Immunization of dissociated spleen cell cultures from normal mice.

A culture system for cell suspensions from mouse spleens has been described. The system provides adequate conditions for in vitro immunization on initial exposure to heterologous erythrocytes. The in vitro response closely parallels that observed in vivo with respect to size, early kinetics, antigen dose, and the inhibitory effect of passive antibody. The response of cultured cells differs in two respects from that seen in vivo. There is an increase in the ability to discriminate between different varieties of homologous erythrocytes and the in vitro response does not appear to be limited by whatever mechanisms regulate the in vivo response.

Cell populations and cell proliferation in the in vitro response of normal mouse spleen to heterologous erythrocytes. Analysis by the hot pulse technique.

The role of proliferation in the development of 19S antibody-forming cells in the primary response has been investigated in an in vitro system. Spleen cell suspensions from normal, unimmunized mice were cultured in vitro in the presence of mammalian erythrocytes and the number of 19S hemolytic plaque-forming cells that arose 4 days later was measured. The hot pulse technique for the selective irradiation of those cells which synthesize DNA during a defined period of time has been described. The effect of such hot pulses administered at various times after the addition of antigen on the subsequent appearance of antibody-forming cells was determined. The results established that: (a) the onset of DNA synthesis does not start for approximately 24-32 hr after the addition of antigen, (b) essentially all the antibody-forming cells arise by cell division, and (c) different cell populations are involved in the response to two non-cross-reacting antigens.

Cellular events in the immune response : analysis and in vitro response of mouse spleen cell populations separated by differential flotation in albumin gradients.

Cell suspensions from the spleens of normal mice or mice injected with sheep erythrocytes were separated on a discontinous bovine serum albumin density gradient. Four bands or subpopulations were obtained and were assayed for antibody-forming cells, and for antigen-sensitive precursor cells. The antibody-forming cells were assayed by the hemolytic plaque assay and the antigen-sensitive precursors were assayed by the number of plaque-forming cells which developed after 3 or 5 day's culture with antigen. It was found that both antibody-forming cells and their precursors were present in the denser region of the gradient when spleen cell suspensions were taken from unimmunized mice. In contrast, both antibody-forming cells and precursors floated to the top in cell suspensions from mice sacrificed 1, 2, or 3 days after antigen injection. The change in density was detectable as early as 12 hr and was complete by 18 hr. The cell which changed in density was specific for the antigen that brought about that change. The significance of these findings is discussed.

Immunological memory in mice. 3. Memory to heterologous erythrocytes in both T cell and B cell populations and requirement for T cells in expression of B cell memory. Evidence using immunoglobulin allotype and mouse alloantigen theta markers with congenic mice.

Using anti-allotype sera and AKR anti thetaC3H sera, a requirement for two cell types has been demonstrated in the adoptive secondary response of mice to heterologous erythrocytes. The cell types have been designated B cells [precursors of plaque-forming cells (PFC)] and T cells (thymus-influenced cells, not providing precursors of detectable PFC). The in vivo indirect PFC response of spleen cells from primed mice is markedly reduced by in vitro treatment of the cells with a mixture of anti-theta serum and guinea pig serum (Anti theta + GPS). This B cell response is fully restored to control levels by thymus cells from normal mice which do not themselves provide precursors of indirect PFC. Thus memory is carried by the B cell lineage but the expression of this memory is dependent on the presence of a cell population which is sensitive to Anti theta + GPS and which is replaced functionally by unprimed T cells. When assayed for T cell activity, thoracic duct cells from specifically primed mice are better than cells from nonspecifically primed mice in restoring the B cell response of spleen cells from immunized mice. Moreover, the T cell activity of a reconstitutive cell population from primed mice is reduced by incubation with Anti theta + GPS. We conclude that memory to heterologous erythrocyte antigens is carried by the T cell lineage as well as the B cell lineage even though unprimed T cells are sufficient for expression of B cell memory.

Immunization of normal mouse spleen cell suspensions in vitro.

Dissociated cells from the spleens of unimmunized mice were cultured with and without various mammalian erythrocytes. Spleen cell suspensions cultured with heterologous red cells developed levels of hemolytic plaque-forming cells only one log(2) less than those seen in vivo. The reaction is specific for the in vitro immunizing erythrocytes. Antibody was demonstrated in the culture fluids.

Cell interaction in an immune response in vitro: requirement for theta-carrying cells.

Cytotoxic antiserum to theta antigen reduces the capacity of mouse spleen cells to generate direct and indirect plaque-forming cells to sheep erythrocytes in vitro but does not affect plaque-forming cells, their precursors, or hemopoietic stem cells. The response of spleen cells treated with antiserum to theta antigen is restored by thymus cells incubated in vivo with sheep erythrocytes.

Differential regulatory effects of accessory cells on the generation of cell-mediated immune reactions.

Conditions for the in vitro generation of cell-mediated immunity to H-2 and non-H-2 alloantigens by mouse spleen cells were compared to conditions for generation of in vitro humoral immunity to sheep red cells. Normal spleen cells grown in standard or allogeneic cell-conditioned media developed strong cellular responses to H-2 antigens but little or no response to non-H-2 antigens. Subpopulations of spleen cells from which the adherent cells had been removed generated strong cellular responses to both H-2 and non-H-2 antigens, provided the cells were cultured in conditioned media. Both cellular responses were shown to be mediated by T cells. Accessory cells from the peritoneum and/or spleen differentially inhibited the non H-2 cellular responses at doses which did not inhibit humoral responses.

Sera and the in vitro induction of immune responses. I. Bacterial contamination and the generation of good fetal bovine sera.

More than 200 samples of pooled fetal bovine sera (FBS) were tested to determine their capacity to support primary immune responses by cultured mouse spleen cells. Less than 10% of the FBS samples were fully supportive; the majority of the samples were moderately to very deficient. Several lines of evidence suggest that bacterial contamination during the processing of sera plays a major role in determining which sera are supportive. Serum samples known to have been temporarily contamined during processing were strongly supportive. Samples of FBS which were likely never to have been contaminated were deficient. Bacteria from a specific lot of supportive serum converted a very deficient serum into one which was supportive. Several mechanicsms by which bacteria could effect sera are discussed.

Differential effects of glucocorticosteroids on the functions of subpopulations of helper T lymphocytes.

The in vitro effects of dexamethasone on the activities of Th1 and Th2 helper cell subpopulations were examined in secondary (IgG) responses to hapten-carrier conjugates under conditions where the selective or dominant expression of their individual activities could be observed. In the induction of IgG anti-hapten responses, carrier-primed Th1 cells cooperate with hapten-primed B cells by linked recognition of antigen, while carrier-primed Th2 cells cooperate with hapten-specific B cells by unlinked recognition of antigen. The function of carrier-primed Th1 cells was resistant to inactivation by dexamethasone. In contrast, the function of carrier-primed Th2 cells was abolished in the presence of pharmacologic concentrations of the steroid. The differential effects of dexamethosone on the activities of the two subpopulations of helper T cells could not be attributed to selective inhibition of a subpopulation of B cells which cooperate with Th2 cells.

Differential effects of glucocorticosteroids on the functions of helper and suppressor T lymphocytes.

The effects of dexamethasone (Dex) on the functions of antigen-primed helper and suppressor T cells were studied in humoral immune responses in vitro. In doses equivalent to elevated physiologic concentrations, the suppressor T cell activity was abolished. In contrast, the helper T cell function was resistant to even pharmacologic concentrations of Dex. The apparent steroid resistance of the helper T cells was found to be mediated by the products of activated macrophages. While macrophage factors protected helper T cells from steroid inhibition, they did not prevent the effects of Dex on suppressor T cells. Because bacterial cell wall and membrane components are potent inducers of the factors that mediate steroid resistance of helper T cells, the combination of physiologically elevated levels of steroids and macrophage factors during acute infections may function to facilitate the expression of host immunity. However, the persistance of these conditions, as in chronic inflammation, may also contribute to the pathogenesis of autoimmunity by perturbing the balance of immune regulation by helper and suppressor T cells.

Black under-representation in United States medical Schools.

To understand why blacks are under-represented in medical schools and how this situation might be changed, we analyzed statistics on medical-school applicants, medical-school students and college undergraduates. The pool of qualified black applicants to medical schools has not been large enough to achieve appropriate black representation. If black under-representation is to be corrected, the pool of qualified black applicants must be increased. Affirmative-action programs for blacks who are already in college are unlikely to be sufficient by themselves to increase the pool of black applicants substantially. Such programs should therefore be developed for high-school students.

Mitogenic effects of purified outer membrane proteins from Pseudomonas aeruginosa.

Three major outer membrane proteins from Pseudomonas aeruginosa PAO1 were purified and tested for their ability to stimulate resting murine lymphocytes to proliferate. It was demonstrated that picomole amounts of all three proteins were mitogenic for both intact and T-lymphocyte-depleted populations of spleen cells from C3H/HeJ mice. In contrast, they had no activity against either mature or immature thymocytes. Since the strain of mice used is unable to respond to lipopolysaccharide, we condlude that the three proteins are B-cell mitogens.

Functional deficiency of splenic adherent cells in New Zealand black mice.

New Zealand Black (NZB) mice hyporespond after in vitro immunization to sheep erythrocytes but not after in vivo immunization. The difference between in vitro and in vivo immunization was found to involve a non-antigen-specific change in the spleen which occurs in vivo soon after priming. Adherent spleen cells from young NZB or control strain mice were compared in their ability to cooperate with nonadherent cells in the induction of a primary immune response to sheep erythrocytes in vitro. The results support the hypothesis that there is a functional deficiency of adherent cells in the spleens of unprimed NZB mice.

Sera and the in vitro induction of immune responses. II. Inhibitory effects of newborn calf sera on primary humoral responses.

The effect of newborn calf (NBC) sera on the in vitro generation of immune responses were studied. NBC sera inhibited the generation of primary humoral responses to SRBC. The inhibitory activity was dose-dependent and diluted out rapidly. Adding inhibitory doses of NBC serum to test cultures at zero or 24 hr resulted in approximately the same amount of inhibition. Pulsing experiments showed that the test cultures were most sensitive to the inhibitory effects of NBC serum during the 24- to 48-hr period. In contrast to its inhibitory effects on primary humoral responses, NBC sera supported the in vitro generation of secondary humoral responses and significantly increased the responses of some of these over control levels. We found that sera obtained from newborn calves before nursing were not inhibitory for primary responses but sera obtained from the same calves 7 days later did inhibit these responses. We postulate that the inhibitory effects of NBC sera may represent a physiologic control mechanism which becomes active when immunologic maturity develops and that it possibly involves the inactivation of cell mediators.

Long-term effects of in vitro sensitization on immune reactivity of lymphocytes: generation of suppressor and helper T cells.

Mouse spleen cells were cultured for 5 days with or without HRBC. Cultured cells were 'parked' in irradiated syngeneic recipients for 3 weeks and then tested for their immunologic reactivity in vitro. We found that spleen cells from recipients of HRBC-sensitized cells (S) as well as spleen cells from recipients of control unsensitized cells (U) possessed radiosensitive suppressor and radioresistant helper activities. Suppressor activity was observed by the capacity of unirradiated S and U spleen cells to inhibit the in vitro generation of IgM and IgG PFC by spleen cells primed in vivo to HRBC or to LacKLH. Helper activity was shown by the capacity of the irradiated S and U cells to restore IgM and IgG PFC responses of in vivo primed, T-depleted spleen cells to HRBC, LacHRBC, and LacCRBC. Both suppressor and helper activities were mediated by T cells. The possibilities that immunologically specific or nonspecific mechanisms account for these phenomena are discussed.

The role of activated accessory cells in preventing immunosuppression by hydrocortisone.

The generation of humoral immunity in vitro by normal and antigen-primed mouse spleen cells was suppressed by in vitro treatment with hydrocortisone. Functions of normal and antigen-activated helper T lymphocytes and of accessory cells were inhibited by the corticosteroids. Spleen cells cultured overnight in medium containing fetal bovine serum became highly resistant to the effects of hydrocortisone. Similar resistance was found to occur when spleen cells were cultured with accessory cells that previously had been activated with bacterial lipopolysaccharide. These studies show that immunologically nonspecific processes significantly alter the effects of the steroids on specific immune responses and suggest that accessory cell products modulate T cells in ways which differ from antigen induction.

Immunodepression in Taenia crassiceps infection: restoration of the in vitro response to sheep erythrocytes by activated peritoneal cells.

The in vitro response to sheep erythrocytes of mesenteric lymph node cells from mice infected with the larval cestode Taenia crassiceps is significantly depressed and can be restored to control levels by addition of activated peritoneal cells depleted of functional T or B lymphocytes. Adherent mesenteric lymph node cells from infected mice are unable to reconstitute the in vitro response to sheep erythrocytes of normal nonadherent cells. The responses of mesenteric lymph node cells from infected mice to the T-lymphocyte mitogens concanavalin A and phytohemagglutinin and the B-lymphocyte mitogen lipopolysaccharide are normal. Mesenteric lymph node cells from infected mice do not suppress the in vitro response to sheep erythrocytes of normal mesenteric lymph node cells. These results suggest that the immunodepression in T. crassiceps-infected mice is primarily the result of alterations in functional accessory cells.