[Use of heat shock of antigen-presenting cells for functional testing of allospecificity memory T-cells]. / Ispol'zovanie teplovogo shoka antigenprezentiruiushchikh kletok dlia funktsional'nogo testirovaniia allospetsivicheskikh T-kletok pamiati.

For many years, the search for the appropriate method of testing the functional activity of the memory T-cells was an urgent problem and determined progress in the study of immunological memory. We proposed simple methods of functional testing the memory of CD8+ T-cells specific to the H-2Kb alloantigen based on measuring their proliferation in response to heat-treated allogenic splenocytes and cells of allogenic tumors in vitro. Primary proliferative response to the alloantigen was shown not to develop when the allogenic antigen-presenting cells were subjected to an acute (45 degrees C, 1 h) or moderate (42 degrees C, 30 min) heat shock. The block of the primary allogenic response of naive T-lymphocytes to the heated splenocytes could not be abrogated by the addition of exogenous IL-2 and was not due to deletion or suppression of antigen-reactive clones. On the contrary, the long-lived memory CD8+ T-cells induced in the course of the primary in vivo response were capable of proliferation in response to heat-treated allogenic stimulators carrying the same immunizing antigen. The different response of the naive T-cells and memory T-cells to the allogenic stimulators subjected to a heat shock might be due to a strict dependence of the naive T-cells on the inducing co-stimulation provided by the B7 ligand, whose expression was suppressed in the cultures containing the heat-treated stimulator cells. These results probably suggest that a specific immunoregulatory mechanism exists that is based on a disorder in costimulatory functions due to the cellular stress-response induced in the antigen-presenting cells.

[The beta-carotene stimulation of cellular immunity reactions in mice]. / Stimuliatsiia beta-karotinom reaktsii kletochnogo immuniteta u myshei.

The immunomodulating properties of synthetic beta-carotene were studied in C57Bl/6 and BALB/c mice using the tests of proliferative, cytotoxic and suppressor activity, and evaluating the adhesive capacity of macrophage lineage cells. Long-term feeding of C57Bl/6 mice with beta-carotene microgranules (0.1-0.5 mg of active substance per mouse) led to enhanced T cell proliferative response to ConA, which lasted for 15-45 days. Administration of beta-carotene oil solution to BALB/c mice previously immunized with alloantigens (0.17-0.34 mg of beta-carotene per mouse) enhanced T-cell cytotoxicity against L-929 and YAC-1 cells and macrophage cytotoxicity against L-929 cells. The treatment also reduced T-suppressor activity as shown in the assays of inhibition of the lymphocyte blast transformation reaction and mixed lymphocyte culture. The treatment with both preparations of beta-carotene enhanced the adhesive properties of macrophages and related cells, and induced the increased production of oxygen active radicals by these cells.

The influence of tumor immunity suppressors on the effector stage of human and animal lymphokine-activated killer cells.

Spleen cells of tumor-bearing mice suppressed the cytolytic activity of syngeneic LAK cells when added to the mixture of LAK cells and target cells at the beginning of the cytotoxicity test. Spleen cells of MC 14 tumor-bearing mice acquired the suppressor potential as early as 10 days after tumor transplantation; the suppressor activity in the EL 4 and X63-Ag8.653 tumor-bearing animals was first revealed at the 30th day and manifested itself up to the 120th day. The suppressor activity was expressed in a dose-dependent manner, both by unfractionated spleen cells and nylon wool-passed and plastic-adherent sub-populations. Similar results were obtained during the analysis of anti-tumor immunity suppressors in bladder cancer patients. MNC, nylon wool-passed and plastic-adherent cells of patients with stages I-II disease suppressed the cytotoxicity of autologous LAK cells in 2/6 cases; all patients [4] with III-IV stage possessed such suppressor activity. Presumably, the tumor growth induces the activity of suppressor T cells and monocytes/macrophages. The suppressor activity can interfere with the antitumor effect of autologous (syngeneic) LAK cells at the effector stage.

[The effect of a clover extract on the proliferation of human and murine lymphocytes in vitro]. / Vliianie ékstrakta klevera na proliferatsiiu limfotsitov cheloveka i myshi in vitro.

The extracts were prepared from meadow clover harvested at the stages of blossoming and budding. The major biological activity of such extracts is represented by flavonoid compounds. The influence of extracts on the proliferation of peripheral blood mononuclear cells obtained from healthy donors and of inbred mouse splenocytes in vitro was analyzed. Both preparations stimulated cellular proliferation. The lever of stimulating activity correlated with the stage-dependent concentration of flavonoids.

[A comparison of the suppressor and cytotoxic activities of the blood mononuclear cells during the adoptive immunotherapy of cancer patients using lymphokine-activated killers with a low dose of recombinant interleukin-2]. / Sravnenie supressornoi i tsitotoksicheskoi aktivnosti mononuklearnykh kletok krovi pri adoptivnoi immunoterapii onkologicheskikh bol'nykh limfokinaktivirovannymi killerami s nizkoi dozoi rekombinantnogo interleikina-2.

The suppressor and cytotoxic activities of mononuclear blood cells (MNC) were studied in 70 cancer patients (melanoma, renal carcinoma) undergoing adoptive immunotherapy (AIT). In the course of AIT the patients' MNC were treated in vitro with the recombinant interleukin-2 (RIL-2) in order to generate the lymphokine-activated killer (LAK) cells. Then patients received i/v 2.5-13.6 10(9) autologous LAK cells and RIL-2 (75000 u). Each course included 2-3 repeated infusions; the patients received 1-5 courses according to their clinical conditions. The cytotoxic activity of MNC was assessed by a routine method; but for evaluation of the suppressor activity we used a new technique based on separation of MNS populations in the Percoll gradient. Twenty-four hours after the completion of each AIT course the suppressor activity of MNC decreased drastically up to the zero level in some patients. The decrease in the suppressor activity inversely correlated with the rise in the cytotoxic activity on Mel-I (LAK-sensitive) and K-562 (natural killer-sensitive) target cells. The level of cytotoxicity in some patients reached 51.2%.

[The preclinical assessment of the activity and pharmacological action of native human interleukin-2]. / Predklinicheskaia otsenka aktivnosti i farmakologicheskogo deistviia nativnogo interleikina-2 cheloveka.

It was demonstrated in in vivo and in vitro experiments that interleukin-2, obtained by cultivation of donor lymphocytes and purified by gel filtration, induces the production of mouse and human killer lymphocytes possessing high cytolytic activity against tumor cells. Interleukin-2 does not cause irreversible changes of the physiological and morphologic indices of vital activity in mice.

Down-regulation of LAK cell-mediated cytotoxicity: cancer and ageing.

A comparative study was made of the generation of lymphokine-activated killer (LAK) cells in patients with melanoma and healthy donors of different age groups. Significant reduction of effector cell cytotoxicity in patients following 72 h culture with 1,000 U/ml or recombinant IL-2 (rIL-2) as well as a decreased ability to generate LAK cells in elderly individuals were shown to be correlated with suppressor cell activation in rIL-2 stimulated cell population. Suppressor effect depends on monocytes and T-lymphocytes: partial abolition of suppression in LAK cells was observed following removal of adherent cells or treatment with OKT8 monoclonal antibodies and complement.

Treatment with interleukin-2 in an immunodepressed state.

Cytotoxic T-lymphocyte (CTL) and lymphokine activated killer (LAK) cell (fraction II) precursors with a density of 1.077-1.067 g/ml and suppressor cells (fraction I) with a density of 1.067-1.056 g/ml were isolated by the separation of cancer patient peripheral blood mononuclear cells (MNC) on a Percoll gradient. Cells from fraction I inhibited the generation of CTL in mixed lymphocyte-tumor culture (MLTC) and LAK cells when added to fraction II lymphocytes at a ratio of 1:1 at the beginning of the culture. The effect was dependent on the dose of added suppressor cells and resistant to mitomycin-C treatment. Treatment of cell fractions prior to culture with monoclonal antibodies and complement showed that CTL precursors and suppressor cells were OKT3+/OKT8+. Cells from fraction I possessed suppressor activity in all patients examined but only in 4 of 10 healthy donors. Studies of monocytes and T-lymphocytes isolated from fraction I demonstrated that in cancer patients both monocytes and T-lymphocytes functioned as suppressors whereas in healthy donors, the monocytes mediated suppression. The data obtained provide evidence for an increased suppressor cell activity in cancer patients which can inhibit the generation of cytotoxic antitumor response with interleukin-2 (IL-2) in vitro.

[Conditions for antitumor cytotoxic T-lymphocyte formation and inhibition of their activity by T-suppressors in mixed culture of human lymphocytes and tumor cells]. / Usloviia obrazovaniia protivoopukholevykh tsitotoksicheskikh T-limfotsitov i podavlenie ikh aktivnosti T-supressorami v smeshannoi kul'ture limfotsitov i kletok opukholi cheloveka.

Conditions for antitumour autolytic T lymphocytes (CTL) induction during human mixed lymphocyte tumour cell culturing (MLTC), as well as the patterns of CTL activation abolition in the experimental system by suppressor cells were investigated. The responders used in MLTC were peripheral blood lymphocytes of colorectal cancer patients fractionated by means of multilayer Percoll gradients centrifugation (the density of layers being 1.077, 1.067, 1.056 g/ml). The cells of the second fraction collected in the density interphase of 1.077-1.067 g/ml (more than 90% of population belonging to T lymphocytes), when used as responders in MLTC, developed an autolytic activity against autologous and allogeneic tumour target cells. The cell of the first fraction (collected in the interphase of 1.067-1.056 g/ml) added to the second fraction cells at the beginning of MLTC, prevented the following CTL induction. The first fraction contained T-suppressor cells capable of strongly interfering with antitumour CTL activity.

[Cytotoxic and suppressor activity of human peripheral blood lymphocytes stimulated by interleukin-2 and phytohemagglutinin before and after fractionation in the percoll gradient]. / Tsitotoksicheskaia i supressornaia aktivnost' stimulirovannykh interleikinom-2 i fitogemaggliutininom limfotsitov perifericheskoi krovi cheloveka do i posle razdeleniia v gradiente perkolla.

The cultivation of peripheral blood lymphocytes (PBL) obtained from patients with colorectal or bladder carcinoma and melanoma and from healthy donors in the presence of interleukin-2 (IL-2) and PHA resulted in the induction of cytotoxic activity against autologous and/or allogeneic tumour cells in 12 out of 13 patients and in 10 out of 10 donors. A higher level of cytolytic activity was achieved when PBL were separated by means of Percoll density gradient (1.077; 1.067 and 1.056 g/ml) centrifugation and the cells of fraction II (1.077-1.067 g/ml) were employed in the experiment, the level of cytotoxicity being elevated in all cases (1.7-fold elevation in donors and 2-fold elevation in patients on the average). The addition of fraction I (1.067-1.056 g/ml) to fraction II prevented (PHA + IL-2)-mediated induction of cytotoxic activity in all the patients, but in 4 out of 10 donors, i.e. cells of fraction I expressed a suppressor activity. The immunofluorescent analysis has shown that fraction II was enriched by T cells (92%) and depleted of monocytes (7%), as compared to unseparated PBL (66% and 27%, respectively). On the contrary, fraction I was characterized by a decreased T cell ratio (36%) and an increased monocyte level (up to 69%).

[Production of monoclonal antibodies to antigens of specific mouse T-suppressors]. / Poluchenie monoklonal'nykh antitel k antigenam spetsificheskikh T-suppressorov myshei.

F1(MSU X WAG) rats were immunized with anti B6 BALB/c specific suppressor T cells (SSTC), purified by absorption/elution technique, with the following fusion of splenocytes to NS-I myeloma cell line. Hybrids were screened for their ability to affect SSTC, cytotoxic T lymphocytes (CTL) and producers of macrophage migration inhibition factor (MIF-producers) all triggered by in vivo priming with allogeneic cells. Two hybridoma cell lines--C1 and C4 inactivated SSTC by approximately 50%, leaving CTL and MIF-producers intact. C4 were also active in vivo, if injected as ascitic fluid from nu/nu mice, though to a lesser extent than in vitro.

[Separation of rat serum antisuppressor antibodies eliminating T-suppressors and stimulating their generation in mice in vivo]. / Razdelenie antisupressornykh antitel syvorotki krysy, éliminiruiushchikh T-supressory i stimuliruiushchikh ikh obrazovanie u myshei in vivo.

Immunization of rats with enriched murine specific T-suppressors (STS) permitted obtaining the antisuppressor serum (ASS) which selectively inactivated in vitro the capacity of the STS to inhibit the proliferation of T-lymphocytes in a mixed lymphocyte culture in response to allo-antigens. Two opposite effects of the ASS in vivo were demonstrated: elimination of T-suppressors (on ASS administration 4 days after immunization) and stimulation of their formation (on ASS administration before immunization or to non-immunized mice). It is assumed that the two opposite effects of the ASS in vivo are caused by two different types of antibodies to unidentified markers of STS to an antigen of differentiated STS and to an antigen expressed on their precursors.

Inactivation of specific anti-H-2 suppressor T cells by antisera to I-J and I-C subregion products of the H-2 complex.

Two antisera to Ia antigens, products of the H-2 complex I-Cd and I-JkEk subregions, respectively, have been obtained by immunization of the F1 hybrids of recombinant strains of mice. These antisera are shown to display a 50% cytotoxic effect in vitro, in the presence of complement, upon lymphocyte populations immune to the H-2-complex antigens and enriched for specific suppressor T cells (SSTC) by fractionation on a monolayer of target cells. The specificity of anti-Ia cytotoxins is shown by cross-antibody absorption with T and B cells of mice originating from the recombinant H-2 haplotypes and bearing either particular I-Cd, I-Jk and I-Ek antigens, or their combinations. Anti-I-Cd cytotoxins were found to react with both B and T cells, but at a different rate, and the anti-I-JkEk serum contains two antibody types directed to I-Ek and I-Jk products, respectively, the latter being able to react preferently with T cells. Although both antisera do inactivate the in vitro SSTC function in the presence of complement to a similar degree, the inactivating action of the anti-I-Cd serum, but not that of the anti-I-JkEk serum, occurs without complement. SSTC are shown to bear both Ia-antigens, I-J and I-C, as shown by both inactivation of the anti-suppressor effect of the antisera absorbed with spleen cells of different H-2 origin, and variation of the H-2 origin of SSTC pretreated with the intact antisera. It is suggested that these two markers, located on the same SSTC, function differently in SSTC immune to the H-2 antigens, and I-C antigen expression on the SSTC surface is presumed to be required for their interaction with the inhibited responder T cells proliferating in MLC.

[Ia antigens: markers of specific T suppressors immune to H-2 complex antigens]. / Ia-antigeny--merkery spetsificheskikh T-supressorov, immunnykh k antigenam kompleksa H-2.

Two antisera to Ia antigens, products of the H-2 complex I-Cd and I-JkEk subregions, respectively, have been obtained by immunisation of the F1 hybrids of recombinant strains of mice. These antisera are shown to display the 50 per cent cytotoxic effect in vitro in the presence of complement upon lymphocyte populations immune to the H-2 complex antigens and enriched for specific suppressor T cells (SSC) by fractionation on the monolayer of target cells. The specificity of anti-Ia cytotoxins is shown by the cross antibody absorption with T- and B-cells of mice originated from the recombinant H-2 haplotypes and bearing either particular I-Cd, I-Jk and I-Ek antigens, or their combinations. Anti-I-Cd cytotoxins are found to react with both B and T cells at a different rate, and the anti-I-JkEk serum contains two antibody types directed to I-Ek and I-Jk products, respectively, the latter being able to react preferently with T cells. Although both antisera do inactivate the in vitro SSC function in the presence of complement at a similar degree, the inactivating action of the anti-I-Cd serum, but not that of the anti-I-JkEk serum, occurs without complement. SSC are established to bear both Ia-antigens, I-J and I-C on the same cell, as demonstrated by the cross antibody absorption and variation of the H-2 origin of SSC. These two markers are suggested to function differently in the SSC immune to the H-2 antigens and the I-C antigen expression on the SSC surface is presumed to be required for their interaction with the inhibited responder T cells proliferating in MLC.

[Selective inactivation of specific T-suppressors immune to H-2 complex antigens and prevention of their generation in vivo by antisuppressor xenogenic antibodies]. / Izbiratel'naia inaktivatsiia spetsificheskikh T-suppressorov, immunnykh k antigenam kompleksa H-2, i predotvrashchenie ikh generatsii in vivo antisupressornymi ksenogennymi antitelami.

Rats were immunized with mouse lymphocytes enriched by the absorption-elution technique with specific suppressor T-cells (STC) immune to antigens of the H-2 complex. The anti-suppressor sera (ASS) obtained being absorbed with mouse erythrocytes and lymph node cells killed in the presence of complement about 30 per cent of the STC-enriched cell population and inactivated the STC in vitro function in a selective fashion, not affecting the function of other T-cell subclasses, killers and MIF-producers, immune to the same H-2 antigens. The STC inactivating ASS action occurred partly in the absence of complement irrespective of the STC strain origin, STC immunological specificity in the H-2 system and the intensity of the STC activity. This ASS action was abolished by exhaustion of antibodies only with STC containing cell suspensions. In contrast, intact (non-enriched) mouse STC appeared to be able to induce a mixture of rat antibodies inactivating partly all three T-cell subclasses assayed. Infections of ASS to mice prevented them from the in vivo STC generation and gave rise to inhibition of the syngeneic tumor growth in the specifically preimmunized mice.

Requirements for induction of specific suppressor T cells and detection of their H-2 antigen-binding receptors by fractionation on target cell monolayers.

The MC-resistant specific suppressor T cells that inhibit DNA synthesis and CTL generation in MLC were induced in vivo by gamma-irradiated allogeneic lymphoid cells in a large dose. MLRs were inhibited only slightly when triggered by third-party cells, even neighbouring with the corresponding stimulators. Unlike the irradiated cells, intact allogeneic lymphoid cells induced a mixture of macrophage-like and T-cell suppressors with a pronounced non-specific component of the action. Syngeneic cells induced low active non-specific suppressors of macrophage type only. The suppression was not due to a cytotoxic effect, since specific T suppressors differed from CTL by conditions of induction and high sensitivity to gamma-irradiation and from CTL precursors by high sensitivity to CY and HC. The specific T-suppressors could be selectively removed by adherence to a macrophage monolayer of the corresponding donor. The subsequent elution of adherent lymphocytes with pronase resulted in enrichment of specific T suppressors by a factor of 30 and 2.6, as judged by reduction in the number of lymphocytes required for 50% inhibition of DNA synthesis and 33% inhibition of CTL generation, respectively. The high specificity of this enrichment is shown by using both syngeneic monolayers for fractionation and third-party stimulators in MLR for testing and by disappearance of slight non-specific suppression caused by non-fractionated suppression. Complete inactivation of the eluted suppressors with anti-Thy-1.2 and anti-T antibodies, their resistance to anti-Mls antibodies, carrageenin, and carbonyl iron, together with the data of autoradiographic study and total DNA synthesis in the population indicate that the eluted highly specific suppressors represent mainly DNA-synthesizing large and medium T lymphocytes.