Early accumulation of suppressor cell precursors in the spleen of Mycobacterium lepraemurium-infected mice and analysis of their in vitro-induced maturation.

Spleen cells harvested from mice infected intraperitoneally with M. lepraemurium 11-17 weeks prior to harvest acquired the capacity to inhibit concanavalin A (Con A) induced proliferation of normal spleen cells when precultured for up to 24 h in mitogen-free medium. The in vivo induced suppressor activity correlated with the length of the preculture period, the time post-infection and the infecting dose. These findings were interpreted as an indication that suppressor cell precursors accumulated in the spleen of infected mice during the early phase of the disease. The interaction of infection-dependent adherent suppressor cell precursors and infection-independent, non-adherent regulatory cells is necessary for the suppressor activity to develop. Both the cells which transmit the inductive signal and the precursor cells which mature into active suppressor cells are radiosensitive, whereas suppressor activity itself is a function of radioresistant adherent cells. Preculture of cells for a short period, before they were cocultured with Con A-stimulated normal spleen cells, allowed the detection of suppressor cells before they were deleterious to the infected host and also turned out to be a relevant in vitro model for characterization of suppressor cell development during M. lepraemurium infection.     

Influence of the Ity/Lsh/Bcg gene on the development of suppressor cell precursors in the early phase of the infection of mice with Mycobacterium lepraemurium.

In vitro inducible suppressor cell precursors were detected in the spleen of BALB/c but not in DBA/2 mice infected intraperitoneally with 10(8) Mycobacterium lepraemurium bacilli, thus suggesting that their development is genetically controlled. Two pairs of mouse strains congenic at the Ity/Lsh/Bcg locus (BALB/c-C.D2 and B10.A-B10.A.Bcgr) were used to investigate whether this phenomenon is influenced by this gene known to control the relative susceptibility of mice to Myco. lepraemurium infection. This seems likely, as the detection of culture-induced suppressor activity was delayed for 5-6 weeks in C.D2 and B10.A.Bcgr mice infected intravenously with 10(4) Myco. lepraemurium bacilli. However, despite the retardation in the detection of suppressor cell precursors, the level of in vitro induced suppressor activity at onset in spleen cell suspensions of mice carrying the resistant allele was higher than in cell cultures derived from susceptible mice. As the resistant allele has a different effect when found on BALB/c or DBA/2 background, other genetic factors are apparently involved in the development of suppressor cell precursors. We finally observed that, in spleen cell cultures from intravenously infected Ity/Lsh/Bcg congenic mice on the BALB/c background, adherent and non-adherent cells were required in the inductive phase of suppressor cell development, whereas in vitro induced suppressor activity was found exclusively in the adherent cell fraction. Given these properties, we thus conclude that suppressor cell precursors detected in the spleen of these intravenously infected mice are similar to those previously observed in C3H mice infected intraperitoneally with a thousand times more bacilli.     

Phenotypic characterization of two cell populations involved in the acquisition of suppressor activity by cultured spleen cells from Mycobacterium lepraemurium-infected mice.

The impairment of cellular immunity in mice infected with Mycobacterium lepraemurium was shown to correlate with the development of suppressor cells. We have previously reported that before suppressor activity is detectable in freshly harvested cell suspensions, suppressor cell precursors accumulate in the spleen of infected mice. Upon overnight culture in the presence of a regulatory cell subset, these precursor cells acquire the capacity to impair the concanavalin A (Con A)-induced proliferation of normal spleen cells. The purpose of this study was to determine the phenotype of the cells involved in this phenomenon. This was done by following the development of suppressor activity in spleen cell suspensions depleted of defined cell subsets of the adherent or the non-adherent cell fractions with selected MoAbs and immunomagnetic beads or by in vivo treatment. Our results indicate that the acquisition of suppressor activity requires the interaction of Ia+CD11b+Fc gamma R+IgG- asialo GM1- adherent cells with Thy1-CD4-CD8-IgG-Ia- asialo GM1-Fc gamma R+CD11b+ non-adherent cells. It is also shown that the development of suppressor activity is impaired by preventing cell-cell contact between these two cell subsets through coculture in 'Transwell chambers'. These observations support the conclusion that the in vitro acquisition of suppressor activity is a consequence of the maturation of suppressor cell precursors of the monocytic lineage induced by a receptor-ligand type interaction with a non-adherent cell subset that is clearly distinct from mature T, B and natural killer (NK) cells.     

Effect of reactive oxygen intermediaries on the viability and infectivity of Mycobacterium lepraemurium

Murine leprosy is a natural disease of the mouse, the most popular model animal used in biomedical research; the disease is caused by Mycobacterium lepraemurium (MLM), a successful parasite of macrophages. The aim of the study was to test the hypothesis that MLM survives within macrophages because it highly resists the toxic effects of the reactive oxygen intermediaries produced by these cells in response to infection by the microorganism. MLM cells were incubated in the presence of horseradish peroxidase (HRPO)-H(2)O(2)-halide for several periods of time. The peroxidative effect of this system was investigated by assessing the changes occurred in (a) lipid composition; (b) viability; and (c) infectivity of the microorganism. Changes in the lipid composition of peroxidated- vs. intact-MLM were analysed by thin layer chromatography. The effect of the peroxidative system on the viability and infectivity of MLM was measured by the alamar blue reduction assay and by its ability to produce an infection in the mouse, respectively. Peroxidation of MLM produced drastic changes in the lipid envelope of the microorganism, killed the bacteria and abolished their ability to produce an in vivo infection in the mouse. In vitro, MLM is highly susceptible to the noxious effects of the HRPO-H(2)O(2)-halide system. Although the lipid envelope of MLM might protect the microorganism from the peroxidative substances produced at 'physiological' concentrations in vivo, the success of MLM as a parasite of macrophages might rather obey for other reasons. The ability of MLM to enter macrophages without triggering these cells' oxidative response and the lack of granular MPO in mature macrophages might better explain its success as an intracellular parasite of these cells.     

Host and bacterial factors control the Mycobacterium avium-induced chronic peritoneal granulocytosis in mice.

Persistent peritoneal granulocytosis and elevated macrophage counts have been found in nine mouse strains from 8 to 90 days after infection with Mycobacterium avium. Peritoneal granulocytosis was higher in M. avium-resistant BALB/c. Bcgr (C.D2) mice, compared with congenic M. avium-susceptible BALB/c (Bcgs) animals. Although maximal granulocytosis values were not related to virulence of the inocula, the kinetics of the granulocytic response varied with the virulence of M. avium. Following infections by avirulent (rough) strains of M. avium, the peritoneal granulocytosis progressively declined in BALB/c and C3H/He mice. A similar decline in granulocyte number was observed in resistant C3H/He mice infected with virulent M. avium (smooth transparent strain). In both instances the decline in the peritoneal granulocytosis was associated with a progressive elimination of the inoculum. In the susceptible BALB/c mice, virulent M. avium strains induced progressive infection accompanied with a rapid decline in granulocyte number, whereas the infection with attenuated M. avium, which caused a chronic infection, induced persistent granulocytosis. The ability to recruit granulocytes following the intraperitoneal inoculation of a phlogistic substance (casein hydrolysate) was decreased in infected susceptible but not in infected resistant mice at 90 days of infection with virulent M. avium.     

A comparison of the T cell delayed-type hypersensitivity epitopes of the 19-kD antigens from Mycobacterium tuberculosis and Myco. intracellulare using overlapping synthetic peptides.

Mycobacterial disease remains a serious international public health concern. Improved methods to rapidly and specifically detect mycobacterial infections would greatly enhance clinical management of these diseases. To define species-specific T cell epitopes that may be useful for the immunodiagnosis of mycobacterial infections, polymerized synthetic peptides from the 19-kD Mycobacterium tuberculosis and Myco. intracellulare protein homologues were tested in guinea pig DTH assays. Five Myco. tuberculosis and eight Myco. intracellulare peptides evoked skin test responses. Although all of the active Myco. tuberculosis and seven of the Myco. intracellulare peptides elicited non-specific DTH reactions, the peptide IN13 induced a Myco. intracellulare-specific skin test reaction, and thus represents a specific Myco. intracellulare T cell DTH epitope. This result suggests that the development of monospecific peptide-based immunodiagnostic reagents may be feasible for future clinical use.     

Seasonal variation in suppressor T cell subsets and non-specific suppressor cell function in hay fever sufferers

The helper/suppressor T cell ratio, as defined by monoclonal antibodies, was significantly higher in hay fever sufferers compared with controls (P less than 0.05), but only during or shortly after the pollen season. This was due to a reduction in the suppressor subset, which returned to control values in the winter. There was no significant difference in the non-specific concanavalin A-induced suppressor cell function compared with controls. The mean summer value was significantly lower than the winter value (P less than 0.05), but we cannot be sure that this was not the result of changes in laboratory conditions. No relationship was found between T cell subsets or suppressor cell function and total or specific IgE levels, or between T cell subsets and suppressor cell function. Our findings suggest that in hay fever, reduction in suppressor cell numbers and function is a secondary phenomenon.     

瘤型麻风动物模型血浆Ig的变化及其对ll—2产生的动态观察

本文报道了瘤型麻风小鼠模型血浆IgG,IgM和IgA含量的动态变化,以及感染鼠的血浆体外对正常鼠脾细胞IL—2生成的影响的动态观察。结果表明,随着感染时间的延长,小鼠血浆中的三种Ig的含量依次递增。进一步的研究发现,随着Ig水平的升高,感染小鼠的血浆对正常小鼠脾细胞IL—2生成呈现明显的抑制作用。加有感染1月、3月和6月的小鼠血浆的正常鼠脾细胞IL—2生成均明显降低(P<0.01),其抑制率分别为26.2％、40.0％和75.8％。结合我们的观察与瘤型麻风独特的免疫偏离进行了讨论。     

Profiles of cell-to-cell interaction of Mycobacterium intracellulare-induced immunosuppressive macrophages with target T cells in terms of suppressor signal transmission

Previously, we have found that immunosuppressive macrophages (M(phi)s) induced by Mycobacterium intracellulare-infection (MI-M(phi)s) required cell contact with target T cells to express their suppressor activity against concanavalin A (Con A)-induced T cell mitogenesis. In this study, we examined the profiles of cell-to-cell interaction of MI-M(phi)s with target T cells. First, MI-M(phi)s displayed suppressor activity in an H-2 allele-unrestricted manner, indicating that MHC molecules are not required for cell contact. The suppressor activity of MI-M(phi)s was reduced markedly by paraformaldehyde fixation or treatment with cytochalasin B or colchicine, indicating that vital membrane functions are required for their suppressor activity. Secondly, the suppressor activity of MI-M(phi)s was independent of cell-to-cell interaction via CD40 ligand/CD40 and M(phi)-derived indoleamine 2,3-dioxygenase, which causes rapid degradation of tryptophan in T cells. Thirdly, precultivation of splenocytes with MI-M(phi)s, allowing cell-to-cell contact, reduced Con A- or anti-CD3 antibody-induced mitogenesis but not phorbol myristate acetate/calcium ionophore A23187-elicited proliferation of T cells. In addition, co-cultivation of T cells with MI-M(phi)s caused marked changes in profiles of the tyrosine phosphorylation of 33 kDa, 34 kDa and 35-kDa proteins and, moreover, the activation of protein kinase C and its translocation to the cell membrane. It thus appears that suppressor signals of MI-M(phi)s, which are transmitted to the target T cells via cell contact, principally cross-talk with the early signalling events before the activation of PKC and/or intracellular calcium mobilization.     

Comparative studies on the roles of mediator molecules in expression of the suppressor activity of Mycobacterium avium complex-induced immunosuppressive macrophages against T cell and B cell mitogenic responses

Mycobacterium avium complex-induced immunosuppressive macrophages (MAC-MPhis) exhibit suppressor activity against concanavalin A-induced T cell mitogenesis (T cell Con A mitogenesis). We examined the profiles of the MAC-MPhi-mediated suppression of lipopolysaccharide-induced B cell mitogenesis (B cell LPS mitogenesis) and found the following. First, although N(G)-monomethyl-L-arginine and carboxy-PTIO effectively blocked the MAC-MPhi's suppressor activity against T cell Con A mitogenesis, MAC-MPhi's action against B cell LPS mitogenesis was only weakly affected by these NO-reducing agents. Second, B cell LPS mitogenesis was remarkably more susceptible to MAC-MPhi-derived reactive oxygen intermediates than T cell Con A mitogenesis. Third, B cell LPS mitogenesis was less susceptible to the inhibitory effects of the other MAC-MPhi-derived suppressor mediators, including free fatty acids, TGF-beta and prostaglandin E(2), than T cell Con A mitogenesis. Fourth, MAC-MPhi's suppressor activity was strongly dependent on B7-1 like molecule-mediated cell contact with target cells only in the case of T cell Con A mitogenesis. Therefore, there are significant differences in the modes of suppressor action of MAC-MPhis against T cell and B cell mitogenesis.     

瘤型麻风动物模型的NK细胞活性的体外测定

本文报道了瘤型麻风小鼠模型中脾脏NK细胞活性的体外测定,以及加入外源性IL-2对这一活性的影响。结果表明,随着感染时间的延长,小鼠脾脏中NK细胞活性逐渐降低。感染1月小鼠的NK细胞活性明显低于正常小鼠,感染3月小鼠的NK细胞活性较感染1月小鼠更低(P值均<0.01)。将含有IL-2的上清液加入NK细胞活性检测系统中,发现外源性IL-2可逆转感染小鼠的NK细胞活性,使其恢复达正常水平。结合IL-2与NK细胞活性在免疫调节中的作用,对瘤型麻风动物模型的特殊表现作了讨论。     

Expression of CD34 on human B cell precursors.

CD34 is a 110-kD glycoprotein previously shown by a variety of monoclonal antibodies (MoAbs) to be expressed selectively on immature hematopoietic cells. However, more detailed characterization of CD34+ cells has been hampered by lack of anti-CD34 MoAbs that can be labelled directly with fluorochromes to facilitate subpopulation analysis by multi-parameter flow cytometry. We have recently isolated a murine anti-CD34 MoAb, designated as 8G12, that can be directly labelled with fluorochromes such as FITC. In this study, we have exploited this property of 8G12 to compare the reactivity of 8G12 and My10 with normal and leukaemic human marrow cells and to characterize normal early human B cell precursors by two- and three-colour immunofluorescence analysis. Comparison of three-colour staining profiles of normal bone marrow cells incubated with both 8G12 and MY10, and either anti-CD10 or anti-CD19 MoAb revealed the reactivity patterns of 8G12 and MY10 to be indistinguishable. This conclusion was confirmed by a similar comparative analysis of 8G12 and MY10 staining of blood and bone marrow cells from 4 patients with B lineage acute lymphoblastic leukaemia (ALL). Of interest, both 8G12 and MY10 detected a CD34+CD10+CD19- population in normal adult bone marrow. To determine whether a CD34+CD10+CD19- precursor population previously reported by others to exist in fetal liver could also be identified, CD10+CD16- marrow cells were first isolated by FACS and the sorted cells then re-analysed for expression of CD19 and CD34. These studies showed that all of the sorted CD10+ cells that expressed CD34 appeared to coexpress CD19. No CD34+CD10+CD19- cells were detected (at a sensitivity of less than or equal to 0.1%). Further studies will be required to determine whether a very minor population of CD34+CD10+CD19- cells may still be generated in the normal development of B cells in adult human marrow.     

Palsy of the rear limbs in Mycobacterium lepraemurium-infected mice results from bone damage and not from nerve involvement

A small but relatively constant proportion (3-5%) of mice chronically infected with Mycobacterium lepraemurium (MLM) develops bilateral paralysis of the rear limbs. The aim of the study was to investigate whether or not the bilateral leg palsy results from nerve involvement. Direct bacterial nerve infection or acute/delayed inflammation might possibly affect the nerves. Therefore, palsied animals were investigated for the presence of: (a) histopathological changes in the leg tissues including nerves, bones and annexes, and (b) serum antibodies to M. lepraemurium and M. leprae lipids, including phenolic glycolipid I from M. leprae. Histopathological study of the palsied legs revealed that the paralysis was not the result of direct involvement of the limb nerves, as neither bacilli nor inflammatory cells were observed in the nerve branches studied. Antibodies to brain lipids and cardiolipin were not detected in the serum of the palsied animals, thus ruling out an immune response to self-lipids as the basis for the paralysis. Although high levels of antibodies to MLM lipids were detected in the serum of palsied animals they were not related to limb paralysis, as the nerves of the palsied legs showed no evidence of inflammatory damage. In fact, nerves showed no evidence of damage. Paralysis resulted from severe damage of the leg bones. Within the bones the bone marrow became replaced by extended bacilli-laden granulomas that frequently eroded the bone wall, altering the normal architecture of the bone and its annexes, namely muscle, tendons and connective tissue. Although this study rules out definitively the infectious or inflammatory damage of nerves in murine leprosy, it opens a new avenue of research into the factors that participate in the involvement or the sparing of nerves in human and murine leprosy, respectively.     

Interferon-gamma independent formation of pulmonary granuloma in mice by injections with trehalose dimycolate (cord factor), lipoarabinomannan and phosphatidylinositol mannosides isolated from Mycobacterium tuberculosis

The mechanisms by which pulmonary granuloma formation is caused by administration of mycobacterial glycolipids such as trehalose dimycolate (TDM), lipoarabinomannan (LAM) and phosphatidylinositol mannosides (PIM) were investigated. When peritoneal and alveolar macrophages were stimulated with TDM, LAM and PIM in vitro, TDM exhibited the strongest tumour necrosis factor (TNF)-inducing activity. Responsiveness of macrophages from mice defected Toll-like receptor 4 (TLR4) was much higher than that of the wild-type mice. Although PIM and LAM also had a significant activity, LAM rather than PIM stimulated higher TNF-alpha production by alveolar macrophage. When mycobacterial glycolipids were injected as water-in-oil-in-water emulsion into mice via the tail vein, development of pulmonary granuloma in response to glycolipids were related closely to their TNF-inducing activity and TDM exhibited the strongest activity. Granuloma formation was observed not only in mice lacking interleukin (IL)-12 signalling but also interferon (IFN)-gamma knock-out mice. Granuloma formation caused by glycolipids correlated with TNF-alpha levels in lungs. Administration of anti-TNF-alpha monoclonal antibody into TDM-injected IFN-gamma knock-out mice decreased in granuloma formation, suggesting that development of pulmonary granuloma by mycobacterial glycolipids such as TDM is due to IFN-gamma-independent and TNF-alpha-dependent pathway.     

Three-dimensional hydrogel scaffolds facilitate in vitro self-renewal of human skin-derived precursors

Skin-derived precursors (SKPs) are multipotent cells with dermal stem cell properties. These easily available cells possess the capacity to reconstitute the skin in vivo, as well as a broader differentiation potential in vitro, which endows them with great prospects in regenerative medicine. However, the present authors’ group and others previously found that adult human SKPs (hSKPs) expanded deficiently in vitro, which largely counteracted their research and practical values. Taking the physiological micro-environment of hSKPs into consideration, the authors sought to establish a hydrogel scaffold-based three-dimensional (3-D) culture system for hSKPs in the present study. After comparing their morphology, growth characteristics, signature gene expression and differentiation potential in different hydrogels, the present authors found that a chemically defined hyaluronic acid and denatured collagen-based hydrogel system that mimicked the natural niche of hSKPs in the dermis could alleviate hSKP senescence, support hSKP proliferation as spheres, while largely retaining their properties and potential. This study suggested that recapitulating the in vivo stem cell niche by providing them with 3-D extracellular matrix environments could help them achieve better self-renewal in vitro. In addition, the animal-origin-free and biocompatible 3-D hydrogel system will certainly benefit fundamental research and clinical applications of hSKPs in the near future.     

Myeloid-derived suppressor cells in parasitic infections

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that share a common property of suppressing immune responses. Several helminth and protozoan parasite species have developed efficient strategies to increase the rate of medullary or extramedullary myelopoiesis and to induce the expansion and accumulation of immature myeloid cells such as MDSC. In this review, we examine current knowledge on the factors mediating enhanced myelopoiesis and MDSC induction and recruitment during parasitic infections and how the MDSC phenotype and mechanism of immune modulation and suppression depends on the factors they encounter within the host. Finally, we place MDSC expansion in the context of the critical balance between parasite elimination and pathogenicity to the host and suggest attractive avenues for future research.     

Mycobacterium bovis with different genotypes and from different hosts induce dissimilar immunopathological lesions in a mouse model of tuberculosis

With the hypothesis that genetic variability of Mycobacterium bovis could influence virulence and immunopathology, five M. bovis strains were selected from an epidemiological study in Argentina on the basis of their prevalence in cattle and occurrence in other species. We then determined the virulence and the immunopathology evoked by these strains in a well‐characterized mouse model of progressive pulmonary tuberculosis. The reference strain AN5 was used as a control. BALB/c mice infected with this M. bovis reference strain showed 50% survival after 4 months of infection, with moderate bacillary counts in the lung. Two weeks after inoculation, it induced a strong inflammatory response with numerous granulomas and progressive pneumonia. In contrast, strain 04‐303, isolated from a wild boar, was the most lethal and its most striking feature was sudden pneumonia with extensive necrosis. Strain 04‐302, also isolated from wild boar but with a different spoligotype, induced similar pathology but to a lesser extent. In contrast, strains 534, V2 (both from cattle) and 02‐2B (from human) were less virulent, permitting higher survival after 4 months of infection and limited tissue damage. Strain AN5 and the cattle and human isolates induced rapid, high and stable expression of interferon (IFN)‐γ and inducible nitric oxide synthase (iNOS). In contrast, the more virulent strains induced lower expression of IFN‐γ, tumour necrosis factor‐α and iNOS. Interestingly, these more virulent strains induced very low expression of murine beta defensin 4 (mBD‐4); whereas, the control strain AN5 induced progressive expression of this anti‐microbial peptide, peaking at day 120. The less virulent strains induced high mBD‐4 expression during early infection. Thus, as reported with clinical isolates of M. tuberculosis, M. bovis also showed variable virulence. This variability can be attributed to the induction of a different pattern of immune response.     

舒尼替尼对转移性肾癌患者髓系来源抑制细胞的影响

舒尼替尼是一种口服的多靶点酪氨酸激酶抑制剂,具有抗血管生成和抑制肿瘤增殖的作用.与IFN-α相比,舒尼替尼明显提高了转移性肾癌(mRCC)患者的无进展生存期,并成为治疗mRGC的一线治疗方案.近期有研究报道,舒尼替尼还可以通过抑制髓系来源抑制细胞(MDSC)调节肿瘤免疫.MDSC是一群来源于髓系细胞谱系的异质细胞,其通...     

A method for analysing the clonal precursors of concanavalin A-induced suppressor cells

Spleen cells from 3 different strains of mice (C57 (H-2^b), CBA (H-2^k) and BALB/c (H-2^d)) were stimulated in vitro with different concentrations of concanavalin A (CA) for 48 h. This resulted in the production of cells capable of inhibiting the generation of alloantigen-specific cytotoxic T lymphocytes (CTL) in a mixed lymphocyte culture (MLC). 1 μg/ml was an effective concentration of CA to induce C57 and BALB/c suppressor cells (SC), but 5 μg/ml CA was required to induce CBA SC. SC precursors (SC-P) were shown to be radiosensitive and the results suggest that SC themselves may be radiosensitive. SC were effective in the presence of added interleukin-2 (IL-2).

SC were then induced at limit dilution in microwells in a volume of 25 μl. A MLC (200 μl) was then (after 48 h) added to each microwell. This resulted in a dilution of the concentration of CA to a level below which it was effective at inducing suppression. Cytotoxicity was then assessed 7 days later. It was thus possible to analyse SC-P at the clonal level and estimate their frequency. The frequency of C57 splenic SC-P (active against a C57 anti-BALB/c MLC) was 14.4 × 10⁻⁶, the frequency of CBA splenic SC-P (active against a CBA anti-BALB/c MLC) was 92.3 × 10⁻⁶, and the frequency of BALB/c splenic SC-P (active against a BALB/c anti-CBA MLC) was 15.8 × 10⁻⁶. It was possible to analyse SC-P at a clonal level whether or not the MLC contained added IL-2. SC and SC-P were shown to be sensitive to anti-Thy-1 and complement.     

T cell clones from a non-leprosy exposed subject recognize the Mycobacterium leprae 18-kD protein.

Although Mycobacterium leprae shares many protein antigens with other mycobacterial species, there is a degree of specificity in the T cell response to the organism. This is evident in the failure of cross-protection between mycobacterial species and the specific unresponsiveness to M. leprae in lepromatous leprosy patients. The antigenic basis of this specificity is unresolved, but the M. leprae 18-kD protein is one candidate because of its restricted distribution and the isolation of M. leprae-specific T cell clones reactive with the protein from M. leprae-vaccinated subjects. In the course of analysing the human T cell repertoire to mycobacteria we have isolated further CD4+ T cell clones reactive with this protein from a subject who had never been exposed to M. leprae. These clones did not respond to other mycobacteria, including M. tuberculosis and M. bovis (BCG). In addition, they were unreactive with the M. tuberculosis 16-kD protein which has recently been shown to have limited amino acid identity with the M. leprae 18-kD protein. Both clones reacted with peptide 38-50 from the M. leprae 18-kD protein, the T cell response to which is restricted by HLA-DR4. Although homologues for the gene encoding the M. leprae 18-kD antigen have been identified in M. avium and M. intracellulare, the clones failed to respond to preparations of M. avium. Both clones secreted interferon-gamma (IFN-gamma) and tumour necrosis factor-beta (TNF-beta) and were cytolytic against autologous targets pulsed with peptide 38-50 or the 18-kD protein. The nature of the antigen which stimulates this apparently 'M. leprae-specific' response is unknown. Nevertheless the recognition of the 18-kD protein by individuals not exposed to leprosy indicates that this protein may not be suitable as a reagent to distinguish between infection with M. leprae and other pathogenic mycobacteria.