Genetically restricted suppressor T-cell clones derived from lepromatous leprosy lesions

Leprosy is a spectral disease in which immune responses to Mycobacterium leprae correlate with the clinical, bacteriological and histopathological manifestations of disease, so study of its pathology provides insights into immunoregulatory mechanisms in man. At the tuberculoid pole, patients have few lesions in the skin which contain rare organisms and are able to mount strong cell-mediated immune responses to M. leprae antigens. In contrast, at the lepromatous pole, patients have disseminated skin lesions containing large numbers of acid-fast bacilli and are selectively unresponsive to antigens of M. leprae. M. leprae-induced suppressor cells derived from peripheral blood have been reported to be active in vitro, yet their in vivo significance has remained unclear. Because the focal point of the immune response to M. leprae is the skin lesion consisting of lymphocytes and macrophages, we have recently developed methods for isolating lymphocytes from skin biopsies of leprosy patients. We report here that two T8 clones derived from lepromatous leprosy skin biopsies, in the presence of lepromin, suppress concanavalin A (Con-A) responses both of peripheral blood mononuclear cells and of T4 clones in an HLA-D (HLA, histocompatibility locus antigen)-restricted manner. Moreover, these T8 clones suppressed responses of HLA-D-matched, but not HLA-D-mismatched antigen-responsive T4 clones to M. leprae antigens, indicating that T-cell suppression is major histocompatibility complex (MHC)-restricted at some level in man.     

Lymphocyte suppression in leprosy induced by unique M. leprae glycolipid

Leprosy remains a significant medical and social problem in many developing countries. The varied forms of the disease form a spectrum. At one pole, tuberculoid leprosy, patients develop high levels of cell-mediated immunity which results in the killing and clearing of bacilli in the tissues. At the lepromatous pole, patients exhibit a selective immunological unresponsiveness to antigens of Mycobacterium leprae so that the organisms inexorably multiply in the skin. We have suggested that in lepromatous leprosy one or a small number of unique antigenic determinants present on M. leprae might induce specific suppressor cells that inhibit the reactivity of helper T-cell clones capable of recognizing other specific or cross reactive determinants. Although unique epitopes have been identified by monoclonal antibodies on a small number of M. leprae proteins, the only unique species of antigen present in M. leprae, and not on any other species of mycobacteria so far examined, is a phenolic glycolipid (gly-I). We show here that this unique antigen of M. leprae is capable of inducing suppression of mitogenic responses of lepromatous patients' lymphocytes in vitro and provide evidence that the suppressor T cells recognize the specific terminal trisaccharide moiety.     

Cloned suppressor T cells from a lepromatous leprosy patient suppress Mycobacterium leprae reactive helper T cells

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. A characteristic feature of the disease is its remarkable spectrum of clinical symptoms correlating with the cellular immune responsiveness of the patient. At one pole of this spectrum are tuberculoid patients displaying both acquired cell-mediated immunity and delayed type hypersensitivity against the bacillus. At the other pole are lepromatous patients which show a specific T-cell unresponsiveness against M. leprae. In between those two poles variable degrees of tuberculoid and lepromatous features may be seen in borderline leprosy patients. Thus far, studies on the mechanism of the antigen specific unresponsiveness in lepromatous leprosy have been contradictory and difficult to interpret, probably because of the use of heterogeneous cell populations in those experiments. We have now succeeded in cloning M. leprae stimulated T-helper (TH) as well as T-suppressor (TS) cells from a borderline lepromatous patient. The TS-clones of this patient specifically suppress responses of peripheral TH cells as well as TH clones induced by both M. leprae and other mycobacteria, but not unrelated antigen or mitogen. These TS cells also completely suppress TH cell responses against a M. leprae specific protein with a relative molecular mass of 36,000 (36K), suggesting the presence of a suppression inducing determinant on this 36K M. leprae protein.     

Mycobacterium leprae-specific protein antigens defined by cloned human helper T cells

Leprosy displays a remarkable spectrum of symptoms correlating with the T-cell-mediated immune reactivity of the host against the causative organism, Mycobacterium leprae. At one pole of this spectrum are lepromatous leprosy patients showing a M. leprae-specific T-cell unresponsiveness; at the other are tuberculoid leprosy patients displaying both acquired immunity and delayed-type hypersensitivity against M. leprae which are thought to be conferred by helper T (Th) cells. Because well-defined M. leprae antigens are crucial for the prevention and control of leprosy, we have cloned M. leprae-reactive T cells (TLC) of the helper phenotype from a tuberculoid leprosy patient. As reported here, these TLC show an unexpected diversity in the recognition of M. leprae and related mycobacteria, which is different from that exhibited by monoclonal antibodies. Half of these TLC are completely or almost M. leprae-specific, whereas the other half are cross-reactive with most or all other mycobacteria. A M. leprae protein of relative molecular mass (Mr) 36,000 (36K) defined by a M. leprae-specific monoclonal antibody stimulates 4 out of 6 TLC tested. Each of these TLC recognizes a different antigenic determinant, one of which is M. leprae-specific. The previous paper describes other M. leprae-specific T-cell clones half of which recognize an epitope on a M. leprae protein of Mr 18 K.     

Human T-cell clones recognize a major M. leprae protein antigen expressed in E. coli

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. As with other intracellular parasites, protective immunity is dependent on T cells and cell-mediated immunity. In animal models, immunization with killed armadillo-derived M. leprae elicits strong T-cell responses, delayed-type hypersensitivity and protection against viable challenge. We have recently shown that killed M. leprae can induce delayed-type hypersensitivity in healthy human volunteers. Identification of the M. leprae antigens that are recognized by T cells and may be involved in protection has been hampered by the inability to cultivate the organism in vitro and by difficulties in antigen purification from limited quantities of armadillo-derived bacillus. Because genes for the major protein antigens of M. leprae as seen by mouse monoclonal antibodies have been isolated, it has become possible to test whether these individual antigens are recognized by T cells. We screened crude lambda gtll phage lysates of Escherichia coli containing individual M. leprae antigens using M. leprae-specific T-cell clones isolated from M. leprae-vaccinated volunteers. Using this method, we find that nearly half of the M. leprae-specific T-cell clones are stimulated to proliferate by lysates containing an epitope of a M. leprae protein of relative molecular mass 18,000 (18K).     

T-cell receptors of human suppressor cells

Cells which can suppress the immune response to an antigen (TS cells) appear to be essential for regulation of the immune system. But the characterization of the TS lineage has not been extensive and many are sceptical of studies using uncloned or hybrid T-cell lines. The nature of the antigen receptor on these cells is unclear. T cells of the helper or cytotoxic lineages appear to recognize their targets using the T-cell receptor (TCR) alpha beta-CD3 complex. TCR beta-gene rearrangements are also found in some murine and human suppressor cell lines but others have been shown not to rearrange or express the beta-chain or alpha-chain genes. We previously established TS clones derived from lepromatous leprosy patients which carry the CD8 antigen and recognize antigen in the context of the major histocompatibility complex (MHC) class II molecules in vitro. We here report the characterization of additional MHC-restricted TS clones which rearrange TCR beta genes, express messenger RNA for the alpha and beta chains of the TCR and express clonally unique CD3-associated TCR alpha beta structures on their cell surface but do not express the gamma chain of the gamma delta TCR on the cell surface. We conclude that antigen recognition by at least some human CD8+ suppressor cells is likely to be mediated by TCR alpha beta heterodimers.     

Genes for the major protein antigens of the leprosy parasite Mycobacterium leprae

Leprosy, a chronic infectious disease afflicting between 10 and 15 million people, is caused by the obligate intracellular parasite Mycobacterium leprae. Although M. leprae was the first identified bacterial pathogen of man, basic biochemical, immunological, diagnostic and therapeutic investigations have been severely limited because it remains one of the few human pathogens that have not been cultured in vitro. An M. leprae recombinant DNA expression library was constructed to provide a source of genes encoding proteins relevant for such studies. Monoclonal antibodies directed against M. leprae specific antigens have been used to isolate the genes encoding the five most immunogenic protein antigens of the leprosy bacillus. We report here that M. leprae specific epitopes recognized by all of 13 monoclonal antibodies tested were produced by recombinant phage in Escherichia coli.     

Importance of IL-2 receptors in intra-thymic generation of cells expressing T-cell receptors

During development, lymphoid stem cells migrate into the thymic rudiment where they proliferate, rearrange their antigen receptor genes and become differentiated into functionally mature T cells. At present, the regulation of these processes is poorly understood, although recent studies have shown that early fetal and adult immature thymocytes express receptors for the T-cell growth factor, interleukin-2 (IL-2). We now present direct evidence that IL-2 receptors have a function in intra-thymic development by demonstrating that proliferation and the generation of cells expressing the T-cell antigen receptor (alpha beta TCR), which is responsible for the recognition of antigens in the context of MHC, are inhibited when antibodies to IL-2 receptors are added to fetal thymus organ cultures. The inhibition is specific in that it does not affect pre-thymic stem cells and can be partially reversed by addition of exogenous recombinant IL-2.     

ICOS co-stimulatory receptor is essential for T-cell activation and function

T-lymphocyte activation and immune function are regulated by co-stimulatory molecules. CD28, a receptor for B7 gene products, has a chief role in initiating T-cell immune responses. CTLA4, which binds B7 with a higher affinity, is induced after T-cell activation and is involved in downregulating T-cell responses. The inducible co-stimulatory molecule (ICOS), a third member of the CD28/CTLA4 family, is expressed on activated T cells. Its ligand B7H/B7RP-1 is expressed on B cells and in non-immune tissues after injection of lipopolysaccharide into animals. To understand the role of ICOS in T-cell activation and function, we generated and analysed ICOS-deficient mice. Here we show that T-cell activation and proliferation are defective in the absence of ICOS. In addition, ICOS -/- T cells fail to produce interleukin-4 when differentiated in vitro or when primed in vivo. ICOS is required for humoral immune responses after immunization with several antigens. ICOS-/- mice showed greatly enhanced susceptibility to experimental autoimmune encephalomyelitis, indicating that ICOS has a protective role in inflammatory autoimmune diseases.     

Accessory cell-dependent selection of specific T-cell functions

Activation of many T-cell functions depends on their interaction with antigen-presenting accessory cells which express I region associated (Ia) products. Cells expressing accessory cell function include those of the monocyte/macrophage lineage and dendritic cells. More recently, B cells and a number of tumour cell lines of macrophage or B-cell origin were shown to act as accessory cells in certain assays. We showed previously that normal peritoneal exudate macrophages (PEC) induced both T-cell proliferation as well as T-cell help, whereas various Ia+ tumour lines of macrophage or B-cell origin, although stimulating antigen-specific T-cell proliferation, did not significantly activate T-cell help. We report here that during the initial T-cell activation in vitro accessory cells (PEC or Ia+ tumour cells) select particular T cells to express previously determined functions. Moreover, some tumour cell lines induce suppressor T cells which inhibit helper activity.     

CD1c-mediated T-cell recognition of isoprenoid glycolipids in Mycobacterium tuberculosis infection

The discovery of the CD1 antigen presentation pathway has expanded the spectrum of T-cell antigens to include lipids, but the range of natural lipid antigens and functions of CD1-restricted T cells in vivo remain poorly understood. Here we show that the T-cell antigen receptor and the CD1c protein mediate recognition of an evolutionarily conserved family of isoprenoid glycolipids whose members include essential components of protein glycosylation and cell-wall synthesis pathways. A CD1c-restricted, mycobacteria-specific T-cell line recognized two previously unknown mycobacterial hexosyl-1-phosphoisoprenoids and structurally related mannosyl-beta1-phosphodolichols. Responses to mannosyl-beta1-phosphodolichols were common among CD1c-restricted T-cell lines and peripheral blood T lymphocytes of human subjects recently infected with M. tuberculosis, but were not seen in naive control subjects. These results define a new class of broadly distributed lipid antigens presented by the CD1 system during infection in vivo and suggest an immune mechanism for recognition of senescent or transformed cells that are known to have altered dolichol lipids.     

Interleukin-2 signals during priming are required for secondary expansion of CD8+ memory T cells

Although interleukin-2 (IL-2) was initially characterized as the primary T-cell growth factor following in vitro activation, less is known about its role in shaping T-cell responses to acute infections in vivo. The use of IL-2- or IL-2-receptor-deficient mice is problematic owing to their early development of autoimmunity, attributable to the central role of IL-2 in the generation, maintenance and function of CD4+CD25+ regulatory T cells. To bypass these inherent difficulties, we have studied the effect of IL-2 on T-cell responses to acute infections by adopting a mixed chimaera strategy in which T cells lacking the high-affinity IL-2 receptor could be studied in an otherwise healthy mouse containing a full complement of regulatory T cells. Here we show that although IL-2 signalling to pathogen-specific CD8+ T cells affects the number of developing effector and memory cells very little, it is required for the generation of robust secondary responses. This is not due to an altered T-cell-receptor repertoire development or selection, and does not reflect an acute requirement for IL-2 during secondary activation and expansion. Rather, we demonstrate a previously unappreciated role for IL-2 during primary infection in programming the development of CD8+ memory T cells capable of full secondary expansion. These results have important implications for the development of vaccination or immunotherapeutic strategies aimed at boosting memory T-cell function.     

Recovery from autoimmunity of MRL/lpr mice after infection with an interleukin-2/vaccinia recombinant virus

Interleukin-2 (IL-2) is a T-cell derived molecule implicated in the clonal expansion of antigen-activated T cells and in T-cell development. IL-2 is also implicated in autoimmune disease, although its role is still controversial. Murine systemic lupus erythematosus (SLE) is a good model for human SLE as most of the immunological abnormalities in the human disease also seem to be operative in the mouse. Among SLE mice, the MRL/lpr strain develops early in life autoimmune diseases such as immune complex-mediated glomerulonephritis, arthritis and arteritis. Lymphoid abnormalities associated with those diseases in this strain are thymic atrophy and abnormal proliferation of CD3+ CD4- CD8- 'double-negative' T cells, resulting in massive generalized lymph node enlargement. We have therefore now examined the effects of IL-2 on the disease progression in MRL/lpr mice using live vaccinia recombinant viruses expressing the human IL-2 gene. Vaccinated mice showed prolonged survival, decreased autoantibody and rheumatoid factor titres, marked attenuation of kidney interstitial infiltration and intraglomerular proliferation, as well as clearance of synovial mononuclear infiltrates. Inoculation with the IL-2/vaccinia recombinant virus led, in addition, to drastic reduction of the double-negative T-cell population, improved thymic differentiation and restoration of normal values of mature cells in peripheral lymphoid organs.     

麻风杆菌多途径接种裸鼠的研究

用麻风杆菌经静脉内、后足垫和耳皮下多途径接种裸鼠。在裸鼠接种菌后307、334、497、和625天分别剖杀1—2只裸鼠,取接种足垫和耳部皮损、胭窝淋巴结、鼻、坐骨神经、尾部皮肤损害、舌、肝、脾、肺、肾和心脏称重后制成匀浆,作菌计数。同时各取部分组织作组织病理学检查。实验结果表明,裸鼠经过多途径接种麻风杆菌后,其菌量增殖明显高于以往报告的单途径接种方法所取得的结果。尤其在接种部位的后足垫及耳部,最高菌量分别达到2.85×10~11/g(497天)和6.28×10~12/g(625天)。虽然麻风杆菌的世代时间近似,但对数生长期时间明显延长。结果也表明,多途径接种方法能使裸鼠发生更严重的病变和系统性播散。组织病理学检查表明,经抗酸染色的足垫组织切片中,在横纹肌、血管壁内皮细胞和血管中均可见到大量抗酸杆菌(AFB)及菌团,皮神经束膜内外和雪旺氏细胞中也找到了许多AFB。最终在整个足垫组织中充满了AFB。HE染色呈瘤型麻风进行性病变,肉芽肿浸润由含大量AFB的泡沫细胞和巨噬细胞构成,直至泡沫细胞组成的麻风瘤病变完全代替了正常结构。各个部位相比较,低体温部位如足垫、耳、鼻及尾部仍然是麻风菌繁殖的优势部位。横纹肌及外周神经是麻风菌好侵犯之处,也显示了麻风菌在裸鼠体内感染的特点。     

Cloning of complementary DNA encoding T-cell replacing factor and identity with B-cell growth factor II

Proliferation and maturation of antigen-stimulated B cells are regulated by several soluble factors derived from macrophages and T cells. These soluble factors are functionally divided into two groups: B-cell growth factor (BCGF), thought to be involved in B-cell proliferation; and B-cell differentiation factor (BCDF), responsible for maturation of activated B cells into immunoglobulin-secreting cells. This classification needs to be re-examined in the light of the recent cloning of complementary DNA encoding IgG1 induction factor (interleukin-4, IL-4) from the 2.19 mouse T-cell line. Recombinant IL-4 has BCGF and BCDF activities and affects B cells, T cells and mast cells (refs 7, 8; our unpublished data). Another well-characterized B-cell factor is T-cell replacing factor (TRF), which, when secreted by the murine T-cell hybridoma B151K12, is defined by two activities: induction of IgM secretion by BCL1 leukaemic B-cell line; and induction of secondary anti-dinitrophenol (DNP) immunoglobulin G (IgG) synthesis in vitro by DNP-prime B cells. Although TRF from B151K12 was classified as BCDF, purified TRF has BCGF-II activity. To elucidate the molecular properties of TRF we isolated cDNA encoding TRF from the 2.19 T-cell line and report here the structure and multiple activities of this lymphokine.     

Mitogens trigger a calcium-independent signal for proliferation in phorbol-ester-treated lymphocytes

The activation of T lymphocytes by mitogens requires at least two signals; the first, delivered to T cells by a mitogen in conjunction with accessory cells (monocytes/macrophages), leads to the generation of the second signal, interleukin-2 (IL-2). The first signal also induces the expression of IL-2 receptors on the surface of a subpopulation of T cells; binding of IL-2 to its receptor then initiates a cascade of events culminating in DNA synthesis by these cells. Certain compounds act synergistically with mitogens in promoting T-cell proliferation by substituting for the activities of interacting cells or their products. For example, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) has been shown to restore the ability of macrophage-depleted T-cell populations to respond to mitogenic lectins. Transmembrane fluxes of calcium, leading to increased free cytosolic calcium concentrations ([Ca2+]), have been demonstrated following mitogen binding to lymphocytes and have been implicated in the initiation of cell proliferation. We show here that the effect of TPA on lymphocyte proliferation occurs in the absence of extracellular Ca2+ or detectable changes in [Ca2+]i, but only in the presence of mitogens. This suggests that in cells which have been incubated with the phorbol ester, mitogens can induce proliferation by a calcium-independent signal.     

Astrocytes present myelin basic protein to encephalitogenic T-cell lines

Astrocyte proliferation and perivascular lymphocyte infiltration are conspicuous among the cellular changes in the active brain lesions of multiple sclerosis patients. Recent observations have indicated that most of the perivascular lymphocytes are T cells which may be actively involved in the generation of the brain lesions. Much less is known about the significance of the proliferative astrocytes, although the fact that they produce an interleukin-1 (IL-1)-like factor that enhances the release of interleukin-2 by T lymphocytes, may provide a clue. We show here that rat astrocytes are able to present antigen to T lymphocytes in a specific manner which is restricted by the major histocompatibility complex (MHC) and that they can in particular activate myelin basic protein (BP)-specific, encephalitogenic T-cell lines. Only on such interaction do astrocytes express Ia antigens in easily detectable amounts. Antigen presentation by astrocytes may have a central role in the generation of immune responses in the brain.