Long-term exposure to gaseous formaldehyde promotes allergen-specific IgE-mediated immune responses in a murine model.

It has long been questioned that whether exposure to formaldehyde in indoor environments may be a risk factor for developing allergen-specific IgE-mediated inflammatory responses, because there is limited clinical or experimental evidence that formaldehyde is involved in the cascade for IgE production. There is no known lower limit, below which there is no threat of serious allergic symptoms. The present study illustrates that the threshold limit of formaldehyde, 0.08 ppm (as defined by the World Health Organization), did not cause ovalbumin-specific IgE inflammatory immune responses, but higher than threshold concentrations of formaldehyde gas result in both enhanced allergen-specific IgE responses and NK (Natural Killer)-cell activity in peripheral blood cells in a murine model. Thus, formaldehyde gas may be involved in promoting allergic inflammatory effects in subjects primed with specific allergens by NK-cell activation. These results indicate that even threshold concentrations of formaldehyde gas may play a regulatory role for 'systemic' cell-mediated immune responses. The extensive use of adhesives for building materials has resulted in higher levels of indoor air pollutants. It is conceivable that increased time indoors may enhance pre-existing allergic symptoms by concomitant exposure to volatile organic compounds and formaldehyde. The affordable limit for formaldehyde might be much lower than currently established levels in indoor environments.

Enhancing effect of tumor necrosis factor (TNF)-alpha, but not IFN-gamma, on the tumor-specific cytotoxicity of gammadeltaT cells from glioblastoma patients.

Adoptive immunotherapy using tumor-specific killer cells can be beneficial in inducing regression of advanced cancer. The roles of cytokines on effector cells in inducing maximal killing activity and the accompanying side-effects should be investigated in vitro and fully understood prior to their clinical use. The present study indicates that the gammadeltaT cells involved in autologous tumor-specific killing consist of several populations in terms of their T cell receptor (TCR) repertoire, but predominantly express the products of the Vgamma9/Vdelta2 gene locus of the TCR. We then examined the effect of TNF-alpha and IFN-gamma on these tumor-specific gammadeltaT cells for possible clinical use in cancer patients. TNF-alpha alone, at concentrations of 0.01-1.0 microg/ml, caused increased gammadeltaT cell cytotoxicity against autologous glioblastoma cells, whereas IFN-gamma alone had no effect. The combination of TNF-alpha (1 microg/ml) with IL-2 (50 units/ml) resulted in further enhancement of cytotoxicity. TNF-alpha, but not IFN-gamma, marginally inhibited the proliferative response of gammadeltaT cells; a similar result was seen when the cytokines were combined. TNF-alpha may, therefore, be one cytokine capable of inducing increased autologous tumor-specific activity in gammadeltaT cells, bearing mainly Vgamma9/Vdelta2 chains, which can be enhanced when combined with other cytokines.

Tumor-specific cytocidal and immunopotentiating effects of relatively low molecular weight products derived from the basidiomycete, Agaricus blazei Murill.

Currently, some natural herbal extracts are believed to have a marked tumoricidal effect and low toxicity for normal tissues. We investigated the effect of relatively low molecular weight products extracted from the basidiomycete, Agaricus blazei Murill, on MethA tumor cell growth with the aim of producing synthetic derivatives based on these products. Inoculation of the low molecule fraction (LM) into the primary tumor of a two-tumor model resulted in the marked inhibition of the tumor, not only in the right flank, but also in the non-injected left flank. Chromatographic purification and physicochemical characterization showed the main tumoricidal activity to be located in a low molecule fraction-3 (LM-3), containing alpha-1,4-glucan-beta-1,6-glucan complex with an average molecular weight of 20 kDa. A11 LM fractions and crude ATF showed in vitro selective cytotoxicity for MethA tumor cells, having no effect on normal cells. Serum levels of immunosuppressive acidic protein (IAP) in mice receiving LM fractions, particularly LM-3, significantly increased, indicating the possible activation of granulocytes. We speculate that the inhibition of the distant tumor might be due to the increased migration of granulocytes, enhanced by the effect of extract injections at the primary tumor site.

Injury to autologous normal tissues and tumors mediated by lymphokine-activated killer (LAK) cells generated in vitro from peripheral blood mononuclear cells of glioblastoma patients.

Activation of peripheral blood mononuclear cells (PBMC) with IL-2 generates lymphokine-activated killer (LAK) cells that show a broad target cell range. In adoptive immunotherapy using in vitro-generated LAK cells, the intensity and specificity of their cytotoxic activity affect the prognosis of cancer patients. The present study was designed to examine the tumor-specific spectrum of T lymphocytes generated from the PBMC of patients with recurrent glioblastoma by in vitro propagation with IL-2 plus either soluble or solid-phase anti-CD3 monoclonal antibody (MAb) in short-term or long-term cultures. Both short-term and long-term culturing with solid-phase anti-CD3 MAb plus IL-2 yielded broad-reactivity CD8+ alphabetaT and gammadeltaT lymphocytes, both of which were non-MHC restricted, as shown by the fact that they were able to lyse autologous glioblastoma cells, MHC class I+II- allogeneic glioblastoma cells, and MHC class I-II-NK-sensitive K562 target cells. More importantly, these cells from patients failed to lyse fresh autologous PBMC. These results demonstrate that cells generated using this approach are non-MHC-restricted LAK cells and exhibit marked tumor specificity. In contrast, incubation with soluble anti-CD3 MAb generated T lymphocytes that after long-term culture, were either CD4+ or CD8+. These caused significant lysis of both allogeneic and autologous glioblastoma target cells, the extent of lysis being greater than that using cells produced by culturing with the solid-phase MAb. However, both the CD4+ and CD8+ cells also caused greater lysis of autologous normal PBMC, indicating that cells generated using this approach may cause significant adverse reactions in cancer patients if used for immunotherapy.

Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood gammadeltaT cells isolated from glioblastoma patients.

GammadeltaT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the activation and proliferation of circulating gammadeltaT cells should be fully understood prior to their adoptive transfer to cancer patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified gammadeltaT cells isolated from glioblastoma patients. GammadeltaT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) alpha betagamma, but the levels of IL-2Rbeta or gamma expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal gammadeltaT cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response, this activity was completely or partially abrogated by anti-IL-2Rbeta, or anti-IL-2Rgamma antibodies, but not by anti-IL-2R alpha antibodies. Incubation of gammadeltaT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific activity could be significantly blocked by anti-IL-2Rgamma and anti-IL-2R-beta mAb, but not by anti-IL-2R alpha mAb. Thus, in contrast to IL-2, IL-15 activates tumor-specific gammadeltaT cells through the components of IL-2Rbeta and IL-2Rgamma, but not IL-2R alpha. These enhanced in vitro tumor-specific and proliferative responses of gammadeltaT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic use of gammadeltaT cells in cancer patients.

Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete, Agaricus blazei Murill, mediated via natural killer cell activation and apoptosis.

We have isolated a novel type of natural tumoricidal product from the basidiomycete strain, Agaricus blazei Murill. Using the double-grafted tumor system in Balb/c mice, treatment of the primary tumor with an acid-treated fraction (ATF) obtained from the fruit bodies resulted in infiltration of the distant tumor by natural killer (NK) cells with marked tumoricidal activity. As shown by electrophoresis and DNA fragmentation assay, the ATF also directly inhibited tumor cell growth in vitro by inducing apoptotic processing; this apoptotic effect was also demonstrated by increased expression of the Apo2.7 antigen on the mitochondrial membranes of tumor cells, as shown by flow-cytometric analysis. The ATF had no effect on normal mouse splenic or interleukin-2-treated splenic mononuclear cells, indicating that it is selectively cytotoxic for the tumor cells. Cell-cycle analysis demonstrated that ATF induced the loss of S phase in MethA tumor cells, but did not affect normal splenic mononuclear cells, which were mainly in the G0G1 phase. Various chromatofocussing purification steps and NMR analysis showed the tumoricidal activity to be chiefly present in fractions containing (1-->4)-alpha-D-glucan and (1-->6)-beta-D-glucan, present in a ratio of approximately 1:2 in the ATF (molecular mass 170 kDa), while the final purified fraction, HM3-G (molecular mass 380 kDa), with the highest tumoricidal activity, consisted of more than 90% glucose, the main component being (1-->4)-alpha-D-glucan with (1-->6)-beta branching, in the ratio of approximately 4:1.

The use of BRM-activated killer cells in adoptive immunotherapy: a pilot study with nine advanced cancer patients.

Adoptive immunotherapy using MHC-nonrestricted-lymphocytes, peripheral blood gammadelta T cells and NK cells was devised. Peripheral blood mononuclear cells (3 x 10(7)) were selected by immobilization to anti-CD3 monoclonal antibody for 4 days and cultured for 2 weeks in the presence of IL-2. Thereafter they were reactivated by 500 U/ml of IFN-alpha and 1000 U/ml of IL-2 for 1 hour. Enhancement of NK and LAK activities was confirmed. Peripheral blood gammadelta T cells proliferated in response to immobilized anti-CD3 antibody (3% to 30%). Approximately 6 x 10(9) BRM-activated killer (BAK) cells composed of CD56+ gammadelta T cells and CD56+ NK cells, were dispensed to cancer patients via intravenous drip infusion. Nine patients were treated with BAK cells every 2 weeks or every month on an outpatient basis. During the course of adoptive immunotherapy, the crossed affinity immunoelectrophoresis (CAIE) pattern of serum immunosuppressive acidic protein (IAP) was analysed. Both the production and glycosylation pattern of IAP is changed in response to tumor enlargement and may therefore act as a marker of the disease progression. During the course of BAK therapy, the glycosylation IAP pattern of 6 patients changed from tumor (T) to normal (N). In addition, the performance status of all patients was maintained at 90-100% of the Karnofsky scale and any side effects including fever were not observed during treatments with BAK cells. Moreover, the overall quality of life (QOL) of the patients, scored at the Face scale was favorable. In addition, blood levels of activated gammadelta T cells producing IFN-gamma were assayed as an indication marker of BAK therapy. The normal range of IFN-gamma producing gammadelta T cells comprised 6.9 +/- 0.9% of peripheral blood mononuclear cells (PBMC), according to a single cell FACScan analyses of PBMCs derived from normal individuals. IFN-gamma producing gammadelta T cells of Patients No. 8 and 9, who received extensive chemotherapy before initiation of BAK therapy, comprised only 0.2% and 2% of PBMC, respectively. These patients died 3 and 6 months after beginning BAK therapy. Peripheral blood gammadelta T cells of Patients Nos. 1-7 proliferated in response to immobilized anti-CD3 antibody and the frequency of IFN-gamma producing gammadelta T cells in PBMC preparation of these patients were over 3% before initiation of BAK therapy. Since our data show a positive correlation between survival time and initial gammadelta T cell counts, a low frequency of these cells may contraindicate BAK therapy.

Antitumor effect of a peptide-glucan preparation extracted from Agaricus blazei in a double-grafted tumor system in mice.

The antitumor effect of extracts obtained from the fruit body of Agaricus blazei Murill was examined in a double-grafted tumor system, in which BALB/c mice received simultaneous intradermal injections of Meth-A tumor cells in both the right (10(6) cells) and left flank (2 x 10(5) cells), and were then injected with 5 mg of extracts of A. blazei in the right tumor on days 3, 4 and 5. Intratumoral administration of ethanol-soluble (Fraction 1), water-ethanol-soluble (Fraction 2), ammonium oxalate-soluble (Fraction 3) and ammonium oxalate-insoluble (Fraction 4) fractions resulted in inhibition of tumor growth, with Fraction 3 showing the most tumoricidal activity, producing regression of the right tumor and inhibition of growth of the left, non-injected tumor. The maximum effect was obtained using 0.5 mg of Fraction 3 and this amount was used in subsequent experiments. The antitumor effect of intratumorally administered Fraction 3 was enhanced by oral ad lib administration of feed containing 0.083% of Fraction 3. When immunized spleen cells from mice that had been cured by intratumoral administration of 0.5 mg of Fraction 3 were directly injected (2 x 10(7) cells/mouse) into the Meth-A tumor, tumor growth was inhibited. The tumor cells on day 7 from the Fraction 3-treated right tumor and from the left tumor were cultured for 24 h and their culture supernatants were assayed for neutrophil or macrophage chemotactic activity. Significant macrophage chemotactic factor activity was detected in the culture media from the left tumor tissue. Serum levels of immunosuppressive acidic protein (IAP), produced by activated macrophages and neutrophils, increased transiently soon after intradermal injection of 0.5 mg of Fraction 3. These results suggest that regression of the left non-injected tumor was due to an immune reaction, involving induction of cytotoxic cells in the spleen, and the release of chemotactic factors in the distant tumor.

A simple method for the propagation and purification of gamma delta T cells from the peripheral blood of glioblastoma patients using solid-phase anti-CD3 antibody and soluble IL-2.

Although gamma delta T cells make up no more than 10% of the peripheral blood mononuclear (PBM) cells, they appear to play an important role in host defense against tumor growth. In order to evaluate their functional activity against tumors and their response to various cytokines, large numbers of cells are required. Here, we describe a newly-devised method for the isolation and expansion of gamma delta T cells from the peripheral blood of cancer patients, in particular those with glioblastoma. Using this approach, a 1000-1500-fold increase in total cell numbers was achieved in two weeks, the proportion of gamma delta T cells in the expanded population being, on average, approximately 30% after 14 days of culture. The method therefore gives a yield of approximately 10-15 x 10(8) gamma delta T cells from only 5 ml of peripheral blood from glioblastoma patients and normal controls. The highly purified gamma delta T cells of glioblastoma patients were shown to bear both a high-affinity interleukin-2 receptor (IL-2R) and a low-affinity IL-12 receptor (IL-12R). They also displayed significant cytotoxicity against autologous tumor cells, but not against autologous fresh or IL-2-treated lymphocytes, and proliferated in response to IL-2, both effects being dependent on the dose of IL-2 used for activation. In addition, overnight incubation with 700 U/ml of IL-2 or 50 ng/ml of IL-12 resulted in significant cytotoxic activity of patients' gamma delta T cells against K562 target cells, the level of activity being almost the same as with similarly-treated gamma delta T cells from normal controls (P > 0.05). These results demonstrate that the patients' gamma delta T cells obtained using this method are intact in terms of cytotoxic function. Thus, this method not only makes it possible to produce large numbers of purified gamma delta T cells but also to produce populations containing both gamma delta T cells and NK cells, both active against tumor targets which might be suitable for clinical trials of adoptive-immunotherapy, especially in cancer patients for whom no effective therapy is available.

In vitro interleukin 12 activation of peripheral blood CD3(+)CD56(+) and CD3(+)CD56(-) gammadelta T cells from glioblastoma patients.

Interleukin (IL)-12 has recently been shown to be directly involved in the activation of natural killer and alphabeta T cells via an IL-2-independent pathway. We show here that another type of human cytotoxic cell, gammadelta T cells activated by solid-phase anti-CD3 antibody and expanded using IL-2, obtained, in this case, from the peripheral blood of glioblastoma patients, displays significant tumoricidal activity. In addition, its cytotoxic activity against K562 or Daudi cells or against autologous glioblastoma targets (but not lymphocytes) is significantly enhanced when costimulated with IL-2 and IL-12. To study this synergistic activation by the two interleukins of the patients' gammadelta T cells, we screened the cells for the presence of the IL-2 receptor (IL-2R) and IL-12 receptor (IL-12R) using both flow cytometric analysis and PCR. The patients' gammadelta T cells constitutively expressed the high-affinity IL-2R; when stimulated with IL-12 plus IL-2, the levels of IL-2Ralpha and IL-2Rbeta increased, whereas that of IL-2gamma did not. They also expressed marginal levels of low-affinity IL-12R both immediately after IL-2 expansion and after 24-h incubation, and significantly higher levels after 72-h incubation, consistent with the level of gammadelta T-cell activation. IL-12 alone induced little proliferation of patients' gammadelta T cells in a 24-h assay and none in a 72-h assay; however, it caused a marked inhibition of the IL-2-induced proliferative response in the 72-h assay. The synergistic action of IL-2 and IL-12 was completely abolished by combined pretreatment with anti-IL-2alpha, beta, and gamma mAbs. IL-12-mediated enhancement of gammadelta T cell cytotoxic activity was inhibited by anti-IL-2Rbeta mAb in a dose-dependent manner but not by anti-IL-2Ralpha or anti-IL-2Rgamma mAbs. Thus, the increased expression of the IL-2Rbeta is critical for the synergistic activation of gammadelta T cells by IL-12 plus IL-2; it is also probable that at least the low-affinity IL-12R contributes to the activation of gammadelta T cells mediated by either IL-12 alone or IL-12 plus IL-2. We have, therefore, demonstrated that IL-12 can stimulate the cytotoxic activity of gammadelta T cells from glioblastoma patients, acting via the IL-2Rbeta component of the IL-2R and low-affinity IL-12R. IL-12 activation of patients' gammadelta T cells could possibly be of potential use in the treatment of glioblastoma patients.

Regulation of the C3 production by gamma interferon from peripheral blood T cells in patients with membranoproliferative glomerulonephritis and poststreptococcal acute glomerulonephritis.

This study assessed the regulatory role of peripheral blood T cells in the C3 production in patients with poststreptococcal glomerulonephritis and branoproliferative glomerulonephritis. Peripheral blood T cells from patients at various stages of disease were cultured and the supernatants tested for gamma interferon (IFN-gamma) content and the capacity to stimulate C3 production by HuH-7 cells. Supernatants from patients with membranoproliferative glomerulonephritis and from convalescent patients with poststreptococcal glomerulonephritis significantly stimulated the C3 production; the degree of stimulation correlated with the IFN-gamma content of the supernatants. Similar results were obtained using recombinant IFN-gamma. In both cases, the effect was blocked by the addition of anti-IFN-gamma monoclonal antibody to the cultures. Interleukin 2 and interleukin 6 levels in supernatants from T cell cultures of patients and controls were essentially the same. In summary, IFN-gamma plays a regulatory role in C3 production by human hepatoma cell lines.

In vivo priming effects of interferon-beta ser on NK activity of peripheral blood mononuclear cells in cancer patients.

Natural killer (NK) activity was assayed in fresh peripheral blood mononuclear cells (PBMs) from cancer patients receiving interferon (IFN)-beta ser. Patients received a single intravenous injection of IFN-beta ser (90 x 10(6) IU m-2) on alternate days for 2 weeks, followed by a higher dose (180 x 10(6) IU m-2) on the same schedule. PBM NK lysis of K562 target cells was significantly increased in PBMs sampled 24 h after the initial injection (P < 0.05). At the end of the first 2 weeks of the protocol, NK cytotoxic activity of PBMs had fallen below the original baseline levels; the higher IFN dose subsequently given was without effect. However, significant increases in the proportion of CD16+ cells were seen following each injection. A positive correlation was also seen between the increased lytic activity of CD16+ NK cells and the proportion of CD38+ NK cells, but not the proportion of CD56+ NK cells. In vitro IFN-treatment of these in vivo-treated PBMs resulted in a further increase in NK activity. Pre-exposure in vivo to IFN-beta ser seems to prime the PBMs to respond to in vitro stimulation by IFN-gamma, which otherwise had no effect. Phenotypic analysis of PBMs after in vitro exposure to IFN-beta ser showed that the levels of CD16+, CD38+ and CD56+ cells did not change. All the NK activity responding to IFN-beta ser was found in the CD16+ enriched population of PBM, suggesting that it is unlikely that in vivo redistribution of CD16+ subsets representative of NK cells has occurred in the peripheral blood.

Spontaneous generation of human CD8+ TCR alpha beta+ cells derived from precursors within the double negative compartment.

Flow cytometric analysis demonstrated that fresh human thymocytes contain only a low level of mature CD8+ TCR alpha beta + or CD8+ TCR gamma delta + cells and they consist consist of approximately 70% double positive (DP) and approximately 10% double negative (DN) cells. These unfractionated thymocytes could be selectively expanded in vitro by stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) and PHA in the presence of IL-2. The majority of the cells expanded from unfractionated thymocytes expressed CD3, TCR alpha beta and CD8 molecules after long-term culture (18 days). When highly purified DN thymocytes were expanded over a period of 18 days in the presence of DP cells, they also co-expressed CD3, TCR alpha beta and CD8 molecules on their surface. However, when purified DN thymocytes were expanded alone, that is, in the absence of DP cells for 18 days, they expressed CD3-associated TCR gamma delta, but not CD8 or TCR alpha beta. Despite the expression of measurable levels of IL-2 alpha and beta receptors, as well as a significant level of TCR alpha beta, purified DP cells failed to proliferate. These findings provide the first evidence, in humans, that the progression of precursor cells in the DN compartment to a later stage of differentiation can be induced outside the thymus and that DP cells can affect the development of TCR expression in proliferating DN thymocytes.

Natural killer cell immunodeficiency in patients with chronic myelogenous leukemia. III. Defective interleukin-2 production by T-helper and natural killer cells.

Even though they possess normal to increased numbers of circulating natural killer (NK) cells, patients with chronic myelogenous leukemia (CML) have a functional NK-cell deficiency which is restorable in vitro in the presence of recombinant IL-2. We therefore measured the level of IL-2 production by both T-helper and NK cells from CML patients as compared to normal controls using PHA-stimulated peripheral blood mononuclear cells (PBMs) as well as FACS-sorted CD4+ (OKT4+) lymphoid cells and FACS-sorted CD16+ (B73.1+) lymphoid cells. Peripheral blood mononuclear cells from CML patients demonstrated markedly defective IL-2 production as compared to normal controls (4.0 +/- 1.6 and 5.9 +/- 1.4 units/ml after 24 hr of 5 and 10 micrograms/ml PHA stimulation as compared with 40.7 +/- 10.3 and 69.3 +/- 15.1 units/ml for normal subjects). In addition to the decreased relative percentage of CD4+ (OKT4+) lymphoid cells in CML patients, FACS-sorted CD4+ (OKT4+) cells also demonstrated a significant defect in IL-2 production, (10.8 +/- 3.6 units/ml as compared to 39.0 +/- 5.8 units/ml after 24 hr stimulation with 10 micrograms/ml PHA). FACS-sorted CD16+ (B73.1+) lymphoid cells from CML patients also demonstrated significantly decreased IL-2 production after 24 hr incubation with increasing concentrations of PHA or with the NK-sensitive target K562 as compared to normal controls. Defective IL-2 production by PBMs, CD4+ (OKT4+), and CD16+ (B73.1+) cells from CML patients was also evident after 48 hr of PHA stimulation. Although the percentages of both T4+2H4+ and T4+4B4+ subsets are significantly decreased in CML patients, CML patients have normal ratios of T4+4B4+/T4+2H4+ subsets as compared to normal controls. These and previous results support the hypothesis that decreased IL-2 production by both T-helper and NK cells from CML patients may be mechanistically related to the observed NK-cell immunodeficiency in CML patients.

Natural killer (NK) cell immunodeficiency in patients with chronic myelogenous leukemia. II. Successful cloning and amplification of natural killer cells.

Defective natural killer (NK) cell populations from patients with chronic myelogenous leukemia (CML), that reacted with both HNK-1+ and B73.1+ antibodies, were obtained by a fluorescence-activated cell sorter (FACS). These fractions, along with NK fractions from normal donors which reacted with both antibodies, were expanded as bulk cultures or clones by limiting dilution, for 4 weeks in the presence of 10% interleukin 2 (IL 2), human type AB plasma, and irradiated human allogeneic mononuclear cells. Successfully established clones from patients with CML, with lytic activity against autologous and more differentiated neoplastic granulocytes, were generated more efficiently from B73.1+ than from HNK-1+ subsets. However, there were no significant differences among the generations of B73.1+ and HNK-1+ clones for both patients and normal donors with lytic activity against NK susceptible K-562 targets. Fresh myeloblast preparations from a blast crisis were found to be more susceptible to lysis by IL 2-proliferative B73.1+ and HNK-1+ clones than were fresh myelocyte preparations from a chronic phase CML patient, which were lytically susceptible to only B73.1+ clones. B73.1+ and HNK-1+ subsets from CML patients demonstrated major histocompatibility complex nonrestricted killing, and showed the following predominant phenotypes: B73.1+T3+T8+ or B73.1+T3+T8- from B73.1+ subsets; and HNK-1-T3+T8+ (initially HNK-1+) from HNK-1+ subsets. In contrast, B73.1+ and HNK-1+ clones from normal donors showed the following predominant phenotypes: B73.1+T3-T8-; and HNK-1-T3-T8- or HNK-1-T3-T8+ (initially all HNK-1+). Short-term in vitro IL 2 or interferon treatment of fresh NK defective subsets from CML patients resulted in minimal cytotoxic augmentation. In contrast, defective NK cells from CML patients, whether HNK-1+ or B73.1+ subsets, proliferated with complete regeneration of cytolytic activity after a 3-4 week exposure to IL 2, but differed in phenotypic profiles as compared to those of normal donors. These observations imply that not only fresh defective NK cells but also the cytotoxically restored clones from CML patients are derived from different NK subsets and may represent undifferentiated forms of NK cells that may be arrested at an early stage of development by yet unknown mechanism(s). In vitro substantiation of autologous leukemia cell killing by IL 2-proliferative NK cell clones is encouraging and may allow for new in vivo immunotherapeutic modalities in CML patients.

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.

Natural killer-cell immunodeficiency in patients with chronic myelogenous leukemia. I. Analysis of the defect using the monoclonal antibodies HNK-1 (LEU-7) and B73.1.

Quantitative evaluation of natural killer (NK) cells using the HNK-1 (Leu-7) and B73.1 monoclonal antibodies (MAbs) was correlated with NK activity in 13 patients with chronic myelogenous leukemia (CML) and compared to normal donor controls. A consistent observation was the presence of normal absolute numbers of B73.1+ lymphocytes as well as normal to increased absolute numbers of HNK-1+ lymphoid cells in the peripheral blood of chronic-phase CML patients. Despite normal to increased numbers of B73.1+ and HNK-1+ lymphoid cells, these patients consistently demonstrated a significant impairment of lymphocyte-mediated NK activity in their peripheral blood. Further experiments demonstrated that chronic-phase CML patients, in contrast to normal controls, had significantly increased percentages of HNK-1+, E+ and B73.1+, E+ lymphoid cells and significantly decreased percentages of HNK-1+, E- and B73.1, E- lymphoid cells, which resulted in significant reversals of the HNK-1+, E+ to HNK-1+, E- and B73.1+, E+ to B73.1+, E- lymphoid cell ratios. (HNK-1+ [E+/E-] greater than B73.1+ [E+/E-]). Furthermore, as compared to normals, both FACS-sorted HNK-1+ and B73.1+ lymphoid cells from the E+ fraction of CML patients were consistently defective in NK activity, and could not be substantially augmented with alpha-interferon preparations. Although markedly defective in their ability to lyse K-562, HNK-1+ lymphoid cells from the E+ fraction of CML patients were not defective in their ability to bind to the NK-sensitive target, K-562. In contrast, NK-defective B73.1+ lymphoid cells were partially defective in their ability to bind to K-562.

Complement-dependent cell lysis assay for quantitation of rubella antibodies.

A comparison of the capacity to detect antibody titers to rubella virus was made between the complement-dependent cell lysis (CDCL) assay and the hemagglutination inhibition (HAI) test. Titers detected by CDCL assay correlated with the HAI test. Although the CDCL assay was less convenient to conduct, it proved to be more sensitive than the HAI test.