Maintenance of lymphocyte surface Ig by mitogen stimulation in vitro.

In 4 to 24 hr cultures of rabbit lymphoid cells in medium supplemented with autologous serum, most B cells lost their surface Ig as assayed by rosette formation with anti-Ig antibody-coated erythrocytes. This loss was prevented by adding selected mitogens such as streptococcal mitogen (SM), lipopolysaccharide, and concanavalin A or by supplementing the medium with fetal calf serum. When SM was added at various times to the cultures (1, 2, 3, and 4 hr), it was effective in maintaining the approximate level of Ig-bearing cells present at the time of its addition but was ineffective in restoring the level of Ig-bearing cells present at the time the cultures were intiated. Very small, submitogenic doses of SM were sufficient to maintain the level of Ig-bearing cells. The data suggest that lymphocytes require continuous stimulation to maintain their surface receptors.     

Immunoregulatory function of T cells activated in the autologous mixed lymphocyte reaction

This study was undertaken to define the functional properties of T cells stimulated in the autologous mixed lymphocyte reaction (MLR) by purified B cells or macrophages. In preliminary experiments, it was found that T cells that had been cultured with autologous non-T cells inhibited pokeweed mitogen- (PWM) stimulated immunoglobulin synthesis by autologous B cells. In addition, the T cell-mediated suppression was eliminated by x-irradiation and hydrocortisone treatment, was mediated by a mechanism that occurred early in the PWM-stimulated cultures, and did not involve killing of mature immunoglobulin-secreting cells. T cells were then cultured with either autologous B cells or macrophages in order to determine whether such autoreactive T cells had a similar capacity to regulate PWM-induced immunoglobulin synthesis. Although T cell populations stimulated either by B cells or by macrophages suppressed proliferative responses and immunoglobulin synthesis, both these populations of autoreactive T cells provided help for immunoglobulin synthesis that was not significantly different from that provided by fresh T cells. These results suggest that the predominant functional consequence of activation of T cells in the autologous MLR is the generation of suppressor T cells capable of inhibiting immunoglobulin synthesis. Thus, the autologous MLR may represent a negative feedback mechanism for the regulation of the immune response.     

Rabbit antisera to human B cell alloantigens: effects on the mixed lymphocyte response.

Antisera were produced in rabbits to two glycoproteins (31,000 MW and 23,000 MW by SDS-gel electrophoresis) isolated from papain digests of membranes from a human B lymphoblastoid cell line (LCL). After minimal absorption with a T LCL the antisera reacted with two glycoproteins (35,000 MW and 27,000 MW) present in detergent-solubilized membranes from human B LCLs. The immunizing molecules are proposed to have arisen by proteolysis of the intact molecules in the detergent-solubilized membranes. The glycoproteins were detectable on human B LCLs and macrophages, but absent from T LCLs and fibroblasts. The molecules were identified as B cell alloantigens by their reactivity with alloantisera specific for human B cells. The rabbit antisera reacted with peripheral blood B lymphocytes, but not with T lymphocytes, platelets or erythrocytes.Pretreatment of human peripheral blood mononuclear cells with the rabbit antisera in a uni- or bidirectional mixed lymphocyte response (MLR) rendered them unable to stimulate, but they were able to respond. Addition of the antisera at various intervals during the MLR to block continuous stimulation indicated that cells were activated at different times. The presence of an F(ab′)₂ or Fab′ preparation of the antisera throughout the MLR did not inhibit the response at the concentrations tested. Further experiments suggest that, while a responding lymphocyte can replicate several times without restimulation, there is a delay between commitment to and commencement of division.     

Induction by an immunogenic immunomodulating agent of nonspecific T cell suppression of lymphocyte responsiveness in MLR but not of antibody production

Summary Spleen cells derived from BALB/c mice that had been repeatedly immunized with the methanol extraction residue (MER) fraction of tubercle bacilli exhibited a depressed capacity to act as responder cells in allogeneic and syngeneic mixed lymphocyte reactions (MLR). Previously reported studies revealed that such spleen cells are also defective in the in vitro generation of antibodies. In order to determine the nature of the cells responsible for the depressed MLR reactivity, purified populations of splenic macrophages, B lymphocytes, T lymphocytes originating from normal and from MER-immunized mice, and cell culture supernatants were added to MLR mixtures consisting of normal mouse splenocytes. Macrophages originating from MER-immunized mice and their culture supernatants exerted a significantly higher suppressive effect on MLR than that of corresponding preparation from normal mice. Splenic T cells originating from MER-immunized mice and their supernatants also significantly suppressed the MLR response. However, the same T cell populations that were inhibitory in MLR failed to suppress the in vitro generation of antibodies against sheep red blood cells in the presence of either MER or 2-mercaptoethanol. These and previously reported findings indicate that a nonspecific immunomodulating agent, MER, can, under certain conditions of treatment, elicit the induction of nonspecific suppressor T cells for MLR but not for antibody production, and, accordingly, can inhibit cellular and humoral immunological responsiveness by different mechanisms.     

T cell recognition in the mixed lymphocyte response. I. Non-T, radiation-resistant splenic adherent cells are the predominant stimulators in the murine mixed lymphocyte reaction.

The ability of subpopulations of murine spleen cells to stimulate a mixed lymphocyte response (MLR) was studied. It was found that T cells (nylon-nonadherent spleen cells) and B cells [G-10 passed and treated with rabbit anti-mouse brain serum (RAMB) and complement (C)] were poor stimulators of an MLR. In contrast, whole spleen cells or B cells plus adherent cells (RAMB +C-treated spleen cells) produced good stimulation. However, a non-T, radiation-resistant splenic adherent cell (SAC) population was up to 20 to 50 times more efficient as a stimulator of an MLR on a per cell basis than an unseparated spleen population. These SAC were shown to express Ia determinants encoded by genes in I-A and I-E/C. These results suggest that Ia+ SAC may be the predominant stimulating cells in spleen cell populations, and the preferential target for T cell recognition in cell interaction events.     

Fractionation of lymphocyte subpopulations which regulate mixed lymphocyte reactions.

Four days after injection of allogeneic lymphocytes BALB/c splenic T cells suppress proliferation of syngeneic cells in mixed lymphocyte reactions (MLR). Conversely, lymph node cells from the same mice amplify MLR responses. To further characterize these functional subpopulations, alloantigen-primed lymphocyte suspensions from both organs were fractionated by velocity sedimentation at unit-gravity. After fractionation MLR suppressor cells from spleens localized exclusively in rapidlly sedimenting fractions of large cells. MLR suppressor activity of cells from these fractions, as well as that of unfractionated spleen cell suspensions, was abolished by treatment with anti-Thy-1.2 serum and complement. Spleen cell fractions of similar sedimentation velocity also secreted a soluble MLR suppressor into culture supernatants. Although inhibitory of MLR, spleen cells of rapid sedimentation velocity did not suppress responses to T cell mitogens. In marked contrast with the effects of spleen cells, large 4-day-alloantigen-primed lymph node cells had no suppressive activity in MLR. MLR amplifier cells of uncertain derivation were found in fractions of medium sedimentation velocity from both spleens and lymph nodes. Fractionation of alloantigen-primed lymph node cell suspensions did reveal, however, a subpopulation of small cells with MLR suppressor acitivty which was unaffected by treatment with anti-Thy-1 serum and complement. The data thus indicate that large alloantigen-activated lymphocytes are not intrinsically suppressive nor are cells which suppress MLR necessarily large. We consequently conclude that regulation of MLR responses by alloantigen-primed lymphocytes involves a complex interaction between distinct functional subpopulations of cells which are separable both by physical and biologic properties.     

T cell recognition in the mixed lymphocyte response. II. Ia-positive splenic adherent cells are required for non-I region-induced stimulation

Ia-positive splenic adherent cells (SAC) have been shown to be the predominant stimulators of a mixed lymphocyte response (MLR) to whole H-2 differences, in which most of the proliferative response is directed against I region-encoded determinants. The present studies were undertaken to examine the ability of several purified lymphoid subpopulations to activate T cells in response to the non-H-2-linked MIs products or to products of the K or D regions of H-2. The results demonstrated that adherent cell-depleted populations of T and B cells were nonstimulatory, whereas SAC were potent stimulators for responses involving each of these genetic differences. Treatment of these SAC with anti-Ia and C abrogated their MLR-stimulating ability. In contrast, whereas treatment of SAC with anti-Ia and C abrogated their ability to stimulate an MLR directed against K or D region-encoded determinants, this treatment had no effect on their ability to generate a cytotoxic T lymphocyte response against these same determinants. These findings suggest that in addition to presenting allogeneic I region-encoded determinants, Ia-positive SAC also play a unique role in the presentation of non-I region-encoded alloantigens to proliferating T cells.     

The formation of stable E-rosettes by human T lymphocytes activated in mixed lymphocyte reactions.

The aim of the present study was to determine whether activation of human T-lymphocytes affects their interaction with sheep red blood cells (SRBC). Less than 3% of the E-rosettes formed by freshly isolated peripheral blood lymphocytes (PBL) and SRBC are stable and do not disintegrate after incubation at 37 degrees C. In contrast, about 30% of PBL kept in culture for 5 days in the presence of mitomycin C-treated allogeneic lymphocytes were found to form stable E-rosettes. Whereas no rosettes were formed by freshly isolated PBL incubated with human red blood cells at 24 degrees C, 15% of the cells recovered from mixed lymphocyte reactions (MLR) formed such rosettes. When responder PBL were maintained in culture in the absence of allogeneic stimuli the proportion of cells forming stable E-rosettes depended on the serum present in the medium. Less than 5% of the responder cells kept in medium containing human serum or in serum-free medium formed stable E-rosettes, whereas 18% of the cells maintained in medium containing fetal calf serum formed stable E-rosettes. The proportion of cells forming stable E-rosettes increased before any increase in DNA synthesis was detectable in MLR. Indeed, a high proportion of cells forming stable E-rosettes appeared in MLR taking place in serum-free medium, without any accompanying increase of DNA synthesis. Depletion of cells forming EAC'-rosettes from responder PBL increased the proportion of cells forming stable E-rosettes in MLR. Exposure of the cells recovered in MLR to specific anti-T sera inhibited the formation of both stable and regular E-rosettes. Exposure of the cells recovered in MLR to anti-Ig serum had no effect on the formation of regular rosettes. Anti-Ig serum strongly inhibited the formation of stable E-rosettes by cells grown in medium containing human serum, but had no effect on the formation of stable E-rossettes by cells grown in either serum-free medium or in serum containing fetal calf serum. It is concluded that activated human T lymphocytes are characterized by their capacity to form stable E-rosettes, resistant to incubation at 37 degrees C, and by their capacity to acquire an immunoglobulin coat, possibly by binding immunoglobulin molecules present in their environment.     

The differentiation of T lymphocytes. Density characterisation of thymic and peripheral T cells responding in syngeneic and allogenic mixed lymphocyte reactions.

Equilibrium density separation on continuous albumin gradients was used to separate and characterise the T cells responding by proliferation to both syngeneic and allogeneic stimulating cells in the one-way mixed leucocyte reactions (MLR). In CBA mouse spleen both light and dense T cells were capable of responding in an allogeneic MLR. No T cells responding to stimulation be syngeneic B lymphocytes could be isolated from adult or 7-day CBA mouse spleen. In adult CBA mouse thymus, cells responding to allogeneic stimuli were enriched in the light density region, along with the low theta subpopulation. Self-reactive cells, responding with proliferation when cultured with syngeneic adult CBA splenic lymphocytes, and found in adult and 4-day CBA mouse thymus, were also enriched in the light density zones. However, in adult thymus syngeneic MLR reactivity was also found in the dense zones, and the density distribution profiles of total syngeneic MLR responding cells revealed a series of peaks extending over the whole density range. It was suggested that these syngeneic MLR responders undergo a complete maturation process, including progressive density increases, within the thymus gland. Such a sterile differentiation pathway could be a censorship process, leading to death of self-reactive cells within the thymus.     

Biological expressions of lymphocyte activation. IV. Concanavalin A-activated suppressor cells in mouse mixed lymphocyte reactions.

Thymus-derived lymphocytes (T cells) from mouse spleen, activated in vitro or in vivo with concanavalin A (Con A), suppress proliferative responses of syngenic lymphocytes in mixed lymphocyte reactions (MLR). Replication in vitro was not required for expression of suppressor activity by Con A-activated cells and was blocked in MLR by treating suppressor cells with mitomycin C or irradiation. Kinetics of MLR responses and viability of cultures were not altered by addition of activated suppressor cells. The data are consistent with a direct inhibitory effect of suppressor T cells on antigen-induced DNA replication. These observations extend a model previously described for regulation of antibody synthesis by Con A-activated T cells to control of cell-mediated immune responses. This model should be particularly useful in further definition of regulatory T cell subpopulations, and in investigation of interactions and relationships between such populations.     

Development of precytotoxic T cells in cyclosporine-suppressed mixed lymphocyte reactions

Cyclosporine (CsA) blocked the generation of cytolytic activity in a primary MLR of mouse spleen cells. As expected from the known mechanism of action of this drug, it also blocked the accumulation of IL-2 during the MLR. Addition of human rIL-2 did not overcome the inhibition of CTL generation, even when it was added daily to keep its level similar to that produced in a normal MLR. Daily addition was necessary, because the CsA-inhibited MLR consumed IL-2, either by utilization or degradation. The outcome of a 5-day MLR in the presence of CsA (CsA-MLR) depended on whether or not IL-2 was continuously present. In the presence of IL-2, there was no generation of CTL activity, probably because such cultures contained IL-2-dependent suppressive elements described previously. However, when day 5 CsA-MLR cells generated in the absence of IL-2 were washed and recultured with human rIL-2, there was a burst of CTL activity, with a more than 50-fold increase in alloantigen-specific cytotoxicity within 24 to 48 h. This increase is not explainable simply by the proliferation of existing effector CTL. The noncytotoxic cells produced in an MLR in the presence of CsA, and which can be rapidly activated to cytotoxic effector cells by IL-2, are termed "precursor-effector CTL" (peCTL). They could be detected by day 3 of a primary CsA-MLR culture. Their conversion to effector CTL by IL-2 was not inhibited by CsA. Exposure of peCTL to IL-4 also generated CTL activity, to a somewhat lesser degree than IL-2, but the IL-4-induced activation was inhibited by CsA, suggesting that it depended on the induction of another CsA-sensitive lymphokine. The intracellular levels of mRNA encoding the CTL-specific serine esterases CCP1 and CCP2 (granzymes B and C, respectively) increased rapidly during the IL-2-driven conversion of peCTL to effector CTL. This study demonstrates that in the presence of CsA precursors for CTL can accumulate, and that these can be rapidly converted to cytotoxic effector cells by IL-2.     

T lymphocyte heterogeneity in the rat. III. Autoreactive T cells are activated by B cells

Evidence has been presented to show that CD4+ autoreactive T cell lines (ATs)2 in the rat require periodic stimulation with syngeneic spleen cells for in vitro proliferation. This proliferation can be blocked by treatment of the stimulator (spleen) cells with mAb to Ia antigens. Although ATs are Ia+ and can activate the allogeneic MLR, they fail to be autostimulatory. Fractionation of the spleen cells revealed that ATs can be stimulated with B cells and not by macrophages, although the latter were efficient in several accessory cell functions, including antigen presentation, lectin-dependent T cell activation and allogenic MLR response. Moreover, B cells proliferated and differentiated in response to AT cells. These data are compatible with a model in which ATs respond to hitherto undetermined B cell membrane antigen(s) in association with MHC class II antigens. These results may have important implications in understanding autoimmune responses.     

Mixed lymphocyte reaction in mice genetically selected for high (Hi/PHA) or low (Lo/PHA) responsiveness to phytohemagglutinin.

Lymph node cells from Hi/PHA and Lo/PHA mice were evaluated for proliferative response after stimulation by allogeneic lymphocytes (MLR) originating from four inbred strains of different H-2 haplotype (C57B1/6, DBA/2, CBA, A). Reactivity to MLR and PHA were compared in these two lines and in the four inbred strains. The high and low responder status of Hi/PHA and Lo/PHA, as determined by T mitogens lymphocyte responsiveness, was also observed when one measured T responsiveness after MLR. Values obtained with the four inbred strains are included in the range of those measured in Hi/PHA and Lo/PHA cells when stimulated by PHA as well as by allogeneic cells. In contrast, when used as stimulator cells, Hi/PHA or Lo/PHA lymphocytes induce an equivalent proliferative response versus every responder inbred strain studied. These experiments support the hypothesis of a common genetic control of proliferative response following PHA or MLR stimulation. The genes implicated would be different from those coding for I region associated antigens.     

Effects of vitamin B6 deficiency on thymic epithelial cells and T lymphocyte differentiation.

Dietary vitamin B6 (pyridoxine) deficiency in young Lewis rats results in a reduction of T lymphocyte numbers and defects of cellular immunocompetence. In vitro studies of thymic epithelial (TE) cells, responsible for inducing T lymphocyte differentiation, revealed that maintenance on a vitamin B6 deficient diet for 2 weeks resulted in a severe defect in TE cell function. When the deficient animals were returned to a normal diet, TE cell function was restored. Exposure of lymphoid precursors from neonatally thymectomized or vitamin B6-deficient donors to normal TE monolayers resulted in their conversion to functional T lymphocytes, as measured by their response in MLR and to mitogens. However, TE monolayers from vitamin B6-deficient animals were unable to effect such a maturation of T lymphocytes. Therefore, it is suggested that the defect in cellular immunocompetence following this dietary deficiency is due, at least in part, to the inability of TE cells to effect the differentiation of T lymphocyte precursors to functional T lymphocytes. The dietary deficiency does not, however, impair lymphoid precursors, which can be stimulated to further differentiation by exposure to normal TE cell monolayers.     

Possible role of neuraminidase in activated T cells in the recognition of allogeneic Ia

In a primary MLR, predominant stimulators in spleen cells are adherent cells and not B cells, although B cells are one of the cell types expressing a large amount of Ia molecules. Our previous experiments showed that T cells treated with neuraminidase (Nase) responded to an allogeneic Ia on B cells. In our experiments, the relationship between the responsiveness to the allogeneic Ia molecules on B cells and Nase activity of T cells was examined. The results showed that T cells increased in Nase activity with the acquisition of the reactivity to Ia on B cells. T cells from normal mice increased in Nase activity after the incubation for 3 days or more in MLR, and these T cells responded to allogeneic Ia on B cells. However, T cells from mice genetically deficient in Nase responded poorly to the Ia on allogeneic B cells even after the incubation in MLR for 3 days. T cells incubated for 3 days in MLR decreased in electrophoretic mobility, indicating the decrease of net negative charge of the cells, and increased in their binding of peanut agglutinin which has been reported to bind to galactosyl residues exposed on T cell surface by removing sialic acids. These results suggest that Nase in T cells was activated by the cultivation in MLR for 3 days, and sialic acids of some molecules on T cell surface were removed by the enzyme and, in turn, T cells acquired the responsiveness to allogeneic B cells in a secondary MLR. Thus, Nase was suggested to play a regulatory role in the recognition of Ia molecules in T cells.     

In vitro analysis of allogeneic lymphocyte interaction. III. Generation of a helper allogeneic effect factor (AEF) across an I-J subregion disparity.

Allogeneic effect factors (AEF) were produced across an I-J subregion incompatibility. The helper activity of these AEFs is H-2 restricted since they help B cells only of the stimulator haplotype and of other haplotypes that carry the same I-J subregion gene(s) as the stimulator haplotype. Immunoadsorption studies demonstrate that they consist of I-J determinants derived initially from the GVHR host and MLR stimulator cells and not the GVHR donor and MLR responder cells used to generate AEF. It is postulated that the genetic restriction of AEF helper activity is mediated in part by the ability of the GVHR activated donor T cells to acquire, in vivo, recipient T cell and/or macrophage derived I-J determinants. Cellular adsorption studies indicate that AEF helper activity may be adsorbed by B cells, but neither T cells nor macrophages, of the stimulator haplotype. The results suggest that an I-J-positive AEF interacts with an I-J subregion controlled complementary recognition structure on a target B cell and, after antigenic stimulation, activates that B cell to IgG antibody synthesis.     

Mixed lymphocyte reactions: absence of stimulation by irradiated allogeneic cells

¹³⁷Cs-irradiated mouse spleen cells, in contrast to mitomycin-blocked cells, do not stimulate a mixed lymphocyte reaction (MLR) when cocultured with normal allogeneic lymphocytes. Attempts to find an irradiation dose which blocks the DNA-synthetic capability of alloantigenic cells, but which does not also render them unstimulatory in mixed cultures, have not been successful. Low-level irradiation (100–500 rad) does not completely block mitogen (PHA) reactivity, hence the target cells may be capable of participating in a two-way MLR. High-level irradiation (> 1000 rad), however, thoroughly blocks PHA-stimulated DNA synthesis and eliminates the capacity of these cells to stimulate an MLR. Potentiation of MLR by irradiated cells syngeneic with the reacting cell population was not possible in these experiments. Cell death and lysis in irradiated suspensions occurred during the 72-hr culture period. It is believed that a combination of cell lysis and irradiation-blocked metabolic events normally necessary for the MLR sufficies to explain the poor or nonexistent MLR's obtained in these experiments.     

The early (18-hr) human mixed lymphocyte reaction: identification and isolation of activated T-cell clones

This study provides direct evidence that de novo expression of the activation antigens B1 49.9 (49.9) (interleukin-2 receptor) and 4F2 enables identification of alloactivated cells within 18 hr of initiation of human mixed lymphocyte reactions (MLR). Using a dual-parameter flow cytometer (simultaneous assessment of immunofluorescence and DNA content on the same cell), it was demonstrated that these activation antigens emerge before activated cells enter into S/G2/M phase of the cell cycle. Family studies illustrate that early activation antigen appearance occurs in response to a mismatch at chromosome 6, and invariably heralds the proliferative outcome at 6 days of MLR. In order to directly study the small alloactivated T-cell population, 49.9-positive cells were isolated using a cell sorter after 18 hr of MLR and cloned by limiting dilution using purified recombinant interleukin-2 (rIL-2). Antigen-specific T4-positive T-cell clones were isolated. Analysis of these clones demonstrates that antigen-specific reactivity is acquired within 18 hr in the MLR. These methods should permit a dissection of the early events of alloactivation.     

H-2 homology requirements for secondary cell-mediated lympholysis and miced lymphocyte reactions to TNP-modified syngeneic lymphocytes.

Secondary cell-mediated lympholysis (CML) and mixed lymphocyte reactions (MLR) were generated in a tissue culture system against trinitrophenyl (TNP)-modified murine syngeneic spleen cells. H-2 homology between primary and secondary TNP-modified stimulating cells was required in order to restimulate in the secondary CML. Strong proliferative responses (MLR) were detected only in the secondary cultures, for which H-2 homology was also required between TNP-modified primary and secondary immunogens. Intra-H-2 mapping for the secondary MLR indicated that the relevant regions of homology were I, D, and K and/or I-A. Homology throughout the entire major histocompatibility complex or at K plus I-A gave stronger MLR than did cultures in which there was homology between the primary and secondary phases at I or D only.     

Trophoblast regulation of maternal-paternal lymphocyte interactions

The modification of immunological responses by murine embryonic trophoblast cells was investigated using the mixed-lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) test. In MLR containing Balb/c (responder) and C57BL/6 (stimulator) splenocytes DNA synthesis was markedly reduced in the presence of ectoplacental cone or placental trophoblast cells. These same trophoblast cell populations inhibited the in vitro generation of cytotoxic lymphocytes while cultures containing nontrophoblast regulator cells expressed normal cytotoxicity. DNA synthesis in MLR and cytotoxic activity in CML were not suppressed in cultures containing $\text{3}\text{1}\text{2}$ day blastocyst outgrowths. The potent immunosuppressive properties of the trophoblast may be important during pregnancy by protecting the genetically dissimilar fetus from potentially harmful maternal immune effector mechanisms.