Disparate cleavage of poly-(ADP-ribose)-polymerase (PARP) and a synthetic tetrapeptide, DEVD, by apoptotic cells.

In the present investigations, we have shown differential cleavage of cellular PARP and a caspase 3-selective synthetic tetrapeptide substrate, Z-DEVD-AFC or Ac-DEVD-AMC using a T lymphoblastoid cell line Jurkat, and its variant clone E6.1(J-E6). Anti-Fas antibody-mediated apoptosis resulted in DNA fragmentation and PARP cleavage in both Jurkat and J-E6 cells. However, unlike Jurkat, J-E6 cells did not cleave a synthetic tetrapeptide substrate efficiently. The failure to cleave the DEVD tetrapeptide by apoptotic J-E6 cells was not due to insufficient expression or processing of caspase 3 in J-E6 cells. Interestingly, when the J-E6 cells were transiently transfected with a cDNA encoding caspase 3, efficient cleavage of Z-DEVD-AFC was achieved. The observations that apoptotic J-E6 cells barely cleaved a synthetic DEVD tetrapeptide, but efficiently cleaved endogenous PARP, potentially at the most preferred DEVD site, suggest that active caspases may have disparate characteristics to recognize substrates presented in different context.

Receptor-mediated gene delivery to human peripheral blood mononuclear cells using anti-CD3 antibody coupled to polyethylenimine.

Gene transfer to primary cells, especially to lymphoid cells, using a nonviral delivery system has been very challenging. In the present studies, we have used a cationic polymer, polyethylenimine (PEI) coupled to an anti-CD3 antibody for achieving receptor-mediated gene delivery to human peripheral blood mononuclear cells (PBMC). Naive, unstimulated PBMC did not express transfected genes, whereas the transgenes were expressed efficiently in PHA activated PBMC. Transiently expressed gene products were detected maximally at 24 and 48 h following transfection. Gene expression was detected until 96 h with a gradual diminution in the signal after 48 h. Receptor-mediated gene delivery was successfully used for freshly isolated, as well as previously frozen lymphocyte samples. The transfections performed using ligands other than anti-CD3 were not as efficient as anti-CD3-PEI. These results suggest that in addition to receptor-mediated endocytosis, signaling subsequent to engagement of the CD3 receptor with anti-CD3-PEI appears to be important for the efficacy of anti-CD3-PEI mediated gene delivery.

Failure in expression of structurally altered (CYS164-->TYR) H-2Kb molecules is mitigated with high affinity peptide-ligand.

A C164Y somatic mutation in the H-2Kb class I molecule causes a disruption of the alpha 2 domain disulfide bond and results in a loss of H-2Kb cell surface expression by the 69.9.15 cell line. In vitro culture of the somatic cell variant at 30 degrees C induced weak, but reproducible, expression of the H-2Kb mutant molecule on the cell surface, which suggests that a temperature-sensitive mutation was contributing to the H-2Kb null phenotype. Based on the inherent structural instability of the mutant H-2Kb molecules synthesized by 69.9.15 cells, we sought to determine the ability of high affinity peptide-ligand to counteract the null expression of H-2Kb. Treatment of 69.9.15 cells was performed with acid-eluted cell-derived peptides, as well as synthetic H-2Kb-restricted peptides, ovalbumin (OVA) p257-264 (YSIINFEKL), and vesicular stomatitis virus-nuclear protein p52-59 (RGYVYQGL). Whereas the endogenous and vesicular stomatitis virus peptides were ineffective at inducing H-2Kb expression at either 37 degrees C or 30 degrees C, treatment with the OVA peptide at 30 degrees C gave rise to dose-dependent enhancement in H-2Kb expression, an effect that was independent of exogenous sources of bovine beta 2-microglobulin at the time of peptide treatment. By comparison, expression of H-2Kb remained unaltered when cells were treated with the OVA peptide at 37 degrees C, consistent with the temperature-sensitive expression of the mutant molecules. Decay of H-2Kb from the cell surface was similar for both 69.9.15 and RMA-S cells, an indication that binding of OVA p257-264 provided the same level of stability for class I molecules with either a cis-(69.9.15) or trans-acting (RMA-S) defect in heavy chain transport. These data provide novel evidence that transport-defective MHC class I molecules, similar in nature to those encoded by class I genes isolated from human genomic libraries, i.e., the 12.4 pseudogene with a polymorphism at amino acid position 164 (C-->F), are subject to high affinity peptide-induced stabilization which reverses the class I null phenotype.

MHC class I heavy chain-dependent expression of discontinuous antigenic epitopes on beta 2-microglobulinb is inducible with peptide-ligand.

Previously, we reported that expression of the murine beta 2-microglobulinb (beta 2mb) antigenic epitopes defined by the mAb S19.8 and 23 (SJL [beta 2ma] anti-B10.S beta 2mb]) was dependent upon association of beta 2m with MHC class I heavy chains. We have further explored the antigenic properties of beta 2m under circumstances requiring the induction of MHC class I surface expression with heavy chain-specific peptide-ligand. For the RMA-S cell line, which is class I surface null due to a defect in the TAP-2 peptide transporter, treatment with the H-2Kb-specific vesicular stomatitis virus-derived N p52-59 peptide resulted in the cell surface expression of the epitopes defined by the anti-H-2Kb mAb Y-3, as well as equally strong expression of the epitopes defined by the anti-beta 2mb mAb S19.8 and 23. Similarly, the FLU-NP p366-374 peptide induced H-2Db on the surface of RMA-S cells as determined by cytofluorometry with the mAb MKQ8; however, expression of the epitope defined by S19.8 was only partially recovered and no reactivity was observed for mAb 23. That the H-2Db heavy chain was assembled with beta 2mb on the cell surface was established from immunoprecipitation experiments with 125I-surface-radiolabeled RMA-S cells treated with FLU-NP p366-374; MKQ8 immunoprecipitated prominent heavy chain and beta 2m bands, whereas S19.8 and 23 isolated a weak beta 2m band (12-15% of that co-immunoprecipitated with MKQ8). These results are consistent with the observation that human beta 2m-deficient cells (designated FO-1) transfected with the B2mb allele were induced, in combination with the endogenous HLA class I heavy chains, to express the epitope defined by S19.8, but not mAb 23, whereas both were expressed when contransfection was performed with the H-2Kb gene. That the determinants recognized by S19.8 and 23 were formed by a discontinuous cluster of amino acids within beta 2m was established from experiments demonstrating that H-2Kb heavy chain assembled with a chimeric beta 2m molecule (comprising human beta 2m from 1-69 and mouse beta 2m from amino acid 70-99, including the polymorphic residue Ala 85) did not lead to expression of the S19.8 and 23 epitopes. The results of this study provide evidence that heavy chain polymorphism can affect the antigenic properties of beta 2m and offer insight into the basis by which CTL may react against beta 2mb when assembled with the H-2Kb molecule.

Replication of human cytomegalovirus in cells deficient in beta 2-microglobulin gene expression.

To study the roles of beta 2-microglobulin (beta 2-m) and major histocompatibility complex (MHC) class I expression in human cytomegalovirus (HCMV) infection, the ability of HCMV strain AD-169 to infect and replicate in a human melanoma cell line (FO-1), which is beta 2-m-deficient and cannot express MHC class I on its cell surface, was examined. Susceptibility of FO-1 cells was compared with human foreskin fibroblasts (HFF) and FO-1H cells (FO-1 cells that have been transfected with the human beta 2-m gene, restoring MHC I expression on the cell surface). As judged by the HCMV immediate early 1 (IE-1) antigen expression, HCMV was able to infect FO-1 cells, although somewhat less efficiently than HFF. However, the expression of HCMV late (L) antigen and the production of virus was significantly less for FO-1 cells than for HFF. Analysis of the FO-1H transfectants revealed that expression of IE-1 and L HCMV antigens was comparable to FO-1 cells, which lack MHC I. Treatment of FO-1 and FO-1H cells with sodium butyrate prior to inoculation did not alter the expression of MHC I in either cell type, but did increase susceptibility of both cell types to HCMV infection, as well as the expression of L antigens and production of virus. These studies indicate that HCMV infection of FO-1 cells is independent of beta 2-m and MHC class I expression.

Differential effect of human and mouse beta 2-microglobulin on the induction and the antigenic profile of endogenous HLA-A and -B antigens synthesized by beta 2-microglobulin gene-null FO-1 melanoma cells.

beta 2-Microglobulin (beta 2-mu) gene-null human melanoma FO-1 cells display lower reactivity with anti-HLA class I monoclonal antibodies (mAb) following transfection with a wild-type mouse beta 2-mu gene (referred to as FO-1C cells) than following transfection with a wild-type human beta 2-mu gene (referred to as FO-1H cells). Furthermore, binding assays with a panel of anti-HLA class I mAb detected higher reactivity of FO-1C cells with mAb TP25.99 than with mAb CR1-S63, CR10-215, CR11-115, TP67, and W6/32 but similar reactivity of FO-1H cells with all the mAb tested. While mAb TP25.99 recognizes a determinant expressed on beta 2-mu-free and beta 2-mu-associated HLA class I heavy chains, the remaining mAb recognize determinants expressed only on beta 2-mu-associated HLA class I heavy chains. The differential effects of mouse and human beta 2-mu on the reactivity with anti-HLA class I mAb of FO-1 cells reflect more than one mechanism. Besides abnormalities in the processing of HLA class I heavy chains associated with mouse beta 2-mu, this molecular complex appears to be unstable on the plasma membrane of FO-1 cells. To analyze the interaction of mouse beta 2-mu with HLA-A and -B antigens, the HLA phenotype of FO-1 cells was determined, using a combination of isoelectric focusing analysis of antigens immunoprecipitated from radiolabeled cells with mAb to monomorphic determinants of HLA class I antigens, binding assays with a limited number of mAb recognizing HLA class I allospecificities, and sequence-specific oligonucleotide probe typing. Although association with mouse beta 2-mu does not cause marked differences in the expression of HLA-A25 and -B8 antigens on the cell surface of FO-1 cells, it causes a selective reduction in the expression of determinants recognized by anti-HLA-A mAb F4/72 and VF19-LL67 and by anti-HLA class I mAb W6/32 on HLA-A25 allospecificities. The differential effect of the association with mouse beta 2-mu on the antigenic profile of HLA-A25 and -B8 antigens may reflect the different characteristics of the amino acids at residue 12, which interact with residue 33 of beta 2-mu. The latter residue is the only one to differ between human and mouse beta 2-mu in the stretch of amino acids interacting with the alpha 1 and alpha 2 domains of HLA class I heavy chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulated expression of the major histocompatibility complex class I genes.

The major histocompatibility complex (MHC) class I gene products are known to play a fundamental role in foreign antigen presentation to the cellular immune system. Much attention has been directed to the study of MHC class I gene expression as a means to understanding the processes by which MHC class I-mediated immune responses are regulated. Two areas of considerable interest have emerged, including regulation at the levels of transcriptional control and antigenic peptide-induced transport of MHC class I molecules. With an emphasis on these major areas of research, we review recent developments on the molecular and biochemical mediators and events of MHC class I gene regulation.

Assembly-dependent expression of antigenic epitopes by beta 2-microglobulin(b).

In this report we provide evidence for the expression of antigenic epitopes on mouse beta 2-microglobulin(b) (beta 2mb) that result from assembly with cognate H-2 class I heavy chains. For the cell line 69.9.15 (beta 2ma x beta 2mb), which expresses a mutant cytosolic form of H-2Kb and wild-type H-2Db, flow cytometry with rabbit antiserum against mouse beta 2m displayed beta 2m expression by cells grown in the presence or absence of fetal calf serum. By contrast, the epitopes identified by the beta 2mb-specific monoclonal antibody (mAb) S19.8 and clone 23 were not expressed by 69.9.15 cells grown in serum-containing conditions, and although S19.8 reactivity was weakly recovered by culture in the absence of serum, no such reactivity was observed with clone 23. Strong expression of these epitopes was achieved following transfection of 69.9.15 cells with the wild-type H-2Kb gene, indicating that the beta 2mb epitopes defined by mAb S19.8 and clone 23 were expressed when beta 2mb was assembled with an appropriate heavy chain. In support of this conclusion, we observed the recovery of the S19.8 and clone 23 epitopes by in vitro assembly of H-2Kb heavy chains with beta 2mb in the presence of the VSV N protein p52-59; however, such epitopes were expressed neither by beta 2mb prior to heterodimer assembly nor by non-conformed beta 2mb present in tissue culture supernatants recovered from H-2 class I surface positive cells. Taken together, these data indicate that in addition to the property of beta 2m to modify the antigenicity of the MHC class I heavy chains, beta 2m epitopes are induced in a reciprocal manner by assembly with MHC class I heavy chain molecules.

The role of beta-2 microglobulin in temperature-sensitive and interferon-gamma-induced exocytosis of HLA class I molecules.

The passage of MHC class I heavy chains through the exocytic pathway is promoted by association with beta 2 microglobulin (beta 2m). In order to analyze the structural basis of this phenomenon, processing and cell surface expression of HLA class I molecules have been investigated in the beta 2m null human melanoma cell line FO-1 transfected with either the human or mouse beta 2m genes. These natural structural variants of beta 2m display 30% amino acid sequence divergence. In comparison with a human beta 2m transfectant of the FO-1 cell line (designated FO-1H), FO-1 cells transfected with the mouse beta 2m gene (FO-1C) express HLA class I molecules that are processed with grossly altered kinetics and are present on the cell surface at reduced levels. The suboptimal expression of HLA class I heavy chains encoded by FO-1C cells reflects a defect in heavy chain stability since cell surface expression of HLA class I antigens was increased following incubation at 30 degrees C. The increased cell surface expression paralleled accelerated processing of HLA class I heavy chains by FO-1C cells. In contrast, no induction in either cell surface expression or processing of HLA class I heavy chains was observed for the beta 2m-negative FO-1 parent cell line, which remained HLA class I antigen null when cultured at 30 degrees C, or the FO-1H human beta 2m transfectant, which expressed equivalent levels of HLA class I antigens on the cell surface at 37 degrees C and 30 degrees C. Further up-regulation of the temperature-sensitive induction of HLA class I antigen expression was accomplished by treatment of the FO-1C transfectant with interferon-gamma; this latter effect appears to be active at a posttranscriptional step for FO-1 cells since IFN-gamma was not as potent a transcriptional activator at 30 degrees C as it was at 37 degrees C. These results indicate that HLA class I heavy chains expressed by FO-1C cells are subject to temperature-sensitive and cytokine-inducible stabilization that increases their affinity for the structural variant of beta 2m and promotes exocytosis of the HLA class I heterodimer to the cell surface. Furthermore, beta 2m non-conformed MHC class I heavy chains undergo stabilization that is not associated with enhanced cell surface expression, indicating that the exocytosis of putative "empty" HLA class I antigens is a process dependent upon association with beta 2m.

Natural killer and lymphokine activated killer cell functions in Hodgkin's disease.

We report the natural killer (NK) and lymphokine activated killer (LAK) cell activities in peripheral blood lymphocytes (PBL) from untreated patients with Hodgkin's disease (HD) and from healthy donors. The frequency of LAK cell precursors was also studied using limiting dilution analysis (LDA). About 75% of the HD patients had normal NK activity. There was a higher percentage of low NK responders (mean percent NK activity of healthy donors--2 SD) in patients with lymphocyte depletion histologic grade of the disease and those who were in clinical stage IV, suggesting a correlation of decrease in NK activity with poor prognosis. We found efficient LAK activity against the NK-sensitive K562 cells and NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumour targets in both low and normal NK responder HD patients, irrespective of the histopathological grade and clinical stage of the disease. In concordance with their good LAK cell activity, HD patients showed a frequency distribution of LAK cell progenitors in the PBL comparable to that of healthy donors.

Antibody dependent cellular cytotoxicity and complement mediated cytotoxicity on leukemic cells mediated by anti K562 monoclonal antibodies.

Three anti K562 monoclonal antibodies (MAb) 4.3, 4.6 and 4.8 reacting predominantly with cells of myeloid lineage, were tested for antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). MAb 4.6 (IgG3k) effectively mediated ADCC against K562 cells and fresh leukemic targets with effectors from healthy donors. However, for ADCC on chronic myeloid leukemia (CML) targets, effectors from CML patients in remission needed modulation with IL-2. All MAb showed significant CDC against peripheral blood (PB) and bone marrow (BM) cells obtained from CML patients in chronic phase, and untreated acute myeloid leukemia (AML) patients. MAb displayed no CDC against PB and BM cells from CML patients in remission and BM cells of Hodgkin's Disease (HD) patients with normal BM cellularity. In clonogenic assay, colony forming units (CFU) in the BM aspirate obtained from CML patients in chronic phase were significantly reduced by treatment with MAb and complement.

Natural killer and lymphokine-activated killer cell functions in chronic myeloid leukemia.

The natural killer (NK) and lymphokine-activated killer (LAK) cell activities of peripheral blood lymphocytes from chronic myeloid leukemia (CML) patients in remission and from healthy donors have been studied. Regression analysis to compare both cytotoxic responses in individual donors and the frequency of LAK cell precursors was also carried out. About 42% of CML patients in remission showed low NK activity (less than the mean percentage NK activity of healthy donors--2 SD) and were categorised as low NK responders. The stage of remission or the drugs used to bring about remission did not influence the NK status. The LAK activity of low NK as well as normal NK responder CML patients was significantly low against the NK-sensitive K562 cell line and the NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumor targets, as assessed by linear regression analysis. Allogeneic leukemic cells were more resistant to killing, especially by patients' LAK cells. The frequency analysis of LAK cell precursors revealed a significant reduction in the LAK cell progenitor frequency in CML patients in remission.

Immunoreactivity of anti K562 monoclonal antibodies with leukemic cells of myeloid lineage.

Three monoclonal antibodies (MAb) raised against K562 cells (erythroleukemic cell line) reacting specifically with myeloid maturation related antigens, were characterized. MAb 4.3 (IgG2bk) reacted with promyelocytes, metamyelocytes and myelocytes, MAb 4.6 (IgG3k) also identified the myeloid blasts, while MAb 4.8 (IgG1k) reacted with metamyelocytes. The affinity constant of the MAbs ranged from 0.8 X 10(7) -3.9 X 10(8) M-1 and the binding sites on K562 cells varied from 8 X 10(4)-4 X 10(5). In competition RIA MAbs 4.6 and 4.8 competed with each other for binding sites on K562 cells. In Western blot analysis the MAbs reacted with 66,000 mol.wt and 84,000 mol.wt peptides on K562 cells and 97,000 mol.wt peptide on chronic myeloid leukemia (CML) cells. These MAbs may help in immunophenotyping of myeloid leukemias.

Studies on natural killer cells in chronic myeloid leukemia patients in remission.

In our earlier studies, the natural killer (NK) cell mediated cytotoxicity was found to be impaired in 55% of chronic myeloid leukemia (CML) patients in remission (low NK responders), while the rest exhibited normal range of cytotoxicity (normal NK responders). In the present investigations probable factors contributing to the impaired NK activity of low NK responder CML patients have been analyzed. Peripheral blood lymphocytes (PBL) from both low and normal NK responder CML patients possessed normal percentages of HNK-1+, CD3+ and CD8+ cells. The proportion of HNK-1+ cells and CD8+ cells in low density fractions (LDF) and high density fractions (HDF) respectively of Percoll separated PBL from low and normal NK responder CML patients and healthy donors was comparable. However, the NK activity of LDF cells of low NK responder CML patients was much lower. Also, HDF cells from low NK responder CML patients exhibited a regulatory effect on NK cytotoxicity of PBL from healthy donors. Therefore, it is likely that the presence of suppressor cells and perhaps an intrinsic inability of HNK-1+ cells may together contribute to the impaired NK cytotoxicity of low NK responder CML patients.

Establishment and characterization of four new squamous cell carcinoma cell lines derived from oral tumors.

Four cell lines were established from squamous cell carcinomas (SCC) of the oral cavity. Cell lines AW 13516 and AW 8507 were derived from poorly differentiated SCC and epidermoid carcinoma of the tongue respectively. Cell line AW 10498 was derived from moderately differentiated SCC of the lower alveolus, and AW 9803 grew from a well-differentiated SCC of a retromolar trigone. The cultures showed typical epithelial cell morphology, numerous mitotic figures, occasional multinucleated giant cells, individual cell diskeratosis and nuclear and nucleolar abnormalities. The cell lines AW 13516 and AW 8507 were fast growers with a doubling time of 35.5 h and 31.9 h, respectively, which was independent of the initial seeding density. Cell lines AW 10498 (doubling time 52.2 h) and AW 9803 (doubling time 66 h) showed slower growth and had shorter doubling times at higher seeding densities. The presence of cytokeratins was detected in all the four cell lines by using polyclonal antikeratin antisera in indirect immunofluorescence and in Western blotting. None of the cell lines expressed major histocompatibility complex (MHC) class II antigens. MHC class I antigens were expressed by three cell lines but not by AW 9803. Flow cytometric analysis of DNA content and chromosomal studies suggested the presence of polyploidy and aneuploidy in all the four cell lines. Ultrastructural studies revealed typical epithelial cell features, such as the presence of desmosomes, tonofilaments and keratin bundles.

Monoclonal antibody against human squamous-cell-carcinoma-associated antigen.

Mouse monoclonal antibody (MAb) 3F8E3 (IgG3k) was developed against the head and neck cancer cell line LICR-LON-HN2. Subjected to indirect immunofluorescence, the MAb reacted exclusively with SCC cell lines and showed no reactivity with normal or transformed mouse and human non-SCC cell lines and hematopoietic cell lines. The radiolabelled MAb showed an affinity constant of 1.8 x 10(8) M-1 with HN2 cells and identified 2.07 x 10(4) sites/cell by Scatchard analysis. It identified 2 peptides from membrane extracts of HN2 cells by Western blotting. Avidin-biotin-complexed immunoperoxidase staining on cryostat sections of tumors from various tissues revealed that 3F8E3 reacted mainly with the membrane antigens of well differentiated SCC cells of oral cavity, larynx, esophagus, lung, uterine cervix, metastatic nodes of patients with oral cancer, and dysplastic cells in oral leukoplakia. The MAb did not react with poorly differentiated cells of Ca esophagus, adenocarcinoma of breast, stomach and colon, renal-cell carcinoma and soft-tissue sarcoma.

Immunoreactivity of lymphocytes from draining lymph nodes, peripheral blood and tumor infiltrates from oral cancer patients.

Lymphocytes from metastatic (met) and nonmetastatic (non-met) regional lymph nodes, LNL peripheral blood (PBL) and tumor infiltrating lymphocytes (TIL) of patients with squamous cell carcinoma of the oral cavity and healthy donors were investigated for CD3, CD4, CD8 and HNK-1 phenotypes, Natural Killer (NK) cell Activity, Antibody Dependent Cellular Cytotoxicity (ADCC) and proliferative response to mitogen (PHA). Modulation of NK cytotoxicity with recombinant Interferon alpha (IFN-alpha) was also investigated in some cases. Lymphocytes from met and non-met lymph nodes showed no variation in the percentages of CD3+, CD4+ and CD8+ cells, when compared with each other and with PBL of oral cancer patients. TIL showed significantly less proportion of CD3+ and CD4+ cells. The percentage of HNK-1+ cells was significantly lower in LNL and TIL when compared to PBL of oral cancer patients. The mitogen responses of met and non-met LNL were comparable to each other and better than that of PBL from the same patients, while, TIL showed significant impairment in mitogen responses. The NK cytotoxicity and ADCC of PBL from oral cancer patients were comparable to healthy donors which could be augmented by rIFN alpha. LNL and TIL showed almost negligible NK and ADCC activities and NK activity could not be modulated by rIFN alpha. The results thus demonstrate that in oral cancer patients, lymphocytes from three compartments viz. PBL, LNL and TIL showed differential effector functions. The metastatic status of LN did not affect the immunoreactivity of LNL.

Lymphokine-activated killer-cell function of lymphocytes from peripheral blood, regional lymph nodes and tumor tissues of patients with oral cancer.

Lymphokine-activated killer (LAK) cells, generated from peripheral blood lymphocytes (PBL) from patients with oral cancer or oral leukoplakia and from healthy donors showed comparable lysis of 6 target tumor cell lines, including 3 derived from head and neck and oral cancers. The tumor burden of the host did not appear to influence the systemic LAK activity. LAK activity of lymphocytes infiltrating the tumor tissues (TIL) was also comparable to that of the PBL. Both TIL and PBL showed a parallel increase in proportion of HNK-I+ and CD-25+ cells upon activation with IL-2. The lymph-node lymphocytes (LNL) from metastatic (met) and non-metastatic (non-met) draining lymph nodes, however, showed reduced LAK activity and an increase in CD8+ cells, in addition to CD25+ and HNK-I+ cells, when cultured with IL-2. When IL-2-activated LNL were co-cultured with autologous PBL during IL-2 activation of the latter, a strong suppressive effect was exerted by LNL. In contrast, IL-2-activated PBL did not suppress autologous LAK generation in spite of an increase in CD8+ cells seen after activation with IL-2. Frequency distribution of LAK precursors was significantly lower in LNL than in PBL from oral cancer patients. LAK precursor frequency in TIL was comparable to that of PBL. The results show that, in oral cancer, regional lymph nodes may not have adequate IL-2-inducible cytotoxic potential, due to a reduced number of LAK progenitors and possible activation of suppressor cells. Alternatively, TIL can be a potential source for LAK cell function.

Natural killer and lymphokine-activated killer cell-mediated cytotoxicity in patients with oral cancer.

Peripheral blood lymphocytes (PBL) from untreated and treated oral cancer patients, lymph node lymphocytes (LNL) from metastatic (met) and nonmetastatic (non-met) lymph nodes, and tumor infiltrating lymphocytes (TIL) were tested for natural killer (NK) and lymphokine activated killer (LAK) cell cytotoxicity using appropriate targets in a short-term chromium release assay. The results showed that while both NK and LAK functions of PBL from oral cancer patients were comparable to those of normal healthy donors, the NK activity of metastatic and nonmetastatic LNL and TIL was highly compromised. On the other hand, potent LAK activity could be generated from all three lymphoid populations. Individual patients showing low NK activity displayed good LAK cytotoxicity, indicating that endogenous cells with low NK potential have adequate ability to respond to interleukin 2 (IL-2). LAK activity tested on autologous tumour targets revealed that TIL were the best source of LAK cells. followed by PBL and LNL.