Tumor-induced immune dysfunction.

Immune system-based approaches for the treatment of malignant disease over the past decades have often focused on cytolytic effector cells such as cytotoxic T lymphocytes (CTL), and natural killer (NK) cells. It has also been demonstrated that tumor-bearing mice can be cured using a wide variety of approaches, some of which involve cytokine-mediated enhancement of CTL and NK cell activity. However, the apparent success in mice stands in contrast to the current situation in the clinic, wherein only a minority of patients have thus far benefited from CTL- or NK cell-based antitumor approaches. The underlying causes of tumor-associated immune suppression of CTL and NK cell activity are discussed, and features of interest shared with HIV infection, leprosy, and rheumatoid arthritis are also be mentioned. Remarkable and very recent observations have shed more light upon the causes of dysfunctional alterations in CTL and NK cells often associated with these diseases, that in turn have suggested new immunotherapeutic approaches for cancer and infectious disease.

Induction of heat shock protein 60 expression in human monocytic cell lines infected with Mycobacterium leprae.

Monocytic cell lines (HL-60 and THP-1) were infected with viable Mycobacterium leprae. Levels of human hsp60 were estimated by Western blot (immunoblot) assay and a sandwich enzyme-linked immunosorbent assay. The results showed that infection of both of the cell lines induced the synthesis of human hsp60, which may be of significance in relation to autoimmune manifestations associated with mycobacterial infections.

In vivo administration of low-dose human interleukin-2 induces lymphokine-activated killer cells for enhanced cytolysis in vitro.

We have examined the effect of the intradermal administration of IL-2 on the generation of natural killer (NK) cell and lymphokine-activated killer (LAK) cell activity. Peripheral blood mononuclear cells (PBMC) obtained from borderline lepromatous (BL) and lepromatous leprosy (LL) patients and normal volunteers prior to and after IL-2 injection were stimulated in vitro with IL-2 and their cytolytic activities compared against 51Cr labeled target K562 cells, Daudi cells, and monocytes. Before IL-2 administration, PBMC obtained from BL/LL patients and normal volunteers possessed similar levels of NK cell activity indicating that the NK cell activity of the BL/LL patients was intact. LAK cell activity was induced with IL-2 in vitro in both BL/LL patients and in normal volunteers. The level of LAK cell activity in BL/LL patients was, however, suboptimal. A single intradermal dose of 25 micrograms IL-2 had no effect on the phenotype of circulating mononuclear cells in either patients or normal volunteers. However, 6-12 days after IL-2 injection and subsequent restimulation of the PBMC with IL-2 in vitro, cytolytic activity of LAK cells obtained from the BL/LL patients was enhanced while cells from normal volunteers expressed the same high levels of activity as observed before IL-2 injection.

A monoclonal antibody to the mycobacterial 65 kDa heat shock protein (ML 30) binds to cells in normal and arthritic joints of rats.

A monoclonal antibody reactive with the mycobacterial 65 kDa heat shock protein (ML 30) was investigated for reactivity with biopsies from normal rat joints and with inflamed joints due to adjuvant arthritis (AA) or collagen induced arthritis (CIA). Immunohistochemical stainings with the anti-hsp 65 antibody on paraffin sections from normal rat joints revealed a weak but exclusive staining of cells within the synovial lining. Also normal chondrocytes and bone marrow cells showed occasional staining. In biopsies from inflamed joints obtained from rats suffering from AA or CIA, an intense staining with ML 30 was seen within the cartilage-pannus junction as well as sites of bone erosion. An increased staining, compared with the normal, was also seen in chondrocytes of the eroded cartilage and in some bone marrow cells. No staining with ML 30 was seen in biopsies from inflammatory lesions due to delayed type hypersensitivity reactions in the skin of rats. Reactivity of ML 30 was also seen in a Western blot assay performed on lysates from inflamed synovia from rats with CIA, preferentially with a component slightly below 60 kDa in molecular weight. The demonstration of epitopes cross-reactive with hsp 65 of mycobacteria in normal and, in higher quantity, in arthritic rat joints, suggests, together with our preliminary biochemical findings, that a recently identified mammalian counterpart to bacterial hsp 65 is both preferentially expressed in normal joints and subject to increased expression in arthritis of different aetiologies.

Mycobacterial-induced cytotoxic T cells as well as nonspecific killer cells derived from healthy individuals and leprosy patients.

Little information is available about the generation and specificity of the cytotoxic cells that eliminate human monocytes/macrophages infected with mycobacteria. To address this we have developed a cytotoxicity assay in which 51Cr-labeled monocytes pulsed with bacillus Calmette Guerin (BCG) or Mycobacterium leprae, were used as target cells in overnight cytotoxicity assays. As effector cells, peripheral blood mononuclear cells from healthy occupational contacts or from leprosy patients stimulated with antigen for 7 days were used. Cytotoxicity against antigen-pulsed monocytes that could be induced by mycobacterial antigens was proportional to the degree of antigen responsiveness in each individual, as measured in lymphocyte transformation tests. The lepromatous leprosy patients tested were often poor responders to BCG as well as M. leprae, both with regard to induction of cytotoxicity as well as in lympho-proliferation. Killing was significantly higher against antigen-pulsed vs. nonpulsed monocytes, although significant killing was induced against the latter as well and paralleled by induction of natural killer activity against the K-562 target cell. Cross-reactivity was observed between BCG and M. leprae, but not with unrelated antigen (tetanus toxoid) or with endogenous stress proteins induced by heat shock. M. leprae- and BCG-activated cytotoxic cells were found in both the CD4-CD8+ and CD4+CD8- populations, whereas in contrast the soluble antigen, purified protein derivative of M. tuberculosis, generated cytotoxic cells that were exclusively of the CD4+ phenotype. The involvement of both specific T cells as well as nonspecific cells in the killing of human macrophages may be important with respect to protection and immunopathology induced by mycobacterial antigens.

Intradermal recombinant interleukin 2 enhances peripheral blood T-cell responses to mitogen and antigens in patients with lepromatous leprosy.

Thirty-one patients with lepromatous leprosy received recombinant interleukin 2 (IL-2) intradermally in doses ranging from 10 to 30 micrograms. Before injection and at time intervals of 2-21 days thereafter, samples of peripheral blood mononuclear cells (PBMC) were obtained. Single or multiple injections (1-3) of IL-2 did not modify the total number of circulating lymphocytes or the number of T cells and the CD4/CD8 T-cell ratio. However, IL-2 had a pronounced influence on the [3H]thymidine incorporation in response to various stimuli 4-8 days after intradermal IL-2. Stimulation indices of three- to sevenfold above pre-IL-2 levels were observed with the polyclonal activator phytohaemagglutinin (PHA) and enhanced thymidine incorporation occurred in the presence of antigens to which the patients were already sensitized, such as purified protein derivative and BCG. IL-2 had no effect on the unresponsive state of lepromatous leprosy patient T cells to the antigens of Mycobacterium leprae.

Serum tumor necrosis factor levels and disease dissemination in leprosy and leishmaniasis.

It has been suggested that tumor necrosis factor alpha (TNF alpha) may serve as an important antigen-independent host defense mechanism against parasitic organisms. Sera from 66 patients with leishmaniasis and 68 patients with leprosy, all from Ethiopia, were tested for TNF alpha using an enzyme-linked immunoassay. Sera from patients with the multi-parasitic/bacillary type of disease (visceral or diffuse cutaneous leishmaniasis and lepromatous leprosy), known to be associated with absent or low specific T cell response, contained significantly higher TNF alpha titers than those of patients with pauci-parasitic/bacillary disease (localized cutaneous leishmaniasis and nonlepromatous leprosy). High titers of TNF alpha in the absence of a functioning T cell response do not appear to confer resistance against Leishmania aethiopica and Mycobacterium leprae.

Induction of antigen-specific CD4+ HLA-DR-restricted cytotoxic T lymphocytes as well as nonspecific nonrestricted killer cells by the recombinant mycobacterial 65-kDa heat-shock protein.

Acquired cell-mediated immunity to intracellular parasites like mycobacteria is dependent on antigen-specific T lymphocytes. We have recently found that mycobacteria not only induce helper T cells but also cytotoxic CD4+ and/or CD8+ T cells as well as nonspecific killer cells that lyse human macrophages in vitro. In addition, we have described that the recombinant heat-shock protein (hsp) 65 of Mycobacterium bovis BCG/M, tuberculosis is an important target antigen for CD4+CD8- cytotoxic T cells. We have now further investigated the cytotoxic effector cells that are induced by the hsp65 of BCG. Purified protein derivative of tuberculin (PPD)- or hsp65-specific cytotoxic T cells specifically lysed PPD, hsp65 of BCG and hsp65 of M. leprae-pulsed macrophages in an HLA-DR-restricted manner. Nonpulsed macrophages were lysed to a much lower but still significant extent. hsp65-induced effector cells expressed CD3, CD5, CD4, CD8 and CD56 markers. Depletion experiments showed that the antigen-specific HLA-DR-restricted killer cell was of the CD5+CD4+CD8-CD56- phenotype. Experiments using N-terminal truncated hsp65 fusion (cro-lacZ) proteins suggested that the N-terminal 65 amino acid residues of the 540 amino acid molecule are critical for the expression of the cytotoxic target epitope(s) in two individuals tested. In addition to inducing antigen-specific cytotoxic effector cells, the hsp65 also triggered nonspecific nonrestricted effector cells with lytic activity against nonpulsed autologous or allogeneic macrophages as well as K-562 and Daudi tumor cells. hsp65-stimulated effector cells produced both interferon and tumor necrosis factor-alpha. An important finding was that hsp65-stimulated effector cells strongly inhibited colony-forming unit formation from live BCG-infected autologous macrophages.

T cell responses to fractionated Mycobacterium leprae antigens in leprosy. The lepromatous nonresponder defect can be overcome in vitro by stimulation with fractionated M. leprae components.

Protective immunity against Mycobacterium leprae is dependent on M. leprae-reactive T lymphocytes. M. lepare-directed T cell reactivity is high in the localized tuberculoid form of leprosy but specifically absent in the disseminated lepromatous type of the disease. Two important questions that are relevant for the understanding of the immune response in leprosy as well as for the design of rational immunoprophylaxis and -therapy strategies are: (a) what are the antigens that trigger T cell responses in tuberculoid patients and thus protect these individuals from developing lepromatous leprosy and (b) is it possible to restore T cell responsiveness to M. leprae in lepromatous patients by rechallenging the immune system with selected antigens that will trigger help but not suppression? We have addressed these question by directly probing the peripheral T cell repertoire of 10 tuberculoid and 18 lepromatous patients with large numbers of different M. leprae and BCG antigenic components that had been separated on the basis of their relative molecular mass (Mr) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. This technique allows the identification of T cell-stimulating antigens independent of the expression of B cell epitopes by these antigens. So far T cell epitopes have only been mapped on M. leprae proteins that had previously been defined by antibodies. Our results show that: (a) tuberculoid patients' T cells responded preferentially to M. leprae and BCG antigens in the lower (i.e. less than 70 kDa) Mr range with a peak in the 10-25 kDa range; (b) 6 out of 18 lepromatous patients that did not respond to whole M. leprae responded strongly to isolated M. leprae components; antigens in the lower Mr. range were recognized by five out of these six patients and thus commonly seen by both tuberculoid and lepromatous patients' T cells; however, antigens in the higher Mr range, in particular greater than 150 kDa, were only recognized by lepromatous patients' T lymphocytes; (c) furthermore, the T and B cell repertoires in leprosy patients are skewed towards different antigenic fractions.

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.

The recombinant 65-kD heat shock protein of Mycobacterium bovis Bacillus Calmette-Guerin/M. tuberculosis is a target molecule for CD4+ cytotoxic T lymphocytes that lyse human monocytes.

Since little is known about Tc cells in the human immune response to intracellular parasites, we have studied the role of Tc cells in response to M. bovis Bacillus Calmette-Guerin (BCG). Donors whose PBMC responded to BCG, purified protein derivative (PPD), and the recombinant 65-kD heat shock protein (HSP) of BCG generated BCG/PPD-specific CD4+ effector T lymphocytes that lysed PPD as well as recombinant 65-kD-pulsed monocytes. Nonpulsed or irrelevant antigen-pulsed target cells were lysed to a much lower but still significant extent. PPD-stimulated effector lymphocytes of a recombinant 65-kD nonresponder lysed PPD but not recombinant 65-kD-pulsed monocytes. Recombinant 65-kD-educated effector lymphocytes lysed both recombinant 65-kD- and PPD-pulsed monocytes. In addition, these effector cells efficiently lysed nonpulsed target cells. These results demonstrate that in recombinant 65-kD responders, the recombinant 65-kD HSP of BCG is an immunodominant target as well as a triggering molecule for BCG/PPD-specific CD4+ cytotoxic T cells that lyse autologous monocytes. The implications of these findings with respect to the role of the 65-kD HSP in autoimmunity are discussed.

Cellular, humoral, and gamma interferon responses to Mycobacterium leprae and BCG antigens in healthy individuals exposed to leprosy.

Protective immunity against mycobacteria is dependent on antigen-specific T cells. The antibodies induced upon immunization with mycobacteria have no apparent role in host protection. Serological techniques have detected some antigens that are also recognized by human T cells but may fail to recognize others. Potentially, there may be differences in the epitopes seen by the T and B cell anti-mycobacterial antigen repertoires. We have screened the different components of sonicated BCG or Mycobacterium leprae that were separated according to their molecular weight (MW) by SDS-PAGE and then electroblotted on nitrocellulose paper. The blots were cut into squares and tested directly in a T cell proliferation assay. Our results indicate that peripheral T cells of healthy leprosy patient contacts respond preferentially to the lower MW (less than 70,000) and not the higher MW fractions of M. leprae and BCG, in contrast to the humoral response of these same individuals. The most important fractions in inducing a lymphoproliferative response were in the regions of 11-16 kDa of BCG and M. leprae and to the 22-26 kDa region of M. leprae. These fractions appeared to represent molecular weight regions that were in some instances clearly distinct from previously defined antigens. It was further shown that lymphoproliferation in response to mycobacterial fractions correlated with the production of gamma interferon, a lymphokine required for macrophage activation and elimination of mycobacteria. These studies allow the direct assessment of antigens involved in protective T cell-mediated immunity, and should be helpful in selecting relevant antigens for skin testing and immunization.