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In the previous paper we showed that the immune response to sheep red cells (SRBC) was degenerate; serum antibodies showed increasing loss of specificity, signalled by cross-reactions with horse RBC (HRBC), with time after immunization and with hyperimmunization. In the present report we have analysed the role T cells may play in this process. To do this we studied the antibodies made by mice deprived of T cells by irradiation as well as those made by normal mice immunized with SRBC in distilled water, which was shown to depress the DNA synthetic response of T cells. In both instances the mice with fewer responding T cells made less antibody which could agglutinate HRBC than did the appropriate controls. This was true even when no difference in anti-SRBC titre was apparent. In addition we showed that the antibody made by mice with reduced numbers of T cells was less effective at passive suppression of the immune response to SRBC than was similarly titred (against SRBC) antibody made by normal mice. Thus, certain sub-populations of antibodies, normally made in response to SRBC, immunization, are particularly thymus dependent. We have discussed why we think they are those of particularly high affinity. [ABSTRACT FROM AUTHOR]

Red cells sensitized with naturally occurring or 'immune' fowl antibody were lysed by fresh fowl serum. This fowl complement could not be replaced by that of human, rabbit or guinea pig. Both naturally occurring and 'immune' antibodies to red cells in the mammalian species tested did not lyse with fowl complement. Lysis by fresh fowl serum of sheep red cells sensitized with mammalian antibody is due to the presence of naturally occurrent antibody to these cells in the fowl serum; the enhancement of this lysis by the mammalian antibody will be described more fully in the next paper. Naturally occurring antibody to sheep red cells present in turkey serum and 'immune' antibody to fowl red cells were not lytic with fresh turkey serum, with fowl complement, with guinea-pig complement or with any combination of these. Some properties of fowl complement are given. [ABSTRACT FROM AUTHOR]

This paper describes a method for simultaneous measurement of the accumulation of plasma protein and erythrocytes in skin reactions of hypersensitivity to bovine γ-globulin (BGG) and tuberculin PPD in the guinea-pig. The procedure consists in giving 125I-labelled plasma albumin and 51Cr-labelled autologous erythrocytes together by intravenous injection into guinea-pigs bearing skin lesions of allergic inflammation at different times and for different periods during the development of the skin reactions. Isotope accumulation in excised skin reactions is measured by scintillation counting at different stages during the evolution of hypersensitivity responses. Skin reactions of combined anaphylactic and Arthus hypersensitivity to BGG were characterized by pronounced increased vascular permeability principally in the first hour. In established 24-hour hypersensitivity reactions to both antigens (BGG and PPD) there was continuing accumulation of both plasma albumin and erythrocytes. During the development of the tuberculin reaction, an intermediate phase of isotope accumulation occurred between 6 and 9 hours after skin testing; serum transfer studies showed that this intermediate peak was not attributable to circulating antibody alone. These isotope tracer techniques were also applied to study vascular permeability in systemically transferred reactions of delayed hypersensitivity and in the vascular response to the intradermal injection of an inflammatory factor generated by antigen-activation of sensitized lymphocytes. It was concluded that isotope tracing provided objective and sensitive methods for analyzing microvascular responses in allergic inflammation. [ABSTRACT FROM AUTHOR]

The effect of an horse anti-mouse thymocyte globulin (ALG) preparation on the primary and secondary immune responses to sheep erythrocytes (RBC) was studied in 2-3-month-old male A/HeJ, C57B1, BALB/c, DBA/1, CBA and C3H mice. A wide variation in the primary and secondary responses of the individual strains was noticed but in ail cases these were significantly suppressed by ALG pretreatment. The capacity of the ALG to suppress the primary immune response was not overcome by increasing the antigen dose from 3 × 107 to 3 × 109 sheep RBC. [ABSTRACT FROM AUTHOR]

Irradiated (NZB × BALB/c)F1 mice were injected with syngeneic bone marrow cells, syngeneic or parental thymus cells, and sheep red cells. The antibody plaque-forming cell response depended on the number of sheep cells and the age and strain of the thymus cells. Young or adult BALB/c thymus, and young hybrid thymus, responded best to low numbers of sheep cells; with higher numbers they became tolerant. Adult hybrid thymus, and young or adult NZB thymus, responded better to high numbers of sheep cells. Hybrid mice irradiated and restored with BALB/c bone marrow developed thymus cells with the reactivity of BALB/c thymus. It is argued that NZB mice, and older hybrids, may develop autoimmunity because of an abnormality of tolerance induction manifested in their thymus cells, but of bone marrow origin. [ABSTRACT FROM AUTHOR]

The induction in mice of a humoral immune response to intact sheep erythrocytes, both in vivo and in vitro, requires participation of thymus-derived (T) lymphocytes. In an in vitro system, spleen cells from both neonatally thymectomized and adult thymectomized irradiated bone marrow protected mice were successfully immunized, using washed sonicated sheep erythrocyte membrane fragments as antigen. This obviation of the requirement for T lymphocytes in the immune response, coupled with previous work on macrophage independence, indicates that sonicated membrane fragments were capable of directly immunizing bone marrow-derived (B) lymphocytes in vitro. These results further confirm the signal importance of antigenic structure in determining the cellular requirements for an immunological response; whereas antigens of particulate or monomeric form require the presence of both T cells and macrophages, polymeric antigens of intermediate size such as polymerized flagellin and sonicated sheep erythrocyte membranes require neither of these accessory cells. The results caution against the use of erythrocytes as models of thymus-dependent antigens. The data further suggest that reports of late antibody responses of relatively normal magnitude in thymectomized animals given larger doses of heterologous erythrocytes may have been due to direct immunization of B lymphocytes by degraded erythrocyte antigen. [ABSTRACT FROM AUTHOR]

Long-term IgM and IgG responses to sheep red blood cells (SRBC) were determined in New Zealand Black (NZB) mice, using the plaque technique. As compared with other strains a prolonged spleen plaque-forming cell (PFC) response was found in young and old NZB mice. This phenomenon was found in the primary as well as in the secondary response and concerned IgM as well as IgG production. These observations are discussed in relation to the production of IgG antibodies of a low avidity. [ABSTRACT FROM AUTHOR]

HgA-positive red cells were treated in vitro with approximately 10 μmoles N-ethylmaleimide (NEM) per ml cells for 1 hour at 37° and then injected into the circulation of HgA-negative rabbits; all the cells were cleared within 24 hours After 1-3 months the animals were given a series of injections of untreated HgA-positive red cells at 3-monthly intervals. Five out of seventeen rabbits showed progressively diminishing survival of HgA-positive red cells with the development of anti-HgA. On the other hand, in eight rabbits red cell survival following three consecutive injections of untreated red cells was normal or almost normal and in four further rabbits survival was initially subnormal but became progressively longer and was almost normal following a third injection of cells. Thus, in as many as twelve out of seventeen animals (70 per cent) the survival of HgA-positive red cells following a third injection of untreated cells was normal or almost normal whereas this pattern was previously observed in only seven out of fifty-one animals (14 per cent) not given an initial injection of NEM-treated cells. It is concluded that the injection of NEM-treated HgA-positive red cells to HgA-negative rabbits induces a degree of immunological tolerance to the HgA antigen. [ABSTRACT FROM AUTHOR]

Attempts were made to immunize F1 hybrid mice to their inbred parental strains by active immunization with either living or dead parental cells and by adoptive transfer of isogenic (F1) lymph node cells to irradiated hybrids that subsequently received parental bone marrow. Cumulative mortality studies and erythrocyte serotyping revealed that many mice survived and retained their parental grafts for long periods of time. In most experiments reported here, 59Fe uptake by erythrocytes and spleens of chimeras 1 week after marrow transplantation was used as a measure of erythropoiesis and thereby of success or failure of the marrow graft. Pre-treatment (‘pre-immunization’)of hybrids with parental spleen cells produced no evidence of specific immunization. However, pre-treatment with spleen cells from parent 1 was detrimental to subse- quently transplanted marrow from parent 2 and vice-versa. Adoptive transfer of viable F1 lymph node cells failed to decrease growth of parental cells in irradiated F1 hybrids. These findings indicate that the poor-growth phenomenon of particular parent-F1 combinations cannot be explained in terms of classical immune rejection of parental cells by F1 hybrids. Instead, growth of parental cells in the F1 environment is inhibited or delayed. [ABSTRACT FROM AUTHOR]

A time-course study of total, 19S and 7S haemolysins showed that Swiss albino mice were able to give a normal secondary response to sheep erythrocytes at an age when the primary response was much reduced and delayed.

India ink or other finely divided agents (trypan blue, saccharated iron oxide, thorotrast, polystyrene latex) were injected intravenously into mice, and sheep erythrocyte stroma injected subsequently by the same route. The splenic uptake of stroma carrying a tag of 131I was consistently higher in mice treated with the 'blockading' agents than in untreated controls, the difference in some experiments being greater than ten-fold. In addition to promoting the splenic uptake of antigen, some of the finely divided agents also enhanced the formation of anti-sheep red cell haemolysin in mice immunized with a single intravenous injection of sheep erythrocyte stroma. India ink injected intravenously caused a significant rise in the splenic weight of mice. [ABSTRACT FROM AUTHOR]

Antibody-forming cells were found in autochthonous spleen colonies, repopulating the spleens of mice which had been exposed to mid-lethal doses of radiation and inoculation of sheep red blood cells. This was true only for the secondary response, where a proportion of colonies contained haemolytic plaque-forming cells in variable numbers, ranging from 1/103 to 1/106 nucleated cells within a given spleen. The frequency distribution of antibody-forming cells in colonies suggests a clonal growth at an exponential rate, with a generation time of about 7 hours. It is therefore possible to cultivate and study single clones of competent cells in readily accessible, anatomically distinct regions of the mouse spleen. The present data do not discriminate between the derivation of the antibody-forming clones from the original colony-forming cells, or from a cell seeded-in from the spleen tissue outside the colony. [ABSTRACT FROM AUTHOR]

Erythrocytes labelled with chromium51 were injected into embryos and young chicks and a similar challenge given soon after the majority of the initial dose had been eliminated. While tolerance was the typical response in embryos and chicks up to 3 days of age, immunity could be induced in most chicks from this time onwards. The intensity of the immune response increased rapidly until the 12th day of age. No maternal antibody could be detected. Pretreatment of the embryo with testosterone impaired the immune response of 14-day-old chicks. [ABSTRACT FROM AUTHOR]

A methanol extraction residue (MER) of BCG has previously been shown to heighten host resistance to a subsequent challenge with microorganisms or syngeneic tumour grafts and to stimulate the antibody response to sheep red blood cells (SRBC) and phage T2. Present investigations on the effect of antigen dose on MER stimulation of the primary and secondary response to SRBC indicated that: (1) Pretreatment by MER stimulates the early primary response most effectively when the immunizing dose of SRBC is less than maximum, and this is due to a greater increase by MER of 19S antibody with low doses of antigen than with high doses of SRBC. Maximum 7S antibody production is higher in MER treated animals at all antigen doses. (2) MER administered after low doses of SRBC but not high doses stimulates the ongoing primary haemagglutinin response. (3) Maximum secondary responses of mice treated with MER before priming are considerably elevated above the corresponding controls and the secondary responses in treated mice are not inhibited by high priming doses of antigen. These results are discussed in relation to the locus of MER activity. [ABSTRACT FROM AUTHOR]

The treatment of Carworth Wistar rats with normal equine IgG or ALG pre-absorbed with sheep erythrocytes sensitized the rats to sheep erythrocytes and prevented ALG from suppressing the primary immune response to this antigen. [ABSTRACT FROM AUTHOR]

Mice homozygous for the mutation nude which Jack a thymus, were found to have a marked lymphopenia. They had a marked granulocytosis following administration of B. pertussis, but only slight lymphocytosis. Unlike littermates they failed to develop an increased cellularity of lymph nodes draining a local injection of PHA. They had reduced levels of immunoglobulin but produced some antibody to sheep- RBC. They accepted allografts. Cell cooperation experiments suggest that the immunological deficits of nu/nu mice can be explained by the absence of a thymus- derived cell population. [ABSTRACT FROM AUTHOR]

Bordetella pertussis is an adjuvant when given to mice immunized with sheep RBC. The adjuvant activity of pertussis is reflected in an increase in the number of cells producing antibody as measured by the localized haemolysis in gel technique. γG-PFC have a more marked response to pertussis than do γM-PFC, and in general γG-PFC are more sensitive than γM to variations in dose or injection schedule of pertussis organisms. Pertussis increases the response to doses of antigen which previously were considered to be maximal. The distribution of PFC between spleen, blood and lymph nodes is altered by pertussis injections. [ABSTRACT FROM AUTHOR]

L-asparaginase was immunosuppressive in mice, A single injection of 1000 I.U. of L-asparaginase markedly suppressed the direct haemolytic plaque forming cell response to immunization with sheep red blood cells. The effect was not due to cytotoxicity but to delayed proliferation of plaque forming cells. Haemagglutinin production was delayed by a single injection and was suppressed when the enzyme was given daily from 3 days before immunization to the day before antiserum collection. L-asparaginase in this dosage form both reduced total antibody prod uction and markedly prolonged 2-ME sensitive antibody production. L- asparaginase treatment was only weakly immunosuppressive and none of the animals studied showed. complete suppression of the immune response. It was suggested that asparaginase would be most effective in the prevention of the development of primary immunity when combined with another agent with an indepen- dent mechanism of action. [ABSTRACT FROM AUTHOR]

The mechanism of immunological tolerance of sheep red blood cells (SRBC) in mice treated with a single dose of cyclophosphamide was studied. Specific tolerance lasted for as long as nine months in some animals, and its maintenance required the repeated administration of SRBC. Anti-SRBC antibody-forming cells were significantly reduced in the tolerant mice, and X-irradiated recipients of their ere specifically tolerant of SRBC. The smallest number of SRBC required for induction of tolerance was 107 this was the smallest number of SRBC that could elicit antibody synthesis within 5 days in normal mice. Since the effects of cyclophosphamide on antibody-forming cells last only 5 days, it was concluded mechanism of tolerance induction involved destruction of antigen- stimulated cells. In support of this is the finding that mice treated with small doses of cyclophosphamide were rendered tolerant only when SRBC were given before the drug. Drug-induced immunological tolerance thus appears to differ significantly from both pneumococcal polysaccharide paralysis and classical, acquired tolerance. A central loss of immunocompetence does not occur in the former, while the latter requires for its induction the administration of antigen in a dose or form that does not stimulate antibody synthesis. [ABSTRACT FROM AUTHOR]

The article analyzes the changes in the antibody responses to bacterial α-amylase and sheep erythrocytes in C57BL strain of mice with ageing and possible mechanisms responsible for these changes. In the C57BL strain of mice an immune response to sheep red cells was markedly reduced, whereas the response to a bacterial α-amylase was not changed with ageing. This reduction in an immune response was found to result from a decrease in the number of antibody-forming cells, not from a decrease in the amount of antibody synthesized by individual antibody-forming cells.

Cell kinetics of the 2,4,6-trinitrophenyl (TNP)-erythrocyte response in non-immune, immune, and vinblastine-treated immune BDF1 mice were studied by measuring hapten- and carrier-RFC and hapten- and carrier-PFC responses at various time intervals. Injection of a single dose of horse red blood cells alone enhances the anti-TNP response. [ABSTRACT FROM AUTHOR]

Incorporation of [³H]thymidine into the spleens of intact mice during the primary and secondary responses to SRC has been studied. In both responses, auto-radiographs show a well marked increased incorporation of [³H]thymidine by cells in the peri-arteholar zone during days 1 and 2. The degree of labelling indicated that many of these cells were in S phase in preparation for mitosis. A similar response occurred in cells situated in the mantle layer on day 3 to day 4 of the primary response, but was much less evident in the secondary response. This was in keeping with the results of scintillation counting which showed that increased uptake of [³H]thymidine was more sustained in the primary than in the secondary response to SRC. Many lightly labelled cells indicating metabolic turnover of DNA developed in the red pulp of the spleen at times which coincided with the maximum development of antibody-producing cells (PFC) which occurred about 24 hours earlier in the secondary than in the primary response. Germinal centres showed many lightly labelled nuclei. In both primary and secondary responses these areas enlarged, becoming maximal in size after the peaks of PFC were achieved. It was concluded that the enlargement of germinal centres was the result of trapping of cells in the areas rather than proliferation. From these studies it appears that metabolic turnover of DNA occurred in areas of the spleen intimately associated with antibody formation. The possible role of this process in antibody synthesis is discussed in the light of these observations. [ABSTRACT FROM AUTHOR]

The plaque-forming cell (PFC) population from spleens of mice at the peak of the primary immune response against sheep red blood cells has been analysed using a plaque-assay technique and automatic photographic equipment which recorded plaque formation and growth within a carboxymethyl-cellulose gel. Preliminary biochemical studies, using tritiated uridine and leucine as markers, and actinomycin D and puromycin as inhibitors of transcription and translation respectively, were performed with normal spleen cells. The effect of each of these inhibitors was then tested by means of the plaque-assay technique. The formation and subsequent growth of a plaque within a normal gel are processes in which both transcription and translation are required for haemolysin secretion. It was found that in PFC populations, the functional lifespan of messenger RNA (m-RNA) specific for haemolysin translation has a mean value of 4 hours, while the functional lifespan of total RNA in whole spleen cell populations is not more than 2 hours. The pool of haemolysin stored in a PFC has a mean secretion time of 1.5 hours. There was considerable heterogeneity with regard to the storage of haemolysin and to the functional lifespan of the haemolysin-specific m-RNA from one PFC to another within the spleen population. Furthermore, some PFC which displayed a high secretion rate of haemolysin were unaffected by actinomycin D, while others were inhibited within a few hours. The short-term action of each inhibitor has been studied. Puromycin was found to have a reversible action on PFC expression, while a 1-hour duration of action of actinomycin D was sufficient to inhibit irreversibly the subsequent PFC expression. [ABSTRACT FROM AUTHOR]

The organ distribution, blood clearance rate, and immune response to intravenous 51Cr-labelled sheep red blood cells (SRBC) have been studied in mice, following administration of a single intravenous dose of colloidal carbon. In normal mice 80-90 per cent of SRBC were found in the liver and 2-6 per cent in the spleen. The blood clearance rate of SRBC was extremely rapid. Colloidal carbon caused a marked depression of hepatic uptake of SRBC and a corresponding increase in splenic uptake. This effect was maximal at 6 hours after carbon administration and recovery of hepatic phagocytosis occurred over 4 days. The rate of clearance of SRBC was greatly reduced while the hepatic uptake of red cells was depressed. At low doses of cells there was an increase in titre of humoral antibody and in numbers of spleen PFCs while, at high doses of cells, a slight depression in immune response occurred. 'Stimulation' of splenic uptake of antigen and of the immune response by colloidal carbon is associated with depression of hepatic phagocytosis. [ABSTRACT FROM AUTHOR]

The present report describes the morphology of lymphoid tissues and the humoral antibody response during primary and secondary immune responses to sheep erythrocytes (SRBC) in Snell-Bagg hereditary pituitary dwarf mice. It has been found that while 19S primary response was depressed to a great extent, 7S secondary response reached practically normal levels. Histological studies of the peripheral lymphoid tissues have shown patterns of cellular depletion in nonthymus-dependent areas in dwarfs killed during primary response, while these areas appeared markedly cellular after a second antigenic stimulation. It is suggested that the cell systems involved in antibody production during primary and secondary responses to SRBC may be separate and different. As a result, the endocrine imbalance of the dwarf mice affects only the primary response to SRBC. [ABSTRACT FROM AUTHOR]

Lepisosteus osseus possesses high titres of natural agglutinins to human group A (HuA) erythrocytes and to Salmonella typhosa 'O' (STO) antigens. Moderate natural haemagglutinin titres to sheep red blood cells (SRBC) were noted. Cold agglutinin (4°) titres exceeded agglutinin titres recorded at 25°. Immunization with STO antigen resulted in a marked increase in corresponding anti-STO agglutinins, as well as increases in anti-HuA and anti-SRBC agglutinins. Animals immunized with SRBC antigen produced elevated anti-SRBC agglutinin titres, while immunization with HuA erythrocytes produced no changes in agglutinin levels. Immunization with STO following previous injections with HuA resulted in increases of corresponding anti-STO agglutinins, as well as marked increases in anti-HuA agglutinins. Absorption studies indicated that anti-STO agglutinins were not cross reactive against HuA antigens. Natural and immune antibodies were all of the same macromolecular class. Rises in antibody titres were reflected in both cold and warm agglutinins. Animals maintained for long periods at low temperatures exhibited preferential ability for increased synthesis of cold agglutinins to HuA. Natural haemolytic titres, as measured by an SRBC indicator system, were also elevated as compared to mammalian systems. These titres increased with stimulation with SRBC. Results suggest that the high levels of natural haemolytic and agglutinin activity in this species are of immunogenic origin. [ABSTRACT FROM AUTHOR]

Initiation of an immune response to sheep erythrocytes by the transplantation of spleen cells into irradiated recipients is thought to involve an interaction between two functionally different lymphoid cells. If such populations actually exist, it should be possible to separate them from each other and to study the properties of each type of cell. To this end, mouse spleen cells have been fractionated using equilibrium centrifugation in density gradients of Ficoll. Two functionally different populations of spleen cells were obtained. Neither population was active in initiating an immune response by itself on transplantation into irradiated mice, but mixtures of the two populations were. One population of spleen cells was also active when transplanted together with normal mouse bone marrow and the other population was active when mixed with thymus cells from normal mice. These results indicate that two different spleen cells must interact to initiate an immune response in vivo. The specificity of the cell that synergizes with thymus ceils was investigated further and shown to have antigen-specific receptors on its surface. These cells can be recognized by their ability to make rosettes with erythrocyte antigens. Thus, the ‘background’ rosette-forming cells found in unimmunized mice are actually progenitors of antibody-producing cells. [ABSTRACT FROM AUTHOR]

The role of macrophages in the immune response against SRBC in spleen cell suspension cultures obtained from mice was studied. 1. The presence of macrophages increases the number of PFC against SRBC in spleen cell cultures. 2. The ratio peritoneal exudate cells to spleen cells is very critical. Two × 105 peritoneal exudate cells preincubated with sheep red blood cells stimulate 1 × 107 mouse spleen cells to an optimal response. This reaction is suppressed when 2 × 106 peritoneal cells are used. 3. Spleen cell populations which had lost the capability of producing antibodies by elimination of cells adhering to glass yielded a strong immune response upon addition of 2 × 105 peritoneal exudate cells. The significance of macrophages for the induction of the immune response is discussed. [ABSTRACT FROM AUTHOR]

Studies were performed on the reconstitution of the immune response to Shigella paradysenteriae and sheep erythrocytes in sublethally irradiated mice by the injection of normal peritoneal macrophages with and without pre-incubation with antigen. The response to Shigella was found to be less radiosensitive than the anti-SRBC response. Sublethally irradiated mice (550 r) injected with peritoneal macrophages from normal donors pre-incubated with Shigella antigen or injected simultaneously with macrophages and Shigella evinced a strong anti-Shigella response. No such reconstitution of the immune response against SRBC was observed, even when lower irradiation doses and phagocytosis-enhancing incubation media were used. Possible reasons for the different response to Shigella and SRBC are discussed. [ABSTRACT FROM AUTHOR]

Summary. Neonatal mice of the inbred strains NZB, Balb/c and C57B1 were injected with sheep, pig or chicken red cells at various ages and antibody plaque- forming cell (PFC) responses in their spleens measured. Marked strain differences were found, notably a high early response by NZB mice to sheep cells. Hybrid and backcross experiments suggested that this was genetically controlled by at least three genes. C57B1 mice were late in developing a response to all of the antigens tested. The kinetics of the PFC responses were interpreted as favouring an explanation not involving cell division in response to antigen. Thymectomy at birth reduced the PFC responses in 1-month-old mice of all three strains to the same low level, while thymectomy after 7 days left the PFC responses intact. It is argued that the thymus must be present for strain-specific immune responses to develop. [ABSTRACT FROM AUTHOR]