Cross reactivity of T cell receptor on memory CD8+ cells isolated after immunization with allogeneic tumor cells.

Experiments on mice deficient in expression of class I major histocompatibility complex molecules showed that memory CD8+ cells recognizing the alloantigen by the direct allogeneic recognition mechanism selectively proliferated in response to heated allogeneic cells. Adoptive transfer of memory cells from mice expressing green fluorescent protein transgene to wild-type animals showed for the first time that long-living memory cells suppress the response of naive T cells and abolish their involvement in the pool of memory cells. The pool of long-living memory T cells was obtained in vitro with heated allogeneic stimulators. Apart from immunizing alloantigen, this clone recognized foreign molecules of the major histocompatibility complex. Cloning and sequencing of rearranged regions in memory T cells showed that two alpha-chains and one functional beta-chain are rearranged in cells of this pool. Only one alpha-chain was capable of forming protein product, which determines expression of only one form of T cell receptor. Experimental data directly confirm the hypothesis about degeneracy of recognition of allelic products of major histocompatibility complex molecules by T cell receptors. Suppression of the response of naive cells by memory cells probably underlies a previously unknown type of polarization of the immune response and determines clonal dominance and peripheral selection of T lymphocytes.

Colorimetric in vitro evaluation of T-cell cytotoxicity.

We propose a new colorimetric method for evaluation of specific T lymphocyte cytotoxicity based on the capacity of viable cells to incorporate alamar blue dye, an indicator of cell metabolic activity. This method is effective for highly specific strains of cytotoxic T lymphocytes and for primary T killers formed in vivo and constituting a small part of analyzed cell population. This test is a simple nonradioactive method for evaluating specific cellular cytotoxicity exhibiting sufficient sensitivity for evaluation of this parameter in the presence of effector cells.

[Multi-functionality of allospecific cytotoxic T-lymphocytes in vitro]. / Polifunktsional'nost' allospetsificheskikh tsitotoksicheskikh T-limfotsitov v sistemakh in vitro.

The data presented demonstrate the capacity of allospecific cytotoxic lymphocytes to fulfill noncytolytic regulatory functions. Cytotoxic lymphocytes suppress cell proliferation induced during development of a response in reactions of a mixed culture of lymphocytes and blast transformation irrespective of haplotypes of the cells involved in these reactions. At the same time, cytotoxic lymphocytes do not practically affect spontaneous proliferation of T-cells. The inhibitory effect of cytotoxic lymphocytes is expressed only upon direct contact with activated T-lymphocytes and is not relate to their apoptosis and death. The results obtained suggest polyfunctionality of allospecific cytotoxic lymphocytes expressed in the capacity of these lymphocytes to fulfill both effector (target lysis) and regulatory functions.

[Characteristics of the cells producing thymic chemotactic factor for bone marrow stem cells]. / Kharakteristiki kletok-produtsentov timusnogo khemotaksicheskogo faktora dlia stvolovykh élementov kostnogo mozga.

It was shown using complement-dependent cytolysis and monoclonal antibodies against CD4, CD8, and NK1.1 antigens that the cortisone-resistant CD3+4-8-NK1.1(-)-thymocytes spontaneously secreted a chemotactic transmitter inducing the release and directed migration of bone marrow cells. When estimating the general profile of the cytokines of these thymocytes by PCR with revertase, it was demonstrated the cells in question did not express cytokines with colony stimulating activities (SCF, IL-3, or GM-CSF) or cytokines affecting the migration of bone marrow stem elements (IL-2, 4, or 7). In addition, an active expression of gene bcl-2 was detected. Thus, the chemotactic cytokine inducing the release of bone marrow stem elements is a product of the cortisone-resistant long-living CD3+4-8-NK1.1(-)-T-cells of the thymus.

[Subforms of transcription factor Oct-1 synthesized in lymphocytes]. / Subformy faktora transkriptsii Oct-1, sinteziruemye v limfotsitakh.

Transcription factor Oct-1 is ubiquitous, participating in expression of the cell housekeeping genes as well as in differentiation of lymphocytes and activation of transcription of immunoglobulin genes in B cells. A new tissue-specific form of Oct-1 (Oct-1L) was found in lymphoid cells of bone marrow, lymph nodes, spleen, thymus, as well as in the cell lines of B and T lymphocytes at different stages of differentiation. This isoform was not found in embryonal and nonlymphoid tissues and cell lines. The complete structure of the oct-1L mRNA was determined, which generally corresponds to the structure of mouse Oct-1b. The difference between oct-1L and isoforms oct-a, b, and c functioning in all cells is replacement of the long first 5'-terminal exon (the 1U exon) encoding 21 amino acid residues with a short one encoding 10 amino acids in the isoforms Oct-1L and Oct-1R. Interestingly, attempts to find mature oct-1 mRNA simultaneously containing the 1L and 1U exons were unsuccessful. The parallel synthesis of "ubiquitous" isoforms Oct-1a, b, and c as well as tissue-specific Oct-1L and Oct-1R in lymphocytes and their precursors may be due either to a high demand of these cells for Oct-1 or to selective participation of different Oct-1 isoforms in regulation of the housekeeping genes and genes involved in the B and T cell differentiation and synthesis of imminoglobulins.

[The induction of the migration of bone marrow stem elements to the periphery by the cytokines of cortisone-resistant thymocytes]. / Induktsiia migratsii stvolovykh élementov kostnogo mozga na periferiiu tsitokinami kortizonrezistentnykh timotsitov.

We present evidence about the functional activity of factors produced by cortisol-resistant thymocytes. Experiments in vivo have shown that the administration of the supernatant prepared from cortisol-resistant thymocytes leads to a strong stimulation of endogenous colony formation, significantly prolongs survival of sublethally irradiated mice, increases the rate of restoration of the number of thymocytes in the thymus after sublethal irradiation, and contributes to the recovery of the humoral immune response of nude mice to T-dependent antigens. The results obtained suggest that along with other cytokines, cortisol-resistant thymocytes spontaneously produce a chemotactic factor inducing migration of stem cells from the bone marrow to the periphery.

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.

The costimulatory and differentiating activity of soluble class I MHC antigens for an autologous thymocyte population.

Combined cultivation of macrophages with syngeneic thymocytes resulted in accumulation of soluble H-2Kk antigens in culture medium. Incubation of intact autologous thymocytes with these soluble class I MHC molecules was shown to induce functional maturation of thymocytes assayed in local graft-vs-host reaction. Similar thymocyte costimulating activity was detected for the papain-solubilized purified H-2Kk antigens. Soluble class I antigens were shown to costimulate IL2 production by thymocytes in response to submitogenic doses of exogenous IL2 and to increase PHA-induced thymocyte proliferation. Soluble class I molecules were shown to increase the level of expression of function-associated membrane antigens, H-2Kk, CD8 and CD4, and to trigger thymocyte differentiation. The expression of I-Ak antigens remained invariable. It was also shown that soluble autologous class I molecules may function as direct amplifiers of thymocyte proliferation in autologous, but not allogeneic, mixed leukocyte reactions. It is concluded that soluble MHC class I molecules are capable of triggering functional and phenotype differentiation of syngeneic thymocytes and acting as restricted coaccessory molecules when thymocyte activation is induced by a submitogenic dose of different stimuli.

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.

[The formation of T-suppressors that suppress the activity of alloreactive T-cells after lymphocyte interaction with 2 different thymus-derived mediators]. / Obrazovanioe T-supressorov, podavliaiushchikh aktivnost' alloreaktivnykh T-kletok, pri vzaimodeistvii limfotsitov s dvumia razlichnymi mediatorami timusa.

The data are presented on simultaneous interaction of thymocytes and T-cells from lymph nodes with two humoral factors produced by macrophages and epithelial cells, main stromal elements of the thymus. Such contact of T-cells with two mediators resulted in the formation of T-suppressors capable of inhibiting the activity of syngeneic allospecific T-lymphocytes. These T-suppressors did not alter the response to another antigen but inhibited proliferation of donor cells. Involvement of such mechanism of T-suppressor formation in regulation of the immune status is discussed.

[Macrophages and the functional differentiation of thymocytes (a review of the authors' own data)]. / Makrofagi i funktsional'naia differentsirovka timotsitov (obzor sobstvenennykh dannykh).

Cellular, humoral, and genetic mechanisms of induction of T-effectors of the transplant vs. host reaction (THR) have been studied in two-cell culture of phagocyte mononuclears and thymocytes. A direct physical contact and similarity of H-2K locus of major histocompatibility complex between the cooperating cells in culture is required for successful induction of T-effectors of THR. Contact interaction of macrophages with thymocytes leads to accumulation of a 67 KDa humoral factor in the culture medium. Incubation of intact thymocytes with this factor leads to functional transformation of immature thymocytes into corresponding effector cells. Similarity of H-2K locus of the factor producers and intact lymphocytes is also required for successful humoral induction of the T-effectors. The surface H-2K antigen is able to induce formation of THR t-effectors from non-reactive thymocytes. The H2-K-specific mediator, affinity-isolated from the supernatant of the macrophage-thymocyte culture can also cause this induction.

[Changes in the expression of the surface antigens of thymocytes during their cultivation with macrophages]. / Izmenenie ékspressii poverkhnostnykh antigenov timotsitov v protsesse ikh kul'tivirovaniia s makrofagami.

The data on changes in expression of H-2 complex and Thy-1 antigens on cell surface of thymocytes resulting from their incubation with peritoneal macrophages has been presented. The process of joint cultivation of thymocytes with macrophages leads to significant decrease in number of cells with Thy-2-antigen and increase in that with H-2 complex antigens. An increase in H-2K+ cells in experimental thymocytes as compared to control ones was observed. No changes in H-2D expression was observed. A significant increase in Ia+ macrophages was observed after interaction with thymocytes as compared with intact mononuclear phagocytes.