cells from patients with leprosy (TT-C and LL-C) had effects of their own on PHA- or ConA-induced proliferation. LL-C depressed the response to ConA and enhanced PHA-induced proliferation of autologous cells. Conversely, TT-C reduced PHA-induced proliferation and increased the ConA response. Suppression of proliferation could not be overcome with exogenous interleukin-2 and was not related to the induction of the Tac antigen. The abilities of LL, TT, ENL, and normal cells to proliferate upon PHA or ConA stimulus were similar, indicating that the defect in the generation of in vitro suppression by M. leprae in LL patients occurred during the induction period (step 1 of assay).

The reconstitution of cell-mediated immunity in the cutaneous lesions of lepromatous leprosy by recombinant interleukin 2.

Human rIL-2 (10-30 micrograms) was injected intradermally into the skin of patients with lepromatous leprosy with high bacillary loads. All patients responded to the lymphokine with local areas of induration that peaked at 24 h and persisted for 4-7 d irrespective of whether the site was "normal skin" or a nodular lesion. Within 24 h there was an extensive emigration of T cells and monocytes into the site. The percentage of the dermis infiltrated by mononuclear cells increased by more than sevenfold, peaking at 4 d and persisting for greater than 15 d. Both CD4+ and CD8+ T cells entered the site. T cells of CD4+ phenotype predominated at 2-7 d but by 11 d, CD8+ cells were predominant. Considerable numbers of T6+ Langerhans' cells appeared in the dermis by 72 h and persisted for 3 wk. By 4 d the thickness of the overlying epidermis had increased twofold, and keratinocytes were expressing MHC class II antigen and the IFN-gamma-induced peptide IP-10. Starting at 48 h, there was an extensive destruction of mononuclear phagocytes that contained structurally intact or fragmented M. leprae observed at the electron microscope level. The organisms, either free or contained within endocytic vacuoles, were discharged into the extracellular space and then reingested by blood-borne monocytes. This was followed by marked reductions in the number of acid-fast organisms in the injected site, evident as early as 4-7 d and more marked at 2-3 wk after injection. 13 of 15 patients exhibited a disposal of acid-fast bacilli ranging from 5- to 1,000-fold with a mean value of approximately 100-fold. The administration of IL-2 leads to the generation of an effective cell-mediated immune response, recapitulating an antigen-driven event and leading to striking local reductions in M. leprae. In comparison with the purified protein derivative of tuberculin reaction, bacilli are cleared more promptly, although emigratory cells persist for a shorter time.

Learning from lesions: patterns of tissue inflammation in leprosy.

The clinical forms of leprosy constitute a spectrum that correlates closely with the degree of cell-mediated immunity. Patients with tuberculoid leprosy develop strong cell-mediated responses and have only a few, localized lesions, whereas patients with multibacillary lepromatous leprosy are specifically unresponsive to antigens of Myobacterium leprae. T cells of the CD4+ subset predominate in tuberculoid lesions, whereas CD8+ cells predominate in lepromatous lesions. Monoclonal antibodies that distinguish subpopulations of CD4+ and CD8+ cells were used to analyze the distribution of T cells infiltrating lesions across the disease spectrum. In lepromatous lesions, T cells of T-suppressor phenotype (9.3-) were the predominant CD8+ cells and suppressor/inducer cells (2H4+, Leu-8+) represented half of the CD4+ subset. In tuberculoid lesions, helper T cells (CD4+4B4+) outnumbered suppressor/inducer T cells by 14:1, compared with a ratio of 1.2:1 in peripheral blood. Analysis of the precursor frequency of antigen-reactive T cells permitted us to estimate that there was a 100-fold enrichment of T cells able to proliferate in response to M. leprae antigens in tuberculoid lesions (2/100), when compared with blood from the same patients. The methods used here to characterize the T-lymphocyte subsets and frequency of antigen-reactive T cells in leprosy may be useful in analyzing immunological reactions occurring in lesions of other inflammatory and autoimmune diseases.

Immunologic unresponsiveness in leprosy is mediated by modulation of E-receptor.

By using an indirect immunofluorescence technique with OKT3 and OKT11 monoclonal antibodies, the percentage of CD2 positive cells was found to be reduced in the peripheral blood of bacterial index positive lepromatous leprosy patients; however, in these patients, CD3 positive cells were at the normal level. Further CD2 positive cells attained the normal proportion in lepromatous patients when mycobacterial load was reduced. Both CD2 and CD3 receptors were expressed at the normal level in tuberculoid leprosy patients. Prior treatment of peripheral blood mononuclear cells from healthy controls with Mycobacterium leprae significantly decreased the percentage of CD2 but not CD3 positive cells. Such a modulation of CD2 on T cells also resulted in blocking the lymphoproliferative response induced by mitogen and antigen. These results suggest that there is a strong correlation between CD2 modulation and immunologic unresponsiveness in leprosy.