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Partial purification of soluble products of guinea-pig lymphocyte activation (LAP) was undertaken by fractional precipitation with ammonium sulphate, ion-exchange and Sephadex chromatography, and by immune precipitation of inducing antigen and of contaminating serum protein. During these purification steps the activity of macrophage migration-inhibition factor (MIF) was concentrated up to 1300-fold and separated from inducing antigen and serum protein. An endpoint assay was devised for expressing antigen-induced MIF activity of LAP fractions as weights of material giving 30% inhibition of migration (MI30 doses). MIF activity precipitated between 50% and 80% saturated ammonium sulphate and eluted from DEAE-cellulose at pH 7-9 at intermediate salt concentrations (0-03-0-2 M phosphate). On Sephadex gel filtration MIF activity was concentrated in fractions of molecular weight range 56,000-82,000 with a smaller amount of activity eluting from 20,000-56,000. After immune precipitation of extraneous protein and elution from DEAE-cellulose, LAP material was found to have an MI30 dose of 0-4 /μg. Materials representative of antigen and serum protein-depleted MIF were selected for intralymphatic injection in order to determine whether MIF-rich LAP fractions were able to induce paracortical distension in guinea-pig lymph nodes (see following paper). [ABSTRACT FROM AUTHOR]

An earlier paper showed that 4 days after immunization with the contact sensitizing agent 'uxazolone' there is a peak in the percentage of cells in the draining lymph nodes that move to sites of inflammation. This was assessed by dissociating the lymph nodes, labelling them with 51Cr and injecting them into mice whose ears were painted within an hour with an unrelated contact sensitizing agent or with croton oil. The ears were then removed at about 18 hours and their radioactivity used as a measure of cell arrival. The cells that move to sites of inflammation are not macrophages as they are not removed by filtration through cotton wool. They are θ-positive and control studies show that the activity of the anti-B serum was indeed due to antibody to the θ-antigen. The cells are large and can be labelled both in vivo and in vitro with [125I]-iododeoxyuridine which is incorporated in the DNA of cells during the S phase of the cell mitotic cycle. It was concluded that the cells in immunized lymph nodes that move to sites of inflammation are T blasts. The unitary hypothesis is put forward that following immunization for delayed hypersensitivity a particular class of T cells proliferates and gives rise to large pyroninophilic blast cells; and that these cells or their immediate descendants possess the properties, either at the same time or at closely related times, of' movement to sites of inflammation and non-specific cytotoxicity as well as the capacity to passively transfer cellular immunity. [ABSTRACT FROM AUTHOR]

The experiments described in this paper extend the observations of previous work in vitro demonstrating a decrease in the frequency of antigenreactive units specific for horse RBC in spleen cells from mice primed with KLH. It was observed that the addition of lymphoid cells having T-cell function reconstituted the anti-HRBC response to normal values. Significant enhancement of the response beyond the normal values could be evoked using two dissimilar methods, (i) addition of allogeneic thymus cells, and (ii) restimulation in vitro with the priming antigen. The latter type enhancement was elicited by the addition of small doses of KLH to cultures of spleen cells from mice recently primed with KLH, including cultures otherwise demonstrating antigen-induced suppression. The stimulus required to enhance the response to HRBC in these cultures was specific for the priming antigen, KLH. These results are discussed in the light of current theories of 'antigenic competition' and specific heterologous enhancement. [ABSTRACT FROM AUTHOR]

This paper describes the course of infection and the immunological response in thymus-deprived and normal CBA mice after intradermal inoculation with promastigotes of L. tropica. Infection of normal mice resulted in the development of a cutaneous ulcer healing within 12 weeks. As the infection progressed and the draining lymph nodes increased in weight, changes in the paracortical and follicular regions were accompanied by the development of delayed hypersensitivity and the production of antibodies detectable by immunofluorescence and a parasite agglutination test. Lesions in thymectomized irradiated mice healed more slowly and the draining lymph nodes were smaller than in normal mice. Follicular reactions were feeble and paracortical activity depressed. The most noticeable feature of these lymph nodes was a persistent and intense macrophage infiltration. Delayed hypersensitivity and antibodies detectable by immunofluorescence were correspondingly low; but parasite agglutinating antibody was not depressed. The course of infection and immunological response of a control group of sham thymectomized, irradiated mice resembled that of normal mice. These experiments indicate that thymus-dependent cell populations play an important role in the response of mice to infection with L. tropica. [ABSTRACT FROM AUTHOR]

An investigation was made of changes in the ether soluble, acid growth substances during the breaking of winter dormancy of terminal buds of longleaf pine saplings. The greatest changes were an increase in tbe amouut of the promoter which cbromatograpbs similarly to indoleacetic acid in iso-propanol : ammonia : water and the decrease of an inhibitor which chromatographs similarly to inhibitor-β of Bennet-Clark and Kefford (2) in the same solvent. The results show that the acid growth promoters and inhibitors regulate the winter rest period of longleaf pine buds or are closely correlated with the regulalion of the rest period. [ABSTRACT FROM AUTHOR]

Fowls which tolerate foreign erythrocytes, but which have not received a transplant of variable cells, eventually lose tolerance when the supply of erythrocytes is discontinued. The loss follows, at an interval, after the foreign erythrocytes have been eliminated by the normal mechanism for removal of aged cells; earlier loss can be brought about by eliminating the foreign cells with passive antibody. The interval required for loss varies, for tolerance of homologous erythrocytes, from less than 5 to over 215 days. The age of the tolerance bird, and hence the duration of exposure to antigen, is the main factor influencing the interval required; the preceding level of tolerance exerts comparatively little effect. An attempt to terminate tolerance by adoptive immunization was only partially successful. [ABSTRACT FROM AUTHOR]

Use of an intra-articular route of injection (a single, hind limb knee joint) and a protein antigen (human serum albumin)in water-in-oil emulsion with added mycobacterial peptidoglycolipid (or Wax D) failed to induce an adjuvant effect upon the resulting immunological response observed three weeks later. Serum antibody levels as measured by the Farr Test (ABC30 μg/ml) and cell-mediated immunity as measured by a quantitative corneal test were consistently lower than those which followed use of a footpad route for the same injection mixture. [ABSTRACT FROM AUTHOR]

A modification of the leucocyte migration inhibition test, in which cells migrate in an agarose layer, was proposed by Clausen (1971). In the present study, this method was applied to PPD and hepatitis B antigen (HBAg), and its advantages were reassessed. From a technical standpoint, the migration in a monolayer facilitated a study of the morphology of the migrating cells. The proportion of the different kinds of migrating leucocytes was calculated and expressed as a function of the distance of migration. On the whole, 92% of the migrating leucocytes were polymorphonuclears and 8% lymphomonocytes. Three to 10% of the incubated cells were shown to migrate in the agarose layer. The surface area of the controls (migration without inhibition) was sufficiently constant and large (on the average 13 mm²) to allow a clear assessment of inhibition when antigen was added. In addition, the small requirement for antigen facilitated the use of this test in experimentation with purified HBAg. Application of the technique to clinical problems revealed that, with PPD as antigen, a highly significant inhibition was obtained in PPD skin test-positive normal individuals (0.0025

The in vitro assay of inhibition of Ieucocyte migration was used in two forms for the study of the specificity of transplantation immunity in mice. In one system thymus cells (antigen) were mixed with peritoneal exudate cells (PEC) from mice immunized with skin grafts and inhibition of migration was detected only when the antigenic cells were of donor origin or were from a third-party strain which shared an H-2 region (K or D) with the graft donor. However, in another form of the assay, in which sensitized lymph node cells (LNC) were mixed with antigenic PEC, inhibition of migration was detected also using antigenic cells from third- party strains which did not share an H-2 region with the donor and possessed only few H-2 specificities of the donor, The inhibition of migration mediated by LNC was abrogated with the use of anti-θ sen. Transplantation immunity was studied in vivo with an assay of resistance to the growth of a third-party tumour (SaI) in mice pre-immunized with skin grafts from congenic strains having different H-2 antigens. Resistance was found in cases where the tumour cells did not share an H-2 region with the graft donor, but was weaker than the resistance obtained upon immunization with grafts from strains sharing an H-2 region with the tumour donor. [ABSTRACT FROM AUTHOR]

Lymphokines have a number of effects in vitro, which may be related to their role as mediators of delayed hypersensitivity. These mediators are also released in vivo. Authors of the article have described the effect of Lymphokines on in vivo and in vitro antibody production. The present article shows the effect of supernatants which contain lymphokines on the development of delayed hypersensitivity to unrelated antigen. Animals were injected into the ear vein with 1ml of 25 percent suspension of three tines washed sheep red blood cells with or without various supernatants.

Humoral antibodies capable of suppressing induction of delayed hypersensitivity to dinitrophenylated proteins in mice were tested for specific absorbability onto chemically insolubilized antigen and for the antigenic determinant specificities of their immunosuppressive (i.e. contrasensitizing) effects. The activity of an antiserum could be completely removed by absorption with homologous antigen, and it could be recovered by dissociating the absorbed antibodies at low pH and high salt concentration. The immunosuppressive antibodies therefore are specific for determinants on the native antigen, and non-antibody serum constituents are non-essential. By selective immunoabsorptions and elutions, antibodies specific for carrier protein, for dinitrophenyl hapten, and for new determinants unique to the hapten-protein complexes were prepared and were compared with unfractionated antiserum for contrasensitizing activity. Nearly all activity could be accounted for by the anti-hapten antibodies, although anti-carrier antibodies also had some. Despite this evidence that immunosuppressiveness was haptenspecific, the delayed hypersensitivity being suppressed by these antibodies was shown to be directed against hapten-carrier complexes or against carrier but not against hapten alone. Hence, humoral antibodies against a portion of an antigen molecule can suppress induction of delayed hypersensitivity specific for other sometimes unrelated parts of this same molecule, and it is possible to induce antibodymediated tolerance to a multi-determinant antigen with an antibody response against just a part of the antigen. The theoretical and practical implications of these findings are discussed. Notable theoretically is the observation that although determinant specificities of humoral antibody and delayed hypersensitivity responses to one antigen usually differ, the former can regulate the latter for a given species of antigen molecule. Most important practically is the implication that control over development of delayed hypersensitivities and cell-mediated immunologic reactions may be easier than hitherto anticipated, because humoral antibodies to only a portion of an antigen molecule will suffice to control sensitization to the entire molecule. [ABSTRACT FROM AUTHOR]

Twenty-eight chimpanzees were divided into six groups according to their history of previous immunization or exposure to hepatitis B antigen (HBAg) and studied for delayed hypersensitivity (DH) to HBAg. Purified HBAg derived from a normal human carrier was used for in vivo skin testing and in vitro leucocyte migration inhibition tests. Of seventeen chimpanzees immunized with HBAg in Freund's complete adjuvant (FCA), nine exhibited positive DH reactions to HBAg with good correlation between the in vivo and in vitro responses. Of the seventeen chimpanzees, fourteen also exhibited positive DH reactions to purified protein derivative of tuberculin (PPD) with marked erythema and induration; the other three exhibited only erythema with no induration. None of the seventeen animals exhibited any immediate reactivity to either HBAg or PPD. Intradermal injection of HBAg-negative human serum failed to elicit DH reactions in four animals who showed positive skin test with purified HBAg; the DH response was thus probably HBAg-specific. Nineteen chimpanzees, including six unimmunized animals, three chronic carriers of HBAg and two which had been injected with HBAg without FCA, failed to show DH response to HBAg. Thus, DH to HBAg was observed only in animals hyperimmunized with HBAg in FCA. [ABSTRACT FROM AUTHOR]

Use of lymphocyte cultures for in vitro studies such as pretransplant histocompatibility testing has established the need for standardization of this technique. A microculture technique has been developed that has facilitated the culturing of lymphocytes and increased the quantity of cultures feasible, while lowering the variation between replicate samples. Cultures were prepared for determination of tritiated thymidine incorporation using a Multiple Automated Sample Harvester (MASH). Using this system, the parameters that influence the in vitro responsiveness of human lymphocytes to allogeneic lymphocytes have been investigated. [ABSTRACT FROM AUTHOR]

A Salmonella typhi glycoprotein preparation is concentrations of 5 to 16 µg/ml strongly inhibited the migration of peritoneal exudates cells (PEC), obtained from guinea pigs immunized with S. typhi vaccine in Freund's complete adjuvant (FCA), PEC from animals injected with saline in FCA were also inhibited but to a lesser extent, whereas cells from guinea pigs that had received only S. typhi vaccine or normal guinea pigs showed variable and weaker inhibition. PEC from all guinea pigs receiving FCA were strongly inhibited by 5 to 15 µg PPD/ml. These PPD concentrations had no inhibitory effect on exudates cells from guinea pigs receiving S. typhi vaccine only. The S. typhi glycoprotein concentration (5 and 10 µg/ml) primarily used in the migration inhibition studies had negligible inhibitory effect on the phytohemagglutinin (PHA)-induced DNA, RNA, and protein syntheses in guinea pig lymph node cells. In the human system only blood leukocytes of donors who had received their third or fourth booster of heat-killed S. typhi vaccine within the latest 2 years were inhibited in their in vitro migration by the S. typhi glycoprotein complex. [ABSTRACT FROM AUTHOR]

Anti-alloantibodies of anti-(Lewis anti-DA) specificity were successfully induced in (Lewis × DA)F1 rats by the IgG fraction, F(ab') 2, and Fab fragments of Lewis anti-DA alloantibodies. Fc fragments as well as separated heavy and light chains from the same alloantibody failed to induce anti-alloantibodies. The idiotypic determinant on alloantibodies seems to be related to a structure formed by the two polypeptide chains. [ABSTRACT FROM AUTHOR]

Rabbit antisera against mouse cells have been absorbed with normal mouse serum and used to detect mouse cell-bound antigens. Mouse ascites tumor cells and their products have mainly been used as source of antigens but those described here are normal mouse components. Seven antigens have been found by agar diffusion precipitin methods and data are given to show that each is a separate molecular entity. Information is provided for the identification of each component. [ABSTRACT FROM AUTHOR]

Haemolysis of sensitized erythrocytes (EA) can be carried out by incubating them successively with R2, followed by R1. When heated guinea-pig serum is present during the incubation of R2 with EA it inhibits haemolysis, though it has no effect when present during the incubation with R1. This inhibition is stronger with aged preparations of R2 which have much lower titres of C′4 than fresh preparations, though this increased inhibition does not occur because of the inactivation of C·4. Pre-incubation of EA with heated serum does not cause inhibition of their subsequent haemolysis by R2 and R1. Guinea-pig serum that has been decomplemented with an antigen-antibody precipitate and then heated is still inhibitory. Heated serum has no inhibitory action when haemolysis is carried out with whole guinea-pig serum in which all the components of complement are present simultaneously. [ABSTRACT FROM AUTHOR]

A new histocompatibility locus, H-4, in the first linkage group of the mouse, has been identified. The gene-order in this linkage group is H-1, c, p, with H-4 very close to p but its position relative to p undetermined. There is 8.5 ± 2.1 per cent crossing over between H-1 and c in the heterozygous female, 5.4 ± 3.7 per cent in the heterozygous male. At least 4 alleles at the H-1 locus have been identified. The distribution of H-1 alleles in various inbred and co-isogenic resistant stocks is shown in Table 11. The mean survival of skin grafts made between various co-isogenic strain pairs, differing at H-1, ranges from 32 to 91 days, with a minimum survival of individual grafts of 20 days and a maximum of 124 days. Ovarian grafts between the same strain pairs show much longer survival. The significance of these results for estimates of gene numbers is discussed; the higher published estimates are favoured. [ABSTRACT FROM AUTHOR]

The capacity of denatured proteins to provoke anaphylactic sensitivity and delayed sensitivity in guinea pigs has been investigated. It has been found that heat-denatured proteins are as effective as native proteins in provoking delayed hypersensitivity to either material, in spite of the fact that such denaturation greatly modifies their antigenicity from the point of view of antibody production. Pure delayed hypersensitivity, moreover, occurs regularly in response to extremely minute amounts of a good antigen, whether native or denatured. It is further shown that immunological cross-reactivity can be demonstrated regularly by means of the delayed hypersensitivity reaction in guinea pigs, at a time when no cross-reacting antibodies of conventional type can be found. The significance of these observations in regard to the relationship between delayed hypersensitivity and antibody production is discussed. [ABSTRACT FROM AUTHOR]

The migration of peritoneal exudate cells (PEC) from rats with delayed skin reactivity to diphtheria toxoid (DT) and tuberculin (PPD) was inhibited in the presence of antigen. Three days after an intravenous injection of 2.5 mg of DT there was no inhibition with DT. Seven and 28 days later migration was inhibited but less than in sensitized controls. In 6 weeks the difference disappeared. Three days after the DT challenge, the migration inhibition with PPD was weaker than in the sensitized controls. From day 7 on, non-specific reduction of migration inhibition was not detected. The 24-hour skin reactivity to DT and PPD closely paralleled the in vitro phenomena. The mechanism of desensitization appears to be the lack or a functional defect of the specific cells or a blocking factor unremovable by washing or produced during the migration assay. [ABSTRACT FROM AUTHOR]

The exact roles played by specific antibody, circulating antigenantibody complexes and cellular immunity in the causation of experimental glomerulonephritis of rats (EGN) need clarification and a sequential study of these factors was therefore undertaken. Rats immunized with renal tubular antigen and studied for humoral response only (group I) produced neither precipitating nor 2- or 48-hour PCA antibodies but did have an antibody titre by an immunofluorescent test against the proximal portion of the proximal convoluted tubular cells by the second to sixth week post-immunization in six of six animals. A Clq test for antigen-antibody complexes became positive in most rats at the time of onset of proteinuria. Rats in group II were studied for cellular immunity to the renal tubular antigen. Skin tests did not become positive until approximately the time of onset of proteinuria when peripheral blood lymphocyte cultures were also stimulated in seven of nine rats tested. Direct immunofluorescence of the kidneys from this group revealed a linear deposition of γG and renal tubular antigen along the glomerular basement membrane. Since both cellular immunity and antigenantibody complexes involving the renal tubular antigen were present at the onset of proteinuria, both modalities may play a significant role in EGN. [ABSTRACT FROM AUTHOR]

The cell types involved in the cellular immune response were studied with the GVH in vitro as a test system. Comparison of the activities of cells of different lymphoid organs in the GVH in vitro showed the cells of peripheral lymph glands and thoracic duct to be the most active. Second in activity were the cells from peritoneal exudate, mesenterial lymph nodes and spleen. Weak activity was found in thymus and bone marrow cells, whereas cells from Peyer's patches showed no activity at all. Treatment of donor animals with ATS, cortisone or cyclophosphamide simultaneously with (acceptor-) transplantation antigen, resulted in spleen cells which were less active in the GVH in vitro. No such results were obtained after treatment of donor animals with dimethylbenzanthracene or cyclo- phosphamide without antigen. Fractionation of donor spleen cells on a bovine serum albumin density gradient resulted in an accumulation of activity in one of the high density fractions (D), whereas this was reduced in the low density fractions (C, B and A) and absent in the highest density fraction (E). Separation of thymus cells on a glass bead column coated with transplantation antigen resulted in augmented activity of the adherent cell fractions, while the effluent cells were less active than the original cells. Fractionation of cells on an uncoated column resulted in adherent and non-adherent cells which were both inactive in the GVH in vitro. [ABSTRACT FROM AUTHOR]

A wide range of different strains of anaerobic coryneforms and classical propionibacteria have been surveyed for some of their macrocytostimulant effects (included under this term are the ability to increase the rate of phagocytic uptake of carbon after intravenous injection of the latter into mice, ability to stimulate an increase in lysosomal hydrolases and ability to exert directly a chcmotactic stimulus on macrophages) and their ability to increase humoral and cellular immunity when admixed with an immunogen. Of twenty-one strains of anaerobic coryneforms tested, fifteen strains were able to produce an increase of the phagocytic index in mice of at least 50 per cent. Micro-organisms that were effective in these tests occurred in each of the four main serological groups. Although no single serological group showed outright superiority in causing an increase of K, three of the five strains which had highest activity belonged to serological group I. Four organisms representing the ‘classical’ propionibacteria were also tested, but none of these caused an increase of phagocytic index in mice. Many of the strains that stimulated a rise in phagocytic index also caused an increase in the weight of the spleen measured at 10 days after injection and a moderately good correlation was apparent between the magnitude of the two effects, Tests in mice and chickens for an adjuvant action on serum levels of antibody failed to show any enhancement of primary responses, although a barely significant elevation of the secondary response in the mouse was observed. In the guinea-pig, a clear adjuvant effect on the levels of serum antibody in a primary response was observed. However, no evidence could be obtained that any of several anaerobic coryneforms was able to enhance cell-mediated hypersensitivity, as shown by delayed-type skin tests or corneal reactions. A study of the histological responses to a footpad injection of various anaerobic coryneforms (in water-oil emulsion with ovalbumin) showed that the regional (popliteal) node underwent a considerable expansion of the lymphoid-cell content of the paracortical (thymus-dependent) area. This was accompanied by extensive sinus plugging by lymphocytes and endothelial hypertrophy of post-capillary venules. [ABSTRACT FROM AUTHOR]

An evaluation of the specificity of antigen binding receptors possessed by cells involved in cellular immune responses and by cells in the antibody synthesizing line was made in order to determine to what extent each cell type could distinguish between closely related mono-ε-DNP-oligo-L-lysines. The antigen binding receptors of cells involved in cellular immune responses were studied by the elicitation of delayed hypersensitivity reactions in vivo and by the stimulation of DNA synthesis by lymph node cells in vitro using 1-ε-DNP-nona-L-lysine and 9-ε-DNP-nona-L-lysine. These experiments demonstrated that closely related compounds could be distinguished by this cell population. On the other hand, serum antibody from animals immunized with 1-ε-DNP-nona-L-lysine did not regularly distinguish between various mono-ε-DNP-oligo-L-lysines of differing immunogenicity or between 1-ε-DNP-nona-L-lysine and 9-ε-DNP-nona-L-lysine. Serum antibody specificity is assumed to reflect the specificity of the receptors possessed by the precursors of antibody producing cells. Thus, it appears likely that the cells involved in cellular immune responses or functioning as 'helper' cells in the stimulation of antibody synthesis by other cells are capable of determining the immunogenicity of a compound. On the other hand, precursors of antibody producing cells, at least in primed animals and in the mono-ε-DNP-oligo-L-lysine response, do not appear to make distinctions between peptides of differing immunogenicity. [ABSTRACT FROM AUTHOR]

A detailed evaluation of the capacity of mono-ε-DNP-oligo-L-lysines to initiate anti-DNP antibody synthesis and a state of delayed hypersensitivity in guinea-pigs is presented. Peptides containing as few as two lysine residues elicit the production of significant amounts of anti-DNP antibody when they are administered as Freund's complete adjuvant emulsions. Under these immunization conditions, the serum concentration of anti-DNP antibody is dependent on the chain length of the peptide, and on the amount and kind of mycobacteria in the adjuvant; guinea-pigs lacking the PLL gene produce amounts of anti-DNP antibody indistinguishable from that produced by guinea-pigs possessing this gene. On the other hand, when guinea-pigs are immunized with either 1-ε-DNP-tetra-L-lysine or 1-ε-DNP-nona-L-lysine without the use of mycobacterial adjuvant, anti-DNP antibody is produced only by guinea-pigs receiving the nona-L-lysine and only by those animals possessing the PLL gene. Delayed hypersensitivity results front immunizing PLL+ guinea-pigs with mono-ε-DNP-octa and nona-lysines but not from immunization with mono-εDNP-hexa-lysine; PLL- animals do not exhibit delayed hypersensitivity to any of these compounds. The data suggest that antibody synthesis to positively charged compounds may proceed as a result of formation of charge complexes with mycobacterial proteins; under such immunization conditions the intrinsic immunogenicity of a compound is more reliably revealed by the induction or elicitation of cellular immune responses. On this basis, a definite discontinuity in the degree of immunogenicity of the mono-ε-DNP-oligo-L-lysines occurs as the peptides are lengthened from six to eight residues. [ABSTRACT FROM AUTHOR]

Rabbit antisera were prepared against mouse leukaemias from several inbred strains, and against a mixture of these leukaemias. The potencies of these antisera were tested against each of the different leukaemias, and against certain normal cells and tissues. As determined by immune cytolysis, the potencies of antisera were strongest against the homologous leukaemias. The potencies against unrelated leukaemias were generally within 50 per cent of those against the homologous leukaemias, with some exceptions. Antisera prepared by injection of a mixture of the leukaemia cells showed remarkably high potencies against all leukaemias tested. As measured by C fixation, the same antisera showed less specificity for the homologous leukaemias. The potencies of antisera for C fixation on a variety of normal tissues was quite strong, and decreased approximately in the following order: testis, kidney, liver, lung, brain, muscle, erythrocytes. For antiserum prepared against DBA/2 leukaemia L1210, the C fixation reaction against normal lymph node lymphocytes was about 20 per cent as strong as the reaction against the homologous leukaemia L1210. A hypothesis concerning the mechanism of immune cystolysis is presented to explain these results. Exhaustive absorption of anti-L1210 sera with normal DBA/2 tissues produced only a slight increase in antileukaemia specificity. [ABSTRACT FROM AUTHOR]

The technique of equilibrium density gradient centrifugation has been used to study the antibody forming cells (AFC) produced in the Lewis rat and the toad Bufo marinus in response to polymerized flagellin protein antigen. In both animals the AFC at the peak of the response were heterogeneous in density, and formed many separate peaks, as has been observed previously with sheep erythrocyte antigen. Two lines of evidence suggested the various peaks were of biological significance. The earliest AFC found in the rat spleen were simpler in density profile than the AFC at the peak, although a complex 'steady-state' situation developed soon after. In the toad, a definite sequence of density peaks appeared progressively during the whole response. In the rat, the AFC making different classes of antibody gave different density profiles, and could be partially separated from each other. The density separation technique may be useful for studying the differentiation and maturation of antibody forming cells. [ABSTRACT FROM AUTHOR]

Sensitization doses of [14C]dinitrofluorobenzene (DNFB) were painted on the dorsum of guinea-pig ears and the total radioactivity in the draining lymph nodes was determined, at various time intervals, in the first 72 hours. Of this radioactivity, 25 per cent was found to be centrifuged off with the cell debris after homogenization of the nodes in saline. The radioactivity in the remaining saline soluble material was shown to be precipitated by trichloracetic acid; of this, the DNFB was found to be conjugated, to approximately the same degree, to both the lipid and the protein components of the nodes. The significance of these findings is discussed. [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]

A method for estimating cell-mediated immunity in the guinea-pig by the spectrophotometric measurement of the corneal test response, is described. The results obtained were found to correlate well with subjective readings of the reaction made by an experienced observer. Also, a much finer discrimination of the degree of corneal response could be made, than was possible by the subjective method. [ABSTRACT FROM AUTHOR]

The time relationships of delayed type hypersensitivity, cellular immunity and protective circulating immunoglobulin production assumed an unexpected pattern in mice responding to sublethal pulmonary infection with Bordetella pertussis. Cellular immunity, characterized by an intracellular bacterial steady state appeared early. Initially, it was not demonstrably associated with either delayed type hypersensitivity or effective levels of circulating protective antibody. The appearance, after 6 weeks or more, of protective levels of antibody was associated with bacteriological clearance accompanied by loss of cellular immunity. [ABSTRACT FROM AUTHOR]

We observed that the volume of any anti-HSA serum needed to bind 50 per cent of the antigen is a linear function of the amount of antigen present in the reaction mixture over three log units. The slope is characteristic of each anti- serum and reflects the ‘avidity’ of the antibodies. By using this quantitative index we found that the avidity of antibodies produced during the secondary adoptive response depends on the number of the cells transferred : few cells will produce antibodies of lower avidity than many cells. The dose of antigen given as a booster also influences the quality of the antibody produced; in the extreme case where some primed cells were rendered ‘tolerant’ by supra-optimal doses of challenging antigen, substantial amounts of low avidity antibodies were synthesized. The results can be explained by competition for antigen among competent cells, favouring those cells which carry antibody sites with high affinity for the corresponding antigenic determinant. [ABSTRACT FROM AUTHOR]

Immune and non-immune mesenteric lymph node and Peyer's patch cells were labelled in vitro with 51Crk and injected intravenously into infected and non-infected syngeneic guinea-pigs (Heston strain). From the distribution of the radioactive label in the small intestine (the site of infection of T. colubriformis) it was concluded that immune lymphocytes: (1) preferentially localize or 'home in' on the infection, (2) come into intimate contact with the parasite in the epithelium, and (3) rapidly undergo 'allergic death' or lysis at the site of infection. [ABSTRACT FROM AUTHOR]

The in vitro reaction between sensitized lymphoid cells and target cells has been studied in an allogeneic transplantation system in mice. Mastocytoma cells of the DBA/2 donor strain were injected into C57BL mice, and the spleens of the recipients harvested 8 days later. The immune lymphoid cells thus obtained were tested for their ability to damage in vitro cultures of target mastocytoma cells. The reaction was followed by two methods: (l) microscopic counts, and (2) ability of target cells to form colonies in a semi-solid medium after various periods of contact with immune lymphoid cells. The results show: (a) that the effect on target cells is an exponential function of the number of immune lymphoid cells employed, and (b) that the reaction becomes noticeable after 3 hours and reaches completion within 12 hours when evaluated by cloning techniques, while it takes 24–48 hours to be microscopically clearly demonstrable. Problems arising from use of both methods are discussed. [ABSTRACT FROM AUTHOR]

The intravenous transfer of mesenteric lymph node cells from intragastrically immunized donors led to the appearance of agglutinins in the sera of recipients. Other lymph node cells from these donors did not impart such a response in the recipients. The serum agglutinin titres in recipients were dependent on the immune state of the donor where the better immunization of the donor gave a high and prolonged response in the recipients. The antibody synthesizing property could be conveyed to the recipients by the intravenous transfer of any of the lymph node cells and spleen tissue using intravenously immunized donors. However, in intragastrically immunized donors, it was only the mesenteric lymph node cells that could effectively transfer this factor. Thus, the sensitization of the regional lymph nodes is important in cell transfer mechanism. The antibody that appeared in recipients after the cell transfer was not due to the transfer of preformed antibody but was due to the active participation of the recipient in the antibody synthesis. The lymph node cells tested in vitro were negative for Vibrio cholerae agglutinins. [ABSTRACT FROM AUTHOR]

Cells obtained from mesenteric lymph nodes of highly inbred guinea-pigs (Heston strain) resistant to Trichostrongylus colubriformis were injected into virgin animals of the same genotype. The adoptivety immunized recipients were challenged with 1000 T. colubriformis larvae 4 days after transfer and slaughtered at intervals which correspond to critical times in the development of the parasite. Differential worm counts carried out on specimens of intestine showed that a sharp decline in the number of parasites occurred between days 7 and 9. This period corresponds to the time required for the parasite to develop to the fourth larval stage. Variation of the time interval between cell transfer and challenge showed that immune cells transferred on the day of challenge and on days 4, 6 and 8 after challenge inhibited the development of infection to potency, while cells injected on day 10 were without effect. This observation confirmed that the fourth larval stage of the parasite is uniquely susceptible to the immunological attack initiated by the transferred cells and showed that these cells are effective within 24-48 hours after injection. This latter finding excludes the possibility of active participation in the response by the recipient. Resistance can be transferred by spleen cells and by cells obtained from lymph nodes other than the mesenteric nodes which drain the site of infection. However the local nodes are more effective and resistance was regularly transferred with as few as 10 × 106 cells injected intravenously. [ABSTRACT FROM AUTHOR]

Guinea-pigs were immunized with DNP-HGG either by direct injection into the lower respiratory tract or intravenously. The distribution of cellular sensitivity was assessed by means of the macrophage migration inhibition test applied to bronchial wash cells (local) and to peritoneal exudate and spleen cell suspensions (systemic). A single low antigen dose given locally via the intratracheal route was sufficient for stimulation of cellular immunity in bronchial wash cells and oil induced peritoneal exudate cells but not in cells obtained from the spleen. Alternatively, a low antigen dose given intravenously resulted in splenic and peritoneal exudate cell sensitivity but not by cells obtained from the bronchus. Under these latter conditions, however, the bronchial wash cell population could be rendered sensitive to the systemic antigen providing sensitization was accompanied by a non-specific local stimulation--such as injection of a non-cross-reacting protein. Although a local low antigen dose does not normally result in systemic cellular immunity as judged by spleen cell assays, cellular sensitivity was induced systemically by increasing the amount of locally administered antigen given as a single injection or increasing the number of low dose injections. Furthermore, under conditions of multiple injection, the onset of a measurable response at any site was accelerated. Experiments to determine the fate of locally injected antigen revealed that proteins do escape from the lung into the general circulation as intact molecules. In addition to other parameters affecting the distribution of cellular immunity, the implication of these findings with special regard to respiratory tract prophylaxis are discussed. [ABSTRACT FROM AUTHOR]

The development of cell-mediated immunity to paternally-derived foetal transplantation antigens in parous mice has been assessed by two techniques: skin grafting in an H-2 incompatible mating and a local graft-versus-host assay in an H-2 compatible mating. (1) Lymphoid cells from BALB/c females, parous one to seven times to C3H/Bi males, were injected i.p. into syngeneic males or females which were then grafted with skin from the paternal strain. A slightly accelerated rejection in the adoptively immunized recipients was observed. (2) Lymphoid cells from CBA females, parous one to five times to AKR males, were injected into the feet of young F1 hybrid recipients. The degree of popliteal lymph node enlargement was significantly greater in recipients of cells from parous than from virgin donors. Results using both techniques demonstrated that the development of cell-mediated immunity to paternal transplantation antigens is a regular feature in parous mice and that the degree of immunity increases with successive interstrain pregnancies. [ABSTRACT FROM AUTHOR]

Pretreatment of mice with Freund's complete adjuvant (FCA) or Corynebacierium parvum depresses the contact sensitivity which otherwise follows immunization with picryl chloride in FCA (PCL/FCA). In contrast, Bordetella pertussis is inactive. The hypothesis that this depression was partly due to the failure of lymph nodes immunized with PCL/FCA to collect lymphoid cells was tested. Mice were pretreated with FCA, C. parvum or B. pertussis, subsequently immunized with PCL/FCA, and injected with 51C-labelled normal lymph node cells. Pretreatment with FCA 10 days before immunization depressed the arrival of labelled cells at draining nodes immunized with PCL/FCA by 10-19%. Pretreatment intravenously with C. parvum 4 days before immunization virtually abolished the increased arrival at draining lymph nodes which is otherwise seen 2 and 4 days after immunization, but had little effect on the increased arrival seen 1 day after immunization. In contrast, B. pertussis caused a slight but significant depression at day 1, but had no effect 2 and 4 days after immunization. The depression of contact sensitivity by these three bacterial adjuvants was paralleled by their depression of the arrival of labelled lymph node cells at recently immunized nodes, and supported the hypothesis that failure of recently immunized lymph nodes to collect Iymphoid cells is part of the mechanism of depression of delayed hypersensitivity by bacterial adjuvants, and in particular. by C. parvum. [ABSTRACT FROM AUTHOR]

The macrophage migration inhibition test was employed to show reactivity of maternal peripheral blood leucocytes to placental antigens. Four to 5 days after delivery, all of forty-one postpartum women tested had leucocytes in their blood that reacted with pooled antigens from five placentas. When antigens from a single placenta were used leucocytes from only the woman who donated that placenta and one of nine unrelated postpartum women were reactive. Leucocytes from seventeen-control subjects, including nulligravidous women, were non-reactive with either the single or the pooled placental antigens. The results indicated that the mothers may be specifically sensitized, as far as the cell mediated immune response is concerned, by the placenta during pregnancy. [ABSTRACT FROM AUTHOR]

A boy aged 6 years with severe chronic mucocutaneous candidiasis of the granulomatous variety has been treated with the dialysable transfer factor. Before the treatment he was unable to express delayed hypersensitivity. His lymphocytes were able, however, to produce migration inhibitory factor following in vitro challenge with soluble candida antigens, but this activation was not accompanied by a significant increase in DNA synthesis. The treatment resulted in a temporary clinical improvement and return to normal of his immune responses. After a relapse he was, 6 months later, once more injected with transfer factor, which again proved effective. Evidence is presented which suggests a defect of monocyte-macrophage function in this patient and the pathogenesis of candida granulomata is discussed. It is postulated that an amplifying mechanism involving lymphocyte-macrophage interaction is required for the normal expression of delayed hypersensitivity reactions and the effective elimination of Candida albicans. [ABSTRACT FROM AUTHOR]

The effect of cortisone acetate on specific cell mediated immunity in mice was investigated using contact sensitivity to oxazolone. A long-acting cortisone acetate preparation was found to be much more effective than a soluble preparation in suppressing temporarily the recall response to oxazolone. The long-acting preparation was also effective in inhibiting the induction of sensitivity when injected before (but not after) oxazolone. Removal of the thymus either before or after sensitization did not significantly enhance the effects of cortisone acetate, and it is therefore suggested that cortisone acetate acts independently of the thymus-derived lymphocyte population. [ABSTRACT FROM AUTHOR]

The experiments reported here were concerned with determining the effects of lymphocyte activation products (LAP) on lymph node architecture, and with assaying afferent lymph for evidence of the peripheral release of LAP during the induction of an immune response. Intralymphatic inoculation of purified homologous LAP into guinea-pigs resulted in increased weight and cellular content of the draining node. Histologically these nodes showed paracortical distension and dense aggregations of lymphoid cells or 'cellular plugs' in the paracortical sinuses. It was suggested that one effect of LAP may be to cause cellular retention in the paracortex of lymph nodes by regulating the rate of cell exit via the sinuses of the node. The peripheral lymph of rabbits was assayed for its ability to inhibit macrophage migration and to accelerate lymphocyte DNA synthesis after stimulation with three different antigens. The antigens were chosen to give a spectrum which ranged from a primarily humoral response (erythrocyte stimulation) through a mixed humoral and cell-mediated response (diphtheria toxoid stimulation) to a predominantly cell-mediated type of response (skin contact sensitization to fuorodinitrobenzene-FDNB). Paracortical distension with lymphoid cell sinus plugging, similar to that observed in the guinea-pig nodes following intralymphatic injection of LAP, were common features of both the diphtheria toxoid and FDNB responses. It was concluded that the development of this type of sinus plugging and paracortical distension might be related to multiple activities of LAP getierated and released either at the peripheral antigen depot or within the draining node. [ABSTRACT FROM AUTHOR]

A patient with chronic mucocutaneous candidiasis and cutaneous anergy has been studied and found to have a circulating plasma factor capable of quenching in vitro lymphocyte responses of leucocytes of clinically well donors to monilia and other specific mitogenic stimulants. After instituting systemic amphotericin B therapy, the patient exhibited rapid clearing of cutaneous and mucosal lesions and the plasma inhibitor was no longer demonstrable. Loss of the plasma inhibitor was followed by appearance of strong cutaneous hypersensitivity and in vitro leucocyte responses to Candida albicans and streptococcal products. These clinical immunologic studies have pointed meaning in relationship to current views regarding immunologic reconstitution vis-à-vis systemic antifungal therapy in treatment of chronic candidiasis. [ABSTRACT FROM AUTHOR]