Stimulator Cell Population Research Articles

A limiting dilution assay to determine the frequency of antigen presenting cells

A limiting dilution assay to measure the functional frequency of antigen presenting cells (APC) has been developed and used to measure the frequency of mouse lymphoid cells which stimulated the mixed leucyte response. The experiments utilised measurements of proliferation in 20 μl hanging drop microcultures of activated T lymphocytes. When the data supported a model of single-hit kinetics, the APC frequency in the titrated stimulating cell population was calculated by the method of maximum likelihood. The assay was validated by measuring the frequency of unpurified spleen cells and enriched spleen dendritic cells which presented alloantigens in mixed leucocyte culture. APC frequency in unpurified spleen cells was 1 : 7254 ( SE ± 2134, 8 experiments) and was increased markedly to 1 : 15 ( SE ± 7, 7 experiments) when enriched populations consisting 70–95% of dendritic cells were used as stimulators.

Analysis of T-T lymphocytes and T-accessory cell interactions using cloned T cells: MHC I restricted cloned T cells activate MHC II restricted T helper cells

We evaluated the role of molecules of the major histocompatibility complex (MHC) involved in the cellular interactions of two T-cell clones by testing the effect of monoclonal antibodies on the responses of the clones in vitro. The two T-cell clones used in the study are specific for minor histocompatibility antigens and restricted to the H-2K k. In the absence of exogenous IL-2 the clones require the presence of Ia +, Thy-1 − accessory cells and of Thy-1 +, Lyt-1 +2 − cells in the irradiated spleen cell suspension used as stimulator. It is also necessary that both the accessory cells and the T cells in the stimulator cell populations are recognized specifically by the clones. Monoclonal antibodies specific for the H-2K product inhibited the lytic effector function of the cytolytic clone. These antibodies when added to cultures of stimulator cells and clones inhibited also the proliferation of this clone and of a nonlytic clone. When antigen recognition was measured by the increase in sensitivity of the clones to IL-2 while confronted with uv-irradiated stimulator cells, both clones were blocked efficiently by anti-H-2K antibodies. Thus, these results suggest that the interaction of monoclonal antibodies with the restricting H-2K molecule is sufficient to block the recognition signal, a prerequisite for proliferation. In contrast, monoclonal antibodies specific for A αA β and/or E αE β had no effect on cytolysis or on restricted recognition. However, they inhibited the proliferative responses as efficiently as the H-2K specific antibodies. Inhibition by class II-specific antibodies was not abolished when stimulator cell populations were depleted of Lyt-2 + cells. The blocking effect, however, was reversed by the addition of IL-2. No inhibition was obtained with antibody specific for E αE β when B10.A(4R) spleen cells, which do not express E αE β, or when B10.A(4R) accessory cells, which were reconstituted with (BALB/c X B10.A(4R)) F1 T cells, were used as stimulators. Stimulator cells heterozygous for H-2 could be inhibited by antibodies to the parental haplotype not encoded in the clones (H-2K d). These and previous results suggest that H-2K-restricted minor histocompatibility antigen-specific recognition transmits an activating signal to the clones and to the stimulator cells. The clones probably are induced to express more IL-2 receptors. The stimulator T cells seem to interact through A αA β and E αE β molecules with syngeneic accessory cells. This interaction results in IL-2 production by the stimulator T cells and thus in the proliferation of the clones.

Cell replication in an immunologically(?) Stimulated cell population in human bone marrow.

In AIDS, although there is a lack of humoral responsiveness in vitro and in vivo, many patients persistently have an increased number of B cells which continue to produce increased amounts of immunoglobulin. An objective, reproducible morphologic classification scheme for B cells was devised. Comparison of cell kinetic parameters in various disease states will require such a classification. Although not immunologically responsive to new stimuli, the marrow B cells in the AIDS patients were shown to be replicating and turning over. The latter may be due to either death in situ or to migration. Plasmacytic lymphocytes and lymphocytic plasma cells, morphologic transitions between lymphocytes and mature plasma cells, had the largest fractions in DNA synthesis. Because of their relative cell numbers, the lymphocytic plasma cells contained most of the cells in DNA synthesis. The position of plasmablasts in the sequential compartments is unclear. Only small numbers are dividing. Within a given morphologic category, large cells were more likely to be in DNA synthesis than smaller cells. These studies can serve as a basis for comparison with marrow B-cell proliferation in other disease states.

The autologous mixed lymphocyte reaction is decreased in Freund's adjuvant-injected rats of arthritis-susceptible and -insusceptible strains.

We examined the T-non-T cell autologous mixed lymphocyte reaction (AMLR) of spleen cells from rats with arthritis induced by Freund's complete adjuvant in an effort to establish an animal model for the study of the relationship between the AMLR and autoimmune disease. We found that the splenic T-non-T AMLR was markedly decreased in rats with adjuvant-induced arthritis and that this decrease was mediated by suppressor cells within the nylon-wool-adherent stimulator cell population. However, we also found a similar decrease in the AMLR of arthritis-resistant Fisher 344 rats that received Freund's complete adjuvant but did not develop arthritis. Control animals with local inflammation induced by turpentine, a non-arthritogenic inflammatory substance, had normal AMLR, whereas other controls given Freund's incomplete adjuvant, also a non-arthritogenic substance, had a modest responder cell-mediated decrease in AMLR. These studies help to clarify the relationship between the decreased AMLR and the pathogenesis of adjuvant-induced arthritis by demonstrating that: 1) the acute-phase inflammatory response does not reduce the AMLR; and 2) the decreased AMLR can occur in the absence of overt autoimmune disease. This latter observation calls into question the proposed pathogenetic relationship between the AMLR and autoimmune disease states.

Lymphokine-like activity of 8-mercaptoguanosine: induction of T and B cell differentiation.

Lymphocyte proliferation and differentiation result from ordered cellular interactions governed by soluble products (lymphokines). Dissecting the individual steps in these processes has been difficult, due to a paucity of pure lymphokines. Recently, it was reported that the derivatized ribonucleoside 8-mercaptoguanosine (8MGuo) has both mitogenic and differentiative effects on murine B cells. In the present studies, we tested 8MGuo for its ability to stimulate both B and T cell responses. In contrast to the murine studies, 8MGuo does not stimulate rat B cells to proliferate and, when tested for B cell growth factor-like activity, no stimulation was observed. The addition of 8MGuo (0.5 to 1 mM final concentration) to mitogen-stimulated B cells led to a marked increase in IgM and a modest increase in IgG secretion. When mixed with conditioned medium, 8MGuo acted synergistically in stimulating secretion of both isotypes, arguing that 8MGuo has both B cell-differentiating factor-mu (BCDF-mu) and BCDF-gamma activity. 8MGuo had no IL 2-like activity when tested on a mouse IL 2-dependent cell line, and no IL 1-like activity on addition to mouse thymocytes with or without submitogenic doses of lectin. However, when added to cultures of murine allogeneic cells in which the stimulating cell populations had been heat-inactivated, 8MGuo induced the generation of specific allogeneic cytotoxic T lymphocytes. Together, these results suggest that a simple derivatized nucleoside can induce both T and B cell differentiation without concomitant proliferation, and thus represent a unique probe for studying events in lymphocyte differentiation.

Role of accessory cell processing and presentation of shed H-2 alloantigens in allospecific cytotoxic T lymphocyte responses.

The present study was undertaken to evaluate the role of accessory cell processing of MHC alloantigens in the initiation of primary allospecific CTL responses. To first determine whether antigen processing by accessory cells is involved in the initiation of allospecific CTL responses, accessory cells were retreated with the lysosomotropic drug chloroquine before their addition to culture. It was found that chloroquine pretreatment abrogated their ability to function as accessory cells only when they were of responder haplotype and had no effect when the accessory cells were of stimulator haplotype. Although accessory cells of either responder or stimulator haplotype can initiate allospecific CTL responses, we have previously demonstrated that they do so by activating distinct classes of T helper TH) cells. Indeed, the differential effects of chloroquine on accessory cells of responder or stimulator haplotypes were shown to reflect the fact that chloroquine pretreatment markedly impaired the ability of accessory cells to activate self-Ia-restricted TH cells, but had little effect on the ability of the same accessory cells to activate either allo-class I- or allo-class II-specific TH cells. We next examined the possibility that accessory cells of responder haplotype mediate alloresponses by acquiring and processing shed MHC alloantigens derived from the stimulator cell population. In these experiments, accessory cell-depleted stimulator cells were fixed with paraformaldehyde to inhibit shedding of their surface MHC alloantigens. It was observed that even though mixed stimulator cells were recognized normally by allospecific CTL precursors, they completely failed to stimulate CTL responses mediated by responder haplotype accessory cells, indicating that the function of such accessory cells is dependent upon their acquisition of shed MHC alloantigens. Taken together, the data presented in this report demonstrate that accessory cells of responder haplotype function in allospecific CTL responses by acquiring and processing shed class I MHC alloantigens, and by then presenting the processed alloantigens in association with self-Ia determinants to self-Ia-restricted TH cells. Thus, these data indicate that the self-Ia-restricted TH cells that are involved in allospecific CTL responses recognize processed class I alloantigens in association with self-Ia determinants.

Suppressor T-cell Activity in Newborns and Mothers

We studied autologous and allogeneic concanavalin A (Con A)-induced suppression of proliferative responses to phytohemagglutinin (PHA) and Con A in peripheral blood mononuclear cells in 24 normal newborns and compared the results with those obtained from 20 normal older children. Three concentrations of PHA and one of Con A were used to stimulate responder cells. Suppressor activity, elicited in the stimulated cell population after 48 h pre-incubation with Con A, was expressed as percentage inhibition of the proliferative response to PHA. There was an inverse relationship between PHA concentration and suppressor activity, and autologous suppression was greater than allogeneic suppression within each group of patients and at each mitogen concentration. In both the autologous and allogeneic systems, older children showed more suppressor activity than newborns. Feto-maternal pairing showed that newborns efficiently suppress their mother's mitogenic responses, but the mothers do not suppress their own or other newborn's lymphocytes, despite having normal autologous suppressor capability. We suggest that suppressor activity by the fetus and it's inhibition in the mother may play a part in the mechanism for controlling maternal-fetal immune rejection.

Regulatory mechanisms in cell-mediated immune responses. Role of I-J and I-C determinants in the activation of H-2I and H-2K/D alloantigen-specific suppressor T cells.

The role of individual H-2I subregion determinants in the activation of H-2I alloantigen-primed mixed leukocyte response suppressor T cells (MLR Ts), as well as their possible expression on stimulator cells required to trigger primed H-2K- or D-specific MLR Ts, was addressed in these studies. Both genetic and serologic studies demonstrated that MLR Ts potentially primed to alloantigens encoded by the entire H-2I region were triggered to MLR Ts factor production only by stimulator cells bearing the priming I-J and/or I-C, but not I-A or I-E alloantigens. The relevant I-J and I-C determinants were demonstrated on a single antigen-presenting cell population that is used in common by independent I-J-specific and I-C-specific MLR Ts. Unexpectedly, the stimulator cell population necessary to trigger MLR Ts primed to class I H-2K or D alloantigens expressed not only the priming class I determinant, but in addition, I-C alloantigens syngeneic with the MLR Ts haplotype. Stimulator populations bearing the appropriate H-2K or D alloantigen but serologically depleted of I-C+ cells or genetically constructed to display MLR Ts-disparate I-C determinants were ineffective stimulators of class I antigen-primed MLR Ts. Thus these data suggest that as allogeneic determinants, I-J- and I-C-encoded molecules are together the major triggering elements for MLR Ts primed to disparate H-2I region determinants. In addition, self-I-C molecule recognition appears to constitute an important feature of the triggering, and by implication, priming process of H-2 class I antigen-specific Ts cells.

Studies of autologous mixed lymphocyte reactions in patients with systemic lupus erythematosus

The autologous mixed lymphocyte reaction (AMLR) measures the proliferative response of peripheral blood T cells to surface antigens of non T cells. The AMLR of SLE patients with active or inactive disease either with (13) or without (6) immunosuppressive treatment was low compared with age and sex-matched controls, confirming previous reports. Only one patient with inactive, untreated SLE and one with drug induced lupus (procainamide) showed normal AMLR. Autologous reactivity was also reduced in 2 patients without treatment who presented with clinically complex disease syndromes, including primary biliary cirrhosis, or polyarteritis nodosa, together with Sjögren's syndrome and serological evidence of lupus. The AMLR could not be increased by changing the ratio of responder to stimulator cells. Patients with decreased AMLR also showed a decreased response to phytohemagglutinin which suggested a general depression of T cells. There was no correlation between the decreased AMLR and age, clinical features or anti-DNA antibody levels of the patients. In allogeneic mixed lymphocyte reactions (MLR) it was shown that non-T cells from SLE patients were poorer stimulators of allogeneic T cells than normal cells, and T lymphocytes from SLE patients were poorer responders to allogeneic non-T cells than were normal T cells. Both effects were much more marked in patients with active disease than in those with inactive SLE. This suggests a defect in both responder and stimulating cell populations in SLE.

I-A antigens on cloned alloreactive murine T lymphocytes are acquired passively.

Cloned lines of alloreactive cytolytic and amplifier T lymphocytes have been examined for expression of I-A antigens. Using flow cytofluorometry and monoclonal antibodies, MHC antigens representing both I-A and H-2K or D determinants were reproducibly detected on the cell surface of these cloned T lymphocytes. All cloned lines examined expressed H-2K or D antigens representing the MHC haplotype appropriate to their strain of origin. However, I-A as well as H-2K or D antigens appeared to be acquired passively from the stimulating cell population. Within the lower limits of detection using the fluorescence-activated cell sorter, evidence suggesting endogenous synthesis of I-A antigens by these cloned T lymphocytes could not be demonstrated. Further, the observed binding appeared to be nonspecific, because material originating from allogeneic, syngeneic, or third-party stimulating cells could be observed under appropriate conditions.

A monoclonal antibody (DT-2) recognizing canine T lymphocytes.

A murine monoclonal antibody (DT-2) is described which has been raised against canine thymocytes. DT-2 activates complement and is reactive with most canine thymocytes, peripheral blood T cells, thoracic duct lymphocytes, bronchoalveolar lymphocytes, and T chronic lymphatic leukemia cells. The antibody is nonreactive with surface immunoglobulin-positive blood lymphocytes, monocytes, bronchoalveolar macrophages, null acute lymphatic leukemia cells, granulocytes, erythrocytes, and platelets. In mixed lymphocyte cultures DT-2 and complement eliminate the responding but not the stimulating cell population. Mitogen stimulation (phytohemagglutinin, concanavalin A) experiments revealed that the responding cell affected by DT-2 is of lymphoid and not of monocyte/macrophage origin. All our data suggest that DT-2 is an antibody reacting specifically with canine T cells.

Suppression of the normal autologous mixed leukocyte reaction by sera from patients with systemic lupus erythematosus.

Sera from 15 patients with active systemic lupus erythematosus and 7 patients with inactive lupus were added to normal autologous mixed leukocyte reaction (MLR) cultures. Twelve of the 22 sera reduced autologous T cell proliferative responsiveness to B plus null (B + N) cells or macrophages by 50% or more. This inhibition correlated with defective autologous MLR in fresh mononuclear cells from those patients. When normal responding and stimulating cell populations were preincubated with suppressive lupus sera, then added to autologous MLR cultures that contained normal serum, proliferative responses were also reduced; inhibitors are directed against participating mononuclear cell subpopulations. The suppressive serum factors were directed variously against monocyte/macrophage (M phi), B + N, or T cell populations; M phi/T interactions were suppressed most frequently. The inhibitors were not removed by high-speed centrifugation or by depletion of antibodies to DNA, and they did not correlate with the presence of lymphocytotoxic antibodies. The presence of serum inhibitors of normal M phi/T and B + N/T autologous MLR may be an immunologic abnormality important in the initiation, perpetuation, or exacerbation of systemic lupus erythematosus.

Antigen-dependent, H-2-restricted cytolytic and noncytolytic T cell lines with specificity for minor histocompatibility antigens.

Several cloned T cell lines were isolated from primed mixed lymphocyte cultures immunized against minor histocompatibility antigens. These lines were selected with irradiated stimulator cells as antigen and require restimulation at intervals to keep growing. They are responsive, as measured by proliferation, to interleukin 2 (T cell growth factor) but cannot be grown in it continuously. These T cell lines have either the H-2d or H-2k haplotype. They all show exquisite H-2 restriction and minor histocompatibility antigen specificity. We did not observe any alloreactivity on 8 different H-2 haplotypes. For the H-2k T cell lines, the restriction element could be mapped to either the K or D end of the H-2 complex. No I-A-restricted cell line was found. It is of interest that all these T cell lines need the presence of T cells in the irradiated stimulator cell population. This suggests a more complex interaction between irradiated stimulators and responder T cells than just H-2K- or D-restricted antigen interaction. This recognition, though necessary, does not seem sufficient to induce the T cell clones to proliferate.

Allogeneic mixed lymphocyte reactions in humans: pretreatment of either the stimulator or the responder cell population with monoclonal anti-Ia antibodies leads to an inhibition of cell proliferation.

We analyzed the effect of 2 hybridoma monoclonal antibodies (Mab), D1-12 and D4-22, with specificity for common determinants of human Ia molecules, on the mixed lymphocyte culture (MLC) response. The results show that addition of either of the 2 Mab as late as day 3 after the onset of the culture completely inhibits the proliferative response generated in MLC. Because the antigenic determinants recognized by the 2 Mab that were used in this study have been shown to belong to distinct Ia molecules, it appears the inhibitory effect observed in MLC containing such Mabs cannot be explained simply by the masking of Ia molecules on the stimulator cell population. In agreement with previous studies by other investigators, treatment of a leukocyte population with the cytolytic D1-12 Mab plus complement strongly reduced its ability to stimulate in MLC. More importantly such a treatment also decreased the ability of a leukocyte population to respond in MLC. In the latter case, the inhibitory effect appears to be directed against T cells since highly purified E-rosetting cells treated with D1-12 plus complement were unable to respond in MLC. The possible implications of these results are discussed.

Alloantigens expressed on activated human T cells different from HLA-A, B, C, and DR antigens.

This study investigates alloantisera containing antibodies directed against antigens which are expressed on alloactivated human T lymphocytes but are absent on resting T and B cells. Among 39 defined anti-HLA-DR sera from multiparous women we found six sera giving positive reactions (more than 25 percent cytotoxicity) on in vitro alloactivated T cells, though negative reactions with resting B or T cells from the donors of either the responding or stimulating cell populations used for alloactivation. Two such sera were submitted to absorption and elution studies. Absorption of these sera with activated T cells did not remove the anti-HLA-DR activity. Furthermore, the antibodies eluted from activated T cells did not react with B cells but were positive only on activated T cells. In addition, we absorbed the sera with B cells and observed that they remained positive on activated T cells. The positive reactions do not seem to be due to either the passive acquisition of antigens from the stimulating population or to low levels of HLA-specific antibodies. As one of the sera we studied intensively gave clear positive and negative reactions on a panel of activated T lymphocytes, we believe it may recognize an antigen of an allogeneic system expressed on alloactivated human T cells.

Generation of immunoglobulin secreting cells in mixed lymphocyte culture

The nature of the cellular interactions and role of the HLA system in the generation of immunoglobulin secreting cells in primary and secondary mixed lymphocyte cultures were investigated. The B lymphocyte response to alloantigen stimulation as measured by a Portein A reverse hemolytic plaque assay, consisted of polyclonal activation with production of IgG. IgM. IgA secreting cells detectable as early as day 4 in a primary and by 24 hr in a secondary mixed lymphocyte culture. B cell activation was shown to be dependent upon collaboration with T helper cells. A disparity at the HLA D/DR region between responding and stimulating cell populations was required for the induction of T helper cells. However, once activated, T helper cells could collaborate with autologous or allogeneic B lymphocytes and, without additional antigen, trigger immunoglobulin production. The mixed lymphocyte culture may now be considered a model of B cell as well as T cell activation.

MURINE AUTOGENEIC MIXED LYMPHOCYTE CULTURE ROLE OF CULTURE CONDITIONS

In an attempt to determine whether culture conditions significantly influence autoreactivity, we tested the effects of fetal calf, syngeneic, and autogeneic serum with and without 2-mercaptoethanol on the murine self-reactive mixed lymphocyte culture. We used both unfractionated and T-enriched lymph node cells as responders and unfractionated and B-enriched irradiated spleen cells as stimulators in a primary mixed lymphocyte culture (MLC). Using unfractionated cells as responders to various stimulator cell populations, we found excellent allogeneic reactions in all media tested but minimal or no self-reactivity in syngeneic of autogeneic serum. When using T-enriched lymph node cells as responders, allogeneic reactivity was excellent but self-reactivity was present only in the cultures supplemented with fetal calf serum and 2-mercaptoethanol. The possibility that the substances in fetal calf serum and/or 2-mercaptoethanol may be needed to enhance minimal, but biologically relevant, self-reactivity is discussed, as well as the possibility that the culture supplements may be inducing "nonspecific" self-reactivity.

Bovine mixed leukocyte response: Effect of selected parameters on cell responses

The bovine mixed leukocyte culture (MLC) system offers potential benefit for the study of genetic and immunologic mechanisms in this species. Selected parameters of the bovine MLC have been investigated to assess their influence. The findings indicate that maximal MLC response was present at days 5 and 6 with 2 × 10 5 responder to 2 × 10 5 stimulator cells per well or greater. Serum concentrations over a wide range effectively supported cell growth. More importantly, the source and treatment of serum influenced the level of MLC response. Serum autologous to the responder which was heat treated appeared to provide maximal MLC values compared to other sources examined. Antisera to serologically-defined lymphocyte antigens of the stimulating cell population did not affect the response. One-way MLC determinations were consistent when the stimulating cell population was irradiated at 1,000 Rads or greater. Both freshly obtained lymphocytes and lymphocytes held at room temperature for 18 hr provided good sources of MLC responder and stimulator cells; however, lymphocytes held for 18 hr consistently gave higher counts and stimulation indices than freshly obtained lymphocytes. The effect of contaminating RBCs was found to enhance MLC reactivity at lower concentrations and inhibit MLC reactivity when in excess of 7 × 10 5 RBC per well. Stimulator cells were present in both macrophage enriched and depleted populations suggesting several cell populations possess antigens which lymphocytes recognize. Consideration of these parameters were essential in providing optimal, reproducible results.

Enhancement of T lymphocyte functions by Fc fragments of immunoglobulins. I. Augmentation of allogeneic mixed lymphocyte culture reactions requires I-A- or I-B-subregion differences between effector and stimulator cell populations.

Fc fragments derived from human Ig were found to be capable of enhancing T cell-mediated, antigen-induced proliferative and mixed lymphocyte culture responses. Maximum enhancement occurred when suboptimal amounts of antigen or suboptimal numbers of stimulator cells were employed. Augmentation of the allogeneic mixed lymphocyte culture reaction requires an I-A and/or I-B subregion difference between effector and stimulator cell populations. Although a significant proliferative response was observed with K- or D- region differences, Fc fragments were unable to enhance the response. The T cell population acted upon by Fc fragments in the potentiation of these responses bears the Lyt-1(+)23(-) phenotype.

T cell lines active in the delayed-type hypersensitivity reaction (DTH).

Two allospecific uncloned T cell lines, C.C3.11.75 (H-2d anti-H-2k) and B6-C.7.76 (H-2b anti-H-2d), were assayed for their ability to elicit a DTH reaction in vivo. It was shown that both cell lines, C.C3.11.75 being cytolytic and B6-C.7.76 being noncytolytic, evoke a strong DTH reaction. The reaction was in tempo and histology typical for a DTH reaction and could not be distinguished from a DTH reaction elicited by in vivo generated T cells active in a GVH reaction. Both cell lines recognize a private specificity encoded in the H-2 IA subregion. Cell proliferation of the responder cells did not appear to be critical in the reaction nor are T cells from the host recruited during the reaction. The DTH reaction can be assayed in allogeneic hosts demonstrating a complete lack of H-2 restriction in the reaction even when presumably antigen presenting macrophages are removed from the stimulator cell population. Furthermore, tumor cells expressing IA antigens could be employed to cause the reaction in both syngeneic and allogeneic recipients. Cloned sublines of one of the lines are also active in the reaction.