Migration of newly formed small lymphocytes from bone marrow to lymph nodes during primary immune response

Selective DNA labelling of bone marrow cells in vivo was used to determine the effect of antigenic stimulation on the migration of small lymphocytes from bone marrow to popliteal lymph nodes. Following footpad injection of keyhole limpet haemocyanin (KLH) in guinea-pigs the regional nodes showed an early increase in weight and cellularity together with a progressive increase in cell proliferation. When [³H]thymidine was injected into tibial and femoral marrow 2 days before KLH administration the DNA radioactivity of the KLH-stimulated nodes increased rapidly and always exceeded that of contralateral nodes. Simultaneously, in radioautographic sections of lymph nodes labelled small lymphocytes, indicative of an origin from marrow precursors, appeared throughout the cortex, post-capillary venules, subcapsular sinus, medullary cords and sinuses. In KLH-stimulated nodes the number of labelled small lymphocytes per section was higher than in contralateral nodes, especially in the cortex, and some of these cells appeared in germinal centres. Labelled large blast cells and macrophages were also increased in numbers. Similar changes were observed in lymph nodes of parental strain rats following intramyeloid [³H]thymidine administration and footpad injection of lymphoid cells from F1 hybrid rats. The results demonstrate that, during the early response of lymph nodes to various antigens, local changes in cell traffic include an enhanced accumulation of newly formed small lymphocytes, putative virgin B lymphocytes, generated in the bone marrow prior to the antigenic stimulation.     

Reduced antigen-induced proliferation and surface Ia expression of peripheral blood T cells following tetanus booster immunization

Immune regulatory changes after tetanus booster immunization were studied by measuring T-cell proliferation and surface Ia expression after cocultivation of T cells with autologous tetanus toxoid pulsed macrophages. Both soluble and aluminium-absorbed tetanus toxoid was used as antigen. T cells proliferated readily prior to immunization, if TTAl was used as an antigen (when proliferation was measured by the uptake of [3H]thymidine: mean +/- SEM 64.5 +/- 11.5 X 10(3) dpm). The proliferative response of the peripheral blood T cells studied decreased significantly to 29.3 +/- 9.3 X 10(3) dpm (P less than 0.01) 1 week postimmunization, returned to preimmunization values after 2 to 4 weeks, and increased further thereafter. Surface Ia expression paralleled the proliferative response at every point pre- and postimmunization. Serum antibody levels increased steadily during the first weeks. These results demonstrate a functional parallel to the previously described changes in T-cell phenotypes (M. M. Eibl, J. W. Mannhalter, and G. Zlabinger, N. Engl. J. Med 310, 198, 1984) after tetanus booster immunization.     

In Vitro Proliferation of T Lymphocytes from Listeria-Infected Rodents: Assay Conditions for Rat Peritoneal Exudate Cells and Characterization of an Inhibitor

Conditions favorable to [³H]thymidine incorporation into antigen-stimulated T lymphocytes from Listeria-infected rats have been established. In cultures of peritoneal exudate (T) lymphocytes purified twice with nylon-wool vigorous antigen-specific proliferation was observed within 2 days. Cultures of lymphocytes from nodes draining a subcutaneous Listeria-infection site differed in that back-ground proliferation was higher than for peritoneal exudate lymphocytes, and [³H]thymidine incorporation was maximal at day 3. A critical factor for the rate of proliferation was the lymphocyte-to-macrophage ratio; optimal cultures of peritoneal exudate lymphocytes contained 2 to 5% macrophages. Macrophages exceeding a proportion of 10% strongly, if not completely, inhibited [³H]thymidine incorporation into antigen-stimulated lymphocytes. Inhibition was associated with mononuclear cells, adherent to plastic or nylon-wool, of the stimulated or unstimulated peritoneal cavity. It was neither attributable to release of cold thymidine from macrophages nor to rapid degradation of particulate antigen by macrophages. The degree of inhibition reflected the metabolic activity of macrophages; on a cell-for-cell basis, heat-killed and glutaraldehyde-fixed macrophages were less inhibitory, and stimulated macrophages were more inhibitory than macrophages from the unstimulated peritoneal cavity.     

The recruitment of recirculating lymphocytes in the antigenically stimulated spleen: specific and non-specific consequences of initiating a secondary antibody response

Antigenic stimulation of the rat spleen to initiate a secondary response to tetanus toxoid (tet. tox.) has been found to have two effects on the recirculating lymphocytes which are migrating through the splenic pulp. Firstly, specific antigen-sensitive cells were selected from a population of immune lymphocytes during migration through an isolated, perfused spleen which was stimulated with tet. tox. This was supported by the substantial, and mostly specific, depression in the ability of such migrated cells to mediate a secondary response to tet. tox. and by the large secondary response produced by transplanted fragments of the perfused spleen without further exposure to antigen.

Secondly the i.v. injection of tet. tox. into rats which had been previously transfused with labelled immune lymphocytes or alternatively labelled non-immune lymphocytes was followed by a transient retention of both populations in the spleen at the expense of the lymph nodes. Any surplus retention of the immune population in the stimulated spleen was not detected which suggests with certain reservations that only a small minority of even the immune population were antigen-sensitive cells.

     

Long-term follow-up of children born to women immunized with tetanus toxoid during pregnancy

Ten years after transplacental immunization with tetanus toxoid, the antibody responses of the immunized children to a booster immunization with 5 Lf tetanus toxoid did not differ from those of the control children. The tetanus toxoid-stimulated lymphocyte proliferative responses showed that there was no tolerance to tetanus toxoid induced by transplacental immunization, and the stimulation indices suggested that there may be some long-term memory for tetanus toxoid among the T lymphocytes in children who had been transplacentally immunized by maternal immunization with a standard dose of tetanus toxoid.     

The role of macrophages in the activation of T lymphocytes by concanavalin A. III. Macrophage-independent activation

Lymphocytes from the mesenteric lymph nodes of Lewis rats were depleted of macrophages by absorption for 60 min at 37°C on 0.1-mm glass beads in the presence of 30% fetal calf serum. Lymphocytes which passed the column (total recovery 30-50%) contained less than 0.2% macrophages, as assessed by neutral red ingestion or nonspecific esterase staining. At cell densities which allowed optimal proliferation of concanavalin A (Con A)-stimulated, untreated lymphocytes (2 × 10⁶ cells/ml), macrophage-depleted lymphocytes were completely incapable of initiating DNA synthesis. The proliferative response to Con A was fully restored by adding back macrophages obtained from the peritoneal cavity. The help of macrophages was optimal at concentrations of 0.8 × 10⁵ cells/ml, which corresponds to about 4% of lymphocytes. Restitution of the mitotic response of macrophage-depleted lymphocytes was also achieved by raising cell densities to 5 × 10⁶/ml. At this cell density, addition of macrophages was without any effect. Early events in lymphocyte activation, such as the increase of the incorporation of ¹⁴C-oleate into membrane phospholipids after 4 h stimulation, was identical in the presence or absence of macrophages independent of the cell density ranging between 1 × 10⁶ and 10 × 10⁶ cells/ml. Similarly, the Con A-stimulated early increase in the incorporation of [³H]uridine into RNA was indistinguishable in macrophage-containing or -depleted lymphocytes. At low cell densities, only macrophage-containing lymphocytes proceeded to increase RNA synthesis, until S-phase, whereas in the absence of macrophages RNA synthesis approached background values after 40 h of Con A stimulation. Macrophage-depleted lymphocytes, too, proceeded with an increase in RNA synthesis, when stimulated at high cell densities. The data suggest that macrophages are not required for the initiation of lymphocyte activation. Their role in supporting mitosis may be the contribution of a second signal close to the G1/S boundary extending the size of the proliferating population.     

Immunoglobulin G (IgG) Subclass Distribution and IgG1 Avidity of Antibodies in Human Immunodeficiency Virus-Infected Individuals after Revaccination with Tetanus Toxoid

In human immunodeficiency virus (HIV)-infected individuals the amount of antibodies formed after vaccination with T-cell-dependent recall antigens such as tetanus toxoid is proportional to the peripheral blood CD4⁺ T-lymphocyte counts. To investigate whether the immunoglobulin G (IgG) subclass distribution and avidity of the antibodies produced after vaccination are affected as well, we gave 13 HIV-infected adults with low CD4⁺ T-lymphocyte counts (<200 × 10⁶/liter; group I), 11 HIV-infected adults with intermediate CD4⁺ T-lymphocyte counts (≥200 × 10⁶/liter; group II), and 5 healthy controls booster immunizations with tetanus toxoid. The prevaccination antibody concentrations against tetanus toxoid were similar in the HIV-infected and healthy adults. After vaccination the total IgG and the IgG1 anti-tetanus toxoid antibody concentrations were significantly lower in group I than in group II and the controls. The avidity of the IgG1 anti-tetanus toxoid antibodies formed by HIV-infected adults was within the range for healthy controls, irrespective of their CD4⁺ T-lymphocyte counts.In human immunodeficiency virus (HIV)-infected individuals the amount of antibodies formed after vaccination with T-cell-dependent recall antigens, such as tetanus toxoid, is impaired in proportion to the number of CD4⁺ T cells and to the in vitro proliferative response of T lymphocytes to anti-CD3 monoclonal antibodies (7, 8). Protection against tetanus will depend on the total amount of antibodies, the subclass distribution, and the avidities of the antibodies that are formed. Avidity reflects the combined functional affinities of antibodies formed during a polyclonal humoral immune response and is considered to be a parameter for the efficacy of the antibodies at eliminating or neutralizing the antigen (12). The aim of the present study was to investigate whether, in addition to the concentration of antibodies, the subclass distribution and the avidity of the antibodies formed by HIV-infected individuals after booster vaccination are affected.(This study was presented in part at the 35th Annual Meeting of the Infectious Diseases Society of America, San Francisco, Calif., 13 to 16 September 1997.)     

Autoanti-idiotypic lymphocytes in man

Cells with anti-idiotypic properties were detected 4–6 months following immunization with tetanus toxoid (TT), and were undetectable 10 days following a booster injection. The presence of these cells concomitantly with the normal drop of anti-TT serum titers, and their specific binding to autologous IgG—F(ab′)₂ anti-TT, suggests a negative feedback at the idiotypic network level.     

Kinetics of macrophage recruitment and turnover in peritoneal inflammatory exudates induced by Salmonella or thioglycollate broth

Kinetics of peritoneal macrophage turnover during infection of mice with Salmonella enteritidis or following injection with thioglycollate broth or other peritoneal stimulants has been studied. Single intravenous injections of tritiated thymidine were given and the cells were examined by autoradiography. Maximum labelling of small adherent peritoneal macrophages occurred when 3H-thymidine was given 1 d after Salmonella and the cells were harvested 1 d later. Labelled cells decreased at later times despite maintenance of high numbers of macrophages in the exudates. Results from experiments in which labelled peritoneal cells were reinjected indicated that small, monocyte-enriched, labelled cells were not the major source of the large macrophages. Similar labelling at 2 d was observed using heat-killed Corynebacterium parvum or lipopolysaccharide (LPS) as ip stimulants. Following injection of thioglycollate broth, labelled peritoneal macrophages were only detectable if 3H-thymidine was given before the stimulant. These labelled cells remained longer in the peritoneal cavity. Labelling of and numbers of blood monocytes were consistent with the promotion of monocytopoiesis by Salmonella but not by thioglycollate. The response to thioglycollate but not Salmonella was dependent on the age of the mice. Animals injected with thioglycollate 1 d before Salmonella also had decreased resistance to bacteria and low numbers of labelled peritoneal macrophages. We propose that thioglycollate may recruit from a subset of preformed monocytes and temporarily block monocytopoiesis or macrophage bactericidal activity.     

In vitro studies of delayed hypersensitivity: inhibition of macrophage spreading in rats sensitive to tuberculin and diphtheria toxoid

Wistar rats were sensitized by footpad injection of BCG in adjuvant, or Mycobacterium butyricum in adjuvant, or diphtheria toxoid in Freund's incomplete adjuvant. It was found that the cell population of the peritoneal washings contained approximately 57 per cent macrophages, 22 per cent lymphocytes, 11 per cent granulocytes and 8 per cent mast cells. The lymphocyte count was significantly reduced and the granulocyte count increased after sensitization. The animals sensitized to M. butyricum exhibited delayed skin reactivity to tuberculin and the spreading of macrophages in vitro was significantly inhibited with the same antigen. On the contrary, the spreading of macrophages obtained from animals sensitized to BCG was not inhibited by tuberculin and there was no cutaneous reactivity. Spreading of macrophages obtained from rats sensitized by diphtheria toxoid was significantly inhibited in the presence of diphtheria toxoid, but not in the presence of tuberculin. These animals displayed delayed cutaneous hypersensitivity to diphtheria toxoid. Spreading of macrophages from normal rats was unaffected by serum antibodies. This was true either when the peritoneal cells were treated with antiserum prior to contact with antigen, or when the antigen—antibody reaction took place in the chamber containing the macrophages ready to spread. These results indicate that the technique of macrophage spreading inhibition is able to detect specifically hypersensitivity of delayed type and offers a convenient method for the in vitro study of delayed hypersensitivity.     

Effect of vitamin A and protein-calorie undernutrition on immune responses

In rats rendered vitamin A-deficient, there was a marked atrophy of the thymus and the spleen. To a large extent, but not completely, these changes were a result of the concomitant protein-calorie undernutrition. In the thymus the cortex was depleted of lymphocytes. The number of germinal centres was reduced in the spleen. The incorporation of [³H-methyl]thymidine into DNA in cells derived from thymus and spleen of deficient animals was six times lower than in normal littermate controls. On administration of sheep erythrocytes (SRBC), the number of plaque-forming cells per million nucleated spleen cells in deficient animals was 73±6, as compared with 141±36 in normal controls. Similar observations were made on the titres of haemagglutinins and haemolysins formed in response to SRBC. The response of vitamin A plus protein-calorie-deficient animals to diphtheria and tetanus toxoid injection was poorer than the pair-fed controls. These studies demonstrate an important influence of vitamin A and protein-calorie nutrition on lymphoid organs and on immune responses.     

Factors influencing the rate of 111indium-labelled lymphocytes after transfer to syngeneic rats

Thoracic duct lymphocytes were labelled in vitro with ¹¹¹indium-oxine or ¹¹¹indium-acetylacetone in order to follow their migration after i.v. injection into syngeneic rats. Under certain conditions both preparations produced results which quantitatively confirmed data obtained by other approaches to the physiological pattern of lymphocyte recirculation. However, three significant difficulties were identified: (1) chemical toxicity by minor contaminants of the preparation; (2) radiation damage indicated by a progressive impairment of the recovery of radioactivity from lymph nodes. A labelling concentration of 20 μCi/10⁸ cells was the highest compatible with survival of most lymphocytes for 24 h in vivo as confirmed by autoradiography; (3) rapid loss of ¹¹¹In in vivo found at labelling concentrations below 1 μCi/10⁸ cells. By one week after the injection of lymphocytes labelled at 20 μCi/10⁸ cells most of the ¹¹¹In had been transferred from lymphocytes to non-recirculating radioresistant cells within the spleen and lymph nodes.     

In Vitro Humoral Immune Response of Human Peripheral Blood Lymphocytes to Tetanus Toxoid Sepharose 4B

The in vitro immune response of unfractionated human peripheral blood lymphocytes (PBL) from immune donors who had not been re-immunized with tetanus toxoid (TT) prior to donation was investigated. In this study we were able to stimulate PBL with tetanus toxoid coupled to Sepharose 4B (STT) for production of anti-tetanus toxoid antibody (Ab). Soluble tetanus toxoid or STT alone did not stimulate production of specific Ab. Pokeweed mitogen (PWM) and STT were required for optimal production of IgG and IgM antibodies specific to tetanus toxoid. Specific Ab responses were reduced in low and high concentrations of STT. Depletion of monocytes had no effect on either total IgG or specific IgG synthesis, but decreased the synthesis of both total and specific IgM. Depletion of E-rosette-forming cells decreased the production of specific Ab, suggesting T-dependency of the immune response to STT. Simultaneous production of total immunoglobulin and specific Ab by Sepharose 4B was negligible in the absence of PWM. In the presence of PWM, total immunoglobulin production was optimal, and specific anti-TT Ab production was undetectable. The specificity of the antiTT Ab was studied by absorption of the culture supernates with an STT column which removed all measurable specific Ab.     

Proliferation and emigration of newly formed lymphocytes from pig spleens during an immune response

Normal young pigs were immunized intravenously with sheep red blood cells (SRBC). At various times after a second SRBC injection the spleens were connected to an extracorporeal perfusion system, and proliferating lymphoid cells in the spleens were selectively labelled with tritiated thymidine. One day later the relative and absolute numbers of spleen-derived lymphocytes were determined by autoradiography in the following organs: various parts of the spleen, mesenteric and cervical lymph nodes, thymus, bone marrow, Peyer's patches, tonsils, intestine, lung, liver and blood. From 1 to 7 days after the second SRBC injection, the spleens produced increasing numbers of lymphocytes, and labelled cells were found especially in the blood and bone marrow. The newly formed splenic lymphocytes migrated preferentially to T- but also to B-cell areas in lymph nodes, Peyer's patches and tonsils. In all organs outside the spleen nearly all labelled spleen-derived lymphocytes were small lymphocytes. However, the bone marrow contained a high proportion of labelled immature and mature plasma cells. The spleen produced large numbers of lymphocytes during the secondary immune response, many of which migrated to different organs probably as memory cells, while others were found in the bone marrow as effector cells from the immune response.     

Quantification of antigen-reactive cells among human T lymphocytes.

The number of antigen-reactive cells among human peripheral blood T lymphocytes was estimated by a limiting dilution analysis. Antigen-induced lymphocyte activation was measured by means of incorporation of tritiated thymidine [3H]dThd. We have studied the frequency of memory T cells for the bacterial antigens tuberculin PPD and tetanus toxoid in immune donors, as well as the frequency of alloantigen-reactive T cells. In 11 different donors, the observed frequencies of the antigen-reactive T cell ranged between 1:300 and 1:16 000 for PPD; for tetanus toxoid values, between 1:750 and 1:11 500 were obtained in 5 different donors. The frequency of alloantigen-reactive T cells was found to be higher: between 1:200 and 1:600 (n = 10). For 3 donors, the estimated frequencies proved to be reproducible over a period of several months. Finally, a correlation could be demonstrated between the frequency of PPD-reactive T cells and the [3H]dThd incorporation of 4 X 10(4) PPD-stimulated lymphocytes.     

Production of colony-stimulating factor in mixed leucocyte cultures

Mixed leucocyte cultures (MLC) were prepared from mouse spleens, lymph nodes or human peripheral blood. After 4 days, media from these cultures contained markedly elevated levels of granulocyte/macrophage colony stimulating factor (CSF). If lymphocyte transformation and proliferation was prevented with mitomycin C or by using cell suspensions from congenitally athymic mice, no increased CSF production occurred. CSF was not detected until after 48 hours and lagged behind RNA, protein, and DNA synthesis. When spleen cells from mice undergoing a graft-versus-host (GvH) reaction were cultured alone there was also production of CSF.

Whether lymphocytes responding to allogeneic cells produce CSF directly or stimulate macrophages to produce it is not clear. However, these results suggest a mechanism by which the granulocyte/macrophage system may be activated in immune responses that involve lymphocyte proliferation.

     

Tetanus Toxoid Reactive T Lymphocytes in the Cerebrospinal Fluid of Multiple Sclerosis Patients

Cerebrospinal fluid (CSF) lymphocytes of 6 multiple sclerosis (MS) patients were cultured with tetanus toxoid (TT) and irradiated autologous antigen presenting cells (APC) followed by propagation of the responding T-cells in Interleukin-2 containing medium. TT-reactive cell lines were recovered from 4 of the 6 CSF samples, even though the patients had not been TT booster immunized in recent years. These findings suggest an active circulation of antigen reactive lymphocytes from the systemic immune compartment(s) into the CSF even without recent activation by booster immunization. Since immune reactions to TT are very unlikely to be pathogenic in MS, these findings also indicate that the presence of CSF lymphocytes reactive to a particular antigen does not necessarily imply a causal role.     

嗜酸性粒细胞可作为抗原呈递细胞

目的探讨ＨＬＡ－ＤＲ＋嗜酸性粒细胞（ＥＯＳ）在体外培养条件下作为抗原呈递细胞（ＡＰＣ）将破伤风类毒素抗原（ＴＴ）呈递给Ｔ淋巴细胞的能力。方法新分离的ＥＯＳ经人重组粒细胞－巨噬细胞集落刺激因子刺激２４小时,以诱导ＨＬＡ－ＤＲ的表达,然后将其暴露于不同浓度的ＴＴ,检测ＨＬＡ－ＤＲ＋ＥＯＳ对自身Ｔ细胞增殖反应的影响,以评价ＥＯＳ呈递抗原的能力。结果ＨＬＡ－ＤＲ＋ＥＯＳ于ＴＴ存在时可以明显促进Ｔ细胞的增殖反应,并与ＴＴ的浓度呈剂量相关性。而抗ＨＬＡ－ＤＲ单克隆抗体则可以明显地抑制ＥＯＳ的抗原呈递过程。结论人ＥＯＳ可以摄入和处理抗原并能将其传递给自身Ｔ细胞,而ＥＯＳ呈递抗原的过程具有明显的ＨＬＡ－ＤＲ依赖性。     

Antigens in immunity: XVI. A light and electron microscope study of antigen localization in the rat spleen*

This paper describes the ultrastructural location of labelled antigens and carbon in the spleens of rats from 4 minutes to 5 days after injection. Particular attention was focused on the sites of deposition 4 minutes after intra-arterial injection of microgram quantities of ¹²⁵I-labelled Salmonella flagellar antigens, crayfish haemocyanin and BSA, using colloidal carbon for comparison. The combination of radioautography with both light and electron microscopy showed the importance of antigen binding by lymphocytes in the marginal zone of the spleen. Macrophage sequestration of antigens was not prominent in the spleen, although it occurred in the liver with the flagellar antigens and haemocyanin.

In the spleen marginal zone, avid antigen-binding cells were found in situ 4 minutes after the injection of labelled haemocyanin. These appear to be the counterpart in vivo of antigen-binding lymphocytes prepared in vitro. Such cells also occurred infrequently after the injection of labelled polymerized flagellin, but were not found with either BSA or carbon.

The apparent movement of flagellar antigen from the marginal zone to the white pulp between 1 and 2 hours after injection was seen to involve lymphocyte-associated antigen. The follicular antigen localization occurring from 1 day onwards after injection was on the dendritic reticular cells of germinal centres, as has been described in lymph nodes after subcutaneous injection.

Carbon particles were rapidly sequestered in macrophages of the spleen and liver, although some particles were found between cells in the marginal zone for as long as 2 hours after injection. By 2 and 5 days, however, all the carbon was in phagocytes, even in the white pulp. Differences between the localization of antigens and carbon were clear, even in the ultrastructural sites of their location in tingible body macrophages of germinal centres.

The unexpected emphasis of lymphocyte association with labelled antigens in the spleen marginal zone has allowed a revison of the mechanism previously proposed for the movement of antigens within the microenvironments of the spleen.

     

Tetanus toxoid reactive T lymphocytes in the cerebrospinal fluid of multiple sclerosis patients

Cerebrospinal fluid (CSF) lymphocytes of 6 multiple sclerosis (MS) patients were cultured with tetanus toxoid (TT) and irradiated autologous antigen presenting cells (APC) followed by propagation of the responding T-cells in interleukin-2 containing medium. TT-reactive cell lines were recovered from 4 of the 6 CSF samples, even though the patients had not been TT booster immunized in recent years. These findings suggest an active circulation of antigen reactive lymphocytes from the systemic immune compartment(s) into the CSF even without recent activation by booster immunization. Since immune reactions to TT are very unlikely to be pathogenic in MS, these findings also indicate that presence of CSF lymphocytes reactive to a particular antigen does not necessarily imply a causal role.