Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity.

The tumor-associated-Ag MART-1 is expressed by most human melanomas. The genes encoding an alphabeta TCR from a MART-1-specific, HLA-A2-restricted, human T cell clone have been efficiently transferred and expressed in human PBL. These retrovirally transduced PBL cultures were MART-1 peptide reactive, and most cultures recognized HLA-A2+ melanoma lines. Limiting dilution clones were generated from three bulk transduced PBL cultures to investigate the function of individual clones within the transduced cultures. Twenty-nine of 29 CD8+ clones specifically secreted IFN-gamma in response to T2 cells pulsed with MART-1(27-35) peptide, and 23 of 29 specifically secreted IFN-gamma in response to HLA-A2+ melanoma lines. Additionally, 23 of 29 CD8+ clones lysed T2 cells pulsed with the MART-1(27-35) peptide and 15 of 29 lysed the HLA-A2+ melanoma line 888. CD4+ clones specifically secreted IFN-gamma in response to T2 cells pulsed with the MART-1(27-35) peptide. TCR gene transfer to patient PBL can produce CTL with anti-tumor reactivity in vitro and could potentially offer a treatment for patients with metastatic melanoma. This approach could also be applied to the treatment of other tumors and viral infections. Additionally, TCR gene transfer offers unique opportunities to study the fate of adoptively transferred T cells in vivo.     

Genotoxic potential of medroxyprogesterone acetate in cultured human peripheral blood lymphocytes

Medroxyprogesterone acetate was studied at three different concentrations (1, 5 and 10 microM), for its genotoxic effects in human peripheral blood lymphocyte culture using chromosomal aberrations and sister chromatid exchanges as parameters. Duplicate peripheral blood cultures were treated with three different concentrations (1, 5 and 10 microM) of medroxyprogesterone acetate. The study was carried out both in the absence as well as in the presence of metabolic activation (S9 mix) with and without NADP. Medroxyprogesterone acetate was found genotoxic at 5 and 10 microM in the presence of S9 mix with NADP. To study the possible mechanism of the genotoxicity of medroxyprogesterone acetate, superoxide dismutase and catalase at different doses were used separately and in combination with 10 microM of medroxyprogesterone at different doses in the presence of S9 mix with NADP. Superoxide dismutase treatment results in an increase of the genotoxic damage but catalase treatment reduce the genotoxic damage of medroxyprogesterone acetate. Catalase treatment in combination with superoxide dismutase also results in the further reduction of the genotoxic damage. The results of the present study reveal that medroxyprogesterone acetate is genotoxic only in the presence of metabolic activation (S9 mix) with NADP. Treatments with superoxide dismutase and catalase suggests the possible generation of reactive oxygen species by redox cycling of various forms of quinones, similar to estrogens, that are the results of aromatic hydroxylation by cytochrome P450s.     

Fibronectin augments anti-CD3-mediated IL-2 receptor (CD25) expression on human peripheral blood lymphocytes.

The extracellular matrix (ECM) is composed of a number of macromolecules that promote cell adhesion, cell migration, and differentiation. Receptors for these molecules have been identified and belong to a superfamily of cell surface proteins, collectively known as the integrins. In this study, we show that the matrix protein fibronectin (FN) acts synergistically with immobilized anti-CD3 antibody to promote proliferation of total human peripheral blood lymphocytes (HPBL) in the absence of exogenous IL-2. Proliferation was inhibited by both the alpha 5 beta 1 and alpha 4 beta 1 recognition peptides. ARG-GLY-ASP (RGD), and GLU-ILE-LEU-ASP-VAL-PRO-SER-THR (EILDVPST), respectively. Expression of CD25 (IL-2 receptor) was significantly higher on cells cultured on anti-CD3 and FN, indicative of T-cell activation. Additionally, cells cultured on immobilized anti-CD3 and FN for 3 days showed increased adhesion to FN and increased forward light scatter/side scatter profile. Synthesis of both IL-1 and to a lesser extent IL-2 was elevated in supernatants from cultures containing both anti-CD3 and FN. These data are consistent with published reports which demonstrate that ECM proteins can act as costimulants of lymphocyte proliferation. Finally, our results show that cells cultured on anti-CD3 antibody and FN have an activated phenotype and that cytokines may be involved in this process.     

Subpopulations of human peripheral blood lymphocytes.

Multiple staining protocols have been developed for the classification of subpopulations of human peripheral blood lymphocytes. Of the non-T (E^?) cells, roughly half (10–20% PBL) have receptors for complement components as detected with complement-coated zymosan particles, but do not show Fc receptors as detected with Ripley IgG-coated human RBC. The other half are C^?, Fc⁺, with a small percentage possessing both receptors. The C⁺, Fc^? cells can be subdivided into cells which are IgM⁺ (75%) or IgM^?. Cells with Fc receptors detected with aggregated IgG were IgM⁺.     

Activation and expansion of tumour-infiltrating lymphocytes by anti-CD3 and anti-CD28 monoclonal antibodies

Summary Cytotoxic T lymphocytes from healthy donors can be expanded to high numbers from the peripheral blood using combinations of anti-CD3 and anti-CD28 monoclonal antibodies (mAb). We investigated whether these antibodies could also be used to induce outgrowth of tumour-infiltrating lymphocytes (TIL) from tumour tissue. In the initiation phase of TIL culture immobilized anti-CD3 antibodies together with anti-CD28 mAb and low-dose interleukin-2 induced a rapid expansion of T cells from various human tumour tissues. The cultured cells showed high levels of cytotoxic T lymphocyte activity, but low levels of lymphokine-activated killer cell activity were generated. This study shows that TIL can be efficiently expanded from tumour tissue by combinations of anti-CD3 and anti-CD28 antibodies. This protocol for cell expansion in vitro may substantially reduce the time required to reach sufficient numbers of TIL for re-infusion to the patient.     

Unusually high level of chromosome variability in cultured human peripheral blood lymphocytes]

A cytogenetic examination carried out in the inhabitants of Seversk (Tomsk oblast) and workers of the Siberian chemical industrial complex (a complex of nuclear-chemical and fuel plants), living in the same town, revealed unusually high level of spontaneous chromosomal variability both in control and industrial groups (total irradiation doses 1.8 to 3.7 and 9.3 to 15.7 Gy, respectively). The frequencies of cells with chromosomal aberrations (estimated per 100 cells) in control and industrial groups were 4.69, 6.04, and 6.64, respectively, and the total number of aberrations constituted 6.93, 8.47 and 12.06, respectively. These frequencies were several times higher compared to the summarized literature data on the control levels. The high average aberration level was caused by the elevated proportion of chromatid-type aberrations and paired fragments. The reasons for this are unclear. The levels of radioactive background and chemical air pollution in the town were not increased.     

Expansion of peripheral blood lymphocytes by culture with immobilized anti-CD3 antibody and IL-2]

Peripheral blood lymphocytes separated from about 20ml of blood were cultured with immobilized anti-CD3 antibody and IL-2. Total T lymphocytes in the culture expanded about 2,000 times by 2 weeks culture. Expanded T lymphocytes were infused to cancer patients, and safety and feasibility was confirmed. This method will be applicable for prevention of cancer recurrence.     

Extracellular domain of 4-1BBL enhanced the antitumoral efficacy of peripheral blood lymphocytes mediated by anti-CD3 x anti-Pgp bispecific diabody against human multidrug-resistant leukemia

Our previous data have shown a significantly higher tumor response to anti-CD3/anti-Pgp bispecific diabody-mediated immunotherapy for P-glycoprotein (Pgp)-overexpressing K562/A02 cells, but a rapid tumor relapse occurred at 1 week after therapy. In an attempt to overcome tumor recurrence, we supplemented the previous therapy with extracellular domain of human 4-1BBL (ex4-1BBL) to regulate the activation of peripheral blood lymphocyte (PBL). As a result, this combination showed enhanced cytotoxicity in vitro and eradicated the multidrug-resistant xenografts of K562/A02 in nude mice. Furthermore, no tumor recurrence was observed within 100 days after the first treatment. Therefore, when used as an adjuvant, ex4-1BBL may improve the outcome of PBL-based immunotherapy.     

Targeted cytotoxic cells in human peripheral blood lymphocytes.

We have isolated subsets of cells from human PBL and have investigated their abilities to mediate lysis targeted by bispecific antibodies. Targeted cytotoxic cells were divided into two distinct types based on buoyant density. The low buoyant density fraction contained all of the targetable cytotoxic activity in unstimulated PBL, including both T and K cells targeted with anti-CD3 and anti-Fc gamma RIII (CD16) containing bispecific antibodies, respectively. Both types of targetable cytotoxic cells required IL-2 for maintenance of cytotoxic activity, expressed the CD56 (NKH1) marker, and mediated MHC-unrestricted lysis. The targetable T cells in low density PBL were exclusively CD8+ and represented only about 2% of the total PBL. The high buoyant density lymphocytes, depleted of NK cells, had no targetable activity, but were able to generate over several days, targetable T cell activity in the presence of a TCR cross-linking signal plus IL-2. Unlike the low-density cells, the activated high buoyant density effector T cells did not express CD56, consisted of both CD4+ and CD8+ cells, and did not mediate MHC-unrestricted lysis. These cells proliferated more rapidly and generated more total lytic activity than the low-density fraction. Our studies show that targetable cytotoxic activity in human PBL is mediated by several subsets of cells with different activation requirements. Presumably all of these activities could be directed against unwanted cells in clinical or preclinical studies involving targeted cytotoxic cells.     

Further characterization of cytotoxic T cells generated by short-term culture of human peripheral blood lymphocytes with interleukin-2 and anti-CD3 mAb

 In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3⁺ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fcγ receptor (FcγRI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab′)₂, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18⁺ ovarian tumor cell line), was also compared in the presence of a bispecific antibody (OC/TR, anti-CD3 × MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 ×antitumoral bispecific antibodies. Received: 6 December 1995 / Accepted: 4 June 1996     

Genotoxicity of pemetrexed in human peripheral blood lymphocytes

Pemetrexed (PMX) is an antineoplastic antifolate used in the treatment of non-small cell lung cancer, mesothelioma and several types of neoplasms. Its toxicity in tumor cells has been linked with the potent inhibition of thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyl transferase, and subsequent depletion of both purine and pyrimidine nucleotides. However, cytogenetic toxicity of PMX in non-diseased cells has not been adequately studied; despite the increasing data on the DNA-damaging potential of antineoplastic agents on normal cells. In the present study, the genotoxic potential of PMX was evaluated in peripheral blood lymphocytes obtained from healthy human subjects using chromosome aberration (CA), sister chromatid exchange (SCE) and micronucleus (MN) assays as the cytogenetic damage markers. Human peripheral blood lymphocytes were exposed to four different concentrations (25, 50, 75 and 100 μg/mL) of PMX for 24- and 48-h treatment periods. PMX significantly increased the formation of CA in 24-h treatment, but not in 48-h treatment. PMX did not increase the mean SCE frequency in 24- and 48-h treatment periods; however, there was a striking increase (although not statistically significant, p > 0.05) in the number of SCEs at 25 μg/mL (24- and 48-h treatment) and 50 μg/mL (24-h treatment) due to an increase of SCE at the single-cell level. Interestingly, PMX did not induce MN formation in either 24- or 48-h treatment periods. PMX strongly decreased the mitotic index (MI), proliferation index (PI) and nuclear division index (NDI) in 24- and 48-h treatment periods. Our results suggest that PMX has a potent cytotoxic effect against human peripheral blood lymphocytes at concentrations which are reached in vivo in the blood plasma.     

Glucocorticoid receptors in purified subpopulations of human peripheral blood lymphocytes.

On the premise that the differential effects of glucocorticoids on various aspects of the immune response may be mediated by differences in the glucocorticoid receptors in the effector cells, subpopulations of human peripheral blood lymphocytes were examined for these receptors as well as for glucocorticoid responsiveness. Purified T and non-T lymphocytes, when studied by a sensitive whole cell assay technique, contained equivalent amounts of specific glucocorticoid receptor, which, by binding affinity and specificity measurements, were indistinguishable from each other. Furthermore, under in vitro incubation conditions, macromolecular synthesis in both of these cell populations was inhibited by glucocorticoid at concentrations which saturated the receptor sites. It is concluded that the putative differential effects of glucocorticoids on T and non-T lymphocyte-associated functions are probably not mediated by differences in the glucocorticoid receptors in these cell populations.     

Shared idiotypes of human peripheral blood B and T lymphocytes.

In a patient with an IgG lambda monoclonal serum component possessing anti-streptolysin O activity, we have demonstrated peripheral blood B and T lymphocytes with shared or similar idiotypes. The idiotypic T lymphocyte membrane structure was capable of binding the specific antigen (SLO). After radioiodination and subsequent detergent solubilization of the same T cell population, immunoprecipitation of the lysate by employing anti-idiotypic antibodies, resulted in the isolation of a polypeptide chain with a m.w. of 70,000 on SDS polyacrylamide gels under reducing conditions. The polypeptide expressed no isotypic immunoglobulin markers. Internal labeling experiments indicated that this membrane structure was actively synthesized by the T lymphocytes.     

Protein phosphorylation in human peripheral blood lymphocytes. Subcellular distribution and partial characterization of adenosine 3'':5''-cyclic monophosphate-dependent protein kinase.

Cytoplasmic and membrane fractions prepared from human peripheral-blood lymphocytes both contained cyclic AMP-dependent protein kinase activity and endogenous protein kinase substrates. Protein kinase activity in the particulate fractions was not eluted with 0.25 M-NaCl, suggesting that it was not derived from non-specifically absorbed soluble cytoplasmic protein kinase. Nor was the particulate protein kinase activity eluted by treatment with cyclic AMP, suggesting that the catalytic subunit is membrane-bound and arguing against cyclic AMP-induced translocation of particulate activity. Cyclic AMP-dependent protein-phosphorylating activity in the cytoplasmic fraction was highly sensitive to inhibition by Mn2+, and was co-eluted from DEAE-cellulose primarily with type-I rabbit skeletal-muscle kinase. Cyclic AMP-dependent phosphorylating activity in the plasma-membrane fractions was stimulated at low [Mn2+] and inhibited only at high [Mn2+]. When solubilized with Nonidet P-40, plasma-membrane protein kinase was co-eluted from DEAE-cellulose with type-II rabbit muscle kinase. These differences, together with the strong association of the particulate kinases with the particulate fraction, suggest the possibility of compartmentalized protein phosphorylation in intact lymphocytes.     

Opposing effects of human peripheral blood lymphocytes on the growth of cultured leukemia cell lines

Natural killing of two human leukemia cell lines (K562 and Molt 4) in a soft-agar, clonagenic assay was shown to be the result of two opposite yet concurrent processes: target cell colony stimulation and inhibition. The stimulatory effect was demonstrable when the effector lymphocytes and target cells were separated in contiguous agar layers, suggesting mediation by a soluble factor. Similarly, stimulation occurred when the effector lymphocytes and target cells were combined at low effector-target cell ratios that do not favor direct cell contact. Target colony inhibition was found to be dominant when large E:T ratios were employed. Both target-effector binding and natural killing were significantly reduced in medium devoid of divalent cations.     

Cyclic nucleotide phosphodiesterase activity in human peripheral blood lymphocytes and monocytes.

cAMP and cGMP phosphodiesterase (PDE) activity was assayed in human peripheral blood lymphocytes purified by isopycnic centrifugation as well as in lymphocyte preparations further purified to remove contaminating platelets and monocytes. The 16,000 X G supernatant from sonicates of each of these cell preparations contained two hydrolytic activities for cAMP with apparent Km of 1.1 to 2.5 microM and 33 to 66 microM, and a single hydrolytic activity for cGMP with an apparent Km of 6 to 25 microM. When lymphocytes were disrupted by Dounce homogenization, there was only a single, low Km cAMP PDE activity in the homogenate; however, the 16,000 X G supernatant demonstrated 2 Km similar to that seen in sonicated lymphocytes. Treatment of the Dounce preparations with 0.5% Triton X-100 or 1.0% NP-40 converted these preparations to activities similar to those seen in sonicated preparations. cGMP hydrolytic activity was low or absent in the Dounce preparations and was not altered by centrifugation; however, it was markedly enhanced by detergent extraction. These data indicate that human peripheral blood lymphocytes and monocytes have PDE activities similar to those seen in other tissues.