Induction Of Killer Research Articles

The Current Application and Future Prospects of Astragalus Polysaccharide Combined With Cancer Immunotherapy: A Review.

So far, immunotherapy has been shown to have impressive effects on different cancers in clinical trials. All those immunotherapies are generally derived from three main therapeutic approaches: immune checkpoint inhibitors, immune cell vaccination, and adoptive cellular immunotherapy. Our research systematically reviewed a wide range of clinical trials and laboratory studies of astragalus polysaccharide (APS) and elucidated the potential feasibility of using APS in activating adoptive immunotherapy. Apart from being effective in adaptive “passive” immunotherapy such as lymphokine-activated killer treatment and dendritic cell (DC)–cytokine–induced killer treatment, APS could also regulate the anti-programmed cell death protein 1 (PD-1)/PD-L1 on the surface of the immune cells, as a part in the immune checkpoint inhibitory signaling pathway by activating the immune-suppressed microenvironment by regulating cytokines, toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) pathways, and immune cells, such as DCs, macrophages, NK cells, and so on. In view of the multiple functions of APS in immunotherapy and tumor microenvironment, a combination of APS and immunotherapy in cancer treatment has a promising prospect.

Adoptive immunotherapy with interleukin-2 & induced killer cells in non-small cell lung cancer: A systematic review & meta-analysis.

Background & objectives:The effectiveness of interleukin-2 (IL-2) and induced killer cells for non-small cell lung cancer (NSCLC) is controversial. This study evaluates the efficacy and safety of interleukin-2 and induced killer cells on NSCLC, so as to provide references for further clinical practice and research.Methods:Relevant randomized controlled trials (RCTs) were searched in Cochrane library (Issue 2, 2013), Web of Science (1980-March 2013), PubMed (1966-March 2013), China Knowledge Resource Integrated database (CNKI) (1994-March 2013), China Biology Medicine database (CBM) (1978-March 2013), VIP (1989-March 2013), and Wan Fang databases (1997-March 2013). There were no language restrictions. After independent quality assessment and data extraction by two authors, meta-analysis was conducted by RevMan 5.1 software.Results:Ten RCTs were included. Odds ratio (OR), 95% confidence intervals (CI), P value expressed as test group (interleukin-2 or induced killer cells combined chemotherapy) versus control group (chemotherapy alone), was 2.02 (1.24, 3.29; P=0.004) for disease control rate. Hazard ratios (HR) (95% CI; P value), expressed as test group (interleukin-2 or induced killer cells) versus control group, were 0.60 (0.46, 0.79; P=0.0003) for overall survival of postoperative treatment, and 0.77 (0.60, 0.99; P =0.04) for overall survival of combination with chemotherapy. Mean differences (MD) (95% CI; P value), expressed as test group (interleukin-2 or induced killer cells) versus control group (after treatment), were 11.32 (6.32, 16.33; P=0.00001) for NK cells, 11.79 (2.71, 20.86; P=0.01) for CD3+ cells, 14.63 (2.62, 26.64; P=0.02) for CD4+ cells, and -4.49 (-7.80, 1.18; P=0.008) for CD8+ cells.Interpretation & conclusions:The meta-analysis showed that IL-2 or induced killer cells combination therapy was efficacious in treating NSCLC and improved overall survival. Further analysis of trials having adequate information and data need to be done to confirm these findings.

INFLUENCE OF SOME TRADITIONAL CHINESE MEDICINES (TCMS) ON CYTOKINE-INDUCED KILLER CELLS PROLIFERATION AND ANTI-TUMOR FEATURES IN VITRO

Some traditional Chinese medical (TCM) formulations shown to have anti - tumor actions in patients with various solid tumours. However, the mechanisms of the potential anticancer actio n are rarely reported. In the present study, we found that extracts from pharmaceutical raw materials consisting of ROR (Red ginseng, Ophiopogon japonicus, Radix Astragali) could significantly improve the proliferation rate and cytotoxic activity of cytoki ne - induced kil ler (CIK) cells, which is a heterogeneous population of polyclonal T lymphocytes sharing NK (Natural Killer) phenotype and functional properties. The extracts were prepared using modified ethanol - water reflux method. Pharmacodynamic test resu lts showed that 5% ROR - adding CIK Cells group performs 2.8 folds proliferation multiples than that of no ROR - adding CIK cells group, meanwhile the cytotoxic activity to K562 tumor cell has also a significant difference between them. In conclusion, ROR - addi ng CIK cells group exhibits stronger proliferation and cytotoxicity than no ROR - adding CIK cells group, and the results provided a new experimental evidence for TCM’s application in CIK - immunotherapy.

Pulsing with Blast Cell Lysate or Blast-Derived Total RNA Reverses the Dendritic Cell-Mediated Increase in Cytotoxic Activity of Cytokine-Induced Killer Cells Against Human Allogeneic Acute Myelogenous Leukemia Cells.

Immunotherapeutic strategies may be a treatment option in patients with refractory acute myelogenous leukemia (AML) or, in cases of complete remission after conventional therapy regimens, may help to reduce disease recurrence or delay time to progression. Because evidence suggests a key role of dendritic cells (DCs) in cancer immunotherapy, we examined cytokine induced killer (CIK) cell responses in vitro after coincubation with autologous peripheral blood monocyte-derived DCs against three cell lines and allogeneic blasts from three patients with de novo AML. Although DCs were unable to enhance CIK cell effects against all three cell lines tested, the cytotoxic activity against the AML cells of the patients increased after coculture with mature DCs, which was significant in two of three patients. However, neither prior pulsing of the DCs with blast cell lysates nor with leukaemic cell-derived total RNA further enhanced the lytic capacity of the CIK cells. On the contrary, pulsing reduced the cytotoxic activity of the effector cells in a concentration-dependent manner. Because this decrease of allogeneic cytotoxicity was observed, we conclude that monocyte-derived DCs may be useful in autologous or allogeneic vaccine strategies for the treatment of AML or in priming donor lymphocytes in vitro, but unfractionated antigens as pulsing agents may have inhibitory effects on T cell efficiency and their employment in immunotherapeutic strategies for AML seems questionable.

Totally synthetic polymer with lectin‐like function: Induction of killer cells by the copolymer of 3‐acrylamidophenylboronic acid with N,N‐dimethylacrylamide

Lectin-like function was demonstrated in this study for a novel water-soluble polymer with phenylboronic acid residues (poly (AAPBA-DMAm)), which induced appreciable proliferation of murine spleen lymphocytes with an increased expression of interleukin-2 (IL-2) receptor on their surface. Consequently, boosted proliferation of lymphocytes with cytotoxic action to YAC-1 cells was achieved by concurrent addition of IL-2 with poly(AAPBA-DMAm) in the medium, indicating this boronate-containing polymer to be worked as an effective immuno-adjuvant for the induction of lymphokine-activated killer (LAK) cells. Flow-cytofluorimetry study revealed that poly(AAPBA-DMAm) competitively inhibited the cellular binding of N-acetylneuraminic acid-specific lectin (Limax Flavus Agglutinin) in a concentration-dependent manner, suggesting that phenylboronate moiety in the polymer may recognize N-acetylneur- aminic acid (sialic acid) residues existing on the plasma-membrane surface of lymphocytes to induce their proliferation. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 307–314, 2001.

INTERLEUKIN (IL-)15 HAS LESS ACTIVITY THAN IL-2 TO PROMOTE TYPE 2 CYTOKINE PREDOMINANCE IN TUMOUR-ASSOCIATED MONONUCLEAR CELLS FROM LUNG CANCER PATIENTS

Deviation of type 1/type 2 cytokine balance to type 2 predominance may contribute to tumour progression. We investigated effect of interleukin (IL-)15 on modulation of type 1/type 2 balance in addition to non-major histo-compatibility complex (MHC)-restricted killer induction in the tumour-growing site. IL-15 induced significant killer activity in mononuclear cells (MNC) in malignant pleural effusion as well as those in peripheral blood. Pleural MNC produced more IFN-γ (type 1 cytokine) by incubation with IL-15 or IL-2 than blood MNC. Moreover, IL-4 and IL-5 (type 2 cytokines) production by pleural MNC were observed only by incubation with IL-2, but not with IL-15. These observations suggest that IL-15 has a potent activity to restore type 1/type 2 balance in addition to killer induction in tumour-growing site.

Totally synthetic polymer with lectin-like function: Induction of killer cells by the copolymer of 3-acrylamidophenylboronic acid withN,N-dimethylacrylamide

Lectin-like function was demonstrated in this study for a novel water-soluble polymer with phenylboronic acid residues (poly (AAPBA-DMAm)), which induced appreciable proliferation of murine spleen lymphocytes with an increased expression of interleukin-2 (IL-2) receptor on their surface. Consequently, boosted proliferation of lymphocytes with cytotoxic action to YAC-1 cells was achieved by concurrent addition of IL-2 with poly(AAPBA-DMAm) in the medium, indicating this boronate-containing polymer to be worked as an effective immuno-adjuvant for the induction of lymphokine-activated killer (LAK) cells. Flow-cytofluorimetry study revealed that poly(AAPBA-DMAm) competitively inhibited the cellular binding of N-acetylneuraminic acid-specific lectin (Limax Flavus Agglutinin) in a concentration-dependent manner, suggesting that phenylboronate moiety in the polymer may recognize N-acetylneur- aminic acid (sialic acid) residues existing on the plasma-membrane surface of lymphocytes to induce their proliferation.

INTERLEUKIN-12-MEDIATED KILLER ACTIVITY IN LUNG CANCER PATIENTS

The authors investigated the interleukin (IL)-12-inducible killer activity of blood mononuclear cells (MNC) from 30 untreated primary lung cancer patients and 24 control subjects. Cytotoxicity was assayed as 4-h 51Cr release from Daudi lymphoma cells or lung cancer cells (H-69, N-291 and PC-9). MNC from lung cancer patients exhibited similar killer activity to those from control subjects after in vitro incubation with IL-12 for 4 days. Effective killer induction by IL-12 was observed even in MNC from advanced lung cancer patients and patients with small cell lung cancer. IL-12 and a suboptimal dose of IL-2 had additive effects in inducing killer activity in MNC from both lung cancer patients and control subjects. On the other hand, with an optimal dose of IL-2, IL-12 suppressed killer induction. Addition of IL-12 alone or in combination with IL-2 resulted in interferon (IFN)-γ production by MNC from lung cancer patients as well as control subjects. These observations suggest that IL-12 could be useful for immunotherapy of lung cancer in humans.

Allo-skin graft rejection, tumor rejection and natural killer activity in mice lacking p56lck.

Mice lacking the p56lck molecule (lck -/-) have a profound block in the maturation of thymocytes and a greatly reduced number of peripheral mature T cells. To analyze further the functions of the T cells developed in lck -/- mice in vivo, we evaluated the ability of lck -/- mice to reject allo-skin grafts and methylcholanthrene (MCA)-induced syngeneic fibrosarcoma, and also examined the biological activity of lck -/- natural killer (NK) cells. Mice lacking p56lck failed to reject skin grafts from either MHC-disparate or minor-histocompatibility-different donors, even after they had been primed with donor spleen cells. They also failed to reject the MCA-induced immunogenic syngeneic fibrosarcoma, MC57X. NK activity in mice lacking p56lck was normal, and there were no differences in the NK cell activation induced by poly(I).poly(C) stimulation or interleukin-2 stimulation (lymphokine-activated killer induction) between mice lacking p56lck and their immunocompetent heterozygous littermates. NK cells lacking p56lck mediated a normal antibody-dependent cell-mediated cytotoxicity (ADCC) response. The results of this study indicate that the loss of p56lck severely impairs the effectors of the immune system which mediate the rejection of allo-skin grafts and syngeneic tumors. The normal NK activity in lck -/- mice suggests that p56lck is not required for the development and activation of NK cells.

Down-regulatory Role of Alveolar Macrophages in IL-2-Stimulated Proliferation and Killer Induction of Lung Lymphocytes in Patients with Active Pulmonary Sarcoidosis

Down-regulatory Role of Alveolar Macrophages in IL-2-Stimulated Proliferation and Killer Induction of Lung Lymphocytes in Patients with Active Pulmonary Sarcoidosis

STUDY OF T-LYMPHOCYTE SUBSETS AND INTERLEUKIN-2 IN GASTRIC CANCER

Peripheral blood, regional lymphnode and spleen lymphocytes were measured by using different monoclonal antibodies and flow cytometry in 20 patients with gastric cancer. The patients were divided in two groups, patients with stage I and II disease and patients with stage III and IV disease, according to the General Rules for Gastric Cancer Study, Japanese Research Society for Gastric Cancer. Such parameters as OKT3, OKT4, OKT8, OKM1, OKT4+ 2H4+, OKT4 + 2H4-, OKT8 + Leu15+ and OKT8 + Leu15- were used as monoclonal antibodies. NK activity, interleukin-2 production and interleukin-2 receptors were also measured.In peripheral blood lymphocytes, IL-2 production was significantly lower in the stage III and IV group, than in the stage I and II group. The percentages of OKT4, OKT4 + 2H4- and the OKT4/OKT8 ratio were lower in stage III and IV group than in stage I and II group. Among the regional lymphnodes in the stage I and II group, the percentages of OKT4 + 2H4- and the OKT4/OKT8 ratio were higher in proximal lymphnodes than in distal nodes. In stage III and IV group, the percentages of OKT3, OKT8 and OKT8 + Leu15- were lower and the OKT4/OKT8 ratio was higher in proximal lymphnodes than in distal nodes. These results suggest that helper T-cell activity in peripheral blood and the induction of killer T cells in regional lymphnodes decrease as gastric cancer progresses.

Assessment of lymphokine-activated killer activity and γ-interferon production in patients with small hepatocellular carcinomas

We have previously reported depressed γ-interferon production and depressed lymphokine-activated killer and natural killer activity in patients with relatively large hepatocellular carcinomas. These parameters were normal in cirrhosis. Some evidence had suggested a γ-interferon production defect as the main cause of depressed lymphokine-activated killer activity in hepatocellular carcinoma, (i.e., γ-interferon enhances lymphokine-activated killer and natural killer activity and γ-interferon antibody inhibits lymphokine-activated killer induction). However, we were unable to clinically define the precise mechanism operating here because γ-interferon production and lymphokine-activated killer activity were both defective in advanced hepatocellular carcinoma. In recent years, it has become possible to detect even small hepatocellular carcinomas on ultrasonography and to confirm them by fine-needle biopsy. In this study, we assessed those immune parameters in 48 patients with hepatocellular carcinomas less than 2 cm in diameter to confirm depressed immune function and to clarify the mechanism of these defects. Lymphokine-activated killer activity was defective in 31 patients (64.6%), whereas γ-interferon production was defective in only 1 of these patients (2.1%). This observation argues against the hypothesis that defective γ-interferon production is the primary defect and provides new insight into the mechanism of progression of defective immune function in hepatocellular carcinoma. Thirty-one of the 48 hepatocellular carcinoma patients were treated surgically, and these immune parameters were followed for 6 mo. The recovery of lymphokine-activated killer activity in all hepatocellular carcinoma patients with low lymphokine-activated killer activity suggests the tumor burden as the cause of defective lymphokine-activated killer activity. (HEPATOLOGY 1993;17:781–787.)

Assessment of lymphokine-activated killer activity and γ-interferon production in patients with small hepatocellular carcinomas

We have previously reported depressed gamma-interferon production and depressed lymphokine-activated killer and natural killer activity in patients with relatively large hepatocellular carcinomas. These parameters were normal in cirrhosis. Some evidence had suggested a gamma-interferon production defect as the main cause of depressed lymphokine-activated killer activity in hepatocellular carcinoma, (i.e., gamma-interferon enhances lymphokine-activated killer and natural killer activity and gamma-interferon antibody inhibits lymphokine-activated killer induction). However, we were unable to clinically define the precise mechanism operating here because gamma-interferon production and lymphokine-activated killer activity were both defective in advanced hepatocellular carcinoma. In recent years, it has become possible to detect even small hepatocellular carcinomas on ultrasonography and to confirm them by fine-needle biopsy. In this study, we assessed those immune parameters in 48 patients with hepatocellular carcinomas less than 2 cm in diameter to confirm depressed immune function and to clarify the mechanism of these defects. Lymphokine-activated killer activity was defective in 31 patients (64.6%), whereas gamma-interferon production was defective in only 1 of these patients (2.1%). This observation argues against the hypothesis that defective gamma-interferon production is the primary defect and provides new insight into the mechanism of progression of defective immune function in hepatocellular carcinoma. Thirty-one of the 48 hepatocellular carcinoma patients were treated surgically, and these immune parameters were followed for 6 mo. The recovery of lymphokine-activated killer activity in all hepatocellular carcinoma patients with low lymphokine-activated killer activity suggests the tumor burden as the cause of defective lymphokine-activated killer activity.

Patent Evaluation: The Use of Interleukin-10 in Adoptive Immunotherapy of Cancer

SummaryNovelty: A method for using interleukin-10 (IL-10) and its compositions in adoptive immunotherapy of human cancers is claimed. Tumour-infiltrating lymphocytes are cultured in the presence of IL-2 and IL-10 so that the tumour-infiltrating lymohocytes proliferate. The cultured tumour-infiltrating lymphocytes (TIL) are then administered to the patient followed by an effective amount of IL-2 and IL-10. The method may reduce side-effects caused by IL-2-induced cytokine production.Biology: The differential effects of IL-4 and IL-10 on IL-2-induced interferon-γ synthesis and cytoxicity in human NK cells were assayed. The results are presented in figures. Both IL-4 and IL-10 (human or viral) suppressed IFNγ and tumour necrosis factor-α synthesis by IL-2-stimulated peripheral blood mononuclear cells. However, only IL-4 inhibited IL-2 induced lymphokine-activated killer activity. No in vivo data are provided.Chemistry: IL-10 is preferably selected from polypeptides exoded by the open reading frames defined by cDNA inserts of pPH5C, pH15C and pBCRF1 (SRα). Expression of cDNA was by standard methods. Full sequence details are provided.

Cancer, cytokines, and cytotoxic cells: Interleukin-2 in the immunotherapy of human neoplasms

Modern immunotherapy of human cancer has evolved as a rapidly expanding field of clinical and experimental research. Employing the systemic application of recombinant interleukin-2 (IL-2) in humans, Rosenberg and colleagues from the National Cancer Institute reported the regression of advanced metastatic tumors in approximately 10%-30% of patients treated. The additional adoptive transfer of autologous patient-derived activated lymphocytes was performed to enhance therapeutic efficacy. While the exact mechanisms of IL-2 based immunotherapy in cancer remain unclear, it has been hypothesized that both the IL-2 activated lymphocyte and its secretory products such as interferon-gamma or tumor-necrosis factor beta may contribute to the lysis of tumor cells in vivo. Accordingly, research has been directed toward enhancing both the activation state and the specificity of IL-2 induced killer cells in humans. Based on in vitro and animal data, the retransfusion of tumor-infiltrating lymphocytes has been shown to mediate the regression of metastatic neoplasms in up to 50% of patients receiving systemic IL-2. Considerable toxicity from the use of high-dose IL-2 has prompted attempts to develop low-dose regimens which allow for the outpatient treatment of patients presenting poor prognosis. While in most clinical trials involving IL-2, patient follow-up has been short, and no or only limited data have become available from controlled prospective and randomized clinical studies, IL-2 has shown some promise in patients with metastatic renal cell cancer or malignant melanoma. Novel approaches toward the improvement of clinical efficacy of IL-2 include local (e.g., intracavitary) application or combinations with other cytokines such as interferon-alpha or cytostatic drugs.

Successful adoptive immunotherapy with OK432-inducible activated natural killer cells in tumor-bearing mice

We had demonstrated that the NK cell mediated cytotoxicity of murine spleen cells could be augmented by in vivo priming and subsequent in vitro challenge with a streptococcal preparation OK432, and the cell surface phenotype of induced killer cells was Thy-1+, asialo GM1+, suggesting that the activated cells were of NK lineage (OK-NK cell). We had also clarified that IL-2 played a major role in inducing the OK-NK cells via the production of IFN-gamma. In this study, we examined the effect of adoptive transfer of OK-NK cells on syngeneic tumors in mice. Mice were implanted with SP2 myeloma cells intraperitoneally (i.p.), or C26 colon adenocarcinoma cells subcutaneously to make the models of peritonitis carcinomatosa or solid tumor, and the OK-NK cells were transferred i.p. or intratumorally, adoptively. By the adoptive transfer of OK-NK cells, 92% of mice bearing SP2-tumor had be cured. The tumor growth of C26-solid tumor was inhibited, and the survival rate of mice bearing C26-tumor was significantly increased. The intratumoral remnants of 125I-labelled OK-NK cells were 61, 27 and 8% at 4, 12 and 36h after intratumoral transfer, respectively. By multiple transfer of OK-NK cells, the antitumor effect was more effectively augmented than that of a single transfer. Results in this study suggested that OK-NK cells could be useful for the therapy of cancer patients.

Lymphokine‐activated Killer Induction and Its Regulation by Macrophages in Malignant Pleural Effusions

Mononuclear cells (MNC) from pleural effusions and peripheral blood of 18 patients with primary lung cancer with malignant pleural effusion were studied. Pleural and blood MNC generated lymphokine‐activated killer (LAK) activity similarly when cultured for 4 days with an optimal concentration of interleukin 2 (IL‐2). Highly purified lymphocytes (>98%) and monocyte‐macrophages (>90%) were isolated by discontinuous Percoll gradient centrifugation from pleural and blood MNC. Pleural macrophages, as well as blood monocytes, showed significant augmenting effects on in vitro LAK cell induction from pleural and blood lymphocytes by IL‐2. During daily intrapleural administration of IL‐2, significant induction of LAK activity in vivo was observed after 3 days, but then this LAK activity in pleural MNC decreased almost to zero by day 15. Daily injections of IL‐2 resulted in reduction in the up‐regulation of LAK induction by pleural macrophages and also in increase in the levels of soluble IL‐2 receptors in pleural effusions. These findings indicate that in vivo LAK induction of lymphocytes in malignant effusions by IL‐2 may be regulated by macrophages in the effusions.

Suppression of lymphokine-activated killer induction by neutrophils.

Peripheral blood polymorphonuclear neutrophils (PMN) suppressed the induction of PBL lymphokine-activated killer (LAK) function by rIL-2 in vitro. The suppression depended on the concentration of PMN in the IL-2 culture, and required intact PMN. However, PMN did not require treatment with immunoregulators such as IL-2, LPS, or TNF to express the suppressive activity, and no direct contact with PBL was needed for the suppression. Addition of anti-TNF antibodies had no effect on the suppression, suggesting that no endogenous TNF in the culture was involved in the suppression. PMN did not inhibit LAK function by preventing utilization of IL-2 by PBL or by selective depletion of NKH-1+ cells which constitute the majority of LAK precursors in PBL. The suppression was reversed by superoxide dismutase but not by catalase, suggesting that superoxide anion, not hydrogen peroxide, was involved in the suppression. No other suppressive factor was detectable in PMN culture supernates. Our results of PMN regulating LAK induction in vitro suggest that PMN may have a role in determining the outcome of immunotherapy with IL-2 in vivo.

Natural killer and interleukin‐2 induced cytotoxicity in asthmatics

Natural killer (NK) cell activity and interleukin-2 (IL-2) induced cytotoxicity to NK-resistant targets T24 in a group of patients with grass pollen seasonal asthma were investigated. Acute bronchial provocation with this antigen was also performed and blood samples were taken before and 15 min after the maximal clinical response. NK activity was elevated in this group but there was a significant decrease after acute challenge. IL-2 induced cytotoxicity was also increased and declined after antigen challenge. Limiting dilution analysis for minimal frequencies of precursors of the IL-2 induced killer cells was introduced and showed increased numbers of lymphokine-activated killing precursors in this group of patients. These data might indicate the aberrations of the IL-2 system in immediate type allergy but the clinical significance of these findings remains to be determined.

Regulatory functions of hapten-reactive helper and suppressor T lymphocytes. III. Amplification of a generation of tumor-specific killer T-lymphocyte activities by suppressor T-cell-depleted hapten-reactive T lymphocytes.

2,4.6-trinitrophenyl (TNP)-reactive T-cell activities were raised in mice by immunization with TNP-isologous mouse gamma globulin. After establishing that TNP-reactive T lymphocytes can serve as amplifier cells for induction of killer T lymphocytes in allogeneic system, we explored the possibility of this hapten-reactive T-cell system to amplify tumor-specific killer T-lymphocyte activity in the syngeneic system. We utlized relatively weak immunogenic syngeneic plasmacytoma X5563 in C3H/He mice. Analysis of the TNP-reactive T-cell activities revealed that such T lymphocytes express the biological functions of both major subtypes of regulatory T cells, namely suppressors and helpers, and that TNP-reactive suppressor and helper T lymphocytes, respectively, differ in their relative susceptibility to specific inactivation by TNP conjugates of the nonimmunogenic D-amino acid copolymer, D-glutamic acid, and D-lysine (D-GL). By taking advantage of the relative susceptibility-difference to TNP-D-GL, selective inactivation of TNP-reactive suppressor T cells was induced by appropriate treatment with TNP-D-GL, and the generation of TNP-reactive helper T-cell activity was amplified. The supplement of augmented TNP-reactive helper T-cell activity to the system at the immunization with syngeneic X5563 with TNP-haptenation, resulted in a striking augmentation of induction of tumor-specific killer T-lymphocyte activity, and a considerable number of hosts survived after the challenge with lethal dose of viable tumor cells. Thus, appropriate manipulations designed to induce potent hapten-reactive helper T-lymphocytes provided the potential for a very effective mode of immunoprophylaxis against tumor.