In Vitro Studies on the Immunological Memory for Antibody Response to Bovine Serum Albumin

Immunological memory for T and B cells was studied in an in vitro culture system with spleen cells from mice primed with bovine serum albumin (BSA). Spleen cells taken from mice immunized at various times previously with a single intravenous injection of alum-precipitated (AP) BSA and bacterial endotoxin (ET) were cultured in Marbrook's system with dinitrophenylated (DNP) BSA as the in vitro antigen. In the cultures of spleen cells obtained from mice primed more than 14 days previously, an IgG-predominant anti-BSA response was generated. However, no anti-BSA response was observed in the culture of spleen cells taken from mice primed 7 days previously (day 7 spleen cells). The failure of day 7 spleen cells to generate an antibody response in vitro was shown to be attributable to both the lack of B memory cells and the effect of “suppressive” macrophages induced by ET. On the other hand, anti-BSA memory in the spleen of mice primed with AP-BSA plus ET and 2 months later challenged with AP-BSA matured within 7 days and declined rather quickly by 30 days after the challenge. The difference in the time course of the generation of memory between the spleen cells from primary and from secondary immunized mice might be attributable to the difference in the maturation of memory B cells, since the time course of the development of memory T cells after the secondary immunization was similar to that observed after primary immunization.     

Regulatory Mechanism of Delayed-Type Hypersensitivity in Mice: III. In Vitro Analysis of Memory T Cells Involved in Augmentation of DTH Responses

The memory of delayed-type hypersensitivity (DTH), manifested by the augmented responsiveness upon challenge with alum-absorbed ovalbumin (OA), was induced in mice primed 7 days, 21 days, or 90 days previously with 1 μg of reduced and alkylated OA. The memory cells involved in the augmentation of DTH responses were analyzed in the in vitro induction system of T cells which mediate DTH against OA. Spleen cells from the primed mice generated DTH-effector T cells (DTH-Te) in a significantly accelerated fashion, compared with unprimed spleen cells, when cultured with OA. The accelerated generation of DTH-Te in vitro was induced antigen specifically and was dependent on a certain T cell population in the primed spleen. The T cell population was found in the spleen of primed mice for at least 3 months after priming, corresponding to the persistence of DTH-memory in vivo. Moreover, it was fractionated in the high-density layer by discontinuous bovine serum albumin gradient centrifugation. The high-density cell population decreased in density with increase in the time of culture and developed into DTH-Te, which were separated in the low-density layer on day 4 of culture. These results indicate that the T cells involved in the accelerated generation of DTH-Te in vitro are long-lived DTH-memory T cells, which are probably precursor cells, capable of differentiating into DTH-Te upon challenge with the antigen.     

Induction of an antibody response in vitro against synthetic antigens in guinea pigs. I. Culture and assay conditions

Guinea pig spleen and lymph node cells were found to produce anti-2,4-dinitrophenyl (Dnp) oligolysine PFC in vivo against 2,4-dinitrophenyl-β-alanyl glycyl glycyl (Dagg-SRBC) but not against trinitrophenyl-SRBC target indicator cells. Furthermore, when sensitized spleen cells or their purified B-cell fractions were cocultured with primed peritoneal exudate lymphocytes (PEL) but not splenic T cells they were able to generate a secondary PFC response in vitro to the synthetic antigens, Dnp oligolysines. PFC were not induced in vitro if these same cultures were pulsed with short-chain peptides (five lysines) or the complex antigen, dinitrophenyl-bovine γ-globulin (DnpBGG). Con A was able to substitute for PEL in triggering spleen cells to mount a secondary in vitro PFC response to homologous Dnp oligolysines. More importantly, the Con A-aided spleen cell cultures were not induced above background values when challenged in vitro with heterologous Dnp oligolysines. This study suggests that spleen cells may lack a nonspecific signal for the development of a secondary in vitro PFC response.     

Nature of T-cells resident in spleens of thymectomized,lethally irradiated,bone marrow-reconstituted mice

Adult thymectomized, lethally irradiated, bone marrow-reconstituted (ATxXB) mice that had been weakly primed with SRBC or HRBC between thymectomy and irradiation were shown to retain antigen-specific immunological memories for at least 1–5 months after bone marrow reconstitution. This could be shown by anamnestic antibody response in vivo as well as by proliferative response of the spleen cells to the test antigens in vitro. Spleen cells taken from ATxXB mice showed a reduced but significant proliferative response to nonspecific T-cell mitogens, in particular to Con A, in vitro. Treatment of the donor bone marrow cells used for reconstitution of ATxXB mice with anti-Thy 1.2 sera + C′ did not affect the generation of immunological memories nor the magnitude of the proliferative response of spleen cells to nonspecific T-cell mitogens in vitro, indicating that the cells responsible for such functions were host derived. Finally, the antibody-forming capacity of spleen cells derived from SRBC-primed ATxXB mice to the test antigen in vitro was completely abrogated by exposure to 450 R, whereas the helper function of the same cell suspension remained unaffected even after exposure to 1000 R. Implication of these findings on the nature of T cells resident in spleens of ATxXB mice was discussed.     

In vitro immune blastogenesis during contact sensitivity in tumor-bearing mice: I. Description of progressive impairment and demonstration of splenic suppressor cells

T-cell responsiveness was measured by the DNA response of disassociated spleen and lymph node cells when exposed to antigen in vitro. Sensitized splenic lymphocytes from fibrosarcoma-bearing mice immunized with 2,4-dinitro-1,5-difluorobenzene (DN2FB) demonstrated a progressive decrease in T-cell responsiveness to the haptenprotein conjugate DNP-BSA. Hyporesponsiveness to the dinitrophenylated-protein conjugate appeared in the spleens but not lymph nodes of tumorous animals. Normal host lymph node cells (LNC) responded strongly 24 to 48 h after sensitization and subsequently declined with a corresponding increase in responsiveness in the spleen. Tumor-bearing hosts (TBH) had similar LNC kinetics during immunization, however, spleen cells were significantly suppressed when compared to normal BALB/c mice sensitization kinetics. Spleen cells from TBH were also capable of suppressing the in vitro response of normal primed lymphocytes to DNP-BSA when admixed. Results from these experiments suggest that in vitro measurement of contact sensitivity was affected by suppressor cells/products existing in the spleens but not lymph nodes of fibrosarcoma-bearing mice.     

In vitro studies of the rabbit immune system: I. Hemolytic plaque-forming response of dissociated spleen cells from normal and primed rabbits

The conditions for the in vitro generation of primary and secondary immune responses by rabbit spleen cells to sheep red blood cell (SRBC) antigen have been examined. Spleen cells from many normal and all previously immunized rabbits are capable of producing in vitro plaque-forming cell (PFC) responses when cultured as dissociated cell suspensions in the presence of antigen. Primed spleen cells generate approximately 100 times the number of PFCs obtained in normal cultures with a shorter lag period. Both types of cultures demonstrate a period of exponential increase in PFCs during which the doubling time is 12–14 hr. This increase occurs after 1 day of culture of spleen cells from primed rabbits and after 4 days of culture of spleen cells from unprimed rabbits. The PFCs which arise in cultures of primed cells appear not to be the progeny of those generated in vivo but to be derived from an increased number of PFC precursors. Repeated immunization of the spleen cell donor is required to produce significant numbers of indirect (IgG) PFC or indirect precursors; most of the PFC found after a single immunization in vivo or in vitro are direct (IgM). There is no evidence for conversion of IgM to IgG PFC in vitro. This system should provide a means for further identification of the cellular interactions involved in the immune response of the rabbit.     

Time-dependent mode of immunization augments suppressed antibody responses in spleen cell cultures from mice experimentally infected with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC). After normal or infected mice were primed with SRBC, their spleen cells were restimulated 4 days later with SRBC in Mishell-Dutton cultures and found to mount hyperaugmented IgM anti-SRBC responses. It was also demonstrated that T-cells derived from normal mice primed in vivo 4 days previously with SRBC, and subsequently added to cultures of spleen cells from T. cruzi-infected mice, enhanced anti-SRBC DPFC responses in a dose-dependent fashion. These results show that functional help provided by T-cells activated during an in vivo priming and exposed to an in vitro challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in the spleen cell cultures from T. cruzi-infected mice.     

The immunoregulatory role of bone marrow. I. Suppression of the induction of antibody responses to T-dependent and T-independent antigens by cells in the bone marrow.

Bone marrow cells (BMC) from normal mice suppressed the in vitro IgM, but not the IgG, antibody (Ab) response of spleen cells. BMC were inhibitory only when added during the first 24 hr of culture, and inhibition was not due to an induced shift in the kinetics of the response. Addition of specifically activated T cells or nonspecific T-cell-replacing factors to normal or T-depleted spleen cell cultures did not abrogate suppression while the response to the T-independent antigen DNP-polymerized flagellin or lipopolysaccharide was also suppressed. BMC did not inhibit background Ab synthesis by normal or primed cells in the absence of antigen and did not inhibit, but stimulated, DNA synthesis in normal spleen cell cultures. In addition, high-avidity Ab synthesis was preferentially suppressed. A possible role for the bone marrow suppressor cell in the induction of B cell tolerance is discussed.     

Cellular requirements for the expression of IgM. Immunological memory in vitro

In vitro culture techniques have been used to compare the direct (IgM) plaqueforming cell (PFC) response to heterologous erythrocytes (RBC) by normal mouse spleen cells and spleen cells from mice injected intravenously with 5 × 10⁴ RBC ten days previously [low dose primed (LDP)]. Although LDP mice fail to undergo a significant primary PFC response, their spleen cells are capable of a secondary or enhanced PFC response in vitro. The secondary PFC response is shown to be a function of: (A) an increase in the frequency of immunocompetent cells or units (IU) due to in vivo priming, and (B) an increased number of PFC generated per IU subsequent to in vitro stimulation. The latter increase is shown to be mediated through a shorter PFC doubling-time during logarithmic expansion of the PFC population. Analysis of nonadherent spleen cell dose response experiments indicate that two nonadherent cell types interact in the secondary response. Subsequent cocultivation experiments suggested that both of these cell types must be “primed” to allow induction of a secondary response. Although adherent cells are required for the secondary response, normal splenic adherent cells serve as equivalent substitutes for LDP adherent cells.     

In vitro response of chicken spleen cells to sheep red blood cells

A modification of the Marbrook system for obtaining both primary and anamnestic responses from chicken spleen cells in vitro is described. The patterns of responsiveness of chicken spleen cell cultures resembled those previously described for the mouse. However, the requirement for normal chicken serum (NCS) to support the response could not be replaced by foetal calf serum (FCS). Cell survival, response kinetics, antigen dose requirements, antibody class, and temporal development of memory are described. The requirement for cooperation of B and T cells in the SRBC response was demonstrated by the use of specific anti-T and anti-B sera.     

The induction in vitro of immunological tolerance in T lymphocytes: an inhibitory role of macrophages.

Hapten-reactive helper T cells were generated in the spleen of C57BL/6 mice primed with sulfanilated syngeneic IgG (S-MGG). Specific immunological tolerance was induced in vitro in these helper T cells, when spleen cell suspension was passed through Sephadex G-10 column to remove adherent cells and cultured in the presence of soluble S-MGG for 21 to 24 hours. On the other hand, tolerance was not inducible in unfractionated, primed spleen cells. When G-10-passed spleen cells were added to the culture dishes containing phagocytic, adherent cells of the spleen, tolerance was no more inducible in these reconstituted cell population. From these experimental results, it was concluded that macrophages played an interfering role in tolerance induction. The experimental data were also discussed in terms of macrophage function in the recognition of antigen by T lymphocytes.     

Separation of various B-cell subpopulations from mouse spleen. I. Depletion of B cells by rosetting with glutaraldehyde-fixed, anti-immunoglobulin-coupled red blood cells.

Mouse spleen cells were depleted of immunoglobulin (Ig)-bearing B cells by rosetting with glutaraldehyde-fixed, tannic acid-treated RBC coupled with antibody to mouse Ig (anti-Ig) and removing the rosetted cells by density gradient centrifugation. The method was routinely greater than 90% effective in removing B cells as assayed by the failure of anti-Ig rosette-depleted primed spleen cells to generate antibody-producing cells in vitro in response to specific antigen or of anti-Ig rosette-depleted nonprimed spleen cells to generate a polyclonal antibody response. T cells were not removed by the rosetting procedure as measured by helper T-cell activity. The greater effectiveness of the rosetting procedure in removing potential IgG-secreting, non-IgM-bearing B cells is shown relative to other commonly used B-cell depletion procedures. Because the RBC in the rosetting reagent are fixed with glutaraldehyde, the rosetting reagent is stable for many months. Such stability makes constantly available a convenient means for B-cell removal, as well as reducing consumption of antisera.     

Immunorestoration of old mice by injection of thymus extract: Enhancement of T cell-T cell cooperation in the in vitro antibody response

The effect of injecting a thymus extract (TP-1 or Thy-5) into immunodeficient old mice on the in vitro antibody response of their spleen cells was investigated by techniques suitable for dissecting out T- and B-cell reactivities. The anti-TNP antibody response of HRBC-primed spleen cells from old mice uninjected or injected with TP-1 or Thy-5 was elicited in vitro by TNP-HRBC or TNP-Ficoll. Treatment with TP-1 or Thy-5 was found to induce only a slight increase in the anti-TNP antibody response to both immunogens. The helper activity of HRBC-primed spleen cells from untreated or treated old mice was titrated by adding graded numbers of these primed cells to cultures containing a constant number of normal spleen cells from young mice and the immunogen TNP-HRBC. Under these conditions it was found that both thymus extracts are very effective in restoring T cell-T cell cooperation in the generation of helper cell activity.     

Antigen-Specific Proliferative Response of Peritoneal Exudate Lymphocytes Primed with Antigen and Bacterial Lipopolysaccharide: The Roles of Ia+ Accessory Cells and IL-2

In vitro antigen-specific proliferation was investigated in a lymphocyte population that had been taken from the peritoneal exudate cells (PEC) of C3H/HeN mice (Ia^k) primed in vivo with both bacterial lipopolysaccharide (LPS) and horse red blood cells (HRBC) and had been purified by passage through a nylon fiber column (Nfc). The proliferative response of the Nfc-passed lymphocytes primed with HRBC and LPS [T(HRBC + LPS) cells] depended on the dose of antigen in the cultures, and the response was higher than that of cells prepared from mice primed with HRBC alone [T(HRBC) cells]. No response was seen in the cells prepared from the LPS-primed mice [T(LPS) cells] or normal mice [T(N) cells]. The response of the T(HRBC) cells was abolished by previous treatment of the cells with anti-Ia^k antibody and complement (C), whereas the response of the T(HRBC + LPS) cells was retained after the same treatment, indicating that the Ia^– T(HRBC + LPS) cells can proliferate in response to antigen in spite of Ia+ accessory cell-depletion. Supernatants from the cultures of Ia^– T(HRBC + LPS) cells in the presence of HRBC showed abundant IL-2 activity, while those of Ia^– T(HRBC) cells did not. The IL-2 should be produced by the L3T4 cell population in T(HRBC + LPS) cells in response to antigen, since the previous treatment of the cells with anti-L3T4 antibody and C abrogated the production. On the other hand, the Ia^– T(HRBC + LPS) cells as well as the Ia^– T(LPS) cells could respond to IL-2 dose-dependently when recombinant IL-2 was added into the cultures, but the response of Ia^– T(HRBC) cells to IL-2 was very weak. The cell population responding to IL-2 in the T(HRBC + LPS) cells as well as T(LPS) cells must be AsGM1-positive or natural killer (NK) cells, since previous treatment of the cells with anti-AsGM1 antibody and C abrogates the response. Together these results suggest that L3T4 lymphocytes capable of producing IL-2 in response to HRBC antigen without Ia⁺ accessory cells are generated in the PEC of the mice after priming with LPS and antigen together, and the IL-2 produced by the L3T4 lymphocytes induces the proliferation of the LPS-primed AsGM1+ cells.     

Composition of the immune system of allophenic mice.

Mice were immunized with antigen (Rabbit Fab' fragments) attached to syngeneic, or f₁ (semi-syngeneic) irradiated spleen cells. Specific anti-rabbit Fab' plaque forming cell numbers showed that the response towards antigen on syngeneic or F₁ cells, was significantly lower than that towards the same antigen on allogeneic cells. By subsequent in vitro incubation of immune spleen cells with antigen followed by plaque assay, it was found that those spleen cells exhibiting lowered plaque forming cell numbers initially, (i.e., those mice immunized with antigen on syngeneic or F₁ cell surfaces) showed, after incubation, a response equal to or greater than those cells which initially (before in vitro incubation) demonstrated a larger response (i.e. those mice immunized with antigen on allogeneic cells).     

Functional relationships between B-cell subsets: evidence for an antigen-induced B1 leads to B2 switch

Priming of mice with the TI-2 antigen TNP-Ficoll results in an increased secondary anti-TNP response to the TD antigen TNP-KLH. Spleen cells obtained from mice primed with TNP-Ficoll or TNP-LPS, but not TNP-KLH, gave augmented responses to DNP-Dextran, TNP-LPS, or LPS. In vitro treatment of spleen cells with DNP-Dextran results in an increased anti-TNP response to TNP-LPS. This effect is also obtained if mitogenic doses of Dextran and LPS are used to induce cell proliferation. We conclude that the B_i2 subset (responding to TNP-Ficoll and DNP-Dextran) switches to the B2 subset (responding to TNP-KLH) after antigen activation. The B_i1 subset (responding to TNP-LPS) may represent an intermediate stage of B-cell differentiation.     

Regulation of antibody response in different immunoglobulin classes. II. Induction of in vitro IgE antibody response in murine spleen cells and demonstration of a possible involvement of distinct T-helper cells in IgE and IgG antibody responses.

In vitro induction of anti-DNP IgE as well as IgG1, IgG2a antibody responses was shown in murine spleen cell culture. Spleen cells primed three times with 1 mug of DNP-OA or DNP-Asc produced significant amounts of anti-DNP IgE as well as IgG antibodies by the in vitro stimulation with DNP-OA or DNP-Asc, respectively. Collaboration between DNP-primed B cells and carrier-primed T cells was required for the induction of both IgE and IgG antibodies with DNP-coupled T-dependent antigen. Carrier-specific T cells induced with a low dose of Asc (0.01 mug) showed helper function only on IgE antibody response, whereas T cells primed with a higher dose of Asc (10 mug) cooperated only with IgG-B cells. T cells primed with Asc in CFA showed helper function mainly on IgG antibody response but not on IgE antibody response. The result indicated the presence of a distinct population of T helper cells for IgE and IgG antibody responses. T-independent antigen (DNP-Ficoll) induced both anti-DNP IgE and IgG antibody responses in DNP-primed spleen cell population without the requirement of the collaboration of helper T cells.     

Suppression of in vitro antibody responses by Listeria primed spleen cells.

Spleen cells from mice primed with virulent Listeria monocytogenes do not develop an anti-SRBC plaque forming cell response to SRBC in culture. Furthermore, when Listeria primed spleen cells are co-cultured with normal spleen cells and SRBC, the anti-SRBC response of the normal cells is suppressed. Listeria primed spleen cells from T cell depleted donors are equally effective at immunosuppression. The immunosuppressive effect does not appear to be due to the presence of the bacterium or its products per se in the cultures. Furthermore, the effect cannot be transferred across a 0.45 μm pore membrane. Kinetic studies show that the immunosuppressive effect develops by 2 days post-Listeria inoculation and peaks by Day 6. Low doses of Listeria are not immunosuppressive and produce some enhancement effect. From these results, it is suggested that a population of non-T cell dependent cells develop in Listeria primed hosts that nonspecifically suppress the response of B cells to an unrelated antigen in culture.     

Hapten-Specific T Cell Response to Azobenzenearsonate-N-Acetyl-L-Tyrosine (ABA-tyr) in Lewis Rats: II. Induction and Suppression of ABA-Specific Helper Cell Activity for Antibody Production

Immunization of Lewis rats with azobenzenearsonate-N-acetyl-l-tyrosine (ABA-tyr) in complete Freund's adjuvant (CFA), produces a hapten-specific helper T cell response measured by an increase in plaque forming cells (PFC) against a different hapten. The response seen is primarily direct (IgM) PFC unless B cells are primed by injection of trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) prior to immunization with ABA-tyr. The response requires both ABA and TNP to be on the same carrier molecule which can be as diverse as bovine serum albumin (BSA), poly l-glutamine-lysine-tyrosine (l-GLT); however, a d-amino acid polypeptide does not work. The in vitro demonstration of such help was successful only with peritoneal exudate lymphocytes, not spleen or lymph node cells. Repeated pretreatment of rats by intraperitoneal injection of ABA-tyr in incomplete Freund's adjuvant (IFA) induced an unresponsiveness for helper activity to subsequent immunization with the same antigen in CFA. Passive transfer of lymphoid cells from spleens and lymph nodes from rats pretreated with ABA-tyr in IFA followed by boosting with ABA-tyr in CFA induced unresponsiveness to subsequent induction of hapten-specific help.     

Suppressive Effect by Cooperation of Splenic and Peritoneal Adherent Cells on Generation of Cytotoxic T Lymphocytes

When spleen cells primed in vivo against allogeneic lymphoid cells were used as responder cells in secondary mixed lymphocyte cultures, a high degree of cytotoxicity was generated even in the absence of splenic adherent cells. However, removal of adherent cells from such primed responder spleen cells reduced the cytotoxicity to some extent. On the other hand, when these responder cells were transferred into the peritoneal cavity of irradiated syngeneic mice together with antigenic cells, unseparated responder cells generated a lower degree of cytotoxicity than did adherent cell-depleted responder cells. In an in vitro system, peritoneal adherent cells also suppressed the generation of cytotoxic T lymphocytes by unseparated responders; however they augmented the cytotoxic T lymphocyte generation by adherent cell-depleted responders. These adherent cell populations with augmenting activity became inhibitory when they coexisted. The mechanism of this inhibitory action remains unclear.