Augmentation of antigen-specific lymphoproliferative responses in vitro by biological response modifiers.

The detection of antigen-specific T cell responsiveness, particularly of resting memory lymphocytes, in cultures of peripheral blood mononuclear cells (PBMC) may be hampered by a less than optimal antigen presentation in vitro. Augmented sensitivity of the test system may be achieved by the addition of reagents with a beneficial effect on lymphocyte and antigen-presenting cell (APC) functions. In this study the effect of several biological response modifiers on antigen-specific T cell proliferation was determined, using nickel sulphate and tetanus toxoid as test antigens. IL-1 alpha (100 U/ml), interferon-gamma (IFN-gamma) (10 U/ml), and indomethacin (2 microM) were found to significantly enhance nickel-induced proliferation in PBMC cultures from nickel-hypersensitive donors (n = 6). Tetanus-induced proliferation (n = 5) was similarly enhanced, both by the above supplements and by the addition of polyethylene glycol (PEG) or a neuraminidase treatment of the PBMC before culture. The addition to PBMC cultures of a combination of IL-1 alpha (30 U/ml), IFN-gamma (10 U/ml), and indomethacin (2 microM) is recommended to specifically enhance antigen-induced lymphoproliferative signals.     

Augmentation in cytotoxicity of lymphnode lymphocytes by OK-432

It has been demonstrated that natural killer (NK) cell activity of lymphnode lymphocytes (LNL) is very low and hardly augmented by interferon. In this study, OK-432 was injected into the gastric cancer mass through endoscopy one week before the operation, through which NK activity of LNL was significantly increased. A single cell assay could divide the OK-432-injected patients into two groups; responder and non-responder. In responders, the increased activity induced by OK-432 was found also in the distal lymphnodes.     

Polysaccharide biological response modifiers

Biological response modifiers (BRMs) are substances which augment immune response. BRMs can be cytokines which are produced endogenously in our body by immune cells or derivatives of bacteria, fungi, brown algae, Aloe vera and photosynthetic plants. Such exogeneous derivatives (exogeneous BRMs) can be nucleic acid (CpG), lipid (lipotechoic acid), protein or polysaccharide in nature. The receptors for these exogeneous BRMs are pattern recognition receptors. The binding of exogeneous BRMs to pattern recognition receptors triggers immune response. Exogenous BRMs have been reported to have anti-viral, anti-bacterial, anti-fungal, anti-parasitic, and anti-tumor activities. Among different exogeneous BRMs, polysaccharide BRMs have the widest occurrence in nature. Some polysaccharide BRMs have been tested for their therapeutic properties in human clinical trials. An overview of current understandings of polysaccharide BRMs is summarized in this review.     

Natural cytotoxicity in man: activity of lymph node and tumor-infiltrating lymphocytes.

Lymphocytes from blood, lymph node and tumor have been tested for cytotoxicity against the K562 cell line which is known to be highly sensitive to lysis by spontaneously reactive cells. Cytotoxicity was found in all 13 samples from healthy donors and in 17/32 cancer patients. By contrast, activity was determined in only 1/18 lymph node and 1/14 preparations of tumor-infiltrating lymphocytes. Lymph node cells were similarly nonreactive against 3 other cell lines known to be sensitive to natural cytotoxicity. Studies of the composition of the effector populations revealed no absolute deficit of a particular cell type although there were differences between them resulting from the different isolation procedures used. Enrichment of the lymph node population for non-T, non-B lymphocyte was ineffective in inducing cytotoxicity in previously nonreactive samples although this procedure uniformly increased the cytotoxic potential of blood lymphocytes. Tests with blood taken during operation showed that the lack of reactivity in these preparations was unlikely to be a result of the effects of anesthesia or surgery. The reason for the low cytotoxicity in the lymph node and tumor-infiltrating lymphocytes is as yet undefined.     

卵巢癌浸润淋巴细胞的生物学特性研究

目的 :研究卵巢癌浸润淋巴细胞 (TIL)在体外扩增后生物学特性 ,评估TIL用于晚期卵巢癌治疗的前景。方法 :利用流式细胞仪分析TIL的细胞表型 ,使用分子生物学和免疫学方法研究TIL分泌细胞因子的能力和杀伤肿瘤细胞的活性。结果 :TIL细胞表型的差异可能与肿瘤的种类、性质、取材部位有关 ,结缔组织、基质中来源的TIL以CD3 CD4 为主 ,肿瘤组织和小血管周围以CD3 CD8 为主 ,体外IL 2的浓度对TIL的免疫学特性有很大的影响。经rIL 2扩增后 ,TIL分泌产生IL 2、TNF α、IFN γ等多种细胞因子能力及杀伤肿瘤细胞的活性均有明显提高。再加入抗CD3单抗或PHA(合适浓度 ) ,TIL细胞因子表达可进一步增强。TIL联合磷酰胺或IL 2治疗晚期卵巢癌患者 ,可显著改善患者外周血细胞表型 ,具有一定的疗效。结论 :TIL在体外经rIL 2扩增后 ,免疫活性有明显提高。体内肿瘤细胞的消退可能主要并不是通过输入的TIL直接杀伤肿瘤细胞 ,而是在很大程度上依靠其分泌多种细胞因子增强了机体细胞免疫活性和免疫调节能力。TIL辅助其它疗法可成为晚期卵巢癌患者的一种有效治疗手段。     

Effects of biological response modifiers on lung natural killer activity.

Natural killer (NK) activity plays an important role in host defense against tumors, especially once augmented by immunomodulators. It is likely that the modulation of NK cells is a reflection of the environment in which they reside. The current study was undertaken to characterize the response profile of lung interstitial lymphocyte natural killer (LLNK) activity to various biological response modifiers (BRM) in vitro after short term incubation (18h). The presented data show that treatment of lung lymphocytes with human recombinant interleukin 2 (rIL-2), purified rat interferon alpha/beta (IFN-alpha/beta), or murine recombinant tumor necrosis factor alpha (rTNF-alpha) resulted in a dose-dependent increase in LLNK activity. The maximum stimulation was similar for rIL-2 and IFN-alpha/beta, although a much higher concentration of IFN-alpha/beta was required to reach this level of stimulation. The maximum response to rTNF-alpha treatment was about half that seen with rIL-2 or IFN-alpha/beta and it, too, required a high concentration. By contrast, rat recombinant interferon gamma (rIFN-gamma) or murine recombinant interleukin 1 (rIL-1) failed to alter LLNK activity significantly when used alone. Furthermore, doses of IFN-alpha/beta and rTNF-alpha that had little enhancing effect were able to synergize with a suboptimal dose of rIL-2, whereas rIL-1 and rIFN-gamma failed to do so. These data demonstrate the response of lung NK activity to BRM treatment, which is important for the responsible and effective use of BRM. However the spectrum of lung NK cell response to BRM is smaller than that previously reported for NK cells from other anatomic compartments.     

Chemiluminescence in a macrophage cell line modulated by biological response modifiers

We have studied a murine macrophage cell line, J774, and found these cells capable of a zymosan-triggered chemiluminescent oxidative burst. Such activity was enhanced by preincubation with Corynebacterium parvum (CP), bacillus Calmette-Guerin, and lipopolysaccharide (LPS). Under similar conditions, CP and LPS were shown to enhance J774-mediated tumor cell lysis. We have also demonstrated that murine interferon alpha + beta rendered J774 cells more sensitive to the actions of CP and LPS. These results indicate that J774 cells may be useful for the in vitro evaluation of biological response modifiers as well as the study of oxygen radical production by macrophages.     

Clonal dynamics of tumor-infiltrating lymphocytes

The presence of tumor-infiltrating lymphocytes (TIL) provides important evidence of anti-tumor immunity in vivo. However, TIL are usually not sufficient for inhibiting tumor growth. We explored the spatial and temporal aspects of clonal accumulation of TIL using RT-PCR/single-strand conformation polymorphism analysis. In CMS5 fibrosarcomas in BALB/c mice, accumulated T cell clones were specific in that dominant TIL were identical between distant tumors. Moreover, dominant TIL in the first tumor appeared consistently in the second tumor inoculated after formation of the first tumor. These results suggest that TIL show a certain level of specific tumor surveillance. When we characterized CD4(+) and CD8(+) TIL separately, CD8(+) TIL were highly concentrated and persistently localized at the tumor site, while most CD4(+) TIL clones were less concentrated and less persistent. A functional analysis showed that TIL had a certain degree of anti-tumor activity when CD4(+) and CD8(+) TIL were co-transferred. Co-transfer of CD4(+) and CD8(+) TIL exhibited equivalent anti-tumor activity, irrespective of tumor stage. However, the numbers of TIL did not increase after the early phase of tumor progression. These data suggest that TIL are specific to the tumor and potentially retain anti-tumor activity, although their accumulation in mice is impaired.     

Suppression of lymphokine-activated killer cell generation by tumor-infiltrating lymphocytes

A functional analysis of tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma (RCC) and malignant melanoma was performed. TILs were expanded in recombinant interleukin-2 (50 U/ml) in Iscoves medium. Phenotypic and functional (cytolytic vs regulatory) analyses were carried out with the fresh and expanded TIL populations after 4 weeks in culture. Only one TIL population from an RCC case (out of six cases studied) was CD8+ and demonstrated MHC class I-restricted tumor-specific cytotoxicity against the autologous RCC target. TIL populations from the other five cases became predominantly CD4+ and they neither killed the respective autologous tumor cells nor killed the NK-sensitive target K-562 cells. When studied for other functions, two CD4+ TIL populations were found to suppress the lymphokine-activated killer cell response by peripheral blood lymphocytes (PBL) in coculture. Of these two, a TIL population from an RCC case (MJ TIL) was used to study the cellular and molecular mechanisms of suppression. The MJ TIL synthesized a supernatant factor that blocked activation of resting PBL as measured by the induction of high-affinity IL-2 receptor (IL-2R) when stimulated by phytohemagglutinin but did not down-regulate the fully expressed IL-2R on activated T cells. The suppression of high-affinity IL-2R induction on T cells did not result from tumor necrosis factor-alpha and beta or from transforming growth factor-beta as these cytokines were not detected in the cell-free supernatant from the MJ TIL culture. The supernatant factor also suppressed IL-2-mediated enhancement of cytotoxicity by natural killer (NK) cells without demonstrating direct toxic effect on the NK cells. Thus, when TIL are used for adoptive immunocytotherapy, it may be useful to fully characterize them functionally, in vitro.     

High proliferative capacity and specific antiautologous melanoma cytotoxicity of a human T-lymphocyte clone derived from tumor-infiltrating lymphocytes

A CD8+ clone, identified by its T-cell receptor gamma- and beta-gene configuration, was shown to preferentially develop, in the bulk culture of melanoma tumor-infiltrating lymphocytes with recombinant interleukin 2 after 1 month. Thirteen CD8+ clones were obtained by limiting dilution culture of tumor-infiltrating lymphocytes from 43-days old culture. Four of these clones, analyzed for T-cell receptor rearrangements, exhibited exactly the same T-cell receptor gene pattern as tumor-infiltrating lymphocytes from the bulk culture, showing, therefore, that all the CD8+ clones were subclones. All the 13 CD8+ subclones were strongly cytotoxic for autologous melanoma cells but did not kill K562. A more complete cytotoxicity analysis showed that the clones did not kill autologous fibroblasts or Con A blasts or allogeneic tumor targets. Furthermore, specific killing was inhibited by monoclonal antibodies against CD3, CD8, T-cell receptors alpha beta, and class I major histocompatibility complex antigens indicating that effector-to-target cell recognition was mediated through the T-cell receptor in a major histocompatibility complex-restricted fashion. These data showed that human melanoma-specific cytotoxic T lymphocytes can be obtained from melanoma TIL and that a single cytotoxic T lymphocyte clone can be expanded to more than 10(10) cells without a loss of autotumor specificity.     

Enhanced superoxide production by rat alveolar macrophages stimulated in vitro with biological response modifiers

The kinetics of superoxide release and the effects of several biological response modifiers (BRM) on superoxide release from rat pulmonary alveolar macrophages (AM) have been studied. These cells produced superoxide anion both spontaneously and in response to phorbol myristate acetate (PMA) in a dose-related manner. The response to PMA peaked in approximately 2 hr and maintained plateau levels for an additional 2-3 hr before subsiding. Pretreatment of the macrophages in vitro with a number of immunostimulants enhanced the production of superoxide above that of controls. The release of superoxide in response to the immunostimulants was a slow phenomenon that took place over a 3-5 hr time period. Lymphokine-containing supernatants from concanavalin A (con A)-stimulated rat spleen cells (LK-Sup), murine recombinant gamma interferon (rMuIFN-gamma), nigeran, and muramyl dipeptide (MDP) enhanced this response in a dose-related manner. Poly I:C and Salmonella typhosa lipopolysaccharide (LPS) stimulated rat alveolar macrophages at low but not high concentrations. In contrast to the alveolar macrophages, rat peritoneal exudate cells were not activated by immunostimulants to produce increased amounts of superoxide.     

TIA-1 positive tumor-infiltrating lymphocytes in nevi and melanomas.

Tumor-infiltrating lymphocytes (TIL) have been shown to be an independent prognostic factor in melanomas. To better characterize the host immune response, we have classified TIL by their immunoreactivity against lymphoid markers in formalin-fixed, paraffin-embedded tissue. Monoclonal antibodies to leukocyte common antigen (LCA) and TIA-1 (a granule-associated protein of cytotoxic T cells and NK cells) were used to immunostain a series of benign nevi, nontumorigenic radial growth phase, and tumorigenic vertical growth phase melanomas and metastases. Among nine nevi, few LCA+ TIL were found, among which rare cells were positive for TIA-1 (mean, 2.0). Five nontumorigenic radial growth phase melanomas also had few total TIL and rare TIA-1+ TIL (mean, 3.4); the nontumorigenic radial growth phase component of seven tumorigenic vertical growth phase melanomas had higher numbers of TIA-1+ TIL (mean, 11). Twelve cases of tumorigenic vertical growth phase melanoma showed a variable but significantly greater number of both LCA+ TIL and TIA-1+ TIL (mean, 30.6). Nine cases of metastatic melanoma had a wide range of variation in LCA as well as in TIA-1+ TIL (mean, 46). Although the mean total number of TIA-1+ TIL increased from nontumorigenic radial growth phase to tumorigenic vertical growth phase to metastases, TIA-1+ as a percentage of TIL declined across these categories of tumor progression (42%, 31%, and 26%, respectively). Our results show that these attributes of TIA-1+ TIL, both increasing total number but decreasing percentage, appear to be a marker of tumor progression of malignant melanomas. In addition, there was significant variability in the number of TIA-1+ TIL among advanced melanomas, raising the possibility that an assessment of TIA-1+ TIL may prove a useful prognostic tool for the evaluation of primary melanomas.     

Augmentation of antibody-dependent cell-mediated cytotoxicity by anti-immunoglobulin. I. Relationship to target cell capping

The functional integrity of the local immune system in vitamin A-deficient (A-) rats was investigated. Secretory IgA levels in the intestinal fluid of A- rats were significantly lower than in controls. This and the decrease in intensity of immunofluorescent staining for secretory component (SC) in the intestinal cells was related to the duration of vitamin A deprivation. IgG levels in the intestinal fluid, and serum IgA and IgG levels were unaffected in deficiency. Moreover, when the response of animals to DNP50-BGG was evaluated, the local anti-DNP response in the intestine was markedly depressed. These defects may result from impaired synthesis of SC by epithelial cells. On the other hand, the serum antibody response in deficient animals was not noticeably different from that of the controls; if any, htere was a slight reduction in the affinity of antibody.     

Cell-mediated immunity and biological response modifiers in insulin-dependent diabetes mellitus complicated by end-stage renal disease

Previous studies have shown several immunoregulatory abnormalities in insulin-dependent diabetes mellitus (IDDM). In this report we compared peripheral blood mononuclear cells (PBMC) from patients with IDDM complicated by end-stage renal disease (ESRD) to those from normal subjects and from patients with ESRD of different etiologies for their: natural killer (NK) and antibody-dependent cell-mediated cytotoxic (ADCC) activities; modulation of NK and ADCC activities by biological response modifiers (BRM) including purified human lymphoblastoid interferon, human recombinant alpha-2 interferon, human gamma interferon and human recombinant interleukin 2; proliferative response of T and B lymphocytes to concanavalin A (Con A), phytohemagglutinin and pokeweed mitogen, and ability to produce T-cell growth factor (interleukin 2; IL-2). PBMC of diabetic patients demonstrated significantly lower NK activity than normal and ESRD subjects. Upon treatment with BRM, NK activity was augmented and achieved normal levels. ADCC activity was not different from that of normal controls and exhibited similar increases when stimulated by BRM. The proliferative responses to Con A, phytohemagglutinin and pokeweed mitogen as well as IL-2 production in response to Con A stimulation were significantly lower in the IDDM group. Our results indicated that NK cells from patients with IDDM can respond to IL-2 with enhanced cytotoxicity, and, because activation of resting T cells by mitogenic stimuli depends on the production of IL-2 as well as the appearance of a receptor for IL-2, our finding of low levels of in vitro IL-2 production by PBMC from patients with IDDM may explain the depressed NK activity and the observed poor response to T-cell mitogens.     

The role of biological response modifiers on apoptosis in HIV infections and AIDS

The vital role played by biological response modifiers especially interferon alpha (IFN) in HIV infections and AIDS is discussed. Apoptosis, a phenomenon of a genetically controlled physiological cell death is also discussed in relation to HIV infections and its response to oropharyngeal IFN therapy. HIV infections induce apoptosis at a stage when CD4-positive T lymphocytes are still immature and weakly expressing CD4 molecules. During IFN therapy, the process of apoptosis is blocked or down-regulated, thus rendering the immature cells to rapidly mature to strongly express CD4 molecules and to perform their expected functional role. These observations are of critical importance in further understanding of the salient features associated with HIV/AIDS, and which may be exploited in the design of strategic intervention measures.     

Therapeutic advances in biological response modifiers in the treatment of cutaneous T-cell lymphoma

Cutaneous T-cell lymphoma (CTCL) is most often a skin-infiltrating malignancy of clonal CD4+ T-cells. Therapy is based on staging and the likelihood of progression. Biological response modifiers and chemotherapeutic agents are used to preserve the integrity of the host antitumour response while selectively targeting the malignant cells. The biological response-modifying treatment options currently used to treat CTCL are bexarotene, denileukin diftitox, interferon-α, interferon-γ and interleukin-12, as well as extracorporeal photopheresis and phototherapy. A combination therapy approach maximises response in patients with advanced CTCL. Biological response modifiers in combination with photopheresis are used for patients with the leukaemic phase of the disease. Among the majority of patients with advanced stage disease so treated, immune response augmentation appears to prolong survival. Future areas of research should assess not only survival and optimal treatment combinations, but also quality of life during the treatment period.     

TIL识别的HLA-A2限制性人黑色素瘤特异抗原肽的研究

目的：探讨TIL识别的人黑色素瘤HLA-A2限制性的肿瘤抗原肽特性。方法：通过亲和层析柱从三种肿瘤细胞系（624-Mel、Chap-Mel和JY）中纯化HLA-A2蛋白和结合的多肽分子，经弱酸高温处理和离心超滤后，应用反相．高效液相色谱层析（RT-RPLC）分离各多肽组份，并将其各组份与T2细胞共同孵育、进行重建TIL识别的人黑色素瘤特异表位试验，鉴定肽分子生物活性，通过质谱分析所获活性肽分子特性，参照活性肽序列，制备人工合成肽加以进一步证实。结果：从RT-RPLC层析的624-Mel的肽组份，经特异表位鉴定发现3个活性峰（P19、P25和P31）。其中P31，TIL杀伤率达67％，质谱分析表明肽分子量为948，氨基酸序列为H^＋Ala Lue Trp Lue Phe Phe Gly Val Lue OH^-的9肽分子。经特异表位测定表明人工合成肽具有纯化活性肽的生物活性特征。结论：分离鉴定的这-9肽分子是一种TIL识别的HLA-A2限制性肿瘤抗原肽，为肿瘤防治、合成肽疫苗的研究奠定了可靠的实验基础。