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The conditions for cooperative interactions between nonresponder B10.S B cells and GAT-primed irradiated (C57BL/6 x SJL)F(1) T cells in the response by cultures of mouse spleen cells to GAT were investigated. GAT-specific antibody responses could be elicited by soluble GAT in cultures of GAT-primed irradiated (C57BL/6 x SJL)F(1) T cells with C57BL/6 B cells but not with B10.S B cells. In contrast, when GAT was presented to the cultures on F(1) macrophages or as aggregates of GAT with MBSA, GAT-specific PFC responses were observed with both B10.S or C57BL/6 B cells. Irradiated GAT-primed T cells were nevertheless essential for the development of these responses. The GAT-specific response of B10.S B cells in these cultures was inhibited by the addition of soluble GAT at culture initiation. These results indicate that genetic disparity at Ir loci is not an absolute barrier to T-B-cell cooperative interactions in the response to antigens under Ir gene control. The significance of these data for the function of Ir gene products in immunocompetent cells is discussed.

A commercially available bacterial colony counter has been modified to permit rapid, highly accurate, semi-automated enumeration of antibody-producing plaque forming cells in semi-solid support medium as well as enumeration of nucleated cells in suspension on a standard hemacytometer chamber. This apparatus should therefore serve as an enormous time-conserving accessory to most modern laboratories involved in immunological research.

Plasmodium berghei yoelii infection in mice severely depressed the splenic antibody response to sheep erythrocytes but had lettle effect on antibody formation in lymph nodes.

It was shown on 5 series of rats by the method of local passive hemolysis in gel that the number of antibody-forming cells (AFC) in the spleen after the immunization with O-antigen from Sal. typhi correlated with the number of background AFC to the given antigen in the animals of the same series. This, possibly, pointed to the fact that the number of spontaneous AFC reflected the number of B-lymphocyte immunocompetent to the given antigen and could be used - to some extent - for the assessment of the immunological reactivity to this antigen before the immunization.

Antibodies to the capsular antigen of plague causative agent were found in the serum of many nonimmunized mice of 6 strains, but none were found in mice of the DBA/2 strain. After the administration of the capsular antigen to mice of different strains cells forming the antibodies to the antigen administered were revealed in a quantitative correlation with the natural antibody level. An association was revealed between the level of natural antibodies and the number of antibody-forming cells in the spleen of nonimmunized mice; it could be described by an equation almost all of whose members represented constant values with a definite quantitative characteristics. To check the suggested equation the concentration of immunoglobulins in the blood of mice was compared with the number of plasma cells in the spleen; a satisfactory correlation was revealed between them.

Normal adult outbred rabbits were immunized intravenously (iv) with sheep erythrocytes (SRBC). At varying times thereafter, the different lymphoid organs were investigated for spontaneous and culture-induced antibody secreting cells by the aqueous hemolytic plaque-forming cell (PFC) technique. During the phase of active antibody formation (Days 3 to 30), immediate PFC, indicative of spontaneous antibody synthesis and secretion, were detected principally in the spleen. In the early postimmune memory period (Days 30 to 90), memory cells capable of generating PFC following secondary immunization in in vitro culture with SRBC were detected only in the spleen. However, by 4 months postimmunization, memory cells were detected in the thymus and popliteal lymph node (PLN) as well as in the spleen. The number of memory cells in the thymus and PLN was significantly higher by 6 months postprimary iv immunization and was even further elevated by 9 months postprimary iv immunization. Following in vivo secondary immunization by the iv injection of SRBC 2 or 6 months postprimary immunization, immediate PFC were detected in large numbers in the spleen, the bone marrow, and the blood, marginally in the PLN and not at all in the thymus. Similar results were obtained at 9 months following primary immunization with SRBC with the exception that large numbers of immediate PFC were detected in the PLN following secondary iv immunization. Following culture of these lymphoid cells for 5 days in vitro with SRBC, the thymus and PLN cells, as well as the spleen cells, generated large numbers of PFC. Since immediate PFC were never detected among the freshly isolated thymus cells whereas thymic cell cultures 6 and 9 months postprimary iv immunization invariably generated large numbers of PFC following secondary immunization in vitro, the thymus memory cells would appear to be inaccessible to particulate antigen injected intravenously; they can only be detected following activation by the antigen in culture. The PFC generated by thymus memory cells (and spleen and PLN) were totally inhibited by the inclusion of sheep anti-rabbit IgG into the PFC assay. This finding demonstrates unequivocally that the plaques induced by thymus cells, just as the plaques induced by spleen and PLN cells, are antibody mediated and not false plaques. Therefore, the thymic PFC cells must be antibody-secreting B-memory cells since T cells do not synthesize or secrete immunoglobulins.

Immunization of dispersed spleen cells from normal mice in vitro with SRBC was suppressed by simultaneous incubation of the spleen cell cultures with splenocytes from mice previously infected with FLV. Cell-free virus preparations alone did not suppress the antibody response. In contrast, relatively small numbers of splenocytes from infected mice, even when present at a ratio of 1-500 normal spleen cells, significantly suppressed the in vitro immune response to SRBC. Viable leukemic splenocytes were necessary for immunosuppression although the leukemic cells did not have to be in direct contact with the normal spleen cells. Specific anti-FLV serum, when added to the leukemic splenocytes or to normal spleen cells separated from infected cells by cell-impermeable membranes, prevented immunodepression.

Passive transfer of thymocytes and spleen cells from donors primed with keyhole limpet hemocyanin (KLH) caused significant decrease in the average avidity of anti-DNP antibodies produced by direct and indirect PFC in the recipients in both primary and adoptive secondary antibody responses against DNP-KLH. The analysis of the avidity distribution of antibodies produced by plaque-forming cells (PFC) indicated that the observed decrease in the average avidity is primarily due to the selective loss of high avidity subpopulation of PFC leaving low avidity subpopulation relatively unaffected. The degree of suppression in antibody avidity did not correlate with the reduction in the number of PFC, and thus causing the "shift" of avidity distribution of PFC to the low avidity end. These results indicate that the "maturation" of antibody in the T-cell-dependent antibody response is influenced by the carrier-specific suppressor T cells with respect to the emergence and selection of B cells having high affinity receptors for hapten. It is suggested that B cells binding antigen with high affinity receptors would be more easily affected than those with low affinity receptors by specific suppressor T cells which are capable of reacting the carrier portion of the same antigen.

It has been proved by diverse experimental methods that cell suspension prepared from the immunocompetent organs and tissues by Jerne's method modified by N. N. Klemparskaya presents a hypertonic medium stimulating formation of false plaques. This evidence suggests that immune-like plaques (aureola) may be formed in the absence of antigen rather than due to lysis of red cells or destruction of plaque-forming cells.

Conditions of long-lasting chimerism induction in adult CBA mice treated with cyclophosphamide and spleen cells (CBAXC57BL/6)F1 were investigated. The necessary requirements are high cell doses (over 50X10(6)), cyclophosphamide dose equal or over 200 mg/kg and a short interval (3-6hr) between their injections. The data obtained were interpreted from the point of view of the capacity of cyclophosphamide to induce reversible lymphocyte DNA damages. After the interaction of lymphocytes with the antigen these damages are fixed with the resultant elimination of the corresponding cell clone.

The synthesis of DNA, as measured by the incorporation of [3H]-thymidine into the nuclei of cells of cultured explants of rabbit spleen, has been studied and correlated with estimation of DNA content of these nuclei using Feulgen densitometry, with and without colcemid arrest. Observations of the proportion of nuclei labelled with [3H]thymidine and their DNA content indicated that many cells were incorporating this precursor for DNA synthesis without the concomitant increases in DNA expected of cells in division cycle. Colcemid arrest failed to produce the changes in DNA content expected of a cell population as rapidly dividing as the labelling kinetics suggested. The results indicated that [3H]thymidine-labelled material, possibly DNA, was being transferred between cells and this accounted for the increased labelling index seen after pulsing with [3H]thymidine. The suppression of [3H]thymidine uptake by hydroxyurea indicated that this was not repair synthesis of damaged DNA.

Neonatal splenectomy and congenital absence of the spleen were shown to be associated with lack of primary IgM antibody synthesis, deficient bone marrow-thymus cell synergism. altered mitogen responsiveness and depressed homing patterns of thymocytes to the spleen. Using congenitally asplenic animals, altered B-T cell cooperation was manifest at the T cell but not B cell level. This correlated with the ability of thymus cells from either congenitally asplenic or neonatally splenectomized (NSx) animals to respond to the B cell mitogen lipopolysaccharide although there were no detectable Ig=ells present. To determine the role played by the spleen in the development of T cell function, NSx animals were given either irradiated or normal spleen grafts. Both irradiated and unirradiated grafts restored normal B-T cooperation and normal mitogenic responses of thymus cells. However, neither type of spleen graft was successful in restoring normal homing patterns to the spleen. It is concluded that the splenic microenvironment influences T cell maturation very early in life and that only some of the effects attributable to the absence of the spleen can be reversed by the reintroduction of this tissue as a graft.

Cell-mediated immune responses were evaluated in genetically obese (C57Bl/6J ob/ob) mice and lean controls. The thymus and spleen weighed less and contained fewer mononuclear cells in obese animals compared with controls. The total number of direct and indirect plaque-forming cells in the spleen of obese mice immunized intraperitoneally with sheep red blood cells was reduced, however, when the results were expressed as plaques/10(6) mononuclear cells, the proportion of antibody-producing cells was higher in the obese. Ob/ob mice immunized in vivo with allogeneic EL-4 lymphoma cells showed markedly diminished capacity to generate cytotoxic cells, whereas such response after in vitro immunization was comparable in the two groups of animals. These observations suggest that altered cell-mediated immune response in obese animals is the result of a deleterious microenvironment.

Studies under controlled conditions of lighting and temperature revealed clear evidence of circadian periodicity with respect to the number of PFC present in the spleens of BALB/c mice 3 or 4 days after immunization with SRBC. Striking differences in proliferative responses of spleen lymphocytes to PHA or Con A were also observed at two different circadian times. Large proliferative responses occurred at the time when injection of antigen and/or sampling for PFC yielded a low PFC formation (early in the daily dark span) and small proliferative responses occurred at the time when antigen injection and sampling yielded high formation of PFC (early in the daily light span). These findings indicate that care should be taken to control the circadian timing of stimulation and sampling in studies of immune responses, and that rhythmically varying aspects constitute a new dimension of immunologic processes awaiting further analysis in both the circadian and weekly spectral regions.

C57BL and CBA mice, with low or high reaction to immunization with sheep erythrocytes, respectively, were given transplantation of the syngenous lymphoid or bone marrow cells. Transplantation of the syngenous cells to C57BL mice intensified the production of antibody-forming cells to sheep erythrocytes and depressed it in transplantation to CBA mice. A different effect of transplantation of the lymphoid and bone marrow cells on the immune response, depending on the recipient's genotype, is discussed.

The influence of purified acetic acid thymus extract (thymarin) and its three fractions isolated by ion-exchange chromatography upon immune response to SRBC in mice was studied. Each fraction was identified as homogeneous individual material of polypeptide nature. Crude thymus extract and one of its fractions with an approximate molecular weight of 5000 exerted a strong stimulating effect on the thymus-dependent response: formation of IgM- and IgG-antibody producing cells and on the circulating antibody level.

The influence of the donor priming upon the recipient's immune response under conditons of syngenic transfer of nucleated cells of the bone marrow, thymus, spleen to the lethally irradiated recipients was studied, the recipients being immunized with sheep erythrocytes. A fall of total number of antibody-forming cells was observed in the recipients in priming. The number of hemolytic foci was increased the first day after priming in case of injection of the bone marrow cells from the intact donors and of the thymus cells from the immunized donors. On the third day of the experiment the number of these foci was three time less in comparison with the control. An increase of the hemolytic foci was discovered in the syngenic transfer of splenic cells affected by priming.

It has been suggested that a regional immune response may be induced by local administration of small doses of antigen, whereas larger doses are capable of inducing not only a local but also a systemic immune response. Except with fluorescence, the study at the cellular level and in the mucosa of specific antibody-producing cells appears difficult. The circulation in the body of lymphocytes from the lamina propria of the gut allows the detection of antibody-producing cells in extra-intestinal lymphoid tissues. The administration of 4 times 10-9 sheep red blood cells (SRBC) by gastric intubation to BALB/c mice (repeated daily for 4 days) was found to cause the appearance in the spleen of plaque-forming cells which predominantly belonged to the IgA class and whose numbers reached a maximum at about day 8 or 9 after the onset of the immunization. This observation is relevant to splenic colonies from cells that had received their antigenic stimulation in the gut wall and thence had seeded out in the body. Booster administration of SRBC performed 3 months after the first antigenic contact induced in the spleen an immunological response similar to that observed in newly primed mice and in no way suggestive of immunologic memory. A secondary response is completely absent when the second intragastric immunization is initiated 10 days after the end of the first. The parenteral administration of 2 times 10-8 SRBC to mice primed by the oral route 15 days earlier is followed by a marked hyporeactivity of the spleen. In any case, the IgA system appears endowed with only a minimum, if any, immunological memory to non-living antigen. In some conditions local immunological stimulation may even cause tolerance.

Data are presented pointing to the principal possibility of using micromodification of the local hemolysis test (suggested by Klemparskaya) for the purpose of characteristics of pollen allergens. In administration to male mice, weighing 18 to 20 g of 0.25 ml of the allergen prepared from a 3% suspension of ambrosia pollen in water-salt Evans-Cock fluid there was a sharp rise in the spleen of cells synthesizing antibodies against own erythrocytes of these animals on the 4th-6th day after the immunization. The water-salt solution included into the composition of the allergen promoted to some extent stimulation of these processes. The allergen possessed a high allergenic activity in the skin test on man and contained 0.3 ml of protein nitrogen per 1 ml. A possibility of autoimmune influence of these preparations in hyposensitizing therapy of man is supposed.

It was previously reported that PHA-incubation of a pool of peripheral-blood lymphocytes before their i.v. injection into rats submitted to total body irradiation, stimulates spleen colony formation and growth. On the same model, the present study has investigated the effect of lymphocyte preincubation with supernates obtained in short term cultures from human lymphocytes prestimulated with PHA (lymphokines). The "in vitro" culture of rat lymphocytes in a medium supplemented with human lymphokines showed either a marked increase of lymphocyte survival rate, or significant stimulation of lymphocyte blastogenesis, but at an extent much lower than that caused by PHA, also because in this particular experiment the culture medium was supplemented with too little amount of lymphokines. These data support the interpretation that human lymphokines may act across the species barriers. Peripheral-blood rat lymphocytes prestimulated with human lymphokines, and then injected i.v. into rats previously submitted to total body irradiation, caused significant increase in the spleen weight, as well as formation of spleen colonies quite larger and with higher erythroid differentiation - though less numerous than in the controls (i.e., in the rats injected just with untreated lymphocytes). This behaviour may be due to the tendency to merge together of two or more colonies in a single giant colony, and supports the interpretation that immune-competent lymphocytes activated either with PHA or with lymphokines may stimulate spleen colony growth and erythroid differentiation, though at different extents.

A relationship was revealed between the number of cells in the suspension of the splenic tissue taken for induction of primary immunization of the reaction outside the organism and the number of the plaque-forming cells (PFC) formed by the end of incubation. It was shown that with increase of the cell density in the culture in the bottom of the incubation flask there was a 10-100 fold decrease of the PFC response; as to the cell viability--it was not affected or decreased but slightly. This effect was observed in using sheep red blood cells and water-soluble antigen extracted from them as an antigen. The effect was independent of shortage of the antigen or of the nutrient substances; it was accompanied by a general reduction of the 3H-thymidine incorporation into cultured cells.

Isologous serum of CBA mice immunized with rabbit immunoglobulins (ARIS) contained a factor capable of inactivating rosette-forming splenocytes (RFC) in vitro from the same strain of mice immunized with SRBC. When mouse SRBC immunization was carried out against the background of ARIS injection the court of RFC to SRBC at the peak of immune response was 30% of that of mice injected with SRBC and normal isologous serum. A decrease of RFC count was the result of disappearance of the theta-negative RFC. Passive ARIS immunization failed to influence the antigen-induced proliferation of the antibody-forming cells and the synthesis of antibodies against SRBC.

Male and female spleen cells were compared before and after culture for their cytotoxicity to autologous-embryo fibroblasts. Cultures of male cells developed significantly greater reactivity than cultures of female cells. Moreover, while the cytotoxicity derived from cultures of male cells was totally abolished by treatment of effector cells with a mouse anti-T cell antiserum, such an antiserum had less affect on the effector cells of female mouse cultures.

The mice of different lines were immunized twice with low doses (10(6) of sheep red blood cells (SRBC) at a 7-day interval. The immune spleen cells (ISC) from syngeneic and allogeneic mice preliminarily (14 days before) given SRBC (5 . 10(8) were transplanted into the animals at varying times before or after primary antigen administration. It was discovered that the administration of the ISC from CBA and DBA/2 mice to syngeneic mice 1 or 2 days after the priming drastically lowers the production of antibody-forming cells in the recipients in response to the repeated antigen injection. The ISC from C57BL/6 mice provided low suppressor effect. ISC transplantation one, three or six days after the priming exerted no effect on the height of the secondary immune response. Suppression of antibody formation was also obtained in the allogeneic combinations CBA ISC leads to leads to C57BL/6 and DBA/2 ISC leads to CBA. It is concluded that the spleen cells from mice immunized with SRBC are capable to suppress the development of the immunological memory of SRBC.

The appearance of antibody- and nonspecific immunoglobulin-forming cells (AFC and nIFC) in spleen cell suspensions from normal mice and from those immunized with sheep red blood cells (SRBC) was studied. The cell suspensions were cultivated in the modified Mishell-Dutton system. The induction of the immune response in vitro resulted in a sharp increase of nIFC formation. Their amount reached 51 218 per 10(6) spleen cells, i. e exceeded the number fo AFC 25-172-fold. Much more was the increase in the absolute number of nIFC during cultivation in the presence of antigen of spleen cells from animals immunized with SRBC 3--4 days before experimentation. In this case, the maximum number of nIFC reached 165 000 per 10(6) spleen cells, i. e. 33% of the total number of B-cells. The kinetics of AFC and nIFC formation and regulatory mechanisms of the populations varied under different conditions. The nature and potential mechanisms of nIFC formation under the influence of the antigen are discussed.

The effect of typhoid bacterial polysaccharide on the primary and secondary immune response to SRBS was studied. The polysaccharide was shown to have both stimulating and depressive effect on the population of antibody-producing cells. This effect depended on the time and the number of polysaccharide injections. Thus, a single polysaccharide injection made on the day preceding immunization resulted in the maximum stimulation in the system of IgM- and IgG-producing cells, while the maximum depression of these cells could be observed after 2 polysaccharide injections: on the day preceding immunization and on the day of immunization. In the secondary immune response considerable stimulation of the populations of antibody-producing cells was observed after polysaccharide injections made on days 2 and 3 after reimmunization.

The transfer of lymphocytes together with sheep erythrocytes from partially hepatectomized mice to syngenous lethally irradiated mice (CAB and C57BL) increased the number of antibody forming cells in the recipient's spleen. The lymphocytes of CBA mice acquired this ability much earlier after the operation (in 4 hours) than those of the C57BL mice (in 17 hours). After the transfer of lymphocytes in the semisyngenous system there was a decrease of antibody forming cells during subsequent recipient's immunization with sheep erythrocytes; this was the result of the graft versus host reaction. The latter reaction was less marked in the operated than in control mice. These changes also occurred earlier after the operation in the CBA than in C57BL mice.