Interaction of T- and B-cells in the secondary response to ram erythrocytes in mice of different ages]

The spleen cells from old primed donors were found to produce lesser secondary response, as compared with the cells of young animals, in the system of adaptive transfer on the CBA mice. In the experiments with extermination of T-or B-cell population in the cell suspension by means of theta-or anti-b-sera it was established that the memory to the antigen used (ram ethyrocytes) was carried by both T- and B-cells but the T-cell component played the leading role in this system. The primed cells from old animals proved to be more sensitive to defects of cellular interactions.     

Priming for a cytotoxic response to minor histocompatibility antigens: antigen specificity and failure to demonstrate a carrier effect.

Spleen cells from normal mice do not give a detectable in vitro cytotoxic T cell (CTL) response to minor H antigens. Spleen cells from animals primed in vivo with minor H antigens give a strong CTL response when boosted in culture with the appropriate stimulating cells. Here I have studied the requirements for priming a CTL response to minor H antigens. It is shown that priming is just as antigen specific as is cytolytic effector function. That is, priming cells have to carry the same minor antigens as the challenge cells. Inducing a graft-vs-host reaction in vivo does not nonspecifically allow spleen cells to respond to minor H antigens in vitro. Using minor H congenic mice (congenic for H-Y and/or H-7) I have also tried, and failed, to demonstrate a carrier effect in priming. Female mice primed to H-Y were challenged in culture with cells bearing H-Y and H-7 antigens in the hope that a helper response to H-Y would augment a CTL response to H-7. This did not happen, however. Such primed and boosted cells gave a strong secondary CTL response to H-Y but none to H-7. It is concluded that in order to prime for a detectable in vitro response to minor antigens it is necessary to expose the CTL precursors to antigen in vivo. This either expands the size of the pool of precursors by cell division or changes them in some qualitative way.     

Failure of NZB spleen to respond to prethymic bone marrow suppressor cells.

Mouse bone marrow contains theta-negative lymphocytes that can suppress an in vitro plaque response by spleen cells primed in vivo with burro red blood cells (BRBC). These bone marrow cells are radiosensitive and can be induced with thymosin fraction 5 or alpha 1 thymic peptides to express the theta antigen. Enrichment for these suppressor pre-T lymphocytes can be achieved by a one-step density centrifugation, macrophage depletion, or a combination of both procedures. NZB mice, which spontaneously develop an autoimmune disorder, have a suppressor abnormality revealed by this assay system. Upon analysis, they have normal BM pre-T suppressor cells but their spleen cells are refractory to the BM suppressor signal. NZB BM suppressor cells inhibit the response by DBA/2 spleen cells, but DBA/2 BM suppressor cells do not inhibit NZB spleen. This resistance to suppression is a property of the B cell fraction recovered from NZB spleen.     

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.     

Neonatal splenic suppressor cells in the chicken. I. In vivo suppression of the immune response to bovine serum albumin by normal and tolerized neonatal spleen cells

The presence of active splenic suppressor cells in neonatal chickens, either normal or tolerant to bovine serum albumin (BSA), was examined by assessment of their effect on both primary and adoptively transferred secondary responses to BSA or sheep red blood cells (SRC). Both normal and BSA tolerized spleen cells were shown to be highly suppressive of secondary anti-BSA responses generated by specifically primed adult spleen cells in inert recipients. Suppression of the secondary anti-BSA response by normal spleen cells was slightly less effective than that seen with BSA tolerant spleen cells. Transfer of BSA tolerant spleen cells into normal recipients, followed by BSA challenge, prevented any significant primary anti-BSA response. In contrast, transfer of normal spleen cells into normal recipients, followed by BSA challenge, failed to show any suppression of the resulting primary response. Neither normal nor BSA tolerant neonatal spleen cells were capable of suppressing either primary or secondary responses to SRC. Thus, chickens tolerized to BSA have suppressor cells specific for the tolerizing antigen. We present evidence that both the tolerance associated suppressors and the suppressors detected in normal neonatal chickens are T cells.     

Induction of nitrite production in mouse spleen cells by immunization.

Changes of nitrite production in mouse spleen cells of in vitro secondary antibody response were investigated. Mouse spleen cells immunized with gamma globulin fraction of rat serum produced nitrite 3 days after in vitro challenging with the same antigen. Nitrite production of rabbit IgG-challenged spleen cells was found to be about 2.9-times higher than that of spleen cells primed with the gamma globulin fraction of rat serum. Nitrite production in this system was completely suppressed by T cell depletion (99.7% inhibition). Furthermore, nitrite production in these cells significantly decreased by addition of anti-interferon gamma antibody (62.9% inhibition). These data indicate that nitrite production in antigen-immunized spleen cells is affected with the immunogenicity of an antigen and regulated by T cells, especially interferon (IFN) gamma.     

Antigen modulation of the secondary immune response. VI. Contribution of cells recruited from precursor pool to expression of memory.

The adoptive transfer system has been used extensively to study the ability of antigen triggered memory cells to become antibody forming cells and/or to proliferate and expand the memory cell population. Selective antigen triggering of the memory cells for low and high affinity antibody formation has also been studied in this way. One of the main counter-arguments to the interpretation of these data is that the presence of antigen in the adoptive host may lead to recruitment of new memory cells from either a host or donor precursor population. In this paper we examined the contribution of both host and donor precursor cells to the total antibody response in adoptive secondary recipients. The following donor-host combinations were used in which the recipients were given 1 mg fluid antigen intravenously: (A) normal (non-immune) donors to normal irradiated recipients; (B) normal donors to carrier primed irradiated recipients; (C) carrier primed donors to normal irradiated recipients; (D) normal donors to carrier primed recipients with challenge and subsequent transfer to additional carrier primed recipients; (E) carrier primed donor to normal recipients to carrier primed recipients; (F) repeat of B and C above with multiple antigen administration; (G) purified immune (DNP-BGG) donor T cells mixed with normal B cells transferred to normal irradiated recipients. In most cases recruitment was seen but this represented less than 4% of the responses seen with immune cells. Thus we conclude that this level of recruitment does not compromise the use of the adoptive transfer system for studying selective antigen triggering of memory cells.     

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.     

Immune response to chemically modified flagellin. IV. Further studies on the relationship between humoral and cell-mediated immunity

Acetoacetylation converts flagellin from an antigen which preferentially induces humoral antibodies to an antigen which exclusively provokes cell-mediated immunity and, under certain circumstances, induces antibody tolerance. Studies reported in this paper revealed that the acetoacetylated flagellins expressed similar immunological properties in flagellin primed rats as in normal rats. Thus, on the one hand, acetoacetylation destroyed the capacity of flagellin to trigger a secondary antibody response, but on the other hand, the acetoacetyl-flagellins very effectively induced delayed-type hypersensitivity reactions in flagellin primed animals. It was concluded from these results that humoral and cell-mediated immunity may be opposing immunological processes in both unprimed and primed animals.Acetoacetylated flagellin induced antibody tolerance in both strain W (low responder) and J (high responder) Wistar rats. Maximum tolerance was induced 12 hr after injection of antigen, but in strain J animals the tolerance had disappeared by 48 hr, whereas in strain W rats tolerance persisted for >28 days. The potential to recover from tolerance in strain J rats appeared to coincide with the level of delayed hypersensitivity at the time of challenge. However, this delayed hypersensitivity disappeared when breaking of tolerance occurred. These results suggest that the T cells which participate in delayed hypersensitivity reactions may also act as “helper” cells in antibody responses. On the other hand, it was found that priming for a secondary antibody response by flagellin appeared to coincide with development of primary antibodies rather than with induction of delayed-type hypersensitivity. The relative importance of specific T and B cells in these phenomena is discussed.     

Immunoregulation of lysozyme-specific suppression. III. Epitope-specific amplification of immunosuppression induced by monoclonal suppressor-T-cell products

The hen egg-white lysozyme (HEL)-specific suppression induced by soluble molecules produced by a monoclonal T-cell lymphoma line (LH8-105) obtained from HEL-specific suppressor T lymphocytes has been examined. Injection of I-J+ molecules from LH8-105 cell culture supernatant (TsFa) in HEL-primed mice during the afferent phase of the response induced Lyt-2+ second order suppressor T (Ts) cells which, upon transfer into HEL-CFA-primed syngeneic recipients, inhibit the delayed-type hypersensitivity (DTH) response to HEL. Transfer of spleen cells from TsFa-injected mice primed with HEL or human lysozyme suppresses the DTH response to HEL in recipient mice whereas this response is not affected by cell transfer from ring-necked pheasant egg-white lysozyme (REL)-primed and TsFa-injected mice, indicating that induction of second order Ts by TsFa is specific for a lysozyme epitope including phenylalanine at position 3. Fine antigenic specificity of second order Ts-cell induction is confirmed by similar results obtained upon injection of TsFa in mice primed with HEL N-terminal synthetic peptide or with an analog in which, as in REL, phenylalanine has been substituted by tyrosine at position 3. The same fine antigenic specificity observed in the induction of second order Ts cells is also present in the expression of TsFe suppressive activity. The similar antigenic specificity of Tsa and Tse suggests that Tse cells could result from amplification of the Tsa cell population or these two cell subsets could reflect different maturation stages of the same cell type rather than distinct T-cell populations activated in cascade.     

The role of cellular proliferation in the induction of experimental autoimmune thyroiditis (EAT) in mice

Experimental autoimmune thyroiditis (EAT) can be induced in susceptible strains of mice by injection of mouse thyroglobulin (MTg) and adjuvant. Lymphocytes from immunized mice develop a proliferative response to MTg which generally correlates with the development of EAT. We utilize a cell transfer system wherein spleen cells from CBA/J mice primed with MTg and lipopolysaccharide (LPS) in vivo are activated by culture with MTg in vitro to transfer EAT to naive recipients. In vivo priming of CBA/J mice is required to develop an antigen specific proliferative response to MTg. This response is optimal between 48 and 90 hr of culture at an MTg concentration of 125-250 micrograms/ml. The correlation between proliferation and transfer of EAT is not absolute as primed Balb/c X CBA/J F1 and AKR lymphocytes do not proliferate detectably in response to MTg but can be activated to transfer EAT; primed Balb/c lymphocytes neither proliferate nor transfer EAT. Proliferation per se is not sufficient to activate cells to transfer EAT as culture with nonspecific mitogens is not effective in activating primed CBA/J spleen cells to transfer EAT. However, lymphoblasts generated during in vitro culture of primed CBA/J spleen cells with MTg are responsible for transfer of EAT; small lymphocytes are ineffective. We conclude that antigen specific proliferation in response to MTg is essential in activating lymphocytes in vitro to transfer EAT.     

An in vitro model for induction of immunologic unresponsiveness to turkey gamma-globulin in primed mouse spleen cells, II. Antigen-specific suppressor cells.

Unresponsiveness induced to turkey γ-globulin (TGG) in cultures of TGG-primed spleen cells by incubation with high concentrations of soluble TGG (sTGG) was shown to involve a state of active suppression. Upon transfer to secondary cultures of primed spleen cells stimulated with an optimal dose of TGG-conjugated erythrocytes, such tolerant spleen cells were able to actively inhibit a secondary plaque-forming cell response to TGG in these cultures. Almost complete inhibition was observed with a tolerant cell to primed cell ratio of as low as 0.1. The suppression was antigen specific in that tolerant spleen cells which were suppressive for the secondary TGG response were unable to inhibit a primary response to sheep erythrocytes. T cells were shown to be required for the suppressor effect, in that (i) suppressor activity could be removed by complement-mediated lysis with an anti-Thy 1.2 antiserum and (ii) suppressor activity was retained in the effluent fraction after passage of suppressor spleen cells over a nylon wool column. Induction of the T-cell suppressor activity was found to be associated with a loss of T-cell helper activity within the TGG-pulsed cell population. The presence of adherent cells was not required for induction of suppressor activity. Furthermore, the suppressor effect was found to be resistant to 1000 R of γ irradiation.     

T cell memory for the cytotoxic response to hapten-modified target cells.

This study describes the development of memory and cytotoxic murine T cells against syngeneic haptne N equals[N-(3-nitro-4-hydroxy-5-iodophenyl-acetyl)-Beta-alanylglycylglycyl] associated antigen. Memory activity in this system had the following characteristics. a) In vitro challenged cells primed in vivo resulted in an augmented cytotoxic response compared to cells primed in vitro. b) The augmented cytotoxic response in vitro was antigen-specific for both target cells in the lytic reaction and stimulator cells in the secondary response. c) Memory activity was long lasting (at least 2 months). d) Memory cells were not cytotoxic. e) Memory activity as well as the cytotoxic cells generated in a secondary response in vitro were T cell dependent, These findings are consistent with the results of others who have investigated T cell dependent memory in other cell-mediated reactions.     

Establishment of unresponsiveness in primed B lymphocytes in vivo

As an approach to examine the influence of the state of cellular activation on the ability to tolerize B cells, the induction of unresponsiveness in human gamma-globulin-(HGG) primed B lymphocytes was studied in an adoptive transfer system. In contrast to transferred normal spleen cells, spleen cells from HGG-primed mice are not readily rendered unresponsive when exposed to the tolerogen, deaggregated HGG (DHGG), in irradiated recipients. A kinetic study showed that unfractionated primed spleen cells do not respond to an antigenic challenge given between 6 and 10 days after cell transfer and injection of DHGG, indicating that they are transiently depressed. In contrast, isolated primed B cells are tolerized when transferred to recipients and treated with DHGG in the absence of T cells. Furthermore, primed B cells exposed to tolerogen in the recipients do not recover the ability to respond to HGG either after a secondary challenge with AHGG given up to 14 days after transfer, or after 2 consecutive challenges given on days 14 and 24 after transfer. The presence of primed T cells at the time of tolerization interferes with the induction of unresponsiveness in these primed B cells. These studies suggest that the presence of primed T cells is responsible for the inability to tolerize unfractionated primed spleen cells populations and that primed B cells themselves are not intrinsically resistant to the induction of unresponsiveness.     

Regulation of anti-hapten antibody response by chemically modified carrier antigen preferentially provoking delayed-type hypersensitivity (DTH). II. DTH-reactivity and carrier-specific suppression of anti-DNP antibody response induced by priming with dodecanoyl-BSA are mediated by functionally distinct T cell subpopulations.

Experiments were carried out to determine whether or not the cell populations involved in DTH and in the suppression of antibody response are identical. The effects of four treatments, i.e., adult thymectomy (ATx), X-irradiation, anti-mouse thymocyte serum (ATS) and hydrocortisone (HC) on the induction of DTH and on the carrier-specific suppression of antibody response were observed in mice immunized with chemically modified antigen, dodecanoyl-BSA (d-BSA), emulsified with complete Freund's adjuvant (CFA), with the following results: 1) DTH induced by immunization with D -BSA remained constant in adult thymectomized mice, whereas the suppression of antibody response was not inducible in these animals. 2) Injection of low doses of ATS caused the depression of DTH in mice primed with D -BSA, but did not affect the suppressive activities of their spleen cells. 3) Sublethal X-irradiation 1 week prior to D -BSA priming inhibited the generation of suppressor cells but did not affect the generation of cells mediating DTH. The suppressive effect was also abrogated by sublethal X-irradiation given 2 days after immunization with DNP-BSA (14 days after priming with D -BSA). 4) The treatment of animals with HC 2 days before the footpad challenge or immunization with DNP-BSA depressed the ability of animals to induce both DTH and the suppression of antibody response. However, the latter was more sensitive to HC than the former. In addition to these results, it was also found that D -BSA-primed spleen cells were capable of suppressing anti-DNP response, but not of inducing DTH-reactivity upon transfer to recipient mice. These results suggest that DTH-reactivity and the carrier-specific suppression of anti-hapten antibody response induced by injection of D -BSA are mediated by different cell populations.     

Augmentation of immune response to syngeneic fibrosarcoma T241 with in vivo protection

Summary Lewis T241 fibrosarcoma, a syngeneic tumor in C57 BL/6J mice, was found to be poorly immunogenic. When tumor-bearing animals (TBA) were challenged with tumor cells either concomitantly or after excision of a growing tumor no protection was observed. In vivo (Winn) neutralization assays also showed a lack of tumor immunogenicity. However, in vitro studies showed that a significant proliferative response could be elicited from the spleen cells of TBA when these cells were cultured with either mitomycin-C-treated tumor cells or KCl tumor extract. Similarly, macrophage migration inhibition factor (MIF) was produced by TBA spleen cells upon incubation with KCl tumor extract, but no cell-mediated cytotoxicity to T241 target cells was observed with various lymphoid cell populations at any stage of tumor growth. Immunization of syngeneic animals with Vibrio cholerae neuraminidase(VCN)-treated, irradiated tumor cells alone or admixed with Freund's complete adjuvant (FCA) resulted in decreased tumor growth and fewer pulmonary metastases following challenge with 10⁶ tumor cells. No complete tumor rejection was observed. In contrast, 13 of 16 animals immunized with irradiated tumor cells admixed with FCA rejected 10⁵ tumor cells. Animals that grew tumors had significantly reduced tumor growths and pulmonary metastases. Lymph node and peritoneal exudate cells (PEC) of immunized animals showed significant cytotoxicity to T241 cells.     

Studies on the mechanism of suppression by T cells in an adoptive secondary response.

Suppression of antibody synthesis by lymphocytes was studied using an adoptive secondary response model in which human serum albumin (HSA)-primed lymphocytes (memory cells) from the thoracic ducts of inbred rats were inhibited in irradiated recipients by nonimmune lymphocytes after mixed cell transfer. This investigation extended earlier work and formally showed that the suppressor cells were peripheral thymus-derived lymphocytes, which could rapidly recirculate from the blood to lymph, were present in spleen but not in bone marrow, and that primed T cells lacked this property to inhibit. The suppressive effect was independent of antigen dose but was markedly influenced by the form of antigen used for challenge in that suppression was significantly abrogated with aggregated HSA or with soluble HSA in the presence of specific antibody. Suppressor cells were found to exert their effect maximally at the time of antigen injection, but became ineffective by 40 hr following challenge. The results are considered within a larger framework of cellular regulation of antibody synthesis.     

Functional heterogeneity of memory B lymphocytes: in vivo analysis of TD-primed B cells responsive to secondary stimulation with TD and TI antigens

The functional heterogeneity of memory B cells induced by a single determinant, consisting of a decapeptide representing amino acid residues 103-112 of tobacco mosaic virus protein (TMVP), was analyzed. Decapeptide specific antibodies were elicited in mice adoptively transferred with TMVP-immune spleen cells when challenged with TMVP, decapeptide conjugated to succinylated human gamma-globulin (SHGG), or decapeptide conjugated to Brucella abortus (BA). Whereas secondary stimulation by either TMVP or decapeptide-SHGG was dependent on appropriately primed T cells, stimulation by decapeptide-BA was independent of conventional T cell help. Furthermore, memory B cells responsive to TMVP (TD), decapeptide-SHGG (TD), or decapeptide-BA (TI. 1 prototype) were shown to consist of overlapping populations because adoptive recipients of TMVP-primed cells challenged simultaneously with TD and TI decapeptide antigens did not result in a higher antibody response than that elicited by one of the TD antigens injected alone. However, decapeptide-BA consistently induced a smaller antidecapeptide response than either TMVP or decapeptide-SHGG. This suggested that only a fraction of the memory B cell population which was activated by the original priming antigen (thymus-dependent) was also responsive to secondary in vivo stimulation by the priming hapten conjugated to Brucella abortus. Detailed analyses of the antibodies induced in the recipients of TMVP-immune spleen cells after secondary challenge with either TMVP, decapeptide-SHGG, or decapeptide-BA failed to distinguish between the responsive memory B cells; the antidecapeptide antibodies induced by all three immunogens shared the same fine specificities and immunoglobulin isotype composition. These data are viewed as further evidence that subsets of TD-primed B cells, which may display differential sensitivity to cross-stimulation with TD and TI forms of the antigen, represent distinct stages of memory B cell maturation within a common B cell lineage. In support of this conclusion, we establish a developmental relationship between TI and/or TD responsive decapeptide memory B cell in the following communication.     

The antigen-binding capacity of mouse lymphocytes. II. Changes during the early immune response

The interaction of antigen with specific receptor sites of primed murine spleen lymphocytes has been estimated quantitatively. The antigen-specific receptors are restricted to a small segment of the entire spleen cell population, one which may be enriched by centrifugation of the cells in a discontinuous density gradient. The specificity of the receptors, their variable affinities for the specific ligand, their quantitative fluctuations following immunization, have been investigated. Attempts have also been initiated for their isolation. The possibility that these receptors may represent passively adherent cytophilic immunoglobulins was excluded. The results of these experiments suggest that the union of antigen with a portion of cell membrane receptors initiates a sequence of reaction steps involving DNA production and multiplication, leading to a small net synthesis of immunoglobulins. The continued presence of antigen during this period leads to complex formation with these immunoglobulins. The resulting immune complexes bind to both adherent and nonadherent cells, thereby amplifying the immune response.     

Propagation of antigen-specific T cell helper function in vitro.

Antigen-induced proliferation of primed lymph node cells was abrogated by treatment with anti-Ly 1 serum and complement (C) but not with anti-Ly 2 serum and C. Lymph node cells from animals primed to ovalbumin were activated with antigen in vitro, followed by propagation in an antigen-free supernatant fluid obtained from lectin-induced normal spleen cells. T cells processed in this manner displayed a stepwise enrichment of helper activity for antibody production as measured in a secondary hapten-carrier response. The sequential increase in antigen-specific help was paralleled by a rise in the antigen-induced proliferative response, a phenomenon whose expression was dependent on the presence of syngeneic or semi-syngeneic irradiated filler cells.