Monocyte function in mixed lymphocyte reactions

Subpopulations of human peripheral blood lymphocytes were isolated by sequential separation techniques. The stimulating and responding capacity of these cells together with the T-cell population remaining after the removal of other populations was studied in one-way allogeneic mixed lymphocyte culture. Incorporation of [³H]thymidine was used as a measure of response. Monocytes, present in the stimulating or responding cell population, were necessary for lymphocyte response. T cells stimulated responding T-cell populations containing monocytes but not B cells. Stimulation by T cells could be inhibited with DRW antisera. Response was also inhibited by sera detecting DRw antigens on the monocytes of the responding cell population. It is concluded that monocytes play an important functional role in mixed lymphocyte reactions. In addition, it appears that the combination of anti-DRw sera and monocytes influences mixed lymphocyte reactions by an active process in that inhibition of response cannot be explained entirely by blocking DRw determinants.     

Stimulation of autologous and allogeneic human T cells by B cells occurs through separate B-cell antigen systems.

Studies were performed to determine the cell types involved in autologous mixed lymphocyte reactions (AMLR). Separation of peripheral blood leucocytes from normal donors into T-cell and B-cell enriched populations and subsequent co-culture of T cells and mitomycin-C inactivated non-T cells resulted in increased DNA synthesis by the responding T cells. Procedures such as removal of plastic-adherent or phagocytic cells enriched the B-cell content of the stimulator population and also enhanced stimulation of both autologous and allogeneic T cells. Velocity sedimentation separation of peripheral blood leucocytes into large and small lymphocyte fractions showed that cells which stimulate in MLR and AMLR are found in the small cell fraction and that large cells, morphologically monocytes, do not stimulate either reaction. Studies with anti-B-cell antisera obtained from multiparous women showed that the antigens of B cells which stimulate AMLR are distinct from those which stimulate MLR.     

Alloantigenicity of human endothelial cells. 1. Frequency and phenotype of human T helper lymphocytes that can react to allogeneic endothelial cells.

To determine the relative ability of allogeneic endothelial cells to stimulate helper T lymphocytes (HTL), human PBMC or purified T cells were incubated in conventional lymphocyte microcultures or in limiting dilution microcultures with allogeneic human umbilical vein endothelia (HUVE), with cytokine-treated allogeneic HUVE, or with allogeneic peripheral blood monocytes. These cultures were tested for IL-2 production as an index of HTL stimulation. Dose-response studies in conventional lymphocyte cultures indicated that allogeneic monocytes were better than allogeneic HUVE at stimulating IL-2 production. Limiting dilution analyses revealed that untreated HUVE and TNF-treated HUVE stimulated small numbers of HTL (approximately 1 HTL/30,000 PBMC), whereas 5 to 10 times more HTL were stimulated by IFN-gamma-treated HUVE and 10 to 20 times more HTL were stimulated by allogeneic monocytes. Serologic deletion studies revealed that most of the high frequency HTL responding to IFN-gamma-treated HUVE were CD4+, whereas most of the low frequency HTL responding to nontreated HUVE or to TNF-treated HUVE were CD8+. Interestingly, mAb to MHC class I and class II molecules, which significantly impaired HUVE-induced proliferation, caused little interference with HUVE-induced IL-2 production. Finally, polymerase chain reaction analysis demonstrated that untreated allogeneic HUVE cells could stimulate PBMC to produce mRNA for IFN-gamma, as well as for IL-2. These data demonstrate the following hierarchy of allogeneic stimulatory capacity for human HTL: monocytes greater than IFN-gamma-treated HUVE much greater than TNF-treated HUVE = nontreated HUVE. Further, these data suggest that non-activated allogeneic endothelial cells can initiate immune responses by inducing IL-2 and IFN-gamma. Because IFN-gamma can induce MHC class II expression by the endothelial cells, this could recruit large numbers of CD4+ T cells for IL-2 production.     

Lymphocyte transformation induced by autologous cells.

Human peripheral blood T lymphocytes are stimulated to proliferate when cultured with autologous B-lymphoblastoid cell lines, autologous mitogen-induced lymphoblasts, or autologous non-T blood lymphocytes. This reaction, the autologous mixed lymphocyte reaction, has attributes of an immune response possessing both memory and specificity. The capacity to stimulate autologous T lymphocyte proliferation depends on the lineage of the lymphoid cell and not on its establishment in continuous culture or carriage of the EB viral genome. The determinant on non-T lymphocytes which stimulates the autologous mixed lymphocyte reaction appears to be an Ia determinant. Thus, allogeneic graft rejection and the allogenic mixed lymphocyte reaction are very likely extensions of an immune response expressed within the host.     

Autologous mixed lymphocyte reaction in man. I. Relationship with age and sex

Coculture of normal human T lymphocytes with autologous cells enriched in B lymphocytes results in increased DNA synthesis. In our system, the responder cells are mononuclear cells stimulated by irradiated autologous non-T cells in the presence of 20% inactivated autologous plasma. The results indicate that the proliferative response is age and sex related; significantly higher levels of thymidine incorporation are observed with female peripheral blood lymphocytes and, moreover, it appears that this strong stimulation is more pronounced in older female subjects. In contrast, the proliferative responses against allogeneic cells are not modified irrespective of the age and sex group considered. The involvement of autorosette-forming cells (A-RFC) in such autologous mixed lymphocyte reaction (A-MLR) is demonstrated by the marked impairment of the A-MLR after removal of A-RFC from responding cells. These data suggest that older healthy subjects, particularly females, exhibit a higher incidence of in vitro autologous reactions.     

Autologous responses to human leukaemic cells in mixed leucocyte culture

Summary Cryopreserved leukaemic blasts and remission non-T cells from 22 patients with acute leukaemia (15 lymphocytic, 7 non-lymphocytic) were tested as stimulators of autologous remission T cells and normal allogeneic T cells in primary and secondary MLC. In most cases the autologous response elicited by leukaemic cells was less than or equal to that elicited by remission non-T cells. However, T cells from 2 patients in long-standing first remission from ANLL displayed greater proliferation in response to leukaemic blasts than to remission non-T cells in both primary and secondary MLC. The results are suggestive of sensitization of these 2 patients to leukaemia-specific antigens, but other possible explanations are discussed. Abbreviations used: MLC, mixed leucocyte culture; ANLL, acute non-lymphocytic leukaemia; ALL, acute lymphoblastic leukaemia; AMLR, autologous mixed lymphocyte reaction; NK cells, natural killer cells; MNC, mononuclear cells     

Induction of suppressor cells in autologous mixed lymphocyte culture (AMLC) in humans

T cells stimulated for 6-7 days in autologous mixed lymphocyte culture (AMLC) showed suppressive effects when added to fresh mixed cultures where autologous lymphocytes (A) were stimulated by Mitomycin C-treated allogeneic lymphocytes (Xm), in a ratio of A:Xm:AMLC-activated cells of 1:1:0.5. Both cytotoxic and proliferative activities in second cultures, as assayed after 6 days of incubation, were significantly inhibited (percentage suppression of cytotoxic activity observed in 17 experiments was 75.3 +/- 22.4; percentage suppression of proliferation was 60.6 +/- 18.2). Suppressor cells (SC) generated in AMLC were Mitomycin C sensitive and nonspecific in their action; not only A/Xm but also X/Am and X/Ym cultures were suppressed to the same extent. AMLC-Activated cells showed a considerable degree of proliferation in response to alloantigens but failed to express any cytotoxic activity against autologous or allogeneic phytohemagglutinin blasts. Thus, the inhibitory effect observed in this system is not due to cytotoxic elimination of responding or stimulating cells in the second culture but rather reflects a true regulatory (suppressive) mechanism.     

Activated human T cells can present alloantigens but cannot present soluble antigens

Human T cells, when activated by antigen or mitogen, express Ia antigens. We have examined the capacity of activated T cells to stimulate autologous and allogeneic T cells and their ability to present soluble antigen. Interleukin 2-dependent T-cell lines (TCL), free of accessory cells, were used for antigen-presenting cells. These activated T cells were potent stimulators in an autologous mixed lymphocyte reaction (AMLR), more so than autologous irradiated non-T mononuclear cells. Activated T cells were also able to stimulate proliferation of allogeneic T cells in the absence of any other accessory cells, and this stimulation was blocked by anti-Ia antibodies. Resting unstimulated T cells were unable to stimulate autologous or allogeneic responses. Thus, activated T cells were able to present self antigens and alloantigens. However, activated T cells could not present soluble antigens to autologous T cells or to antigen-specific TCL even if exogenous interleukin 1 was added to cultures. The ability of activated T cells to stimulate an AMLR in vitro may reflect an important immunologic amplification mechanism in vivo. The ability of activated T cells to present alloantigens but not soluble antigens suggests an inability to process antigen, and this may provide further insights into the complexities of antigen presentation.     

T cell proliferation induced by autologous non-T cells is a response to apoptotic cells processed by dendritic cells

Self-reactive T cells are present in the mature immune repertoire as demonstrated by T cell proliferation induced by autologous non-T cells in the autologous mixed lymphocyte reaction. This reaction generates regulatory T cells in vitro and may reflect immune regulatory pathways in vivo, but the antigenic peptides recognized remain uncharacterized. We revisited this issue in light of the importance of apoptosis in immune regulation. We found that apoptosis among peripheral blood non-T stimulator cells is associated with augmented induction of autologous T cell proliferation. Our data show that caspase activity in the non-T stimulator population is essential for induction of autologous T cell proliferation, suggesting that cellular components in the non-T cell fraction are enzymatically modified, most likely by effector caspases, and have a direct or indirect effect on autoreactive T cell activation. Furthermore, exposure of macrophage-derived dendritic cells to apoptotic non-T cells augments autologous T cell proliferation, and blockade of alpha(v)beta(5) integrin, but not alpha(v)beta(3), inhibits the capacity of irradiated non-T cells or dendritic cells to stimulate autologous T cell proliferation. These experiments, using an entirely autologous system, suggest the interpretation that autoreactive T cells may recognize self-Ags modified through the actions of caspases and presented to T cells by dendritic cells. Induction of an in vivo autologous mixed lymphocyte reaction by caspase-modified self-Ags present in apoptotic cells may represent a mechanism to maintain peripheral immune tolerance.     

Lymphocyte transformation induced by autologous cells. III. Lymphoblast-induced lymphocyte to stimulation does not correlate with EB viral antigen expression or immunity.

Human mitogen-induced and cell line B lymphoblasts stimulate the proliferation of allogeneic and autologous lymphocytes in culture. The role in thes reaction of EB viral determinants on the stimulating cells and immunity of the lymphocyte donor to the EB virus has been studied. The stimulatory capacity of cultured cell line lymphoblasts is not inhibited by incubating lymphoblasts with antisera to EB viral determinants. Cultured cell line B lymphoblasts stimulate as much thymidine incorporation by lymphocytes from donors with or without immunity to the EB virus. Further, a B lymphoblast cell line (U-698) which lacks the EB viral genome stimulated as much lymphocyte proliferation as did B lymphoblasts with the EB genome. Cultured T lymphoblast cell lines do not stimulate allogeneic lymphocyte proliferation. These cells appear to lack the determinants which stimulate lymphocyte transformation. No evidence was found that cultured cell line T lymphoblasts suppressed allogeneic lymphocyte proliferation. Mitogeninduced lymphoblasts from EB-immune and non-immune subjects stimulated the proliferation of autologous lymphocytes comparably. It is concluded that neither immunity to the EB virus nor expression of EB viral antigens on mitogen-induced on cell line lymphoblasts is necessary for the stimulation of lymphocyte proliferation.     

Enhancive and suppressive effects of cytomegalovirus on human lymphocyte responses in vitro.

Virus-specific lymphocyte proliferation in the presence of cytomegalovirus (CMV) without and with monocytes was studied in healthy persons. Three categories of lymphocyte response could be distinguished: seropositive low responders, naturally high responders, and lymphocyte populations responding well to CMV antigen in the presence of added CMV-incubated autologous monocytes. This latter category could be identified by preincubating autologous monocytes with CMV. CMV-seronegative persons were nonresponders. Early CMV antigens were produced in monocytes but not in lymphocytes by all CMV isolates. Infection of monocytes as detected by antibody to early viral protein did not appear to abort the antigen-presenting ability. The virus-specific responding lymphocytes were mainly of the T4+ phenotype. In contrast, addition of CMV to polyclonal mitogens significantly suppressed total lymphocyte DNA synthesis. CMV thus may have an enhanced virus-specific stimulatory effect on lymphocytes together with monocytes but a suppressive effect on the total lymphocyte population.     

Weak mixed lymphocyte culture response in the Mongolian gerbil (Meriones unguiculatus).

A study was made to determine whether the mixed lymphocyte culture test could detect histocompatibility differences between two strains of the Mongolian gerbil, Meriones unguiculatus. A semi-micro mixed lymphocyte culture was developed using 6 X 10(5) stimulating and responding cells per 0.12 ml culture volume at a ratio of 1:1. A culture period of 120 hours was found to be optimal. Although a weak allogeneic response was demonstrated with the Uclp:(MON) strain responding to stimulating cells from the MON/Tum strain, the reverse was not seen. A mixed lymphocyte reaction to xenogeneic (mouse) cells was demonstrated, and response to the mitogen, phytohemagglutinin, was strong. These data and the low stimulation index obtained in allogeneic culture supported the view that histocompatibility differences among different strains of the Mongolian gerbil are weak.     

Human monocytes as intermediaries between allogeneic endothelial cells and allospecific T cells: a role for direct scavenger receptor-mediated endothelial membrane uptake in the initiation of alloimmunity

Recipient monocytes, T cells, and donor endothelial cells (ECs) are recognized as critical components of allograft rejection. We have recently shown that human monocytes infiltrate vascularized allografts before clinical rejection and have thus hypothesized that monocytes, rather than costimulation-poor ECs, initiate an alloimmune response. However, the nature of the interactions between ECs, monocytes, and T cells has been incompletely defined. Specifically, it is not clear whether these cells interact in a hierarchical manner, nor is it apparent what constitutes an interaction. We therefore studied human ECs, monocytes, and T cells in various isolated in vitro combinations to define the salient features of their contact and to determine whether their interactions were sequential in nature. We find that T cells proliferate poorly to allogeneic ECs and autologous monocytes but well to autologous monocytes following allogeneic EC contact. We show that monocytes gain their stimulatory capacity by phagocytizing allogeneic but not autologous EC membranes in a process governed by scavenger receptors. This process facilitates the subsequent presentation of intact donor HLA molecules to T cells (semidirect presentation). Moreover, monocytes are receptive to T cell help only after exposure to ECs and require CD4+ T cells to optimally express costimulatory molecules and foster Ag presentation. Our results indicate that monocytes engage allogeneic ECs through scavenger receptors and are then primed to facilitate T cell activation in a codependent manner. This reciprocal codependence allows for monocytes to serve as a regulated bridge between the allograft and T cells.     

Functional involvement of the LFA-1/ICAM-1 adhesion system in the autologous mixed lymphocyte reaction

The integrin surface molecule termed lymphocyte functional antigen-1 (LFA-1), and its physiological ligand intercellular adhesion molecule-1 (ICAM-1), have been proven to play a relevant role in several immune reactions where cell-to-cell contact is required: these reactions include allogeneic mixed lymphocyte reaction (MLR) and direct cytotoxicity. In the present study, we show that monoclonal antibodies (mAbs) directed to LFA-1 as well as to ICAM-1 molecules are able to inhibit T cell proliferation in autologous MLR (AMLR). Such an in vitro reaction is generally considered a functional model of Ia-mediated immunocompetent cell cooperation, and is impaired in several pathological conditions. It is noteworthy that the LFA-1 molecule is largely represented on the T cell surface, whereas ICAM-1 is poorly expressed on resting T cells: autologous stimulation slightly increases ICAM-1 expression. Pretreatment studies indicate that the inhibitory effect of anti-ICAM-1 mAb on T cell proliferation in AMLR is exerted on responder T cells.     

Influence of sex hormones on Coxsackie B-3 virus infection in Balb/c mice

Background and “spontaneous” proliferation are terms often used for the proliferative activity normally exhibited by peripheral blood mononuclear cells (MNC) in vitro. In this report, we show that Interleukin-2 (IL-2) added to unfractionated MNC but not to isolated T or non-T cells significantly increased their proliferative activity. The cells responding to IL-2 stimulation from MNC were OKT3 positive lymphocytes. In addition, treatment of MNC with either a monoclonal anti-HLA-DR antibody (in the absence of C′) or Cyclosporin-A strongly suppressed the “background” whereas treatment of MNC with the 3A1 monoclonal anti-human T cell antibody did not modify “spontaneous” proliferation of these cells. IL-2 could not restore or increase the proliferative activity of MNC exposed to the anti-HLA-DR antibody or Cyclosporin-A while the T cell growth factor significantly enhanced proliferation of MNC cultured in the presence of the OKT4 antibody. Taken together these results strongly suggest that IL-2 responding T cells from MNC become sensitive to IL-2 by interacting with HLA-DR antigens on B lymphocytes and/or monocytes contained in MNC (resting T cells are Dr^?). By a similar mechanism we have previously shown that T cells acquire responsiveness to IL-2 in the autologous mixed lymphocyte reaction (AMLR). Since all the cells that participate in AMLR are present in MNC, we postulate that a “mini” AMLR taking place within MNC may explain the “spontaneous” proliferation of peripheral blood mononuclear cells.     

Cytotoxic T cells generated in the autologous mixed lymphocyte reaction. I. Primary autologous mixed lymphocyte reaction

In the course of the culture of an autologous mixed lymphocyte reaction (AMLR), T cells proliferated in response to autologous non-T cells, and differentiated to cytotoxic T cells (AMLR killers). DNA synthesis was necessary to generate AMLR killers, as the elimination of autoreactive proliferating cells with BUdR and UV light completely abrogated AMLR killer cytolysis. Amlr killers lysed various lymphoid cell lines, including autologous B cell lines, autologous or allogeneic mitogen blasts stimulated by Con A, PHA, or pokeweed mitogen, variious nonlymphoid cell lines derived from human, mouse, or rat, and weakly normal autologous or allogeneic non-T cells. KMT-17, methylcholanthrene-induced rat fibrosarcoma, was the only resistant cell line to have been tested. AMLR killers had characteristics similar to NK cells, Major histocompatibility antigens were not the target antigens for AMLR killers. AMLR killers distinguished the blasts stimulated by alloantigens as self from the blasts stimulated by mitogens as non-self.     

Simple sugars inhibit proliferation of human T lymphocytes in autologous and allogeneic mixed lymphocyte reactions

Several oligo- and monosaccharides were studied for their capacity to modulate lymphocyte proliferation in human allogeneic and autologous mixed lymphocyte reactions (MLR). A defined subset of sugars showed a marked inhibitory effect on lymphocyte proliferative response in the majority of the allogeneic MLR combinations studied. The inhibitory effect disappeared when sugars were added to allogeneic MLR 96 hr after the beginning of culture. These sugars also showed a significant inhibitory power on autologous MLR, performed by using T- and non-T-enriched lymphocytes from the same donor. The reported data suggest that carbohydrate determinants are involved in the proliferative response of human lymphocytes in both autologous and allogeneic MLR.     

The differentiation of suppressor cell populations as revealed by studies of the effects of mitogens on the mixed lymphocyte reaction and on the generation of cytotoxic lymphocytes.

The regulation by concanavalin A (Con A) and bacterial lipoloysaccharide (LPS) of the mixed lymphocyte reaction (MLR) and of the generation of cytotoxic lymphocytes (CL) was studied in congenic resistant mice using cortisone resistant thymocytes as the responding cells. LPS enhances the generation of CL selectively when suboptimal numbers of allogeneic cells are present in mixed lymphocyte cultures and also results in the augmentation of the MLR. Mitogenic concentrations of Con A on the other hand suppress the generation of CL regardless of alloantigen dose. The mechanism of suppression cannot be ascribed to the presence of suppressor T cells, since the addition to the cultures of syngeneic cortisone resistant thymocytes activated by Con A does not change the immune response. However, prospective suppressor cells that can be activated by Con A are located in secondary lymphoid organs such as spleen and lymph node. Suppressor activity by those cells is abolished by anti θ plus complement. Con A activated spleen cells suppress the MLR, whereas Con A activated thymocytes amplify the proliferation of responding cells.     

Immature Dendritic Cells Generated from Cryopreserved Human Monocytes Show Impaired Ability to Respond to LPS and to Induce Allogeneic Lymphocyte Proliferation

Dendritic cells play a key role in the immune system, in the sensing of foreign antigens and triggering of an adaptive immune response. Cryopreservation of human monocytes was investigated to understand its effect on differentiation into immature monocyte-derived dendritic cells (imdDCs), the response to inflammatory stimuli and the ability to induce allogeneic lymphocyte proliferation. Cryopreserved (crp)-monocytes were able to differentiate into imdDCs, albeit to a lesser extent than freshly (frh)-obtained monocytes. Furthermore, crp-imdDCs had lower rates of maturation and cytokine/chemokine secretion in response to LPS than frh-imdDCs. Lower expression of Toll-like receptor 4 (at 24 and 48 h) and higher susceptibility to apoptosis in crp-imdDCs than in fresh cells would account for the impaired maturation and cytokine/chemokine secretion observed. A mixed leukocyte reaction showed that lymphocyte proliferation was lower with crp-imdDCs than with frh-imdDCs. These findings suggested that the source of monocytes used to generate human imdDCs could influence the accuracy of results observed in studies of the immune response to pathogens, lymphocyte activation, vaccination and antigen sensing. It is not always possible to work with freshly isolated monocytes but the possible effects of freezing/thawing on the biology and responsiveness of imdDCs should be taken into account.     

Dissociation of autologous and allogeneic mixed lymphocyte reactivity by using a monoclonal antibody specific for human T helper cells

A monoclonal antibody defining a population of human T helper cells was developed and shown to specifically block the autologous mixed lymphocyte reaction (AMLR). This antibody, termed KT69-7 (IgG1), recognized 62% of peripheral blood E rosette-positive (E+) cells while demonstrating negligible reactivity with E- cells, monocytes, granulocytes, EBV-transformed B cell lines, and mouse splenocytes. Separation of E+ cells into KT69-7+ and KT69-7- populations revealed that KT69-7+ T cells provided helper function in PWM-driven B cell differentiation, whereas KT69-7- T cells provided no help and may suppress this response. Modulation of membrane moieties by using KT69-7 or OKT4 plus goat anti-mouse IgG removed reactivity to both these antibodies, suggesting an association between these molecules recognized by these antibodies. In functional studies, KT69-7 selectively blocked the AMLR while demonstrating minimal or no effect on the allogeneic MLR (allo-MLR). Blocking of the autoreactivity occurred when either autologous B lymphocytes or macrophages were used as stimulators. The failure of KT69-7 to block the allo-MLR was not attributable to excessive allogeneic stimulus; KT69-7 failed to block even under conditions of limiting numbers of stimulator cells. KT69-7 thus appears to recognize a molecule on the surface of T helper cells required for recognition of autologous class II antigens.