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Immunization of BALB/c mice with Group A streptococcal vaccines leads to the induction of high levels of monoclonal antibody populations. Subsequent immunization of these mice with Group A-variant streptococcal vaccines induces a significant level of monoclonal antibody of A-variant antigen specificity revealed by labeled group A-variant polysaccharide. During these course Av and Av' immunizations, the monoclonal Group A-specific antibodies were also restimulated to levels usually higher than the variant-specific antibodies. With two exceptions, these homogeneous antibody populations were not cross-reactive in vitro with the related antigen. Such cross-stimulation of monoclonal antibodies was interpreted as a function of particular membrane properties of the Ig receptor-bearing memory cells which, for restimulation, would only require the structurally closely related antigen which serves as a backbone to the original antigen, and not necessarily the exact fit of the homologous immunodominant group.

After total splenectomy, a portion of the spleen was autotransplanted into subcutaneous ectopic sites of DBA/2 mice. These implanted fragments regenerated into splenic tissue that are microscopically indistinguishable from the structure of the original spleen. These implants are also immunologically functional as demonstrated by their ability to produce antibodies to sheep red blood cells (SRBC) and SRBC inhibition of splenic (implant) cell migration.

Spleens of adult mice immunized with either RSA or RGG responded in vitro to DNP-RSA or DNP-RGG, respectively, at a significantly higher rate than spleens of untreated mice. Stimulation in vitro could be achieved by short pulses of the antigen (5–15 min). It was found that thymectomy prior to injection of the carrier protein interfered with the subsequent response in vitro to the hapten-carrier conjugate, and that this was much more pronounced in 8- to 10-day-old mice than in older mice. It is suggested that antigen stimulation in vivo triggers thymus cell migration. Although this is by no means the only mechanism accountable for manifestation of the carrier effect, it may represent a device for amplification of the immune response in vivo .

It has been previously demonstrated that alloantisera can specifically block the activation of T lymphocytes by antigens, the response to which is linked to the presence of histocompatibility (H) types against which the alloantisera are directed. Thus, strain 13 anti-2 serum can inhibit the activation of (2 x 13)F1 T lymphocytes by a DNP derivative of a copolymer of L-glutamic acid and L-lysine (DNP-GL), an antigen the response to which is controlled by a 2-linked Ir gene. It was proposed that alloantisera can inhibit T-lymphocyte antigen recognition through interference with the activity of immune response (Ir) gene products. In order to further study whether the inhibitory antibodies within the alloantisera are directed against H antigens or against the products of the Ir genes, we have examined whether the anti-2 serum can inhibit the function of an Ir gene (the L-glutamic acid and L-alanine [GA] gene), which is normally linked to strain 2 H genes when this gene occurs in an outbred animal lacking strain 2 H genes. In the majority of cases, the anti-2 serum was capable of inhibiting the in vitro proliferative response to GA of T cells derived from animals that were GA+2+, but the serum had little if any effect on the GA response of T cells from GA+2- animals. Furthermore, an antiserum prepared in strain 13 animals against the lymphoid cells of a GA+2- outbred animal was devoid of inhibitory activity on the GA response of cells from a (2 x 13)F1, while an antiserum prepared in strain 13 animals against the lymphoid cells of a GA+2+ outbred animal was capable of specifically inhibiting the response to GA. It thus appears that the inhibition of the GA response by the anti-2 serum is primarily mediated via antibodies directed toward strain 2 H antigens rather than antibodies specific for the product of the GA Ir gene. The mechanism of alloantiserum induced suppression of Ir gene function would then be by steric interference with the Ir gene product on the cell surface, rather than by direct binding to it. This conclusion implies that the products of both the H genes and the Ir genes are physically related on the cell surface. The implications of such a relationship in terms of the fluid-mosaic model of the lymphocyte surface are discussed.

Mice treated with divinyl ether-maleic anhydride (DVE/MA) 7 days before, 2 days before, or simultaneously with foot-and-mouth disease (FMD) viral antigens (aqueous or oil emulsified) were more resistant to virus infection and released antibody earlier than mice treated with antigen alone. Simultaneous treatment of swine with these antigens plus DVE/MA resulted in a slightly enhanced early antibody response, which, in a test with oil-emulsified antigen, was not associated with an early induced antiviral resistance to FMD. In general, the primary and secondary antibody responses to oil antigen plus DVE/MA were the same as in control swine receiving the antigen alone. However, swine sensitized with aqueous antigen plus DVE/MA responded faster and developed higher antibody titers after a secondary treatment with aqueous antigen than swine treated similarly but without DVE/MA.

Using immunodiffusion methods it has been shown that purified hemagglutinin (HA) extracted from two related strains of influenza A viruses (A/PR8/34 and A/FM1/47) have two distinct antigenic determinants, or groups of determinants. One determinant is cross-reactive while the other is strain-specific. Antisera raised in normal mice against HA were shown to contain two populations of antibody molecules, each directed against one of the determinants. Immunization of thymus-deprived (TXBM) mice showed a strong thymus dependence of antibody formation to HA. However, the thymus dependence of antibody formation against the cross-reactive determinant could be overcome by repeated inoculations of HA in TXBM mice, indicating a different handling of two portions of the same molecule by the immunological system. Strong, secondary-type responses to the strain-specific determinant were observed in primed thymus-deprived mice after reconstitution with virgin thymus cells, showing that specific immunological memory was elicited by this determinant despite the absence of detectable antibody secretion. These findings are interpreted as examples of immunological recognition and memory mediated by B lymphocytes and discussed in terms of mechanisms of T and B lymphocyte co-operation. It is suggested that the helper effect of T lymphocytes is exerted at a late stage in the differentiation of specific populations of B cells into antibody-secreting cells.

The peak of blast response in primary, one-way mixed lymphocyte culture of CBA plus DBA -m took place on culture day 6 to 7 whereafter the number of Masts per culture declined. Concomitantly with the disappearance of blast cells from these cultures, the number of lymphocytes increased, indicating that a certain number of blasts reverts back to lymphocytes (‘secondary lymphocytes’). The number of antigen-reactive cells in primary mixed culture (quantitated by hydroxyurea block) was on the order of 3% to 5%, whereas when the same cultures were reprimed with the original antigen, the number of antigen-reactive cells was on the order of 90% . Thus most lymphocytes surviving prolonged primary mixed lymphocyte culture are in fact secondary lymphocytes–that is, blast-derived. The secondary lymphocytes thus obtained responded promptly and in a biphasic manner to the original and H-2 cross-reactive stimulator cells in secondary mixed culture The first, major response peak took place on culture day 2 to 3 and the second, minor peak on culture day 5 to 6. Although the secondary lymphocytes were sensitive to anti-θ plui complement, they did not respond to phytohemagglutinin The secondary lymphocytes were themselves cytotoxic to relevant allogeneic target cells in vitro, and they were promptly reinduced to maximal cytotoxicity by a second priming with the original antigen. We suggest that these functions are character-istic to T memory cells primed against histoincompatible cells.

BALB/c mice repeatedly immunized with Pneumococcus R36A vaccine produce antibodies to phosphorylcholine having the TEPC-15 myeloma idiotype (murine IgA myeloma protein that binds phosphorylcholine). The plaque-forming cell response to phosphorylcholine shows a decrease with repeated immunizations. In contrast, spleen cells from multiply immunized mice responded better in vitro than spleen cells from nonimmunized mice. The serum of animals immunized four or five times agglutinates TEPC-15-coated sheep erythrocytes. Inhibition of hemagglutination shows that the agglutinating activity is directed against the TEPC-15 idiotype. Sera from these mice, when added to cultures of normal spleen cells, specifically suppress the response to phosphorylcholine. The suppressive activity in the serum can be removed by solid absorption with TEPC-15. Evidently, repeated immunization with antigen induces two kinds of antibody responses: one directed against antigen and the other directed against the antibody to the antigen. It is proposed that this “auto” antibody against receptor is involved in the regulation of the immune response.

Die phylogenetische Entwicklung des Immunsystems last sich anhand des Stammbaumes der Vertebraten deutlich verfolgen. Bei den Urfischen entstanden nacheinander intestinale Lymphocytenaggregate, die Milz, der Thymus und die ersten Plasmazellen. Transplantate werden chronisch abgestosen und Antikorper der IgM-Klasse gebildet. Das Erscheinen von „jugular bodies“ und intestinalen Plasmazellansammlungen bei den Amphibien geht einher mit akuter Transplantatverwerfung, der Bildung von IgG-Antikorpern mit groserer Affinitat zum Antigen als die IgM-Antikorper und erstmals auch einem schwachen immunologischen Gedachtnis. Mit dem Auftreten keimzentrenhaltiger Lymphknoten bei den Reptil-Saugern erscheint ein ausgepragtes immunologisches Erinnerungsvermogen. Das komplizierte lymphatische System der Vogel und Saugetiere ermoglicht schlieslich durch zahlreiche lymphatische Filter und Lymphknoten in allen Regionen des Organismus einen optimalen Kontakt zwischen immunkompetenten Zellen und korperfremden Substanzen und Erregern. Die besondere Struktur der verschiedenen Immunglobulinklassen ist auf molekularer Ebene das Ergebnis von Genduplikationen, zufallsverteilten Punktmutationen und Deletionen verschiedener Primordialgene. Dabei begunstigte der Selektionsdruck im Verlauf der Evolution die Entwicklung multipler Gene fur die variablen Bereiche gegenuber einer geringeren Anzahl an Genen fur die konstanten Bereiche der Immunglobulinpolypeptidketten. Die einheitliche phylogenetische Entwicklung von der Stufe der Amphibien bis zu den Saugetieren sowie die fast identische ontogenetische Entwicklung des Immunsystems der hoheren Saugetiere rechtfertigen es, tierexperimentelle Ergebnisse auf den Menschen zu ubertragen und daraus prophylaktische und therapeutische Konsequenzen zu ziehen.

The thymus of mice intravenously immunized with SRBC contains a subpopulation of B memory cells. Cell transfer experiments were clone to study whether these B memory cells respond like normal thymocytes to treatment with corticosteroids in vivo and to treatment with anti theta serum and complement in vitro . Corticosteroid treatment reduced the amount of thymic IgM, IgG, and IgA memory cells to about 50%. Anti theta treatment in vitro did not affect the thymic B memory cells.

Functional separation of the two signals involved in stimulating immunological responses was achieved through the judicious use of two natural bacterial antigens. Native protoplasmic polysaccharide (NPP) extracted from Escherichia coli was immunochemically identical to the lipopolysaccharide (LPS) extracted from the same organism. However, NPP was not endotoxic, not mitogenic, did not fix complement, and was immunologically independent of T cells. The NPP, which appeared to contain only the antigenic signal, could induce a primary antibody response in mice and could sensitize mice for a secondary response. However, the antigenic signal contained in NPP was insufficient to trigger a secondary response in mice primed with either NPP or LPS. LPS, containing both the antigenic and second signals, was required to trigger a secondary response in primed mice.

It has been suggested that dominant monoclonal responses in mice after transfer of small numbers of spleen cells are the result of strong selection for cell clones producing antibody of high affinity. We have attempted to measure the affinity of anti-DNP (2,4dinitrophenyl) and anti-NIP (4hydroxy5iodo3nitrophenacetyl) antibodies produced in monoclonal responses by inhibition of plaque-forming cells (PFC) with free hapten. PFC from individual monoclonal spleen foci and from spleens after adoptive transfer of 5 × 105 to 2 × 106 primed spleen cells were generally inhibited at lower free antigen concentrations than the PFC found in a heterogeneous secondary response. It was possible to compare several individual cell clones, forming monoclonal antibody to DNP, with respect to the free antigen concentration needed for 50 % plaque inhibition. These comparisons did not correlate well with the relative affinities of serum antibody estimated by the Stupp-Farr technique. Published work analyzing PFC from plasma cell tumor MOPC 315 forming antibody to DNP might have predicted that the curve of PFC inhibition by free hapten for a monoclonal PFC population would be steep. Usually this was not the case, and 20–80 % PFC inhibition occurred over 100-fold concentration range of free hapten. Some of the anti-DNP clones formed large plaques and when these PFC were inhibited by free antigen, the plaque diameter decreased progressively as the hapten concentration increased. Large plaques consequently require more free antigen for inhibition than small plaques. The poor agreement between apparent affinity measured by plaque inhibition and that measured in serum antibody for monoclonal antibody responses may well be a result of the observed differences in plaque size between individual clones. We therefore have strong reservations about the use of plaque inhibition by free antigen to estimate antibody affinity at the cellular level.

Neonatally and adult thymectomized (Tx) mice of strains C57BL/10Sn and A. BY (H-2b) were immunized at the age of 5 – 8 weeks with lethally irradiated spleen and lymph node cells from adult B 10. A and A (H-2a) donors, respectively. The titer of H-2 hemagglutinins and cytotoxins was determined at different intervals after immunization. Both untreated antibodies and antibodies reduced with 2-mercaptoethanol (2-Me) were tested. The non-Tx control mice responded with production of both 2-Me-sensitive and 2-Me-resistant H-2 antibodies. In the Tx mice, only 2-Me-sensitive H-2 antibodies were detected. Thus, the anti-H-2 IgM response appears to be thymus-independent and the anti-H-2 IgG response thymus-dependent.

In vitro cultivation of murine spleen cells resulted in a spontaneous release of receptors for alloantigens. This was revealed by the capacity of cell-free culture supernates to recognize alloantigens as measured in the PAR assay. Qualitatively, recognition responses obtained with these supernates reproduced faithfully those found with the corresponding cells. Large amounts of receptors were released by untreated spleen cells and by spleen cells treated with a rabbit antimouse immunoglobulin serum and complement, smaller amounts were released by bone marrow cells, and native thymus cells released none. Spleen cells from nude mice and spleen cells from normal mice treated with anti-θ serum and complement showed no release of receptors. It was concluded that receptors active in the PAR test were of T-cell origin. Release of T-cell receptors was found to be discontinuous and proceeded in waves. The amount of released receptors depended on the number of cells cultivated. Release occurred at 37°C but not at 4°C. Interaction with antigen, however, was temperature-independent. In contrast to T-cell receptors, a release of H-2 antigens could not be detected with the culture conditions employed.

A secondary local and splenic cell-mediated immune response was observed and compared to the primary response. Previous studies have demonstrated cell-mediated immunity (CMI) by lymphocytes from bronchopulmonary washings and have shown that its appearance is to a large extent inedpendent of splenic CMI. This study evaluated the secondary as compared to the primary response, with respect to both cellular and humoral immune responses. Guinea pigs were immunized with influenza virus vaccine either nasally or parenterally, booster immunizations were given by the same route, and animals were killed at various times after immunization or booster. The inhibition of macrophage migration was used to assess CMI. As in previous studies, local application of antigen led to mainly local appearance of CMI, whereas parenteral immunization led to mainly systemic CMI. Both pulmonary and splenic lymphocytes showed an inhibition of macrophage migration that appeared 2 to 3 days sooner after the booster, as compared to the primary immunization. There was no evidence, however, for the earlier production or increased amount of antibody in the bronchial secretions in the boosted animals. The results suggest that pulmonary as well as splenic T lymphocytes exhibit memory, but that pulmonary B lymphocytes do not.

Nearly normal secondary tetanus antitoxin responses were produced by young adult female mice subjected to 600 rads of 60Co gamma whole-body radiation 4 days after secondary antigenic stimulation via the hind leg footpads. In an attempt to correlate the rapid transition from radiosensitivity of the antibody-forming system on day 3 to relative radioresistance on day 4 after booster, we carried out studies on cytokinetics in regional (popliteal) lymph nodes and on incorporation of 3H-l-histidine into circulating antitoxin. Analyses of tritium radioactivity in antigen-antibody precipitates of pooled sera 2 hr after i.v. injection of the labeled amino acid revealed maximum incorporation into antibody around day 7 after booster in nonirradiated controls, and around day 12, i.e., 8 days after radiation, in experimental mice, respectively. Relative radioresistance of the antibody-forming system on day 4 after booster, as opposed to day 3, was attributed to the finding that plasmacellular proliferation in medullary cords reached a marked and narrow peak on day 3 and had almost subsided to control levels between days 4 and 5. Accordingly, many medullary plasma cells survived and continued to proliferate after exposure to radiation. In contrast, germinal centers, which largely determine the overall maximum DNA synthetic activity in the lymph node and attain their full development in non-irradiated animals only 5 to 6 days after booster, were completely destroyed by radiation within 1 day. Since a) antibody production persisted despite the disappearance of germinal centers; and b) mice irradiated 4 days after booster have previously been shown to lack tertiary responsiveness, the present findings lend strong support to the hypothesis that germinal center cells were involved more in the generation of memory cells than in active antibody secretion. The results do not favor the assumption of a marked contribution of germinoblasts to the antibody-forming plasma cell population elicited by the same stimulus which had triggered germinal center formation.

We have previously demonstrated that alloantisera prepared by reciprocal immunization of strain 2 and strain 13 guinea pigs specifically block stimulation of in vitro DNA synthesis in genetically controlled systems. In order to determine whether this blockade extends to other T-lymphocyte functions, we examined the effect of alloantisera on the production of migration inhibition factor (MIF). (2 x 13)F1 guinea pigs were immunized with a DNP derivative of the copolymer of L-glutamic acid and L-lysine (DNP-GL) and with DNP guinea pig albumin (GPA). The response to the former is controlled by a 2-linked Ir gene while that to the latter is mainly controlled by a 13-linked Ir gene. MIF production was assayed by an indirect procedure in which the migrating cell population lacked the histocompatibility antigen against which the alloantiserum was directed. Our results showed that anti-2 serum blocked MIF production by F1 cells in response to DNP-GL but not DNP-GPA while anti-13 serum had the opposite effect. These experiments show that expression of a second major T-cell function is specifically blocked by alloantisera and suggest that Ir-gene products may act as antigen recognition substances on more than one type of T cell.

The quantitative response of coelomic cells associated with first- and second-set Eisenia xenografts transplanted to Lumbricus hosts at 20 ° C was compared with autografts and nonspecific wounds. Coelomocyte numbers were significantly lower in response to first than second-set xenografts. Coelomocytes also increased in association with autografts and nonspecific wounds, but the reaction is short lived, and essential for early wound healing and repair. Such nonspecific increases are different from subsequent specific immunologic longer-lasting coelomocyte responses. First-set xenografts induced a relatively slow increase in coelomocytes, which declined after 3–4 days postgrafting. By contrast, second-set xenografts caused an accelerated rise in coelomocytes, usually 20 to 30% greater than the maximum coelomocyte response induced by first-set xenografts. The mean survival time for first-set xenografts ( non-self ) was 17 ± 1 days, but repeat second-sets were rejected in an accelerated time of 6 ± 1 days. Autografts ( self ) are never destroyed. After priming with a first-set xenograft, this heightened coelomocyte reaction, to a second-set xenograft, was interpreted as an anamnestic response. The memory response is measurable in two ways: grossly as accelerated rejection of repeat xenografts, and at the cellular level, heightened coelomocyte numbers. Specific cellular immunity is demonstrable phylogenetically at the level of annelid worms.

Mice immunized sequentially with two related influenza virus hemagglutinins (HA) produced a secondary antibody response with two different specificities. Some antibodies were specific for determinants common to both HA's. Paradoxically, some antibodies were directed to determinants existing only in the HA first encountered. Primed spleen cells treated with anti-θ serum and complement were transferred from animals immunized with the first HA to either normal, irradiated, or thymus-deprived recipients. These memory cells were boosted in the recipients with either the homologous or the heterologous cross-reacting HA. B-memory lymphocytes were shown to be directly triggered by both HA's and to be able to secrete, independently of T lymphocytes, antibodies to both kinds of determinants. However, T cells were shown to modulate this secondary response by either enhancing or suppressing antibody secretion by B-memory cells, depending on experimental conditions. These results are discussed in terms of antigen recognition by B cells and of kinetics of development of immunological memory.

We have used the “allogeneic effect” as a model system for investigating the effects of augmented thymus-derived lymphocyte function on secondary antibody responses of primed inbred guinea pigs. A transient graft-versus-host reaction was induced by infusing nonimmune strain 13 lymphoid cells into strain 2 guinea pigs which had been primed with dinitrophenylated (DNP) ovalbumin. Six days later the recipients were challenged with the priming antigen intradermally. Four days after boosting with antigen, sera from these “allogeneic” guinea pigs had significantly higher affinity for e-DNP-L-lysine than sera from “control” guinea pigs which were primed, but had not received allogeneic lymphoid cells. Spleen cells taken from “allogeneic” guinea pigs three to five days after boosting were shown to have both higher numbers of plaque-forming cells and a predominance of high avidity antibody-producing cells in comparison to spleen cells from “control” guinea pigs. Thus, increased donor thymus-derived lymphocyte function brought about by the transient graft-versus-host reaction causes an enlargement of the host antibody-producing cell pool and preferentially expands the subpopulation of cells producing high affinity antibody. The effects of this preferential expansion are reflected in the sera of “allogeneic” guinea pigs as higher affinity antibody in comparison to serum antibody of “control” guinea pigs.

The antibody response to the hapten 2,4dinitrophenyl (DNP)lysine coupled to two thymus-independent carrier molecules (pneumococcal polysaccharide SIII and levan) has been studied in mice. The characteristics of the anti-hapten response are essentially similar to the response to the carrier itself: both conjugates elicit a strongly dose-dependent IgM response (with little or no IgG antibody), and supraoptimal doses paralyze both anti-hapten and anti-carrier antibody responses. The anti-hapten response to DNPlyslevan is thymus-independent; it is also carrier-independent, since levan-paralyzed mice give a normal antihapten response to the conjugate and, conversely, paralysis of the antihapten reactive lymphocyte population fails to affect the response to the carrier. The results do not support the concept of B cell-B cell cooperation that has been proposed to explain the immunogenicity of haptenpolysaccharide conjugates. Instead, they indicate that these conjugates immunize separate and noninteracting B cell populations, the one hapten-specific and the other carrier-reactive. Both the numbers and the size of anti-DNP hemolytic plaques were related to the immunizing dose of DNPlyslevan. Preliminary experiments suggest that the reduction in plaque size in partially tolerant mice may be caused by persisting antigen on the surface of immunized lymphoid cells.

Specific immunological tolerance was induced in CBA mice by a single injection of deaggregated fowl immunoglobulin G (FγG). The unresponsive state was stable on adoptive transfer and irreversible by pretreatment of tolerant cells with trypsin. Tolerant spleen cells could suppress the response of normal syngeneic recipients. They also suppressed the adoptive primary response of spleen cells to FγG in irradiated hosts. The inhibitory effect was on the indirect (7S) plaque-forming cell (PFC) response. Incubation of the tolerant cell population with anti-θ serum and complement reversed the suppressor effect. Furthermore, the addition of purified T cells from normal donors restored the capacity of the anti-θ serum-treated tolerant cells to transfer an adoptive response to FγG. The existence of FγG-reactive B cells was supported by the demonstration of normal numbers of antigen-binding cells in the spleen and thoracic duct lymph from tolerant animals. Moreover, the formation of caps by these cells implied that they could bind antigen normally. These experiments provided direct evidence for the existence of suppressor T cells in the tolerant population. Further evidence was derived from examination of the effect of antigen "suicide". Tolerant spleen cells were treated with radioactive FγG under conditions known to abrogate T-cell helper function. When these cells were transferred together with normal spleen cells into irradiated hosts, suppression of the primary adoptive response to FγG was no longer observed. Inhibition of an adoptive secondary response to FγG was obtained by transferring tolerant spleen cells with primed B cells provided high doses of tolerant cells were used. By contrast low doses exerted a helper rather than a suppressor effect in this system.

Chickens were bursectomized surgically during the first week after hatching and immunized subsequently with human serum albumin (HSA). Bursectomy (Bx) 4 days posthatching suppressed the IgG response, but did not affect the IgM response when tested at 2 weeks. The overall responsiveness had declined by 4 weeks, however, probably due to the nonreplicating character of the early peripherally seeded B cells. Furthermore, these chickens failed to give a memory response. B cells with self-replicating and memory potential are seeded later than 4 days posthatching. However, 7 day Bx still produced a selective suppression of the primary IgG response while the IgM response, although delayed, reached a normal peak titer. Treatment of memory cells in vitro with anti-μ serum interfered with the adoptive transfer of both IgM and IgG responses. The data are interpreted in terms of sequential seeding of IgM and IgG precursors of the anti-HSA clones, both of which maintain membrane-bound μ-chain antigen.

Some parameters of the development of immunological memory to B. abortus (BA) and sheep erythrocytes (SE) in the mouse have been compared. The thymus-independence of the BA response allowed evaluation of B-cell memory in vivo and in adoptive immune responses. A reduced responsiveness to BA was seen during the first few days after the primary injection, whereas enhanced ability to give responses to SE (thymus dependent) occurred at that time. The ability of primed spleen cells to transfer 19S and 7S memory responses to SE developed in parallel. In contrast, the earliest appearance of 19S memory to BA on Days 5–7 after priming was not yet accompanied by memory for the 7S response, but by Day 10 both 19S and 7S memory were present. At 1–2 months after priming, 100-fold fewer cells than needed for transfer of the primary response still transferred excellent 19S and 7S memory responses to BA. Anti-θ treatment of long-term memory 19S and 7S spleen cells did not affect their ability to respond to challenge even with limiting BA doses. It is suggested, however, that the T-independency of the response to BA applies only to the specific induction by antigen of preexisting B cells into antibody secreting cells, whereas optimal B cell memory formation to any antigen may be a separate T-dependent function. Serial spleen cell transfers into lethally irradiated recipients at 1–2 week intervals with antigen challenge at each transfer, appeared to interfere with the development of memory to BA, particularly for the 7S response. No such effect was seen on the responses to SE.

The secondary immune response patterns of young adult male and female hamsters injected with SRBC were studied and compared. Females were shown to produce higher number of PFC per spleen than did males. No major differences were shown to exist between males and females using serum hemagglutinin and hemolysin titers as parameters of comparison. The spleens of all animals were evaluated in terms of organ weight, total splenocyte count per spleen and spleen weight expressed as percent of total body weight. A partial but indirect explanation of female advantage over the male is reflected in a significantly greater splenic mass, i.e., lymphoid tissue, for females than for males of equivalent age and weight.

Antibody synthesis was spontaneously induced in lymphoid cell cultures prepared from the draining lymph nodes of immunized rabbits. Induction of this response was attributed to cell-associated persisting antigen. The identity of the cell with which the antigen was associated was sought. Removal of the macrophage-rich adherent cell population did not diminish spontaneous induction. However, removal of a different cell population by the carbonyl iron method markedly inhibited spontaneous antibody synthesis. In addition, treatment of cultured lymph node cells with ethylenediaminetetraacetate, which is known to remove antigen bound to cell surfaces, also inhibited spontaneous induction. Induction of antibody synthesis could be restored in either of these situations by the addition of a suitable quantity of specific antigen. Although removal of the cell type which adhered to carbonyl iron depressed spontaneous antibody synthesis, the treatment had no effect on concanavalin A-mediated lymphocyte transformation. The combined evidence indicates that the persisting antigen responsible for induction of the spontaneous response is bound to the surface of an accessory cell which is probably a sessile macrophage or a dendritic-type cell.

The suppression of antibody formation by passively administered antibody is influenced by the dose and nature of the antigen, type of immunization procedure, ratio of antibody to antigen, species origin and characteristics of the antiserum used, as well as the species selected for immunization. In guinea pigs, diphtheria antitoxin formation can be effectively suppressed by an intravenous injection of excess homologous or heterologous antitoxin as long as 5 days after toxoid immunization and after delayed-type hypersensitivity to toxoid has developed. Following the period of antibody suppression which lasts 2 to 7 weeks, serum antibody can usually be demonstrated. It is proposed that this delayed immunization results from dissociation of antigen, since diphtheritic paralysis and death can be produced in guinea pigs and rabbits by the intravenous injection of toxin-antitoxin precipitates formed in antitoxin excess. This syndrome is prevented by injection of excess horse antitoxin 1 hour after injection of the toxin-antitoxin complexes.

Using guinea pigs of strains 2 and 13 and their F1 hybrids as experimental subjects, various lines of evidence have been obtained that in this species, as in all others tested, the only significant cellular antigens with which donor lymphocytes engage when normal and immune lymphocyte reactions are incited are radiosensitive leukocytes. Constitutive cells of the skin are unimportant. (a) The intensities of these reactions in irradiated subjects are dependent upon the peripheral leukocyte concentration. When this falls below a certain threshold no reactions are incitable. (b) Highly leukopenic animals are capable of developing immune lymphocyte transfer (ILT) reactions if normal lymphoid cells of their own genetic constitution are mixed with the putative attacking donor cells, as "supplementing antigen," before inoculation. (c) Radiation-chimeric strain 13 animals having F1 hybrid leukocytes in their bloodstream give typical ILT reactions when challenged intradermally with strain 13 anti-2 node cells. Exposure of strain 2 animals to 600 R does not prevent their becoming actively immunized if, 24 hr later, they are injected intradermally with strain 13 lymphocytes. However, this sensitization, revealed by the host's capacity to give delayed hypersensitivity reactions, wanes as leukopenia progresses. On the basis of this and other findings it is argued that the flare-up stage of the NLT reaction in preirradiated hosts is mainly an expression of host sensitivity against the transferred alien cells. Two unexpected observations have been made in the course of this study: (a) F1 hybrid animals developed what appeared to be a strong delayed hypersensitivity after intradermal inoculation with parental strain lymphoid cells or antigenic extracts prepared from them. (b) If strain 13 guinea pigs which had been sensitized against strain 2 tissue antigens by intradermal injection of lymphocytes 7 days beforehand were inoculated intravenously with strain 2 antigenic extract a significant proportion of the animals developed severe delayed necrotizing reactions, recall flares, at some or all of the healed skin inoculation sites.

Addition of polyadenylic-polyuridylic acid in complex form (poly A:U) without antigen to a suspension of spleen cells obtained from BALB/Aj mice primed 6 wk previously with human γ-globulin (HGG) resulted in an immediate fourfold increase over background number of anti-HGG rosette-forming cells (RFC). Culture of similar cells in the presence of puromycin for 1–6 hr before poly A:U did not significantly reduce the response. Continued culture of primed spleen cells in the presence of poly A:U, resulted in a decrease of RFC to background levels within an hour followed by an increase again 6 hr later. This later increase in RFC was inhibited by addition of puromycin to the culture medium. The nonspecific stimulation by poly A:U of antibody production by primed spleen cells also was induced in vivo. Increases in splenic RFC were detectable 6 hr after intravenous injection of poly A:U alone, without antigen, into primed mice. The response peaked at 18 hr and had dissipated completely within 3 days. A second injection of poly A:U 24 hr or later after the first injection resulted in a second response, similar to the first with respect to kinetics and intensity. Rosette formation by poly A:U-stimulated cells could not be inhibited by mitotic poisons, but was inhibited by treatment of the cells with goat anti-mouse γ-globulin serum, suggesting that the antibody involved was a 7S γ-globulin. The decrease in RFC induced by culture of primed cells for 1 hr in poly A:U paralleled a decrease in secondary responsiveness of the cells to antigen. This poly A:U-induced inhibition of secondary responsiveness could be reversed by suspending the treated cells in supernatant fluids derived from poly A:U-stimulated cultures. The reversal was specific in that supernatant fluids removed from bovine serum albumin (BSA)-primed cells by poly A:U did not stimulate the response of HGG-primed cells to HGG. However supernatant fluids from BSA-primed cells caused the production of anti-HGG RFC if BSA rather than HGG was used as triggering antigen. The active factor in the supernatant fluids appeared to be a 7S γ-globulin since activity was lost after 45 min incubation of the supernatant fluids in the presence of goat anti-mouse 7S γ-globulin serum.

Unstable chromosome aberrations (acentrics, dicentrics, rings) have been observed in tuberculin-stimulated leukocyte cultures from three patients up to six months after therapeutic irradiation. This is considered evidence that human blood contains immunologically committed, long-lived, nondividing small lymphocytes which retain the latent capacity to proliferate when re-exposed to the sensitizing antigen. "Immunologic memory" may reside in such cells, which, because they are not continually dividing, need not have undergone genetic alteration in becoming immunologically committed.

SUMMARY Early cutaneous allograft rejection was found to be characteristic of wild specimens of Chromis caeruleus and Dascyllus aruanus, two common species of advanced bony fishes of the order Perciformes from the tropical Indo-Pacific. Development and persistence of substantial immunological memory was indicated by the invariably accelerated rejection of second-set allografts made some 3 weeks after first-set grafting. Bv contrast, control autografts healed promptly and remained fully viable under the normal environmental conditions maintained, including a temperature of 25-27 C. Strong histocompatibility barriers are now known to be characteristic of diverse orders of advanced Osteiehthyes in contrast with the weaker, altough no less discriminating alloimmune reactions of more primitive fishes. This highly developed capacity for transplantation immunity in teleost fishes holds regardless of the geographical origin or natural habitat of the many species tested thus far. The evolutionary success of the class Osteichthyes and especially the Teleostei as the predominant group of aquatic vertebrates may be at least partly attributable to prompt and vigorous immunoresponsiveness

The effect of preincubation with anti-θ or anti-mouse immunoglobulin (Ig) and complement (C') on immune responsiveness of spleen cells from BALB/c mice immunized with sheep erythrocytes (SE) was investigated. Both treatments greatly depressed the remaining ability to produce a secondary response to SE in vitro. Normal BALB/c spleen cells were far less effective in reconstituting the responses of such depleted cell populations than were much smaller numbers of untreated immune spleen cells. Thymus-derived cell (T cell) memory appeared early after immunization and showed specificity for the immunizing antigens. Recombination of anti-Ig-treated with anti-θ-treated immune spleen cells resulted in virtually complete reconstitution of responsiveness. The presence of immunological memory in T cells and the nature of their surface receptors are discussed.

There have been many attempts to determine the effect of adrenal cortical hormones on the resistance of the host to various noxious and infectious agents.1 These studies generally have shown a beneficial effect of these hormones on the host's reactivity to harmful agents,1-2 but have not provided an understanding of the mechanisms involved. In 1945, Dougherty and White reported the results of studies on the physiological functions of these hormones.3-5 They noted that the injection of adrenal cortical steroids or pituitary ACTH into mice, rats, and rabbits was followed in several hours by a reduction in the number of circulating lymphocytes, presumably as a result of the dissolution of these cells in the lymphoid organs. A similar effect could be produced upon subjection of these animals to various stressful stimuli which caused increased adrenal cortical secretion. During the destruction of the lymphocytes portions of their cytoplasm were shed into the blood, producing an in

Allotypic suppression of adult mouse spleen cells has been investigated by treating the cells with anti-allotype serum directed against the Ig-1 specificities of the γG2a immunoglobulin class. Suppression of precursor, memory and primed cells in the anti-sheep red blood cell response, assayed in terms of the number of plaque-forming cells producing γM, γG1 and γG2b immunoglobulins has been investigated. The results are discussed in terms of the stage of differentiation of cells when class restriction and allotypic exclusion occurs. It is concluded that γM or γG commitment occurs at an early precursor stage, and that γG class and allotype restriction occurs sometime after contact of precursor cells with antigen.

The carrier specificity of secondary anti-hapten responses using different haptenic densities on autologous or heterologous carrier proteins was studied. In addition, the T cell requirements for anti-hapten antibody synthesis were investigated. The results demonstrated a clearcut decrease in carrier specificity as hapten density goes up. Also, memory against autologous hapten carrier conjugates at the T cell level waned with time in contrast to the memory against heterologous hapten-carrier conjugates. Specific helper cells against autologous conjugates were demonstrated by filtration through anti-immunoglobulin coated columns.

The DNA-synthesizing capacity of single antibody-forming cells was tested by a combination of micromanipulatory and autoradiographic techniques. Rats were immunized with S. adelaide flagellin, a protein antigen known to contain significant contamination with somatic (O) antigen. Single cells from secondarily immunized rats were tested for production of anti-H and anti-O antibodies by previously described and newer techniques. Positive antibody producers were transferred onto clean dry slides by micromanipulation, and autoradiographs were performed. When rats had received tritiated thymidine 1 hour before killing, labeling of antibody-forming cells was taken to imply that the cell was preparing for further mitotic division. It was found that on the 2nd and 3rd day of a secondary response, many of the antibody-producing cells in the nodes (chiefly plasmablasts) were incorporating tritiated thymidine. At the height of the cellular response, however, at 4 and 5 days, the majority of active antibody producers (chiefly mature plasma cells) were incapable of DNA synthesis. There appeared to be an inverse relationship between the antibody-forming and DNA-synthesizing capacities of the cell population under study; as more of the cells studied formed detectable antibody, fewer of them incorporated the DNA precursor. The age of plasma cells was also studied. Animals were killed at the height of the cellular immune response, having previously received an injection of tritiated thymidine 1 to 48 hours before killing; i.e., at 63 to 110 hours after their secondary stimulus. As the interval between isotope injection and killing increased, the proportion of antibody-forming cells showing labeling increased. With an interval of 30 hours, about half the antibody-forming cells were labeled and of 48 hours, over 95 per cent were labeled. This was taken as evidence that, few, if any, antibody-forming cells found at the height of a secondary response were more than 48 hours old. On the basis of these experiments and those reported in the accompanying paper, a simplified scheme showing the development of an antibody-forming clone in the secondary response was proposed.