Decreased suppressor cell activity in disseminated granulomatous infections

The effect of granulomatous infections upon the activity of a T lymphocyte subclass in human peripheral blood that can be induced by concanavalin A (Con A) to function in a suppressor mode was studied. Peripheral blood lymphocytes (PBL) from eleven patients with disseminated mycotic or mycobacterial infections or from controls were preincubated with and without Con A, washed and cultured with allogeneic PBL freshly drawn from healthy donors sensitive to histoplasmin. DNA synthesis was then measured in co-cultures stimulated by Con A, histoplasmin, or by the mixed lymphocyte culture (MLC) reaction alone. As compared with cells preincubated without Con A, the Con A-pretreated cells were significantly less effective in suppressing the responses of normal PBL to histoplasmin (P < 0.01), and in a one-way MLC reaction (P < 0.05). The Con A-induced suppressor activity of PBL from nine patients with localized granulomatous infections did not differ significantly from that exerted by PBL of normal controls in two of the three co-culture systems employed. These studies suggest that either dysfunction or a reduction of the Con A-inducible T-suppressor cell subpopulation in peripheral blood is frequent among patients was disseminated granulomatous infections.     

The Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) as stimulating cells in mixed lymphocyte culture (MLC)

The MLC response of normal lymphocytes to different kinds of EBV-transformed LCL was investigated. The response to allogeneic LCL cells remarkably exceeded the response to xenogeneic and autologous LCL cells. Stimulation performed with EBV-transformed and normal cells obtained from the same donors showed a correlation in the reaction levels (Q = 0.434). This incomplete correlation was not improved by the reduction of the number of stimulating LCL cells. In MLC stimulated with LCL no statistically significant differences between responses to 2,1 or alloantigens were found.     

HLA–D and –DR antigens on human amniotic fluid cells

Human amniotic fluid cells, known to express HLA-A, -B, and -C antigens, were tested for the presence of lymphocyte-stimulating antigens (LD or HLA-D) using modifications of the mixed lymphocyte culture (MLC) and primed lymphocyte typing (PLT) tests. Peripheral blood lymphocytes were co-cultured with various concentrations of allogeneic amniotic fluid cells, either growing as a monolayer culture in microtiter plates or suspended in medium following treatment with trypsin. The kinetics of such mixed lymphocyte amniotic fluid cell culture (MLAC) reactions were followed during days 3 to 8. Under none of these conditions did amniotic fluid cells significantly stimulate allogeneic lymphocytes, even after lymphocytes were specifically primed in the PLT assay to the HLA-D antigens segregating in the family of the amniotic fluid cell donor. Furthermore, in three-cell experiments, amniotic fluid cells failed to inhibit an ongoing MLC reaction, indicating that the absence of proliferative response to amniotic fluid cells is not due to active suppression. Taken together, these data strongly suggest that amniotic fluid cells either do not express HLA-D antigens or do not express them in a form that is detectable in either primary or secondary MLC.     

Rapid responses of lymphocytes in an optimized mixed lymphocyte culture

When adherent mononuclear cells are removed from whole blood, the separated lymphocytes respond rapidly by proliferation to foreign cocultured lymphocytes. The reaction of responding cells against a pool of foreign stimulating cells can be detected in this rapid mixed lymphocyte culture (r-MLC) within 28 h and antigenic disparity between family members, differing for only one HLA haplotype, can be measured within 36 h. These early responses show genetic specificity corresponding to those observed in a standard MLC at 96 h. The involvement of similar cell populations in the regulation of early responses to allogeneic cells and increased responses to autologous cells in r-MLC, as well as accelerated responses in secondary MLC, is discussed.     

Con A-induced suppressor cells in man II. Role of monocytes and their interaction with soluble inhibitory factors

Monocyte dependence of Con A-induced suppression was studied in co-cultures of normal human lymphocytes. Con-A-pretreated (25 µg/ml for 48 hours) peripheral blood mononuclear (MN) cells inhibited the ³H-thymidine uptake by fresh autologous or allogeneic lymphocytes stimulated with Con A. Monocyte-depleted lymphocytes pretreated with 25 µ/ml of Con A failed to exhibit the suppressor properties. Supernatants of MN cell activated with 25 µ/ml Con A for 72 hours inhibited the mitogenic response of fresh allogeneic cells when added in proportion 1 : 1 to the culture medium. No inhibition was observed when supernatants of monocyte-depleted, Con A activated cultures were tested. The short preincubation with suppressive supernatants rendered blood adherent mononuclear cells suppressive for ³H-thymidine incorporation by autologous MN cells stimulated with Con A.These studies indicate that monocytes are essential for induction of suppressor cells in Con A stimulated cultures. The suppressor cells produce a soluble inhibitory factor(s) which has the property of binding to the surface of fresh monocytes. Such monocytes become responsible for suppression of mitogenic response of autologous lymphocytes.     

Autologous mixed lymphocyte reaction in man. III. Regulation of autologous MLR by theophylline-resistant and -sensitive human T-lymphocyte subpopulations

Human peripheral blood T lymphocytes were separated into theophylline-resistant (TR) and -sensitive (TS) subpopulations. Proliferative responses TR, TS and unfractionated T cells were studied, using irradiated autologous or allogeneic non-T cells as stimulators. TR cells proliferated vigorously in both autologous and allogeneic mixed lymphocyte cultures (MLC). TS cells. which constitute about 20% of unfractionated T cells, exhibited poor proliferative responses to autologous and allogeneic stimulation. The magnitude of proliferation in autologous and in allogeneic MLC was found to be directly dependent on the number of TR cells in the culture. Mitomycin-C (MMC)-treated TR cells augmented and MMC-treated TS cells suppressed (P less than 0.05) the autologous and allogeneic MLC responses of unfractionated T cells. However, the response of TS cells did not increase in autologous or allogeneic MLC when co-cultured with MMC-treated TR cells. MMC-treated TS cells, when co-cultured with TR cells, suppressed the responses of TR cells (P less than 0.05). The enhancing effect of TR cells was radiation-resistant. Suppressor influence of TS cells, in contrast, was abolished by irradiation (P less than 0.05). These findings demonstrate that TR cells are the responding cell in autologous MLR and augment the MLC responses of unfractionated T cells. TS cells, on the other hand, respond poorly in autologous or allogeneic MLC and suppress the response of TR cells.     

Effects of monocytes from human neonates on lymphocyte transformation

Adherent cells (approximately 75% monocytes, 25% lymphocytes) obtained from neonates and from adults were studied to compare their effects on mitogen-induced lymphocyte transformation. The response of autologous lymphocytes to concanavalin A (Con A) was enhanced significantly by adherent cells from neonates and from adults. Whereas the addition of 2-mercaptoethanol (2-ME) did not enhance the response of unseparated peripheral blood mononuclear cells or fully restore the response of adherent cell-depleted lymphocytes from neonates, both of these effects were observed when 2-ME was added to cell preparations from adults. In fact, the response to Con A of lymphocytes from adults was significantly greater in the presence of 2-ME than in the presence of autologous adherent cells. Equivalent enhancement of the response to Con A was observed when adherent cells from neonates were added to lymphocytes from adults or when adherent cells from adults were added to lymphocytes from neonates.

Adherent cells from neonates consistently inhibited the autologous lymphocyte response to the specific B-cell mitogen, NWSM, a water-soluble extract of Nocardia opaca. Lymphocytes from five out of nine neonates failed to respond to NWSM unless adherent cells were depleted. The presence of adherent cells did not prevent the response of lymphocytes from any of the eight adults tested. This difference in response to NWSM between lymphocytes from neonates and adults was significant. Inhibition of the response of autologous lymphocytes to NWSM by adherent cells from adults was of lesser magnitude and could be demonstrated consistently only when 2-ME was added to adherent cell-depleted lymphocyte preparations. We conclude that the effects of adherent cells which were observed were due to monocytes. The enhancing effect of monocytes from adults on lymphocyte response to Con A could be replaced by 2-ME, whereas this was not true for neonates. In contrast to their effects on response to Con A, monocytes from neonates inhibited the response to NWSM more consistently and to a greater degree than did monocytes from adults.

     

Suppression of DNA synthesis by Con A-activated human lymphocytes: Stimulation by con A bound to non-T cells unless removed after activation

The capacity of human peripheral blood lymphocytes to suppress DNA synthesis of other lymphocytes was studied in an assay consisting of two steps: firstly, activation by Con A during 24 hr followed by alpha-methylglucoside and mitomycin treatment; secondly, incubation of these Con A-activated 'suppressor' cells with autologous responder cells and stimulants, or incubation with allogeneic responder cells. The results were compared with cells similarly treated but not incubated with Con A. If alpha-methylglucoside treatment is omitted, stimulation of T and non-T cells occurs by Con A bound to the Con A-activated cells. Con A is especially bound to non-T lymphocytes and even gives a T cell-independent proliferation of non-T cells without differentiation to plasma cells. With alpha-methylglucoside treatment, 'suppressor' cells, activated by high Con A concentrations, are able to suppress DNA synthesis of autologous lymphocytes stimulated by allogeneic cells or soluble antigens to about 50%. In a one-way MLC, in which the cell suspension containing the suppressor cells is also used as a stimulator cell suspension, a similar suppression was observed. Suppression of DNA synthesis was correlated with suppression of proliferation without evidence of cytotoxicity.     

Cloning of the MHC class II DRB cDNA from the brushtail possum (Trichosurus vulpecula)

The cell-surface glycoproteins encoded by the major histocompatibility complex (MHC) bind to processed foreign antigens and present them to T lymphocytes. Two classes of MHC molecules and their corresponding gene sequences have been extensively studied in eutherian mammals and birds, but data on the marsupial MHC are limited. Marsupials split from eutherian mammals about 125 million years ago and represent a distinct branch in mammalian evolution. Here the cDNA cloning of MHC class II genes of the brushtail possums (Trichosurus vulpecula) is reported. The sequences obtained were found to be relatively conserved when compared to the red-necked wallaby (Macropus rufogriseus) and an South American marsupial, Monodelphis domestica. The T. vulpecula sequence shared an average overall sequence identity of 75.4% at the deduced amino acid level with M. rufogriseus and M. domestica, respectively.     

Antigen Presenting Cells in Human Decidual Tissue: III. Role of Accessory Cells in the Activation of Suppressor Cells

ABSTRACT: Human decidual antigen presenting cells (DAPCs) exposed to fetal cells in vitro induce generation of suppressor T cells among a population of peripheral blood lymphocytes. Human lymphocyte antigen (HLA)-class II positive antigen presenting cells were isolated from early normal human decidual tissue and from peripheral blood (PAPCs) by adhering Ficoll-Pa-que separated cell suspensions to fibronectin. In contrast to PAPCs, DAPCs pulsed with fetal antigens induced a radio-sensitive, Leu 1,2-positive T suppressor cell population. A nylon wool adherent B cell population is required during the in vitro induction of the suppressor cells. These suppressor cells impair primary mitogen and mixed lymphocyte culture (MLC) responses, generation of anti-trinitrophe-nyl (TNP) cytotoxic T lymphocytes, and antibody response of autologous and allogeneic lymphocytes. Only intact viable embryonic cells can effectively confer upon DAPCs the ability to induce T suppressor cells. The T suppressor cell induction by DAPCs primed with fetal antigens is restricted by the major histocompatibility complex. Our results show that the HLA-DR molecules are the most prominent restriction elements.     

Immune function in aged mice. III. Role of macrophages and effect of 2-mercaptoethanol in the response of spleen cells from old mice to phytohemagglutinin, lipopolysaccharide and allogeneic cells.

Spleen cells from old mice responded less well to phytohemagglutinin (PHA), lipopolysaccharide (LPS), lipid A and allogeneic cells than those from young mice. The defect in each case resided with the nonadherent responding lymphocyte population rather than adherent accessary cells (macrophages). This conclusion was reached by showing that macrophages from old mice functioned normally in each of their known roles in lymphocyte responses in vitro; namely, presentation of antigen or mitogen to responding lymphocytes, support of lymphocyte responses and regulation of responses to mitogens and antigens. Normal presentation function was demonstrated by the comparable ability of peritoneal exudate cells from old and young mice to furnish the macrophage requirement for PHA responsiveness in vitro. This conclusion was confirmed by the finding that old, nonadherent cells reconstituted with macrophages from young mice, responded to PHA like unfrationated old cells. Old macrophages were also shown to be normal in their ability to provide the 2-mercaptoethanol (2ME) replaceable support function to young lymphocytes in vitro. Furthermore, replacement of macrophage supportive activity with 2ME did not rejuvenate the response of old cells to PHA, LPS, lipid A or alloantigens. Finally, no evidence of adherent cell suppression of responses of old cells could be obtained by two different approaches. First, although depletion of adherent cells increased, to a similar degree, the responses of both old and young cells to allogeneic stimulation, this procedure did not reduce the difference in responsiveness between them. That is, the decreased response of old cells in a mixed lymphocyte culture (MLC) could not be explained by excessive adherent cell suppression. Second, no evidence for suppression could be obtained in co-cultures of old and young cells responding to PHA, LPS or allogeneic cells. Taken together, these results suggest that macrophage function, in contrast to lymphocyte function, does not decline with age. It is suggested that this difference may result from the different half-lives of macrophages and lymphocytes in the intact animal. In contrast to the above results, old spleen cells acting as stimulators in an MLC were not always less effective than young cells. Furthermore, any difference that did exist between young and old cells was largely ablated by depletion of adherent cells. This result suggests that adherent cells from old mice may determine the ability of old cells to stimulate allogeneic lymphocytes in an MLC. It is not known whether this adherent cell is a macrophage or some other adherent (perhaps regulatory) cell.     

Function of surface-adherent cells in the induction of the human mixed leucocyte reaction. I. The transient surface adherence of responding lymphocytes in the human mixed leucocyte response

The aim of this study was to establish the temporal requirement for adherent cells in the mixed leucocyte reaction (MLR). At various intervals after co-cultivation, mixed leucocytes from two unrelated donors were separated into adherent and nonadherent cells and the MLR response of each population assayed on day 6. After 1 hr of co-cultivation adherent cells had a high MLR response, whereas the nonadherent cells were unresponsive and could not be restimulated by the addition of allogeneic leucocytes or syngeneic adherent cells. In contrast, the MLR was positive when syngeneic adherent cells were added to nonadherent cells which had not been co-cultured prior to the initiation of culture. The MLR of adherent cells declined progressively as a function of time of co-cultivation prior to separation. Conversely, there was a progressive increase in the MLR of nonadherent cells obtained after 12, 18 and 24 hr of co-cultivation. Morphological studies revealed a higher number of lymphocytes in the early adherent cell population than at later times. We conclude that during the first few hours of co-cultivation lymphocytes with the potential of responding to allogeneic stimulation are retained in the adherent cell population. These lymphocytes are released from the adherent cells and can generate a positive MLR without further support from adherent cells. The transient adherence of responding lymphocytes after short exposure to allogeneic cells could provide a mean of separating a subpopulation of lymphocytes capable of reacting to allogeneic stimuli.     

Autologous Mixed Lymphocyte Reaction in Man

Human peripheral blood T lymphocytes were separated into theophylline-resistant (T_R) and -sensitive (T_S) subpopulations. Proliferative responses of T_R, T_S and unfractionated T cells were studied, using irradiated autologous or allogeneic non-T cells as stimulators. T_R cells proliferated vigorously in both autologous and allogeneic mixed lymphocyte cultures (MLC). T_S cells, which constitute about 20% of unfractionated T cells, exhibited poor proliferative responses to autologous and allogeneic stimulation. The magnitude of proliferation in autologous and in allogeneic MLC was found to be directly dependent on the number of T_R cells in the culture. Mitomycin-C (MMC)-treated T_R cells augmented and MMC-treated T_S cells suppressed ( P <0.05) the autologous and allogeneic MLC responses of unfractionated T cells. However, the response of T_S cells did not increase in autologous or allogeneic MLC when co-cultured with T_R cells. MMC-treated T_S cells, when co-cultured with T_R cells, suppressed the responses of T_R cells ( P <0.05). The enhancing effect of T_R cells was radiation-resistant. Suppressor influence of T_S cells, in contrast, was abolished by irradiation ( P <0.05). These findings demonstrate that T_R cells are the responding cells in autologous MLR and augment the MLC responses of unfractionated T cells. T_S cells, on the other hand, respond poorly in autologous or allogeneic MLC and suppress the response of T_R cells.     

IFN-alpha-induced modulations of the events in human mixed lymphocyte cultures

Autologous mixed lymphocyte cultures (AMC) with T-enriched subset of human blood lymphocytes as responders and B-cells or plastic adherent cells as stimulators and allogeneic mixed lymphocyte cultures (MLC) were assayed for blastogenesis and generation of cytotoxic potential. The activated cells lyzed K562 and Daudi, autologous and allogeneic PHA-blasts. The AMC population affected the autologous and allogeneic blasts at a similar strength and there was no indication for selective effects. B-Blasts induced with Staphylococcus aureus were not lyzed. The MLC populations had a stimulation-specific cytotoxic component. This was revealed by the stronger effect against the stimulator PHA-blasts and by the lysis of the stimulator B-blasts. Short-time interferon (IFN) treatment prior to the lytic assay enhanced the anti-Daudi and anti-K562 lytic activity of the AMC and MLC populations. With AMC the lytic efficiency against the autologous and allogeneic PHA-blasts were not changed while with MLC they were also elevated. This increase was confined to the non-specific component of the cytoxicity. The proliferation of lymphocytes was suppressed when interferon was added at the initiation of the mixed cultures. On a per-cell basis the cytotoxic potential of these cultures were stronger. In the MLC the stimulation-specific component increased more substantially than the effect against the non-specific targets. It is possible that the IFN-induced modification of the culture conditions such as suppression of the initial proliferation favored the growth of the specific clone. Re-exposure of these cells to another dose of interferon prior to the lytic assay had no effect on the lytic potential.     

ADCC against human erythrocyte target cells: role of the anti-target cell antibodies in determining lymphocyte killer activity.

Studies were undertaken to investigate the role of anti-target cell antibodies in determining whether lymphocytes can mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro. Trinitrophenyl (TNP) modified Chang liver cells and human erythrocytes were employed as target cells and were coated with xenogeneic and allogeneic antibodies against TNP and natural cell surface antigens. Two cytotoxic effector cell populations were used: human peripheral blood mononuclear cells (PBMC) containing both lymphocytes and monocytes, and monocyte-depleted peripheral blood lymphocytes (PBL). With Chang targets, both PBMC and PBL mediated ADCC with xenogeneic anti-Chang and xenogeneic anti-TNP sera. With human erythrocyte targets, PBMC but not PBL mediated ADCC with human anti-blood group B serum, while both PBMC and PBL mediated ADCC with xenogeneic anti-TNP sera and also with a human anti-CD serum. These results demonstrate that the source of anti-target cell antibodies employed in ADCC reactions may determine whether or not lymphocytes are capable of mediating cytotoxicity.     

Deficiency of immunity to Mycobacterium avium that can be restored by allogeneic lymphocytes.

A 3-year-old girl developed a disseminated Mycobacterium avium infection despite treatment with eight antimycobacterial drugs. She had no pre-existent general humoral or cellular immunodeficiency. In the course of the disease B lymphocyte areas in the lymphoid tissues were replaced by histiocytes and an IgM and IgA deficiency evolved. The patient still made antibodies to concomitant micro-organisms and to transfused blood cells. Peripheral blood mononuclear cells (PBMC) had normal responses to mitogens and various antigens in vitro. However, she lacked any response to mycobacterial antigens, in vivo and in vitro. The defect appeared not to be dependent on immunosuppression by lymphocytes or monocytes or on deficient antigen presentation by monocytes. because a genetic origin could not be substantiated, acquired immunological paralysis for mycobacterial antigens was the most likely explanation. Addition of irradiated PBMC from her HLA-A, -B, -C and -DR phenotypically identical father, transferred a response to mycobacterial antigens of the patient's PBMC in vitro. We concluded that the disseminated M. avium infection was accompanied by a selective deficiency of the lymphocyte response to mycobacterial antigens which could be restored by allogeneic antigen responsive lymphocytes.     

Further characterization of T cell receptor chains of marsupials

cDNA clones encoding T cell receptor alpha (TCRalpha) and beta (TCRbeta) from the South American opossum, Monodelphis domestica were isolated and characterized. A single clone isolated encoding a TCRalpha chain was full length, containing the complete V (variable), J (joining) and C (constant) regions. Three partial cDNA clones were isolated for TCRbeta which contained complete C sequences. Phylogenetic analysis of the TCR Valpha revealed that the M. domestica sequence and a sequence from the Australian brushtail possum, Trichosurus vulpecula, belong to separate Valpha families and intersperse with sequences from eutherian mammals. Similar to results described for marsupial and eutherian light chains, diversity at the V region of the TCR is ancient and maintained. In contrast phylogenetic analysis of the TCR Calpha and Cbeta sequences from M. domestica, T. vulpecula, and other vertebrates revealed that the marsupial TCR C grouped together forming a sister group to eutherian mammals.     

Cell-mediated immune responses between HLA-identical siblings: recognition of antigenic changes associated with acute myelogenous leukaemia.

Cell-mediated immune reactions between a patient suffering from acute myelogenous leukaemia (AML) and an HLA-identical sibling were studied in order to characterize the in vitro reactions in MLC and CML prior to bone marrow transplantation. Our results indicated that antigenic differences were detectable between the blasts and the remission lymphocytes. While the normal sibling did not respond in MLC to her HLA-identical sister's remission lymphocytes, there was an anti-blast response. This proliferative response, however, did not lead to the development of detectable cytotoxic cells capable of destroying blast cells. Unrelated individuals, on the other hand, responded strongly both in MLC and CML to the allogeneic tumour blasts and remission lymphocytes of the patient and the lymphocytes of the healthy sibling. The kinetics and magnitude of the MLC response to blast cells was different from that to remission lymphocytes. This response indicated that the blast cells expressed antigenic differences which were recognized in MLC by both the HLA-identical sibling and unrelated individuals. Furthermore, these tumour cells were capable of sensitizing allogeneic, but not syngeneic lymphocytes to become cytotoxic, though they seemed to be more resistant to destruction in CML than normal cells.     

Cell interaction during lymphocyte activation.

Adherent cells were separated from stimulated lymphocytes by a layer of agarose to determine the requirement for lymphocyte interaction with adherent cells. The results demonstrated that the presence of plastic adherent cells was obligatory for oxidative activation, but contact of plastic adherent cells with oxidized nonadherent lymphocytes was not. A similar relationship was found to exist between adherent and nonadherent populations during PHA and Con A stimulation. In contrast, the responses of PWM- and ZnCl2-stimulated lymphocytes were not augmented by the presence of adherent cells unless the two populations were in contact.     

Lymphocyte-potentiating factor from human peripheral blood adherent cells.

Low-molecular-weight factor, which can restore the diminished concanavalin A (Con A) response of adherent-cell-depleted human peripheral blood lymphocytes, was partially purified from the culture supernatant of human peripheral blood adherent cells by ion-exchange column chromatography, gel filtration and preparative paper chromatography. This factor apparently has a mechanism of action distinct from that of 2-mercaptoethanol. It affects the cells just before the onset of DNA synthesis possibly as one of the second signals in the Con-A-induced activation of human peripheral blood lymphocytes. However, it was demonstrated that the effective activation of lymphoyctes also required the presentation of Con A by adherent cells to lymphocytes at the triggering stage of the activation, indicating that adherent cells have two distinct functions in the Con-A-induced activation of lymphocytes, i.e. the the presentation of Con A to lymphoyctes and the elaboration of soluble factors.