Effect of RES `blockade' on antibody formation: III. Proliferation capacity of antigen-reactive stem cells in carbon-treated mice after transfer to irradiated recipients*

The number of antigen-sensitive stem cells in spleens of mice treated with carbon particles to suppress the reticuloendothelial system was studied by a cell transfer system. X-irradiated recipient mice were injected with varying quantities of spleen cells from control and carbon-treated donor mice, followed immediately by challenge immunization with sheep erythrocytes. The number of individual haemolysin forming cells and antibody foci in spleens of recipient animals was determined.

There was usually a maximum of 10–20 per cent fewer foci in the spleens of irradiated mice treated with spleen cells from carbon-injected as compared to normal donor mice. Furthermore, the number of individual antibody forming cells appearing in spleens of irradiated mice restored with spleen cells from carbon-treated mice was only moderately less than that occurring in spleens of mice receiving cells from normal donors. Similarly, serum antibody titres were only moderately suppressed in recipient mice when the donors were carbon-treated.

The day of administration of carbon into the donor affected the number of antibody forming cells and foci, as well as serum titres, in the recipient mice. Maximum inhibition occurred when the donors were injected with the carbon 1–3 days before sacrifice and cell transfer.

Since each focus of antibody activity in a recipient spleen is considered due to proliferation of an individual antigen-reactive stem cell in the donor cell population, the results obtained suggest that only a portion of the immunodepression occurring in carbon-blockaded mice is due to stem cell inhibition.

     

Antibody formation by transferred peritoneal cells and spleen cells of mice: I. Transfer of cells from immunized non-irradiated donors to syngeneic recipients with and without antigen

Peritoneal cells and spleen cells from LAF₁ mice given three intraperitoneal immunizations of sheep red blood cells synthesized haemagglutinins after transfer to X-irradiated syngeneic recipients, either with or without a concomitant injection of antigen. Antibody formation by cells transferred with antigen resembled a secondary antibody response in intact animals. Haemagglutinins appeared rapidly and in high titre. Approximately 50 per cent of the antibodies were resistant to treatment with 2-mercaptoethanol.

Antibody formation by cells transferred without further exposure to antigen differed in several respects. Haemagglutinin titres were lower. Throughout the period of observation, only 6–25 per cent of the antibodies formed were mercaptoethanol-resistant. In recipients injected with spleen cells, antibodies appeared rapidly, suggesting that mature antibody-forming cells had been transferred. However, in recipients injected with peritoneal cells from the same donors, antibodies were detected only after a delay of several days, which suggested that mature antibody-forming cells had not been transferred. Haemagglutinins were synthesized equally well after transfer of all types of peritoneal cells or of a fraction consisting almost entirely of lymphoid cells. Recipients of peritoneal cells from donors which had been given either one intraperitoneal immunization or three intravenous immunizations had no or only low haemagglutinin titres, in contrast to recipients of cells from donors given three intraperitoneal immunizations.

These observations suggest that antibody formation in recipients of peritoneal cells only, i.e. without further stimulation by antigen, can be attributed to cells which are present in the peritoneal cavity of donors immunized repeatedly by the intraperitoneal route, but which are not part of the active antibody-synthesizing apparatus of the donors at the time of cell transfer. The development of these cells into antibody-forming cells can be detected only in an environment devoid of antigen and of mature antibody-forming cells. It is postulated that these cells are distinct from cells which respond on re-exposure to antigen.

     

Effect of RES `blockade' on antibody formation: II. Cytokinetics of the secondary haemolysin response and suppressed immunological `memory' in mice treated with carbon particles

Suppression of the primary immune response by treatment of mice with carbon 1 day before initial immunization markedly interfered with development of immunological `memory', since such mice responded to a subsequent challenge injection of RBCs by formation of mainly IgM PFCs and serum antibody. Appearance of IgG PFCs and 2-ME resistant antibody was delayed several days in these carbon treated animals, indicating failure of a typical secondary response. The immune response of these animals was similar to that of a primary response of control animals to a single injection of red cells.

Reticulo-endothelial cell blockade with colloidal carbon suspensions interfered with development of a normal secondary type immune response to sheep red blood cells, as assayed on both the cellular and humoral levels. Fewer antibody PFCs, mainly 19S IgM but also 7S IgG, appeared in spleens of antigen primed mice treated with carbon 1–3 days prior to a challenge injection of red cells, as compared to control primed mice injected with erythrocytes alone. However, the peak day of antibody response was the same for both control and carbon treated animals.

Mice treated with carbon 1–3 days before secondary immunization had much lower peak serum titres, mostly susceptible to 2-ME inactivation.

The time of inoculation of carbon in relation to immunization was important since carbon treatment 1–3 days before secondary RBC immunization resulted in maximum suppression. Injection of carbon 5–7 days before resulted in only a slight effect, whereas injection 30 days before had no detectable effect. Injection of carbon simultaneously or after RBC injection had little effect.

The dose of carbon used for immunosuppression, as well as the concentration of sheep erythrocytes used for immunization affected the number of antibody PFCs and the serum titres in control as well as carbon treated animals.

     

Antibody formation against Vibrio cholerae by mesenteric lymph node cell transfer

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.

     

Shigella-: Rabbit Gamma-Globulin Allotypes as Genetic Markers for the Source of Antibody Produced in Recipients of* • Incubated Lymph Node Cells*

Rabbits homozygous for each of an allelic pair of allotypes of γ globulin (A4 and A5) were used as donors and recipients of transferred antigen-incubated lymph node cells, the cells of donors of one allotype being in each case transferred to recipients of the other. When agglutinins to Shigella (the source of the antigen with which the lymph node cells were incubated) appeared in the sera of the recipient animals, the allotype of the agglutinin was determined by adsorbing it to Shigella on a glass slide, then treating the preparations with fluorescein-conjugated rabbit anti-A5-γ-globulin and anti-A4-γ-globulin antisera, respectively. In each case the reactions of the recipients' sera were positive for γ globulin of the donors' allotype but not of their own. Positive reactions were given only by sera above a certain range of agglutinin titre.

After sufficient decline of the agglutinin level in the sera of the recipients, these animals were actively immunized with Shigella. The agglutinins that now appeared in these rabbits gave positive reactions with the fluorescent antibody against their own allotype.

These data indicated that in moderately X-irradiated rabbits given antigen-incubated rabbit lymph node cells, the antibody that subsequently appears in the recipient's serum has been synthesized by the donor's cells.

These experiments also illustrate the use of allotypes as genetic markers in lymph node cell transfer experiments.

     

Studies on globulin and antibody production in mice thymectomized at birth

Serum globulin levels and antibody against sheep red cells, Salmonella typhi (`O' and `H'), haemocyanin and pneumococcus type 3 capsular polysacharide (SSS III) were studied in C3H/Bi and C57BL×C3H/Bi F₁ hybrid mice thymectomized within 18 hours of birth. Thymectomized mice tend to recover more slowly than intact mice from the physiological hypo-γ-globulinaemia present 3–4 weeks after birth, but the majority of mice that survived beyond 6 weeks had serum globulin levels comparable to those in intact animals. Increased γ_1A-globulin levels were observed in some thymectomized mice which survived for 7 weeks or more and the immuno-electrophoretic patterns sometimes resembled those seen in mice with γ_1A myelomas. Macroglobulin (γ_1M) was detected in the sera of thymectomized mice which contained normal amounts of γ-globulin.

Total serum protein levels of groups of thymectomized and intact mice were not significantly different. The turnover rate of plasma albumin was also closely similar in both groups, but that of γ-globulin was accelerated in most of the thymectomized mice examined.

Antibodies against SSS III, sheep red cell agglutinins and typhoid `H' agglutinins behaved as 7S globulins, in contrast with sheep red cell haemolysins which belonged to the 19S group.

Antibody levels following primary stimulation with sheep red cells and Salmonella antigens were usually, but not always, much lower in thymectomized mice than in intact control mice. However antibody levels against haemocyanin and SSS III were within the range of controls in more than half the thymectomized mice. Few thymectomized mice after stimulation with a mixture of three antigens failed to give a detectable antibody response to all the antigens, and some responded as well as did intact mice.

The significance of these findings is discussed in relation to current theories of the function of the thymus in the development of immune responses.

Many of the older thymectomized mice showed a characteristic wasting syndrome, but it is unlikely that a general failure of antibody response can account for this. Evidence for the presence of auto-antibodies against red cells or nuclear or cytoplasmic constituents was sought but was not found.

     

Experimental delayed type allergy without demonstrable antibodies: I. Absence of cytophilic antibodies

In guinea-pigs a delayed type allergy against sheep erythrocytes (SE) without detectable circulating antibodies (haemolysins) could be produced by immunization with erythrocyte—antibody complexes. Cytophilic antibodies against SE as measured by adherence of SE to macrophages could not be detected on the macrophages or in the serum of these animals as long as they had no haemolysins. Conversely skin tests were often negative in animals with cytophilic antibodies in serum. These antibodies do not seem to be involved in the allergic reaction.

Though cytophilic antibodies only occur in sera with haemolysin titres above 50–200 no constant relation between the titres of both antibodies was found in individual sera. Complete Freund's adjuvant stimulates the production of cytophilic antibodies but they are also produced by immunization without adjuvant.

     

Radioautographic study of cellular mechanisms in delayed hypersensitivity: IV. Distribution of injected lymph node, spleen, thymus and bone marrow cells

Mononuclear cells from various organs of sensitized donor rats were labelled in vitro with tritiated leucine and injected intravenously into sensitized, syngeneic recipients. The localization of injected cells was studied in radioautographs.

Bone marrow cells were the most frequent of the labelled cells in skin reactions. Equal numbers of bone marrow cells were found in specific and non-specific delayed reactions and irritation reactions induced with turpentine.

Lymph node cells were found in delayed but not in turpentine reactions. Spleen cells were less frequent than lymph node cells in delayed skin reactions; small numbers were found in the turpentine reactions. Lymph node and spleen cells did not show detectable antigenic specificity.

Thymus cells were not found in the skin sites, even when they were allowed to circulate 2–3 days in the recipients before biopsies were taken.

Irrespective of the source of the cells injected few or no labelled cells were found in the recipient thymuses. Lymph node and spleen cells migrated equally into lymph nodes and spleen, but were infrequent in bone marrow. Only bone marrow cells had a decided preference for their organ of origin.

     

Clearance of Rh-positive red cells by low concentrations of Rh antibody

Ten previously untransfused Rh(D)-negative subjects were given an intravenous injection of approximately 0.3 ml of Rh(D)-positive red cells and at about the same time, or 24–48 hours previously, were given an intramuscular injection of 1–1000 μg of anti-D.

Following the injection of the red cells there was a variable period before the onset of red cell destruction; when the antibody was injected at the same time as the red cells the delay was due partly to the time taken for the antibody to reach the circulation; when the antibody was injected at least 24 hours before the red cells there was still some delay due to the time required for the antibody to be taken up by the red cells; the delay before the onset of a maximum rate of red cell destruction varied from about 0.2 hours following the injection of 1000 μg of antibody to approximately 100 hours following the injection of 1 μg.

The maximum rate of red cell destruction was calculated to be approximately proportional to the square root of the amount of antibody on the cells. After the injection of the smallest amount of antibody (1 μg) the amount on the red cells was calculated to be about 0.03 μg/ml corresponding to about ten molecules of antibody per cell. This dose produced clearance with a T_½ of the order of 100 hours.

The significance of these observations in relation to protection against Rh-immunization is briefly discussed.

     

A simple method for the cryopreservation of lymphocytes. Retention of specific immune effector functions by frozen-stored cells.

A simple, quick and inexpensive method for cryostorage of lymphocytes is discribed. When injected into appropriate normal recipients, frozen-stored rat and sheep lymphocytes caused GVH and NLT reactions respectively. Sheep lymphocytes remained viable after several months storage and functioned satisfactorily as 51Cr-labelled target cells in assays for cytotoxic antibodies. Lymphocytes, including immunoblasts, drained from sheep efferent lymphatics during immune responses to injected murine P815 cells or allogeneic lymphocytes survived freezing; when thawed the cells retained specific immune cytotoxic effector functions including the ability to secrete complement-dependent and leucocyte-dependent antibodies     

On the induction of immunological tolerance to a self-reproducing antigen

Inoculation of a dose of 1000 LD₅₀ of LCM virus intraperitoneally into newborn mice within the first 18 hours of life always resulted in the development of a complete and permanent state of tolerance to the virus as judged by a life-long viraemia with titres of [unk] 10^2.3 and complement fixation (CF) titres of <4.

Sixteen litters of outbred white Swiss mice were inoculated intraperitoneally with this virus dosage at an age of 2–9 days. A total of 103 babies were inoculated. Of these babies sixty survived the first 2 weeks of life. They were followed for 24 weeks with regard to the course of the viraemia and CF-antibody production.

On the basis of the values obtained in this manner in the individual mouse seven types of course could be distinguished. These represented the following immunological phenomena: (1) complete and permanent tolerance; (2) disappearance of the viraemia and antibody formation; (3) disappearance of the viraemia without antibody formation; (4) permanent viraemia with antibody formation; and (5) temporary, incomplete tolerance as illustrated both by temporary antibody formation with constant viraemia and by temporary viraemia elimination without antibody formation.

The results indicate that a dissociation of the immunological response of the mouse to LCM virus can take place, presumably representing a state of split tolerance. Furthermore the results indicate that specifically reactive cells can be present in an apparently completely tolerant animal. These findings are discussed in the light of the hypothesis that complete tolerance represents the maximum degree of specific immunosuppression.

     

A kinetic study of antibody producing cells in the spleen of mice immunized intravenously with sheep erythrocytes

The number of antibody producing cells, i.e. rosette forming cells (RFC) has been studied in the spleen of mice injected intravenously with a full immunizing dose of sheep RBC.

The spleen of a non-immunized mouse contains a background of about 70,000 RFC (normal RFC), which do not appear to be the `target cells' for antigens of sheep RBC. Our findings suggest that the spleen of a mouse contains about 4000 `target cells' which initiate the immune response to sheep RBC.

In the primary response, the rise of RFC is exponential for about 96 hours with a doubling time of 13 hours involving seven to eight consecutive doubling periods. The peak value of RFC is 1·6 × 10⁶ per spleen.

In the secondary response, the doubling time of RFC is 6–7 hours. The exponential rise lasts 72 hours and includes nine to eleven doubling periods leading to a peak of 3·5 × 10⁶ RFC/spleen.

Adjuvant in the primary response leaves the doubling time of RFC unaltered but prolongs the exponential rise until the 120th hour leading to a peak of 6·3 × 10⁶ RFC/spleen after ten doubling periods. The effects of priming and adjuvant are not fully additive.

     

Inflammatory lymphoid cells: Cells in immunized lymph nodes that move to sites of inflammation

The arrival of cells from normal and immunized lymph node cells at sites of inflammation was studied. Mice were immunized with `oxazolone' or picryl chloride and 3–4 days later the draining lymph node cells were dissociated, labelled in vitro with ⁵¹Cr and injected intravenously into recipients.

Sites of inflammation were produced in the recipients by painting the ear with chemically reactive contact sensitizing agents or croton oil. The net arrival of normal lymph node cells at sites of inflammation was 0.1 per cent. In contrast the arrival of cells from immunized lymph nodes was 4–8 times greater. The peak number of cells that moved to sites of inflammation occurred 4 days after immunization with `oxazolone'.

An increase in the percentage of cells that moved to sites of inflammation occurred in lymph nodes after immunization with a number of agents including contact sensitizing agents, skin grafts and Freund's complete adjuvant. There was little or no increase in mice rendered unresponsive to picryl chloride and then immunized with picryl chloride or in mice injected with aluminium hydroxide, alum precipitated mouse serum or pneumococcal polysaccharide. This suggested that immunization and possibly the induction of delayed hypersensitivity was necessary for the generation of these cells.

The arrival of the cells did not depend on antigenic similarity between the agent used to immunize the lymph node and the agent used to produce inflammation. For example increased arrival of cells from immunized lymph nodes occurred at sites of inflammation produced in the ear by croton oil or in the peritoneal cavity by paraffin oil.

     

Effect of RES `Blockade' on antibody-formation: I. Suppressed cellular and humoral haemolysin responses in mice injected with carbon particles

Antibody formation to sheep erythrocytes, as detected both at the cellular and humoral level, was suppressed in adult mice after reticulo-endothelial cell (RES) blockade induced by intraperitoneal injection of colloidal carbon. Fewer antibody plaque-forming cells (PFC) appeared in spleens of carbon pretreated mice as compared to normal controls following intraperitoneal immunization with sheep erythrocytes. The day of peak antibody response was the same, however, for both control and carbon treated animals.

There was no compensatory increase in the number of PFCs in other lymphoid organs of carbon treated animals. Similarly, carbon inoculation had no detectable effect on the number of `background' PFCs in the spleens of unstimulated mice.

The time of injection of carbon in relation to time of immunization influenced the effect since injection of carbon 24–48 hours prior to RBC injection resulted in maximum immunosuppression. Injection of carbon 4–5 days before red blood cells resulted in only partial immunosuppression. Treatment with carbon 1–2 weeks prior to immunization had no detectable effect. Similarly, injection of carbon 1 or 2 days after immunization had little or no effect on the peak plaque response.

The decrease in the amount of serum antibody to sheep red blood cells in carbon treated mice was not as marked as that which occurred on the individual cell level. However, most of the antibody in the sera of carbon treated animals was susceptible to 2-mercaptoethanol, even 1 or 2 weeks after immunization. On the other hand, serum antibody from control mice was mainly 2-mercaptoethanol sensitive only during the first week after immunization.

Immunosuppression seemed to be related to a direct effect of carbon since the supernatant fluid obtained from carbon suspensions was not suppressive. Also, washed or dialysed carbon preparations were just as effective as the original preparation in suppressing antibody responses.

     

Lung alveolar histiocytes engaged in antibody production

After introduction of antigen (sheep red blood cells) into rabbit lung alveoli by intrapulmonary or intratracheal injection antibody forming and releasing cells are found in subsequently evoked alveolar exudates. Intratracheal immunization results in slightly delayed antibody formation with a remarkably low involvement of mediastinal lymph nodes. At peak appearance of plaque-forming cells (PFC) in alveolar exudates 20–26 per cent of PFC are histiocytes or monocytes by microscopic and ultrastructural criteria. Plaque-forming histiocytes are less differentiated forms, different from typical macrophages. Their antibody production is sensitive to puromycin and to a lesser degree to actinomycin-D. In contrast to lymphoid PFC, some of these histiocytes are sensitive to a phagocyte-destructive agent-silica.

Among the PFC, plasma cells and (early after immunization) activated lymphocytes and blasts are conspicuous in comparison with the overall composition of the cell populations of the alveolar exudate and mediastinal lymph node.

It is calculated that 1 in 100–500 plasma cells and 1 in 20,000–100,000 histiocytes give demonstrable haemolysin formation during the peak of cellular response. This is suggested as the difference between the specialized and occasional or `primitive' antibody forming cell.

     

The cellular transfer of immunity to Trichostrongylus colubriformis in an isogenic strain of guinea-pig: II. The relative susceptibility of the larval and adult stages of the parasite to immunological attack

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 adoptively 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 patency, 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.

     

IgA antibodies in the bile of rats. I. Some characteristics of the primary response

About a week after suspensions of sheep red blood cells (SRBC) or killed Brucella abortus organisms were injected into the Peyer's patches of Wistar rats specific agglutinins of the IgA class appeared in the bile of titres which equalled or exceeded those of the IgG and IgM agglutinins in the blood serum. The injection of these antigens by conventional routes was relatively ineffective in inducing biliary antibodies. The relationship between the dose of B. abortus injected into the Peyer's patches and ensuing humoral response in the bile was investigated; a single dose of 5x10(5) organisms caused no detectable biliary response, while a dose of 10(9) organisms caused a substantial response in which specific antibodies persisted in the bile for several months, even though immunogenic material could not be recovered from the injection sites (the Peyer's patches) after a few weeks. A haemolytic plaque assay showed that many antibody-forming cells occurred in the mesenteric nodes and that up to half of them were synthesizing IgA. Few antibody-forming cells were found in Peyer's patches, and although some IgA-forming cells were found in the spleen they were less numerous, both in absolute terms and relative to cells producing other isotypes, than in the mesenteric nodes. The active production of biliary antibody was transferred to unimmunized recipients by thoracic duct lymph cells collected a few days after immunization of the donors when their lymph contained an increased percentage of immunoblasts. Athymic (nude) rats produced normal amounts of specific, biliary, IgA antibodies after immunization in the Peyer's patches with B. abortus but made no detectable response to similar injections of SRBC.     

Heterophile antibodies, M-antiglobulins and immunoglobulins in experimental trypanosomiasis

Monkeys (Macaca mulatto) were infected with different strains of Trypanosoma rhodesiense, T. gambiense and T. brucei. The titres of non-sensitized sheep cell agglutinins (NSCAT), M-antiglobulins (rheumatoid factor-like globulins) and the levels of immunoglobulins (IgG, IgA and IgM) were determined in sera before and during the course of infections controlled by treatment with Suramin.

Persistently high titres of NSCAT-agglutinins developed in monkeys infected with all three organisms.

IgM levels increased in all animals reaching levels more than ten times the original values. The levels of IgA remained unchanged and of IgG slightly increased in some animals.

Slightly elevated M-antiglobulin titres were occasionally found without any relation to the progress of infection.

NSCAT-agglutinins were found to be macroglobulins (Sephadex G-200 immunoelectrophoresis, S₂₀ = 18·5) sensitive to β-mercaptoethanol treatment. Their activity was absorbed by sheep red blood cells, guinea-pig kidney cells and T. rhodesiense antigen.

     

Malignant changes in New Zealand black mice

Ageing, Coombs positive, NZB mice, may spontaneously develop a neoplasia of the reticulum cell type which can be transferred by serial passage of their lymphoid tissues in young intact or neonatally thymectomized syngeneic recipients. The recipients (103/117) of cell suspensions prepared from the enlarged spleens and/or lymph nodes of four such donors developed an extensive and lethal reticulum cell neoplasia affecting the spleen, lymph nodes, lungs and liver but the bone marrow, thymus and kidneys were seldom involved. The recipients (16/17) of spleen cells from a fifth donor showed massive proliferations of eosinophils in all the organs examined.

Prematurely positive antiglobulin (Coombs) reactions were detected in only two recipients. Although there was an indication that the IgM content of the sera decreased as one of the passages progressed, the levels of IgG and IgA were not seriously distorted.

Particles resembling murine leukaemia virus were identified by electron microscopy in the spleen and in plasma or serum pellets of passage recipients. However, similar particles were also seen in the thymus and/or spleen or bone marrow of untreated NZB mice including an 18-day embryo and animals aged 1–56 weeks, although no particles were found in plasma and serum pellets of mice aged 6–70 weeks.

The theory that autoimmunity, malignancy and virus infection are directly related is discussed.

     

Response of rat blood, spleen, and lymph node leucocytes to soluble and insoluble antigen

Solubilized sheep erythrocyte stroma was found to be antigenic in rats. Spleen and lymph nodes of rats injected with this antigen contained more 7S than 19S plaque-forming cells throughout the primary and secondary responses. When compared to the primary response, secondary immunization with this antigen elicited increased numbers of both 19S and 7S plaque-forming cells. Antibody synthesizing leucocytes in the blood during the primary and secondary responses were predominantly 7S producers during the first few days. Later 19S producers predominated.

Intact sheep erythrocytes elicited the same pattern of 19S and 7S antibody-forming cell development in the lymph nodes and blood of intravenously injected rats, but during the early primary response in the spleen there was a predominance of 19S over 7S plaque-forming cells. The 7S cells were in a majority during the entire secondary response of the spleen to intact erythrocytes. The secondary response of spleen and lymph node to intact erythrocytes showed an elevated 7S plaque forming cell response but the number of 19S cells was similar to that detecred after primary immunization.

The appearance of haemolytic and haemagglutinating 19S and 7S antibody to sheep erythrocytes or solubilized stroma generally reflected the cellular picture of the spleen. By using an anti-7S globulin it was found that 19S and 7S antibody appeared simultaneously in the serum.

After immunization of rats with intact erythrocytes or solubilized stroma the number of lymphoid cells that took up tritiated thymidine was about one hundred-fold greater than the number of antibody-forming cells as determined by localized haemolysis in gel. The number of lymphoid cells positive in an immunocyto-adherence assay was more closely related to the number of cells taking up tritiated thymidine.

The passive transfer of spleen cells from rats immunized to sheep erythrocytes showed the number of circulating antibody-forming cells in the normal and irradiated recipients to be related to the concentration of antibody-forming cells localizing in the recipient spleen. The number of antibody-forming cells in the peripheral blood was greater in splenectomized recipients. Irradiation had no effect on the number of antibody-forming leucocytes in the circulation of the splenectomized recipients.