Role of cyclic AMP on differentiation of T- and B-lymphocytes during the immune induction.

Spleen cell cultures from genetically thymus-deficient nude mice were restored with a T-cell replacing factor obtained from normal spleen cells of Balb/c-Ig^b mice stimulated with concanavalin A. Treatment of these cultures with an inhibitory dose of cyclic AMP did not result in reduction of the number of specific antibody-forming cells after stimulation by antigen, whereas the same treatment led to inhibition in cultures restored with normal hydrocortisone-resistant thymus lymphocytes. Further experiments lead to the conclusion that the early effect of cAMP on the immune induction seen in vitro reflects inhibition of the production or secretion of a T-cell factor which is a prerequisite for triggering B-cells with a thymus-dependent antigen.     

B-cell suppression of antibody response to sheep red blood cells in mice of high- and low-responding genotypes.

CBA and C57B1 mice (high and low responders to sheep red blood cells, respectively) were injected intravenously with syngeneic lymph node, marrow, spleen, or thymus cells together with sheep red blood cells (SRBC), and the production of antibody-forming cells (AFC) was assayed in the spleen. Transfer of lymph node, marrow, spleen, or thymus cells led to a significant enhancement of immune responsiveness in low-responding C57B1 mice. In contrast, transfer of marrow, lymph node, or spleen cells to high-responding CBA mice was accompanied by a decline in AFC production. These effects were magnified if syngeneic cell donors had been primed with SRBC; suppression in CBA mice and stimulation in C57B1 mice were especially pronounced after transfer of SRBC-primed lymphoid cells. Pretreatment of CBA donors with cyclophosphamide in a dose causing selective B-cell depletion completely abrogated the suppression of immune responsiveness. A large dose (10⁷) of syngeneic B cells injected together with SRBC suppressed the accumulation of AFC in both CBA and C57B1 mice. No suppression of immune responsiveness was observed after transfer of intact thymus cells, hydrocortisone-resistant thymocytes, or activated T cells. We conclude that suppression of the immune response to SRBC is induced by B cells. At the same time, there is a possibility that the addition of “excess” B cells acts as a signal, triggering suppressor T cells.     

Transfer Agent of Immunity

Using erythrocytes as antigen particles, number of antibody-forming cells was enumerated by immunocytoadehesion technique, in which formation of rosette was shown to be inhibited by anti-mouse immunoglobulin sera. This number increased in vitro after treatment of spleen cells of mice for 60 min with RNA fraction extracted from spleen of mice immunized with erythrocytes used in the enumeration, and incubation of cells for 12 hr at 37 C. Response of cells treated with immune RNA fraction was immunologically specific and was inhibited by puromycin or cycloheximide. The activity of immune RNA capable of converting nonimmune cells to antibody-forming cells was shown to be sensitive to ribonucleases but resistant to deoxyribonuclease and proteolytic enzyme.     

Immune cytolysis of human tumor cells mediated by xenogeneic "immune" RNA

Normal, nonimmune, human peripheral blood lymphocytes, when incubated with RNA extracted from lymphoid organs of guinea pigs or sheep immunized with human tumor cells, mediated the immune cytolysis of those tumor cells in vitro. Lymphocytes incubated without RNA, or with control RNA preparations, failed to evidence cytotoxic activity. Treatment of the active RNA preparations with ribonuclease abrogated the cytotoxic activity, but treatment with deoxyribonuclease or pronase did not effect activity.     

Effects of marrow donor and recipient age on immune responses

This report describes treatments to restore diminished splenic immune responses of old mice. Lethal irradiation, followed by young bone marrow and infant thymus transplants, restored the T cell mitogen response and the antibody-forming cell response against sheep red blood cells in the old mice. Although old bone marrow cells restore these immune responses in young recipients, as well as do young bone marrow cells, old bone marrow in old recipients did not improve their levels of response. Longevities of old recipients with rejuvenated responses were not increased, and aging of tail tendon collagen was not affected. The effect of lethal irradiation before the marrow transplant was shown to be minimal, by the use of unirradiated old W-anemic recipients. Parabiosing young mice with old partners caused impairment of these two immune responses in the young partners without enhancing them in the old partners. The old partners did not have increased longevities. To explain these results, we suggest the following hypothesis: old bone marrow contains precursors that produce suppressive factors or cells when in an old environment but not when in a young environment. However, these factors, if allowed to develop in an old environment, can function in a young parabiosed partner.     

A model for exploring afferent signals from the immune system to the nervous system

Mechanisms of information transmission from the immune system to the nervous system have been studied. The results of the studies support the assumption that these signals can be transmitted by oligopeptides (the products of limited proteolysis) which are the fragments found in the active sites of many regulatory peptides of the nervous and immune systems. The testing of a synthesized tripeptide (Ser-Lys-Asp) has shown that it inhibits the antibody-forming cells in intact mice only in response to the administration of large antigen doses and exerts a protective effect against viral infection. When added to the culture of the incubated leukocytes from the peripheral blood of the oncological patients, the tripeptide lowers an increased or normal functional activity of natural killers. In rabbits, tripeptide administration brings about a complex long-lasting reorganization of bioelectrical activity in subcortical structures of the brain.     

Selective concurrent suppression of the process of rosette-forming cell accumulation during the immune response]

Repeated injections to mice of normal rabbit immunoglobulins preceding immunization with sheep erythrocytes inhibited the accumulation of rosette-forming cells (RFC) in the spleen, without influencing the proliferation of the antibody-forming cells and hemaggutinin production. Reduction of the RFC under these conditions occurred on account of B-cells whose antigen-binding receptors could be blocked by antibodies against the aggregated mouse immunoglobulins and a complex of polyadenylic-polyuridylic acids. Repeated injections of the competitive antigen enhanced the formation of the immunological memory to the second antigen. The problem of the origin of the immune rosette-forming B-cells and their influence on the formation on the immunological memory is discussed.     

Thymus, peripheral lymphoid tissues and immunological responsiveness of the pituitary dward mouse (author's transl)]

Snell's pituitary dwarf mice (dw) were used for studies on the relationship between hypophysis and lymphoid organs. The age-dependent changes of thymus or spleen weights of dwarf mice were compared with those of normal littermates. The suppression of growth of the thymus or spleen in dwarf mice was recognized at 5th day of age. Although involution of the thymus varied among animals, a strong positive correlation was demonstrated between relative thymus weight and body weight in 30 approximately 40 days old dwarf mice. Lymphoid organs of dwarf mice were reconstituted by injection of growth hormone and or thyroxin. Relative thymus weight significantly increased in dwarf mice when the treatment with growth hormone started at 7 days of age, but the same treatment at 3 months of age did not show any effect on the increment of relative thymus weight. On the other hand, the antibody-forming capacitiy against sheep erythrocytes of dwarf mice was significantly increased even when the treatment with growth hormone was started at 3 months of age. A marked increase in the number of lymphoid cells in dwarf mice was observed by treatment with thyroxin, even if treatment was started either at 7 days or 3 months of age. Similar changes were also obtained in the antibody-forming capacity.     

Regualtion of the immune response to tumor antigens. I. Immunosuppressor cells in tumor-bearing hosts.

A/Jax mice were rendered immune to the syngeneic and transplantable methylcholanthrene-induced Sarcoma 1509a by the surgical removal of the tumor 7 days after implantation; subsequent injection i.v. transfer of 10(7) to 10(8) washed thymus or spleen cells of tumor-bearing animals (TBA) to immune animals significantly inhibited the rejection of the tumor; this suppressive effect was entirely abolished by the treatment of these lymphocytes with anti-theta serum or anti-thymocyte serum (ATS) and complement before adoptive transfer. On the other hand, an equal number of thymus or spleen cells of normal animals or of animals bearing an unrelated tumor had no suppressive effect. Treatment of normal syngeneic animals with ATS after tumor cell inoculation or splenectomy of TBA resulted in the suppression of the tumor growth. The serum of TBA had no effect on tumor growth in immune syngeneic mice. Together these results suggest that TBA possess immunosuppressor T cells regulating negatively their immune response to the tumor.     

Mechanisms of action of synthetic polyelectrolytes and polyampholytes on the immune response]

We investigated the effect of polyacrilic acid (PAA) on the immune response in mice of various strains on sheep red blood cells and also the influence of poly-2-methyl-5-vinyl-pyridine (PMVY), PAA and their statistical copolymers on antibody-forming cells (AFC) production in cultures of T- and B-lymphocytes in vivo. PAA was seen to increase accumulation of AFC in the spleen of mice depending on their genotypes. PMVP and PAA were found to intensify the cooperating interaction of T- and B-lymphocytes, whereas their copolymers exert quite an opposite effect. The injection of copolymers to the recipients of cooperating T- and B-lymphocytes practically results in the complete elimination of the cooperation effect between T- and B-lymphocytes in the immune response to sheep erythrocytes without cytostatic action of cell proliferation.     

Role of immune RNA in the interaction of T- AND B-lymphocytes]

"Immune" RNA preparations were obtained from the total population and also from the T- and B-lymphocytes of the spleens of the QBA line. Intact bone marrow cells or splenic cells activated with antigen served as target cells for the "immune" RNA. Investigations were carried out in the system of syngenic transfer. To study the target cells in the activated population of the spleen elimination of T-or B-lymphocytes was realized immediately after the incubation of the suspension of the splenic cells with the RNA preparations with the aid of anti-theta-or anti-B-antilymphocytic sera. T-lymphocytes served as the source of the biologically active RNA in the total preparation. B-lymphocytes of the spleen and the bone marrow served as target cells for the RNA of the cells of thymus origin. However, to detect the inducing action of the RNA simultaneous presence in the population of T- and B-lymphocytes is necessary.     

Effect of highly purified thymus factor on the cellular and humoral indices of immunity in thymectomized mice]

Effect of homogeneous polypeptide thymus factor of mol weight about 5000 (thymarin-III) on cellular and humoral immune responses of thymectomized adult CBA mice was studied. Thymectomy proved to greatly decrease the number of T-cells in the spleen. Accordingly, the capability of these mice to produce both IgM and IgG antibody-forming cells in response to the thymus-dependent antigen (sheep red blood cells) was significantly depressed. Subcutaneous injections of thymarin-III (1 microgram per g of body weight) for 7 days completely restored the T-cell spleen population and normalized the animals' immune response.     

Transfer Agent of Immunity

Antibody formation in vitro was studied using erythrocytes (RBC) as antigen and immunocytoadhesion as the technique for detection of antibody-forming cells. Spleen cells (SPC) of nonimmune mice gained the ability to produce antibody after treatment with ribonucleic acid (RNA) preparation extracted from allogeneic mice immunized with xenogeneic or allogeneic RBC. It was also found that a small proportion of SPC from individual mice of certain strains formed antibody against autologous RBC when the cells were treated in vitro with RNA preparation obtained from the spleen of an allogeneic mouse immunized with RBC of that individual. No converting ability was observed in the RNA preparation from spleen of nonimmune autologous or allogeneic mice. The converting activity of immune RNA preparation was shown to be sensitive to ribonuclease treatment. These evidences exclude the possible contribution of antigen or fragments thereof in the RNA preparation to the induction of antibody formation in RNA recipient cells.     

Intensification of cellular immune response by immunization of mice with the cells preincubated in interferon]

Mice F1(CBA X C57BL/6) were immunized intraperitoneally with a single injection of L-1210 cells preincubated in interferon or control "false" preparation. CBA mice were injected with MX-11 cells: similarly treated according to the same scheme. Injection of interferon-treated cells was accompanied by the enhancement of the cellular immune response. The greatest cytotoxic activity was possessed by the lymphocytes of mice to which M-11 cells were administered together with interferon in which the cells were formerly incubated.     

Studies on the maturation of immune responsiveness in the mouse. II. Role of the spleen.

Experiments were designed to investigate the role of the spleen in the development of the murine immune system. By using mice splenectomized within 24 hr of birth, as well as mice with a hereditary, congenital absence of the spleen, the primary immune response to sheep erythrocytes was examined. The immunocompetence of lymph node cells from spleenless or control mice was assessed in vitro, in organ and in cell suspension cultures, and in vivo, by transfer into lethally irradiated syngeneic recipients followed by antigenic stimulation. The immunologic capacities of thymus and bone marrow cells were similarly tested by injection separately or in combination into irradiated syngeneic mice. Lymph node cells from spleenless animals appeared fully competent both in vitro and in transfer experiments. Neither neonatal splenectomy nor congenital absence of the spleen significantly reduced the capacity of bone marrow or thymus cells to participate in the immune response to sheep erythrocytes.     

Indirect effects of leptin receptor deficiency on lymphocyte populations and immune response in db/db mice

Leptin-deficient ob/ob and leptin receptor (Ob-rb)-deficient db/db mice display a marked thymic atrophy and exhibit defective immune responses. Lymphocytes express leptin receptors and leptin exerts direct effects on T cells in vitro. In addition, ob/ob and db/db mice display multiple neuroendocrine and metabolic defects, through which leptin deficiency may indirectly affect the immune system in vivo. To study the relative contributions of direct and indirect effects of leptin on the immune system in a normal environment, we generated bone marrow chimeras (BMCs) by transplantation of leptin receptor-deficient db/db, or control db/+, bone marrow cells into wild-type (WT) recipients. The size and cellularity of the thymus, as well as cellular and humoral immune responses, were similar in db/db to WT and db/+ to WT BMCs. The immune phenotype of db/db mice is thus not explained by a cell autonomous defect of db/db lymphocytes. Conversely, thymus weight and cell number were decreased in the reverse graft setting in WT to db/db BMCs, indicating that expression of the leptin receptor in the environment is important for T cell development. Finally, normal thymocyte development occurred in fetal db/db thymi transplanted into WT hosts, indicating that direct effects of leptin are not required locally in the thymic microenvironment. In conclusion, direct effects of leptin on bone marrow-derived cells and on thymic stromal cells are not necessary for T lymphocyte maturation in normal mice. In contrast, leptin receptor deficiency affects the immune system indirectly via changes in the systemic environment.     

Depressed Cellular Formation of Antibody to Shigella Antigens In Vitro by Spleen Cell Cultures from Unresponsive Mice

Specific antibody plaque-forming cells (PFC) to Shigella-soluble antigen did not appear in spleen cell cultures from Shigella-tolerant mice, as occurred with similar cultures prepared from normal mice immunized with Shigella antigen prior to sacrifice. Cultures from tolerant mice also failed to form serologically detectable amounts of agglutinins in vitro. Exposure of cell cultures from tolerant mice in vitro to additional antigen had little or no effect on appearance of plaque-forming cells to Shigella. Spleen cells from normal control mice formed readily detectable levels of antibody, as well as specific antibody plaque-forming cells, after similar stimulation with antigen either in vivo or in vitro. The absence of antibody-forming cells in cultures prepared from spleens of tolerant mice was specific since such cultures, as well as those from normal control mice, formed numerous antibody plaques to unsensitized sheep erythrocytes in vitro after in vivo challenge of the mice with sheep erythrocytes. Tolerance to Shigella antigen, as assessed by absence of antibody-forming cells in vitro, persisted for several months. Spleen cell cultures from tolerant mice less than 3 to 4 months of age did not form significant numbers of antibody plaques, even after in vitro exposure to specific antigen. However, spleen cultures prepared from neonatally treated mice, approximately 6 to 8 months old, formed essentially normal numbers of specific PFC in vitro, indicating that the animals had "recovered" from tolerance and that their lymphoid cells were capable of responding to Shigella antigen in vitro. Absence of specific PFC in cell cultures from tolerant animals supports the concept that tolerance is due to a central failure of specific immunocompetent cells and not due to an inhibitory effect caused by either "excess" antigen or humoral antibody.     

The role of protein kinase A anchoring via the RII alpha regulatory subunit in the murine immune system

Intracellular cAMP may inhibit T cell activation and proliferation via activation of the cAMP-dependent protein kinase, PKA. PKA signaling is maintained through interactions of the regulatory subunit with A-kinase anchoring proteins (AKAPs). We demonstrated that T cells contain AKAPs and now ask whether PKA anchoring to AKAPs via the RIIalpha regulatory subunit is necessary for cAMP-mediated inhibition of T cell activation. We studied the immune systems of mice lacking the RIIalpha regulatory subunit of PKA (-/-) and the ability of cells isolated from these mice to respond to cAMP. Dissection of spleen and thymus from wild-type (WT) and -/- mice, single cell suspensions generated from these organs, and flow cytometry analysis illustrate that the gross morphology, cell numbers, and cell populations in the spleen and thymus of the -/- mice are similar to WT controls. In vitro, splenocytes from -/- mice respond to anti-CD3/anti-CD28 and PMA/ionomycin stimulation and produce IL-2 similar to WT. Cytokine analysis revealed no significant difference in Th1 or Th2 differentiation. Finally, equivalent frequencies of CD8(+) IFN-gamma producing effector cells were stimulated upon infection of WT or -/- mice with Listeria monocytogenes. These data represent the first study of the role of RIIalpha in the immune system in vivo and provide evidence that T cell development, homeostasis, and the generation of a cell-mediated immune response are not altered in the RIIalpha -/- mice, suggesting either that RIIalpha is not required for normal immune function or that other proteins are able to compensate for RIIalpha function.     

Secondary immune response of mice immunized with a protein-cellulose complex

It has been previously established that an intravenous injection of a protein antigen solution into mice primed with the same antigen in the form of a protein-cellulose complex induces an intensive antibody production (up to 10,000 antibody-forming cells/10(6) splenocytes and up to 3 mg of antibodies/ml of serum). The present study has shown that secondary immune response can be considerably enhanced if large amounts of the antigen are administered intraperitoneally in a protein-cellulose complex during secondary immunization. In these experiments the mean number of antibody-forming cells was 50.000/10(6) splenocytes and the antibody serum level averaged 10 to 12 mg/ml. The effect persisted for a long time: as late as on day 80 the antibody concentration was 2 mg/ml of serum.     

Homeostasis and effector function of lymphopenia-induced "memory-like" T cells in constitutively T cell-depleted mice

Naive T lymphocytes acquire a phenotype similar to Ag-experienced memory T cells as a result of proliferation under lymphopenic conditions. Such "memory-like" T (T(ML)) cells constitute a large fraction of the peripheral T cell pool in patients recovering from T cell ablative therapies, HIV patients under highly active antiretroviral therapy, and in the elderly population. To generate a model that allows characterization of T(ML) cells without adoptive transfer, irradiation, or thymectomy, we developed genetically modified mice that express diphtheria toxin A under control of a loxP-flanked stop cassette (R-DTA mice). Crossing these mice to CD4Cre mice resulted in efficient ablation of CD4 single-positive thymocytes, whereas double-positive and CD8 single-positive thymocytes were only partially affected. In the periphery the pool of naive (CD44(low)CD62L(high)) T cells was depleted. However, some T cells were resistant to Cre activity, escaped deletion in the thymus, and underwent lymphopenia-induced proliferation resulting in a pool of T(ML) cells that was similar in size and turnover to the pool of CD44(high)CD62L(low) "memory phenotype" T cells in control mice. CD4Cre/R-DTA mice remained lymphopenic despite the large available immunological "space" and normal Ag-induced T cell proliferation. CD4Cre/R-DTA mice showed a biased TCR repertoire indicating oligoclonal T cell expansion. Infection with the helminth Nippostrongylus brasiliensis resulted in diminished effector cell recruitment and impaired worm expulsion, demonstrating that T(ML) cells are not sufficient to mediate an effective immune response.