Cytotoxicity Of Peripheral Blood Lymphocytes Research Articles

Pinus roxburghii constituents augment human lymphocytes mediated cytotoxicity towards cancer cells.

Background: Pinus roxburghii has been used in the Himalayan region as folkloric remedy while nothing is yet known about its immunomodulatory potential. Methods: The crude extracts of green and fallen pine needles were subjected to sequential fractionation to get partially purified fractions. Human peripheral blood lymphocytes (PBL) were used to analyze immunoenhancing potential of these fractions. Microcytotoxicity assay was employed to investigate improved cancer cells killing capabilities of PBL against various cancer cell lines. GC-MS was carried out to identify the major compounds in the bioactive fractions. Results: The lymphocyte proliferation assay depicted the immunoenhancing potential of extracts and fractions of fallen and green needles of P. roxburghii. The ethyl acetate fractions of both fallen and green needles displayed highest mitogenic activity on human PBL. Both fractions heightened the expression of cell surface markers (CD3, CD8, and CD56) and significantly increased the production of cytokines (IL-2, IFN-γ and TNF-α). The enhanced intracellular granulysin (immunomarker for activated CTLs and NK cells) expression also confirmed immune stimulatory potential of these fractions on human lymphocytes. The ethyl acetate fractions of pine needles enhanced the cytotoxicity of PBL towards various cancer cells (HCT-116, HeLa, PC-3 and A549) as compared to untreated PBL. GC-MS analysis showed presence of major compounds like 3-α-mannobiose, octakis (trimethylsilyl) ether, methyloxime in ethyl acetate fraction of the green needles and cyclodecasiloxane, eicosamethyl in ethyl acetate fraction of the fallen needles. Conclusion: The bioactive fractions of the fallen and green needles of P. roxburghii stimulate cancer cells killing potential of human lymphocytes.

Rhein enhances the cytotoxicity of effector lymphocytes in colon cancer under hypoxic conditions.

The immunosuppressive tumor microenvironment limits the application of adoptive immunotherapy for solid tumors. Hypoxia is closely associated with the formation of the immunosuppressive tumor microenvironment. Hypoxia-inducible factor-1 (HIF-1) is an oxygen-sensitive transcriptional activator that drives the transcription of several immunosuppressive molecules. In addition, previous studies confirmed that rhein downregulated the expression of HIF-1α, a subunit of HIF-1, in pancreatic cancer cells. The present study established correlations between mRNA expression levels of HIF-1α and six immunosuppressive molecules in colorectal cancer (CRC) tissue samples. This study examined the effect of rhein on the expression levels of HIF-1α and six immunosuppressive molecules in CRC cell lines under hypoxic conditions by western blot analysis and reverse transcription-quantitative polymerase chain reaction. This study demonstrated that rhein downregulated the expression of HIF-1α and immunosuppressive molecules in CRC cells under hypoxic conditions. In addition, the present study analyzed the cytotoxicity of peripheral blood lymphocytes in vitro using a non-toxic cytotoxicity assay. This study demonstrated that in vitro, rhein enhanced the cytotoxicity of effector lymphocytes toward tumor cells under hypoxic conditions, and therefore rhein may be used in combination with effector lymphocytes for the treatment of CRC.

Effects of dietary conjugated linoleic acid on cytotoxicity of peripheral blood lymphocytes in piglets

The aim of this study was to investigate the effects of dietary conjugated linoleic acid (CLA) on the cytotoxicity of peripheral blood lymphocytes in piglets. A total of forty 21-d-old Duroc × Large White × Landrace weaned piglets were randomly allocated into two treatment groups and fed with control diet or 2% CLA diet for 6 weeks. Dietary CLA significantly increased the number of peripheral blood CD8+ T lymphocytes (P < 0.05). Compared with the control group, cytotoxicity, perforin, and granzyme B mRNA expression of peripheral blood lymphocytes in piglets from the CLA-supplementation group increased by 29.98% (P < 0.01), 32.87% (P < 0.01), and 43.36% (P < 0.05), respectively. The piglets on the CLA diet had higher mRNA expression of Lck and Fyn in peripheral blood lymphocytes (P < 0.05). The content of C16:0 significantly increased in piglets from the dietary CLA-supplementation group (P < 0.05), whereas the content of C18:1 significantly decreased (P < 0.05). The CLA content of peripheral blood lymphocytes in piglets in the CLA diet group ranged up to 0.24% (P < 0.01). These results suggest that dietary CLA enhanced cytotoxicity of peripheral blood lymphocytes perhaps through changing its fatty acid composition.

Effects of dietary conjugated linoleic acid on cytotoxicity of peripheral blood lymphocytes in piglets

Effects of dietary conjugated linoleic acid on cytotoxicity of peripheral blood lymphocytes in piglets

Combination effects of chronic cadmium exposure and gamma-irradiation on the genotoxicity and cytotoxicity of peripheral blood lymphocytes and bone marrow cells in rats

This work investigated the effects of chronic cadmium (Cd) exposure combined with γ-ray irradiation on the cytotoxicity and genotoxicity of peripheral blood cells and bone marrow cells in rats. Results showed that when the rats were exposed to low dose (LD) Cd of 0.1mg CdCl2/(kgd) for 8 and 12 weeks, the Cd concentration in blood reached to 135–140μg/L and no toxic effects on peripheral blood lymphocytes, white blood cells (WBC) and granulocyte–monocyte (GM) progenitor cells were observed except polychromatic erythrocytes (PCE) of bone marrow. Moreover, this chronic LD Cd exposure significantly decreased irradiation-induced micronucleus (MN) formation and hypoxanthine–guanine phosphoribosyl transferase (hprt) mutation in lymphocytes and PCE, while the combination of LD Cd exposure and irradiation induced the additive metallothionein (MT) protein expression in bone marrow cells. When the rats were exposed to a high dose (HD) Cd of 0.5mg CdCl2/(kgd) for 8 and 12 weeks, the blood Cd level approached to 458–613μg/L and an inflammatory response was induced, meanwhile, MN formation and hprt mutation were markedly increased, and the ratio of PCE/NCE (normochromatic erythrocyte) was significantly decreased. Furthermore, when the rats were exposed to HD Cd plus 2Gy irradiation, additive toxic effects on MN formation, hprt mutation, PCE damage and GM progenitor cell proliferation were observed, while this combination treatment resulted in an obvious reduction of MT protein compared to HD Cd group. In conclusion, chronic exposure to LD Cd induced the adaptive response to irradiation in the genotoxicity of peripheral blood lymphocytes and PCE of bone marrow by the up-regulation of Cd-induced MT protein, but the combination of HD Cd exposure and irradiation generated the additive effects on the cytotoxicity and genotoxicity in peripheral blood lymphocytes and bone marrow cells.

Analysis of granulysin-mediated cytotoxicity in peripheral blood of patients with psoriatic arthritis

The objective of the present study was to investigate possible changes in granulysin (GNLY)-mediated cytotoxicity of peripheral blood lymphocytes in psoriatic arthritis (PsA) patients with respect to different phases of the disease. We prospectively enrolled 25 PsA patients in the active phase, 26 PsA patients in remission and 24 healthy controls. The simultaneous detection of intracellular GNLY and cell surface antigens (CD3 and CD56) was performed with flow cytometry. GNLY apoptotic protein was visualised by immunocytochemistry. Natural killer (NK) cell cytotoxicity was analysed with a cytotoxicity assay against human erythroleukaemia K-562 cells. The percentage of GNLY(+) cells did not differ significantly between PsA patients in the acute phase and those in remission; however, it was always higher than in healthy examinees due to the increased percentage of GNLY(+) cells within T cells, NKT cells, and both, and in the CD56(+dim) and CD56(+bright) NK subsets. The mean fluorescence intensity for GNLY was higher in all lymphocyte subpopulations in the acute phase than in remission and in healthy controls. Accordingly, GNLY-mediated NK cell cytotoxicity against K-562 cells of active phase PsA patients was significantly higher than that in patients in remission or in healthy controls. These findings demonstrated the involvement of GNLY in the worsening of PsA and suggested that GNLY mediated the development of joint lesions.

Titanium dioxide nanoparticles trigger p53‐mediated damage response in peripheral blood lymphocytes

Titanium dioxide nanoparticles (nano-TiO2) are widely used as a photocatalyst in air and water remediation. These nanoparticles are known to induce toxicity; however, their cytotoxic mechanism is not fully understood. In this study, we investigated the underlying mechanism of nano-TiO2-induced cytotoxicity in peripheral blood lymphocytes. We examined the genotoxic effects of nano-TiO2 in lymphocytes using alkaline single-cell gel electrophoresis (Comet) and cytokinesis-block micronucleus (CBMN) assays. Lymphocytes treated with nano-TiO2 showed significantly increased micronucleus formation and DNA breakage. Western-blot analysis to identify proteins involved in the p53-mediated response to DNA damage revealed the accumulation of p53 and activation of DNA damage checkpoint kinases in nano-TiO2-treated lymphocytes. However, p21 and bax, downstream targets of p53, were not affected, indicating that nano-TiO2 does not stimulate transactivational activity of p53. The generation of reactive oxygen species (ROS) in nano-TiO2-treated cells was also observed, andN-acetylcysteine (NAC) supplementation inhibited the level of nano-TiO2-induced DNA damage. Given that ROS-induced DNA damage leads to p53 activation in the DNA damage response, our results suggest that nano-TiO2 induces ROS generation in lymphocytes, thereby activating p53-mediated DNA damage checkpoint signals.

Natural cell-mediated cytotoxicity of peripheral blood lymphocytes against target cells transfected with epidermodysplasia verruciformis-specific human papillomavirus type 8 L1 DNA sequences

The aim of the present study was to characterise natural cell-mediated cytotoxicity against COS-7 cells transfected with potentially oncogenic HPV-8 L1 DNA sequences cloned in sense and antisense orientation and to evaluate their lysis by peripheral blood lymphocytes (PBL) from patients with epidermodysplasia verruciformis (EV), a rare disease associated with life-long infection by specific HPV types. COS-7 cells were transfected with HPV-8 Hinc II restriction fragment (nucleotide positions 5434-7654) cloned in sense (COS-L1S) and antisense (COS-L1A) orientation into pCB6 expression vector. Cytotoxic activity of isolated PBL against COS cell lines as well as K562 erythroleukaemic cells was evaluated by 51Cr-release assay. We found that lymphocytes responsible for natural lysis of COS and K562 cells are CD3-negative CD56-positive natural killer (NK) cells. Analysis of NK cell cytotoxic activity against different COS cell lines has revealed that lymphocytes from healthy subjects killed COS-L1S cells significantly more efficiently than wild COS-7 and COS-L1A cells. Significantly more efficient lysis of COS-L1S cells was also observed in EV patients. Thus, expression of HPV L1 renders target cells more susceptible to NK-mediated cytotoxicity that may enable more effective elimination of transformed cells.

Interleukin-12 augments cytolytic activity of peripheral and decidual lymphocytes against choriocarcinoma cell lines and primary culture human placental trophoblasts.

Human trophoblasts are tolerant to the maternal immune system, but susceptible to interleukin (IL)-2-activated lymphocytes. IL-12 is also a key cytokine in the induction of cytotoxic responses. We administered IL-12 to peripheral blood lymphocytes (PBLs) and to decidual lymphocytes (DLs) and studied resulting cytotoxicity against trophoblasts. PBLs and DLs were stimulated with rIL-2 and/or rIL-12 for 48 hr in vitro. Cytotoxicity against the choriocarcinoma cell line JEG-3, JAR, and primary culture trophoblasts were examined by LDH release assay. The proliferative response was estimated by MTT assay. Expression of cytotoxic factors was studied by reverse transcriptase-polymerase chain reaction (RT-PCR). Whereas IL-12 alone produced a modest enhancement in cytotoxicity of PBLs and DLs, the combination of IL-2 and IL-12 was most effective in trophoblast cell lysis. IL-12 enhanced the mRNA expression of T-cell specific serine protease (TSP, granzyme B) and FasL in DLs, but the expression of perforin was unchanged. Expression of these cytotoxic factors in PBLs was up-regulated by IL-12. Our findings indicate critical roles of IL-12 in the activation of maternal lymphocytes, which could possibly result in pregnancy failure syndromes.

Enhancement of cytolytic activity of human peripheral blood lymphocytes by sodium periodate (IO4) possible involvement of protein kinase C

Sodium periodate (IO 4) exerts a number of biological effects including the enhancement of lymphocyte activation. In this study, we investigated its effects on cytotoxicity of human peripheral blood lymphocytes (PBL) and explored the mechanism whereby it exerted these effects. In vitro treatment of human PBL with IO 4 augmented their cytotoxicity against K562 myelogenous leukemia cells. IO 4 oxidative treatment increased the frequency of effector-to-target cell binding. It also increased cellular ATP levels in effector cells, suggesting that the post-binding cytolytic functions of these cells were also enhanced after treatment with IO 4. Moreover, IO 4 treatment significantly increased the protein kinase C (PKC) activity of effector cells and induced the translocation of activity in the membrane fraction from the cytosol. H-7, a potent PKC inhibitor, significantly reduced this enhancement of membrane-associated PKC activity at 10 μM and significantly reduced the enhanced cytotoxicity of PBL at the same concentration. These results indicated that IO 4 enhanced the binding capacity and post-binding cytolytic functions of PBL and that PKC activation was one mechanism to explain the IO 4-induced cellular activation.

Dietary supplementation with L-arginine: Modulation of tumour-infiltrating lymphocytes in patients with colorectal cancer

Dietary supplementation with L-arginine enhances natural cytotoxicity in peripheral blood lymphocytes but its effect on infiltrating lymphoreticular cells within a tumour microenvironment is unknown. The effects of dietary supplementation with L-arginine on tumour-infiltrating lymphocytes (TILs) in patients with colorectal cancer were evaluated. Eighteen patients received either a standard hospital diet (controls) or a standard diet supplemented with 30 g per day of L-arginine for 3 days before surgery. Tumour biopsies were taken at surgery and lymphocyte subsets (CD antigens) and macrophages examined immunohistochemically. Tumours from patients receiving L-arginine contained increased numbers of specific cell subsets within the tumour which expressed CD16 (P = 0.004) and CD56 (P = 0.001) surface markers, when compared with tumours from control patients. There were no differences in the total number of T and B cells, T helper and T suppressor cells. Dietary supplementation with L-arginine significantly alter the spectrum of TILs in human colorectal cancers in vivo. These findings have important implications for new strategies in anticancer treatment.

Limited antitumor T cell response in melanoma patients vaccinated with interleukin-2 gene-transduced allogeneic melanoma cells.

We have immunized advanced melanoma patients with a HLA-A2-compatible human melanoma line genetically modified to release interleukin-2 (IL-2), to elicit or increase a T cell-mediated anti-melanoma response that may affect distant lesions. Twelve stage-IV patients were injected subcutaneously at days 1, 13, 26, and 55 with IL-2 gene-transduced and irradiated melanoma cells at doses of 5 or 15 x 10(7) cells. Both local and systemic toxicities were mild, consisting of transient erythema at the vaccination site; fever occurred in a minority of patients. Three mixed responses were recorded. Seven patients were evaluable for immunological studies. Mixed tumor-lymphocyte cultures carried out with different allogeneic HLA-A2-matched melanoma lines as stimulators and targets revealed an increase in the MHC-unrestricted, but no changes in the MHC-restricted, cytotoxicity in peripheral blood lymphocytes (PBL) obtained after vaccination as compared with those obtained before vaccination. Increased recognition of the tyrosinase 368-376 peptide occurred in post-vaccination PBL of one patient, whereas a weak increase in recognition of the gp100 280-288 peptide was detectable in another patient; these 2 patients also recognized the gp100 457-466 peptide. After in vitro, stimulation with the only available autologous melanoma line, CD4+ cells with autologous tumor-specific cytotoxicity and ability to release interferon-gamma (IFN-gamma) were found in post- but not in pre-vaccination PBL. In the same patient, as well as in another patient, limiting dilution analysis showed that vaccination resulted in an increased frequency of melanoma-specific cytotoxic T lymphocyte (CTL) precursors. These results indicate that vaccination with cells releasing IL-2 locally can expand a T cell response against antigen(s) of autologous, untransduced tumor, although this response occurred in a minority of the melanoma patients studied.

Natural killer cells and cancer

Natural cytotoxicity, mediated by natural killer (NK) cells and cell with lymphokine-activated killer (LAK) activity, is believed to play an important role in host anti-cancer mechanisms. The authors critically review recent publications on the role of natural cytotoxicity in patients with cancer. In patients with cancer, several studies have noted variations in the numbers and activity of NK and cells with LAK activity in different body compartments. NK cell activity in the peripheral blood lymphocytes (PBLs) is higher than that found in lymph nodes and within tumors, and this appears to be due to the presence of suppressor factors. The natural cytotoxicity of PBLs in patients with different types of cancers varies. However, there appears to be a trend for natural cytotoxicity to be reduced in certain cancer patients, possibly related to tumor volume or dissemination. Anti-cancer treatments (e.g., surgery, hormonal modulation, radiotherapy and chemotherapy) can also result in suppression of natural cytotoxicity, although the long-term effect on response to treatment and development of metastases is at present unknown. NK and LAK cells, through the use of immune biologic modifiers, have been demonstrated to have a therapeutic role in the treatment of human cancers. Further studies are required to determine the optimal dosages and combinations of chemotherapeutic agents, the timing of surgery, and the adjuvant use of immune biologic response modifiers. An increasing awareness and understanding of this field, may allow for the future development of anti-cancer therapies.

Immune cell circulating subsets are affected by gonadal function

Influence on the immune system activity by sex hormones has been widely reported. Fertile women are proner to the onset of autoimmune diseases than men, but this increased susceptibility disappears after menopause. The hormonal changes are very likely to be responsible for this event, but precise correlations between sex hormone levels and immune functions have not been defined. For this reason we have analyzed phenotype and natural cytotoxicity of peripheral blood lymphocytes (PBL) from 35 women in menopause, comparing them with the same parameters of 28 fertile and 8 postmenopausal women and correlating them with the hormonal pattern of each group. We have also considered 8 women with premature menopause. Hormonal levels have been detected by radioimmune assays, while PBL phenotype has been studied by immunofluorescence and FACS analysis. The natural killer (NK) cell activity has been calculated on the basis of a chromium release assay. Postmenopausal women showed a reduction of the number of total lymphocytes (1650±215 cells/mmc) in comparison to fertile women (2081±200 cells/mmc, P<0.01). The decrease mainly involved B and CD4+ T lymphocyte subpopulations (P<0.05 and P<0.01, respectively). Women with premature menopause had lower percentage of CD4 lymphocytes (34% vs 47%, P<0.01) and higher percentage of CD8 (30% vs 22%, P<0.02) and NK cells (32% vs 14%, P<0.009) than fertile women of the same age. The percentage of circulating lymphocytes expressing HLA class II antigens also resulted as being increased (22% vs 9%, P<0.01). The number of total, CD2, CD4 T lymphocytes, B and NK cells correlated positively with LH and negatively with FSH serum levels (P<0.05 and P<0.002, respectively). PRL positively influenced CD2, CD4 and B lymphocyte numbers (P<0.001). FSH and 17β-estradiol inversely affected CD8 and B lymphocyte numbers (P<0.005 and P<0.02, respectively). In conclusion, the increase of FSH and the decrease of PRL levels appear to be involved in the reduction of B and CD4 T lymphocytes thus lowering the risk for the onset of autoimmune diseases during and after menopause. Generalized activation of the immune system (raised expression of HLA class II antigens) with elevated numbers of cytotoxic subpopulations (CD8 and NK lymphocytes) is present in women affected by premature menopause suggesting the involvement of autoimmune dysregulation in the pathogenesis of this syndrome.

Eisenin (L-pyroGlu-L-Gln-L-Ala), a new biological response modifier.

Eisenin (L-pyroGlu-L-Gln-L-Ala), a tripeptide extracted from a brown marine alga (Eisenia bicyclis Setchell) showed the immunological activity to augment natural cytotoxicity of peripheral blood lymphocytes (PBLs) in humans. This activity could be seen when it was added directly to 51Cr release assay, and also when PBLs alone were incubated with this compound for 0.5-1 h before addition to the 51Cr release assay. The resulting augmented cytotoxicity can be attributed to natural killer cells because treatment of eisenin-stimulated PBLs with anti-Leu 11b monoclonal antibody (mAb) plus complement completely abolished the augmented cytotoxicity, and, moreover, deletion of Leu 19-positive cells with anti-Leu 19 mAb (mouse IgG1) and anti-mouse IgG-coated magnetic beads showed the same effect. Eisenin could not augment natural cytotoxicity of Sephadex G-10 column-eluted cells. Eisenin rendered K-562 target cells resistant to lysis by PBLs stimulated by eisenin. Both amino acids, L-pyroglutamic acid and L-alanine, as well as a mixture of the three component amino acids of eisenin could augment the natural cytotoxicity. Therefore, it is considered that the structure responsible for the augmentation of natural cytotoxicity by eisenin may be that of the amino acids.

Retargeting of human lymphocytes against human ovarian carcinoma cells by bispecific antibodies: from laboratory to clinic

We have selected a monoclonal antibody (MOv18) reactive with ovarian carcinoma, which exhibits a restricted tumor specificity, a high affinity constant and which recognizes a 38-kDa glycoprotein homogeneously expressed on the cell surface of 90% of human ovarian carcinomas. Localization studies with radiolabelled MOv18 showed that MOv18 could specifically reach ovarian carcinoma cells growing in the peritoneal cavity of nu/nu mice. MOv18 did not mediate antibody-dependent cellular cytotoxicity via Fc and could not be used as a carrier for toxins due to poor internalization of the antigen-antibody complex. Bispecific F(ab')2 antibodies made with MOv18 and an antibody reactive with CD3 were able to redirect the cytotoxicity of peripheral blood lymphocytes (PBL) against ovarian carcinoma cells both in vitro and in vivo in an animal model. The treatment of athymic mice bearing a human ovarian carcinoma intraperitoneally, with human PBL coated with the bispecific F(ab')2, significantly prolonged survival of the animals compared with tumour-bearing untreated and control mice treated with PBL alone. Four ovarian cancer patients have been treated with autologous lymphocytes coated with this bispecific F(ab')2 in a phase I clinical trial. No serious side-effects were observed but patients developed human anti-murine antibodies mainly directed against the idiotype of MOv18. We have now begun phase II clinical trials in ovarian cancer patients.

Lysis of autologous tumor cells by peripheral blood lymphocytes treated with IFN-alpha in patients with renal cell carcinoma

Immunotherapy, consisting mainly of interferon (IFN) therapy, has been used to treat renal cell carcinoma refractory to radiotherapy and chemotherapy ever since IFN was reported to be effective against renal cell carcinoma in 1982. The efficacy of IFN is low, with the response rate being only about 20% and the method and duration of its administration have yet to be established. Using viable tumor cells obtained at nephrectomy, we discovered that IFN-alpha induced killer cells have a cytocidal effect on autologous renal cell carcinoma cells and that assaying the cellular immunity of the peripheral blood lymphocytes of 25 patients with renal cell carcinoma, using ACHN derived from human renal cell carcinoma as the target cells, is useful as a monitoring method during IFN-alpha therapy. Augmentation of the cytotoxicity of peripheral blood lymphocytes in response to administration of IFN-alpha was observed in all 25 cases. Cytotoxicity was activated to 5-426 LU compared with the control value of 1 LU or less before IFN therapy, when autologous renal cell carcinoma cells were used as the target cells. Cytotoxicity for ACHN cells in the control value before IFN-alpha therapy and 4 weeks after the institution of IFN-alpha administration and autologous tumor lysis in the induction assay were more strongly correlated than in the case of K562 cells. A strong correlation was also found between cytotoxicity for ACHN cells in the induction assay and 4 weeks after the institution of IFN-alpha administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Human allogeneic melanoma‐reactive T‐helper lymphocyte clones: Functional analysis of lymphocyte‐melanoma interactions

Lymphocyte clones were isolated from CD4+ peripheral-blood lymphocytes (PBL) of melanoma (Me) patient 9923 (HLA-DR7, DQw2, w6), co-cultured for 30 days with autologous accessory cells, allogeneic Me (Me 1811) (HLA-DR7, DQw1, w2), IL-1 beta (2 U/ml) and IL-2 (15 IU/ml). The 55 clones tested displayed a CD3+, CD4+, CD8-, T-cell receptor (TCR) alpha/beta+, gamma/delta- phenotype. Twenty clones were assayed for proliferation in the presence of Me 1811 and B-lymphoblastoid cell line (LCL) 1811, both expressing HLA-class-I and -II (DR7 and DQw2 shared with patient 9923), intercellular adhesion molecule-1 (ICAM-1) and lymphocyte-function-associated antigen-3 (LFA-3) molecules. Eight clones were found to be reactive to Me 1811 but not to LCL 1811. Specificity analysis of these 8 clones revealed that each of them proliferated only to Me 1811, not to other 14 Me and 12 different LCL, suggesting recognition of melanoma-associated antigen (MAA) expressed on the stimulating Me. One clone (103) was analyzed in more detail. A wider specificity analysis showed that it reacted to Me 1811 but not to 10 other Me expressing or not HLA-DR7, 5 normal melanocyte cultures (2 of them typing HLA-DR7-positive when exposed to interferon-gamma--IFN-gamma), 4 tumors other than Me and 20 different LCL. Clones did not show proliferation in the presence of autologous Me cells. Clone proliferation in response to Me 1811 was significantly inhibited by monoclonal antibodies (MAbs) directed to CD3, TCR alpha/beta, TCR beta chain V12, CD4 and HLA-DR. Moreover, following stimulation with Me 1811, clone 103 showed increased surface expression of CD25 (IL-2 receptor) and CD71 (transferrin receptor) and produced significant amounts of IL-2 and IFN-gamma. The supernatant taken from co-culture of clone 103 with Me 1811 augmented the cytotoxicity of PBL 9923 and other allogeneic PBL against K562 and Me 1811. Thus, the lymphocyte clone 103 is a CD4+ Th clone which uses its CD3/TCR alpha/beta complex to recognize an MAA in conjunction with HLA-DR7. Availability of this type of reagent may prove useful to identify and characterize MAA recognized by T lymphocytes.

BLT-esterase activity followingin vitro andin vivo activation of human lymphocytes with interleukin-2

BLT-esterase and cytolytic activity by human in vitro and in vivo generated Lymphokine Activated Killer (LAK) cells were measured. Lysates made from peripheral blood lymphocytes (PBL) of both normal donors and cancer patients receiving IL-2 therapy were assayed for BLT-esterase activity in a spectro-photometric assay. Cytotoxicity of PBL was measured in a 51Cr-release assay. Both BLT-esterase activity and cytotoxicity increased when normal-donor PBL were stimulated in vitro with IL-2, with greater activities at higher IL-2 concentrations. The activities also increased over time, peaking at 6 days of in vitro stimulation. Patient PBL had increased BLT-esterase and cytotoxic activities after 4 weeks of in vivo IL-2 treatment. This association of BLT-esterase activity and cytotoxicity with IL-2 activation is consistent with the model that LAK cytotoxicity is mediated by secretion of BLT-esterase associated cytolytic granules. Lymphocytes obtained after in vivo IL-2 treatment and cultured for 3-4 hours in IL-2 show markedly augmented cytotoxic activity but no increase in their BLT-esterase activity. These results indicate that the increased cytotoxicity observed after this brief pulse of in vitro IL-2 following in vivo IL-2 treatment must result from effects of IL-2 other than the production of more esterase-containing cytolytic granules.

Effect of calcitriol on peripheral bood lymphocyte cytotoxicity

To clarify the biochemical mechanism responsible for the inhibition by ealcitriol (1,25-dihydroxyvitamin D3) of eytotoxiciiy in peripheral blood lymphocytes (PBL), the human NK sensitive K562 cell line and the human tumor necrosis factor-sensitive murine L929 cell line were used as targets and subsequently compared. The cytotoxicity of PBLs for K562 cells was not changed by preincubation for 4 h with 10 ng ml phorbol myristate acetate (PMA), but was reduced after an overnight preincubation with 10 −9 or 10 −8 M ealcitriol. Using L929 cells, preincubation of PBLs with 10 ng ml PMA for 4 h increased their cytotoxicity. Overnight incubation with ealcitriol significantly reduced PBL cytotoxicity for L929 cells in a dose related manner and suppressed the enhancement of this cytotoxicity by PMA. Pretreatment of PBLs with cycloheximide reduced their eytolysis for L929 cells but did not change their cytotoxicity towards KS62 cells. Con- sequently, the natural cytotoxicity of PBLs for K562 cells does not involve the same mechanism as their cytotoxicity for L929 cells and is therefore subject to different forms of regulation. However, calcitriol reduced PBL cytotoxicity towards both target cells.