RU2514019C2 - Method of evaluating cytotoxic activity of lymphocytes of natural killers - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to clinical immunology, and can be applied for evaluating cytotoxic activity of lymphocytes of natural killer (NK) by target cells K-562, which remain non-degraded after contact with NK lymphocytes. For this purpose NK lymphocytes in composition of mononuclear cells are separated from peripheral blood of patients. Cytotoxic test, based on incubation of NK lymphocytes and target cells K-562 is set. At that target cells K-562 are not labeled with ³H-uridine. Cytotoxic activity of NK lymphocytes is evaluated by calculation of quantity of remaining non-degraded target cells on automatic cell counter and analyser, adjusted for detection of cells with diameter from 15 to 40 mcm. After that, index of cytotoxicity is calculated by formula $$\text{IC}=(1-\frac{\text{Number of cells in the experiment cell}}{\text{Number of cells in the reference cell}})\times 100\%.$$

EFFECT: invention ensures safety for researcher's health, as well as reduction of research duration.

3 tbl, 12 ex

Description

The invention relates to medicine, in particular to clinical immunology, and relates to a method for assessing the cytotoxic activity (CA) of natural killer lymphocytes, (NK) lymphocytes in the peripheral blood of patients and healthy, based on counting the number of undegraded target cells K-562 after their contact with NK lymphocytes on an automatic counter and cell analyzer. The invention can be used in medical immunodiagnostics with a comprehensive examination of patients in order to detect changes in the cytotoxic activity of natural killer lymphocytes in them; its use is also possible in medical practice in order to correct the revealed changes in this immunity in patients with psychopathological symptoms and various somatic diseases, primarily malignant neoplasms and diseases of an infectious nature.

A known colorimetric method for evaluating the CA of NK lymphocytes in peripheral blood is based on staining dead target cells with trypan blue or alamar blue as a result of increasing the membrane permeability after contact with NK lymphocytes in a cytotoxic test (Anfalova T.V., Kazan DB ., Khromykh L.M. Bulletin of Experimental Biology and Medicine. 2003; (9): 3 56-360). This method is inaccurate, since when counting stained cells, dead blood cells can be taken into account together with target cells.

A radioactive evaluation method is also known, which is based on measuring the amount of radioactive N-RNA in target cells that remained undegraded after contact with NK lymphocytes in a cytotoxic test. As target cells adapted to NK, human K-562 line myeloid leukemia cells are used, which are treated with ³ H-uridine before setting up a cytotoxic test (Rykova M.P., Spirande I.V., Zedgenidze M.S. et al. Immunology. 1981; 3: 88-90, prototype). The essence of this method is that when setting up a cytotoxic test, mononuclear cells isolated from the peripheral blood of patients and healthy and consisting of monocytes and a pool of lymphocytes, 10-15% of which are NK lymphocytes, add target cells K-562, which are preliminarily labeled for 3 hours with a ³ H-uridine radioactive label and washed with large volumes of medium 199 from the unincorporated radioactive tag for three hours more. After incubation for 16-18 hours, the cell suspension is sedimented using vacuum a pump and a harvester designed to collect cells by filtration on filters, on special fiberglass filters. The filters are dried for 6-18 hours, then transferred to bottles with scintillation liquid and left for another 6-18 hours for the release of β-particles into the liquid. After that, the level of radioactivity of the samples on the β-counter is counted.

The disadvantage of this method is the duration and complexity of its implementation, as well as the danger to the health of the performer. Namely, the assessment of CA NA is multi-stage, takes 2-3 days and requires the use of a large number of expensive instruments and specific materials, such as a harvester, vacuum pump, β-counter, scintillation bottles, fiberglass filters, and also involves the use of harmful for the body of substances such as scintillation fluid and a radioactive label, to work with which it is necessary to use a fume hood. In addition, to work with radioactivity, it is necessary to authorize the sanitary and epidemiological station to equip a special radioactive unit, which consists of a separate room with a fume hood, a refrigerator and a safe for storing radioisotopes.

The aim of the invention is to simplify the method for evaluating the CA of NK lymphocytes, namely, to reduce the evaluation time, its cost and complexity, as well as the ability to make it safe for the researcher, since in this case the use of scintillation fluid and radioactive components is excluded. The absence of a radioactive label also eliminates the need to obtain permission from a sanitary and epidemiological station to work with radioactive substances.

This is achieved by the fact that, firstly, in contrast to the known method, in the proposed method, in the cytotoxic test, K-562 target cells not treated with ³ H-uridine and RNase are added to the mononuclear cells. Secondly, to assess the CA of NK lymphocytes, only one device is used instead of several devices - an automatic counter and a cell analyzer, configured to detect cells whose diameter is in the range from 15 to 40 microns. This corresponds to the diameter of the target cells K-562, which exceeds the diameter of the largest blood cells - monocytes, whose diameter does not reach 15 microns. Using this device, the level of CA of NK lymphocytes is estimated by the number of K-562 target cells that are not degraded after contact with NK lymphocytes. Thirdly, the stage of assessing the cytotoxic activity of NK takes only 10-15 minutes instead of 2-3 days when evaluated in a known manner. Fourth, the proposed method for assessing the cytotoxic activity of NK lymphocytes completely eliminates the use of a radioactive label that is hazardous to the health of the researcher, and therefore the need for special radioactive unit equipment disappears.

A method of evaluating the cytotoxic activity of NK lymphocytes according to the prototype is as follows.

1) For the production of a cytotoxic test, target cells for NK lymphocytes are human myeloid leukemia cells of the K-562 line (DI Ivanovsky Institute of Virology, RAMS), supported in vitro in RPMI-1640 medium (FSUE PIPVE named M .P. Chumakova RAMS), with the addition of 10% fetal serum (Defined Fetal Bovine Serum company Hyclone, USA) and 10% glutamine.

2) Before staging a cytotoxic test, 3 ml of suspension of K-562 target cells at a concentration of 1-4 × 10 ⁶ in 1 ml of medium 199 are placed in a centrifuge tube, 0.3 ml of working solution of ³ N-uridine (3 μCi / ml, specific radioactivity 24 Ci / mM, Institute of Molecular Genetics, Russian Academy of Sciences). Cells are placed in a thermostat at 37 ° C for one hour. After this time, the cells are washed three times from the radioactive label not included in them with large volumes of 199 medium by centrifugation at 1100-1200 rpm and 4 ° C for 10 min; then diluted to a concentration of 10 ⁵ / ml. In time, this procedure takes another hour.

3) For the degradation of K-562 ³ H-uridine that was not included in the target cells, pancreatic ribonuclease A (RNase, REAHIM NPO Biolar) is added to them, which cuts the 3'-end of unpaired cytidyl and uridyl nucleotides, catalyzing their degradation ^

4) Mononuclear cells, which include monocytes and a common pool of lymphocytes, 10-15% of which are NK lymphocytes, are isolated from the peripheral blood of patients in a density gradient of ficoll-verographin with beats. pl. 1,077, dilute them with medium 199 (FSUE PIPVE named after MP Chumakov RAMS) to a concentration of 4 × 10 ⁶ / ml.

5) The staging of the cytotoxic test is carried out in special 96-well round-bottom polystyrene plates, in which the volume of the hole is 0.25 ml (Medpolimer plant, St. Petersburg); 0.05 ml of medium 199, 0.05 ml of complete medium 199 enriched with 10% fetal calf serum and 10% glutamine solution are added to wells with experimental samples, then 0.05 ml of mononuclear cell suspension and 0 are added in a 20: 1 ratio. 1 ml treated with ³ H-uridine and RNase target cells K-562. Instead of mononuclear cells, 0.05 ml of 199 medium is added to the wells with control samples. Each experimental and control sample is placed in 2 or 3 parallels.

6) The plates are incubated in a thermostat for 16-18 hours at a temperature of 37 ° C and 5% CO ² .

7) After incubation, the cells are transferred using a multichannel harvester, a device designed to collect cells by filtering on filters (Combi cell Harvester Skatron, England), and a vacuum pump (Millipore) from the plates onto special fiberglass filters (Skatron, Filter Mat for 12 well Cell Harvesters, England) and washed with sodium phosphate buffer from the unincorporated label.

8) The filters are dried in air for 6-18 hours, after which they are placed in scintillation vials (Beckman, USA). In a fume hood, using a dispenser (Brinkmann, Germany), 3 ml of ZhS-8 scintillation liquid (Monocrystal Institute, Ukraine) is poured into each vial and left for another 6-18 hours for β-particles to enter the scintillation liquid.

9) After that, the scintillation vials are placed in a liquid β-particle scintillation counter (MicroBeta trilux, PerkinElmer Inc.) and the number of radioactive pulses in each vial is counted.

10) In the formula for calculating the cytotoxic activity of NK lymphocytes, the radioactivity values obtained in bottles with experimental and control samples are added, and the result is expressed as a cytotoxicity index (IC) in%, which is calculated by the formula:

$$\mathrm{AND}\mathrm{Ts}=(\mathrm{one}-\frac{H\mathrm{and}\mathrm{from}l\mathrm{about}\mathrm{and}mP\mathrm{at}lb\mathrm{from}\mathrm{about}\mathrm{at}\mathrm{at}\mathrm{about}Pstn\mathrm{about}\mathrm{th}te\mathrm{from}t-\mathrm{I\; am}he\mathrm{th}\mathrm{to}e}{H\mathrm{and}\mathrm{from}l\mathrm{about}\mathrm{and}mP\mathrm{at}lb\mathrm{from}\mathrm{about}\mathrm{at}\mathrm{at}\mathrm{to}\mathrm{about}ntR\mathrm{about}lbn\mathrm{about}\mathrm{th}te\mathrm{from}t-\mathrm{I\; am}he\mathrm{th}\mathrm{to}e})\times \mathrm{one}00\%$$

The proposed method. Evaluation of the cytotoxic activity of NK lymphocytes on an automatic cell counter and analyzer by counting the number of K-562 target cells not degraded after contact with NK was carried out as follows.

1) For the production of a cytotoxic test, target cells for NK lymphocytes were human myeloid leukemia cells of the K-562 line (DI Ivanovsky Institute of Virology, RAMS) supported in vitro on RPMI-1640 medium (FSUE PIPVE named after MP Chumakova RAMS), with the addition of 10% fetal serum (Defined Fetal Bovine Serum company Hyclone, USA) and 10% glutamine.

2) The target cells were not labeled with N-uridine before staging the cytotoxic test, and as a result, the need to treat them with RNase disappeared; washed once with a large amount of medium 199 (FSUE PIPVE named after MP Chumakov RAMS) by centrifugation at 1100-1200 rpm and 4 ° C for 10 min and diluted to a concentration of 1x10 / ml with medium 199.

3) Mononuclear cells, which included monocytes and a common pool of lymphocytes, 10-15% of which are NK lymphocytes, were isolated from the peripheral blood of patients in a density gradient of ficoll-verographin with beats. pl. 1,077, was diluted with medium 199 to a concentration of 4 × 10 ⁶ / ml.

4) The cytotoxic test was carried out in special 96-well round-bottom polystyrene plates with a volume of 0.25 ml (Medpolimer plant, St. Petersburg). 0.05 ml of complete medium 199 enriched with 10% fetal calf serum and 10% glutamine solution was added to the wells with experimental samples to 0.05 ml of medium 199; then, 0.05 ml of a suspension of mononuclear cells and 0.1 ml of untreated ³ H-uridine and RNase K-562 target cells were added in a 20: 1 ratio. Instead of mononuclear cells, 0.05 ml of 199 medium was added to the wells with control samples. Each experimental and control sample was placed in 2 or 3 parallels.

5) The plates were incubated in a thermostat for 16-18 hours at a temperature of 37 ° C and 5% CO ² .

6) After incubation, the contents of the wells of two or three parallels of each sample were transferred using an automatic pipette from the culture plate into Ependorf plastic tubes and mixed thoroughly.

7) 100 μl of cell suspension was taken from each tube and diluted in 10 ml of sample dilution liquid in special 20 ml aquuets (aquuets and sample dilution liquid are supplied with the device).

8) Akuvet was placed in an automatic cell counter and analyzer (Beckman Coulter, USA), with the help of which the number of K-562 target cells not degraded after contact with NK lymphocytes was counted.

9) When analyzing the counted cells, only those cells whose diameter was in the range from 15 to 40 μm were taken into account. Since the diameter of the largest mononuclear blood cells, namely monocytes, is not more than 15 μm, and the diameter of the K-562 target cells is more than 15 μm, the cells counted on the device in the range from 15 to 40 μm were assigned specifically to K- cells 562.

10) The values obtained on the instrument, that is, the number of target cells in the samples with experimental and control samples, were entered into the formula for calculating the CA of NK, and the result was also expressed as the cytotoxicity index (IC) in%, which was calculated by the formula:

$$\mathrm{AND}\mathrm{Ts}=(\mathrm{one}-\frac{H\mathrm{and}\mathrm{from}l\mathrm{about}\mathrm{to}let\mathrm{about}\mathrm{to}-m\mathrm{and}wene\mathrm{th}\mathrm{at}\mathrm{about}Pstn\mathrm{about}\mathrm{th}te\mathrm{from}t-\mathrm{I\; am}he\mathrm{th}\mathrm{to}e}{H\mathrm{and}\mathrm{from}l\mathrm{about}\mathrm{to}let\mathrm{about}\mathrm{to}-m\mathrm{and}wene\mathrm{th}\mathrm{at}\mathrm{to}\mathrm{about}ntR\mathrm{about}lbn\mathrm{about}\mathrm{th}te\mathrm{from}t-\mathrm{I\; am}he\mathrm{th}\mathrm{to}e})\times \mathrm{one}00\%$$

The procedure for assessing CA NA on the device is less time-consuming, economical and safe for the health of the researcher, since it takes 10-15 minutes, does not require the use of a large number of devices, radioactive substances and scintillation fluid, as in the prototype.

Subjects and research parameters.

In 16 patients with schizophrenia with the first attack of endogenous psychosis aged 18 to 25 years (average age - 20.8 ± 0.4), a comparative analysis of two methods for assessing the cytotoxic activity of NK lymphocytes was carried out: by measuring the level of ³ H-uridine in the cells targets K-562, that is, the prototype, and the number of target cells K-562, as in the proposed method. All patients were examined once before prescribing psychotropic therapy. Studies were performed on mononuclear cells isolated from peripheral venous blood of patients in a density gradient of ficoll-verographin with a density of 1.077 and consisting of monocytes and a pool of lymphocytes, 10-15% of which are NK lymphocytes.

Since both methods for assessing the CA of NK lymphocytes are associated with K-562 target cells remaining undegraded as a result of the cytolytic effect of NK lymphocytes on them, the aim of the study was to demonstrate that the results obtained using the proposed method are similar to those obtained using the known way. To confirm the identity of the results obtained, the cytotoxic test with both methods for assessing the CA of NK lymphocytes was set in different ways: in cell culture in the general group of patients and in subgroups of patients with low and high levels of NK activity; in cell culture without monocytes, which were removed by adhesion in plastic cups for one hour at 37 ° C and 5% CO ² ; in cell culture with the addition of serotonin at a concentration of 10 ^-6 and 10 ^-7 M.

Statistical Computing

All statistical studies were performed using StatSoft (USA), version 6. When comparing different groups, the U Mann-Whitney test was used. Correlation analysis was performed using Spearman Rank R.

Examples.

Example 1 (prototype).

Example 2 (the proposed method).

Example 3

Similarly to examples 1 and 2, but with the addition of serotonin at a concentration of 10 ^-6 M before suspension of the cytotoxic test to suspension of target cells K-562 and mononuclear cells of patients in the general group.

Example 4

Similarly to examples 1 and 2, but with the addition of serotonin at a concentration of 10 ^-7 M before suspension of the cytotoxic test to suspension of target cells K-562 and mononuclear cells of patients in the general group.

Example 5

Similarly to examples 1 and 2, but with the removal of monocytes from the suspension of mononuclear cells of patients in the general group before staging a cytotoxic test.

Example 6

Similarly to examples 1 and 2, but with an assessment of CA NK in a subgroup of patients with a high level of CA NK.

Example 7

Analogously to example 6, but with the addition of serotonin at a concentration of 10 ^-6 M before suspension of the cytotoxic test to suspend K-562 target cells and mononuclear cells in a subgroup of patients with high levels of CA NK.

Example 8

Analogously to example 6, but with the addition of serotonin at a concentration of 10 ^-7 M before suspension of the cytotoxic test, to suspend K-562 target cells and mononuclear cells in a subgroup of patients with high levels of CA NK.

Example 9

Similarly to examples 1 and 2, but with an assessment of CA NK in a subgroup of patients with a low level of CA NK.

Example 10

Analogously to example 9, but with the addition of serotonin at a concentration of 10 ^-6 M before suspension of the cytotoxic test, to suspend K-562 target cells and mononuclear cells in a subgroup of patients with low CA NK.

Example 11

Analogously to example 9, but with the addition of serotonin at a concentration of 10 ^-7 M before suspension of the cytotoxic test, to suspend K-562 target cells and mononuclear cells in a subgroup of patients with low CA CA.

Example 12

Analogously to example 9, but with the removal of monocytes from a suspension of mononuclear cells in a subgroup of patients with a low level of CA NK before staging a cytotoxic test.

A brief description of the drawings.

Table 1 presents the data of a comparative analysis of two methods for assessing the cytotoxic activity of NK lymphocytes in the general group of patients. You can see that the values of the level of cytotoxic activity of NK, determined by the radioactive method, as in example 1, and counting the number of target cells K-562 on the analyzer, as in example 2, are almost the same values. During statistical processing of the results, these data were fully confirmed, that is, significant differences between the average values of the determined indicators were not found (p> 0.05). Confirmation of the correctness of the results was the identification of statistically significant positive correlations between the level of cytotoxic activity of NK lymphocytes, determined in non-degraded K-562 targets by the radioactive method, on the one hand, and their number (Spearman r = 0.6; p <0.05), on the other hand.

Table 2 presents the data of a comparative analysis of two methods for assessing the cytotoxic activity of NK in the general group of patients with the addition of serotonin at a concentration of 10 ^-6 and 10 ^-7 M before removal of monocytes from the suspension of mononuclear cells before staging the cytotoxic test. The table shows that the values of CA of NK lymphocytes, determined by the level of radioactivity in non-degraded target cells K-562 on the β-counter and counting their number on the analyzer, are almost identical in all variants of the cytotoxic test. Statistical processing of the results fully confirmed these data, that is, significant differences between the average values of the determined indicators were not revealed (p> 0.05). Correlation analysis also confirmed the similarity of the results obtained by assessing the CA of the NK by the radioactive method and on the analyzer in different versions of the cytotoxic test. Namely, a high level of positive correlation was found between the results obtained by the two methods in cell cultures with the addition of serotonin at a concentration of 10 ^-6 and 10 ^-7 M (Spearman r = 0.81; p <0.001) and with the removal of mononuclear from the suspension monocyte cells (r = 0.95; p <0.001).

Table 3 presents the results of a comparative analysis of two methods for assessing the CA of NK lymphocytes in subgroups of patients with high and low levels of this activity, as well as when serotonin at a concentration of 10 ^-6 and 10 ^-7 M is added to the cell cultures of patients in each a subgroup and removal of monocytes from a suspension of mononuclear cells of patients with a low level of CA NK before staging a cytotoxic test of monocytes. The table shows that the values of the level of CA of NK lymphocytes, determined by the radioactive method on the β-counter and counting their number on the analyzer, are almost the same in all versions of the cytotoxic test. Statistical processing of the results also confirmed these data, that is, significant differences between the average values of the indicators determined by the radioactive method and on the analyzer were not detected in different versions of the cytotoxic test (p> 0.05). At the same time, a high level of positive correlation was found between the values of the cytotoxic activity of NK obtained by assessing the level of ³ H-uridine in undegraded targets and on the analyzer by the number of remaining targets (Spearman r = 0.81; p <0.001).

Thus, a new safe and economical time and cost method for assessing the cytotoxic activity of NK lymphocytes has been proposed. A comparative analysis of two methods for assessing the cytotoxic activity of NK lymphocytes in the general group of patients by counting the level of radioactivity in non-degraded K-562 target cells and counting their numbers on an automatic counter and cell analyzer revealed the complete identity of the results, which was confirmed in all variants of cytotoxic test. Moreover, the proposed method is simpler and shorter in execution, since it requires the use of only one device instead of several and takes 10-15 minutes against the 2-3 days required to evaluate the cytotoxic activity of NK in a radioactive manner. An important advantage of the proposed evaluation method is also the health safety of the researcher, since its implementation does not require the use of a radioactive label and scintillation fluid. It is known that NK lymphocytes are the first line of defense of the human body and animals from all sorts of foreign influences, which include infectious agents and their own transformed cells. Based on this, it can be assumed that the use of a simple and safe method for assessing the CA of NK lymphocytes will be widely used as a test system to detect a decrease in the activity of this important link of natural immunity in patients with psychopathological symptoms and various somatic diseases, primarily malignant neoplasms and diseases of an infectious nature, as well as when conducting screening tests to identify groups of people at risk of development among the population silent disease.

Table 1 A method for assessing the cytotoxic activity of NK The number of patients in the general group CA NK, IC,% By level ³ H-uridine in target cells 16 31.4 ± 3.5 (Prototype Example 1) By the number of target cells 16 37.1 ± 5.6 (The proposed method Example 2)

table 2 Experience Options The number of patients in the general group CA NK, IC,% Assessment of the level of ³ H-uridine in target cells (Prototype) Assessment on the analyzer by the number of target cells (The proposed method) Example 1 Example 2 With the addition of serotonin at a concentration of 10 × 10 ^-6 M fourteen 28.3 ± 4.7 29.5 ± 6.5 Example 3 With the addition of serotonin at a concentration of 10 × 10-7 M fourteen 34.8 ± 4.5 33.6 ± 5.2 Example 4 No monocytes 21.0 ± 4.4 Example 5 9 20.8 ± 5.2

Table 3 Experience Options Number of patients CA NK, IC,% Assessment of the level of ³ H-uridine in target cells (Prototype Example 1) Assessment on the analyzer by the number of target cells (The proposed method Example 2) In the subgroup of patients with high levels of CANK With monocytes 7 43.8 ± 4.2 46.7 ± 8.7 Example 6 With the addition of serotonin in concentration
10 ^-6 M 6 45.4 ± 2.6 43.8 ± 9.7 Example 7 With the addition of serotonin in concentration
10 ^-7 M 6 46.1 ± 3.9 47.6 ± 6.7 Example 8 In a subgroup of patients with low levels of CANK With monocytes 9 27.9 ± 6.1 21.7 ± 2.1 Example 9 With the addition of serotonin in concentration
10 ^-6 M 8 18.5 ± 6.0 15.5 ± 3.6 Example 10 With the addition of serotonin in concentration
10 ^-7 M 8 20.3 ± 5.4 26.4 ± 5.7 Example 11 Monocyte-free Example 12 7 20.4 ± 5.1 19.3 ± 5.5

Claims (1)

A method for assessing the cytotoxic activity of natural killer lymphocytes is carried out on K-562 target cells that remain undegraded after contact with NK lymphocytes, including the isolation of NK lymphocytes in mononuclear cells from the peripheral blood of patients, staging a cytotoxic test based on incubation of NK lymphocytes and K-562 target cells, characterized in that K-562 target cells are used that do not label ^{3 with} H-uridine, and the cytotoxic activity of NK lymphocytes is evaluated by counting the number of remaining undegraded target cells on an automatic cell counter and analyzer configured to detect cells with a diameter of 15 to 40 μm, after which the cytotoxicity index (IC) is calculated, in%, according to the formula: