Search

The pinocytosis of radioactive colloidal gold by peritoneal exudate cells from guinea-pigs sensitized against tuberculin or ovalbumin was enhanced by specific antigen. The effect was mediated by soluble material (gold uptake stimulator (GUS) produced by lymphocytes and acting upon macrophages. GUS production began within the first 18 hours of culture and required 24 hours contact to maximally stimulate normal peritoneal exudate cells. Comparison of the gold uptake and migration inhibition assays on identical populations of sensitive exudate cells showed in terms of minimum effective antigen dose that the gold uptake assay was considerably more sensitive. Similarly, when sensitive lymph node cells were incubated with antigen, activity could be detected in the culture supernatants at about sixty-four-fold greater dilution by the uptake of gold. At low concentrations of GUS a log-linear dose response relationship held. At high concentrations, gold uptake levelled off or even fell, and this was true for concentrations of antigen greater than optimal when added to sensitive exudate cells. Chromium uptake varied little at the concentrations of antigen and supernatant factors tested. Hence the effects of gold uptake are unlikely to be indirect effects on cell proliferation or viability. [ABSTRACT FROM AUTHOR]

Evidence is presented to substantiate an earlier report that macrophages are the operative cells in the initiation of lymphocyte trapping. Several procedures which increase phagocytosis in mice of a soluble antigen (BGG), including transfer of specific serum or of adherent and non-adherent immune cells, or the transfer of in vitro sensitized macrophages, enhance the trapping potential of BGG. Animals tolerant to BGG trapped effectively when challenged with aggregated BGG, or when passively immunized and challenged with soluble BGG. The trapping defect in tolerant animals is thus due to a specific defect in immune responsiveness rather than an inherent failure of the trap mechanism. The dissociation between trapping and immune responsiveness is discussed. [ABSTRACT FROM AUTHOR]

Horse anti-rabbit bone marrow cell antiserum was tested for its cytotoxic activity with respect to the lymphocytes of the various lymphoid organs. The unabsorbed antiserum was highly cytotoxic with respect to the circulating WBC and cells of the bone marrow and thymus but demonstrated low cytotoxic activity with respect to spleen, lymph node and SAPP cells (sacculus rotundus, appendix and Peyer's patches). However, following absorption with thymocytes, lymph node cells or SAPP cells, cytotoxic activity directed toward any of these cell types disappeared without affecting the cytotoxic activity with respect to bone marrow and circulating lymphocytes. On the other hand, bone marrow and spleen cells and circulating white blood cells were capable of absorbing out completely the cytotoxic activity directed toward these cells, On the basis of a comparison of efficiency of absorption of anti-bone marrow cell activity by cells of the different lymphoid organs and cytotoxicity assays of the absorbed antiserum, it is concluded that approximately 15-25 per cent of the spleen lymphocytes and 20-40 per cent of the circulating lymphocytes in the rabbit are bone marrow-derived cells. The other lymphoid organs do not normally appear to possess these cells. [ABSTRACT FROM AUTHOR]

Sheep erythrocyte (SRBC) antigen was delivered directly into the lymphoid component of the appendix or via the appendicular artery, followed by ligation or clamping of appendicular vessels. Antigen was also injected intravenously, into the spleen, or into the thymus. Counts of anti-SRBC plaque-forming cells (PFC) in the appendix were compared with counts in the spleen and thymus. None of the injection routes employed resulted in an elevation of PFC activity above background in the appendix. However, delivery of antigen via the appendicular artery caused a marked increase of PFC activity in the spleen. These results and other available evidence point towards a helper-cell function of lymphocytes in the appendix, and therefore suggest a central role of this organ in humoral immunity. [ABSTRACT FROM AUTHOR]

The time course of the relative distribution of small lymphocytes binding 125I-labelled human thyroglobulin (HTg) in cell suspensions from the peripheral blood and various lymphoid organs was studied in guinea-pigs at progressive intervals up to 28 days after immunization with an emulsion of HTg and BCG in Freund's incomplete adjuvant (FIA). Small lymphocytes binding 125I-labelled HTg were first detected in peripheral blood, popliteal (draining) lymph node, spleen and bone marrow preparations on the 10th day, and in mesenteric (distant) lymph node and thymus preparations on the 14th day after primary immunization. In general, the percentage of these cells increased progressively thereafter until the end of the period of study. Blocking experiments with unlabelled antigens indicated that the binding of 125I-labelled HTg by small lymphocytes was specific. An anti-HTg antibody cytophilic for guinea-pig small lymphocytes was demonstrated by the passive transfer of antigen-binding capacity to lymphocytes of unimmunized animals with hyperimmune guinea-pig serum. It is proposed that, in these experiments, anti-HTg cytophilic antibody was bound first to small lymphocytes in tile tissues participating actively in the immune response (popliteal node, spleen and bone marrow) before spilling over into the general circulation to coat lymphocytes at other sites (mesenteric node and thymus). [ABSTRACT FROM AUTHOR]

A mean of 6.9 per cent of small lymphocytes in peripheral blood preparations and between 1.8 and 2.4 per cent of small lymphocytes in lymph node, spleen, bone marrow and thymus preparations from unimmunized guinea-pigs bound 125I-labelled purified protein derivative of mammalian tuberculin (mammalian PPD). The percentage of these cells fluctuated but did not alter substantially after immunization with BCG or with BCG emulsified with human thyroglobulin (HTg) in Freund's incomplete adjuvant (FIA). Blocking experiments indicated that the binding of 125I-labelled mammalian PPD was specific and there was tentative evidence that the lymphocyte receptors may be IgG. A comparison is drawn between the observed time course of 125I-labelled mammalian PPD- binding small lymphocytes and the response of lymphocytes sensitive to strong histocompatibility antigens, and it is proposed that the propensity of certain anti- gens to induce a delayed hypersensitivity-type response is related to the presence of substantial numbers of antigen-binding cells in unimmunized animals. A noteworthy incidental finding was an unexplained depression in the cellular and humoral responses to mammalian PPD in guinea-pigs that had been immunized with HTg-BCG-FIA emulsion. [ABSTRACT FROM AUTHOR]

Thymus-dependent cells from thymus and peripheral lymphoid organ were preparatively separated by mean of free flow electrophoresis into various subpopulations which were defined in terms of θ (theta) antigen content, negative surface charge, graft-versus-host (GvH) reactivity, hydrocortisone sensitivity, cell volume and morphological details. Most thymocytes in the cortex have a low negative surface charge, high θ antigen content, are hydrocortisone-sensitive and immuno-incompetent. On the basis of electronic cell sizing this group consists of large population of 90 μm³ cells (T1) and a small population of 175 μm³ cells (T2), the latter being less hydrocortisone-sensitive than the former. A minority of thymocytes resides in and around the medulla and has high negative surface change, a medium θ antigen content, is hydrocortisone-resistant and reveals low GvH reactivity. These cells are medium sized (125 μm³)1 electrophoretically bimodal (T3 had a medium and T4 a high negative surface charge) and on the basis of morphological criteria are metabolically more active than the thymocytes of low negative surface charge. In the peripheral lymphoid organs, all thymus-dependent cells show high negative surface charge and have the lowest observed θ antigen content and the highest observed GvH reactivity. These cells fall into two populations of which one is 125 μm³ with lower negative surface charge and the other is 90 μm³ with a somewhat higher negative surface charge. These 125 μm³ cells (T4), which morphologically resemble the 125 μm³ thymocytes, are less GvH-reactive than the 90 μm³ (T5) cells, which seem to be resting cells. On tile basis of these data, a possible sequence of steps in the maturation of T cells was constructed as follows: in the cortex of the thymus T1 thymocytes are transformed into T2 and these develop into T3 and T4 thymocytes which have higher negative surface charge, lower θ antigen content and are in an advanced stage of maturity. After further loss of θ antigen these cells, which are in the medulla, emigrate into the periphery and are finally transformed into highly immunocompetent T5 cells possessing the highest observed negative surface charge. [ABSTRACT FROM AUTHOR]

The effect of a single injection of 300 mg/Kg on the lymphoid cells of normal mice has been studied. The proportions of θ-positive cells in the thymus, lymph nodes and spleen were examined in vitro using a Trypan Blue exclusion cytotoxicity test. Two anti-θ sera of different titre were employed in an attempt to evaluate changes in θ-antigenicity of these cells following azathioprine treatment. In the thymus the drug was found to deplete a particular population of cortical thymocytes without affecting the hydrocortisone-resistant cells. The ability of azathioprine-resistant thymocytes to absorb out the cytotoxic capacity of both antisera was not better than that of hydrocortisone-resistant thymocytes. This result was taken to indicate that, following azathioprine, the residual cells had a decreased frequency of the θ antigen and were in this respect more like hydrocortisone-resistant cells. It is also postulated that the two antisera were of different avid- A decrease in antigenicity was also seen in lymph node cell populations from azathioprine-treated animals. It is therefore suggested that azathioprine given in heavy doses might have at least two effects on lymphoid cells: (i) it depletes certain populations; and (ii) it decreases the θ-antigenicity of others. [ABSTRACT FROM AUTHOR]

Seven murine lymphomas and three plasmacytomas were examined for the distribution of the theta antigen, immunoglobulin determinants, the receptor for Fc portion of antigen-antibody complexes and the receptor for the third component of complement. The theta-bearing tumours lacked C3 and Fc receptors and easily detectable surface immunoglobulin. The theta-negative lymphomas, while being morphologically lymphocytic, lacked all but the Fc receptors. One of the plasmacytomas possessed clearly detectable surface immunoglobulin. All three lacked the receptors for Fc and C3. [ABSTRACT FROM AUTHOR]

The progeny of mothers subjected to marginal lipotrope or moderately low protein diets exhibit deficient humoral immune responses. Serum antibody titres and numbers of antibody-forming cells to sheep erythrocytes (SRBC) are lower in the offspring of mothers whose diets were limited in either percentage protein or percentage lipotropes than in offspring whose mothers were fed a complete diet. Immune lymphocyte stimulation by SRBC antigens was marginal and not measurably different between the progeny of malnourished mothers and controls. The in vitro response of' spleen cells from offspring of nutritionally deprived mothers to the T-cell mitogen phytohaemagglutinin (PHA) is considerably less than that of controls. Thymus cells from marginal lipotrope offspring respond well to PHA, suggesting that PHA-reactive cell migration may be inhibited in these animals. These results, obtained using a T-dependent antigen (SRBC) and a T-dependent mitogen (PHA), support the conclusion that maternal dietary deficiency can adversely affect T-cell function in rat offspring. [ABSTRACT FROM AUTHOR]

We investigated the cellular requirements for lymphocyte trapping. Depletion of lymphocyte populations selectively or indiscriminately did not affect the ability of animals to trap. The variety of materials which initiate trapping was also studied and this information, coupled with the resistance of 'trapping' to severe lymphocyte depletion, is consistent with the hypothesis that the macrophage may be the central cell in initiating the trapping of lymphocytes after antigen stimulation. [ABSTRACT FROM AUTHOR]

Lymphocytes which were incubated with Erwinia carotovara L-asparaginase for only 1 hour were found to be markedly non-responsive to soluble antigens and moderately non-responsive to allogeneic cells or phytohaemagglutinin. When lymphocytes were exposed to antigen first, the inhibitory effect of L-asparaginase was markedly diminished, whereas exposure of lymphocytes to the enzyme prior to contact with antigen resulted in almost complete inhibition of blastogenesis. These observations suggest that L-asparaginase acts on the cell membrane causing impairment of antigen or mitogen binding, and that this is responsible for its inhibitory effect. [ABSTRACT FROM AUTHOR]

We have confirmed that adherence of antibodies to spleen lymphocytes is stabilized when the antibodies form complexes with antigen. Furthermore, there is evidence that antibodies having both binding sites occupied are less easily supplanted from the lymphocyte receptor by unspecific IgG than antibodies carrying one antigen only. Based on these results a hypothetical model for a feed-back control of the humoral immune response is put forward. [ABSTRACT FROM AUTHOR]

Experiments were carried out to test whether a humoral factor capable of restoring immunological competence to neonatally thymectomized animals was released by the isolated perfused rabbit thymus. New Zealand white rabbits thymectomized within 24 hours of birth received twice weekly injections of cell-free thymic perfusate from the second week of life. Their body weights, peripheral blood lymphocyte counts, and delayed hypersensitivity responses to skin test antigens were compared with those of two control groups of thymectomized rabbits, one that received injections of cell-free perfusate that had been run in a perfusion circuit with no thymus, and another that received no injections, and also with those of normal or sham-thymectomized rabbits. The results suggest that a humoral factor was liberated from the thymus during perfusion which restored delayed hypersensitivity responses to thymectomized rabbits, but had no effect on the depressed lymphocyte count resulting from thymectomy. [ABSTRACT FROM AUTHOR]

Lymph node cells from preimmunized rabbits were cultured with varying concentrations of antigen in autologous serum which had been collected before immunization. [3H]Thymidine was added after 18 hours of culture and the cells were harvested at 24 or 66 hours for the determination of the radioactive labelling of acid-precipitable material. Preheating serum (56°, 20 minutes) enhanced labelling in both control and antigen-treated cultures. This 'heat effect' had an early (24 hour) non-specific component, independent of antigen concentration, and a late (66 hour) specific component which was most evident at high antigen concentrations. The conditions of preheating serum (temperature and time) required to produce the heat effect were similar to those required to remove haemolytic activity against rat erythrocytes. However, at certain temperatures and times there were discrepancies. These discrepancies, and data from experiments in which preheated and unheated sera were mixed in varying proportions, or interchanged in different sequences, were explicable on the basis of (i) a requirement for complement in stoichiometric quantities dependent on the number of cells being inhibited, (ii) the involvement of the majority of the cultured cells in the early non-specific component of the heat effect, but only cells capable of proliferating in response to added antigen in the late specific component, (iii) the secretion of complement by cultured cells. Preheating serum (66&deg:, 20 minutes) depressed labelling in control and antigentreated cultures and reduced agglutinating activity against both autologous and heterologous erythrocytes. The results are discussed in relationship to models which require that the size of a specific lymphocyte clone be positively or negatively regulated by the concentration of antigen specific for that clone. With increasing antigen concentration three effects on cells bearing specific receptor sites are distinguished. (i) Cell stimulation under conditions of antigen concentration and cell receptor specificity such that only a few antigen molecules can bind to cells. (ii) Complement-dependent inhibition under conditions such that more antigen molecules can bind to cells. (iii) Complement-independent inhibition under conditions, possibly unphysiological, such that very large quantities of antigen molecules can bind to cells. [ABSTRACT FROM AUTHOR]

Lymph node cells from immunized rabbits were cultured with varying concentrations of antigen in preheated (56°, 30 minutes) autologous serum which had been collected before immunization. [3H]Thymidine was present for the last 6 hours of the 24-hour culture period and the radioactive labelling of acid-precipitable material was then determined. Changes in labelling due to variations of culture conditions were interpreted according to whether they were specific for control or antigen-treated cultures or non-specific. Cell concentration and serum concentration were predominantly non-specific variables influencing the labelling in control and antigen-treated cultures to a proportionate extent. However, at serum concentrations below 5 per cent labelling was disproportionately inhibited in antigen-treated cultures; there were further minor disproportionate inhibitions at higher serum concentrations. Labelling was inhibited by increasing the concentration of serum from 25 to 50 per cent, mainly due to a non-diffusible competitive inhibitory activity. Isotope-dilution analysis of the effects of serum on labelling over a wide range of serum concentrations indicated that the relationship was a complex one with at least three step-wise stimulations of the maximum labelling rate being produced by increasing the serum concentrations from 0 to 25 per cent. Labelling in antigentreated cultures containing post-immunization serum was less than labelling in cultures containing an equal volume of preimmunization serum, but labelling in control cultures was enhanced by post-immunization serum. These results are shown to be compatible with the proposals (i) that labelling in control cultures reflects the response of cells to low concentrations of endogenous antigens, and (ii) that preimmunization serum and post-immunization serum contain ‘natural’ and ‘acquired’ antibodies respectively, which normally buffer cell-borne receptor sites against reaction with endogenous and exogenous antigens. [ABSTRACT FROM AUTHOR]

Tritiated mouse alloantibodies were prepared by reaction of the gamma globulin fractions of antisera with DL-[3-³H]alanine-N-carboxy anhydride. The uptake of [³H]alanylated alloantibody against H-2, TL or &thea\ta; on mouse cells proved specific. On the basis of the known specific radioactivity the number of alloantibody molecules taken up per mouse lymphocyte was calculated for the three alloantigenic systems. In comparisons of cell populations from lymph nodes and thymus it was shown that there is a striking difference in alloantigen representation, qualitative as well as quantitative, implying extensive changes in alloantigenic surface patterns during the maturation of thymocytes into lymphocytes. [ABSTRACT FROM AUTHOR]

The immunological significance of lipopolysaccharide (LPS) transfer from macrophage into lymphocytes was studied in an in vitro system in which the lymphocytes were incubated during 24-72 hours with macrophages which had previously ingested S. enteriditis LPS (during 1 hour). Our results show that the in vitro immune response to LPS requires co-operation of lymphocytes with macrophages. After 24-72 hours of contact of the lymphocytes with macrophages containing ingested antigen, we observed within lymphocyte population a time dependent increase of the number of LPS-binding cells. This result was obtained by means of two different methods: rosettes formation and autoradiography. Autoradiographies performed on thick and thin sections showed that after contact with macrophages which had ingested 14C-LPS, most rosettes forming cells (RFC) exhibited radioactivity. All these findings strongly argue in favour of the idea that LPS transfer from macrophages to lymphocytes is highly relevant for specific antibody synthesis by lymphocytes. [ABSTRACT FROM AUTHOR]

Activation of fresh blood lymphocytes has been measured by uptake of tritiated thymidine, following exposure to irradiated cells from autochthonous or allogeneic cell lines (LCL cells). Nine autochthonous combinations were studied. In every case activation was observed and in one the rate and peak level of activation were comparable to those found in control allogeneic mixtures. Neither foetal calf serum nor antibiotics appeared to be important factors in the activation process. There was no correlation between the number of identified HL-A histo-incompatibilities in a given allogeneic mixture and the rate or peak level of activation achieved. Activation appears to be influenced by the metabolic state of the LCL cells and the rate of the reaction is sensitive to the precise culture conditions under which it proceeds. Some distinction can be drawn between the antigens responsible for activation in autochthonous and in allogeneic mixtures but the former may prove to be modified histocompatibility determinants. [ABSTRACT FROM AUTHOR]

Cells from thirty-three human lymphoid cell lines and sublines have been typed for HL-A antigens by a microcytotoxicity test. Similar patterns of HL-A antigens were found for the cell line cells and for the fresh lymphocytes of the donor of the line (eleven cases). However, certain typing sera gave positive reactions with the cell line cells which were not found with the fresh lymphocytes. No correlation was noted between the pattern of these 'extra' reactions and the HL-A typing of the cells. These same typing sera often gave positive reactions with blood lymphocytes cultured for several days in conventional media. These positive reactions were quantitatively more pronounced and sometimes qualitatively different if the cells had been stimulated. A range of normal sera failed to react with cell line cells suggesting that the HL-A typing sera giving 'extra' reactions are detecting antigens in some way related to a histocompatibility system. Absorption studies performed with two of the lines confirmed the HL-A typing by the direct cytotoxicity test. Two sera giving 'extra' reactions were also tested in the absorption experiments. The results indicated that antibodies other than those of the HL-A specificity designated for these sera were responsible for the 'extra' reactions. It is suggested that 'extra' reactions indicate a change in the apparent antigenic expression of lymphoid cells reflecting altered membrane characteristics as they adapt to a culture environment. [ABSTRACT FROM AUTHOR]

The time of appearance and counts of antigen-binding cells, using radioiodine-labelled flagellin and hemocyanin, was studied in the human and mouse foetus at different gestational ages: the capacity for binding radioiodinelabelled antigens was equated with acquisition of immunological function. Cells resembling lymphocytes from human and mouse liver and bone marrow showed antigen binding at early gestational ages, but tiffs binding could not be prevented with species-specific antisera to immunoglobulins. Specific antigen binding to lymphocytes was detected first with thymic lymphocytes, at gestational ages of 12 weeks in humans and 14 days in mice, then with splenic lymphocytes, at 16 weeks in humans and 17 days in mice, and still later with gut lymphocytes. Relative counts of antigen-binding cells in human foetal thymus were maximal at 16-22 weeks and decreased thereafter. Lymphocyte immunocompetence, as judged by the capacity specifically to bind antigen, develops rapidly after the appearance in thymus of cells with the morphology of lymphocytes; this seemed to occur at the equivalent foetal stage in the species studied. [ABSTRACT FROM AUTHOR]

Migration patterns of 51Cr-labelled murine lymph node cells were studied in syngeneic, allogeneic, semi-allogeneic and congenic strain combinations. In certain allogeneic and semi-allogeneic combinations with strong H-2 histoincompatibility, reduced migration of a donor population representing recirculating lymphocytes into recipient lymph nodes was observed. This phenomena could be reproduced in congenic strains differing only at the H-2 locus, and was not seen in congenic strains differing at the Ly-A, Ly-B, RZ, M, or θ loci. The kinetics of reduced lymphocyte homing to allogeneic lymph nodes and the role of host and donor recognition were investigated. These studies are discussed in relation to possible mechanisms of action, and relevance to factors regulating lymphocyte recirculation. [ABSTRACT FROM AUTHOR]

A micro-immune adherence assay employing direct microscopic observation was used to detect the presence of thymus-derived cell antigenic specificities on guinea-pig lymph node cells, purified peritoneal exudate lymphocytes, and thymocytes using a heterologous burro anti-guinea-pig thyrnocyte serum. A method is described for the direct determination of thymus-derived cell specificity of anti-lymphocyte sera. [ABSTRACT FROM AUTHOR]

This study has investigated the charge of IgG involved in antibody dependent lysis of cells by lymphocytes. The following conclusions are drawn: 1. That under certain conditions the physical alteration of IgG can result in molecules which compete with antibody bound to target cell antigens for receptors on cytotoxic lymphocytes. This phenomenon is used in a cytotoxicity inhibition test to determine the charge distribution of antibodies, which are capable of interacting with cytotoxic lymphocytes. This test is independent of the antigenic specificity of the inhibiting antibody. 2. The charge distribution of rat antibodies capable of reacting with cytotoxic lymphocytes, as assessed by cytotoxicity inhibition is similar to that of specific rat antibody capable of inducing cytotoxic activity against a xenogeneic target cell. By both criteria the isoelectric points ranged between pH 5 and 10. 3. Spontaneously occurring human antibody against a human line of target cells was found to have a highly restricted range of isoelectric points separating between pH 8 and 9.5. On the other hand assay of these sera by cytotoxicity inhibition showed a normal distribution of total antibody capable of reacting with cytotoxic lymphocytes. It is suggested that a likely explanation of these findings is that the human antibody with specificity for an allogeneic strain of target cells is cross reacting with a single antigen on the target cells and that the antibody from specifically immunized rats is likely to be against multiple antigens. [ABSTRACT FROM AUTHOR]

The localization of HL-A histocompatibility antigens at the surface of human lymphocytes in electron microscopy has been studied using hybrid antibodies to bind electron-dense particles (ferritin and plant viruses) to anti-HL-A antibody. A discontinuous distribution of the markers is observed at the cell surface, which is identical with that described for H-2 antigens on mouse lymphocytes with the same technique. Double labelling experiments suggest that the areas of the cell surface where HL-A antigens are detected contain also the heterologous lymphocyte antigens detected by an anti-thymocyte serum and that HL-A antigens are not renewed at a detectable level during the period of the labelling procedure in the areas of the cell surface which are not labelled primarily with ferritin-anti-IgG-anti-HL-A complexes. The interpretation of the discontinuous labelling of HL-A antigens with direct immunoferritin techniques is discussed. [ABSTRACT FROM AUTHOR]

Human lymphocytes were fractionated on gradients of bovine serum albumin (BSA). Cells obtained from the more dense layers bound antigen specifically. Cells capable of interacting with antigen contained antibody which was separable with the nuclear components of the cell. Immune cells contained antibody which was specific for the immunizing antigen. Cell-bound antibody mediated and, in the presence of isologous antibody, slightly enhanced the uptake of antigen. Specific antibody passively bound to cell membranes did not promote uptake of antigen. Antigen which bound to lymphocytes in vitro was not enzymatically digested within the cell but became bound to acid-insoluble nuclear DNA. [ABSTRACT FROM AUTHOR]

Certain concentrations of antigen stimulated DNA synthesis in sensitized human lymphocytes cultivated in vitro, higher and lower concentrations being less stimulatory. The simultaneous addition of two antigens in low concentrations to the same cells caused an additive response. The decreased response to a high antigen dose did not affect the capacity of the cells to respond to the simultaneous addition of another antigen, as determined at the population level as well as at the cellular level by autoradiography. Presumably specific immunological paralysis was induced by high antigen doses. Addition of low antigen doses for 1–3 days to human .sensitized lymphocytes cultivated in vitro resulted in decreased DNA synthesis as a response to the same antigen added in an optimal dose. Suppression of DNA synthesis was not caused by induction of tolerance or antibody suppression, because the cells also failed to respond to an unrelated antigen and to non-specific mitogens, such as PHA and ALS. Most likely the suppressed response after antigen pretreatment represents a phenomenon analogous to antigenic competition, although this term is not appropriate, since there need not be competition between antigens for a detectable effect. No soluble mediators of suppression could be demonstrated in the supernatant of suppressed cultures. [ABSTRACT FROM AUTHOR]

Peritoneal exudate lymphocytes (PEL) of guinea-pigs injected with Freund's complete adjuvant (FCA) were capable of liberating skin reactive factor (SRF) and macrophage migration-inhibitory factor (MIF) when cultured for 24 hours in the presence of sheep red cells (SRC) preincubated with lymph node cells (LNC) of guinea-pigs immunized with SRC in FCA. Similar release of soluble mediators was found in the following situations: (a) incubation of non-immune PEL with SRC sensitized with the exclusion peak of Sephadex G-200 fractionated immune LNC supernatant; (b) incubation of non-immune LNC with complexes of SRC and anti-SRC IgG, prepared from serum of animals immunized with SRC in CFA, and (c) incubation of non-immune LNC with a mixture of SRc urea extract and anti-SRC IgG. SRC incubated for 24 hours with immune LNC and freed of lymphocytes by selective filtration through a Millipore filter, were able to provoke an inflammatory reaction in the skin of normal guinea-pigs, which was delayed in character. These findings are interpreted as suggestive evidence for the non-identity of the mediator-producing and antigen-reactive cells and as support for the hypothesis that a complex composed of antigen and an antibody-like material, secreted by stimulated antigen-reactive lymphocutes, is responsible for the release of soluble mediators in cell-mediated immune reactions. [ABSTRACT FROM AUTHOR]

The onset of ability to produce haemolytic antibody to sheep erythrocytes does not appear in hamsters until 11 days after birth. However, when mouse erythrocytes were used as antigen an earlier onset of immunocompetence at 6 days after birth was detected. The hamster can make haemolytic antibody only 9.5 days after the appearance of lymphocytes in the thymus—this is sooner than any other species. This capacity is compared with other species on the basis of physiological development by age-equivalence. [ABSTRACT FROM AUTHOR]

Guinea-pigs immunized with sheep red cells (SRC) in Freund's complete adjuvant (FCA) developed delayed type hypesensitivity to a SRC urea extract 7 days after sensitization and showed combined Arthus and delayed reactivily from the 14th day on. Animals immunized by an intracardiac injection of SRC in saline and skin tested 4-5 days later with SRC urea extract, responded with an erythematous and indurated lesion reaching its peak 24 hr after testing and resembling a typical delayed hypersensitivity reaction. Lymphocytes from lymph nodes, peritoneal exudate and peripheral blood of guinea-pigs immunized with SRC in FCA released skin reactive factor (SRF) and macrophage immunized inhibitory factor (MIF) when cultured for 24 hr with SRC. No SRF was formed in the absence of divalent cations in the cultured medium. No SRF or MIF was produced by lymphocytes of guinea-pigs immunized by thc intracardiac route and cultured under identical conditions SRF obtained after in vitro exposure of sensitized lymphocytes to SRC was elutd from Sepbadex G-200 in the region of serum albumin. Lyric anti-SRC antibody of the IgM class was liberated into the supernatant of spleen cultures after intracardiac immunization and antibody of the IgG class was produced by lymph node cultures after immunization with SRC in FCA, in both cases in the absence of antigenic stimulation in vitro. It is considered improbable that the latter antibody is involved in the release of soluble mediators. [ABSTRACT FROM AUTHOR]

When viable cells sensitized in excess cytotoxic alloantibody are washed and resuspended in antibody-free medium, they spontaneously release appreciable quantities of antibody. The amount released is directly proportional to the concentration of alloantibody during sensitization. Spontaneous release was observed from all cell types tested (thymocytes, lymphocytes, leukaemia cells and ascites sarcoma cells) and with all alloantibodies tested (H-2, θ and Ly-B). In preliminary tests with radio-labelled H-2 antibody, the quantity of antibody released in a period of 2½ hours was 29 per cent of the antibody originally absorbed. Dissociation at 37° was greater (or more rapid) than at 1°. When washed sensitized cells were suspended in antibody directed to an antigen closely adjacent on the cell surface to the site of attachment of the first antibody, release of the first antibody was impeded. [ABSTRACT FROM AUTHOR]

Changes in the lysosomal enzyme acid phosphatase have been assayed in mouse spleen cells after injection of the animal with various antigens. Disruption by freezing and thawing or sonication of spleen cells or the large granular fraction showed, 48 hours after injection of antigen, an increase in enzyme level irrespective of the antigen used. In contrast, if non-disrupted lysosomes were examined only cells from animals treated with aggregate-free antigen (BSA) showed an increase in level. Spleen cells from animals treated with the other antigens, heat-aggregated BSA, haemocyanin (KLH) and sheep red cells, showed that the enzyme was less accessible to its substrate, reflected by lower levels of enzyme. The results are discussed in relation to possible changes in the stability of the lysosome membrane and the involvement of lymphocytes. [ABSTRACT FROM AUTHOR]

This paper describes the ultrastructural location of labelled antigens and carbon in the spleens of rats from 4 minutes to 5 days after injection. Particular attention was focused on the sites of deposition 4 minutes after intra-arterial injection of microgram quantities of 125I-labelled Salmonella flagellar antigens, crayfish haemocyanin and BSA, using colloidal carbon for comparison. The combination of radioautography with both light and electron microscopy showed the importance of antigen binding by lymphocytes in the marginal zone of the spleen. Macrophage sequestration of antigens was not prominent in the spleen, although it occurred in the liver with the flagellar antigens and haemocyanin. In the spleen marginal zone, avid antigen-binding cells were found in situ 4 minutes after the injection of labelled haemocyanin. These appear to be the counterpart in vivo of antigen-binding lymphocytes prepared in vitro. Such cells also occurred infrequently after the injection of labelled polymerized flagellin, but were not found with either BSA or carbon. The apparent movement of flagellar antigen from the marginal zone to the white pulp between 1 and 2 hours after injection was seen to involve lymphocyte-associated antigen. The follicular antigen localization occurring from 1 day onwards after injection was on the dendritic reticular cells of germinal centres, as has been described in lymph nodes after subcutaneous injection. Carbon particles were rapidly sequestered in macrophages of the spleen and liver, although some particles were found between cells in the marginal zone for as long as 2 hours after injection. By 2 and 5 days, however, all the carbon was in phagocytes, even in the white pulp. Differences between the localization of antigens and carbon were clear, even in the ultrastructural sites of their location in tingible body macrophages of germinal centres. The unexpected emphasis of lymphocyte association with labelled antigens in the spleen marginal zone has allowed a revision of the mechanism previously proposed for the movement of antigens within the microenvironments of the spleen. [ABSTRACT FROM AUTHOR]

Thoracic duct cells and spleen cells from normal Lewis rats were tested for their ability to restore the antibody response of X-irradiated hosts to diphtheria toxoid (DTAP) or DNP-diphtheria toxoid (DNP-DT)adsorbed to alum. Although spleen cells restore a measurable response to both DTAP and DNP- DT, thoracic duct cells do not. However, thoracic duct cells from normal donors combined with small numbers of thoracic duct cells from donors primed to DTAP restore the primary anti-DNP response to DNP-DT. These findings indicate that lymphocytes which initiate the antibody response to the carrier play an important role in the initiation of the primary response to the hapten coupled to that carrier. Further experiments show that the primary response of neonatally thymectomized rats to both DTAP and DNP-DT is markedly reduced as compared to that of age-matched normal rats. Thoracic duct cells can specifically restore the response of the thymectomized rats to DTAP, and, therefore, contain cells which can recognize and interact with the determinants of diphtheria toxoid. A combination of the latter cells and cells which can initiate the primary response to DNP is found in the thoracic duct lymph of normal rats. Yet, ‘normal’ thoracic duct cells cannot restore the response to DNP-DT in irradiated hosts. These results suggest that the complete immunologically competent unit for the carrier is required for the initiation of the primary response to a hapten-protein conjugate. [ABSTRACT FROM AUTHOR]

Antilymphocyte serum was prepared in several species and its effect on the production of migration inhibitory factor (MIF) studied. Antilymphocyte sera in vitro in dilutions as high as 1/3200 inhibited the liberation of MIF by sensitized lymphocytes in the presence of antigen. In contrast antipolymorph and antiglobulin sera were inactive. Antilymphocyte serum also blocked the formation of MIF when the lymphocytes were preincubated with ALS and then washed before the addition of antigen. The antilymphocyte serum and sera prepared against supernatants containing MIF did not block MIF activity by direct neutralization. [ABSTRACT FROM AUTHOR]

Experimental glomerulonephritis (EGN) was produced in Sprague-Dawley rats with a single intradermal injection of a renal tubular fraction called Fx1A. Presence of disease was confirmed by proteinuria and by fight and electron microscopic changes. This antigen and also the non-nephritogenic antigen, Fx1B, were used to study cellular immunity in nephritic and three groups of control rats. 64 per cent (9/14) of the Fx1A immunized rats gave delayed hypersensitivity skin tests to Fx1A. The difference between the experimental and the three control groups was highly significant. Peripheral blood lymphocyte culture responses to Fx1A, Fx1B, phytohaemag- glutinin-P, were studied in the nephritic and control groups. 77 per cent (10/13) of Fx1A immunized animals showed stimulation of their lymphocyte cultures with Fx1A. None of the control groups showed any significant stimulation. These experiments have demonstrated cellular immunity in EGN and support the possibility that these mechanisms may play a role in the production of this disease. [ABSTRACT FROM AUTHOR]

Optimum conditions have been found for a specific quantitative absorption of mouse lymphocytes on to allogeneic target cells. Using this technique, it has been established that C3H anti-A lymphocytes immune to the specificities of a single H-2 sub-locus (D) were absorbed neither on cells bearing only some of the components of the immunizing complex (DBA/1 and I/St targets) nor on their mixture. Conversely, C57BL anti-A lyrnphocytes immune to the specificities of two H-2 sub-loci (D and K) reacted separately with each of the components on corresponding third-party targets (B10.D2(H-2d) and C3H (H-2k) as shown both by the direct cytotoxic effect and by the quantitative absorption technique. The results of absorption of these lymphocytes on mixtures of B10.D2 and CSH target cells used in various proportions indicate that C57BL anti-A lymphocytes represent a mixture of two 'polyvalent' populations in ratio of 1:3. This, together with the previous data indicates that 'committed' lymphocytes may be 'polyvalent' and that the initial recognition step of H-2 antigens may result from a direct contact between membranes of grafted cells and 'unprimed' host lymphocytes. Structural matching of the membrane antigenic complex and of normal and immune lymphocytes is suggested as a decisive factor of immunological recognition initiation in a transplantation context. [ABSTRACT FROM AUTHOR]

The delayed-type iso-hypersensitivity that guinea-pigs acquire spontaneously towards a β-globulin antigen of other guinea-pigs has been measured in vitro. Serum containing the antigen specifically triggers lymphocytes from naturally iso-hypersensitive animals to undergo blastogenic response. This reaction is similar to that of lymphocytes cultured in the presence of antigen to which cell donors have been intentionally immunized. Thus, the in vitro technique of DNA synthesis may be used to detect both artificially induced and naturally occurring delayed-type hypersensitivities. In addition, naturally existing delayed hypersensitivity offers a plausible explanation for reports of spontaneous transformation of lymphocytes by homologous or heterologous serum and of certain other in vitro phenomena involving interaction of lymphoid cells from non-immunized donors. [ABSTRACT FROM AUTHOR]

A markedly positive reaction for IgG at the endothelial cell borders in post-capillary venules of human tonsils was obtained using modified fluorescent antibody techniques. This observation is discussed in relation to lymphocyte recirculation at this site. [ABSTRACT FROM AUTHOR]

Both differentiating plasma cells and modified lymphocytes (lympho-plasmocytes) synthesize specific humoral antibody in rabbits, rats, mice and guinea-pigs. They do so during both primary and secondary responses, after subcutaneous injection of three enzymic antigens: horse radish peroxidase, glucose oxidase and alkaline phosphatase, administered as soluble or insolubilized proteins or alum precipitated or mixed with Freund's adjuvant. [ABSTRACT FROM AUTHOR]

Immunofluorescent staining has been used to detect antigenic determinants on the surface of living lymphoid cells of mice. It has been possible to distinguish two distinct populations of peripheral lymphocytes: one population carries the theta isoantigen and is thymus-dependent; the other has naturally-occurring immunoglobulin determinants on the cell surface and appears to be thymus-independent. The relative proportions of these two populations of cells is characteristic for each type of lymphoid tissue and these proportions have been determined for thymus, lymph node, spleen, and thoracic duct cells. [ABSTRACT FROM AUTHOR]

Human blood lymphocytes stimulated in vitro with non-specific mitogens, such as PHA, ALS and streptolysin, responded with DNA synthesis only to certain optimal doses of the mitogens, higher and lower doses failing to stimulate the cells. With ALS only a 100-fold difference in concentration covered the entire dose range. Specific antigens on the other hand were stimulating over a 10,000-fold difference in concentration. The simultaneous stimulation of lymphocytes with two non-specific mitogens resulted in antagonistic or synergistic effects. Antagonism was found when two mitogens were added in optimal concentrations, whereas synergism occurred when both were present in suboptimal concentrations. Non-specific mitogens in high concentrations abolished induction of DNA synthesis by specific antigens, whereas they increased the response to antigen when they were added in low, by themselves non-stimulating, concentrations. The mixed lymphocyte culture reaction was found to abolish stimulation caused by nonspecific mitogens added 3 days later. The findings suggest that individual lymphocytes respond with DNA synthesis after contact with a stimulus only when a threshold number of sites on the surface has been triggered. When the number of triggered sites is increased above a certain higher threshold, the lymphocytes fail to respond. Furthermore, the lymphocytes do not discriminate between the quality of the sites triggered, but only between the number of triggered sites, disregarding the nature of the stimulus. Finally, sensitized and presumably thymus-processed lymphocytes, confronted with antigen appear to influence other non-antigen recognizing cells, presumably by releasing some factors. These factors are capable of stimulating non-antigen-recognizing cells or to make them refractory to stimulation by subsequently added mitogens. [ABSTRACT FROM AUTHOR]

Guinea-pigs were immunized with emulsions of protein antigens in Freund's complete adjuvant. Draining lymph node cells were cultured for 20 hours together with the immunizing antigen; after harvesting, the culture supernatants were added to fresh lymphocytes from allogeneic or autochthonous guinea-pigs, and these 'recipient' cells were maintained in culture for 3 days. Uptake of [³H]thymidine was measured during the last 18 hours of culture, and revealed that the lymphocyte culture supernatants induced greater thymidine uptake by the 'recipient' lymphocytes than could be expected from the amount of antigen present in the cultures. The soluble factor which mediates this effect was termed 'mitogenic factor'. Analysis of this phenomenon revealed that the mitogenic factor was only generated by interaction between sensitized lymphocytes and the specific antigen used for sensitization; but that once induced, the soluble mitogenic factor was equally active on non-sensitized and sensitized lymphocytes. It is suggested that the mitogenic factor contributes towards the expression of cell-mediated immunity and that it may participate in regulation of the immunological response. [ABSTRACT FROM AUTHOR]

The histological accompaniments of adjuvant activity were studied in popliteal nodes of CBA mice at various intervals after footpad injection. Substances tested possessed either extrinsic adjuvanticity but little or no antigenicity (alum, vitamin A alcohol), antigenicity but no extrinsic adjuvanticity (bovine γA-like globulin), both (Freund's complete adjuvant, killed B. pertussis organisms, alum-precipitated BGG), or neither (paraffin oil BP). Substances possessing extrinsic adjuvanticity produced marked persistent enlargement of the draining lymph node, with prominent expansion, hypercellularity and blast transformation in the paracortical areas by the 4th day after injection, This was followed by germinal centre formation and medullary plasmacytosis occurring between the 4th and 10th day. Substances possessing little or no antigenicity produced a feeble germinal centre response and minimal medullary expansion. Bovine γA-like globulin, which lacks extrinsic adjuvanticity, produced a prominent germinal centre and medullary response but a minimal paracortical response. Only mild, transient lymph node enlargement, lasting 1-2 days, was seen alter paraffin oil BP. Passive immunization of CBA mice with CBA anti-pertussis hyperimmune serum inhibited the adjuvant action of pertussis, and diminished and delayed the histological changes in the draining lymph node. These data suggested that paracortical expansion and hyperplasia were characteristic histological accompaniments to the injection of substances with extrinsic adjuvanticity. Because paracortical hypercellularity was observed in some instances to precede blast cell transformation, it is suggested that paracortical expansion induced by adjuvants is partly due to an augmented cellular traffic, with a net flux of recirculating lymphocytes into these areas. [ABSTRACT FROM AUTHOR]

The purpose of the present experiments was to determine whether populations of lymphocytes from mouse or rat thoracic duct lymph deprived of the larger, dividing cells (large and medium lymphocytes) could still transfer adequate primary or secondary adoptive immune responses using syngeneic irradiated hosts. The transfer system used involved the antigen polymerized flagellin from Salmonella adelaide and assay of anti-H antibody levels in the recipients. Two methods of preparation of ‘small lymphocytes’ were used, namely the glass bead column filtration method of Shortman and the agitated culture method of Gowans and Uhr (1966). Both of these yield lymphocyte fractions essentially free of dividing cells. Unfractionated thoracic duct lymphocytes gave satisfactory adoptive immune responses in both species, primed cells being more effective than normal cells. In both species and for both primary and secondary immune responses, the Shortman-column fractionated small lymphocytes (col-SL) were grossly impaired in their capacity to transfer adoptive responses. In contrast, the Gowans-type post-incubation small lymphocytes (inc-SL) gave rather better adoptive immune responses. Gowans and Uhr (1966) had found some enhancement of reactivity of inc-SL in comparison with normal lymph cells in another transfer system. We, however, found a mild impairment with unstimulated mouse and rat inc-SL and primed rat inc-SL and a slightly more marked impairment with primed mouse inc-SL. We have confirmed the finding of Gowans and Uhr (1966) that lymphocyte populations free of dividing cells can mount an adoptive antibody response, showing that a proportion, at least, of the antigen-reactive cells can be described as ‘small lymphocytes’. The study also revealed the functional heterogeneity of morphologically similar lymphocytes, in that col-SL, morphologically the same as inc-SL, are functionally quite different; they appear to contain very few antigen- reactive cells. In the Salmonella system, therefore, the antigen-reactive thoracic duct cells can be described as lymphocytes which are fairly resistant to agitated culture but which fail to pass through Shortman columns. [ABSTRACT FROM AUTHOR]

Peritoneal exudate lymphocytes (PEL), purified on glass beads and lymph node (LN) cells from guinea-pigs immunized with tubercle bacilli were cultured for 24 hours, in serum-free medium, without and with various concentrations of Tuberculin PPD. Supernatants obtained from cultures with 10 μg PPD/107 lymphocytes provoked an intense inflammatory reaction, when injected into the skin of normal guinea- pigs. PEL were more active than LN cells from the same animals. The reaction was characterized by erythema and induration, with a peak between 3 and 6 hours and histologically a mixed polymorphonuclear-mononuclear infiltrate in the dermis was seen. When fractionated on Sephadex G-200, skin activity of both PEL and LN supernatants was concentrated in a peak corresponding to the molecular weight of serum albumin, while in LN material some activity was also present in a small molecular weight peak. The active material could be separated from albumin by chromatography on DEAE-cellulose. Immunoelectrophoretic analysis of skin reactive peaks detected a slow α-globulin in both PEL and LN supernatants. PPD in the form of a complex with a protein precipitated by anti-γG antiserum, was detected in the skin-active Sephadex peak III of LN supernatants, by radio- immunoelectrophoresis. Skin activity was precipitated with ammonium sulphate at 66 per cent saturation and was destroyed by pepsin treatment. Formation of the skin-active material was depressed by Puromycin and Actinomycin-D and the development of skin inflammation was suppressed by pretreatment of the recipient with anti-lymph node extract serum. Evidence for antigen induced specific synthesis and release of an α-globulin in PEL and LN cultures was found but its relation to the skin active material is unknown. [ABSTRACT FROM AUTHOR]

When lymphocytes from hypersensitive animals are incubated with antigen, biologically active substances are formed which inhibit the migration of mesenchymal cells from normal animals. These substances were tested by intradermal injection in guinea-pigs and rabbits. The supernatants from incubation of lymphocytes with a high dose of antigen caused immediate pallor which lasted several hours. Later there was a macroscopic inflammation maximal at 24 hours. The histology was characteristic of a delayed hypersensitivity reaction. The injection of the supernatant from hypersensitive lymphocytes incubated with a small dose of antigen caused little or no pallor and was not followed by a delayed inflammatory reaction. Injection of this supernatant together with the antigen did not potentiate or alter the reaction, in contrast to in vitro experiments where the inhibition of the migration by this supernatant was potentiated by antigen. Besides this factor a distinct factor occurs in extracts and supernatant fluids of lymphocytes cultivated without antigen and those from control animals, which increases vascular permeability. This substance is probably identical with the lymph node permeability factor (LNPF). The possible role of these biologically active substances in the mechanism of delayed type hypersensitivity is discussed. [ABSTRACT FROM AUTHOR]

Summary. A comparison was made between the number of cells forming or carrying antibody and those stimulated to transform in vitro by antigen, by using blood lymphocytes from rabbits immunized against sheep erythrocytes. In blood samples collected 7-77 days after a single immunization, the number of transformed cells was always markedly higher than that of cells forming plaques or rosettes. It is suggested that unsensitized cells are induced to transform by a stimulus transferred from other reacting cells. [ABSTRACT FROM AUTHOR]

The serum haemolysin response was studied in rats which had been subjected to a suppressive whole-body dose of γ-irradiation followed after 24 hours by an intravenous injection of sheep erythrocytes. A standard restorative dose of syngeneic thoracic duct lymphocytes was given immediately after the antigen or after a variable delay. Delays of 12, 24, 48 and 72 hours were followed by progressively smaller haemolysin responses. These observations suggested that the recruitment of lymphocytes begins very soon after the injection of sheep erythrocytes and that the inductive influence of the antigen wanes steadily so that the effective period of recruitment probably lasts for 1–2 days. [ABSTRACT FROM AUTHOR]

The distribution of a lymphoid-specific antigen has been studied by immunofluorescence in the thymus and other lymphoid organs of the rat and in the thymuses of other vertebrate species. It was demonstrable in all rat lymphocytes except those of the bone marrow. The thymic lymphocytes of all warm-blooded vertebrates also reacted with the lymphoid-specific serum, Plasma cells in anti- genically stimulated lymph nodes did not seem to possess the antigen but similar cells appearing in lymph nodes of rats which had been irradiated and injected with marrow cells of the same strain showed a strong reaction with the antiserum. The antigen is depleted in human and murine leukaemic lymphocytes. X-irradiation did not appear to affect the antigen significantly in cells surviving the treatment.