The functional transformation of cytotoxic lymphocytes into T-suppressors under the influence of two mediators.

A set of CTL-lines in the system differing in the whole H-2 complex, as well as in distinct subregions of H-2, was established. These lines were routinely tested in Cr51-release assay to insure that they retained lytic activity and antigen-specificity. The result of the interaction of CTL with H-2Kb-molecule and alpha1-thymosine was transition of CTL into a reversible state of anergy. A high concentration of alpha1-thymosine is necessary for this transition. Anergic CTL caused specific suppressor activity that is functional if transformation of CTL into T-suppressors took place.

Peptides of a major histocompatibility complex class I (Kb) molecule cause prolongation of skin graft survival and induce specific down-regulatory T cells demonstrable in the mixed lymphocyte reaction.

Six individual peptides of the major histocompatibility complex (MHC) class I molecule H-2Kb were synthesized. Intravenous injection of peptide 6 into mice prolonged the survival of Kb (BL/6 or B10.MBR) skin grafts on allogeneic R101 and B10.AKM mice, respectively. This was specific, as control skin grafts from Kk (B10.BR) or Kd (DBA/2) donors, respectively, were rejected at the same time in both control and peptide-treated mice. The optimal doses for peptide 6, which is from the alpha 2 domain, were defined. The test system was the inhibition of proliferation in vitro of naive lymph node cells by syngeneic mitomycin c-treated spleen cells from R101 mice preimmunized with irradiated stimulator splenocytes of Kb (BL/6) origin. Down-regulation was specific, as proliferation in response to third-party allogeneic stimulator Kk (B10.BR) splenocytes was not inhibited. Of the six peptides of H-2Kb tested, potent down-regulatory cells were induced by peptides 2 (alpha 1 domain) and 5 and 6 (alpha 2 domain). The greatest down-regulatory activity was obtained by giving peptide 2 to mice that had already been immunized against H-2Kb by injecting EL4 cells. Under the same conditions, injecting peptide 2 did not induce any cytotoxic T cells. In contrast, specific cytotoxic lymphocytes (CTL) were induced when cells from primed mice were incubated for 4 days with heated stimulator cells from BL/6 mice. The data suggest that peptides from MHC class I molecules activate precursors of down-regulatory T cells, but not of CTL, and this may explain their ability to prolong skin allograft survival.

Immunosuppressive factor from liver induces apoptosis in thymoma EL-4 cells but not normal MHC class II-specific T lymphocytes.

An endogenously produced immunosuppressive factor (ISFnp, immunosuppressive factor-neutral protein), inducing a decrease in viability of thymoma EL-4 cells in vitro, was isolated from murine liver using ion exchange, gel filtration and hydrogen-bonding chromatography. Polyclonal rabbit antibodies against this factor were developed and attached to periodate-activated Sepharose CL-6B. The immunoaffine sorbent obtained significantly depleted the biological activity of ISFnp from tested fractions. The factor shows liver-specific location, an M(r) of about 70-80 kDa and consists of 2 subunits (40 and 42 kDa) as determined by SDS-PAGE and Western blotting. ISFnp induced DNA degradation in EL-4 cells similar to the cleavage of DNA onto olygonucleosomal fragments in dexamethasone-treated thymocytes. This DNA degradation preceded lysis of thymoma cells, suggesting an induction of apoptosis in ISFnp-treated EL-4 cells. Addition of the factor into primary mixed lymphocyte culture (MLC) strongly inhibited proliferative response but failed to induce any decrease in the ability of normal MHC class II-specific alloreactive cells to respond in the secondary MLC. Moreover, addition of ISFnp into primary MLC on the peak of proliferative response resulted in augmentation of secondary responses of primed cells as compared with the same quantities of primed cells from untreated cultures. These results suggest a possible role of liver both in deletion of transformed clones of T lymphocytes and formation of allospecific memory T cells.

[Use of histocompatibility class I molecule peptides for in vivo induction of specific T-suppressors in allogenic systems and prolongation of mouse skin graft life]. / Ispol'zovanie peptidov molekuly gistosovmestimosti klassa I dlia induktsii spetsifichnykh T-supressorov in vivo v allogennoi sisteme i prodleniia zhizni transplantata kozhi myshei.

Six synthetic peptides were selected for the individual molecule H-2Kb of the major histocompatibility complex class 1. Optimum conditions were elaborated for the induction of specific suppressor T cells in vivo by peptide 6 (alpha 2 domain) in an intravenous dose of 33 micrograms to the tail vein or 100 micrograms to the orbital sinus, followed by testing the suppressor activity in inhibiting in vitro proliferation in the three-cell MLC in response to irradiated cells of the stimulator Kb (BL/6) in the absence of suppressed response to the foreign stimulator Kk (B10.BR). Out of 6 different peptides of the same molecule H-2Kb, suppressor T cells were induced effectively by peptides 2 (alpha 2-domain), 5 and 6 (alpha 2-domain), while the high efficiency of suppressors is realized by memory cells along with peptides 2. Under the same conditions, in vivo peptide immunization did not induce cytotoxic T lymphocytes at all. In contrast, CTL were specific in their high efficiency where memory cells were in vitro pretreated with stimulators of warmed BL/6 cells. Intravenous administration of peptide 6 to mice gave rise to prolongation of Kb (BL/6 or B10.MBR)-induced skin transplantation during the transfer from allogenic murine R101 and B10.AKM, respectively, but without the terms of skin prolongation at all with the stranger donors Kk (B10.BR) or Kd (DBA/2).

[Isolation, study of activity, and characteristics of an immunosuppressive liver factor causing apoptosis of EL-4 thymoma cells in vitro]. / Vydelenie, izuchenie biologicheskoi aktivnosti i kharakteristika immunosupressornogo faktora pecheni, vyzyvaiushchegi apoptoz kletok timomy EL-4 in vitro.

Immunosuppressive factor (ISFnp) which inhibits proliferation and viability of thymoma EL-4 cells was isolated from mouse liver. The testing procedure based on the biotransformation of MTT-tetrazolium by mitochondrial enzymes of viable cells allowed us to purify this factor as individual peak of protein, that allowed to obtain polyclonal rabbit antibodies to this factor. By the methods of double immunodiffusion, gel-filtration and SDS-PAGE with subsequent immunoblotting we shown that this factor specifically localized in liver and consists two subunits of 40 and 42 kDa which form dimers with apparent M(r) about 70-80 kDa. This factor induced olygonucleosomal DNA cleavage in EL-4 cells in vitro similar to dexamethasone-induced DNA-degradation in thymocytes. This cleavage preceded to lysis of EL-4 cells assessed by 51Cr-release, that strongly suggested an involvement of apoptosis in cell death mechanism. ISFnp strongly inhibited blast-transformation and proliferation in MLC-responses to mutant MHC class 2 molecule. This effect was not due to deletion of allo-reactive clones, because removing of this factor from MLC cultures treated with one for 4 days resulted in blast-transformation without any reduction of the number of viable cells as well as their capacity for secondary responses to the same antigen as compared with control cultures.

L3T4+ but not LYT2+ T-helper cells are required for in vitro maturation of in vivo primed T cells in the cytotoxic response to MHC class I disparate cells following footpad immunization.

The induction characteristics of major histocompatibility complex (MHC) class I-specific cytotoxic T lymphocytes (CTL) after footpad immunization were studied. Primary CTL were generated in the regional lymph nodes of C57Bl/6 mice by footpad injection with 10(7) irradiated (2000 rad) spleen cells from MHC class I mutant mouse strain (bm1) followed by a short in vitro culturing without antigen. The requirement of accessory cells and L3T4+ T cells during in vitro maturation of in vivo primed CTL precursor (CTLp) was shown. Moreover, using inhibitory antibodies, the need for IL-2 and IL-4 for in vitro maturation of CTL was established. We have suggested that accessory cells act at the level of L3T4+ T cells which in turn non-specifically up-regulate the CTL response through the production of growth and differentiation factors. Thus, the T-helper population of L3T4+ but not Lyt2+ phenotype appears to be recruited in the in vitro maturation of in vivo primed CTLp in a given system. Possible mechanisms of this phenomenon are discussed.

[Use of peptides of major histocompatibility type I molecules for induction of specific T-suppressors in vivo in an allogeneic system and extending the life of a murine skin transplant]. / Ispol'zovanie peptidov molekuly gistosovmestimosti klassa I dlia induktsii spetsifichnykh T-supressorov in vivo v allogennoi sisteme i prodleniia zhizni transplantata kozhi myshei.

Six synthetic peptides represented as variable sequences of the MHC class 1 molecule H-2Kb were synthesized. Appropriate conditions were elaborated for induction of specific suppressor T cells in vivo by peptide 6 (alpha 2 domain) that is introduced intravenously in a dose of 33 micrograms into the tail vein or 100 micrograms into the orbital venous sinus with subsequent testing for inhibition of proliferation in vitro in response to irradiated stimulator cells Kb (BL/6), whereas the stimulator Kk (B10.BR) did not induce any suppressor activity. Of six different peptides tested, suppressor T cells were induced efficiently by peptides 2 (domain alpha 1), 5, and 6 (domain alpha 2), while the highest suppressor efficiency was realized by i/v injection of peptide 2 into mice preimmunized with EL-4 cells. In the same conditions in vivo, immunization by peptides did not induce any cytotoxic T lymphocytes (CTL). In contrast, specific CTL of high efficiency were induced when memory cells were incubated in vitro with heated BL/6 stimulator cells for 4 days. Intravenous injections of peptide 6 into mice gave rise to prolongation of (BL/6 or B10.MBR) skin graft retention Kb when transferred into allogeneic R101 and B10.AKM mice, respectively, but had no influence on rejection of skin grafts from foreign donors of Kk (B10.BR) or Kd (DBA/2) haplotype, respectively.

[The use of monoclonal antibodies for detecting the influenza virus]. / Ispol'zovanie monoklonal'nykh antitel dlia vyiavleniia virusa grippa.

The possibilities of using influenza A (Leningrad) 385/80 (H3N2) virus matrix protein-specific FITC-labeled D8 monoclonal antibodies in immunofluorescence assays were investigated. The virus antigen accumulation was detected in chorioallantoic cells of chick embryos. Exhibiting the type-specific properties, the fluorescent antibodies stain the perinuclear space, cytoplasmic membrane, and granular structures in the cytoplasm of infected cells. The haemagglutination test tires in the corresponding specimens were at least 1:16.

[The induction of alloantigen-specific cytotoxic T-lymphocytes by the intravenous immunization of mice]. / Induktsiia alloantigen-spetsificheskikh tsitotoksicheskikh T-limfotsitov pri vnutrivennoi immunizatsii myshei.

It is shown possible to induce specific cytotoxic T-lymphocytes (CTL) in the course of intravenous (i.v.) immunization of Balb/c mice by 2000-rad-irradiated allogeneic C57Bl/6 splenocytes in a dose of 9 x 10(7). The induced CTL express Thy1.2+L3T4-Ly2+ cell surface markers. No correlation was observed between the level of cytotoxic activity and the ability to inhibit proliferation in the population of lymphocytes primed by i.v. immunization.

Generation of specific antitumor cytotoxic T-lymphocytes in monoculture can be inhibited by T-suppressors from tumor-bearing mice.

A new model for the generation of specific antitumor cytotoxic T lymphocytes (CTL) was proposed. In contrast to other models, it allows to generate secondary effector CTL (CTL-2) without tumor stimulator cells in vitro (in monoculture). C57BL/10 mice or/and C57BL/6 mice were immunized by injection with gamma-irradiated syngeneic tumor cells into the footpads. For estimation of cytotoxic activity, chromium-51 release assay was used. It has been shown that effector CTL were absent in the lymph nodes after 1-fold as well as 2-fold immunization. Cytotoxic cells have not been found in 1-fold immunization even after maturation of the lymphocytes in monoculture. Specific CTL were detected only after secondary immunization and subsequent cultivation in vitro. Effector cells had Thy1.2, CD8+, CD4- phenotype. Presence in vitro of exogenous recombinant interleukin 2 (rIL-2) was needed for the generation of CTL-2 against Mech-11 sarcoma but not against EL4 lymphoma. The spleen cells from B10 mice with progressively growing Mech-11 tumor specifically suppressed the maturation of CTL-2 against Mech-11 in monoculture. Since suppression took place in the presence of exogenous rIL2 in monoculture, it was suggested that suppression was not resulted by negative influence of the suppressor cells upon endogenic IL-2 production. The treatment of the suppressor cells with monoclonal antibody (Mab) against Thy1.2 as well as against CD4 or CD8 markers plus complement (C') considerably decreased Ts activity. Obviously, two distinct subsets of T-lymphocytes were required for suppression.

Induction of alloantigen-specific cytotoxic and suppressor activities of lymphocytes by intravenous immunization of mice when donor and recipient differ in class II MHC molecule.

Induction of alloantigen-specific cytotoxic and suppressor activities of lymphocytes was studied upon intravenous immunization of recipients C57Bl/6 with B6.C-H-2bm12 donor's splenocytes irradiated with 2000 rads and introduced in a dose of 9 x 10(7). A possibility was shown to induce a specific cytotoxic and suppressor activities may be induced individually. We studied a possibility to detect the suppressor activity of immune splenocytes in the reaction of skin graft rejection. No significant prolongation of the B6.C-H-2bm12 skin graft life was noted in C57Bl/6 recipients, irradiated with 2000 rads, under the action of adoptive transfer of the immune syngenic splenocytes in comparison with the grafts life in recipients received the adoptive transfer of intact splenocytes.

[Use of a series of synthetic peptides and purified major histocompatibility complex I (H-2K(b)) molecules for inhibiting T-suppressors, specific for the K(b) molecule, and their induction by peptides in vivo]. / Ispol'zovanie nabora sinteticheskikh peptidov i ochishchennoi molekuly glavnogo kompleksa gistosovmetimosti klassa I (H-2K(b)) dlia ingibirovaniia T-supressorov, spetsifichnykh k toi zhe molekule K(b), i ikh induktsiia peptidami in vivo.

Six synthetic peptides of the MHC class 1 molecule corresponding to individual H-2kb participants in amino acid sequences of domains alpha 1 (peptide 1 and 2) and alpha 2 (peptide 3, 4, 5, 6) were selected. Kb-specific suppressor T cells (Ts) were in vivo in mice, then pretreated with a set of peptides and assayed by proliferation decrease in the third-partial mixed lymphocyte culture (MLC). Effector function of Ts was abolished by the complex of the alpha 2-domain peptides (but not by the alpha 1-domain peptides) and decreased by each peptide (4, 5, 6) of the alpha 2-domain. Both alpha 1 and alpha 2 domain peptides, added at high concentrations, decreased the otherwise efficient enrichment of Ts during the absorption-elution procedure on the syngeneic macrophage (MP) monolayers. A similar significant effect was observed the purified Kb molecule (100 mg/ml) on the allogeneic MP monolayer. Interaction between Ts receptors with some MHC peptides indicates effector Ts activation in vivo by induction with peptides 5+6 of the alpha 2 domain. The fine mechanisms of interaction between MHC class I molecule epitopes and T cell receptors (TCR) of each of the T cell subsets separately are under study now.

[The stimulation of the proliferative reaction of immune splenocytes by monoclonal antibodies to the beta-chain of the LFA-1 molecule can lead to an increase in their cytotoxicity]. / Stimuliatsiia proliferativnoi reaktsii immunnykh splenotsitov monoklonal'nymi antitelami k beta-tsepi molekuly LFA-1 mozhet privodit' k usileniiu ikh tsitotoksichnosti.

A system for investigation of relationship between proliferation and cytotoxicity of immune splenocytes fractions stimulated by monoclonal antibodies (mAb) to beta-chain LFA-1 molecule was elaborated. To obtain different proportional contents of effector cells in populations, the immune splenocytes were eluted from donor, third-party, and recipient macrophage monolayers. Cytotoxic indices of effector splenocyte fractions were compared before and after their proliferation in mixed lymphocyte culture. The results do not exclude the role of proliferation in increasing the effector cells proportions in populations stimulated by mAb to beta-chain LFA-1 molecule.

[Monoclonal antibodies to the beta-chain of the LFA-1 molecule promote an increase in the cytotoxic index of effector lymphocytes and stimulate their proliferation]. / Monoklonal'nye antitela k beta-tsepi molekuly LFA-1 sposobstvuiut povysheniiu tsitotoksicheskogo indeksa éffektornykh limfotsitov i stimuliruiut ikh proliferatsiiu.

The influence of monoclonal antibodies (mAb) to LFA-1 molecule beta-chain on the functional activities of immune splenocyte fractions which were eluted from donor, third party and recipient macrophage monolayers was studied. The splenocyte fractions were treated with the mAb and tested for cytotoxicity and proliferation. MAb to LFA-1 molecule beta-chain (without co-stimulation by other antibodies) were found to stimulate the immune response of the splenocyte fractions in both functional tests. The observed effect might be non-specific. The relationship between the stimulation of proliferative and cytotoxic responses of the immune splenocyte fractions and the treatment with mAb of LFA-I molecule beta-chain remains obscure.

Utilization of the MHC class I (H-2Kb) purified molecule and its synthetic peptides for inhibition of Kb-specific suppressor T cells and their induction in vivo by the MHC peptides.

Six synthetic peptides of the MHC class I molecule corresponding to individual H-2Kb participants in amino acid sequences of domains alpha 1 (peptide 1 and 2) and alpha 2 (peptides 3, 4, 5, 6) were selected. Kb-specific suppressor T cells (Ts) were induced in vivo in mice, then pretreated with a set of peptides and assayed by proliferation decrease in a three-cell lymphocyte culture (MLC). The effector function of Ts was abolished by the complex of the alpha 2-domain peptides (but not by the alpha 1-domain peptides) and decreased by particular peptides separately (4, 5, 6) of the alpha 2-domain. Both alpha 1- and alpha 2-domain peptides, added in high concentration, decreased otherwise efficient enrichment of Ts during the absorption-elution procedure on the syngeneic macrophage (M psi) monolayers. A similar significant effect was observed using the purified Kb molecule (100 micrograms/ml) in the allogeneic M psi monolayer. Interaction between Ts receptors and some MHC peptides indicates in effector Ts activation in vivo by induction with peptides 5 and 6 of the alpha 2-domain. The fine mechanisms of interaction between MHC class I molecule epitopes and T-cell receptors of each of the T-cell subsets separately are presently being studied.

[Effect of the Ly2 molecule on the function of alloantigen-specific effector cytotoxic T-lymphocytes as a function of the variability of their receptors' affinity]. / Vliianie molekuly Ly2 na funktsiiu alloantigenspetsifichnykh éffektornykh tsitotoksicheskikh T-limfotsitov v zavisimosti ot variabel'nosti srodstva ikh retseptorov.

To study T cell receptors' affinity alloantigen-specific anti-K cytotoxic T-lymphocytes (CTL) were divided on fractions by elution from donor K and third-party macrophage monolayers. The functional activity of CTL was suppressed by anti--Ly2 monoclonal antibodies (mAb), 51-Cr-labelled transformed fibroblasts (L-cells) transferred with the H-2K gene were used as targets. The results demonstrate that primary CTL enriched on third-party macrophage monolayers are the most sensitive to anti-Ly2 mAb. T-cells exhausted on third-party monolayer and then enriched on donor monolayer were resistant to treatment with the mAb. Secondary CTL enriched on donor monolayer were resistant to treatment with anti-Ly2 mAb even should they were not exhausted on third-party monolayer. These results show that Ly2 (CD8) molecule plays an essential role in the interaction of CTL with MHC class I molecule only if T-cell receptor has low affinity.