Antigen-presenting capacity of rheumatoid synovial fibroblasts.

In normal, healthy joints, synovial fibroblasts do not express major histocompatibility complex (MHC) class II molecules. However, in inflamed joints of rheumatoid arthritis (RA) patients, synovial fibroblasts show an abundant expression of MHC class II. Does this increase in expression have functional consequences for antigen presentation to T cells? To date, the precise role of synovial fibroblasts in antigen presentation has not been documented. Here, we show by three different examples that cultured synovial fibroblasts with interferon-gamma (IFN-gamma)-induced MHC class II expression are capable of processing soluble protein for presentation to CD4+ T cells. First, the antigen-presenting cell (APC) function of synovial fibroblasts was studied in an autologous model. From synovial tissue of a RA patient both a fibroblast cell line and a tetanus toxoid (TT)-specific CD4+ T-cell line were generated. A dose-dependent TT response was observed only when TT was presented by IFN-gamma-pretreated synovial fibroblasts. As more direct evidence for MHC class II-restricted antigen presentation, the response of a Mycobacterium tuberculosis-specific CD4+ T-cell clone isolated from rheumatoid synovial fluid was demonstrated in the presence of synovial fibroblasts. The response was DR4Dw4-restricted and could be inhibited by monoclonal antibody (mAb) to HLA-DR. In addition, the lymphokine secretion pattern of the synovial T-cell clone did not differ qualitatively upon antigen-specific stimulation using peripheral blood mononuclear cells (PBMC) or synovial fibroblasts as APC. In order to provide evidence for intracellular antigen processing we next examined the response of a M. leprae-specific T-cell clone with known epitope specificity. Our data suggest that synovial fibroblasts are not passive bystanders, but can become active participants in the development and maintenance of chronic inflammation.

[Immunohistochemical analysis of cell surface markers in leprosy patients].

Nine biopsy specimens from 3 tuberculoid (TT), 1 borderline tuberculoid (BT), 2 borderline (BB), 2 borderline lepromatous (BL), and 1 lepromatous (LL) patients were studied using immunoperoxidase procedures with monoclonal antibodies. In TT patients 4B4+, TCR alpha beta+, CD4+ T cells were dominant in dermal granulomas. In LL patient, dermal granulomas with 4B4+, TCR alpha beta+, CD4+ T cells and 2H4+, TCR alpha beta+, CD4+ T cells also had CD8+ T cells. The proportion of 4B4+ T cells increased in granulomas from LL to TT and 2H4+ T cells proportionately increased from TT to LL. TCR gamma delta+ T cells were detected only in BB and BL patients. However, there is no difference of staining pattern of ICAM-1 or LFA-1 antigen in any type of leprosy. These results indicate that the immunohistochemical pattern of T cells (CD4, CD8, 4B4, 2H4, TCR) may be useful in the diagnosis of the spectrum of leprosy.

Evidence that the capsule around mycobacteria grown in axenic media contains mycobacterial antigens: implications at the level of cell envelope architecture.

The intracellular growth of pathogenic mycobacteria has been linked to the presence of an electron transparent zone (ETZ or capsule), which surrounds the phagocytized bacteria and prevents the diffusion of lysosomal enzymes in infected macrophages. Recently, it was suggested that this capsule may be a bacterial structures, even being present in test tube-grown pathogenic mycobacteria (FEMS Microbiol. Lett. 1988, 56, 225-230). In the present paper, we show that under special fixation and embedding conditions, this capsule was clearly observed among 7 strains of mycobacteria grown in axenic media and also in M. leprae extracted and purified from experimentally infected armadillo or nude mice. In the case of bacteria treated likewise but subject to a prior dehydration step, this capsular structure disappeared suggesting its lipidic nature. Ultrathin sections of M. intracellular after immunolabelling showed for the first time that this capsule obtained mycobacterial antigens confirming its mycobacterial origin. It is suggested that the mycobacterial capsule may be formed of inert lipids, in which surface antigens are embedded.

CD1 positive epidermal Langerhans cells in indeterminate leprosy.

Langerhans cells (LC) in the skin lesions of 10 untreated indeterminate leprosy patients were defined by indirect immunofluorescence using monoclonal antibodies. No difference was observed in the numbers and distribution of epidermal LC in the indeterminate lesions and controls. However, the infiltrates of these lesions contained CD1+ cells. Most cells in the infiltrates HLA DR antigens.

Quantitative analysis of contrasuppressor T lymphocytes in leprosy; induction of Ia antigens with gamma interferon.

Lepromatous leprosy is characterized by immune anergy and abnormal suppressor T-cell function. Contrasuppressor cells are a subset of CD8+, vicia villosa-adherent T lymphocytes. T-contrasuppressor (Tcs) cells act on T-helper cells to cause them to become unresponsive to the action of T-suppressor cells. In 8 lepromatous (LL) and 7 tuberculoid (TT) patients, and 6 healthy contacts we studied the percent of the following lymphocyte subsets: CD3+, CD4+, CD8+, Ia+, vicia villosa+ (VV+), CD8, VV+, VV, Ia+, and Ia, Tac+. This was done in baseline status as well as post-stimulation with recombinant gamma interferon (rIFN-gamma). We found that peripheral blood mononuclear cells from LL and TT patients and controls exhibit a similar number of putative contrasuppressor lymphocytes (CD8, VV+ cells). However, in the contrasuppressor subset from LL patients we found a low percent of Ia+ (p less than 0.05 compared to controls or TT). In the three groups studied, the rIFN-gamma enhanced the percent of Ia+ lymphocytes in the CD8, VV+ cell subpopulation. However, the CD8, VV+ lymphocytes from LL patients, despite the effect of rIFN-gamma, continue to have a low percent of Ia+ cells (p less than 0.05 compared to controls or TT). These findings suggest that LL patients might have abnormalities in the contrasuppressor immune circuit. Future functional studies on the role of Tcs cells in the anergy seen in LL will be required in order to define the apparent dysfunction occurring in this disease.

Haemophilus influenzae type b lipooligosaccharide: stability of expression and association with virulence.

Spontaneous antigenic and phenotypic variations in the lipooligosaccharide (LOS) of two strains of Haemophilus influenzae type b (Hib) were previously shown to be associated with changes in virulence (A. Kimura and E.J. Hansen, Infect. Immun. 51:69-79, 1986). The goal of the present study was to define further the stability of LOS expression by this pathogen and the role of Hib LOS in virulence. Variation in LOS antigenic reactivity, as detected with LOS-specific monoclonal antibodies, was observed in 3 of 30 Hib strains after single-colony passage. When large numbers of individual colonies from seven other Hib strains were screened, however, spontaneous LOS antigenic variation was detected in all of the strains. Antigenic variation was not consistently associated with an altered LOS phenotype, as determined by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and silver staining of LOS preparations. Changes in the LOS antigenic phenotype were correlated with altered virulence potential in two strains. In these strains, acquisition of reactivity with certain LOS-directed monoclonal antibodies was associated with the synthesis of a higher-molecular-weight LOS, enhanced virulence, and increased resistance to serum killing involving the classical complement pathway.

In situ identification of activated Ta1+ T lymphocytes in human leprosy skin lesions.

Using a monoclonal antibody, anti-Ta1, that identifies antigen-activated T lymphocytes in vitro, we sought to identify activated T lymphocytes in leprosy skin lesions. Greater numbers of Ta1 positive T lymphocytes were observed in tuberculoid leprosy, lepromin skin tests, and reversal reactions as compared with lepromatous leprosy or erythema nodosum leprosum (ENL) (p less than 0.001). With a double-staining technique, we found that the majority of these activated T lymphocytes were of the helper/inducer phenotype. No differences of Ta1 positive lymphocytes were observed in the peripheral blood. The defective cell-mediated immune response in lepromatous and ENL patients correlates with, and may be related to the failure of T-helper/inducer activation or proliferation in the presence of Mycobacterium leprae.

Mycobacterium leprae antigen-induced suppression of T cell proliferation in vitro.

The extent to which M. leprae and its products induced suppression of T lymphocyte proliferation in vitro was evaluated. M. leprae antigens suppressed T cell proliferation in response to mitogens and antigens in both lepromatous and tuberculoid patients, as well as controls never exposed to M. leprae or M. leprae endemic areas. Both soluble and particulate fractions of M. leprae were found to suppress proliferation in a dose-dependent manner. The extent of suppression was inversely related to the proliferative response of the donors mononuclear cells to M. leprae. Evidence indicates that M. leprae contains both stimulatory and suppressive molecules for T cells. One such suppressive antigen, Lipoarabinomannan (LAM)-B of M. leprae, also suppressed the proliferative response of tuberculoid patients. Suppression was also observed with the LAM-B of M. tuberculosis. The suppressive effects observed were not due to the toxicity of the antigen. Some of the suppressive activity was mediated by T8+ suppressor cells and was expressed in both lepromatous and tuberculoid patients. We suggest that previous sensitization to M. leprae and other cross-reactive mycobacterial antigens determines the sensitivity of T cells to the suppressive effects of M. leprae antigens.

Study of DTH and resistance in Mycobacterium lepraemurium infection using a T-cell line isolated from mice infected with Mycobacterium bovis (BCG).

A T-cell line of mixed phenotype (60% L3T4+, 40% Lyt-2+) was isolated from mice infected with Mycobacterium bovis (BCG). This line responded to M. lepraemurium and BCG but not to M. leprae and produced TCGF spontaneously. It also produced factors which stimulated macrophages to secrete hydrogen peroxide and superoxide anion. In vivo studies showed that only L3T4+ cells were required to transfer DTH responses and that Lyt-2+ cells suppressed this response. Both L3T4+ and Lyt-2+ cells were required to inhibit M. lepraemurium multiplication in vivo.

Mycobacterium leprae-specific Lyt-2+ T lymphocytes with cytolytic activity.

Mice were immunized intradermally with 10(7) irradiated Mycobacterium leprae organisms, and draining lymph nodes were collected after 4 weeks. Lymph node cells were restimulated in vitro with soluble M. leprae antigen and accessory cells. The resulting T-cell line was propagated in vitro in the presence of M. leprae antigen, accessory cells, and interleukin-2-containing supernatants from concanavalin A-stimulated rat spleen cells. Long-term cultured T cells were Thy-1+ L3T4- Lyt-2+ as revealed by analysis with the fluorescence-activated cell sorter. From this line, T-cell clones with the same phenotype were established. The T-cell clone A4 failed to secret interleukin-2 after stimulation with antigen and accessory cells, and its growth depended on exogeneous interleukin-2. A4 T cells produced gamma interferon in an antigen-specific, H-2-restricted, and interleukin-2-dependent way. Importantly, this T-cell clone was capable of lysing bone marrow macrophages presenting M. leprae antigen. Other T-cell clones as well as native Lyt-2+ T cells from M. leprae-immunized mice were also capable of lysing bone marrow macrophages expressing M. leprae antigens. These findings suggest that specific Lyt-2+ T cells participate in the immune response to M. leprae. It is postulated that cytolysis of M. leprae-infected macrophages or Schwann cells contributes to protection against and pathogenesis of leprosy.

Suppressor T lymphocytes from lepromatous leprosy skin lesions.

The immune response in leprosy forms a spectrum with lepromatous leprosy patients exhibiting specific unresponsiveness to antigens of Mycobacterium leprae. This unresponsiveness is thought to be related to the prevalence of T8-positive lymphocyte in these lepromatous lesions. To analyze the immunoregulatory function of these T8 cells, we developed simple procedures to extract lymphocytes from skin biopsy specimens of patients with leprosy. These lymphocytes were sorted for T8 and T4 positive cells, and cell lines were established by expansion with interleukin 2 (IL 2) and irradiated feeder cells. All T8 positive lines tested were positive for IL 2 receptors and HLA-DR determinants. These lines were additionally assayed for lepromin-induced suppression of the normal peripheral blood lymphocyte Con A proliferative response. Thirteen of 32 lines from six lepromatous patients showed significant suppressor activity, whereas nine lines from six tuberculoid patients and one line from normal peripheral blood failed to show suppression (p less than 0.001). Taken together, the finding of M. leprae-triggered suppressor cells within lepromatous skin lesions may in part explain the M. leprae unresponsiveness of lepromatous leprosy patients.

T90/44 (9.3 antigen). A cell surface molecule with a function in human T cell activation.

T90/44 is a cell surface antigen which is present on human T cells of the helper and cytotoxic subsets and which binds the 9.3 monoclonal antibody (9.3 mAb). It is expressed in the form of 90-kDa disulfide-bonded dimers of a 44-kDa polypeptide and of free 44-kDa subunits. The function of T90/44 was investigated in a series of T cell function assays. 9.3 mAb was found to inhibit the activation of class II-restricted cloned T helper cells derived from leprosy patients and reactive with M. leprae antigens. The inhibition was first found at 1-10 ng/ml 9.3 mAb and regularly increased with the antibody concentration. The extent of the inhibition varied among different T cell clones in proportion to the respective different levels of T90/44 expression at their cell surface. The proliferative responses of peripheral blood lymphocytes (PBL) to purified protein derivative of M. tuberculosis (PPD) and tetanus toxoid were enhanced by the 9.3 mAb resulting in up to 20-30-fold increase of [3H]-thymidine incorporation. After phytohemagglutinin-induced activation of PBL, the number of T90/44 molecules per cell expressed at the cell surface rose from day 0 to day 7 by a factor of about 10. High concentrations of 9.3 mAb (5-10 micrograms/ml) at low cell densities and in the presence of monocytes in culture media supplemented by fetal calf serum were directly mitogenic for resting lymphocytes. The cytolytic effector functions of class I-restricted cytotoxic T lymphocytes (CTL) were not modulated by 9.3 mAb. The mixed lymphocyte reactions of three class I-restricted CTL to their specific target cells were found not to be significantly influenced by 9.3 mAb. In conclusion it is proposed that an antigen-independent T cell activation pathway can be entered at T90/44.

M. leprae and PPD-triggered T cell lines in tuberculoid and lepromatous leprosy.

Proliferative responses of peripheral blood mononuclear cells (PBMC) to Mycobacterium leprae and bacillus Calmette Guerin-derived purified protein derivative (PPD) were studied in the presence or absence of interleukin 2 (IL 2) in high M. leprae responders (tuberculoid leprosy patients and healthy subjects) and low M. leprae responders (lepromatous leprosy patients). High responders in most cases developed a strong proliferative response to both antigens in the absence of IL 2. Additional IL 2 and restimulation with antigen plus autologous antigen-presenting cells (APC) allowed the derivation of antigen-specific T cell lines. The lines were assayed for proliferative responses to several mycobacterial antigens. Both PPD and M. leprae-triggered T cell lines exhibited a good proliferative response to either antigen and showed in addition a broad cross-reactivity with other mycobacteria, suggesting a preferential T cell response to epitopes shared by several mycobacterial species. Within the lepromatous group, 50% of the patients studied could mount a proliferative response to PPD antigen in the absence of IL 2, but none of them was able to do so with M. leprae antigen. The addition of IL 2 increased the number of positive responders to PPD in this group, and in some patients IL 2 was able to restore M. leprae reactivity as well, suggesting that IL 2 had overcome a suppressor mechanism. PPD and M. leprae-triggered T cell lines were obtained from these subjects (with IL 2 added from the beginning of the culture when required). M. leprae lines exhibited variable and unstable pattern of specificity, most lines exhibiting, at least transiently, a cross-reactive response to other mycobacteria, but some displaying only M. leprae-specific response. In contrast, PPD lines from these subjects consistently exhibited a good response to PPD, a lesser response to various other mycobacteria and no response to M. leprae, a pattern differing from that obtained with PPD lines of high M. leprae responders. Co-cultures of irradiated lepromatous PPD triggered T cell lines with fresh autologous PBMC non-specifically reduced the proliferative response of the latter to PPD, as well as to unrelated antigens. A similar suppression was also observed when PPD lines from one of the tuberculoid patients were assayed. PPD and M. leprae T cell lines from both high and low responders initially exhibited the same CD4+ CD8- phenotype. In all cases, antigenic specificity declined and could not be maintained after 5 to 8 wk of continuous culture, a change associated with the progressive appearance of CD8+ and Leu8+ cells.

Influence of delayed immune reactions on human epidermal keratinocytes.

The epidermal changes that occur in human cutaneous immune responses have been investigated in the tuberculin reaction and in the lesions of tuberculoid and lepromatous leprosy and cutaneous leishmaniasis. In each situation, there was a dermal accumulation of monocytes and T cells, and the epidermis exhibited thickening. In the tuberculin response, the thickness of the epidermis sometimes doubled in 48-72 hr, and this was attributed to increases in both size and number of keratinocytes. In addition, the phenotype of the keratinocytes changed from Ia- to Ia+. Similar changes in keratinocyte Ia-antigen expression occurred in the epidermis overlying untreated tuberculoid leprosy and cutaneous leishmaniasis lesions, but not in lepromatous leprosy. We suggest that one or more epidermal growth factors may be generated in the course of a delayed immune reaction in the dermis.

Human T-cell clones with reactivity to Mycobacterium leprae as tools for the characterization of potential vaccines against leprosy.

T-cell clones with the T4 phenotype were established from patients with tuberculoid leprosy. The antigen reactivity of these clones ranged from stringent specificity for Mycobacterium leprae to broad cross-reactivity with other mycobacteria. Killed M. leprae had a weak stimulatory capacity which could be enhanced by ultrasonication. Among the three candidate antileprosy vaccines, M. leprae, M. bovis BCG, and the ICRC (Indian Cancer Research Center) strain, the last was superior in stimulating cross-reactive T4 clones. This finding argues for a differential masking of similar or identical membrane antigens in various mycobacterial species. T-cell clones with defined reactivity patterns for mycobacterial antigens could be helpful tools for the characterization of an antileprosy vaccine.

In situ and in vitro characterization of the cellular immune response in erythema nodosum leprosum.

We sought to evaluate cell-mediated immune responses in erythema nodosum leprosum (ENL), a reactional state occurring in lepromatous leprosy. Skin biopsies from patients with leprosy were studied with monoclonal antibodies against T lymphocyte antigenic determinants, interleukin 2 (IL 2), and IL 2 receptors (Tac) by using immunoperoxidase staining of frozen sections. Peripheral blood lymphocytes from 18 ENL patients were tested in vitro for lepromin-induced suppression of Con A stimulation. Serial studies of seven lepromatous patients who developed ENL during the course of the study showed increases in both the Leu-3a:Leu-2a ratio and the number of IL 2-positive cells. IL 2-positive cells comprised 0.3% of the cells in all of the ENL lesions studied as compared with the 0.03% found in nonreactional lepromatous lesions (P less than 0.001). Lepromin-induced suppression of the Con A response, present in nonreactional lepromatous patients, significantly decreased in patients developing the ENL reaction, but returned after recovery from ENL. These changes in tissues and peripheral blood suggest that the pathogenesis of ENL is related to cell-mediated immune processes. Despite these immunologic changes, however, ENL patients do not recover antigen-specific skin tests or eliminate Mycobacterium leprae.

HLA class-II-restricted Mycobacterium leprae-reactive T-cell clones from leprosy patients established with a minimal requirement for autologous mononuclear cells.

This report describes an effective method for the cloning of Mycobacterium leprae-reactive T lymphocytes with Epstein-Barr-virus transformed autologous B cells as antigen-presenting cells. The two advantages of this method are that it drastically reduces the number of autologous peripheral blood mononuclear cells (less than 10(7) cells) needed to obtain and propagate these T-cell clones (TLC), and that it enables us to expand individual TLC to large numbers of cells (greater than 10(8)). Thus the major obstacles for the cloning of T lymphocytes--especially important with regard to patients--are bypassed. Thus far, TLC from three leprosy patients have been established. These TLC are HLA class II restricted in their M. leprae-directed response. A marked enhancement in antigen responsiveness was observed after further expansion of several TLC, some of which turned from nonresponder into responder TLC. Four tested TLC display strikingly different antigen recognition patterns when tested against a number of other mycobacterial antigens; one TLC so far recognizes only M. leprae antigens.