Allotypic suppression of adult mouse spleen cells

Adult mouse spleen cells were transferred into irradiated adult mice with xenogeneic erythrocytes. Antibody-producing cells were measured by the localized haemolysis in gel (LHG) assay. Anti-allotype serum directed against the γG_2a allotype of the donor cells, given to the recipients at the same time as antigen and spleen cells, suppressed the plaque-forming cells (PFC) producing antibody of that allotype, and had little effect on the other classes investigated.     

Allotype suppression in the chicken II. Suppression in homozygous chickens with antiallotype antibody and allotype-disparate B cells

Injection of heterozygous (M-1³/M-1^b, G-1^g/G-1ⁱ) B 14-line chickens with antisera directed against either IgM-1a or IgM-1b induced suppression of the relevant IgM-1 and genetically linked IgG-1 allotypes, whereas a mixture of anti-M-1a and anti-M-1b antibodies failed to produce allotype suppression. Injection of anti-M-1 antiserum into M-1 homozygous chickens induced only a transient delay of a few days in the appearance and rise of serum IgM-1 levels. However, suppression of host allotypes was induced by injecting M-1, G-1 homozygous neonatal or embryonal recipients with anti-M-1 antisera together with B locus-histocompatible allotype-disparate spleen, bone marrow or bursal cells. The active cell type were donor B cells, which established chimerism in the injected hosts, whereas peripheral blood T lymphocytes from agammaglobulinemic donors were ineffective. Allotype suppression was attributed to a homeostatic control mechanism which is exerted by normal B cells (but not T cells) over B cell recruitment in anti-M-1 antibody-treated, immature hosts.     

Transformation of rabbit lymphocytes by anti-allotype serum: ultrastructure of transformed cells and suppression of responding cells by foetal exposure to anti-allotype serum

The transformation of rabbit lymphocytes by anti-allotype serum (AAS) has been confirmed. The transformed cells closely resembled at the ultrastructural level those obtained by phytohaemagglutinin stimulation. The cytoplasm contained abundant free ribosomes with virtually no rough surfaced endoplasmic reticulum. A feature peculiar to AAS stimulated cultures was the presence of an amorphous material in and around the blast cells which might represent immune complexes of AAS with its antigen. The suppression of immunoglobulin allotype in animals heterozygous at a given locus by foetal exposure to AAS has been confirmed. It was further shown that this allotypic suppression was accompanied by gross deficiency in the number of peripheral blood lymphocytes which transformed in the presence of the AAS to which the animal had initially been exposed in utero.     

Allotype suppression in the chicken. IV. Deletion of B cells and lack of suppressor cells during chronic suppression

Embryonally induced allotype suppression in M-1,G-1 heterozygous chickens was stable for at least 18 months after hatching. Suppression was established rapidly since injection of antigen only 4 days after anti-IgM-1 antiserum failed to abrogate its effect. Injected chickens had undetectable serum levels (i.e. less than 40 micrograms/ml) of the suppressed IgM-1 and low (0.3-0.6 mg/ml) levels of the linked IgG-1 allotype. This correlated with a complete depletion of cells bearing the relevant IgM-1 allotypes and a compensatory increase in the alternative nonsuppressed IgM-1 allotype-bearing cells in the spleen, peripheral blood and bursa. Cell transfer studies suggested that suppression could not be attributed to allotype-specific suppressor cells.     

The effect of antigen dosage on the response of adoptively transferred cells

Mice were immunized with BSA or HSA in Freund's adjuvant, and their lymph node and spleen cells transplanted into syngeneic hosts, which in most experiments had been irradiated. After transplantation the cells do not synthesize much antibody if left without stimulation, but can be stimulated to do so by injection of BSA or HSA in solution. The response has been studied over a dose range of 10^-3–10⁵ μg. antigen. Stimulation can be detected down to 10^-3 μg. antigen, and reaches a maximum at middling doses. Middling doses stimulate proliferation of the primed cells to an extent which can be measured by ¹³¹IUdR uptake. At high doses both antibody production and IUdR uptake are inhibited. The conclusion is drawn that high concentrations of antigen can paralyse the immunological reaction of primed cells.     

Active suppression masks an underlying enhancement of antibody production in vitro by spleen cells from BCG-infected mice.

The depressed antibody responses resulting from the administration of live BCG i.v. to mice have been investigated. The antibody response of spleen cells to SRBC or DNP-Ficoll in vitro was followed using Marbrook culture vessels. Depressed responses were also found in vivo confirming the results obtained in vitro. The response in vitro of normal spleen cells was suppressed by the addition of spleen cells from mice injected with BCG but not by the medium in which they had been growing for 2 days. The response of the normal spleen cells was also not suppressed by freeze/thaw disrupted BCG spleen cells, suggesting that the depressed responses in the mice injected with BCG are due to an active suppression by intact cells. This was confirmed by the cell-depletion experiments. Removal of cells from the BCG-primed cell populations using carbonyl iron or adherence to plastic not only abrogated the depressed responses but revealed an underlying enhancement of the immune response. The data suggest that the suppressive cell might be a macrophage.     

Cellular requirements for the in vitro induction of specific suppression of antibody responses by immune spleen cells

Feedback regulatory signals to an intermediary suppressor cell may provide the mechanism of the suppression of antibody responses which has been described in cocultures of primed (immune) and unprimed (normal) spleen cells. The active cell(s) in the unprimed spleen population is nonadherent to nylon wool, Sephadex G-10 and glass beads. Lymph node cells and cortisone-resistant thymocytes from normal animals act similarly to normal spleen cells in this coculture system. Spleen cells from homozygous nude mice, unlike their heterozygous thymus-bearing littermates, do not produce a high degree of suppression in coculture with immune spleen cells. These data strongly suggest that the normal cell which interacts with primed cells in the cocultures to produce suppression is of thymic origin. However, spleen cells from neonatally thymectomized mice are suppressive in the presence of spleen cells immune to sheep red blood cells. The unprimed cell(s) active in the suppression are sensitive (in the presence of complement) to antisera directed against the surface markers, Thy-1, Qa-1, Lyt-1, Lyt-2, and Ia. Most of the antiserum treatments abrogated the suppressive capacity of the normal spleen cells only partially. Only treatment with anti-Qa-1 and complement routinely eliminated the ability of these cells to suppress in the cocultures suggesting either low concentration of or inaccessible surface alloantigen on the active cells. Alternatively, more than one Qa-1-positive cell set from the unprimed population may be involved. It is postulated that there is at least one subset of Qa-1-positive T lymphocytes present in unprimed spleen and lymph node cell populations capable of participating in the suppression of specific secondary antibody responses when cocultured with spleen cells from specifically primed animals.     

Visualizing the dynamic of adoptively transferred T cells during the rejection of large established tumors

Adoptive T-cell therapy (ATCT) can result in tumor rejection, yet the behavior and fate of the introduced T cells remain unclear. We developed a novel bioluminescence mouse model, which enabled highly sensitive detection of T-cell signals at the single-cell level. Transferred T cells preferentially accumulated within antigen-positive tumors, relative to the unaffected areas in each mouse, and remarkably, expanded within both lymphopenic and P14 mice. This expansion was controlled and efficient, as evaluated by bioluminescence imaging (BLI) of the T-cell signals and by tumor rejection respectively. Analysis of the population dynamics of transferred T cells in ATCT of large tumors revealed that proliferation did not always follow a simple linear pattern of expansion, but showed an oscillating pattern of expansion and contraction that was often followed by a rebound, until full tumor rejection was achieved. Furthermore, visualizing the recall response showed that the transferred T cells responded expeditiously, indicating the ability of these cells to survive, establish memory and compete with endogenous T cells for as long as 1 year after rejecting the tumor.     

Longer local retention of adoptively transferred T-LAK cells correlates with lesser adhesion molecule expression than NK-LAK cells.

The local retention of adoptively transferred lymphokine (IL-2)-activated killer (LAK) cells was examined in 11 patients with head and neck carcinoma. Unseparated lymphocytes, T and natural killer (NK) cells isolated from patients were cultured with IL-2 for 7 days, labelled with 99mTc-HMPAO, and immediately injected back into the respective donors via the superficial temporal artery or locally into the tumour tissue. The injected LAK cells were periodically traced using a gamma camera, and the LAK cell retention rate was calculated from the radioactivity. One hour after the injection, about 70% of the locally infiltrated LAK cells remained in the tumour tissue, while about half of the LAK cells transferred via the regional artery were dislodged from the tissue. LAK cells induced from T cells (T-LAK) were retained in the tissue for a longer time than LAK cells induced from NK cells (NK-LAK). T-LAK were less chemotactic and less adherent to human umbilical vein endothelial cells (EC), and showed lesser migration through EC. Flow cytometric analysis revealed higher expression of CD11a, CD11b, CD18 and CD49d on NK-LAK compared with T-LAK. MoAbs against these adhesion molecules suppressed adhesion and migration of LAK cells. These results indicate that the rapid disappearance of NK-LAK from the tissue is associated with their greater chemotactic and adhesive as well as migratory activities depending on differing expression of adhesion molecules.     

Allotype suppression in the chicken III. Analysis of the recovery from suppression by neonatally injected or maternal antibodies

Injection of M-1 (Cμ), G-1 (CμgM) heterozygous chickens on the day of hatch with anti-IgM-1 antiserum induced allotype suppression from which chickens recovered over a period of approximately 4 months. The suppression of the serum IgM-1 levels was matched by a decrease in the number of splenic and peripheral blood B cells bearing the relevant IgM-1 allotype, and a compensatory increase in the number of cells bearing the alternative nonsuppressed IgM-1 allotype. However, the proportion of IgM-1-bearing bursal cells was only marginally altered. The recovery from suppression was due to B cell recruitment and could be abrogated by bursectomy. Allotype suppression induced in ovo or maintained by repeated injections of anti-IgM-1 anti-serum resulted in chronic suppression and depletion of the relevant peripheral as well as bursal IgM-1-bearing cells. Antibody titers of the relevant allotype in partially suppressed chickens generally correlated with serum allotype levels without clonal restriction in antibody response of the suppressed allotype.     

Suppression of myosin-induced and adoptively transferred myocarditis by prior treatment with complete Freund's adjuvant.

BACKGROUND: We evaluated the effect of pretreatment with complete Freund's adjuvant (CFA) on experimentally induced myocarditis concomitant with the assessment of antigen-specific properties of disease-triggering lymphocytes. METHODS AND RESULTS: Rats pretreated with CFA a week prior to myosin immunization developed a significantly attenuated myocardial inflammation as compared to control-treated animals. Furthermore, prior administration of CFA virtually abolished histological evidence of myocarditis induced by transfer of antimyosin lymphocytes. CFA administered subcutaneously prior to myosin immunization resulted in a significant reduction in lymph node cell reactivity to myosin. Assessment of cultured medium from lymphocytes obtained from CFA-pretreated myosin-immunized rats revealed reduced levels of interferon gamma but an increased production of IL-10, suggesting an induction of a Thl to Th2 switch. CONCLUSIONS: Thus, CFA treatment suppressed both myosin-induced as well as adoptively transferred myocarditis concomitant with induction of antigen-specific unresponsiveness to myosin and skewing of the immune response in favor of a Th2-dominated profile.     

过继转输的胚胎抗原耐受T、B细胞在受体孕鼠体内的归巢和分析

目的 :观察过继转输的胚胎抗原耐受T、B细胞在受体孕鼠体内的归巢和分布 ,以探讨免疫耐受T细胞在诱导受体母 胎免疫耐受中的作用机制。方法 :于孕 4天 (着床期 )给小鼠自然流产模型CBA J×DBA 2孕鼠腹腔注射抗CD80和CD86mAb ,以诱导母 胎免疫耐受。孕 9天应用免疫磁珠分选孕鼠脾脏T、B细胞 ,并用CFSE体外荧光标记。将标记的T、B细胞分别转输至孕 4天的CBA J×DBA 2孕鼠 ,36小时后在双光子共聚焦显微镜下观察T、B细胞在受体体内脾脏、子宫引流淋巴结及母 胎界面组织中的分布。结果 :过继转输的T、B细胞分布在孕鼠体内脾脏和子宫引流淋巴结 ,但并不留驻在母 胎界面。结论 :过继转输的胚胎抗原耐受T细胞定居于外周免疫器官 ,从而介导受体孕鼠对相应父系抗原的免疫耐受。     

Quantification of in vitro antibody secretion by immune spleen cells.

A simple method to study 19S anti-SRBC antibody production is described. This technique, performed by incubation of immune spleen cells, SRBC and C in the fluid phase, utilises photomeric estimation of antibody mediated haemoglobin release. The test is quicker and more accurate than the two-step haemolytic plaque assay (Jerne technique). In combination with the Jerne technique this method gives information about haemolysin production per PFC. This was maximal on day 2 and on day 6 after immunisation, and least on day 4. The steady state value was reached again at day 9. In principle this method is suitable for the estimation of IgG production.     

Allotype suppression induced in the adoptive transfer system: the variables of the system and an apparent absence of a role for T cells.

A study has been made of the variables concerned in allotype suppression of adult spleen cells in the adoptive transfer system. These are; SRBC (antigen) dose; the dose and timing of injection of anti-allotype serum IgG; the number of spleen cells transferred and whether these cells were taken from primed or unprimed donors. Adoptively transferred primed cells are considerably less susceptible to suppression by concomitantly injected anti-allotype serum IgG than are unprimed spleen cells. Injection of anti-allotype serum during the period after adoptive transfer, has shown that primed cells loose their susceptibility sooner (2 days) than the unprimed cells (4 days). Allotype heterozygous CBA spleen cells are less susceptible heterozygous CBA spleen cells are less susceptible to allotype suppression than either allotypically homozygous or heterozygous non-H-2k cells (H-2b,d, or s). Allotype suppression of the TI IgG response to DNP-Ficoll was measured 7 days after adoptive transfer of allotype-homozygous cells from both normal and nude CBA mice (unprimed). The results indicate that T cells do not play a role in the initiation of short-term allotype suppression in the adoptive transfer system.     

Marginal metallophilic cells of the mouse spleen identified by a monoclonal antibody.

A monoclonal antibody, MOMA-1, is described that recognizes the marginal metallophilic macrophages in the mouse spleen. The cells are localized at the marginal sinus forming a ring around the periarteriolar lymphocyte sheath and follicular areas at the inner side of the marginal zone. They show high non-specific esterase activity and can be distinguished from the marginal zone macrophages by antibody staining and the lack of FITC-Ficoll uptake. The relationship of MOMA-1-positive cells with other macrophage populations is discussed.     

Protective immunity in murine histoplasmosis: functional comparison of adoptively transferred T-cell clones and splenic T cells.

In this study, I examined whether a murine T-cell line and three clones that recognize Histoplasma capsulatum antigens in vitro could confer protection in vivo against a challenge of Histoplasma yeasts. C57BL/6 mice were each inoculated with 5 X 10(4) yeasts intravenously; 1 h later, 5 X 10(6) or 2 X 10(7) resting T cells were inoculated intravenously. At week 1 of infection, the T-cell line and all clones failed to reduce the number of H. capsulatum CFU in the spleens of mice compared with numbers in infected controls. Administration of recombinant interleukin 2 or cyclophosphamide to infected mice did not potentiate the functional activity in vivo of either the T-cell line or the clones. In contrast, inoculation with 2 X 10(7) CD4+ but not CD8+ cells isolated from the spleens of mice immunized with 10(6) viable yeast cells sharply diminished the number of CFU in the spleens of infected animals. Moreover, splenic CD4+ cells from immune mice transferred a delayed-type hypersensitivity response, whereas the T-cell line and clones did not. Injection Injection of an equal number of cloned T cells and CD8+ splenocytes from immune mice did not transfer resistance to infected mice. Additional studies were undertaken to determine if the ineffectiveness of cloned T cells was associated with a failure to migrate to and survive within spleens of infected mice. B6.PL Thy-1a/Cy mice, which are genetically identical to C57BL/6 mice except that T cells of the former bear Thy-1.1 rather than Thy-1.2, were inoculated with Histoplasma yeasts and then injected with immune CD4+ splenocytes or a T-cell clone. At days 1 and 7 of infection, virtually no Thy-1.2+ cells were detected in the spleens of infected mice given cloned T cells. However, the spleens of animals inoculated with immune CD4+ cells contained a small but significant (P less than 0.01) proportion of Thy-1.2+ cells at both day 1 and day 7 postinoculation of H. capsulatum. Thus, the failure of T-cell clones to transfer protection against H. capsulatum may be explained by defective trafficking or poor survival in vivo or both.     

Immunosuppression induced by attenuated Salmonella: effect of LPS responsiveness on development of suppression.

A live, avirulent strain of Salmonella typhimurium, SL3235, was previously shown to afford protection against virulent Salmonella challenge in three mouse strains of the C3H lineage, C3H/HeJ, C3HeB/FeJ, and C3H/HeNCrlBR, which differ in their innate susceptibility to Salmonella infection, as well as in their responsiveness to lipopolysaccharide (LPS). Concurrent with protection, however, SL3235 was found to induce greater than 90% reduction in proliferative responses of splenocytes from immunized mice to a panel of B and T cell mitogens. Suppression appeared to be independent of susceptibility to Salmonella infection, since the mitogenic responses of hypersusceptible C3H/HeJ and C3HeB/FeJ, as well as resistant C3H/HeNCrlBR mice, were suppressed. The suppressor cell population in immunized C3HeB/FeJ mice was recently shown to be of monocytic lineage. Using transwell plates, co-culture studies indicated that suppression was mediated by soluble factors. In the present study, the effect of LPS responsiveness on susceptibility to SL3235-induced suppression was evaluated in C3H mice by studying their ability to mount plaque-forming cell (PFC) responses to sheep red blood cells (SRBC) and in vivo antibody responses to tetanus toxoid. Comparison of PFC responses as a function of SL3235 dose in C3HeB/FeJ and C3H/HeJ mice, revealed that the latter strain was markedly more resistant to the development of suppression, as evidenced by the significantly higher (10-35-fold) SL3235 doses needed to achieve comparable suppression to those seen in C3HeB/FeJ mice. In contrast to C3HeB/FeJ mice, suppression in C3H/HeJ mice required direct cell-cell contact. In both mouse strains, suppression was alleviated by pre-treatment of immune splenocytes with either mitomycin C or x-irradiation, indicating that actively proliferating cells are required for suppressor function. Resistance of C3H/HeJ mice to SL3235-induced suppression was not due to a lesser bacterial load in vivo, since a higher number of SL3235 organisms were seen in C3H/HeJ spleens compared to C3HeB/FeJ mice. Rather, resistance of C3H/HeJ mice correlated with their reduced ability to recruit macrophages and other inflammatory cells into the spleen, as evidenced by the significantly smaller degree of splenomegaly induced in these mice following immunization with SL3235.     

Specific suppression of the local GVH reaction by F1 spleen cells

The local graft-versus-host (GvH) reaction in (C57BL/6 X BALB/c) F1 hybrid mice, assayed by popliteal lymph node enlargement, was specifically depressed by an injection of parental lymphocytes mixed with spleen cells from F1 mice pretreated with the same parental lymphocytes. Suppressor activity of CBF1 spleen cells was obtained 7 days after inoculation of parental lymphocytes, and peaked on day 10. The suppressive activity was induced by only spleen cells from CBF1 which was inoculated Balb/c lymphocytes, but not C57BL/6 lymphocytes. The lymphocyte subpopulation responsible for the suppressive activity was noticed in T cell population.     

Allotype suppression in the chicken. I. Generation of chronic suppression in heterozygous but not in homozygous chickens.

The injection of antibody directed against either the IgM-1 a or IgM-1 b allotype into heterozygous (M-l^a/M-l^b, G-l^g/G-lⁱ) B 14-line chickens produced suppression of the relevant IgM-1 and genetically linked IgG-1 allotypes in their serum, as quantitated by single radial immunodiffusion. Suppression of the IgM-1 and IgG-1 allotypes was associated with a compensatory increase in the alternative IgM-1 and IgG-1 serum allotype levels. This suppression was induced (a) by passive injection of anti-allotype antiserum into 13-day-old embryonal or neonatal recipients, or (b) by egg yolktransmitted antibody in chickens hatched from homozygous B 14 A (M-l^a, G-l^g) hens immunized against the IgM-l b of the homozygous B14C (M-l^b, G-lⁱ) rooster. Heterozygous chickens injected embryonally with anti-allotype antiserum were profoundly suppressed for at least 16 weeks after hatching, while neonatally injected chickens showed a gradual recovery of both IgM-1 and IgG-1 allotypes over the same period. Suppression of the IgM-1 b allotype could be induced in heterozygotes which had inherited the M-l^b allele either maternally or paternally. However, no suppression of either IgM-1 or IgG-1 levels could be detected in homozygous chickens injected with the relevant anti-allotype antiserum. Hence, allotype suppression only occurred in M-1 heterozygous chickens which had an alternative source of B cells available. The involvement of a B cell surveillance mechanism in allotype suppression is postulated and the possible role of suppressor cells is discussed.