Daudi Tumor Cells Research Articles

Therapy of Small Subcutaneous B-Lymphoma Xenografts with Antibodies Conjugated to Radionuclides Emitting Low-Energy Electrons

Radionuclides emitting low-energy electrons (Auger and conversion electrons of <50 keV) are potentially useful for cancer therapy when conjugated to an antibody, because they can irradiate the cell to which they bind while producing relatively little irradiation of surrounding cells and tissues. We showed previously the ability of such antibody conjugates to treat micrometastatic, disseminated human B-lymphoma in a severe combined immunodeficient mouse model using an anti-CD74 antibody. In this study, we have evaluated the ability of such conjugates to treat s.c. tumors. Severe combined immunodeficient mice were injected s.c. with Raji, Daudi, or RL B-lymphoma human tumor cells. Antibodies to CD74, CD20, or HLA-DR were radiolabeled with (111)In or (125)I and injected i.v. at various times starting at day 5, and tumor growth was monitored. Controls included the testing of unlabeled antibodies, labeled nonreactive antibodies, and a combination of the two. Therapy of s.c. B-lymphoma was more difficult than therapy of tumor cells that had been injected i.v. Although large, macroscopic tumors were not effectively treated, therapy was effective on s.c. Daudi tumors on day 36 after injection of this slowly growing tumor, with an (111)In anti-CD74 antibody given in two doses. An anti-CD20 antibody labeled with either (111)In or (125)I was able to effectively treat s.c. RL tumors when given as late as day 16 after tumor inoculation. The largest tumors that were effectively treated were macroscopic thin discs (<2 mm in diameter) growing on the mesentery. These results extend previous evidence that antibody conjugates with emitters of low-energy electrons can be effective therapeutic agents for micrometastatic cancer.

Antitumor Activity of Expanded Human Tumor-Infiltrating γδ T Lymphocytes

Background: The objective of this study was to investigate the antitumor activity of selectively expanded γδ T cells in tumor-infiltrating lymphocytes (γδTILs) or tumor ascites lymphocytes (γδTALs) from patients with colorectal and ovarian epithelial carcinoma (OEC) in vitro and in vivo. Methods: γδTILs/TALs were expanded by the solid-phase antibody method; their cytolytic and proliferative activities in vitro were detected by the MTT method and ³H-TdR incorporation and their effect in vivo was evaluated by the nude mice model. Results: Expanded γδTILs from colorectal tumors demonstrated marked cytotoxicities to allogeneic human colon adenocarcinoma HR8348 and lymphoma Daudi cells, as well as xenogeneic murine thymoma EL-4 cell lines. Cytokines, including IL-2, IL-4, IL-12, IL-15, TNF-α and INF-γ, could promote the cytotoxicities of γδTILs to tumor cells, whereas IL-10, GM-CSF and TFG-β had no effect on such killing activities. Rested γδTILs could proliferate strongly in response to mitomycin C-treated Daudi and EL-4 tumor cells, but not to HR8348 tumor cells, suggesting that the latter might possess only cytotoxicity-related antigen recognized by γδTILs. Either αβTILs or γδTILs from patients with OEC displayed cytotoxicities to allogeneic or autologous OEC cell lines at a similar strength in vitro. Transferring γδTILs into Daudi cell-bearing BALB/c nude mice with an injection of IL-2 was able to maintain a high survival rate of the mice for 30 days, when compared with mice treated with αβTILs or without any treatment (p < 0.05). Without coinjection of IL-2, after 3 months of Daudi tumor inoculation, a high survival rate was observed in γδTIL-treated mice. Similarly, adoptive γδTALs from the ascites of patients with OEC transferred into nude mice displayed a stronger antitumor response to OEC SKOV3 cells than αβTALs in vivo. Tumor volumes in γδTAL-treated mice were smaller than in αβTAL-treated or non-TAL-treated mice within the period from day 23 to day 50 after tumor inoculation (p < 0.05). Fifty days after SKOV3 tumor inoculation, a decreasing trend of carcinogenic rate was observed in γδTAL-treated nude mice. Conclusion: Taken together, our results suggest that γδT cells could be a new candidate for adoptive immunotherapy in the future treatment of patients with cancer.

Human gamma delta T cells recognize heat shock protein-60 on oral tumor cells.

In the present investigations, gammadelta T cells were isolated from the peripheral blood of oral cancer patients and analyzed for their immunophenotype and cytotoxic potential. Flow-cytometric analysis revealed a dominant population expressing Vgamma9 and Vdelta2 T-cell receptors. In a 4-hr 51Cr-release assay, activated gammadelta T cells showed specific cytotoxicity against Daudi Burkitt's lymphoma cells and fresh oral tumor cells. Cold target competition assays demonstrated that gammadelta T cells recognize a common ligand on Daudi and oral tumor cells. Expression of heat shock protein 60 (hsp60) molecules was detected on the surface of Daudi as well as oral tumor cells by flow cytometry and immunoprecipitation of surface biotinylated cells by anti-hsp60 monoclonal antibody (MAb). Such MAbs brought about a significant inhibition of cytotoxicity of gammadelta T cells against Daudi and oral tumor cells. The results suggest that gammadelta T cells isolated from the peripheral blood of oral cancer patients have the ability to lyse oral tumor cells. The lysis of oral tumor cells occurs via recognition of hsp60 on the surface of oral tumor cells.

Restoration of the immunocompetence by IL-2 activation and TCR-CD3 engagement of the in vivo anergized tumor-specific CTL from lung cancer patients.

The present study investigates the nature of the immunosuppressed state of the lymphocytes obtained from the malignant pleural effusion (effusion associated lymphocytes, EAL) of lung cancer patients. The immunocompetence of EAL from 13 patients was assessed by determining their T-helper cell phenotype, proliferative response to alpha CD3-activation, and their cytolytic activity against three tumor targets: the autologous tumor, Daudi, and K562. Flow cytometry analysis showed that the lymphocytes in EAL were predominantly T cells with < 1% natural killer cells. The T-helper cell phenotype was found to be predominantly of Th2 type, but could be readily converted to Th1 type by culturing the EAL in vitro, and this conversion was augmented by interleukin-2 (IL-2) or IL-2 plus alpha CD3. To test the cytolytic activity of EAL, it was found that after 6-day culturing, the EAL remained in an immunosuppressed state so that they failed to kill any of the three tumor targets. Stimulation with IL-2 partially restored the immunocompetence of EAL. Further engagement of TCR-CD3 by alpha CD3 fully restored the cytolytic activity of the EAL to kill the autologous tumor target but not Daudi or K562 tumor cells, and thus seemed to be tumor specific. The specificity was further confirmed by testing the activated EAL and normal donor peripheral blood lymphocytes against a variety of tumor targets and control targets. Furthermore, the killing by EAL against the autologous tumor target seemed to be major histocompatibility complex-restricted and was inhibited by anti-human leukocyte antigen class I antibody. The EAL from lung cancer patients also showed much reduced responsiveness to the alpha CD3 stimulation to induce proliferation, and addition of IL-2 restored the responsiveness. These results suggest that, through close contact with tumor cells, anergy of cytotoxic T lymphocytes (CTLs) was induced in vivo at a localized site. IL-2 stimulation and TCR-CD3 engagement could reverse the anergic state and restored the full competence of CTLs in EAL to mediate the specific anti-tumor killing against the autologous tumor. Proper manipulation of EAL may prove useful as a source of anti-tumor effectors for cancer adoptive immunotherapy.

Correlation of Class I MHC Antigen Levels on Some Human Tumor Cell Lines with Susceptibility to LAK Cells and Performance in Cold Target Inhibition Assays

Relationships between (a) levels of class I MHC antigens, (b) susceptibility to LAK cells, and (c) ability to perform in cold target competition assays were studied using K562, MOLT-4, Raji, Daudi, and HR7 human tumor cell lines. The hypothesis of inverse correlation between class I MHC antigen expression and susceptibility to LAK cells was concurrent with results using K562, HR7, and Daudi cell lines. For Raji cells, IFN-γ had no effect on the levels of class I MHC antigen but induced resistance to LAK cells, whereas for MOLT-4 cells, enhanced MHC I expression was not accompanied with any change in susceptibility to LAK cells. In competition experiments, all five tumor cell lines were used as competitors as well as targets. Different specificities of competition were observed for different tumor cells. These results indicated that the different tumor cell lines could share one or more target structures recognized by LAK effector cells. IFN-γ treatment lowered the ability of several tumor cell lines to inhibit target lysis in cold competition assays. Changes in the competition efficacy of tumor cells correlated better with their susceptibility to LAK cells rather than levels of class I MHC antigens.

Bispecific anti-CD22/anti-CD3-ricin A chain immunotoxin is cytotoxic to Daudi lymphoma cells but not T cells in vitro and shows both A-chain-mediated and LAK-T-mediated killing.

We have generated a bispecific anti-CD22/anti-CD3 immunotoxin (IT) and determined whether it would exert both lymphokine-activated killer (LAK) T cell-mediated and ricin A chain (dgA)-mediated toxicity to Daudi tumor cells but not to T cells in vitro. One parental IT, Fab'-anti-CD22-dgA makes a potent immunotoxin for B cells, but not T cells, while the other, Fab'-anti-CD3-dgA, kills neither T nor B cells. Three mouse quadromas were generated and the bispecific Abs (BsAbs) were purified by double affinity chromatography. Two of the three purified BsAbs induced significant proliferation and IL-2 production in T cells. All three BsAbs induced LAK-T cell-mediated specific lysis of CD22+ Daudi cells. Two of the purified Ab were conjugated to dgA. Using a 51Cr release assay in the presence of LAK-T cells and Daudi target cells, the IC50s of the BsAbs were 3.5 x 10(-10) M and 9 x 10(-11) M, as compared to 2.1 x 10(-11) M and 3.2 x 10(-11) M, for their respective ITs. Hence, in the presence of LAK-T cells, the BsITs were 3- to 17-fold more cytotoxic than unconjugated BsAbs in 51Cr-release assays. Daudi cells were also treated in vitro with different mixtures of LAK-T cells, BsAbs, and BsITs and then adoptively transferred into SCID mice. As determined by the mean paralysis time of the recipients, in the presence of LAK-T cells the BsITs had impressive anti-tumor activity.

Lymphokine-Activated Killer Cell Activity in Lung Cancer

This study evaluates local pulmonary immune effector cell lytic activity. Purified lymphocyte populations were isolated from BALF obtained from 18 patients with bronchogenic carcinoma, six patients with lung disorders other than cancer, and ten normal control volunteers matched for age and smoking history. These cells were evaluated for NK and LAK cell lytic activity against NK-resistant LAK-sensitive tumor targets (A549 pulmonary tumor and Daudi tumor cells) and an NK-sensitive tumor (K562); LAK activity was detected in BALF from 6 of the 18 patients with cancer. The remaining patients with cancer, the subjects with pulmonary disease other than cancer, and the normal volunteers had no detectable lytic activity. Peripheral blood lymphocytes from all subjects had only NK lytic activity and did not kill the pulmonary tumor target; AMs were not tumoricidal. Interleukin-2, which is required for LAK cell activation, was detected only in BALF recovered from the six patients with pulmonary LAK lytic activity. These results demonstrate that activated LAK cells, capable of killing pulmonary tumor cells, are present in BALF of some patients with bronchogenic carcinoma. This lytic LAK cell population represents a local pulmonary response against the lung cancer in the absence of systemic tumoricidal activity. The functional status of pulmonary immune effector cells, as well as the type and quantities of cytokines in the lung determine local responsiveness to bronchogenic carcinoma and may well control the course of this disease.

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.

Selective proliferation of natural killer cells among monocyte-depleted peripheral blood mononuclear cells as a result of stimulation with staphylococcal enterotoxin B.

In vitro stimulation of monocyte-depleted peripheral blood mononuclear cells with staphylococcal enterotoxin B (SEB) resulted in selective proliferation of cells which express the phenotypic and functional characteristics of natural killer (NK) cells. This culture system provides an easy method for obtaining highly purified NK cells, by sequential incubation of monocyte-depleted cells with SEB and then with interleukin-2 (IL-2). After culture for 4 to 5 days in the presence of SEB, 98 to 100% of the cells expressed the CD16 (Leu11) and HNK-1 (Leu19) antigens. This purification occurred through the death of lymphocytes lacking NK cell markers and marked proliferation of NK cells themselves, which leads to an enrichment of the NK cell population. Activation of NK cells was detected by the appearance of the gamma interferon receptor and IL-2 receptor antigens. This homogeneous population showed the morphology of large granular lymphocytes, were potent effectors of cell-mediated cytotoxicity against K562 and Daudi tumor cell lines, and were able to kill gram-positive and gram-negative bacteria. IL-2 was necessary to maintain the activation and proliferation after SEB stimulation for 4 days. Moreover, the maximum frequency of binding to K562 cells (60.6%) was similar to that recently found (58 +/- 3%) (P. Garcia-Peñarrubia, F. T. Koster, and A. D. Bankhurst, J. Immunol. Methods 118:199-208, 1989) with fresh and highly purified NK cells. This method can be used as a source of highly purified NK cells to study their functional properties and applications to the treatment of cancer.

Ti (WT31)-Negative, CD3-Positive, Large Granular Lymphocyte Leukemia With Nonspecific Cytotoxicity

Abstract A case of WT31-, CD3+ large granular lymphocyte leukemia is reported. On surface marker analysis, the proliferating cells were found to be CD3+4–8–16+ and WT31-. By two-color immunofluorescence staining, CD3+4- 8- cells were found to be WT31-, and a small population of WT31+ cells expressed either CD4 or CD8. WT31-, CD3+ cells were also identified in a bulk culture of lymphocytes expanded in vitro. Because WT31 monoclonal antibody (MoAb) reacts with the nonpolymorphic epitope of the disulfide-linked heterodimer of the T cell antigen receptor (Ti), the absence of the WT31-reactive Ti determinant may represent an expression of different CD3-associated polypeptides. The rearrangement of the Ti-beta and Ti-gamma genes but not the immunoglobulin gene was demonstrated, and the single pattern of rearrangement indicated the monoclonal origin of the lymphocytes. When the lymphocytes were assayed for their cytotoxicity against K562, MOLT-4, Daudi, and Raji tumor cell lines, a broad spectrum of cytotoxicity for these tumor cells was observed, and the lymphocytes also exhibited antibody- and lectin- dependent cellular cytotoxicity and lymphokine-activated killer activity. Treatment with anti-CD2 and anti-CD3 MoAbs inhibited their nonspecific cytotoxicity. The anti-CD3-mediated inhibition of nonspecific cytotoxicity suggested that an as yet unidentified Ti, present in association with the CD3 molecule on these lymphocytes, serves as a specific receptor for target tumor cell recognition.

Ti (WT31)-negative, CD3-positive, large granular lymphocyte leukemia with nonspecific cytotoxicity

A case of WT31-, CD3+ large granular lymphocyte leukemia is reported. On surface marker analysis, the proliferating cells were found to be CD3+4-8-16+ and WT31-. By two-color immunofluorescence staining, CD3+4-8- cells were found to be WT31-, and a small population of WT31+ cells expressed either CD4 or CD8. WT31-, CD3+ cells were also identified in a bulk culture of lymphocytes expanded in vitro. Because WT31 monoclonal antibody (MoAb) reacts with the nonpolymorphic epitope of the disulfide-linked heterodimer of the T cell antigen receptor (Ti), the absence of the WT31-reactive Ti determinant may represent an expression of different CD3-associated polypeptides. The rearrangement of the Ti-beta and Ti-gamma genes but not the immunoglobulin gene was demonstrated, and the single pattern of rearrangement indicated the monoclonal origin of the lymphocytes. When the lymphocytes were assayed for their cytotoxicity against K562, MOLT-4, Daudi, and Raji tumor cell lines, a broad spectrum of cytotoxicity for these tumor cells was observed, and the lymphocytes also exhibited antibody- and lectin-dependent cellular cytotoxicity and lymphokine-activated killer activity. Treatment with anti-CD2 and anti-CD3 MoAbs inhibited their nonspecific cytotoxicity. The anti-CD3-mediated inhibition of nonspecific cytotoxicity suggested that an as yet unidentified Ti, present in association with the CD3 molecule on these lymphocytes, serves as a specific receptor for target tumor cell recognition.

Selective reduction of c-myc mRNA in Daudi cells by human beta interferon.

Under normal growth conditions, the human lymphoblastoid cell line Daudi expresses high levels of c-myc mRNA. These cells are also sensitive to growth inhibition by interferons. We have compared the levels of mRNA for the c-myc in untreated and human beta interferon (IFN-beta)-treated Daudi cells by RNA dot-blot and blot-hybridization analysis methods. Using a synthetic oligonucleotide complementary to the human c-myc mRNA as the probe, we detected a more than 75% reduction in the c-myc hybridizable poly(A)+ RNA in the IFN-beta-treated cells. This reduction in the c-myc mRNA appears to be selective because the level of actin mRNA is not significantly affected by the IFN-beta treatment. In addition, neither in vitro translation of mRNA extracted from IFN-beta-treated cells nor in vivo synthesis of cellular proteins in IFN-beta-treated cells are quantitatively affected. We surmise that the selective reduction in the amount of c-myc mRNA in IFN-beta-treated Daudi cells may be related to the IFN-induced inhibition of the Daudi tumor cell growth.

Anomalous killer cells: Thymus cell dependency, precursor frequency, and response to immunosuppressive therapy

Human peripheral blood lymphocytes (HPBL) cultured with the Daudi tumor cell line develop into nonspecific cytotoxic cells. These cytotoxic cells, anomalous killer (AK) cells, are dependent upon thymus-derived cells and monocytes in the generation phase and bear serologically determined T-cell antigens in the effector cell stage. Precursor frequency determinations of these killer cells range from 1 166 to 1 689 in untreated patients and drop considerably after treatment with fractionated X irradiation or cytoreductive therapy. Lymphokine (LK)-containing supernatants partially restore cytotoxic activity of AK cells after X irradiation, indicating a second signal requirement for precursor differentiation into lytic effector cells.