Enhancement of human lymphocyte proliferative response to purified protein derivative by an anti-interleukin-2 receptor alpha chain antibody (CD25).

While it is clear that the beta subunit of interleukin-2 receptor (IL-2R) plays a pivotal role in IL-2-induced signal transduction, the function of the alpha subunit, other than modulating the association rate of IL-2, is still unknown. It has been reported that the interaction between IL-2 and the IL-2R alpha subunit of several IL-2-dependent murine T-cell lines may result in a negative regulatory signal. To confirm this finding, we investigated the effect of an anti-IL-2R alpha antibody, CD25-8D8, on the proliferative response of human peripheral blood lymphocytes. Lymphocytes from purified protein derivative (PPD)-positive donors were cultured with PPD and various concentrations of CD25-8D8 for up to 9 days, and [3H]thymidine uptake was measured. Whereas the proliferative response of human lymphocytes to PPD was suppressed by high concentrations of CD25-8D8, subinhibitory amounts of CD25-8D8 enhanced lymphocyte proliferation by 3.5-fold (range 2.2-6.2-fold) on the second day after maximal [3H]thymidine uptake had occurred. By itself, CD25-8D8 could not induce proliferation of washed 5-day PPD-activated lymphocytes during reculturing; instead, growth enhancement by CD25-8D8 was dependent on the presence of PPD-activated culture supernatant or moderate levels of exogenous IL-2. The enhancing effect of anti-IL-2R alpha antibody, observed in both murine and human systems, reinforces the possibility that binding of IL-2 to the IL-2R alpha chain plays a negative regulatory role in signal transduction.

Enumeration of interleukin-1 producing monocytes from human peripheral blood mononuclear leukocytes by agar plating technique.

An agar plating technique was developed for enumeration of IL-1-producing monocytes based on the principle that when IL-1-producing monocytes were cocultured with mouse thymocytes and PHA in semisolid agar medium in a plate, mouse thymocytes proliferated around IL-1-producing monocytes resulting in the clusters or colonies of cells. The IL-1-produced clusters or colonies of cells can be counted under a dissecting microscope. Optimal conditions were established for induction and development of IL-1-producing monocytes. The numbers of IL-1-producing monocytes ranged from 819 to 1930 cells/10(5) monocytes, with mean +/- SEM = 1344 +/- 182 cells/10(5) monocytes; the IL-1 activity ranged from 11.7 to 85.9 U/10(5) monocytes/ml, with mean +/- SEM = 42.8 +/- 11.2 U/10(5) monocytes/ml in seven normal subjects. The IL-1 activity per one monocyte ranged from 12.7 to 86.5 mU, with mean +/- SEM = 33.5 +/- 9.8 mU. The mean numbers of IL-1-producing monocytes and the mean IL-1 levels produced by monocytes from the same normal subjects were highly correlated (r = 0.981). The numbers of IL-1-produced colonies resulting from IL-1-producing monocytes could be completely abolished by incorporation of rabbit anti-human IL-1 in the semisolid agarose but not by rabbit anti-human IL-6 or anti-human TNF-alpha.

Comparison of IgM, IgG and IgA responses to M.leprae specific antigens in leprosy.

Antibodies of IgM, IgG and IgA classes against M.leprae specific antigens (PGL-I, ND-O-BSA, and NT-O-BSA) were determined in the sera of 80 leprosy patients (28 untreated, 34 treated lepromatous and 18 tuberculoid), 25 tuberculosis patients and 33 normal individuals of Northern Thailand. No strong distinction in reactivity could be found between the three antigens. The IgM antibody assay yielded more positive results than assays for IgG and IgA. It was found that the positivity rates of IgM antibodies to all three antigens were highest in untreated lepromatous leprosy (82%). In tuberculoid leprosy, the positivity rates of IgM, IgG and IgA to the antigens were more variable, ranging from 22 to 50 percent. Patients with tuberculosis and normal individuals did not produce IgM antibodies against the antigens. The results suggested that the determination of IgM against the three antigens is a more sensitive and specific test for active leprosy than those of IgG and IgA. The relationship between the duration of treatment and IgM antibody levels in lepromatous leprosy (LL) was studied. Untreated LL patients had significantly higher IgM and IgA antibody levels than treated patients. There was no difference in IgG antibody levels between the two groups, and the levels of both groups were higher than normal controls. Serial determination of IgM antibodies in 7 LL patients revealed that treatment was strongly associated with progressive decrease in IgM antibody levels against all three antigens.

Stimulation by anti-idiotype antibody of murine T cell responses to the 38 kD antigen of Mycobacterium tuberculosis.

Rabbit antisera raised against eight monoclonal antibodies (MoAb) binding to distinct mycobacterial antigens revealed individual anti-idiotype specificities following cross-absorption with normal mouse globulin. Only one of these antibodies (Rb71), directed towards an M. tuberculosis-specific epitope on the 38 kD protein antigen was able to stimulate mouse T cell responses which had 'internal image' characteristics. This was demonstrated by anti-Id induced in vitro proliferation of Lyt 1.2+ T cells and the elicitation of delayed type hypersensitivity (DTH) foot pad reactions in antigen stimulated BALB/c mice. The DTH reaction was equally strong in mice sensitized with either M. tuberculosis or M. bovis soluble extracts, thus showing a greater degree of cross-reactivity than antibody binding of the corresponding TB71 MoAb. The immunizing potency of the anti-Id in vivo was demonstrable in three strains of mice following injection of Rb71 emulsified with incomplete Freund's adjuvant. These mice showed 38 kD antigen induced DTH-reactions as well as in vitro proliferation of spleen cells. The display of T cell stimulatory internal image by anti-Id may be interpreted as a consequence of binding specificity of TB71 MoAb of a T cell epitope present in the 38 kD antigen.

Study of idiotypes expressed by monoclonal antibodies to the 35 kD and 12 kD antigens of Mycobacterium leprae.

Rabbit antisera were raised against four monoclonal antibodies (MoAb) binding with the 35 kD protein and four MoAb binding with the 12 kD protein antigen of Mycobacterium leprae. Antisera showed idiotype (Id) specificity following cross-absorption with normal mouse globulin. One Id on a single MoAb and another Id shared between three MoAb were identified for each group. Functional studies were carried out with the Rb04 anti [anti-35 kD] specificity. The expression of this Id and paratope in antigen immunized mice was associated with Igh alleles. Inoculation of mice with anti-Id Rb04 induced an 'Ab3' serum response of corresponding Id specificity only when the anti-Id was given in emulsion with incomplete Freund's adjuvant (IFA). Conversely, prior injection of soluble anti-Id inhibited the subsequent Ab3 response to Rb04/IFA. Moreover, the suppressive effect of soluble anti-Id was abrogated by prior injection of 50 mg/kg cyclophosphamide. These results indicate that regulatory mechanisms similar to those involved in antigenic stimulation may explain the stimulatory or suppressive potency of anti-Id antibodies. Finally, the Ab3 responses to the two tested anti-Ids did not contain any antigen binding activity.

T-cell activation by anti-idiotypic antibody: evidence for the internal image.

Human lymphoproliferative responses to a rabbit anti-idiotypic antibody (anti-Id TB71) and the corresponding mycobacterial protein antigen [38,000 molecular weight (MW)] have been investigated in a number of donors. It was found that responsiveness to anti-Id TB71 correlated with responder and non-responder (four subjects each) status to the 38,000 MW antigen. Furthermore, the induction of T-cell proliferation by both the 38,000 MW antigen and the anti-Id TB71 was dependent on accessory cells. When taken together with the concordance between the 38,000 MW antigen and anti-Id responsiveness, this implies that the 38,000 MW antigen and anti-Id TB71 stimulate related, or at least partially overlapping, repertoires of T cells. This was confirmed by the finding that cloned T cells reactive with the 38,000 MW antigen also proliferated in response to the anti-Id TB71. These observations are readily explained if the anti-idiotypic antibody contains an internal image of, and can therefore mimic, the antigen.

T cell activation by anti-idiotypic antibody: mechanism of interaction with antigen-reactive T cells.

It has been shown that the activation of T cells by an anti-idiotypic antibody (anti-Id) TB71 containing an internal image of the corresponding mycobacterial antigen (38 kDa) was achieved by the interaction of anti-Id TB71 with the T cell receptor complex (CD3/Ti). The accessory cell requirement in this response could not be replaced by anti-Id TB71 coupled to Sepharose beads and was not inhibited by Fc receptor blockade. When taken together with the finding that anti-Id TB71-induced proliferation of a T cell clone was restricted by determinants encoded by the major histocompatibility complex, these findings suggested that anti-Id TB71 was presented to 38-kDa antigen-reactive T cells by the same mechanisms as conventional antigenic determinants. That is, both stimulated T cells through the CD3/Ti complex and had to be presented in the context of class II molecules on accessory cells. The finding that the disruption of the integrity of the anti-Id TB71 combining site did not affect T cell responsiveness although antibody binding was ablated implied that anti-Id TB71 may be partially degraded and re-expressed with MHC class II determinants.

Detection of an antigen (MY4) common to M. tuberculosis and M. leprae by 'tandem' immunoassay.

A novel 'tandem' immunoassay for the detection of mycobacterial antigen was devised using a monoclonal antibody (ML 34) both as solid phase 'capture' and as the 125I- or enzyme-labelled 'tracer' antibody. This antibody binds to the repeating epitopes (MY4b) of a water-soluble protease-resistant antigen from M. tuberculosis, M. leprae and some other species of mycobacteria. Optimal binding results could be obtained within 4 h by the consecutive incubation of ML34-coated microtitre plates with antigen followed by the labelled ML34 antibody. The binding of intact bacilli was positive for M. tuberculosis but not for M. leprae. These results suggested that the MY4 antigen is expressed on the surface of M. tuberculosis and internally within M. leprae. Analysis of subcellular fractions suggested that this antigen is a constituent of cell walls.