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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.

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.

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.

A new model for the generation of specific antitumor cytotoxic T-lymphocytes (CTL) was proposed. In contrast to other models, it allows to generate effector CTL without immunization in vitro. 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 in 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+, Lyt2+, L3T4- phenotypes. Presence in vitro of exogenous IL-2 was needed for the generation of CTL against MX-11 sarcoma but not against EL4 lymphoma. We suggest that the release of IL-2 from lymphomas cells could stimulate generation of the effector cells through activation of the endogenous production of IL-2, or due to some other factors.

The generation of specific antitumor cytotoxic T-lymphocytes (CTL) via 2-fold immunization in vivo and subsequent cultivation without tumor cells (in monoculture) was previously described. The spleen cells from B10 mice bearing progressively growing MX-11 sarcoma suppressed the maturation of CTL specific to MX-11 but not to EL-4 lymphoma in monoculture. It was observed, that the suppression was not the result of the inhibitory effect of suppressor cells upon the IL-2 production, because suppression took place in the presence of the exogenous IL-2 in monoculture. Since the treatment of the spleen cells with MoAb against both L3T4 and Lyt2.2 antigens plus C' considerably decreased the suppressive activity, it was suggested, that two distinct subsets of T-lymphocytes were required for suppression. It might be possible, that the presence of anti-idiotype on the effector suppressors was the cause of the suppressive specificity in the absence of tumor antigens in vitro.

The in vivo induced pCTL-2 with phenotype L3T4- Lyt2- specific to the H-2Kb molecule, turn into effector CTL during 4 days in the mixed lymphocyte culture (with heat-treated donor stimulators) much more efficiently when donor and recipient are different from one another not only in MHC class I (anti-BIO, MBR BIO.AKM) but in I + II (Kb + Ib) ahti-C57BL/6 BIAD2(RIOI). The initial pCTL-2 differentiation in enhanced as a result of synergistic effect between the Kb alloantigen and rIL2. The anti-Kb pCTL-2, being separated from helper T cells by means of absorption onto the macrophage donor monolayer and elution from it, give rise to pronounced differentiation in simplified conditions, irrespective of the stimulator presence and without external rIL2. It is supposed that these phenomena are raised to secretion of the CTL differentiation factor by the eluted pCTL-2 themselves, and besides, rIL2 may promote for secretion of this factor additionally.

Lymphocytes of inbred mice immunized with allogenic tumour cells were labelled in vitro or in vivo by 3H-thymidine, washed out and incubated with target cells in the presence of "cold" thymidine. A fraction of labelled small and medium lymphocytes was demonstrated by autoradiography to be absorbed rapidly and specifically onto the corresponding target cells. When the non-labelled immune lymphocytes were preincubated with target cells for 2 hours, and 3H-thymidine was added to the medium at various time periods, the percentage of labelled small and medium lymphocytes which adhered to the target cells appeared to be reduced in the course of the incubation, with the target reaching the initial value after an 8-hour contact. Small and medium immune lymphocytes which synthesize DNA are supposed to be the effector killer fraction which do not transform into blasts during the interaction with the target cells.

Conditions are selected for the killer induction during primary and secondary responses in the one-way MLC, stimulated by x-irradiated or killed allogenic lymphocytes. MIF is found in the MLC culture medium as a sharp peak on the 2nd or the 3rd day of the primary reaction, or as a plato from the 1st day of the secondary reaction. In contrast, the killers are shown to be induced much later reaching their maximum on the 5th and the 4th days, respectively. If heated, instead of irradiated, allogenic lymphocytes were used for stimulation of the primary MLC reaction, the MIF-producer kinetics was not changed, but the killer induction was found to be delayed.

The immunological memory T cells assayed by the cytotoxic T lymphocyte (CTL) generation in the secondary mixed lymphocyte culture bear H-2 antigen-binding receptors as shown by the technique of the specific lymphocyte absorption on target cell (TC) monolayers of different H-2 origin. Memory T cells and specific suppressor T cells are demonstrated to be capable of adhering to native and fixed TC in a similar fashion, whereas CTL absorption appears to be two-fold reduced when the TC monolayer is fixed. The primary CTL precursors differ from memory T cells by a poor adherence to native TC which is not demonstrable at all when TC are fixed. The findings evidence the differences in receptor affinity (or structure) among the primary and secondary CTL precursors and the CTL themselves.

The nature of suppressor cells which are contained in lymphocyte suspension of mice immunized with allogeneic tumor cells and inhibit the DNA synthesis activation in mixed normal lymphocyte cultures has been studied. The suppressor cells are shown to be resistant to mitomycin C, X-irradiation and are not inactivated by the treatment of anti-O-serum or anti-Ig-serum in the presence of the complement. They adhere to the plastic and are inactivated by carrageenan in vitro. The macrophage nature of the suppressor cells is suggested.

The in-vivo-induced memory T cells (MC) of mice, specific to H-2 antigens, are assayed by the generation of the secondary cytotoxic T lymphocytes (CTL) in mixed lymphocyte culture (MLC) activated by heat-killed stimulator cells. The MC are shown to adhere selectively to the corresponding target monolayer that gives rise to both the loss of MC activity in the population of non-adherent lymphocytes and gain in MC activity in the population adherent and eluted from the same monolayer. In addition to the revealing of MC H-2 antigen-binding receptors, the absorption-elution technique allows the separation of the MC into two categories: secondary CTL precursors bearing these receptors, and secondary amplifier cells non-adherent to the monolayer and assayed by promotion of the CTL generation from the primary precursors activated in MLC by heated stimulators. The difference in the receptor properties between the primary and secondary CTL precursors raises the possibility that the MC are generated not only in the amplifier cell population but also in the independent CTL precursor population.

Cytotoxic T-lymphocytes (CTL) obtained by in vivo immunization were enriched by the absorption-elution technique, using the relevant allogeneic target cell (TC) monolayers. After the separation of C57BL anti-A (anti-KkDd) lymphocytes into anti-Kk and anti-Dd subpopulations they displayed cross killing cytotoxic effect on H-2d and H-2kTC, respectively. B10D2 anti-B10 (anti-KbDd) lymphocytes cross reacted to H-2a and H-2q TC. The results are discussed in the light of heterogeneity of CTL clones or their receptors.

Cytotoxic T-lymphocytes (CTL) and suppressors induced by immunization in the H-2 system and inhibiting DNA synthesis activation in mixed lymphocyte culture were studied in parallel. Unlike CTL, suppressors did not adhere specifically to the target cell monolayer, they were not inactivated by anti-theta serum plus complement, and their action was not specific: they inhibited the DNA synthesis activation stimulated by any stimulant cells, as well as by phytohemagglutinin and concanavaline A. CTL and suppressors proved to represent different effector cell populations which could be separated from one another.

A hybridoma producing monoclonal antibodies (McAb) NATF9.9 (F9) was obtained from fusion of murine myeloma X63 and splenocytes of AKR mice immunized with a single intravenous injection of 5 X 10(7) thymocytes of CBA mice. F9 McAb were cytotoxic for 80% thymocytes, 10% splenocytes, 20% lymph node cells, 85% cortical and 32% medullary thymocytes of CBA, C57BL/6, BALB/c, DBA/2 and SJL but not for the cells of C58 and AKR mice. F9 McAb reacted only with T cells and did not react with B cells and EL4 thymoma cells (Thy-1.2+, Lyt-1+2-3-). The proportion of F9+ cells accounts for about 40% among T lymphocytes of the lymph nodes and spleen as tested by flow-type cytometry. Lymph node cells treated with F9 McAb plus complement completely lost their reactivity with rat anti-Lyt-2 McAb and only partly (by 30%) with anti-Lyt-1 McAb. The reactivity pattern of F9 McAb attests to their specificity for Lyt-3.2 antigen.

Suppressor cells are generated in allogeneic or syngeneic one-way mixed lymphocyte culture (MLC) for 1 to 5 days, the suppressor activity being tested by inhibition of proliferation of 10 to 100-fold number responders in the fresh allogeneic MLC. In both allogeneic and syngeneic MLC the suppressors are found to be neither B cells, nor macrophages, resistant to mitomycin C, and their precursors are shown to be cyclophosphamide sensitive. Their properties differ from those previously shown for T killers or T suppressors generated in the lymphocyte monoculture. The allogeneic suppressors are T cells that are heterogeneous in respect to radiosensitivity and specificity. Generation of the nonspecific T suppressors is reduced if human or bovine serum is added to the MLC medium instead of fetal calf serum. The syngeneic suppressors are heterogeneous as well and differ from the allogeneic ones by greater stability to the effect of anti-T bodies and irradiation, and by greater resistance of their precursors to hydrocortisone.

Antigen-binding T cells of mice immunized with low doses of syngenic spleen cells modified by 2,4,6-trinitrophenyl sulphonic acid, were fused with BW 5147.3.13 thymoma subclone. Suppressor hybridomas were not identical in their differentiating antigens and functional activity. Extract from Lyt 1-2+ hybridoma suppressed efferent and afferent limbs of delayed type hypersensitivity (DTH) in recipients sensitized with subcutaneous injection of 3 X 10(7) TNP-SC. Extract from Lyt 1+2+ I-hybridoma suppressed only afferent DTH limb. It is suggested that during DTH induction with low doses of hapten-modified cells the generation of different types of antigen-binding DTH T suppressors takes place.

Monoclonal antibodies (MoAb) against specific T-suppressors CI and C4 are characterized by their reactivity with normal lymphoid cells and some tumour cell lines cultivated in vitro. MoAb CI and C4 react with T and B cells from spleen and lymph nodes. The amount of CI and C4 T and B subsets are equal in the spleen (25-29%), while lymph node T-lymphocytes contain twice as much CI and C4 cells than B-lymphocytes (40 and 20%, respectively). In the thymus CI is expressed mostly on immature (cortical) thymocytes and C4--on the mature (medullary) thymocytes. CI is expressed on some T-lymphoma cell lines (BW 5147, EL4, LBRM33), but not on thymoma RDM4 and mastocytoma P815. C4 is not found on the above cell lines but is expressed on the intermediate filament of mouse and quail fibroblasts and in lymphoid cells. This cross-reactivity may result from the existance of similar determinants in cytoskeleton proteins and lymphocyte membranes or from the intermediate filament expression on T-suppressor cellular membranes, but not on other functional T subsets.

Following appropriate absorption rabbit serum to CBA anti-C57BL/6 lymph node cells (rabbit ISS) and rat serum to enriched CBA anti-C57BL/6 T killers (rat ISS) selectively inhibited the activity of K-anti-b T killers but did not affect T killers of other specificities (K-anti-d, d-anti-b). Rabbit ISS did not suppress the capacity of CBA anti-C57BL/6 (K-anti-b) T cells to produce MIF. Under the same conditions, anti-Thy-1.2 serum inactivated killers and K-anti-b MIF-producers. The findings indicate that the killers and MIF-producers immune to the antigens of H-2 complex constitute different subpopulations of T cells, which appear to carry nonidentical sets of idiotypes.

Spleen cells from SJL mice immunized with rat immunoglobulin light chains were fused with X-63 myeloma. Seven hybridoma lines were obtained. Monoclonal antibodies (McAB) secreted by lines L1G9, L2B2 and L3E8 were purified and labeled by 125I. McAB L1G9 did not bind directly to murine lymphocytes but reacted in a different manner with rat splenocytes. McAb L2B2 and L3E8 but not L1G9 reacted with murine lymphocytes treated with rat McAb against Lyt-1, Lyt-2, C1-9, C4-20 antigens whatever the class of heavy chains of rat McAb used. The binding levels obtained with 125I-McAb reflect the quantitative differences in the expression of respective antigens.

Gain in the specific cytotoxic effect of immune lymphocytes after their absorption on the corresponding target cells (TC) and subsequent elution with pronase was caused not by the increase of the cytotoxic activity of individual cells, but by the quantitative enrichment of the population with the effector T-cells. Eluted and the initial immune lymphocytes failed to differ by the kinetics of absorption on the TC. The eluted lymphocyte fraction was characterized by a two fold increase in the T-cell content and a 4-5-fold increase in the number of DNA-synthesizing cells on account of an increase in the content of medium and large lymphocytes.

Receptor repertoire analysis of in vivo induced secondary cytotoxic T lymphocyte precursors (pCTL-2) was performed, using the technique of their specific adherence to macrophage monolayers with subsequent elution of the adherent pCTL-2 and their activation by heat-killed donor stimulator cells. The capacity of anti-H-2Kb pCTL-2 receptors to contact H-2Kbm has been revealed only in a minor pCTL-2 component whose progenitors were able to lyse mutant bm1 target cells (TC). Unlike poor cross-reactivity of CTL descended from anti-Kb, pCTL-2 which were eluted from the donor monolayer, CTL-progenitors of anti-Kb pCTL-2 eluted from bm 1 or B10. A (4R) third-party monolayers lyse in equal quantities the donor TC and those third-party TC from which they have been eluted, but fail to lyse other TC. Enrichment of pCTL-2 eluted from bm 1 or B10. A (4R) monolayers is 6- or 12-fold lower, respectively, than after their elution from the donor monolayer. The findings indicate that anti-class I MHC pCTL-2 are separated into fractions, with their receptors being strongly specific (with high affinity) to the particular fragment of the same complex epitope without cross-reactivity to other fragments. These data differentiate pCTL-2 receptors from effector CTL ones which are homogenous in specificity to a whole (single) complex epitope with variable degree of complementarity. A cardinal distinction of receptor repertoire between CTL, pCTL-2 and suppressor T cells specific to the same class I MHC molecule and alteration of the active site during pCTL-2 differentiation have been suggested.

The magnitude of the cytotoxic effect (CE) of immune lymphocytes R101 anti-Kb (C57BL/6) and R101 anti-Dd (B10.D2) upon target cells (TC) of mutants Kba (Hzl) and Kbd (M505), as well as Dda (M504) was considerably less than that on the TC of the original C57BL/6 and B10.D2 strains, respectively. A decrease of the CE on the H (zl) and M504 TC was more prominent than that on the M505 TC. "Monospecific" lymphocytes anti-Kb and anti-Dd were separated into two distinct subpopulations by absorption on the monolayers of macrophages from the mutant strains of mice. One of the subpopulations reacted with the TC of the original strain only, whereas the other one - with the TC of both the original and mutant strains.

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.

The differences in the generation of specific suppressor T cells (SSTC) against H-2Kb wild type were investigated in H-2Kbm1, H-2Kbm3 and H-2Kbm4 mutants. Anti-Kb SSTC were produced only by bm3 mutant and F1(BALB/c X bm3) hybrid. T-cell nature of SSTC of bm3 mutant was confirmed by anti-Thy 1.2 monoclonal antibodies described in the same study.

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.

Substitution of normal lymphocytes by immune ones as responding cells in the "one-way" mixed lymphocyte culture (MLC) results in inhibition of the DNA synthesis. This phenomenon is reproduced irrespective of the mouse strains used, immunization method (in vivo or in vitro) and various MLC techniques (macro- or microcultures, the presence of mercaptoethanol in the medium, control variants). Unlike the cytotoxic effect of immune lymphocytes showing a sharp peak on the 8th day after in vivo immunization, inhibition of the DNA synthesis activation in MLC occurred as the second day, and persisted for several weeks after immunization; this inhibition was reproduced on addition of a small fraction (5 to 10%) of immune lymphocytes to normal ones.

Quantitative enrichment of the immune lymphocyte fraction with H-2 antigen-specified suppressor cells was obtained by their elution from the relevant allogeneic monolayer of target cells. This was accompanied by 2-3-fold gain in the content of T lymphocytes and DNA synthetizing cells, as well as in the total (3)H-thymidine incorporation resulting from an increase in the percentage of medium and large lymphocytes in the population and also in the proportion of DNA synthesizing small and medium lymphocytes. Complete abolition of the suppressor effect by treatment with anti-Thy-1 and anti-T antibodies rather than with anti-B serum, and the resistance of this effect to carrageenan and carbonyl iron indicate the T cell nature of the eluted suppressor cells.

Intravenous immunization of mice with a large dose of gamma-irradiated allogenic spleen cells gives rise to specific suppressor T cells and cytotoxic lymphocytes (CTL). However, being optimal form suppressor T cell induction, these conditions of immunization are not conducive to identification of CTL unless they are enriched by elution from the allogenic target monolayer. Unlike CTL, specific suppressor T cells are highly susceptible to gamma-irradiation while their precursors differ from those of CTL by high susceptibility to cyclophosphamide and hydrocortisone.

Rabbit antisera were obtained against cytotoxic small peritoneal lymphocytes (IPEL) of CBA (H-2k) mice immune to alloantigens C57BL/6 (H-2b) and to the enriched 5-day MLC cytotoxic blast lymphocytes (MLC--CL). After appropriate absorption by cells and tissues of intact mice the cytotoxicity of the sera was lost relative to normal lymphoid cells. The absorbed anti-CPL serum inhibited, in the presence of complement, the cytotoxic effect of CPL but not that of MLC--CL on 51Cr-labeled allogeneic macrophages. This inhibition was restricted by idiotypic and strain specificity. Conversely, the absorbed anti-MLC--CL serum inhibited the cytotoxic effect of both CPL and MLC--CL of various mouse strains, irrespective of their immunologic specificity. It is supposed that the effect of the anti-CPL serum is mainly caused by antibodies againts idiotypic determinants of the killer T receptors, whereas the effect of the anti-MLC--CL serum is due to antibodies against differentiation antigens of the proliferating lymphocytes.

High immunological specificity of the direct and indirect macrophage migration inhibition tests was demonstrated in the H-2 system. The capacity of immune lymphocytes for the MIF production was revealed under their incubation with splenic cells of the congenic or recombinant strains of mice sharing particular private or public H-2 specificities with the donor strains. Selective removal of cytotoxic fraction of lymphocytes resulting from their absorption on the corresponding target cells failed to reduce the capacity of the non-adherent cell population for the MIF production. A fraction of the MIF-producing lymphocytes was found to adhere to the target cells and thereafter to be eluated with the cytotoxic lymphocytes. MIF-producing and target-destroying T-lymphocyte populations are supposed to have antigen-binding receptors differing in their affinity and structural arrangement on the cell surface.

Migration inhibition factor (MIF) was found to be produced by T-cells rather than B-cells as shown by the indirect macrophage migration inhibition test in the H-2 system performed with fractionated lymphocytes. MIF-producers appeared to be more sensitive than T-killer cells to the cytotoxic action of anti-theta-antibodies, plus complement. MIF synthesis by lymph node cells developed earlier after immunization than the cytotoxic activity of these cells. These findings indicated that T-cell subpopulation producing MIF differed from the cytotoxic T-lymphocytes.

Cellular immunity to alpha-fetoprein (AFP) was studied in rats by the macrophage migration inhibition (MMI) test after breakage of tolerance to homologous AFP of rat (RAFP). A single injection of cross-reacting AFP of mouse (MAFP) has induced the capacity of peritoneal exudate cells of rats to react in the MMI test to both MAFP and RAFP. The second injection of MAFP results in reduction of the MMI reaction to both antigens and the appearance of stimulation in the macrophage migration together with further elevation of antibody titer to MAFP and RAFP. The fractionation of rat peritoneal cells has shown the T-cell nature of MMI reaction to both MAFP and RAFP.

The nature of suppressor cells induced by immunization with the allogenic spleen cells and inhibiting the DNA synthesis activation in the mixed lymphocyte culture was studied. Suppressor cells are resistant to mitomycin C and carrageenan. They are not inactivated by the treatment with rabbit anti-B- and anti-Ig- as well as with mouse antibodies (anti-Mls serum) against B lymphocytes in the presence of complement but eliminated by rabbit anti-lymphocyte and anti-T globulins and by mouse anti-theta serum. These T suppressor cells are concentrated in the large lymphocyte fraction in the ficoll gradient. Their blocking of the DNA synthesis activation is evidently non-specific.